NZ618079B2 - Compositions containing, methods involving, and uses of non-natural amino acid linked dolastatin derivatives - Google Patents

Abstract

The disclosure relates to non-natural amino acids and dolastatin analogs (formula (I)) that include at least one non-natural amino acid, and methods for making such non-natural amino acids and polypeptides. The dolastatin analogs can include a wide range of possible functionalities, but typically have at least one oxime, carbonyl, dicarbonyl, and/or hydroxylamine group. Also disclosed herein are non-natural amino acid dolastatin analogs that are further modified post-translationally, methods for effecting such modifications, and methods for purifying such dolastatin analogs. Further disclosed are methods for using such non-natural amino aciddolastatin analogs and modified non-natural amino acid dolastatin analogs, including therapeutic, diagnostic, and other biotechnology use. ve at least one oxime, carbonyl, dicarbonyl, and/or hydroxylamine group. Also disclosed herein are non-natural amino acid dolastatin analogs that are further modified post-translationally, methods for effecting such modifications, and methods for purifying such dolastatin analogs. Further disclosed are methods for using such non-natural amino aciddolastatin analogs and modified non-natural amino acid dolastatin analogs, including therapeutic, diagnostic, and other biotechnology use.

Description

COMPOSITIONS CONTAINING, METHODS INVOLVING, AND USES OF NON-NATURAL AMINO ACID LINKED DOLASTATIN DERIVATIVES CROSS REFERENCE This application claims priority to US. Provisional Application No. 61/491,146, entitled, “Compositions Containing, Methods Involving, and Uses of Non-Natural Amino Acid Linked Dolastatin tives,” filed May 27, 2011, the contents of Which are incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION The ability to incorporate non-genetically encoded amino acids (i.e., “non-natural amino ) into proteins permits the introduction of al functional groups that could provide valuable alternatives to the naturally-occurring onal groups, such as the epsilon —NH2 of , the sulfliydryl —SH of cysteine, the imino group of histidine, etc. Certain chemical functional groups are known to be inert to the functional groups found in the 20 common, genetically-encoded amino acids but react cleanly and efficiently to form stable linkages With functional groups that can be orated onto non-natural amino acids.

Methods are now available to selectively introduce al functional groups that are not found in proteins, that are chemically inert to all of the functional groups found in the 20 common, genetically- d amino acids and that may be used to react efficiently and selectively with reagents comprising certain functional groups to form stable covalent linkages.

SUMMARY OF THE INVENTION Disclosed herein are toxic moieties with one or more linker(s), toxic groups linked to non-natural amino acids, and methods for making such non-natural amino acids and polypeptides.

Some embodiments of the present invention describe a compound, or salt f, comprising Formula (1): may,R70 /\ Me OMeO wherein: Z has the structure of: €Yé\(KAr R5 ; R5 is H, CORg, C1-C6alkyl, or thiazole; R8 is OH or —NH—(alkylene—O)n—NH2; R6 is OH or H; Ar is phenyl or pyridine; R7 is C1-C6alkyl or hydrogen; Y is selected from the group consisting of an hydroxylamine, methyl, aldehyde, ted aldehyde, ketone, protected ketone, thioester, ester, dicarbonyl, hydrazine, amidine, imine, diamine, azide, keto-amine, keto-alkyne, alkyne, cycloalkyne, and ene-dione; L is a linker selected from the group consisting of —alkylene—, —alkylene—C(O)—, lene—O)n— alkylene—, —(alkylene—O)n—alkylene—C(O)—, —(alkylene—O)n—(CH2)n~NHC(O)—(CH2)nv~ —S—S—(CH2)nu~NHC(O)—(alkylene—O)nmv—alkylene—, —(alkylene—O)n—alkylene—W—, — alkylene—C(O)—W—, —(alkylene—O)n—alkylene—U—alkylene—C(O)—, and lene—O)n— alkylene—U—alkylene—; W has the structure of: ILWJUCVJK; U has the structure of: COZH ,nmiwr or L is absent, Y is methyl, R5 is CORg, and R8 is —NH—(alkylene—O)n—NH2; and each n, n', n", n'" and nH" are independently integers greater than or equal to one.

In some embodiments, R5 is le. In other embodiments, R6 is H. In n embodiments, Ar is phenyl. In further or additional embodiments, R7 is methyl. In some embodiments, n is an integer from Ot020,0to 10 orOtoS.

In some embodiments, a compound is described comprising Formula (II): Me Me H2N\ /L\ N O N a N R7 0 /_\ Me OMeO Me Me MeO NH In certain embodiments, L is —(alkylene—O)n—alkylene—. In specific embodiments, each alkylene is — CHQCH2—, n is equal to 3, and R7 is methyl. In other embodiments, L is —alkylene—. In specific embodiments, each alkylene is —CH2CH2— and R7 is methyl or hydrogen. In certain embodiments, L is — (alkylene—O)n—alkylene—C(O)—. In certain specific embodiments, each alkylene is 2—, n is equal to 4, and R7 is methyl. In further or alternative embodiments, L is —(alkylene—O)n—(CH2)nv—NHC(O)— (CH2)nu—C(Me)2—S—S—(CH2)nu~NHC(O)—(alkylene—O)nmv—alkylene—. In specific ments, each alkylene is —CH2CH2—, n is equal to l, n' is equal to 2, n" is equal to l, n '" is equal to 2, n"" is equal to 4, and R7 is methyl.

In some embodiments, Y is azide. In other ments, Y is ctyne. In specific embodiments, the cyclooctyne has a structure of: |©—§ (R19)q each R19 is independently selected from the group consisting of C1-C6 alkyl, C1-C6 alkoxy, ester, ether, thioether, aminoalkyl, halogen, alkyl ester, aryl ester, amide, aryl amide, alkyl halide, alkyl amine, alkyl sulfonic acid, alkyl nitro, thioester, sulfonyl ester, halosulfonyl, nitrile, alkyl nitrile, and nitro; and qis 0,1, 2, 3, 4, 5, 6, 7, 8, 9,10 or 11.

Some embodiments of the present invention be a compound, or salt thereof, comprising Formula (III), (IV), (V) or (VI): L2—N:[IfeN\)J\18%:0 “'9 R7 0 MAMeMe OMeOM Y’ (III) L3—N:[IMfNfiJJi/IN Me R7 0 Me/_\MeMe OMeOM Me\N MeNjN Me Me O Me OMeO MeO NH O 348%HN—L2 Me (IV) Me\N MeNjLN Me O Me/\MeMe OMeOM mNHHN—L3 Rw¥Q>R70 /\ Me OMeO Y] Me L3_N meNi/IN Me R7 0 Me/\MeMe OMeOM N\H J o 2 L4—N Mai/IN Me R7 0 Me/\MeMe OMeOM NH /\ Me OMe O Me Me MeO NH HN—L4 wherein: Z has the structure of: €Yé\(KAr R5 ; R5 is H, CORg, C1-C6alkyl, or thiazole; R8 is OH; R6 is OH or H; Ar is phenyl or pyridine; R7 is C1-C6alkyl or hydrogen; Y and V are each selected from the group consisting of an ylamine, methyl, aldehyde, protected aldehyde, ketone, protected ketone, thioester, ester, dicarbonyl, hydrazine, amidine, imine, diamine, azide, keto-arnine, lkyne, alkyne, lkyne, and ene-dione; L1, L2, L3, and L4 are each linkers independently selected from the group consisting of a bond, — alkylene—, —(alkylene—O)n—alkylene—J—, ene'—J—(alkylene—O)n—alkylene—, —J— (alkylene—O)n—alkylene—, —(alkylene—O)n—alkylene—J—(alkylene—O)n'—alkylene—J'—, — (alkylene—O)n—alkylene—J—alkylene'—, —W—, —alkylene—W—, alkylene'—J—(alkylene—NMe)n— alkylene—W—, —J—(alkylene—NMe)n—alkylene—W—, —J—alkylene—NMe—alkylene'—NMe— alkylene"—W—, and —alkylene— —alkylene'—NMe—alkylene"—NMe—alkylene'"—W—; W has the structure of: Me Me k5; o KW\H 04;\NH2 ; each I and J' independently have the structure of: zit/iiN/‘Zi fiNiO/‘Zi or ‘fiNié’; H H H , , ,mm each n and n' are independently integers greater than or equal to one.

In n embodiments, a compound is described sing a (VII): Me Me 0M9 M H e\'\ll ; ".1 Me Me O /_\ Me OMeO Me Me MeO NHoHNL2 ”Mgr“?fry WU) Me O Me/\MeMe OMeOM NHeReoL1>7VHN—L3 In certain embodiments, L1 is —(alkylene—O)n—alkylene—J—, L2 is —alkylene'—J'—(alkylene—O)n'— alkylene—, L3 is —J"—(alkylene—O)n"—alkylene—, alkylene is 2—, alkylene' is —(CH2)4—, n is l, n' and jiNfl; J5: NiO/EL n" are 3, J has the structure of H J' and J" have the structure of H and R7 is methyl.

, , In other embodiments, L1 is —J—(alkylene—O)n—alkylene—, L2 is —(alkylene—O)nbalkylene—J'—alkylene'—, L3 is —(alkylene—O)nu—alkylene—J"—, alkylene is —CH2CH2—, alkylene' is —(CH2)4—, n is l, n' and n" are 4, and all; I, J' and I" have the ure of H In some embodiments, Y is azide. In other embodiments, Y is cyclooctyne. In specific embodiments, the cyclooctyne has a structure of: |©—§ (R19)q each R19 is independently selected from the group consisting of C1-C6 alkyl, C1-C6 alkoxy, ester, ether, thioether, aminoalkyl, n, alkyl ester, aryl ester, amide, aryl amide, alkyl halide, alkyl amine, alkyl sulfonic acid, alkyl nitro, thioester, sulfonyl ester, halosulfonyl, nitrile, alkyl e, and nitro; and qis 0,1, 2, 3, 4, 5, 6, 7, 8, 9,10 or 11.

Certain ments of the present invention describe a compound comprising Formula (VIII) or (IX): (VIII) wherein: A is optional, and when present is lower alkylene, substituted lower alkylene, lower lkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene, B is optional, and when present is a linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower alkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, - S-, -S-(alkylene or substituted alkylene)-, -S(O)k- where k is l, 2, or 3, -S(O)k(alkylene or substituted ne)-, , -C(O)-(alkylene or tuted alkylene)-, -C(S)-, -C(S)- (alkylene or substituted alkylene)-, -N(R’)-, -NR’-(alkylene or substituted alkylene)-, -C(O)N(R’)-, -CON(R’)-(alkylene or substituted alkylene)-, -CSN(R’)-, -CSN(R’)-(alkylene or substituted alkylene)-, -N(R’)CO-(alkylene or substituted ne)-, -N(R’)C(O)O-, -S(0)kN(R’)-, C(O)N(R’)-, _N(R’)C(S)N(R’)_a -N(R’)S(O)kN(R’)-, -N(R’)-N=, - C(R’)=N—, -C(R’)=N—N(R’)-, -C(R’)=N—N=, -C(R’)2-N=N-, and -C(R’)2-N(R’)-N(R’)-, where each R’ is independently H, alkyl, or substituted alkyl, R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R1 is H, an amino protecting group, resin, at least one amino acid, polypeptide, or cleotide; R2 is OH, an ester protecting group, resin, at least one amino acid, polypeptide, or polynucleotide; R3 and R4 are each independently H, halogen, lower alkyl, or substituted lower alkyl, or R3 and R4 or two R3 groups optionally form a cycloalkyl or a heterocycloalkyl; Z has the ure of: €Yé\(KAr R5 ; R5 is H, COZH, C1-C6alkyl, or thiazole; R6 is OH or H; Ar is phenyl or pyridine; R7 is C1-C6alkyl or hydrogen; L is a linker selected from the group consisting of ene—, —alkylene—C(O)—, —(alkylene—O)n— alkylene—, —(alkylene—O)n—alkylene—C(O)—, —(alkylene—O)n—(CH2)n~NHC(O)—(CH2)nv~ C(Me)2—S—S—(CH2)nu~NHC(O)—(alkylene—O)nmv—alkylene—, lene—O)n—alkylene—W—, — alkylene—C(O)—W—, —(alkylene—O)n—alkylene—U—alkylene—C(O)—, and —(alkylene—O)n— ne—U—alkylene—; W has the structure of: Lwflfifk“; U has the structure of: COZH Mimi 2012/039472 each n, n', n", n'" and nH" are independently integers greater than or equal to one; or an active metabolite, or a pharmaceutically acceptable prodrug or solvate thereof.

In some embodiments, R1 is a polypeptide. In specific embodiments, the ptide is an antibody. In certain specific embodiments, the antibody is herceptin. In other embodiments, R2 is a polypeptide. In specific embodiments, the polypeptide is an antibody. In n specific embodiments, the antibody is herceptin.

Some embodiments of the present invention describe a nd, or salt thereof, comprising Formula (X), (XI), (XII) or (XIII): ._Mag, RrSLNltwfifir B\A R7 0 /\ Me OMeO R3 R04 R3 R2 Me O Me/_\MeMe OMeO OMeOAMfLMfl—Z R2 31 L (XI) 1 R4 \ R3 (I) ’i‘ R3 Me\NWNJLN NYE Me o Me/KMeMe OMeO OMeOA R L_NIM;H91:];WNMNR7 0 Me/\MeMe OMeOM 9 Me MeH 0 Me R / N NyLM -“H \r N (X11) L3_M Me B\A 0 R7 0 /\ Me OMeO R4 Me Me MeO N\H R3 2 Me Me 9 HN1 H R1 N N Lrw $N Me R7 O /\ Me Me Me MeO NH 0 Z ; Me Me Me Me Me 0 Me\ NHi N N '1“ ; N R7 0 eMe OMeO OMe OArfiM_L2 R6 R2 81 |_ (XHD Me 1 R4 (P A | R3 R7 OMe/\MeMe OMeO OMeOA R Me Me Me e\M ; N R7 O /\ R 12 OMeO Nj\)R:N_L4H Me Me OMeOAr R6 wherein: A is optional, and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted alkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, tuted alkarylene, aralkylene, or substituted aralkylene; B is optional, and when t is a linker selected from the group consisting of lower alkylene, tuted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, kylene or substituted alkylene)-, - S-, -S-(alkylene or substituted alkylene)-, -S(O)k- where k is l, 2, or 3, -S(O)k(alkylene or substituted alkylene)-, -C(O)—, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)- (alkylene or substituted alkylene)-, -, -NR’-(alkylene or substituted alkylene)-, -C(O)N(R’)-, -CON(R’)-(alkylene or substituted alkylene)-, -CSN(R’)-, -CSN(R’)-(alkylene or substituted ne)-, -N(R’)CO-(alkylene or substituted alkylene)-, -N(R’)C(O)O-, -S(0)kN(R’)-, -N(R’)C(O)N(R’)-, _N(R’)C(S)N(R’)_a -N(R’)S(O)kN(R’)-, -N(R’)-N=, - C(R’)=N—, -C(R’)=N—N(R’)-, -C(R’)=N—N=, -C(R’)2-N=N-, and -C(R’)2-N(R’)-N(R’)-, where each R’ is independently H, alkyl, or tuted alkyl, R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R1 is H, an amino protecting group, resin, at least one amino acid, polypeptide, or polynucleotide; R2 is OH, an ester ting group, resin, at least one amino acid, polypeptide, or polynucleotide; R3 and R4 are each independently H, halogen, lower alkyl, or substituted lower alkyl, or R3 and R4 or two R3 groups optionally form a cycloalkyl or a heterocycloalkyl; Z has the structure of: €Yé\(KAr R5 ; R5 is H, COZH, C1-C6alkyl, or le; R6 is OH or H; Ar is phenyl or pyridine; R7 is C1-C6alkyl or hydrogen; L1, L2, L3, and L4 are each linkers independently selected from the group consisting of a bond, — alkylene—, —(alkylene—O)n—alkylene—J—, —alkylene'—J—(alkylene—O)n—alkylene—, —J— (alkylene—O)n—alkylene—, —(alkylene—O)n—alkylene—J—(alkylene—O)n'—alkylene—J'—, — ene—O)n—alkylene—J—alkylene'—, —W—, —alkylene—W—, alkylene'—J—(alkylene—NMe)n— alkylene—W—, —J—(alkylene—NMe)n—alkylene—W—, —J—alkylene—NMe—alkylene'—NMe— alkylene"—W—, and —alkylene—J—alkylene'—NMe—alkylene"—NMe—alkylene'"—W—; W has the structure of: Lwflfifk“; o NH2 each J and J' independently have the structure of: 1 éiNiO/E‘ or J’S\Nj\e§ H H , , ; and each n and n' are independently integers greater than or equal to one.

In some embodiments, R1 is a polypeptide. In specific embodiments, the polypeptide is an antibody. In certain specific embodiments, the antibody is tin. In other embodiments, R2 is a polypeptide. In specific embodiments, the ptide is an antibody. In certain c embodiments, the antibody is herceptin.

In some embodiments, provided herein is a method for derivatizing a dolastatin analog comprising a (I), (III), (IV), (V), or (VI), the method comprising contacting the dolastatin analog with a reagent of Formula (XXXVII), wherein Formula (I), (III), (IV), (V), or (VI) correspond to: Italian}R7 0 /\ Me OMeO L2—NMeNjLM;:E|\/WNeOO R7 0 MAMeMe OMeOM Y! filwwk (H1) R7 0 Me/\MeMe OMeOM NH Me\NMeN\_)0J\NINWA/I/WNeOO Me O Me/\MeMe OMeOM NHOHN—L2 (IV) |\/|e\NIrreNjLN Me L1 Me O Me/\MeMe OMeO MO NH HN—L3 Ar 0 Lena;R70 /\ Me OMeO Y] IWNJQ;W (W JL4<3e Me R7 0 Me/\MeMe OMeOM N\H 0 Z _NIWNJ1]:JW Me R7 0 Me/\MeMe OMeOM NH 0 Z ; MeleeWi/I Me O MAMeMe OMeOM floHN—L2 JigsJ1: R6 Me L1 _ V 0Me/\ Me Me OMeOM NH HN—L3 Ar 0 MeganJ1: R6 0Me/:\ Me Me OMeOM NH HN—L4 Ar 0 wherein: Z has the ure of: €Yé\(KAr R5 ; R5 is H, CORg, C1-C6a1kyl, 0r thiazole; R8 is OH or —NH—(a1kylene—O)n—NH2; R6 is OH or H; Ar is phenyl or pyridine; WO 66560 R7 is C1-C6 alkyl or en; Y is NHg—O— or methyl; L, L1, L2, L3, and L4 are each linkers selected from the group consisting of a bond, —alkylene—, — alkylene—C(O)—, —(alkylene—O)n—alkylene—, —(alkylene—O)n—alkylene—C(O)—, —(alkylene— O)n—(CH2)n~NHC(O) (CH2)nu C(Me)2 S S (CH2)nm NHC(O)—(alkylene—O)nmv—alkylene—, — (alkylene—O)n—alkylene—W—, —alkylene—C(O)—W—, —(alkylene—O)n—alkylene—J—, —alkylene'— J—(alkylene—O)n—alkylene—, —(alkylene—O)n—alkylene—J—alkylene', —J—(alkylene—O)n— alkylene—, —(alkylene—O)n—alkylene—J—(alkylene—O)n'—alkylene—J'—, —W—, —alkylene—W—, alkylene'—J— (alkylene—NMe)n—alkylene—W—, and J— (alkylene—NMe)n—alkylene—W—, — (alkylene—O)n—alkylene—U—alkylene—C(O)—, —(alkylene—O)n—alkylene—U—alkylene—; —J— alkylene—NMe—alkylene'—NMe—alkylene"—W—, and —alkylene—J—alkylene'—NMe—alkylene"— NMe—alkylene'"—W—; W has the structure of: flLWJUCVk; U has the ure of: COZH Mink each J and J' independently have the structure of: agiN/ksz éiNiO/E‘ or fimief H H or L is absent, Y is methyl, R5 is CORg, and R8 is lkylene—O)n—NH2; and eachn n' n" n'" andnH" are inde endentl inte ers eater than or e ual to one‘ 7 7 7 7 wherein Formula (XXXVII) corresponds to: R3 A‘B’K‘R RbN R2 H R40 wherein: A is optional, and when present is lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, e, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene; B is optional, and when present is a linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or tuted alkylene)-, -S(O)k- where k is 1, 2, or 3, -S(O)k(alkylene or substituted ne)-, , -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R’)-, -NR’-(alkylene or substituted alkylene)-, -C(O)N(R’)-, -CON(R’)-(alkylene or substituted alkylene)-, ’)-, -CSN(R’)-(alkylene or substituted alkylene)-, -N(R’)CO-(alkylene or substituted alkylene)-, -N(R’)C(0)0-, -S(0)kN(R’)-, -N(R’)C(0)N(R’)-, _N(R’)C(S)N(R’)_a S(O)kN(R’)-, -N(R’)-N=, -C(R’)=N—, -C(R’)=N—N(R’)-, -C(R’)=N—N=, 2-N=N-, and -C(R’)2-N(R’)-N(R’)-, where each R’ is independently H, alkyl, or substituted alkyl, each R’ is independently H, alkyl, or substituted alkyl, My S ”L Ki, 120%“ 1‘?"ka X i: $5? “ah/[ko/L“L.

R is H, alkyl, substituted alkyl, cycloalkyl, or tuted cycloalkyl; R1 is H, an amino protecting group, resin, at least one amino acid, or polynucleotide; R2 is OH, an ester protecting group, resin, at least one amino acid, or polynucleotide; and R3 and R4 are each independently H, halogen, lower alkyl, or substituted lower alkyl, or R3 and R4 or two R3 groups ally form a cycloalkyl or a heterocycloalkyl.

In some embodiments, the derivatized dolastatin analog comprises at least one oxime containing amino acid haVing the structure of Formula (VIII), (IX), (X), (XI), (XII), or (XIII): Me O MAMeMe OMeO OMeOO R3 R3 31 A)ZNH| R4 “#30 R2 Me Me 0M9 Me H_ .\\H L2—| N Me R7 0 Me/\MeMe OMeOM NH ,L1 Z <9 Me (X) R /N \l/ L3—N MeNfi/IN B\A 0 R7 0 Me/\MeMe OMeOM RRMRZR4 NH 3 o z HNNR Me\NR1J1N HMOYKWH5:th Me O Me/\MeMe OMeO OMeOAAr R2 81 Me b R3 I I R3 Me\ MHJ1: HOW/CNNNfiwN N\ B N Y Me O Me OMeO OMeOAAr L_N MHjL: R7 0 Me/\MeMe OMeOM , Me 9 Me MeH 0 Me R / N -“H \r (X11) N L3—N NyLM Me B\A 2\ 0 R7 0 Me OMeO Me Me MeO N\H R3RMRzR4 J o z Me Me 0M9 Me HN\ H \\ H R1 N N Lrw $N He R7 0 A Me OMeO Me e MeO NH 0 Z ; R7 OMe/\M 6Me 0MeO OMeOA R2 81 |_ (XHD Me 1 R3 Me Q 4‘ R3 M A”OH/Mpg 3\ N\ B N Y R eMe OMeO OMeoA R Me Me Me MeMMETNflNwe%$:fifi R7 OMe/\MeMe OMeO OMeOA In specific embodiments, the dolastatin analog is contacted With the reagent of Formula (XXXVH) in aqueous solution under mildly acidic conditions.

Certain embodiments of the present invention describe a compound comprising Formula (XXV), (XXVI), , (XXVIH), (XXIX), orM(XXX):M (R16)nl— H ' \7/ N\ R7 R 0Me/\MeMe OMeoM (XXV) 0 R2 R7 OMe/\MeMe OMeO OMeOO L\—H _’ N \I—/) RO4 (R16)n Me MeH Me 0 Me NA “‘ H H N N\ L2—N g I?! R7 0Me/\MeMe OMeO OMe O (R 16) @NVMI— (XXVII) WKNRL, H MIN“J1Mpg/WW“ W R4 _N N\z 0 R2 R7 OMe/\MeMe OMeO OMeO UmmfimJim—L2 R7 OMe/\MeMe OMeO OMeOA R , 1 LNAC/H _ (XXVIH) Me R40 Me\l\|l MeNjLN (R16)n R7 0Me/\MeMe OMeO OMeOAWEN—L3 _ RENE“?WAT/WR7 OMe/\MeMe OMeOM (Rush—(y Hv1L M9 V N\ (XXIX) R4 L3_N 0 R2 R7 eMe OMeOM NH J Z LEZIWMUENQW R7 OMe/\M Me OMeO MUL: H R7 OMe/\MeMe OMeO OMeOAr R6 R1 HN R2 L1 — Me Me \_ R O Me 0 4 Me\ “vii H N9H N a fiMfl—s (R16)n (XXX) R7 0 /_\ Me OMeO OMeO Me e Ar R5 Me Me Me 0 ‘ H Me\ N ‘ I}! 3 ”_L4 R7 OMe/\MeMe OMe O OMe OAr R6 - wherein: Z has the structure of: ‘7;\KKAr R5 ; R5 is H, COZH, C1-C6alkyl, or thiazole; R6 is OH or H; Ar is phenyl, or pyridine; R1 is H, an amino protecting group, resin, at least one amino acid, polypeptide, or polynucleotide; R2 is OH, an ester protecting group, resin, at least one amino acid, polypeptide, or polynucleotide; R4 is H, halogen, lower alkyl, or substituted lower alkyl; R7 is C1-C6alkyl or hydrogen; L, L1, L2, L3, and L4 are each linkers selected from the group consisting of a bond, —alkylene—, — alkylene—C(O)—, —alkylene—J—, —(alkylene—O)n—alkylene—, lene—O)n—alkylene—C(O)—, — ene—O)n—J—, —(alkylene—O)n—J—alkylene—, —(alkylene—O)n—(CH2)n~NHC(O)—(CH2)nu— C(Me)2—S—S—(CH2)nu~NHC(O)—(alkylene—O)nmv—alkylene—, —(alkylene—O)n—alkylene—W—, — alkylene—C(O)—W—, —(alkylene—O)n—alkylene—J—, —alkylene'—J—(alkylene—O)n—alkylene—, — (alkylene—O)n—alkylene—J—alkylene', —J—(alkylene—O)n—alkylene—, —(alkylene—O)n—alkylene— ylene—O)n'—alkylene—J'—, —W—, ene—W—, alkylene'—J—(alkylene—NMe)n—alkylene— W—, —J—(alkylene—NMe)n—alkylene—W—, —(alkylene—O)n—alkylene—U—alkylene—C(O)—, — (alkylene—O)n—alkylene—U—alkylene—; —J—alkylene—NMe—alkylene'—NMe—alkylene"—W—, and —alkylene—J—alkylene'—NMe—alkylene"—NMe—alkylene'"—W—; W has the structure of: LWJLKTJK“; U has the structure of: COZH QM”? each I and J' independently have the structure of: Erin/571 fiNiO/‘zi or Jimif each n and n' are independently integers greater than or equal to one; and each R16 is independently selected from the group consisting of hydrogen, halogen, alkyl, N02, CN, and substituted alkyl.

In some embodiments, R1 is a polypeptide. In specific embodiments, the polypeptide is an dy. In certain specific ments, the dy is herceptin. In other embodiments, R2 is a ptide. In specific embodiments, the polypeptide is an antibody. In certain specific embodiments, the antibody is herceptin.

Some embodiments of the present invention describe a compound comprising Formula (XXXI), (XXXII), (XXXIII), (XXXIV), (XXXV), or (XXXVI): RNf,B\/L\:gf\)J\RRH 33 R7 O /\ Me OMeO R2 MeO NH 0000) WMSLrHflMI%%N Ar (XXXH) R7 OMe/\MeMe OMeO OM30 :ro IH R3 R3 H L\ / \ D B\A)ZN R1 Me Me Me Me M6 H "‘H N H Lz—w ; N \2 R7 0 /_\ Me OMeO OMeO Me Me H R3 R3 N /B\ R1/ /L1 A D Me Me Me R4 Me (XXXIH) H 3 NNxH H L3—'\Il ; N Z R7 OMe/\MeMe OMeO OMeO NeNN R7 NMHJ1M;%@\J§NI:N_LZOMe/\MeMe OMeO OMeOA R3 R3N L1\ DA/B\ Me H|V|e%NNfLWI—s AR4 NR1 I) R7 OMe/\MeMe OMeO OMeOA _NIMMN:NJfiwR7 OMe/\MeMe R3 R3 OMeOM R1/HfA31D/L1 L_:W%%OMe/\MeMe OMeOM R7 OMe/\M Me OMeO “AelfilflN .\ NfiNH R7 OMe/\MeMe OMeO OMeO r R5 R3 R3 H L1\ /B\ N\ Me 0 R4 N 0 R2 u—Ls OM6 0 Ar R5 (XXXVI) i Me O _\ H R7 OMe/\MeMe OMe 0 OMe OAr R6 - wherein: Z has the structure of: ‘7;\KKAr R5 ; R5 is H, COZH, C1-C6alkyl, or thiazole; R6 is OH or H; Ar is phenyl or pyridine; R1 is H, an amino protecting group, resin, at least one amino acid, polypeptide, or polynucleotide; R2 is OH, an ester protecting group, resin, at least one amino acid, polypeptide, or polynucleotide; R4 is H, halogen, lower alkyl, or substituted lower alkyl; R7 is lkyl or hydrogen; L, L1, L2, L3, and L4 are each s selected from the group consisting of a bond, —alkylene—, — alkylene—C(O)—, —alkylene—J—, —(alkylene—O)n—alkylene—, —(alkylene—O)n—alkylene—C(O)—, — (alkylene—O)n—J—, —(alkylene—O)n—J—alkylene—, —(alkylene—O)n—(CH2)n~NHC(O)—(CH2)nu— C(Me)2—S—S—(CH2)nu~NHC(O)—(alkylene—O)nmv—alkylene—, —(alkylene—O)n—alkylene—W—, — alkylene—C(O)—W—, —(alkylene—O)n—alkylene—J—, —alkylene'—J—(alkylene—O)n—alkylene—, — (alkylene—O)n—alkylene—J—alkylene', —J—(alkylene—O)n—alkylene—, —(alkylene—O)n—alkylene— ylene—O)n'—alkylene—J'—, —W—, —alkylene—W—, alkylene'—J—(alkylene—NMe)n—alkylene— W—, —J—(alkylene—NMe)n—alkylene—W—, —(alkylene—O)n—alkylene—U—alkylene—C(O)—, — (alkylene—O)n—alkylene—U—alkylene—; —J—alkylene—NMe—alkylene'—NMe—alkylene"—W—, and —alkylene—J—alkylene'—NMe—alkylene"—NMe—alkylene'"—W—; LWJLKTJK“; U has the structure of: COZH QM”? each J and J' independently have the structure of: Erin/571 fiNiO/‘zi or Jimif ! a each n and n' are independently integers greater than or equal to one; D has the structure of: each R17 is ndently selected from the group consisting of H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, alkylalkoxy, substituted lkoxy, polyalkylene oxide, substituted polyalkylene oxide, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkaryl, substituted l, aralkyl, tuted l, -(alkylene or substituted alkylene)-ON(R”)2, -(alkylene or substituted alkylene)-C(O)SR”, -(alkylene or substituted alkylene)-S-S-(aryl or tuted aryl), -C(O)R”, -C(O)2R”, or -C(O)N(R”)2, Wherein each R” is independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, alkaryl, substituted alkaryl, aralkyl, or substituted aralkyl; each 21 is a bond, CR17R17, O, S, NR’, CR17R17-CR17R17, CR17R17-O, O-CR17R17, CR17R17-S, S-CR17R17, 7-NR’, or NR’-CR17R17; each R’ is H, alkyl, or substituted alkyl; each 22 is selected from the group consisting of a bond, -C(O)-, -C(S)-, optionally substituted C1-C3 alkylene, optionally tuted C1-C3 alkenylene, and optionally substituted heteroalkyl; each 23 are independently selected from the group consisting of a bond, optionally substituted C1-C4 alkylene, optionally substituted C1-C4 alkenylene, optionally substituted heteroalkyl, -O-, -S-, , , and -N(R’)-; each T3 is a bond, C(R”)(R”), O, or S; With the proviso that When T3 is O or S, R” cannot be halogen; each R” is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; m and p are 0, l, 2, or 3, provided that at least one ofm or p is not 0; (b) (b) | ‘A'I'V‘ /R3 if; nlnr /C\— E03) /C—C\(b—E) (577R4 /C=C\—E(b) mlnn (b) /C\—5—§ (b) M2 is (a) R3 R4 (a5; R4 M3 (201K: (b) (b) (b) (b) M J‘ri R fi R 3 3 m R3 I / / R3/i—d—é /C=C—E (b) O—T—E (b) s—T—E (b) (b) \R4 >44“ R4 mIJu‘ w m (a) (a) (a) or (a) Where (a) indicates a , , , bonding to the B group and (b) tes bonding to respective positions Within the heterocycle group; (b) (b) (b) (b) “if” /R3 mi: 3(1)) —c/R3—§(b) T—o——(b)§ c—s—E (b) (f—ng (b) / / R M3 is (3)3? (a)$??/||— CR\R4 (352% (3)3/ 4 Where (a) indicates bonding to the B group and (b) indicates bonding to respective positions Within the heterocycle group; (b) (b) ”$3“ (b) (b) “I: “l” R—c'2—c=§ I— —g '— :31) /C\_q=g (b) 3 (b) 0 1|?4 >5: C}; (b) S /C—§(b) CE‘Nm) R3 M41s (3)19 (a) R3 (a) (a) or (a) , , a , 7 Where (a) indicates bonding to the B group and (b) indicates bonding to respective ons Within the heterocycle group; each R19 is ndently selected from the group consisting of C1-C6 alkyl, C1-C6 alkoxy, ester, ether, thioether, lkyl, n, alkyl ester, aryl ester, amide, aryl amide, alkyl halide, alkyl amine, alkyl sulfonic acid, alkyl nitro, thioester, sulfonyl ester, halosulfonyl, nitrile, alkyl nitrile, and nitro; qis 0,1, 2, 3, 4, 5, 6, 7, 8, 9,10 or 11; and each R16 is ndently selected from the group consisting of hydrogen, halogen, alkyl, N02, CN, and substituted alkyl.

In some embodiments, R1 is a polypeptide. In specific embodiments, the polypeptide is an antibody. In certain specific embodiments, the antibody is herceptin. In other embodiments, R2 is a polypeptide. In specific embodiments, the polypeptide is an antibody. In certain specific ments, the antibody is herceptin.

In some embodiments, a|Vlcompound is described comprising Formula (XXXI-A): =N R7 MeMe OMeO MeO N\H (XXXI'A) R 0 40 Z R1 R2 In certain embodiments, a pharmaceutical composition is provided comprising any of the compounds described and a pharmaceutically acceptable carrier, excipient, or binder.

In further or alternative embodiments are s for detecting the presence of a polypeptide in a patient, the method comprising administering a polypeptide comprising at least one heterocyclecontaining non—natural amino acid and the resulting cycle-containing non-natural amino acid polypeptide modulates the immunogenicity of the polypeptide relative to the homologous naturally- occurring amino acid polypeptide.

It is to be understood that the methods and compositions described herein are not limited to the particular ology, protocols, cell lines, constructs, and reagents described herein and as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the methods and compositions bed herein, which will be limited only by the appended claims.

As used herein and1n the appended claims, the singular forms 44a 39 ccan,” and “the” include plural nce unless the context clearly indicates otherwise.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ry skill in the art to which the inventions described herein belong.

Although any methods, devices, and materials similar or equivalent to those bed herein can be used in the ce or testing of the inventions described herein, the preferred methods, deVices and materials are now described.

All publications and patents mentioned herein are incorporated herein by reference in their entirety for the e of describing and sing, for example, the constructs and methodologies that are described in the publications, which might be used in connection with the presently described ions. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. g herein is to be construed as an admission that the inventors described herein are not entitled to te such disclosure by virtue of prior invention or for any other reason.

The terms “aldol-based linkage” or “mixed aldol-based linkage” refers to the acid— or base- catalyzed condensation of one carbonyl nd with the enolate/enol of another carbonyl compound, which may or may not be the same, to generate a B-hydroxy carbonyl compound—an aldol.

The term “affinity label,” as used herein, refers to a label which reversibly or irreversibly binds another molecule, either to modify it, destroy it, or form a compound with it. By way of example, affinity labels include enzymes and their substrates, or antibodies and their antigens.

The terms “alkoxy,” “alkylamino” and “alkylthio” (or thioalkoxy) are used in their conventional sense, and refer to those alkyl groups linked to molecules via an oxygen atom, an amino group, or a sulfur atom, respectively.

The term “alkyl,” by itself or as part of r molecule means, unless otherwise stated, a straight or branched chain, or cyclic hydrocarbon radical, or combination thereof, which may be fully saturated, mono- or polyunsaturated and can include di- and alent radicals, having the number of carbon atoms designated (Le. C1-C10 means one to ten carbons). Examples of saturated hydrocarbon radicals e, but are not limited to, groups such as methyl, ethyl, n-propyl, pyl, l, t-butyl, isobutyl, sec-butyl, cyclohexyl, (cyclohexyl)methyl, cyclopropylmethyl, homologs and isomers of, for example, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. An unsaturated alkyl group is one having one or more double bonds or triple bonds. Examples of unsaturated alkyl groups e, but are not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(l,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and the higher homologs and isomers. The term “alkyl,” unless otherwise noted, is also meant to include those derivatives of alkyl defined in more detail , such as “heteroalkyl”, “haloalkyl” and “homoalkyl”.

The term “alkylene” by itself or as part of another molecule means a divalent radical derived from an alkane, as exemplified, by (—CH2—)n, wherein n may be 1 to about 24. By way of example only, such groups include, but are not limited to, groups having 10 or fewer carbon atoms such as the structures — CH2CH2— and —CH2CH2CH2CH2—. A “lower alkyl” or “lower alkylene” is a shorter chain alkyl or alkylene group, generally having eight or fewer carbon atoms. The term “alkylene,” unless ise noted, is also meant to include those groups described herein as “heteroalkylene.” The term “amino acid” refers to lly occurring and tural amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally encoded amino acids are the 20 common amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, ine, phan, ne, and valine) and pyrolysine and selenocysteine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, by way of example only, an (x-carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group. Such analogs may have modified R groups (by way of example, norleucine) or may have modified e backbones while still retaining the same basic chemical structure as a naturally occurring amino acid. Non-limiting examples of amino acid analogs include homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium.

Amino acids may be referred to herein by either their name, their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Additionally, nucleotides, may be referred to by their commonly accepted single-letter codes.

An “amino terminus modification group” refers to any molecule that can be ed to a terminal amine group. By way of example, such al amine groups may be at the end of polymeric les, wherein such polymeric molecules include, but are not limited to, polypeptides, polynucleotides, and polysaccharides. Terminus modification groups include but are not limited to, various water soluble polymers, peptides or proteins. By way of example only, terminus modification groups include polyethylene glycol or serum n. Terminus modification groups may be used to modify therapeutic teristics of the polymeric molecule, including but not limited to increasing the serum half-life of peptides.

By “antibody fragment” is meant any form of an antibody other than the full-length form.

Antibody nts herein include dies that are smaller components that exist within ength antibodies, and antibodies that have been engineered. dy fragments include but are not limited to Fv, Fc, Fab, and (Fab')2, single chain Fv (scFv), diabodies, triabodies, tetrabodies, bifunctional hybrid antibodies, CDRl, CDR2, CDR3, combinations of CDR’s, variable regions, framework regions, constant regions, heavy chains, light chains, and variable regions, and ative scaffold non-antibody molecules, bispecific antibodies, and the like rd & Georgiou, 2000, Annu. Rev. Biomed. Eng. 2:339-76; Hudson, 1998, Curr. Opin. Biotechnol. 9:395-402). Another functional ucture is a single chain Fv (scFv), comprised of the variable regions of the immunoglobulin heavy and light chain, covalently connected by a e linker (S-z Hu et al., 1996, Cancer Research, 56, 3055-3061). These small (Mr ,000) proteins generally retain specificity and affinity for antigen in a single polypeptide and can provide a convenient building block for larger, antigen-specific molecules. Unless cally noted otherwise, statements and claims that use the term “antibody” or “antibodies” specifically includes ody fragment” and “antibody fragments.” By ody-drug conjugate, or “ADC”, as used herein, refers to an antibody molecule, or fragment thereof, that is covalently bonded to one or more biologically active molecule(s). The biologically active molecule may be conjugated to the antibody through a linker, polymer, or other covalent bond.

The term “aromatic” or “aryl”, as used herein, refers to a closed ring ure which has at least one ring haVing a conjugated pi electron system and includes both carbocyclic aryl and heterocyclic aryl (or oaryl” or “heteroaromatic”) groups. The carbocyclic or heterocyclic aromatic group may contain from 5 to 20 ring atoms. The term includes clic rings linked covalently or fused-ring polycyclic (i.e., rings which share nt pairs of carbon atoms) groups. An ic group can be unsubstituted or substituted. Non-limiting examples of “aromatic” or “aryl”, groups include , 1-naphthyl, 2- naphthyl, 4-biphenyl, anthracenyl, and thracenyl. Substituents for each of the above noted aryl and aryl ring systems are selected from the group of acceptable tuents described herein.

For brevity, the term “aromatic” or “aryl” when used in combination with other terms (including but not limited to, aryloxy, arylthioxy, aralkyl) includes both aryl and heteroaryl rings as defined above.

Thus, the term yl” or yl” is meant to include those radicals in which an aryl group is ed to an alkyl group (including but not limited to, benzyl, phenethyl, pyridylmethyl and the like) ing those alkyl groups in which a carbon atom (including but not limited to, a methylene group) has been replaced by a heteroatom, by way of example only, by an oxygen atom. Examples of such aryl groups include, but are not limited to, phenoxymethyl, 2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl, and the like.

The term “arylene”, as used herein, refers to a divalent aryl radical. Non-limiting examples of “arylene” include phenylene, pyridinylene, pyrimidinylene and thiophenylene. Substituents for arylene groups are selected from the group of acceptable substituents described herein.

A “bifunctional polymer”, also referred to as a “bifunctional linker”, refers to a polymer comprising two functional groups that are capable of reacting specifically with other moieties to form covalent or non-covalent linkages. Such moieties may include, but are not limited to, the side groups on l or non-natural amino acids or peptides which contain such natural or tural amino acids. The other moieties that may be linked to the bifunctional linker or bifunctional polymer may be the same or different moieties. By way of example only, a bifunctional linker may have a functional group reactive with a group on a first peptide, and another onal group which is reactive with a group on a second peptide, whereby forming a conjugate that includes the first peptide, the bifunctional linker and the second peptide. Many procedures and linker molecules for ment of various compounds to peptides are known. See, e.g., European Patent Application No. 188,256; US. Patent Nos. 4,671,958, 4,659,839, 4,414,148, 784; 4,680,338; and 4,569,789 which are incorporated by reference herein in their entirety. A “multi-functional polymer” also referred to as a “multi-functional linker”, refers to a polymer comprising two or more functional groups that are capable of ng with other es. Such moieties may include, but are not limited to, the side groups on natural or non-natural amino acids or peptides which contain such natural or non-natural amino acids. (including but not limited to, amino acid side groups) to form covalent or non-covalent linkages. A bi-functional polymer or multi-functional polymer 2012/039472 may be any desired length or molecular weight, and may be selected to provide a ular desired spacing or conformation between one or more molecules linked to a nd and molecules it binds to or the compound.

The term “bioavailability,” as used herein, refers to the rate and extent to which a substance or its active moiety is delivered from a pharmaceutical dosage form and becomes available at the site of action or in the general circulation. Increases in bioavailability refers to increasing the rate and extent a substance or its active moiety is delivered from a pharmaceutical dosage form and becomes available at the site of action or in the general circulation. By way of e, an increase in bioavailability may be indicated as an increase in concentration of the substance or its active moiety in the blood when compared to other substances or active moieties. A non-limiting example of a method to evaluate increases in bioavailability is given in examples 21-25. This method may be used for evaluating the bioavailability of any polypeptide.

The term “biologically active molecule”, gically active moiety” or “biologically active agent” when used herein means any nce which can affect any physical or biochemical properties of a biological , pathway, molecule, or interaction relating to an sm, including but not limited to, viruses, bacteria, bacteriophage, transposon, prion, insects, fungi, plants, animals, and humans. In particular, as used herein, ically active molecules include but are not limited to any nce intended for diagnosis, cure, mitigation, treatment, or tion of disease in humans or other animals, or to otherwise enhance physical or mental well-being of humans or animals. Examples of biologically active molecules e, but are not limited to, peptides, proteins, enzymes, small molecule drugs, hard drugs, soft drugs, prodrugs, ydrates, inorganic atoms or molecules, dyes, lipids, nucleosides, radionuclides, oligonucleotides, toxins, cells, viruses, liposomes, microparticles and micelles. Classes of biologically active agents that are le for use with the methods and compositions described herein include, but are not limited to, drugs, prodrugs, radionuclides, imaging agents, polymers, antibiotics, fungicides, anti-viral agents, anti-inflammatory agents, anti-tumor agents, cardiovascular agents, anti- y agents, hormones, grth s, steroidal agents, microbially derived , and the like.

By “modulating ical activity” is meant increasing or decreasing the reactivity of a polypeptide, altering the selectivity of the polypeptide, enhancing or decreasing the substrate selectivity of the polypeptide. Analysis of modified biological activity can be performed by comparing the biological activity of the non-natural polypeptide to that of the natural polypeptide.

The term “biomaterial,” as used herein, refers to a biologically-derived material, including but not limited to material obtained from bioreactors and/or from recombinant methods and techniques.

The term “biophysical probe,” as used herein, refers to probes which can detect or monitor ural changes in les. Such molecules include, but are not limited to, proteins and the “biophysical probe” may be used to detect or monitor interaction of ns with other macromolecules.

Examples of biophysical probes include, but are not limited to, spin-labels, a fluorophores, and photoactivatible groups.

The term nthetically,” as used herein, refers to any method utilizing a translation system (cellular or non-cellular), including use of at least one of the following components: a polynucleotide, a codon, a tRNA, and a ribosome. By way of example, tural amino acids may be “biosynthetically orated” into non-natural amino acid ptides using the methods and techniques described herein, “In vivo tion of polypeptides comprising non-natural amino acids”, and in the non-limiting example 20. onally, the methods for the selection of useful non-natural amino acids which may be “biosynthetically incorporated” into non-natural amino acid polypeptides are described in the non-limiting examples 20.

The term “biotin analogue,” or also referred to as “biotin mimic”, as used herein, is any molecule, other than biotin, which bind with high affinity to avidin and/or streptavidin.

The term “carbonyl” as used herein refers to a group containing at a moiety selecting from the group consisting of -C(O)-, -S(O)-, -S(O)2—, and —C(S)-, including, but not limited to, groups containing a least one ketone group, and/or at least one aldehyde groups, and/or at least one ester group, and/or at least one carboxylic acid group, and/or at least one thioester group. Such carbonyl groups include ketones, aldehydes, carboxylic acids, esters, and thioesters. In addition, such groups may be part of linear, branched, or cyclic molecules.

The term “carboxy terminus modification group” refers to any molecule that can be attached to a terminal carboxy group. By way of example, such terminal carboxy groups may be at the end of polymeric molecules, wherein such polymeric molecules include, but are not d to, polypeptides, polynucleotides, and polysaccharides. Terminus modification groups include but are not limited to, various water soluble polymers, peptides or ns. By way of example only, terminus modification groups include polyethylene glycol or serum n. Terminus modification groups may be used to modify therapeutic characteristics of the polymeric molecule, including but not d to increasing the serum half-life of peptides.

The term “chemically cleavable group,” also referred to as “chemically labile”, as used herein, refers to a group which breaks or cleaves upon exposure to acid, base, oxidizing , reducing agents, chemical inititiators, or radical initiators.

The term “chemiluminescent group,” as used herein, refers to a group which emits light as a result of a chemical reaction without the on of heat. By way of e only, luminol (5-amino-2,3- dihydro-l,4-phthalazinedione) reacts with oxidants like hydrogen peroxide (H202) in the presence of a base and a metal catalyst to produce an excited state product (3-aminophthalate, 3-APA).

The term “chromophore,” as used herein, refers to a molecule which absorbs light of visible wavelengths, UV ngths or IR wavelengths.

The term tor,” as used herein, refers to an atom or molecule essential for the action of a large molecule. Cofactors include, but are not limited to, inorganic ions, coenzymes, proteins, or some other factor necessary for the activity of enzymes. Examples include, heme in obin, magnesium in phyll, and metal ions for proteins. ding,” as used herein, refers to refolding processes, reactions, or methods which employ at least two molecules which interact with each other and result in the transformation of unfolded or improperly folded molecules to properly folded molecules. By way of example only, “cofolding,” employ at least two ptides which interact with each other and result in the transformation of unfolded or improperly folded polypeptides to native, properly folded polypeptides. Such ptides may contain natural amino acids and/or at least one non-natural amino acid.

A “comparison window,” as used herein, refers a segment of any one of contiguous positions used to compare a sequence to a reference ce of the same number of uous positions after the two sequences are optimally aligned. Such uous positions e, but are not d to a group consisting of from about 20 to about 600 sequential units, including about 50 to about 200 sequential units, and about 100 to about 150 sequential units. By way of example only, such sequences include polypeptides and polypeptides containing non-natural amino acids, with the sequential units include, but are not limited to l and tural amino acids. In addition, by way of example only, such sequences include polynucleotides with nucleotides being the corresponding sequential units. Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted, including but not limited to, by the local homology algorithm of Smith and Waterman (1970) Adv. Appl. Math. 2:482c, by the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, by the search for similarity method of Pearson and Lipman (1988) Proc. Nat’l. Acad. Sci. USA 852444, by computerized entations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection (see, e. g., Ausubel et al., Current Protocols in Molecular Biology (1995 supplement)).

By way of e, an algorithm which may be used to determine percent sequence identity and ce similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1997) Nuc. Acids Res. 25:3389-3402, and Altschul et al. (1990) J. Mol. Biol. 215:403-410, respectively.

Software for performing BLAST analyses is publicly available through the National Center for hnology ation. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for tide ces) uses as defaults a wordlength (W) of 11, an expectation (E) or 10, M=5, N=-4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1992) Proc. Natl. Acad. Sci. USA 89:10915) alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands. The BLAST algorithm is typically performed with the “low complexity” filter turned off.

The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarity ed by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, or less than about 0.01, or less than about 0.001.

The term “conservatively modified variants” s to both natural and non-natural amino acid and natural and non-natural nucleic acid sequences, and ations thereof. With respect to particular nucleic acid sequences, “conservatively modified variants” refers to those natural and non-natural nucleic acids which encode identical or essentially identical natural and tural amino acid sequences, or where the natural and non-natural nucleic acid does not encode a natural and non-natural amino acid sequence, to ially cal sequences. By way of example, because of the racy of the genetic code, a large number of onally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the d polypeptide. Such nucleic acid variations are “silent variations,” which are one species of conservatively modified ions. Thus by way of example every natural or non-natural nucleic acid sequence herein which encodes a natural or tural polypeptide also describes every possible silent variation of the natural or non-natural nucleic acid. One of ordinary skill in the art will recognize that each codon in a natural or non-natural nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a natural and non-natural nucleic acid which encodes a natural and non-natural polypeptide is implicit in each described sequence.

As to amino acid sequences, individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein ce which alters, adds or deletes a single natural and non-natural amino acid or a small percentage of l and non-natural amino acids in the encoded sequence is a rvatively modified variant” where the alteration results in the deletion of an amino acid, addition of an amino acid, or substitution of a natural and non-natural amino acid with a ally similar amino acid. Conservative substitution tables providing functionally similar natural amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the methods and itions described herein.

Conservative substitution tables ing functionally similar amino acids are known to those of ordinary skill in the art. The following eight groups each contain amino acids that are vative substitutions for one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); ) lsoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g., ton, Proteins:Structures and Molecular Properties (W H Freeman & Co.; 2nd edition (December 1993) The terms alkyl” and “heterocycloalkyl”, by themselves or in combination with other terms, represent, unless otherwise stated, cyclic versions of “alkyl” and “heteroalkyl”, respectively. Thus, a cycloalkyl or heterocycloalkyl include saturated, partially unsaturated and fully unsaturated ring linkages. Additionally, for heterocycloalkyl, a heteroatom can occupy the position at which the heterocycle is attached to the remainder of the molecule. The heteroatom may include, but is not limited to, oxygen, nitrogen or sulfur. Examples of cycloalkyl include, but are not limited to, cyclopentyl, cyclohexyl, l-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples of heterocycloalkyl include, but are not limited to, l—(l,2,5,6-tetrahydropyridyl), l-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4- morpholinyl, 3-morpholinyl, tetrahydrofuran—2-yl, tetrahydrofuran—3-yl, tetrahydrothienyl, tetrahydrothien—3-yl, l—piperazinyl, 2-piperazinyl, and the like. Additionally, the term encompasses multicyclic structures, ing but not limited to, bicyclic and tricyclic ring ures. Similarly, the term “heterocycloalkylene” by itself or as part of another molecule means a divalent radical derived from heterocycloalkyl, and the term “cycloalkylene” by itself or as part of another molecule means a divalent radical derived from cycloalkyl.

The term dextrin,” as used herein, refers to cyclic carbohydrates consisting of at least six to eight glucose molecules in a ring formation. The outer part of the ring contains water e ; at the center of the ring is a relatively nonpolar cavity able to accommodate small molecules.

The term “cytotoxic,” as used herein, refers to a compound which harms cells.

“Denaturing agent” or “denaturant,” as used , refers to any compound or material which will cause a reversible unfolding of a polymer. By way of example only, “denaturing agent” or “denaturants,39 may cause a reversible unfolding of a protein. The strength of a denaturing agent or denaturant will be determined both by the properties and the concentration of the particular denaturing agent or denaturant. By way of example, ring agents or denaturants include, but are not limited to, chaotropes, detergents, c, water miscible solvents, phospholipids, or a combination thereof. Nonlimiting examples of chaotropes include, but are not limited to, urea, guanidine, and sodium anate.

Non-limiting examples of ents may include, but are not limited to, strong detergents such as sodium dodecyl sulfate, or polyoxyethylene ethers (e. g. Tween or Triton detergents), Sarkosyl, mild non-ionic detergents (e. g., digitonin), mild ic detergents such as N—>2,3-(Dioleyoxy)-propyl-N,N,N— trimethylammonium, mild ionic detergents (e. g. sodium cholate or sodium deoxycholate) or zwitterionic ents including, but not limited to, sulfobetaines (Zwittergent), 3-(3- midopropyl)dimethylammonio-l -propane e (CHAPS), and 3 -(3 - chlolamidopropyl)dimethylammoniohydroxy-l-propane sulfonate (CHAPSO). miting examples of organic, water miscible ts include, but are not limited to, acetonitrile, lower alkanols (especially C2 - C4 ls such as ethanol or isopropanol), or lower alkandiols (C2 - C4 alkandiols such as ethylene-glycol) may be used as denaturants. Non-limiting examples of olipids include, but are not limited to, naturally occurring phospholipids such as phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, and phosphatidylinositol or tic olipid derivatives or variants such as dihexanoylphosphatidylcholine or diheptanoylphosphatidylcholine.

The term ed functionality” as used herein refers to any group selected from a label; a dye; a polymer; a water-soluble polymer; a tive of polyethylene glycol; a photocrosslinker; a cytotoxic compound; a drug; an affinity label; a ffmity label; a reactive compound; a resin; a second protein or polypeptide or polypeptide analog; an antibody or antibody fragment; a metal chelator; a cofactor; a fatty acid; a carbohydrate; a cleotide; a DNA; a RNA; an antisense polynucleotide; a saccharide, a water-soluble dendrimer, a cyclodextrin, a biomaterial; a nanoparticle; a spin label; a fluorophore; a metal-containing moiety; a ctive moiety; a novel functional group; a group that covalently or noncovalently interacts with other molecules; a photocaged moiety; an actinic radiation excitable moiety; a ligand; a photoisomerizable moiety; biotin; a biotin analogue; a moiety incorporating a heavy atom; a chemically cleavable group; a photocleavable group; an ted side chain; a carbon-linked sugar; a redox-active agent; an amino id; a toxic moiety; an isotopically labeled moiety; a biophysical probe; a phosphorescent group; a chemiluminescent group; an electron dense group; a magnetic group; an intercalating group; a chromophore; an energy transfer agent; a biologically active agent (in which case, the biologically active agent can include an agent with therapeutic activity and the non-natural amino acid polypeptide or modified tural amino acid can serve either as a co-therapeutic agent with the ed therapeutic agent or as a means for delivery the therapeutic agent to a desired site within an organism); a detectable label; a small molecule; an inhibitory ribonucleic acid; a radionucleotide; a neutron-capture agent; a derivative of biotin; quantum dot(s); a ansmitter; a radiotransmitter; an , an activated complex activator, a Virus, an adjuvant, an aglycan, an allergan, an angiostatin, an antihormone, an antioxidant, an aptamer, a guide RNA, a saponin, a shuttle vector, a macromolecule, a mimotope, a receptor, a reverse micelle, and any combination thereof.

The term “diamine,”as used herein, refers to groups/molecules comprising at least two amine functional groups, including, but not d to, a hydrazine group, an amidine group, an imine group, a l,l-diamine group, a 1,2-diamine group, a 1,3-diamine group, and a l,4-diamine group. In addition, such groups may be part of linear, branched, or cyclic molecules.

The term “detectable label,” as used herein, refers to a label which may be observable using analytical techniques including, but not limited to, fluorescence, chemiluminescence, electron-spin resonance, ultraviolet/visible absorbance spectroscopy, mass spectrometry, nuclear magnetic resonance, magnetic resonance, and electrochemical methods.

The term bonyl” as used herein refers to a group containing at least two moieties selected from the group consisting of -C(O)-, -S(O)-, -S(O)2-, and , including, but not limited to, 1,2- dicarbonyl groups, a 1,3-dicarbonyl groups, and l,4-dicarbonyl groups, and groups containing a least one ketone group, and/or at least one aldehyde groups, and/or at least one ester group, and/or at least one carboxylic acid group, and/or at least one thioester group. Such dicarbonyl groups include diketones, ketoaldehydes, ketoacids, ketoesters, and ketothioesters. In addition, such groups may be part of linear, branched, or cyclic molecules. The two moieties in the dicarbonyl group may be the same or different, and may include substituents that would produce, by way of example only, an ester, a ketone, an aldehyde, a thioester, or an amide, at either of the two moieties.

The term “drug,” as used herein, refers to any nce used in the prevention, diagnosis, alleviation, treatment, or cure of a disease or condition.

The term “dye,” as used herein, refers to a soluble, coloring substance which contains a chromophore.

The term “effective amount,” as used herein, refers to a sufficient amount of an agent or a compound being administered which will relieve to some extent one or more of the symptoms of the disease or condition being d. The result can be ion and/or alleviation of the signs, symptoms, or causes of a e, or any other desired alteration of a biological system. By way of example, an agent or a compound being administered includes, but is not limited to, a natural amino acid polypeptide, non- natural amino acid polypeptide, modified natural amino acid ptide, or modified non-amino acid ptide. Compositions containing such natural amino acid polypeptides, non-natural amino acid ptides, modified natural amino acid polypeptides, or modified tural amino acid ptides can be administered for prophylactic, enhancing, and/or therapeutic treatments. An riate “effective” amount in any individual case may be determined using techniques, such as a dose escalation study.

The term “electron dense group,” as used herein, refers to a group which scatters electrons when irradiated with an electron beam. Such groups include, but are not d to, ammonium molybdate, h subnitrate cadmium iodide, 99%, carbohydrazide, ferric chloride hexahydrate, thylene tetramine, 98.5%, indium trichloride anhydrous, lanthanum e, lead acetate trihydrate, lead citrate trihydrate, lead nitrate, ic acid, phosphomolybdic acid, phosphotungstic acid, potassium ferricyanide, potassium ferrocyanide, ruthenium red, silver nitrate, silver proteinate (Ag Assay: 80-85%) “Strong”, silver tetraphenylporphin (S-TPPS), sodium chloroaurate, sodium tungstate, thallium nitrate, thiosemicarbazide (TSC), uranyl acetate, uranyl nitrate, and vanadyl sulfate.

The term “energy transfer ” as used herein, refers to a molecule which can either donate or accept energy from another molecule. By way of example only, fluorescence resonance energy er (FRET) is a dipole-dipole coupling s by which the excited-state energy of a fluorescence donor molecule is non-radiatively transferred to an unexcited acceptor molecule which then fluorescently emits the donated energy at a longer wavelength.

The terms “enhance” or “enhancing” means to increase or prolong either in potency or duration a desired effect. By way of example, “enhancing” the effect of therapeutic agents refers to the ability to increase or g, either in potency or duration, the effect of therapeutic agents on during ent of a disease, disorder or condition. An “enhancing-effective amount,” as used herein, refers to an amount adequate to enhance the effect of a therapeutic agent in the treatment of a disease, disorder or condition.

When used in a patient, amounts effective for this use will depend on the severity and course of the disease, disorder or condition, previous therapy, the patients health status and response to the drugs, and the judgment of the treating physician.

As used herein, the term “eukaryote” refers to sms belonging to the phylogenetic domain Eucarya, ing but not limited to animals (including but not limited to, mammals, insects, reptiles, birds, etc.), ciliates, plants (including but not limited to, ts, dicots, and algae), fungi, yeasts, flagellates, microsporidia, and protists.

The term “fatty acid,” as used , refers to carboxylic acids with about C6 or longer hydrocarbon side chain.

The term “fluorophore,” as used herein, refers to a molecule which upon excitation emits photons and is y fluorescent.

The terms “functional 9 ccactive moiety:9 ccactivating 9 cc ccreactive , , , leaving group:9 , site”, “chemically reactive group” and “chemically reactive ,” as used herein, refer to portions or units of a molecule at which al reactions occur. The terms are somewhat synonymous in the chemical arts and are used herein to indicate the portions of molecules that perform some function or activity and are reactive with other molecules.

The term “halogen” includes fluorine, chlorine, iodine, and e.

The term “haloacyl,” as used herein, refers to acyl groups which contain halogen moieties, including, but not limited to, -C(O)CH3, F3, H20CH3, and the like.

The term “haloalkyl,” as used herein, refers to alkyl groups which contain halogen moieties, including, but not limited to, -CF3 and —CH2CF3 and the like.

The term oalkyl,” as used herein, refers to straight or branched chain, or cyclic hydrocarbon radicals, or combinations thereof, consisting of an alkyl group and at least one heteroatom selected from the group consisting of O, N, Si and S, and wherein the nitrogen and sulfur atoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized. The heteroatom(s) O, N and S and Si may be placed at any interior position of the heteroalkyl group or at the position at which the alkyl group is attached to the remainder of the molecule. Examples include, but are not limited to, -CH2-CH2-O-CH3, - CHZ-CHZ-NH-CHg, -CH2-CH2-N(CH3)-CH3, -CH2-S-CH2-CH3, -CH2-CH2,-S(O)-CH3, -CH2-CH2-S(O)2- CH3, -CH=CH-O-CH3, -Si(CH3)3, -CH2-CH=N-OCH3, and —CH=CH-N(CH3)-CH3. In addition, up to two atoms may be consecutive, such as, by way of e, H-OCH3 and —CH2-O-Si(CH3)3.

The terms “heterocyclic-based linkage” or “heterocycle linkage” refers to a moiety formed from the reaction of a dicarbonyl group with a diamine group. The resulting reaction product is a cycle, including a heteroaryl group or a heterocycloalkyl group. The resulting heterocycle group serves as a chemical link between a non-natural amino acid or non-natural amino acid polypeptide and another functional group. In one ment, the heterocycle linkage includes a nitrogen-containing heterocycle linkage, including by way of example only a pyrazole linkage, a pyrrole e, an indole linkage, a benzodiazepine linkage, and a pyrazalone linkage. rly, the term “heteroalkylene” refers to a divalent radical derived from heteroalkyl, as exemplified, but not limited by, -CH2-CH2-S-CH2-CH2- and —CH2-S-CH2-CH2-NH-CH2-. For heteroalkylene groups, the same or different heteroatoms can also occupy either or both of the chain termini (including but not limited to, alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, aminooxyalkylene, and the like). Still further, for ne and heteroalkylene linking groups, no orientation of the linking group is implied by the direction in which the formula of the g group is written. By way of e, the formula —C(O)2R’- represents both —C(O)2R’- and —R’C(O)2-.

The term “heteroaryl” or “heteroaromatic,” as used herein, refers to aryl groups which contain at least one heteroatom selected from N, O, and S; wherein the nitrogen and sulfur atoms may be optionally ed, and the nitrogen atom(s) may be optionally quatemized. Heteroaryl groups may be substituted or unsubstituted. A heteroaryl group may be attached to the remainder of the molecule through a heteroatom. Non-limiting examples of heteroaryl groups include l-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3- pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyloxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3- l, 2-pyridyl, 3-pyridyl, 4-pyridyl, midyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl, 2- benzimidazolyl, 5-indolyl, l-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and 6- quinolyl.

The term “homoalkyl,” as used herein refers to alkyl groups which are hydrocarbon groups.

The term “identical,” as used herein, refers to two or more sequences or subsequences which are the same. In addition, the term “substantially identical,” as used herein, refers to two or more sequences which have a percentage of tial units which are the same when compared and aligned for maximum correspondence over a ison window, or designated region as measured using comparison algorithms or by manual alignment and Visual inspection. By way of example only, two or more sequences may be “substantially identical” if the sequential units are about 60% cal, about 65% identical, about 70% cal, about 75% identical, about 80% identical, about 85% identical, about 90% cal, or about 95% cal over a ied region. Such percentages to describe the “percent identity” of two or more sequences. The identity of a sequence can exist over a region that is at least about 75-100 sequential units in length, over a region that is about 50 sequential units in length, or, where not specified, across the entire ce. This definition also refers to the complement of a test sequence. By way of example only, two or more polypeptide sequences are identical when the amino acid residues are the same, while two or more polypeptide sequences are “substantially identical” if the amino acid es are about 60% identical, about 65% identical, about 70% identical, about 75% cal, about 80% identical, about 85% identical, about 90% identical, or about 95% identical over a specified region. The identity can exist over a region that is at least about 75 to about 100 amino acids in length, over a region that is about 50 amino acids in length, or, where not ed, across the entire ce of a polypeptide sequence. In addition, by way of e only, two or more polynucleotide sequences are identical when the nucleic acid es are the same, while two or more polynucleotide sequences are “substantially cal” if the nucleic acid residues are about 60% identical, about 65% identical, about 70% identical, about 75% identical, about 80% identical, about 85% identical, about 90% identical, or about 95% identical over a specified region. The identity can exist over a region that is at least about 75 to about 100 nucleic acids in length, over a region that is about 50 nucleic acids in length, or, where not specified, across the entire sequence of a polynucleotide sequence.

For sequence comparison, lly one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison thm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if ary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison thm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

The term “immunogenicity,” as used herein, refers to an antibody response to administration of a therapeutic drug. The immunogenicity toward therapeutic non-natural amino acid polypeptides can be obtained using quantitative and qualitative assays for detection of anti-non-natural amino acid ptides dies in biological fluids. Such assays include, but are not limited to, Radioimmunoassay (RIA), Enzyme-linked immunosorbent assay ( ELISA), luminescent immunoassay (LIA), and fluorescent immunoassay (FIA). Analysis of immunogenicity toward therapeutic non-natural amino acid ptides involves ing the dy response upon administration of therapeutic non-natural amino acid ptides to the antibody response upon administration of therapeutic natural amino acid polypeptides.

The term calating agent,” also referred to as calating group,” as used herein, refers to a chemical that can insert into the intramolecular space of a molecule or the intermolecular space n molecules. By way of example only an intercalating agent or group may be a molecule which inserts into the stacked bases of the DNA double helix.

The term “isolated,” as used herein, refers to separating and removing a ent of interest from components not of interest. Isolated substances can be in either a dry or semi-dry state, or in solution, including but not limited to an aqueous solution. The isolated component can be in a homogeneous state or the isolated ent can be a part of a pharmaceutical composition that ses additional pharmaceutically acceptable carriers and/or excipients. Purity and homogeneity may be determined using analytical chemistry techniques including, but not limited to, polyacrylamide gel electrophoresis or high performance liquid chromatography. In addition, when a component of interest is isolated and is the predominant species present in a preparation, the ent is described herein as substantially purified. The term “purified,” as used herein, may refer to a component of interest which is at least 85% pure, at least 90% pure, at least 95% pure, at least 99% or greater pure. By way of example only, nucleic acids or proteins are “isolated” when such nucleic acids or proteins are free of at least some 2012/039472 of the cellular components with which it is associated in the natural state, or that the nucleic acid or n has been concentrated to a level greater than the concentration of its in vivo or in vitro production.

Also, by way of example, a gene is isolated when separated from open reading frames which flank the gene and encode a protein other than the gene of interest.

The term “label,” as used herein, refers to a substance which is incorporated into a compound and is y detected, whereby its physical distribution may be detected and/or monitored.

The term “linkage,” as used herein to refer to bonds or chemical moiety formed from a chemical reaction between the functional group of a linker and another molecule. Such bonds may include, but are not limited to, covalent linkages and non-covalent bonds, while such chemical es may include, but are not limited to, esters, carbonates, imines phosphate esters, hydrazones, acetals, sters, peptide linkages, and oligonucleotide linkages. Hydrolytically stable es means that the linkages are ntially stable in water and do not react with water at useful pH values, including but not limited to, under physiological conditions for an extended period of time, perhaps even indefinitely. Hydrolytically unstable or degradable linkages mean that the es are degradable in water or in aqueous ons, ing for e, blood. Enzymatically unstable or able linkages mean that the linkage can be degraded by one or more enzymes. By way of example only, PEG and related polymers may e degradable linkages in the polymer backbone or in the linker group between the polymer backbone and one or more of the terminal fimctional groups of the polymer molecule. Such degradable linkages include, but are not limited to ester linkages formed by the reaction of PEG carboxylic acids or activated PEG carboxylic acids with alcohol groups on a biologically active agent, wherein such ester groups generally hydrolyze under physiological conditions to release the biologically active agent. Other hydrolytically degradable linkages include but are not limited to carbonate linkages; imine linkages resulted from reaction of an amine and an aldehyde; phosphate ester linkages formed by ng an alcohol with a phosphate group; hydrazone linkages which are on product of a hydrazide and an aldehyde; acetal linkages that are the reaction product of an aldehyde and an l; orthoester linkages that are the reaction product of a formate and an alcohol; peptide linkages formed by an amine group, including but not limited to, at an end of a polymer such as PEG, and a yl group of a peptide; and ucleotide linkages formed by a oramidite group, including but not limited to, at the end of a polymer, and a ' hydroxyl group of an oligonucleotide.

The terms “medium” or “media,” as used herein, refer to any culture medium used to grow and harvest cells and/or products expressed and/or secreted by such cells. Such “medium” or ” include, but are not limited to, solution, solid, semi-solid, or rigid supports that may support or contain any host cell, including, by way of example, bacterial host cells, yeast host cells, insect host cells, plant host cells, eukaryotic host cells, mammalian host cells, CHO cells, prokaryotic host cells, E. coli, or Pseudomonas host cells, and cell contents. Such “medium” or “media” includes, but is not limited to, medium or media in which the host cell has been grown into which a polypeptide has been secreted, including medium either before or after a proliferation step. Such “medium” or “media” also includes, but is not limited to, buffers or reagents that contain host cell lysates, by way of example a polypeptide produced intracellularly and the host cells are lysed or disrupted to release the polypeptide.

The term “metabolite,” as used herein, refers to a derivative of a compound, by way of example natural amino acid ptide, a non-natural amino acid polypeptide, a modified natural amino acid polypeptide, or a modified non-natural amino acid polypeptide, that is formed when the compound, by way of example natural amino acid ptide, non-natural amino acid polypeptide, modified natural amino acid polypeptide, or modified non-natural amino acid polypeptide, is lized. The term “pharmaceutically active metabolite” or “active metabolite” refers to a biologically active derivative of a nd, by way of example natural amino acid polypeptide, a non-natural amino acid polypeptide, a modified natural amino acid ptide, or a modified non-natural amino acid polypeptide, that is formed when such a compound, by way of example a natural amino acid polypeptide, non-natural amino acid polypeptide, modified natural amino acid polypeptide, or modified non-natural amino acid polypeptide, is lized.

The term “metabolized,” as used , refers to the sum of the processes by which a particular substance is changed by an organism. Such processes include, but are not limited to, hydrolysis reactions and ons catalyzed by enzymes. Further information on metabolism may be obtained from The Pharmacological Basis of Therapeutics, 9th Edition, McGraw—Hill (1996). By way of example only, metabolites of natural amino acid polypeptides, non-natural amino acid polypeptides, modified l amino acid polypeptides, or modified non-natural amino acid polypeptides may be fied either by administration of the natural amino acid ptides, tural amino acid polypeptides, modified natural amino acid polypeptides, or modified non-natural amino acid ptides to a host and analysis of tissue samples from the host, or by incubation of natural amino acid polypeptides, non-natural amino acid polypeptides, d natural amino acid polypeptides, or d non-natural amino acid polypeptides with hepatic cells in vitro and analysis of the resulting nds.

The term “metal chelator,” as used herein, refers to a molecule which forms a metal complex with metal ions. By way of example, such molecules may form two or more coordination bonds with a central metal ion and may form ring structures.

The term “metal-containing moiety,” as used herein, refers to a group which contains a metal ion, atom or particle. Such es include, but are not limited to, cisplatin, chelated metals ions (such as nickel, iron, and platinum), and metal nanoparticles (such as , iron, and platinum).

The term “moiety incorporating a heavy atom,” as used , refers to a group which incorporates an ion of atom which is usually heavier than carbon. Such ions or atoms include, but are not limited to, silicon, tungsten, gold, lead, and uranium.

The term “modified,” as used herein refers to the presence of a change to a natural amino acid, a non-natural amino acid, a natural amino acid polypeptide or a non-natural amino acid polypeptide. Such changes, or modifications, may be obtained by post synthesis modifications of natural amino acids, non- natural amino acids, natural amino acid polypeptides or non-natural amino acid polypeptides, or by co- translational, or by post-translational modification of natural amino acids, non-natural amino acids, natural amino acid polypeptides or non-natural amino acid polypeptides. The form “modified or unmodified” means that the natural amino acid, non-natural amino acid, natural amino acid polypeptide or non-natural amino acid polypeptide being sed are optionally d, that is, he natural amino acid, non-natural amino acid, natural amino acid polypeptide or non-natural amino acid polypeptide under discussion can be modified or unmodified.

As used , the term “modulated serum half-life” refers to positive or negative changes in the circulating half-life of a modified biologically active molecule relative to its non-modified form. By way of example, the modified biologically active molecules include, but are not limited to, natural amino acid, non-natural amino acid, natural amino acid polypeptide or non-natural amino acid ptide. By way of e, serum half-life is ed by taking blood samples at various time points after stration of the biologically active molecule or modified biologically active molecule, and determining the tration of that molecule in each sample. Correlation of the serum concentration with time allows calculation of the serum half-life. By way of example, modulated serum half-life may be an increased in serum half-life, which may enable an improved dosing regimens or avoid toxic effects. Such ses in serum may be at least about two fold, at least about three-fold, at least about five-fold, or at least about ten-fold. A non-limiting example of a method to te ses in serum half-life is given in example 33. This method may be used for ting the serum half-life of any polypeptide.

The term “modulated therapeutic half-life,” as used herein, refers to positive or negative change in the half-life of the therapeutically effective amount of a modified biologically active molecule, relative to its non-modified form. By way of example, the modified biologically active molecules include, but are not limited to, natural amino acid, non-natural amino acid, natural amino acid polypeptide or non-natural amino acid polypeptide. By way of example, therapeutic half-life is measured by measuring pharmacokinetic and/or pharmacodynamic properties of the molecule at various time points after administration. Increased therapeutic half-life may enable a particular beneficial dosing n, a particular beneficial total dose, or avoids an undesired effect. By way of example, the increased therapeutic half-life may result from increased potency, increased or decreased binding of the modified molecule to its target, an increase or decrease in another parameter or mechanism of action of the non- modified molecule, or an increased or sed breakdown of the molecules by enzymes such as, by way of example only, proteases. A non-limiting example of a method to evaluate increases in therapeutic half- life is given in example 33. This method may be used for evaluating the therapeutic half-life of any polypeptide.

] The term “nanoparticle,” as used herein, refers to a le which has a particle size between about 500 nm to about 1 nm.

The term “near-stoichiometric,’ 9 as used , refers to the ratio of the moles of nds participating in a al reaction being about 0.75 to about 1.5.

As used , the term “non-eukaryote” refers to non-eukaryotic organisms. By way of example, a non-eukaryotic organism may belong to the Eubacteria, (which includes but is not d to, Escherichia coli, Thermus thermophilus, or Bacillus thermophilus, Pseudomonas fluorescens, Pseudomonas aeruginosa, Pseudomonas putida), phylogenetic domain, or the Archaea, which es, but is not limited to, Methanococcus jannaschii, Methanobacterium thermoautotrophicum, Archaeoglobus fulgidus, ccus furiosus, Pyrococcus horikoshii, Aeuropyrum pemix, or Halobacterium such as Haloferax volcanii and Halobacterium species NRC-1, or phylogenetic domain.

A “non-natural amino acid” refers to an amino acid that is not one of the 20 common amino acids or pyrolysine or selenocysteine. Other terms that may be used synonymously with the term “non-natural amino acid” is “non-naturally encoded amino acid,” “unnatural amino acid,” “non-naturally-occurring amino acid,” and variously hyphenated and non-hyphenated versions thereof. The term “non-natural amino acid” includes, but is not d to, amino acids which occur naturally by modification of a lly encoded amino acid (including but not limited to, the 20 common amino acids or ysine and selenocysteine) but are not themselves incorporated into a growing polypeptide chain by the translation complex. Examples of naturally-occurring amino acids that are not naturally-encoded include, but are not limited to, N—acetylglucosaminyl-L-serine, N—acetylglucosaminyl-L-threonine, and O- phosphotyrosine. Additionally, the term “non-natural amino acid” includes, but is not limited to, amino acids which do not occur naturally and may be obtained synthetically or may be obtained by modification of non-natural amino acids.

The term “nucleic acid,” as used herein, refers to deoxyribonucleotides, deoxyribonucleosides, ribonucleosides or ribonucleotides and polymers thereof in either single- or double-stranded form. By way of example only, such c acids and nucleic acid polymers include, but are not limited to, (i) ues of natural nucleotides which have similar binding properties as a reference nucleic acid and are metabolized in a manner similar to naturally ing nucleotides; (ii) oligonucleotide analogs including, but are not limited to, PNA donucleic acid), analogs of DNA used in antisense technology (phosphorothioates, phosphoroamidates, and the like); (iii) conservatively modified variants thereof ding but not limited to, degenerate codon substitutions) and complementary sequences and sequence explicitly indicated. By way of example, rate codon substitutions may be achieved by generating ces in which the third position of one or more selected (or all) codons is substituted with mixed- base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 ; Ohtsuka et al., J.

Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 ).

The term “oxidizing agent,” as used herein, refers to a compound or material which is capable of removing an electron from a compound being oxidized. By way of example oxidizing agents include, but are not limited to, oxidized glutathione, e, cystamine, oxidized threitol, oxidized erythreitol, and oxygen. A wide variety of oxidizing agents are suitable for use in the methods and compositions described herein.

The term “pharmaceutically acceptable”, as used herein, refers to a material, including but not d, to a salt, carrier or diluent, which does not abrogate the biological activity or properties of the compound, and is relatively nontoxic, i.e., the material may be administered to an individual without causing rable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.

The term “photoaffinity label,” as used herein, refers to a label with a group, which, upon exposure to light, forms a linkage with a molecule for which the label has an affinity. By way of e only, such a linkage may be covalent or non-covalent.

The term caged moiety,” as used herein, refers to a group which, upon illumination at certain wavelengths, covalently or non-covalently binds other ions or molecules.

The term “photocleavable group,” as used herein, refers to a group which breaks upon exposure to light.

The term “photocrosslinker,” as used herein, refers to a compound comprising two or more functional groups which, upon exposure to light, are reactive and form a covalent or non-covalent linkage with two or more monomeric or polymeric molecules.

The term “photoisomerizable moiety,” as used herein, refers to a group wherein upon illumination with light changes from one ic form to another.

The term “polyalkylene glycol,” as used herein, refers to linear or branched polymeric polyether s. Such polyalkylene glycols, including, but are not limited to, polyethylene glycol, polypropylene glycol, polybutylene glycol, and derivatives thereof. Other exemplary embodiments are listed, for example, in commercial supplier catalogs, such as ater Corporation's catalog “Polyethylene Glycol and Derivatives for Biomedical Applications” (2001). By way of example only, such polymeric polyether s have average molecular weights between about 0.1 kDa to about 100 kDa. By way of example, such polymeric polyether s include, but are not d to, between about 100 Da and about 100,000 Da or more. The molecular weight of the polymer may be between about 100 Da and about 0 Da, including but not limited to, about 100,000 Da, about 95,000 Da, about 90,000 Da, about 85,000 Da, about 80,000 Da, about 75,000 Da, about 70,000 Da, about 65,000 Da, about 60,000 Da, about 55,000 Da, about 50,000 Da, about 45,000 Da, about 40,000 Da, about 35,000 Da, about 30,000 Da, about 25,000 Da, about 20,000 Da, about 15,000 Da, about 10,000 Da, about 9,000 Da, about 8,000 Da, about 7,000 Da, about 6,000 Da, about 5,000 Da, about 4,000 Da, about 3,000 Da, about 2,000 Da, about 1,000 Da, about 900 Da, about 800 Da, about 700 Da, about 600 Da, about 500 Da, 400 Da, about 300 Da, about 200 Da, and about 100 Da. In some embodiments molecular weight of the polymer is n about 100 Da and about 50,000 Da. In some embodiments, the molecular weight of the polymer is between about 100 Da and about 40,000 Da. In some embodiments, the molecular weight of the polymer is between about 1,000 Da and about 40,000 Da. In some embodiments, the molecular weight of the r is between about 2,000 to about 50,000 Da. In some embodiments, the molecular weight of the r is between about ,000 Da and about 40,000 Da. In some embodiments, the molecular weight of the polymer is between about 10,000 Da and about 40,000 Da. In some embodiments, the poly(ethylene glycol) molecule is a branched polymer. The molecular weight of the ed chain PEG may be between about 1,000 Da and about 100,000 Da, including but not limited to, about 100,000 Da, about 95,000 Da, about 90,000 Da, about 85,000 Da, about 80,000 Da, about 75,000 Da, about 70,000 Da, about 65,000 Da, about 60,000 Da, about 55,000 Da, about 50,000 Da, about 45,000 Da, about 40,000 Da, about 35,000 Da, about 30,000 Da, about 25,000 Da, about 20,000 Da, about 15,000 Da, about 10,000 Da, about 9,000 Da, about 8,000 Da, about 7,000 Da, about 6,000 Da, about 5,000 Da, about 4,000 Da, about 3,000 Da, about 2,000 Da, and about 1,000 Da. In some embodiments, the molecular weight of the branched chain PEG is between about 1,000 Da and about 50,000 Da. In some embodiments, the molecular weight of the branched chain PEG is between about 1,000 Da and about 40,000 Da. In some embodiments, the molecular weight of the branched chain PEG is between about 5,000 Da and about 40,000 Da. In some embodiments, the molecular weight of the branched chain PEG is between about 5,000 Da and about 20,000 Da. In other embodiments, the molecular weight of the branched chain PEG is between about 2,000 to about 50,000 The term “polymer,” as used herein, refers to a molecule composed of repeated subunits. Such molecules include, but are not limited to, polypeptides, cleotides, or polysaccharides or polyalkylene glycols.

The terms eptide,” “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. That is, a description directed to a ptide applies equally to a description of a e and a description of a protein, and vice versa. The terms apply to naturally occurring amino acid rs as well as amino acid polymers in which one or more amino acid residues is a non-natural amino acid. Additionally, such “polypeptides,39 66peptides” and “proteins” include amino acid chains of any length, including full length proteins, n the amino acid es are linked by covalent peptide bonds.

The term “post-translationally modified” refers to any cation of a natural or non-natural amino acid which occurs after such an amino acid has been translationally incorporated into a polypeptide chain. Such modifications include, but are not d to, co-translational in vivo modifications, co- translational in vitro modifications (such as in a cell-free translation ), post-translational in vivo modifications, and post-translational in vitro ations.

The terms “prodrug” or “pharmaceutically acceptable prodrug,” as used herein, refers to an agent that is converted into the parent drug in vivo or in vitro, wherein which does not abrogate the biological activity or properties of the drug, and is relatively nontoxic, i.e., the material may be administered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any of the ents of the composition in which it is contained. Prodrugs are generally drug precursors that, following administration to a subject and subsequent absorption, are converted to an active, or a more active species via some process, such as sion by a metabolic pathway. Some prodrugs have a chemical group present on the prodrug that renders it less active and/or confers solubility or some other property to the drug. Once the chemical group has been cleaved and/or modified from the prodrug the active drug is ted. Prodrugs are converted into active drug within the body through enzymatic or non-enzymatic reactions. gs may provide improved physiochemical properties such as better solubility, enhanced delivery characteristics, such as specifically targeting a particular cell, tissue, organ or ligand, and improved therapeutic value of the drug. The benefits of such prodrugs include, but are not limited to, (i) ease of administration compared with the parent drug; (ii) the prodrug may be bioavailable by oral stration whereas the parent is not; and (iii) the prodrug may also have improved solubility in ceutical compositions ed with the parent drug. A pro-drug includes a pharmacologically inactive, or reduced-activity, derivative of an active drug. Prodrugs may be designed to modulate the amount of a drug or biologically active molecule that reaches a desired site of action through the manipulation of the properties of a drug, such as physiochemical, biopharmaceutical, or pharmacokinetic properties. An example, without limitation, of a prodrug would be a non-natural amino acid ptide which is administered as an ester (the “prodrug”) to facilitate transmittal across a cell membrane where water solubility is detrimental to ty but which then is metabolically hydrolyzed to the carboxylic acid, the active , once inside the cell where water solubility is ial. Prodrugs may be designed as reversible drug tives, for use as modifiers to enhance drug transport to site-specific tissues.

The term “prophylactically effective amount,” as used herein, refers that amount of a composition containing at least one tural amino acid polypeptide or at least one modified non-natural amino acid polypeptide prophylactically applied to a patient which will relieve to some extent one or more of the symptoms of a disease, condition or disorder being treated. In such prophylactic applications, such s may depend on the patient's state of health, weight, and the like. It is considered well within the skill of the art for one to determine such prophylactically effective amounts by routine experimentation, including, but not limited to, a dose escalation al trial.

The term “protected,” as used herein, refers to the presence of a “protecting group” or moiety that prevents reaction of the chemically reactive onal group under certain reaction conditions. The protecting group will vary depending on the type of chemically reactive group being protected. By way of example only, (i) if the chemically reactive group is an amine or a hydrazide, the protecting group may be selected from tert-butyloxycarbonyl ) and 9-fluorenylmethoxycarbonyl (Fmoc); (ii) if the chemically reactive group is a thiol, the protecting group may be orthopyridyldisulfide; and (iii) if the chemically reactive group is a carboxylic acid, such as butanoic or propionic acid, or a hydroxyl group, the ting group may be benzyl or an alkyl group such as methyl, ethyl, or tert-butyl.

By way of e only, blocking/protecting groups may be selected from: :2 0 H (1312 O \ C\O El ,0 \El/ \ O \(+312 Y Hzc/, “39/ 2 O allyl Bn Cbz alloc Me H2 H3C\ [CH3 0 ch/c\ (HsC)sC/ (Hsc)3<:’s'\ (HsC>sSi\/\OJ\ Et t—butyl TBDMS T900 H2 )1 0/ o HZC’O (CH3)3C/ \n/ (C6H5)3C‘ H ck ° 3 O.0 B00 pMBn trityl acetyl Fmoc Additionally, protecting groups include, but are not limited to, including photolabile groups such as Nvoc and MeNvoc and other protecting groups known in the art. Other protecting groups are described in Greene and Wuts, Protective Groups in Organic sis, 3rd Ed., John Wiley & Sons, New York, NY, 1999, which is incorporated herein by reference in its entirety.

The term active moiety,” as used herein, refers to a group whose nuclei spontaneously give off nuclear radiation, such as alpha, beta, or gamma les; wherein, alpha particles are helium nuclei, beta particles are electrons, and gamma particles are high energy photons.

The term ive nd,” as used herein, refers to a compound which under appropriate conditions is reactive toward another atom, molecule or compound.

The term “recombinant host cell,” also referred to as “host cell,” refers to a cell which includes an exogenous polynucleotide, n the methods used to insert the exogenous polynucleotide into a cell include, but are not limited to, direct uptake, transduction, f-mating, or other methods known in the art to create recombinant host cells. By way of example only, such exogenous polynucleotide may be a nonintegrated vector, including but not limited to a plasmid, or may be integrated into the host genome.

The term “redox-active ” as used herein, refers to a molecule which oxidizes or reduces another molecule, whereby the redox active agent becomes reduced or oxidized. Examples of redox active 2+/3+ complexes, and agent include, but are not limited to, ferrocene, quinones, Ruw3+ complexes, Co Osw3+ complexes.

The term “reducing agent,” as used herein, refers to a compound or material which is capable of adding an on to a compound being reduced. By way of example reducing agents e, but are not d to, threitol (DTT), 2-mercaptoethanol, dithioerythritol, cysteine, cysteamine (2- thanethiol), and reduced glutathione. Such reducing agents may be used, by way of example only, to maintain sulfhydryl groups in the reduced state and to reduce intra- or intermolecular disulfide bonds.

“Refolding,” as used herein describes any process, reaction or method which transforms an improperly folded or unfolded state to a native or properly folded conformation. By way of example only, refolding transforms disulfide bond containing polypeptides from an improperly folded or unfolded state to a native or properly folded conformation with respect to de bonds. Such disulfide bond ning polypeptides may be natural amino acid polypeptides or non-natural amino acid ptides.

The term “resin,” as used herein, refers to high molecular , ble polymer beads. By way of example only, such beads may be used as supports for solid phase e synthesis, or sites for attachment of molecules prior to purification.

The term “saccharide,” as used , refers to a series of carbohydrates including but not limited to sugars, ccharides, accharides, and polysaccharides.

The term “safety” or “safety profile,” as used herein, refers to side effects that might be related to administration of a drug relative to the number of times the drug has been administered. By way of example, a drug which has been administered many times and produced only mild or no side effects is said to have an ent safety profile. A non-limiting example of a method to evaluate the safety profile is given in example 26. This method may be used for evaluating the safety profile of any polypeptide.

The phrase tively hybridizes to” or “specifically hybridizes to,” as used herein, refers to the binding, duplexing, or hybridizing of a molecule to a particular nucleotide sequence under stringent hybridization conditions when that sequence is present in a complex mixture including but not limited to, total cellular or library DNA or RNA.

The term “spin label,” as used herein, refers to molecules which contain an atom or a group of atoms exhibiting an unpaired electron spin (i.e. a stable paramagnetic group) that can be detected by electron spin resonance spectroscopy and can be attached to another molecule. Such spin-label molecules include, but are not limited to, nitryl radicals and nitroxides, and may be single spin-labels or double spin- labels.

The term “stoichiometric,” as used , refers to the ratio of the moles of compounds participating in a chemical on being about 0.9 to about 1.1.

The term “stoichiometric-like,” as used herein, refers to a chemical reaction which becomes stoichiometric or near-stoichiometric upon changes in reaction ions or in the presence of additives.

Such s in reaction conditions include, but are not limited to, an increase in temperature or change in pH. Such additives include, but are not limited to, accelerants.

The phrase “stringent hybridization conditions” refers to hybridization of sequences of DNA, RNA, PNA or other nucleic acid mimics, or combinations thereof, under conditions of low ionic th and high temperature. By way of example, under stringent conditions a probe will hybridize to its target subsequence in a complex mixture of nucleic acid (including but not limited to, total cellular or library DNA or RNA) but does not hybridize to other ces in the complex mixture. Stringent conditions are sequence-dependent and will be different in different circumstances. By way of example, longer sequences ize specifically at higher temperatures. Stringent ization conditions include, but are not limited to, (i) about 5-10 0C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH; (ii) the salt concentration is about 0.01 M to about 1.0 M at about pH 7.0 to about pH 8.3 and the temperature is at least about 30 0C for short probes (including but not limited to, about 10 to about 50 nucleotides) and at least about 60 0C for long probes (including but not d to, greater than 50 nucleotides); (iii) the addition of destabilizing agents including, but not limited to, formamide, (iv) 50% formamide, 5X SSC, and 1% SDS, incubating at 42 0C, or 5X SSC, about 1% SDS, incubating at 65 0C, with wash in 0.2X SSC, and about 0.1% SDS at 65 0C for between about 5 s to about 120 minutes. By way of e only, detection of selective or ic hybridization, includes, but is not limited to, a positive signal at least two times background. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Laboratory ques in Biochemistry and Molecular Biology-- Hybridization with Nucleic Probes, “Overview of principles of hybridization and the strategy of nucleic acid assays” (1993).

The term ct” as used herein, refers to an animal which is the object of treatment, observation or experiment. By way of example only, a subject may be, but is not limited to, a mammal ing, but not limited to, a human.

The term “substantially purified,” as used herein, refers to a component of interest that may be substantially or essentially free of other components which ly accompany or interact with the component of st prior to cation. By way of example only, a component of interest may be “substantially purified” when the preparation of the component of st contains less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% (by dry weight) of contaminating components. Thus, a “substantially purified” component of interest may have a purity level of about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or greater. By way of example only, a natural amino acid polypeptide or a non-natural amino acid polypeptide may be purified from a native cell, or host cell in the case of recombinantly produced natural amino acid polypeptides or non-natural amino acid polypeptides. By way of example a preparation of a natural amino acid polypeptide or a tural amino acid polypeptide may be “substantially purified” when the preparation contains less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% (by dry weight) of contaminating al. By way of example when a natural amino acid polypeptide or a non-natural amino acid polypeptide is recombinantly produced by host cells, the natural amino acid polypeptide or non-natural amino acid polypeptide may be t at about 30%, about 25%, about 20%, about 15%, about 10%, about 5%, about 4%, about 3%, about 2%, or about 1% or less of the dry weight of the cells. By way of example when a natural amino acid polypeptide or a non-natural amino acid polypeptide is recombinantly produced by host cells, the natural amino acid polypeptide or non-natural amino acid polypeptide may be present in the culture medium at about 5g/L, about 4g/L, about 3g/L, about 2g/L, about 1g/L, about 750mg/L, about 500mg/L, about 250mg/L, about 100mg/L, about 50mg/L, about 10mg/L, or about 1mg/L or less of the dry weight of the cells. By way of e, “substantially purified” natural amino acid polypeptides or non-natural amino acid polypeptides may have a purity level of about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or greater as determined by appropriate s, including, but not limited to, SDS/PAGE is, RP-HPLC, SEC, and capillary electrophoresis.

The term “substituents” also referred to as “non-interfering substituents39 66refers to groups which may be used to replace another group on a molecule. Such groups include, but are not limited to, halo, C1- C10 alkyl, C2-C10 alkenyl, C2-C10 l, C1-C10 alkoxy, C5-C12 aralkyl, C3-C12 cycloalkyl, C4-C12 cycloalkenyl, , substituted phenyl, toluolyl, xylenyl, biphenyl, C2-C12 alkoxyalkyl, C5-C12 aryl, C5-C12 aryloxyalkyl, C7-C12 oxyaryl, C1-C6 alkylsulfmyl, C1-C10 alkylsulfonyl, -(CH2)m-O-(C1- C10 alkyl) wherein m is from 1 to 8, aryl, substituted aryl, tuted alkoxy, fluoroalkyl, heterocyclic radical, substituted heterocyclic radical, nitroalkyl, -N02, -CN, -NRC(O)-(C1-C10 alkyl), (C1-C10 alkyl), C2-C10 oalkyl, -C(O)O-(C1-C10 , -OH, -S02, =S, -COOH, -NR2, carbonyl, -C(O)-(C1- C10 alkyl)-CF3, -C(O)-CF3, -C(O)NR2, -(C1-C10 aryl)-S-(C6-C10 aryl), -C(O)-(C6-C10 aryl), -(CH2)m-O- (CH2)m-O-(C1-C10 alkyl) wherein each m is from 1 to 8, -C(O)NR2, -C(S)NR2, -S02NR2, -NRC(O)NR2, - NRC(S)NR2, salts thereof, and the like. Each R group in the ing list includes, but is not limited to, H, alkyl or substituted alkyl, aryl or substituted aryl, or alkaryl. Where substituent groups are specified by their conventional chemical formulas, written from left to right, they equally encompass the chemically identical substituents that would result from writing the structure from right to left; for example, -CH20- is equivalent to —OCH2-.

By way of example only, substituents for alkyl and heteroalkyl radicals (including those groups referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) includes, but is not limited to: -OR, =0, =NR, =N—OR, -NR2, -SR, - halogen, -SiR3, -OC(O)R, -C(O)R, -C02R, -CONR2, -OC(O)NR2, -NRC(O)R, -NRC(O)NR2, -NR(O)2R, - NR-C(NR2)=NR, -S(O)R, -S(O)2R, -S(O)2NR2, -NRS02R, -CN and —N02. Each R group in the ing list includes, but is not limited to, hydrogen, tuted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, including but not limited to, aryl substituted with 1-3 halogens, substituted or unsubstituted alkyl, alkoxy or thioalkoxy groups, or aralkyl groups. When two R groups are attached to the same nitrogen atom, they can be ed with the nitrogen atom to form a 5-, 6-, or 7-membered ring. For example, -NR2 is meant to e, but not be d to, l-pyrrolidinyl and 4-morpholinyl.

By way of example, substituents for aryl and aryl groups include, but are not limited to, - OR, =0, =NR, =N-OR, -NR2, -SR, -halogen, -SiR3, -OC(O)R, -C(O)R, -C02R, -CONR2, NR2, - NRC(O)R, -NRC(O)NR2, -NR(O)2R, -NR-C(NR2)=NR, -S(O)R, -S(O)2R, -S(O)2NR2, -NRS02R, -CN, — N02, -R, -N3, -CH(Ph)2, C1-C4)alkoxy, and fluoro(C1-C4)alkyl, in a number ranging from zero to the total number of open valences on the aromatic ring system; and where each R group in the preceding list includes, but is not limited to, hydrogen, alkyl, heteroalkyl, aryl and heteroaryl.

The term “therapeutically effective amount,” as used , refers to the amount of a composition containing at least one non-natural amino acid polypeptide and/or at least one modified non-natural amino acid polypeptide administered to a patient already suffering from a disease, condition or disorder, sufficient to cure or at least partially arrest, or e to some extent one or more of the symptoms of the disease, disorder or condition being treated. The effectiveness of such compositions depend conditions including, but not limited to, the severity and course of the disease, er or condition, us therapy, the patient's health status and response to the drugs, and the judgment of the treating physician.

By way of example only, therapeutically effective amounts may be determined by routine experimentation, ing but not limited to a dose escalation clinical trial.

The term “thioalkoxy,” as used herein, refers to sulfur containing alkyl groups linked to les Via an oxygen atom.

The term “thermal melting point” or Tm is the temperature (under def1ned ionic strength, pH, and nucleic concentration) at Which 50% of probes complementary to a target hybridize to the target sequence at equilibrium.

The term “toxic moiety” or “toxic group” as used herein, refers to a compound Which can cause harm, disturbances, or death. Toxic moieties include, but are not limited to, auristatin, DNA minor groove binding agent, DNA minor groove alkylating agent, enediyne, lexitropsin, duocarmycin, taxane, puromycin, dolastatin, maytansinoid, Vinca alkaloid, AFP, MMAF, MMAE, AEB, AEVB, atin E, paclitaxel, docetaxel, CC-1065, SN—38, topotecan, morpholino-doxorubicin, rhizoxin, cyanomorpholino- doxorubicin, dolastatin-lO, echinomycin, combretatstatin, chalicheamicin, maytansine, DM-l, netropsin, podophyllotoxin (e.g. etoposide, teniposide, etc.), baccatin and its tives, anti-tubulin agents, cryptophysin, combretastatin, auristatin E, Vincristine, Vinblastine, Vindesine, Vinorelbine, VP-l6, camptothecin, lone A, epothilone B, zole, colchicines, colcimid, ustine, cemadotin, discodermolide, maytansine, eleutherobin, mechlorethamine, cyclophosphamide, lan, carmustine, lomustine, semustine, streptozocin, chlorozotocin, uracil mustard, chlormethine, ifosfamide, chlorambucil, pipobroman, triethylenemelamine, triethylenethiophosphoramine, busulfan, dacarbazine, and temozolomide, ytarabine, cytosine arabinoside, fluorouracil, floxuridine, 6-thioguanine, 6- mercaptopurine, pentostatin, 5-fluorouracil, methotrexate, lO-propargyl-5,8-dideazafolate, 5,8- dideazatetrahydrofolic acid, leucovorin, fludarabine phosphate, pentostatine, gemcitabine, Ara-C, paclitaxel, docetaxel, deoxycoformycin, cin—C, raginase, azathioprine, brequinar, antibiotics (e. g., anthracycline, gentamicin, cefalotin, vancomycin, telavancin, daptomycin, azithromycin, erythromycin, rocithromycin, furazolidone, illin, ampicillin, carbenicillin, acillin, methicillin, penicillin, ciprofloxacin, moxifloxacin, ofloxacin, doxycycline, minocycline, racycline, tetracycline, streptomycin, rifabutin, ethambutol, rifaximin, etc.), ral drugs (e. g., abacaVir, acyclovir, ampligen, cidofovir, rdine, didanosine, efavirenz, entecaVir, fosfonet, ganciclovir, ibacitabine, imunovir, idoxuridine, inosine, lopinaVir, methisazone, r, neVirapine, miVir, penciclovir, stavudine, trifluridine, truvada, valaciclovir, zanamiVir, etc.), daunorubicin hydrochloride, ycin, rubidomycin, cerubidine, icin, doxorubicin, epirubicin and morpholino derivatives, phenoxizone biscyclopeptides (e.g., dactinomycin), basic glycopeptides (e.g., bleomycin), anthraquinone glycosides WO 66560 (e. g., plicamycin, mithramycin), anthracenediones (e. g., mitoxantrone), azirinopyrrolo indolediones (e. g., mitomycin), macrocyclic immunosuppressants (e.g., cyclosporine, FK-506, tacrolimus, prograf, rapamycin etc.), navelbene, CPT-ll, anastrazole, letrazole, capecitabine, reloxafine, cyclophosphamide, ifosamide, droloxafine, allocolchicine, Halichondrin B, colchicine, colchicine derivatives , maytansine, rhizoxin, paclitaxel, paclitaxel derivatives, docetaxel, thiocolchicine, trityl cysterin, Vinblastine sulfate, Vincristine sulfate, cisplatin, carboplatin, hydroxyurea, N—methylhydrazine, epidophyllotoxin, procarbazine, mitoxantrone, leucovorin, and tegafur. “Taxanes” e paclitaxel, as well as any active taxane derivative or ug.

The terms “treat,” “treating” or “treatment”, as used herein, include alleviating, abating or ameliorating a disease or condition symptoms, preventing additional symptoms, ameliorating or preventing the underlying metabolic causes of symptoms, inhibiting the disease or condition, e.g., arresting the development of the disease or condition, ing the disease or condition, causing regression of the disease or condition, relieving a condition caused by the disease or condition, or stopping the symptoms of the e or condition. The terms “treat,39 66treating” or “treatment”, include, but are not d to, lactic and/or therapeutic treatments.

As used herein, the term “water soluble polymer” refers to any polymer that is soluble in aqueous ts. Such water e polymers include, but are not limited to, hylene glycol, hylene glycol propionaldehyde, mono C1-C10 alkoxy or aryloxy tives thereof (described in US. Patent No. ,252,714 which is incorporated by reference herein), monomethoxy—polyethylene glycol, polyvinyl pyrrolidone, polyvinyl alcohol, polyamino acids, diVinylether maleic anhydride, N—(2-Hydroxypropyl)- methacrylamide, dextran, dextran derivatives including dextran sulfate, polypropylene glycol, polypropylene oxide/ethylene oxide copolymer, polyoxyethylated , heparin, n fragments, polysaccharides, oligosaccharides, glycans, cellulose and cellulose derivatives, including but not limited to methylcellulose and carboxymethyl cellulose, serum albumin, starch and starch derivatives, ptides, polyalkylene glycol and tives thereof, copolymers of polyalkylene glycols and derivatives thereof, polyvinyl ethyl ethers, and alpha-beta-poly[(2-hydroxyethyl)-DL-aspartamide, and the like, or mixtures thereof By way of example only, coupling of such water soluble polymers to natural amino acid polypeptides or non-natural ptides may result in changes including, but not limited to, increased water solubility, increased or modulated serum half-life, sed or modulated therapeutic half-life relative to the unmodified form, increased bioavailability, modulated biological ty, extended ation time, modulated immunogenicity, modulated physical association characteristics including, but not limited to, aggregation and multimer formation, altered receptor binding, altered binding to one or more binding partners, and altered receptor dimerization or multimerization. In addition, such water soluble polymers may or may not have their own biological ty.

Unless otherwise indicated, conventional methods of mass spectroscopy, NMR, HPLC, protein try, biochemistry, recombinant DNA techniques and pharmacology, within the skill of the art are employed.

WO 66560 2012/039472 Compounds, (including, but not d to non-natural amino acids, non-natural amino acid polypeptides, modified non-natural amino acid polypeptides, and reagents for producing the aforementioned compounds) presented herein include isotopically—labeled compounds, which are identical to those recited in the various formulas and structures presented herein, but for the fact that one or more atoms are replaced by an atom haVing an atomic mass or mass number ent from the atomic mass or mass number usually found in nature. Examples of es that can be incorporated into the present compounds include isotopes of en, carbon, en, oxygen, fluorine and chlorine, such as 2H, 3H, 13C, 14C, 15N, 18O, 17O, 35 S, 18F, 36Cl, respectively. Certain isotopically-labeled compounds described herein, for example those into which ctive isotopes such as 3H and 14C are incorporated, are useful in drug and/or substrate tissue distribution assays. Further, substitution with isotopes such as deuterium, i.e., 2H, can afford certain therapeutic advantages resulting from greater metabolic stability, for example increased in Vivo half-life or reduced dosage requirements.

Some of the compounds herein (including, but not limited to tural amino acids, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides, and reagents for producing the aforementioned compounds) have asymmetric carbon atoms and can therefore exist as omers or diastereomers. Diasteromeric mixtures can be separated into their indiVidual diastereomers on the basis of their physical chemical differences by methods known, for example, by chromatography and/or fractional crystallization. Enantiomers can be separated by ting the omeric mixture into a diastereomeric mixture by reaction with an appropriate optically active compound (e. g., alcohol), separating the diastereomers and converting (e. g., hydrolyzing) the dual diastereomers to the ponding pure enantiomers. All such isomers, including diastereomers, enantiomers, and mixtures thereof are considered as part of the compositions described herein.

In additional or further embodiments, the compounds described herein (including, but not limited to tural amino acids, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides, and reagents for producing the aforementioned compounds) are used in the form of prodrugs.

In additional or further embodiments, the compounds described herein ((including, but not limited to tural amino acids, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides, and reagents for producing the aforementioned compounds) are metabolized upon stration to an organism in need to produce a metabolite that is then used to produce a desired effect, including a desired therapeutic effect. In further or additional embodiments are active metabolites of non- natural amino acids and “modified or unmodified” non-natural amino acid polypeptides.

The methods and formulations described herein include the use of es, crystalline forms (also known as polymorphs), or pharmaceutically acceptable salts of non-natural amino acids, non-natural amino acid ptides and modified non-natural amino acid polypeptides. In certain embodiments, non- natural amino acids, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides may exist as tautomers. All tautomers are ed within the scope of the non-natural amino acids, non-natural amino acid polypeptides and modified non-natural amino acid ptides presented herein. In addition, the non-natural amino acids, non-natural amino acid polypeptides and modified non- natural amino acid polypeptides described herein can exist in unsolvated as well as solvated forms with pharmaceutically acceptable solvents such as water, ethanol, and the like. The solvated forms of the non- natural amino acids, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides presented herein are also considered to be disclosed herein.

] Some of the compounds herein (including, but not limited to non-natural amino acids, non-natural amino acid polypeptides and d non-natural amino acid polypeptides and ts for producing the aforementioned nds) may exist in several tautomeric forms. All such tautomeric forms are considered as part of the compositions described herein. Also, for example all eto forms of any compounds (including, but not limited to tural amino acids, non-natural amino acid polypeptides and d non-natural amino acid polypeptides and reagents for producing the aforementioned compounds) herein are considered as part of the compositions described herein.

Some of the compounds herein (including, but not limited to non-natural amino acids, non-natural amino acid ptides and modified non-natural amino acid polypeptides and reagents for producing either of the entioned compounds) are acidic and may form a salt with a pharmaceutically acceptable cation. Some of the nds herein (including, but not limited to non-natural amino acids, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides and reagents for ing the aforementioned compounds) can be basic and accordingly, may form a salt with a pharmaceutically able anion. All such salts, including di-salts are within the scope of the compositions described herein and they can be prepared by conventional methods. For example, salts can be prepared by contacting the acidic and basic es, in either an s, non-aqueous or partially aqueous medium. The salts are recovered by using at least one of the following techniques: filtration, precipitation with a non-solvent followed by filtration, evaporation of the solvent, or, in the case of aqueous solutions, lyophilization.

Pharmaceutically acceptable salts of the non-natural amino acid polypeptides disclosed herein may be formed when an acidic proton present in the parent non-natural amino acid polypeptides either is replaced by a metal ion, by way of example an alkali metal ion, an alkaline earth ion, or an aluminum ion; or coordinates with an organic base. In addition, the salt forms of the disclosed non-natural amino acid polypeptides can be prepared using salts of the ng materials or intermediates. The tural amino acid polypeptides described herein may be prepared as a pharmaceutically acceptable acid addition salt (which is a type of a pharmaceutically acceptable salt) by reacting the free base form of non-natural amino acid polypeptides described herein with a pharmaceutically acceptable inorganic or organic acid.

Alternatively, the non-natural amino acid polypeptides described herein may be prepared as pharmaceutically acceptable base addition salts (which are a type of a ceutically acceptable salt) by reacting the free acid form of non-natural amino acid polypeptides described herein with a pharmaceutically acceptable inorganic or organic base.

The type of pharmaceutical acceptable salts, include, but are not limited to: (1) acid addition salts, formed With inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or formed With c acids such as acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, ic acid, c acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, ic acid, citric acid, benzoic acid, ydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, thalenesulfonic acid, 4-methylbicyclo- [2.2.2]octene-l-carboxylic acid, glucoheptonic acid, 4,4’-methylenebis-(3-hydroxyene-l -carboxylic acid), 3-phenylpropionic acid, trimethylacetic acid, tertiary cetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid, and the like; (2) salts formed When an acidic proton present in the parent compound either is replaced by a metal ion, e. g., an alkali metal ion, an alkaline earth ion, or an aluminum ion; or coordinates With an organic base.

Acceptable organic bases include ethanolamine, diethanolamine, triethanolamine, tromethamine, N- methylglucamine, and the like. Acceptable inorganic bases e aluminum hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate, sodium hydroxide, and the like.

The corresponding counterions of the non-natural amino acid polypeptide pharmaceutical acceptable salts may be analyzed and identified using various methods ing, but not limited to, ion exchange chromatography, ion chromatography, capillary electrophoresis, inductively coupled plasma, atomic absorption spectroscopy, mass spectrometry, or any combination thereof. In addition, the therapeutic ty of such non-natural amino acid polypeptide pharmaceutical acceptable salts may be tested using the techniques and methods described in es 87-91.

It should be tood that a reference to a salt includes the solvent addition forms or crystal forms thereof, ularly solvates or polymorphs. Solvates contain either stoichiometric or non- stoichiometric amounts of a solvent, and are often formed during the process of crystallization With ceutically acceptable solvents such as water, l, and the like. Hydrates are formed When the solvent is water, or alcoholates are formed When the solvent is alcohol. Polymorphs include the different crystal packing arrangements of the same elemental composition of a compound. Polymorphs usually have ent X-ray diffraction patterns, infrared spectra, melting points, density, hardness, crystal shape, optical and electrical properties, stability, and solubility. Various factors such as the recrystallization solvent, rate of crystallization, and storage temperature may cause a single crystal form to dominate.

The ing and characterization of non-natural amino acid polypeptide pharmaceutical acceptable salts polymorphs and/or solvates may be accomplished using a variety of techniques including, but not limited to, thermal analysis, x-ray diffraction, spectroscopy, vapor sorption, and microscopy.

Thermal analysis methods address thermo chemical degradation or thermo al processes including, but not d to, polymorphic transitions, and such methods are used to analyze the relationships between polymorphic forms, determine weight loss, to find the glass transition temperature, or for excipient compatibility studies. Such methods include, but are not limited to, ential scanning calorimetry (DSC), Modulated Differential ng Calorimetry (MDCS), Thermogravimetric analysis (TGA), and Thermogravi-metric and Infrared analysis (TG/IR). X-ray diffraction methods include, but are not limited to, single crystal and powder ctometers and synchrotron sources. The various spectroscopic techniques used include, but are not limited to, Raman, FTIR, UVIS, and NMR (liquid and solid state). The various microscopy techniques include, but are not limited to, polarized light microscopy, Scanning Electron Microscopy (SEM) with Energy sive X-Ray Analysis (EDX), Environmental Scanning Electron Microscopy with EDX (in gas or water vapor atmosphere), IR copy, and Raman microscopy.

BRIEF DESCRIPTION OF THE DRAWINGS The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be ed by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which: Figure 1 presents a graphical illustration of Her-Tox binding to the Her2 receptor.

Figure 2 presents a graphical illustration of the expression of Anti-Her2 variants determined by ELISA analysis.

Figure 3 presents a cal ration of the expression of Anti-Her2 variants determined by ELISA analysis.

Figure 4 presents a graphical illustration of the cell proliferation assay with HCC 1954 breast cancer cell line and dolastatin linker derivatives.

Figure 5 presents a graphical ration of the analysis of the cell eration assay with HCC 1954 breast cancer cell line and trastuzumab-tox conjugates.

Figure 6 presents a cal illustration of the analysis of the cell proliferation assay with SKOV-3 ovarian cancer cell line and dolastatin linker derivatives.

Figure 7 presents a graphical illustration of the analysis of the cell eration assay with SKOV-3 ovarian cancer cell line and trastuzumab-tox conjugates.

Figure 8 presents a graphical illustration of the analysis of the cell proliferation assay with MDA- MB-468 breast cancer cell line and dolastatin linker derivatives.

Figure 9 presents a graphical illustration of the analysis of the cell proliferation assay with MDA- MB-468 breast cancer line and trastuzumab-tox conjugates.

Figure 10 presents a cal ration of tumor volume measurement (mm3) after a single IC dose (3.3 mg/kg, 10 mg/kg, g) of trastuzumab-linked dolastatin tives.

Figure 11 presents assay formats used to measure trastuzumab-linked dolastatin tives tration in SD rat serum Figure 12 presents graphical illustrations of serum concentrations (ng/mL) of trastuzumab-linked dolastatin derivatives after single IV injections.

Figure 13 presents a graphical illustration of serum concentrations (ng/mL) of trastuzumab-linked dolastatin derivatives after single IV injections. This assay detects antibody binding to the ErbB2 Figure 14 presents a graphical illustration of of serum concentrations (ng/mL) of trastuzumab- linked dolastatin derivatives after IV ion. The in Vivo stability measurements detect at least two dolastatin derivatives linked to umab.

Figure 15 presents graphical illustrations of the change in rat body weight and tumor volume after treatment with trastuzumab-linked dolastatin derivatives.

Figure 16 presents graphical illustrations of anti-tumor efficacy of trastuzumab, Her2-HSl22- NCDl and Her2-HSl22/LK145-HCD1 against established tumors of HCC1954 in SCID-bg mice. Mice were administered a single IV ion on day l ). Data points ent group e tumor volume and error bars represent standard error of the mean (SEM).

Figure 17 presents graphical rations of umor efficacy of dolastatin linker derivatives in the MDA361DYT2 Breast (2--) Xenograft model.

Figure 18 presents graphical illustrations of anti-tumor efficacy of dolastatin linker derivatives in the MDA361DYT2 Breast (2--) Xenograft model.

DETAILED DESCRIPTION OF THE INVENTION While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those d in the art that such embodiments are provided by way of example only.

Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that s and structures within the scope of these claims and their equivalents be covered thereby.

I. Introduction Recently, an entirely new technology in the n sciences has been ed, which promises to overcome many of the limitations associated with site-specific cations of proteins. Specifically, new components have been added to the protein biosynthetic machinery of the prokaryote Escherichia coli (E. 6011') (e. g., L. Wang, et al., (2001), m 292:498-500) and the eukaryote Sacchromyces cerevisiae (S. cerevisiae) (e. g., J. Chin et al., m 301 :964-7 (2003)), which has enabled the incorporation of non-natural amino acids to proteins in vivo. A number of new amino acids with novel chemical, physical or biological ties, including ff1nity labels and somerizable amino acids, keto amino acids, and glycosylated amino acids have been incorporated efficiently and with high fidelity into proteins in E. coli and in yeast in response to the amber codon, TAG, using this methodology.

See, e.g., J. W. Chin et al., (2002), Journal of the American Chemical Society 124:9026-9027 (incorporated by reference in its entirety); J. W. Chin, & P. G. Schultz, (2002), ChemBioChem 3(1 1):] 135-1 137 (incorporated by reference in its entirety); J. W. Chin, et al., (2002), PNAS United States of America 99(17):]1020-11024 (incorporated by reference in its entirety); and, L. Wang, & P. G.

Schultz, (2002), Chem. Comm. l-ll (incorporated by reference in its ty). These studies have demonstrated that it is possible to selectively and routinely introduce al functional groups that are not found in ns, that are chemically inert to all of the functional groups found in the 20 common, genetically-encoded amino acids and that may be used to react ently and selectively to form stable covalent linkages.

II. Overview ]At one level, described herein are the tools ds, itions, techniques) for creating and using atin linker derivatives or analogs comprising at least one carbonyl, dicarbonyl, oxime, hydroxylamine, de, protected aldehyde, ketone, protected ketone, thioester, ester, dicarbonyl, hydrazine, azide, amidine, imine, diamine, keto-amine, keto-alkyne, alkyne, cycloalkyne, or ene-dione.

At another level, described herein are the tools (methods, compositions, techniques) for creating and using dolastatin linker derivatives or analogs comprising at least one non-natural amino acid or modified non- l amino acid With an oxime, aromatic amine, cycle (e. g., indole, quinoxaline, phenazine, pyrazole, triazole, etc.).

.Such dolastatin linker derivatives comprising non-natural amino acids may contain further functionality, ing but not limited to, a polymer; a water-soluble polymer; a derivative of polyethylene glycol; a second protein or polypeptide or polypeptide analog; an antibody or antibody fragment; and any combination thereof Note that the various aforementioned functionalities are not meant to imply that the members of one functionality cannot be classified as members of another functionality.

Indeed, there Will be overlap depending upon the particular circumstances. By way of example only, a soluble polymer overlaps in scope With a derivative of polyethylene glycol, however the overlap is not complete and thus both functionalities are cited above.

Provided herein in some embodiments, is a toxic group linker derivative comprising a yl, dicarbonyl, oxime, hydroxylamine, aldehyde, protected de, ketone, protected ketone, thioester, ester, dicarbonyl, hydrazine, azide, amidine, imine, diamine, keto-amine, keto-alkyne, alkyne, cycloalkyne, or ene-dione. In some embodiments, the toxic group derivative comprises any of the linkers disclosed herein. In other embodiments, described herein are the tools (methods, compositions, techniques) for creating and using toxic group derivatives or s comprising at least one non-natural amino acid or modified tural amino acid With an oxime, aromatic amine, heterocycle (e. g., indole, quinoxaline, phenazine, pyrazole, triazole, etc.).

] In some embodiments, such toxic tives comprising non-natural amino acids may contain further functionality, including but not limited to, a polymer; a water-soluble polymer; a derivative of polyethylene glycol; a second protein or polypeptide or polypeptide ; an antibody or dy fragment; and any combination thereof In specific embodiments, the toxic group is dolastatin or auristatin. In certain specific embodiments, the toxic group is dolastatin-10. Note that the various aforementioned functionalities are not meant to imply that the members of one functionality cannot be classified as members of another functionality. Indeed, there will be overlap depending upon the particular circumstances. By way of e only, a soluble polymer overlaps in scope with a derivative of polyethylene glycol, however the p is not complete and thus both functionalities are cited above.

Certain embodiments of the present invention describe preparations of certain toxic moieties with linkers that reduce the toxicity of the moiety in vivo while the toxic moiety retains pharmacological activity. In some embodiments, the toxicity of the linked toxic group, when administered to an animal or human, is reduced or eliminated compared to the free toxic group or toxic group derivatives comprising labile linkages, while retaining pharmacological activity. In some embodiments, increased doses of the linked toxic group (e. g., atin linker derivatives, non-natural amino acid linked dolastatin tives) may be administered to animals or humans with greater safety. In certain embodiments, the non-natural amino acid ptides linked to a toxic moiety (e. g., dolastatin derivative) provides in vitro and in vivo stability. In some embodiments, the non-natural amino acid ptides linked to a toxic moiety (e. g., dolastatin-10 derivative) are efficacious and less toxic compared to the free toxic moiety (e. g., dolastatin- ).

III. Dolastatin Linker Derivatives ]At one level, described herein are the tools (methods, compositions, techniques) for creating and using a dolastatin linker tives or analogs sing at least one non-natural amino acid or modified non-natural amino acid with a carbonyl, dicarbonyl, oxime or hydroxylamine group. Such dolastatin linker derivatives comprising non-natural amino acids may contain further functionality, including but not limited to, a polymer; a soluble polymer; a derivative of polyethylene ; a second n or polypeptide or polypeptide analog; an antibody or antibody fragment; and any ation thereof Note that the various aforementioned functionalities are not meant to imply that the members of one onality cannot be classified as members of another functionality. Indeed, there will be overlap depending upon the particular circumstances. By way of example only, a water-soluble polymer overlaps in scope with a tive of polyethylene glycol, however the overlap is not complete and thus both functionalities are cited above.

In one aspect are methods for selecting and designing a dolastatin linker derivative to be d using the methods, compositions and techniques described herein. The new dolastatin linker derivative may be designed de novo, including by way of example only, as part of high-throughput screening s (in which case numerous polypeptides may be designed, synthesized, characterized and/or tested) or based on the interests of the researcher. The new dolastatin linker derivative may also be designed based on the structure of a known or partially characterized polypeptide. By way of example only, dolastatin has been the subject of intense study by the scientific community; a new compound may be designed based on the structure of dolastatin. The ples for selecting which amino acid(s) to substitute and/or modify are described separately herein. The choice of which modification to employ is also described , and can be used to meet the need of the experimenter or end user. Such needs may include, but are not limited to, manipulating the therapeutic iveness of the polypeptide, improving the safety profile of the ptide, adjusting the pharmacokinetics, pharmacologics and/or pharmacodynamics of the polypeptide, such as, by way of example only, increasing water solubility, bioavailability, increasing serum half-life, increasing therapeutic half-life, modulating immunogenicity, modulating biological actiVity, or extending the circulation time. In addition, such modifications include, by way of example only, providing additional functionality to the polypeptide, incorporating an antibody, and any combination of the aforementioned modifications.

Also described herein are dolastatin linker derivatives that have or can be modified to contain an oxime, carbonyl, dicarbonyl, or hydroxylamine group. Included with this aspect are methods for producing, purifying, characterizing and using such atin linker tives ] The dolastatin linker derivative may contain at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or ten or more of a carbonyl or dicarbonyl group, oxime group, hydroxylamine group, or protected forms thereof. The dolastatin linker derivative can be the same or different, for e, there can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, , 16, 17, 18, 19, 20, or more different sites in the derivative that se 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more different ve groups.

A. Structure and Synthesis of Dolastatin Linker Derivatives: Electrophilic and Nucleophilic Groups Dolastatin derivatives with linkers containing a hydroxylamine (also called an aminooxy) group allow for reaction with a variety of electrophilic groups to form conjugates (including but not limited to, with PEG or other water soluble rs). Like hydrazines, hydrazides and semicarbazides, the enhanced nucleophilicity of the aminooxy group permits it to react efficiently and selectively with a variety of molecules that contain carbonyl- or dicarbonyl-groups, including but not d to, s, aldehydes or other functional groups with similar chemical reactivity. See, e. g., Shao, J. and Tam, J., J.

Am. Chem. Soc. 117:3893-3899 (1995); H. Hang and C. Bertozzi, Acc. Chem. Res. 34(9): 727-736 (2001). Whereas the result of reaction with a hydrazine group is the corresponding hydrazone, however, an oxime results generally from the reaction of an aminooxy group with a carbonyl- or dicarbonyl- ning group such as, by way of example, a ketones, aldehydes or other functional groups with similar chemical reactiVity. In some embodiments, dolastatin derivatives with linkers comprising an azide, alkyne or lkyne allow for linking of molecules Via cycloaddition reactions (e. g., polar cycloadditions, azide-alkyne Huisgen cycloaddition, etc.). ibed in US. Patent No. 7,807,619 which is incorporated by reference herein to the extent relative to the reaction).

Thus, in certain embodiments described herein are dolastatin derivatives with linkers comprising a hydroxylamine, de, protected aldehyde, ketone, protected ketone, thioester, ester, dicarbonyl, hydrazine, amidine, imine, diamine, keto-amine, keto-alkyne, and one hydroxylamine group, a ylamine-like group (which has reactiVity similar to a hydroxylamine group and is structurally similar to a hydroxylamine group), a masked hydroxylamine group (which can be readily converted into a hydroxylamine group), or a protected hydroxylamine group (Which has vity r to a hydroxylamine group upon deprotection). In some embodiments, the dolastatin derivatives with linkers comprise azides, alkynes or cycloalkynes. Such dolastatin linker tives include compounds having the structure of Formula (I), (III), (IV), (V), and (VI): Y N NdLLT/W e (1) R7 0 Me/_\MeMe OMeOM LZ—N | Mtg R7 0 Me/\MeMe OMeOM v' _N MULN (111) R7 0 MAMeMe OMeOM Me\NMeN\_)0J\MNWA/I/WNeOO Me O Me/\MeMe OMeOM +1r4-[.2NH0 (1‘1) |\/|e\NfireNjLN Me L1 Me O Me/\MeMe OMeOM NH HN—L3 Ar 0 _NIMNQHAfiNNKN R7 0 /\ Me OMeO v' INVL W J4<LMMeR7 0 Me/\MeMe OMeOM _NIMN8H¢LNgi: R7 0 Me/\MeMe OMeOM MeMe:grNET Me O Me/\MeMe OMeOM HN—L2 Me O /\ Me Me Me OMeOM NH HN_’ L3 04H (VD M3\NMeNjL% OMe MeM/:\ OMeOM NH HN—L4 Arfie wherein: Z has the structure of: R5 ; R5 is H, CORg, C1-C6a1kyl, 0r thiazole; R8 is OH or —NH—(a1ky1ene—O)n—NH2; R6 is OH or H; Ar is phenyl or pyridine; R7 is C1-C6a1kyl or hydrogen; Y and V are each selected from the group ting of an hydroxylamine, methyl, aldehyde, protected aldehyde, ketone, protected ketone, thioester, ester, dicarbonyl, hydrazine, azide, e, imine, diamine, keto-amine, keto-alkyne, alkyne, cycloalkyne, and ene-dione; L, L1, L2, L3, and L4 are each linkers ed from the group consisting of a bond, —alkylene—, — alkylene—C(O)—, —alkylene—J—, —(alkylene—O)n—alkylene—, —(alkylene—O)n—alkylene—C(O)—, — (alkylene—O)n—J—, —(alkylene—O)n—J—alkylene—, —(alkylene—O)n—(CH2)n~NHC(O)—(CH2)nu— C(Me)2—S—S—(CH2)nu~NHC(O)—(alkylene—O)nmv—alkylene—, —(alkylene—O)n—alkylene—W—, — alkylene—C(O)—W—, —(alkylene—O)n—alkylene—J—, —alkylene'—J—(alkylene—O)n—alkylene—, — (alkylene—O)n—alkylene—J—alkylene', —J—(alkylene—O)n—alkylene—, lene—O)n—alkylene— J—(alkylene—O)n'—alkylene—J'—, —W—, —alkylene—W—, alkylene'—J—(alkylene—NMe)n—alkylene— W—, —J—(alkylene—NMe)n—alkylene—W—, —(alkylene—O)n—alkylene—U—alkylene—C(O)—, — (alkylene—O)n—alkylene—U—alkylene—g —J—alkylene—NMe—alkylene'—NMe—alkylene"—W—, and —alkylene—J—alkylene'—NMe—alkylene"—NMe—alkylene'"—W—; W has the structure of: ILWJUCVJK; U has the ure of: COZH mink each I and J' independently have the structure of: agiN/ksz éiNiO/E‘ or fimief H H each n, n' n", n'" and n"" are independently integers greater than or equal to one; and or L is absent, Y is methyl, R5 is CORg, and R8 is —NH—(alkylene—O)n—NH2.

Such dolastatin linker tives may be in the form of a salt, or may be incorporated into a non-natural amino acid polypeptide, r, polysaccharide, or a polynucleotide and optionally post translationally modified.

In certain embodiments of compounds of Formula (I), (III), and (V), R5 is thiazole or ylic acid. In certain embodiments of compounds of Formula (I), (III), and (V), R5 is hydrogen. In certain embodiments of compounds of Formula (I), (III), and (V), R5 is methyl, ethyl, propyl, iso-propyl, butyl, iso-butyl, sec-butyl, tert-butyl, pentyl, or hexyl. In certain embodiments of compounds of Formula (I), (III), and (V), R5 is —NH—(alkylene—O)n—NH2, n alkylene is —CH2—, —CH2CH2—, —CH2CH2CH2—, — CH2CH2—, —CH2CH2CH2CH2CH2—, —CH2CH2CH2CH2CH2CH2—, —CH2CH2CH2CH2CH2CH2CH2— , —CH2CH2CH2CH2CH2CH2CH2CH2—, —CH2CH2CH2CH2CH2CHQCHQCHZCH2—, CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2—, —CH2CHZCHZCHZCHZCHZCHZCH2CH2CH2CH2—, or — CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2—. In certain embodiments of compounds of Formula (IV) and (VI), R5 is —NH—(alkylene—O)n—NH2, Wherein n is O, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, , 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100.

In some embodiments, Y is azide. In other embodiments, Y is cycloalkyne. In specific embodiments, the cyclooctyne has a structure of: |©—§ (R19)q each R19 is ndently selected from the group consisting of C1-C6 alkyl, C1-C6 alkoxy, ester, ether, thioether, lkyl, halogen, alkyl ester, aryl ester, amide, aryl amide, alkyl , alkyl amine, alkyl sulfonic acid, alkyl nitro, thioester, sulfonyl ester, lfonyl, nitrile, alkyl nitrile, and nitro; and qis 0,1, 2, 3, 4, 5, 6, 7, 8, 9,10 or 11.

In certain embodiments of compounds of Formula (I), (III), and (V), R6 is H. In some ments of compounds of Formula (I), (III), and (V), R6 is hydroxy.

In certain embodiments of nds of Formula (I), (III), and (V), Ar is phenyl.

In certain embodiments of nds of Formula (I), (III), (IV), (V), and (VI), R7 is methyl, ethyl, propyl, iso-propyl, butyl, sec-butyl iso-butyl, tert-butyl, pentyl, or hexyl. In n embodiments of compounds of Formula (I), (III), (IV), (V), and (VI), R7 is hydrogen.

] In n embodiments of compounds of Formula (I), (III), and (V), Y is hydroxylamine, aldehyde, protected aldehyde, ketone, protected ketone, thioester, ester, dicarbonyl, hydrazine, amidine, imine, diamine, keto-amine, keto-alkyne, or ene-dione.

In certain embodiments of compounds of Formula (IV) and (VI), V is a hydroxylamine, , aldehyde, protected aldehyde, ketone, protected ketone, thioester, ester, dicarbonyl, hydrazine, amidine, imine, diamine, keto-amine, keto-alkyne, and ene-dione.

In certain embodiments of compounds of Formula (I), (III), (IV), (V), and (VI), each L, L1, L2, L3, and L4 is independently a cleavable linker or non-cleavable linker. In certain embodiments of compounds WO 66560 of Formula (I), (III), (IV), (V), and (VI), each L, L1, L2, L3, and L4 is independently a oligo(ethylene ) derivatized linker.

In certain embodiments of compounds of Formula (I), (III), (IV), (V), and (VI), each alkylene, ne', alkylene", and alkylene'" independently is —CH2—, —CH2CH2—, —CH2CH2CH2—, — CH2CH2—, —CH2CH2CH2CH2CH2—, —CH2CH2CH2CH2CH2CH2—, 2CH2CH2CH2CH2CH2— —CH2CH2CH2CH2CH2CH2CH2CH2—, —CH2CH2CH2CH2CH2CH2CH2CH2CH2—, — CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2—, —CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2—, or — CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2—. In certain embodiments of compounds of Formula (XIV), (XV), (XVI), (XVII), and (XVIII), each n, n', n", n'", and n"" is O, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100.

B. Structure and Synthesis 0fDolastatin Linker Derivatives: ylamine Groups Thus, in certain embodiments described herein are dolastatin derivatives with linkers comprising a hydroxylamine group, a hydroxylamine-like group (Which has reactivity similar to a hydroxylamine group and is structurally similar to a ylamine group), a masked hydroxylamine group (Which can be readily converted into a hydroxylamine group), or a protected hydroxylamine group (which has reactivity similar to a hydroxylamine group upon deprotection). Such dolastatin linker derivatives include compounds having the structure of Formula (I): mm,R70 /\ Me OMeO wherein: Z has the structure of: R5 ; R5 is H, CORg, C1-C6alkyl, or thiazole; R8 is OH or —NH—(alkylene—O)n—NH2; R6 is OH or H; Ar is phenyl or pyridine; R7 is C1-C6alkyl or hydrogen; Y is NHg—O— or methyl; 2012/039472 L is a linker selected from the group consisting of —alkylene—, —alkylene—C(O)—, —(alkylene—O)n— alkylene—, —(alkylene—O)n—alkylene—C(O)—, —(alkylene—O)n—(CH2)n~NHC(O)—(CH2)nv~C(Me)2— S—S—(CH2)nm—NHC(O)—(alkylene—O)nvm—alkylene—, —(alkylene—O)n—alkylene—W—, —alkylene— C(O)—W—, —(alkylene—O)n—alkylene—U—alkylene—C(O)—, and —(alkylene—O)n—alkylene—U— alkylene—; W has the ure of: ILWJUCVJK; U has the structure of: COZH or L1s absent, Y1s methyl, R5 is CORg, and R8 is —NH—(alkylene—O)n—NH2; and each n, n', n", n'" and n"H are independently integers greater than or equal to one. Such dolastatin linker derivatives may be in the form of a salt, or may be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and ally post translationally modified.

In certain embodiments of compounds of Formula (I), R5 is thiazole. In certain embodiments of compounds of Formula (I), R5 is hydrogen. In certain embodiments of compounds of Formula (I), R5 is methyl, ethyl, , iso-propyl, butyl, iso-butyl, sec-butyl, tert-butyl, pentyl, or hexyl. In certain embodiments of compounds of Formula (I), R5 is —NH—(alkylene—O)n—NH2, wherein alkylene is —CH2—, — CHZCH2—, 2CH2—, —CH2CH2CH2CH2—, —CH2CH2CH2CH2CH2—, —CH2CH2CH2CH2CH2CH2—, — CHZCHZCHZCHZCHZCHZCHE —CH2CHZCHZCHZCHZCHZCHZCH2—, — CHZCHZCHZCHZCHZCHZCHZCHZCHE —CH2CHZCHZCHZCHZCHZCHZCHZCHZCH2—, — CHZCHZCHZCHZCHZCHZCHZCHZCHZCHZCHE or —CH2CH2CH2CH2CHZCHZCHZCHZCHZCHZCHZCHF In n embodiments of compounds of Formula (I), R5 is —NH—(alkylene—O)n—NH2, wherein n is O, 1, 2, 3, 4, 5, 6, 7, 8, 9,10,11,12,13,14,15,16,17,18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100.

In n ments of compounds of Formula (I), R6 is H. In some embodiments of compounds of a (1), R6 is hydroxy.

In certain embodiments of compounds of Formula (I), Ar is phenyl.

In n ments of compounds of Formula (1), R7 is , ethyl, propyl, iso—propyl, butyl, tyl iso-butyl, tert-butyl, pentyl, or hexyl. In certain embodiments of compounds of Formula (1), R7 is hydrogen.

In certain embodiments of compounds of Formula (I), Y is hydroxylamine, de, protected aldehyde, ketone, protected ketone, thioester, ester, dicarbonyl, hydrazine, e, imine, diamine, keto- amine, keto-alkyne, or ene-dione. In certain ments of compounds of Formula (I), V is a hydroxylamine, methyl, aldehyde, protected aldehyde, ketone, protected ketone, ter, ester, dicarbonyl, hydrazine, amidine, imine, diamine, keto-amine, keto-alkyne, and ene-dione.

In certain ments of compounds of Formula (I), each L is independently a cleavable linker or non-cleavable linker. In certain embodiments of compounds of Formula (I), each L is independently a oligo(ethylene glycol) derivatized .

In certain embodiments of compounds of Formula (I), alkylene is —CH2—, -—CH2CH2—, -— CH2CH2CH2—, —CH2CH2CH2CH2—, —CH2CH2CH2CH2CH2—, —CH2CH2CH2CH2CH2CH2—, — CH2CH2CH2CH2CH2CH2CH2—, —CH2CH2CH2CH2CH2CH2CH2CH2—, — CH2CH2CH2CH2CH2CH2CH2CH2CH2—, —CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2—, — CH2CHZCH2CH2CHZCHZCHZCHZCHZCHZCH2—, or —CH2CH2CH2CHZCH2CHZCHZCHZCHZCHZCHZCH2—.

In certain embodiments of compounds of Formula (I), each n, n', n", n'", and n"" is O, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11,12, 13, 14, 15, 16, 17,18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100.

In certain embodiments, dolastatin linker derivatives e compounds haVing the structure of Formula (II): H2N\O/L\NMeNfi-JLNI:§WNO R7 0 Me/\MeMe OMeOM OK/s In some embodiments of compounds of Formula (11), L is —(alkylene—O)n—alkylene—. In some embodiments, each alkylene is 2—, n is equal to 3, and R7 is methyl. In some embodiments, L is — alkylene—. In some embodiments of compounds of Formula (11), each alkylene is —CH2CH2— and R7 is methyl or hydrogen. In some embodiments of compounds of a (II), L is —(alkylene—O)n—alkylene— C(O)—. In some embodiments of compounds of Formula (11), each alkylene is —CH2CH2—, n is equal to 4, and R7 is . In some embodiments of compounds of Formula (II), L is —(alkylene—O)n—(CH2)n~ NHC(O) (CH2)nu C(Me)2 S S (CH2)nm NHC(O)—(alkylene—O)nmv—alkylene—. In some embodiments of 2012/039472 nds of Formula (11), each alkylene is —CH2CH2—, n is equal to 1, n' is equal to 2, n" is equal to 1, n '" is equal to 2, "H ' n 1s equal to 4, and R7 is methyl. Such dolastatin linker derivatives may be in the form of a salt, or may be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.

In certain embodiments of compounds of Formula (11), each L is independently a cleavable linker or non-cleavable linker. In certain ments of compounds of Formula (11), each L is independently a oligo(ethylene glycol) derivatized linker.

] In certain embodiments of compounds of Formula (11), R7 is , ethyl, propyl, iso-propyl, butyl, sec-butyl tyl, tert-butyl, pentyl, or hexyl. In certain embodiments of compounds of Formula (II), R7 is hydrogen.

In certain embodiments of compounds of Formula (II), alkylene is —CH2—, -—CH2CH2—, -— CH2CH2CH2—, —CH2CH2CH2CH2—, —CH2CH2CH2CH2CH2—, —CH2CH2CH2CH2CH2CH2—, — CH2CH2CH2CH2CH2CH2CH2—, —CH2CH2CH2CH2CH2CH2CH2CH2—, — CH2CH2CH2CH2CH2CH2CH2CH2CH2—, —CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2—, — CH2CHZCH2CH2CHZCHZCHZCHZCHZCHZCH2—, or —CH2CH2CH2CHZCH2CHZCHZCHZCHZCHZCHZCH2—.

In certain embodiments of nds of Formula (II), each n, n', n", n'", and n"" is O, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11,12, 13, 14, 15, 16, 17,18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100.

Such dolastatin linker derivatives include compounds having the ure of Formula (111), (IV), (V) or (VI): L_NI“”;N51”;];WNO N.

R7 0 MAMeMe OMeOM Y’ (111) L3—N:[IMfNfiJJi/IN Me R7 0 Me/\MeMe OMeOM Me\N MeNjN Me Me O Me/\MeMe OMeO MeO NH O 348%HN—L2 Me (IV) Me\N MeNjLN Me O Me/\MeMe OMeOM mNHHN—L3 NI?“$§WQF§F R7 0 /\ Me OMeO Yl Me L3—N meNJN Me R7 0 Me OMeOM NNH J o 2 L4_N “Ami/IN Me R7 0 Me/\MeMe OMeOM NH wherein: Z has the structure of: €Yé\(KAr R5 ; R5 is H, CORg, C1-C6alkyl, or thiazole; R8 is OH; R6 is OH or H; Ar is phenyl or pyridine; R7 is C1-C6alkyl or hydrogen; Y is NH2—0—; V is —O—NH2 L1, L2, L3, and L4 are each linkers ndently selected from the group consisting of a bond, — alkylene—, —(alkylene—O)n—alkylene—J—, —alkylene'—J—(alkylene—O)n—alkylene—, —J— (alkylene—O)n—alkylene—, —(alkylene—O)n—alkylene—J—(alkylene—O)n'—alkylene—J'—, — (alkylene—O)n—alkylene—J—alkylene'—, —W—, ene—W—, alkylene'—J—(alkylene—NMe)n— alkylene—W—, —J—(alkylene—NMe)n—alkylene—W—, —J—alkylene—NMe—alkylene'—NMe— alkylene"—W—, and ene— —alkylene'—NMe—alkylene"—NMe—alkylene'"—W—; W has the structure of: each I and J' independently have the structure of: zit/iiN/‘Zi ‘Zi or ‘fiNié’; H H H , , ,and each n and n' are independently integers r than or equal to one.

Such dolastatin linker derivatives may be in the form of a salt, or may be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a cleotide and optionally post translationally modified.

In certain embodiments of compounds of Formula (III), (IV), (V) or (VI), R5 is thiazole. In certain embodiments of nds of Formula (III), (IV), (V) or (VI), R6 is H. In certain embodiments of compounds of Formula (III), (IV), (V) or (VI), Ar is phenyl. In certain embodiments of nds of Formula (III), (IV), (V) or (VI), R7 is methyl. In certain embodiments of compounds of Formula (III), (IV), (V) or (VI), n and n' are integers from O to 20. In certain embodiments of compounds of a (III), (IV), (V) or (VI), n and n' are integers from O to 10. In certain embodiments of compounds of a (III), (IV), (V) or (VI), n and n' are integers from O to 5.

In certain embodiments of nds of Formula (III) and (V), R5 is thiazole or carboxylic acid.

In certain embodiments of compounds of Formula (III) and (V), R5 is hydrogen. In certain ments of compounds of Formula (III) and (V), R5 is methyl, ethyl, propyl, iso-propyl, butyl, iso-butyl, sec-butyl, tert-butyl, pentyl, or hexyl. In certain embodiments of compounds of Formula (III) and (V), R5 is —NH— (alkylene—O)n—NH2, Wherein alkylene is —CH2—, -—CH2CH2—, —CH2CH2CH2—, —CH2CH2CH2CH2—, — CH2CH2CH2CH2CH2—, —CH2CH2CH2CH2CH2CH2—, —CH2CH2CH2CH2CH2CH2CH2—, — CH2CH2CH2CH2CH2CH2CH2CH2—, —CH2CH2CH2CH2CH2CH2CH2CH2CH2—, — CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2—, —CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2—, or — CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2—. In certain embodiments of compounds of Formula (III) and (V), R5 is lkylene—O)n—NH2, Wherein n is O, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100.

In certain embodiments of compounds of Formula (III), (IV), (V) and (VI), R6 is H. In some embodiments of compounds of Formula (III), (IV), (V) and (VI), R6 is hydroxy.

In certain embodiments of compounds of Formula (III), (IV), (V) and (VI), Ar is phenyl.

In certain embodiments of compounds of Formula (III), (IV), (V) and (VI), R7 is methyl, ethyl, propyl, opyl, butyl, sec-butyl iso-butyl, tert-butyl, pentyl, or hexyl. In certain embodiments of compounds of Formula (III), (IV), (V) and (VI), R7 is en.

In certain embodiments of compounds of Formula (III) and (V), Y is hydroxylamine, aldehyde, protected aldehyde, ketone, ted ketone, thioester, ester, dicarbonyl, hydrazine, amidine, imine, diamine, keto-amine, lkyne, or ene-dione. In certain embodiments of compounds of Formula (IV) and (VI), V is a hydroxylamine, methyl, aldehyde, ted aldehyde, ketone, protected , thioester, ester, dicarbonyl, hydrazine, amidine, imine, diamine, keto-amine, keto-alkyne, and ene-dione.

In certain embodiments of compounds of a (XIV), (XV), (XVI), (XVII), and (XVIII), each L, L1, L2, L3, and L4 is independently a cleavable linker or non-cleavable linker. In certain embodiments of compounds of Formula (XIV), (XV), (XVI), (XVII), and (XVIII), each L, L1, L2, L3, and L4 is independently a oligo(ethylene glycol) tized linker.

] In certain embodiments of nds of Formula (III), (IV), (V) and (VI), each alkylene, alkylene', alkylene", and alkylene'" independently is —CH2—, -—CH2CH2—, -—CH2CH2CH2—, — CH2CH2CH2CH2—, —CH2CH2CH2CH2CH2—, 2CH2CH2CH2CH2—, —CH2CH2CH2CH2CH2CH2CH2— , —CH2CH2CH2CH2CH2CH2CH2CH2—, —CH2CH2CH2CH2CH2CH2CH2CH2CH2—, CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2—, —CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2—, or — CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2—. In n embodiments of compounds of Formula (III), (IV), (V) and (VI), alkylene is methylene, ethylene, propylene, butylenes, pentylene, hexylene, or heptylene.

In certain embodiments of nds of Formula (III), (IV), (V) and (VI), each n and n' independently is 0,1, 2, 3, 4, 5, 6, 7, 8, 9,10,11,12,]3,14,15,16,17,18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100.

In certain embodiments, dolastatin linker derivatives include nds having the structure of Formula (VII): _ I Me O /\ Me OMeO Me Me MeO NHOHN‘L2 Me (VII) Me\ " Ii] 3 I Me O /\ Me Me Me OMeOM In certain embodiments of compounds of Formula (VII), L1 is —(alkylene—O)n—alkylene—J—, L2 is —alkylene'—J'—(alkylene—O)n'—alkylene—, L3 is —J"—(alkylene—O)n"—alkylene—, alkylene is —CH2CH2—, swig alkylene' is —(CH2)4—, n is 1, n' and n" are 3, J has the structure of H J' and I" have the structure §é\Ni0/15. of H and R7 is methyl. In certain embodiments of compounds of Formula (VII), L1 is —J— (alkylene—O)n—alkylene—, L2 is —(alkylene—O)n~alkylene—J'—alkylene'—, L3 is —(alkylene—O)nv~alkylene— J"—, alkylene is —CH2CH2—, alkylene' is —(CH2)4—, n is l, n' and n" are 4, and J, J' and I" have the ure of H . Such atin linker derivatives may be in the form of a salt, or may be incorporated into a non-natural amino acid ptide, polymer, polysaccharide, or a polynucleotide and ally post translationally modified.

In certain embodiments, compounds of Formula (I)-(VII) are stable in aqueous solution for at least 1 month under mildly acidic conditions. In certain embodiments, compounds of Formula (I)-(VII) are stable for at least 2 weeks under mildly acidic conditions. In certain embodiments, compound of Formula (I)-(VII) are stable for at least 5 days under mildly acidic conditions. In certain embodiments, such acidic conditions are pH 2 to 8.

The methods and compositions provided and described herein e polypeptides comprising dolastatin linker derivative containing at least one carbonyl or onyl group, oxime group, hydroxylamine group, or protected or masked forms f. Introduction of at least one ve group into a dolastatin linker derivative can allow for the application of conjugation chemistries that involve specific al reactions, ing, but not limited to, With one or more dolastatin linker derivative(s) While not reacting With the commonly occurring amino acids. Once incorporated, the dolastatin linker derivative side chains can also be modified by utilizing chemistry methodologies described herein or suitable for the particular functional groups or substituents present in the dolastatin linker derivative. 2012/039472 The dolastatin linker derivative methods and compositions described herein provide conjugates of substances having a wide variety of functional , substituents or moieties, with other substances including but not limited to a polymer; a soluble polymer; a derivative of polyethylene glycol; a second protein or polypeptide or polypeptide analog; an antibody or antibody fragment; and any combination thereof.

In certain embodiments, the dolastatin linker derivatives, linkers and reagents described herein, including compounds of Formulas (I)-(VII) are stable in aqueous solution under mildly acidic ions (including but not limited to pH 2 to 8). In other embodiments, such compounds are stable for at least one month under mildly acidic conditions. In other embodiments, such compounds are stable for at least 2 weeks under mildly acidic ions. In other ments, such compounds are stable for at least 5 days under mildly acidic conditions.

In another aspect of the compositions, methods, techniques and strategies described herein are methods for studying or using any of the aforementioned “modified or unmodified” non-natural amino acid dolastatin linker derivatives. Included within this aspect, by way of example only, are therapeutic, diagnostic, assay-based, industrial, cosmetic, plant biology, environmental, energy-production, consumer- products, and/or military uses which would benefit from a dolastatin linker derivative comprising a “modified or unmodified” non-natural amino acid ptide or protein.

Non—limiting examples of dolastatin linker derivatives include: WO 66560 H2N\O/Vo\/\NW/SWNMo/Vowo/VowMENfiRQI/RKHUS OMe O OMe O HzN—O ,wwwwwéxfigw OMe o OMe o N/NS\:/ Mbwmwxnxama OMe O OMe O —/ N,_,3 wowowowcmo$KHf%go051w”fi%%“OMe O OMe O CF3COOH \:/ HNJ/ o NH2 i 21 o O O N .

H | ; | N O /\ OMeO OMeO H N’0 N N 2 N/ H i H \_7$ H of O NH2 o O 0 H 'I‘ OMe O H N/o\/\O/\)J\N N\)J\N H : CF3COOH of o NH2 HZINOJKN\_/U\N/©/\00 OMeO OMeON CF23C00H HNf 0 NH2 COZH 0 0 0 HZN,O\/\o/\/N\n/\NJJ\/\/\/U\NH 3 NgLNH El: 5?2:: H I I o o A ”ZN/OwONNEN/ELA/mc02H ARI/g1" E: if2:: PCT/US 12/39472 072012 HOf 0*:Ao El M” 2‘ a zra-o VOW3;;3 W4); K H «0% NOWN" N N H_N7 o ‘4’]; hat/0v: s 0 NH2 0 f”” 5 ° HJL cal? = ? [JO HNYVNV‘NA/H‘N NE N A H H HEN-D 0 of O‘LNHZ SUBSTITUTE SHEET (RULE 26) PCT/U312/39472 032012 IV. Non-Natural Amino Acid Derivatives The non-natural amino acids used in the s and compositions described herein have at least one of the following four properties: (1) at least one functional group on the sidechain of the non- natural amino acid has at least one characteristics and/or activity and/or reactivity orthogonal to the chemical reactivity of the 20 common, cally-encoded amino acids (i.e., alanine, arginine, asparagine, ic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and ), or at least orthogonal to the chemical reactivity of the naturally occurring amino acids present in the ptide that includes the non-natural amino acid; (2) the introduced non-natural amino acids are substantially chemically inert toward the 20 common, genetically-encoded amino acids; (3) the non-natural amino acid can be stably incorporated into a polypeptide, preferably With the stability commensurate With the naturally-occurring amino acids or under typical physiological conditions, and further preferably such oration can occur via an in vivo system; and (4) the tural amino acid includes an oxime SUBSTITUTE SHEET (RULE 26) functional group or a functional group that can be ormed into an oxime group by reacting with a reagent, preferably under conditions that do not y the biological properties of the polypeptide that includes the tural amino acid (unless of course such a destruction of biological properties is the purpose of the modif1cation/transformation), or where the transformation can occur under aqueous conditions at a pH between about 4 and about 8, or where the reactive site on the non-natural amino acid is an electrophilic site. Any number of non-natural amino acids can be introduced into the polypeptide. tural amino acids may also include protected or masked oximes or protected or masked groups that can be transformed into an oxime group after deprotection of the protected group or unmasking of the masked group. Non-natural amino acids may also e protected or masked yl or dicarbonyl groups, which can be transformed into a carbonyl or dicarbonyl group after ection of the ted group or unmasking of the masked group and thereby are available to react with hydroxylamines or oximes to form oxime groups.

Non-natural amino acids that may be used in the methods and compositions bed herein include, but are not limited to, amino acids comprising a amino acids with novel functional groups, amino acids that covalently or noncovalently interact with other molecules, glycosylated amino acids such as a sugar substituted serine, other carbohydrate modified amino acids, keto-containing amino acids, aldehyde- containing amino acids, amino acids comprising polyethylene glycol or other polyethers, heavy atom substituted amino acids, chemically cleavable and/or photocleavable amino acids, amino acids with an elongated side chains as compared to natural amino acids, including but not limited to, polyethers or long chain arbons, including but not limited to, greater than about 5 or greater than about 10 carbons, carbon-linked sugar-containing amino acids, redox-active amino acids, amino id containing amino acids, and amino acids comprising one or more toxic moiety.

In some embodiments, non-natural amino acids comprise a saccharide moiety. Examples of such amino acids include N—acetyl-L-glucosaminyl-L-serine, N—acetyl-L-galactosaminyl-L-serine, N- acetyl-L-glucosaminyl-L-threonine, N—acetyl-L-glucosaminyl-L-asparagine and O-mannosaminyl-L- serine. Examples of such amino acids also e examples where the naturally-occurring N— or O- linkage between the amino acid and the saccharide is replaced by a covalent linkage not commonly found in nature — including but not limited to, an alkene, an oxime, a thioether, an amide and the like. Examples of such amino acids also include saccharides that are not commonly found in naturally-occurring proteins such as 2-deoxy-glucose, 2-deoxygalactose and the like.

] The chemical moieties incorporated into polypeptides Via incorporation of non-natural amino acids into such polypeptides offer a y of advantages and manipulations of polypeptides. For example, the unique reactivity of a carbonyl or dicarbonyl onal group ding a keto- or aldehyde- functional group) allows selective modification of proteins with any of a number of hydrazine- or hydroxylamine-containing reagents in vivo and in vitro. A heavy atom tural amino acid, for example, can be useful for phasing x-ray structure data. The site-specific introduction of heavy atoms using non-natural amino acids also provides selectiVity and flexibility in choosing positions for heavy 2012/039472 atoms. Photoreactive non-natural amino acids (including but not limited to, amino acids with benzophenone and arylazides (including but not limited to, phenylazide) side chains), for example, allow for efficient in vivo and in vitro photocrosslinking of polypeptides. Examples of photoreactive non-natural amino acids include, but are not limited to, p-azido-phenylalanine and p-benzoyl—phenylalanine. The polypeptide with the photoreactive non-natural amino acids may then be crosslinked at will by excitation of the photoreactive group-providing temporal control. In a non-limiting example, the methyl group of a non-natural amino can be substituted with an ically labeled, including but not limited to, with a methyl group, as a probe of local structure and dynamics, including but not limited to, with the use of nuclear magnetic resonance and vibrational spectroscopy.

A. Structure and Synthesis ofNon-Natural Amino Acid Derivatives: Carbonyl, yl like, Masked Carbonyl, and Protected Carbonyl Groups ] Amino acids with an electrophilic reactive group allow for a variety of reactions to link molecules via s chemical reactions, ing, but not d to, nucleophilic addition reactions. Such electrophilic reactive groups include a carbonyl- or onyl-group ding a keto- or aldehyde group), a carbonyl-like- or dicarbonyl-like-group (which has reactivity similar to a carbonyl- or dicarbonyl-group and is urally similar to a carbonyl- or dicarbonyl-group), a masked carbonyl- or masked dicarbonyl-group (which can be readily converted into a carbonyl- or dicarbonyl-group), or a protected carbonyl- or ted dicarbonyl-group (which has reactivity r to a carbonyl- or dicarbonyl-group upon deprotection). Such amino acids include amino acids having the structure of Formula (XXXVH): R3 A\ , K \ R3 B R o<xxv11) R1\N R2 H R4o wherein: A is optional, and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted alkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, tuted alkarylene, aralkylene, or tuted aralkylene, B is al, and when present is a linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O)k- where k is 1, 2, or 3, -S(O)k(alkylene or substituted alkylene)-, -C(O)-, -NS(O)2-, -OS(O)2-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -, -NR’-(alkylene or tuted alkylene)-, -C(O)N(R’)-, -CON(R’)-(alkylene or substituted alkylene)-, -CSN(R’)-, -CSN(R’)-(alkylene or substituted alkylene)-, -N(R’)CO-(alkylene or substituted alkylene)-, C(O)O-, -S(O)kN(R’)-, -N(R’)C(O)N(R’)-, -N(R’)C(S)N(R’)-, -N(R’)S(O)kN(R’)-, -N(R’)-N=, -C(R’)=N—, -C(R’)=N—N(R’)-, -C(R’)=N—N=, -C(R’)2-N=N-, and 2-N(R’)-N(R’)-, where each R’ is independently H, alkyl, or substituted alkyl; 0 120%?“ s E" 5:" 0R" SR" Ff" a. ”\l/ “A, o fikwfiwks,thaw/”\o/imkf.

R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; each R” is independently H, alkyl, substituted alkyl, or a ting group, or when more than one R” group is present, two R” optionally form a cycloalkyl; R1 is H, an amino ting group, resin, amino acid, polypeptide, or polynucleotide; and R2 is OH, an ester protecting group, resin, amino acid, polypeptide, or cleotide; each of R3 and R4 is independently H, halogen, lower alkyl, or substituted lower alkyl, or R3 and R4 or two R3 groups optionally form a cycloalkyl or a heterocycloalkyl; or the —A-B-K-R groups together form a bicyclic or tricyclic cycloalkyl or heterocycloalkyl comprising at least one carbonyl group, including a dicarbonyl group, protected carbonyl group, including a protected dicarbonyl group, or masked carbonyl group, ing a masked dicarbonyl group; or the —K-R group together forms a monocyclic or bicyclic lkyl or heterocycloalkyl comprising at least one carbonyl group, including a dicarbonyl group, protected carbonyl group, including a protected dicarbonyl group, or masked carbonyl group, ing a masked dicarbonyl group; with a proviso that when A is phenylene and each R3 is H, B is present; and that when A is —(CH2)4- and each R3 is H, B is not —NHC(O)(CH2CH2)-; and that when A and B are absent and each R3 is H, R is not methyl. Such non-natural amino acids may be in the form of a salt, or may be incorporated into a non- natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally d.

] In certain ments, compounds of Formula (XXXVII) are stable in aqueous solution for at least 1 month under mildly acidic conditions. In certain embodiments, compounds of Formula (XXXVII) are stable for at least 2 weeks under mildly acidic conditions. In certain embodiments, compound of Formula (XXXVII) are stable for at least 5 days under mildly acidic conditions. In certain embodiments, such acidic conditions are pH 2 to 8.

In certain embodiments of nds of Formula (XXXVII), B is lower alkylene, substituted lower alkylene, -O-(alkylene or substituted alkylene)-, -C(R’)=N—N(R’)-, -N(R’)CO-, , -C(R’)=N—, -C(O)-(alkylene or substituted alkylene)-, ’)-(alkylene or substituted alkylene)-, -S(alkylene or substituted alkylene)-, -S(O)(alkylene or substituted alkylene)-, or -S(O)2(alkylene or substituted alkylene)-. In certain embodiments of compounds of Formula (XXXVII), B is —O(CH2)-, -CH=N—, -CH=N—NH-, -NHCH2-, -NHCO-, -C(O)-, -C(O)-(CH2)-, -CONH-(CH2)-, -SCH2-, -S(=O)CH2-, or - S(O)2CH2-. In certain embodiments of compounds of Formula (XXXVII), R is C1_6 alkyl or lkyl. In certain embodiments of compounds of Formula (XXXVII) R is —CH3, -CH(CH3)2, or cyclopropyl. In certain embodiments of compounds of Formula (XXXVII), R1 is H, tert-butyloxycarbonyl (Boc), 9- Fluorenylmethoxycarbonyl , N—acetyl, tetrafluoroacetyl (TFA), or benzyloxycarbonyl (Cbz). In certain ments of compounds of Formula (XXXVII), R1 is a resin, amino acid, polypeptide, antibody, or polynucleotide. In certain embodiments of nds of Formula I), R2 is OH, 0- methyl, O-ethyl, or O-t—butyl. In certain embodiments of compounds of Formula (XXXVII), R2 is a resin, amino acid, polypeptide, antibody, or polynucleotide. In certain embodiments of compounds of Formula (XXXVII), R2 is a polynucleotide. In certain embodiments of compounds of Formula (XXXVII), R2 is ribonucleic acid (RNA).

In certain embodiments of nds of Formula (XXXVII), flog/A is selected from the group ting of: (i) A is tuted lower ne, C4-arylene, substituted arylene, heteroarylene, substituted arylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene, B is optional, and when present is a divalent linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S(O)-, -S(O)2-, -NS(O)2-, - OS(O)2-, -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -N(R’)-, -C(O)N(R’)-, ’)-(alkylene or tuted alkylene)-, -CSN(R’)-, -N(R’)CO-(alkylene or substituted alkylene)-, -N(R’)C(O)O-, -N(R’)C(S)-, (R’), -S(O)2N(R’), -N(R’)C(O)N(R’)-, -N(R’)C(S)N(R’)-, -N(R’)S(0)N(R’)-, -N(R’)S(O)2N(R’)-, -N(R’)- N=, -C(R’)=N—N(R’)-, -C(R’)=N—N=, -C(R’)2-N=N-, and -C(R’)2-N(R’)-N(R’)-; (ii) A is optional, and when present is substituted lower alkylene, C4-arylene, substituted arylene, heteroarylene, substituted heteroarylene, lene, substituted alkarylene, aralkylene, or substituted aralkylene, B is a nt linker selected from the group consisting of lower alkylene, substituted lower ne, lower alkenylene, substituted lower alkenylene, -O-, -O-(alkylene or tuted alkylene)-, -S-, -S(O)-, -S(O)2-, -NS(O)2-, -OS(O)2-, -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -N(R’)-, -C(O)N(R’)-, -CON(R’)-(alkylene or substituted alkylene)-, -CSN(R’)-, -N(R’)CO-(alkylene or tuted alkylene)-, -N(R’)C(0)O-, -N(R’)C(S)-, (R’), -S(O)2N(R’), -N(R’)C(O)N(R’)-, -N(R’)C(S)N(R’)-, -N(R’)S(O)N(R’)-, -N(R’)S(O)2N(R’)-, -N(R’)-N=, -C(R’)=N—N(R’)-, -C(R’)=N—N=, -C(R’)2-N=N-, and -C(R’)2-N(R’)-N(R’)-; (iii) A is lower alkylene; B is optional, and when present is a divalent linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower lene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S(O)-, -S(O)2-, -NS(O)2-, - OS(O)2-, -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -N(R’)-, -C(O)N(R’)-, -CSN(R’)-, -CON(R’)-(alkylene or substituted alkylene)-, -N(R’)C(O)O-, -N(R’)C(S)-, -S(0)N(R’), -S(0)2N(R’), _N(R’)C(O)N(R’)_a -N(R’)C(S)N(R’)-, _N(R’)S(O)N(R’)_a - (O)2N(R’)-, -N(R’)-N=, -C(R’)=N—N(R’)-, -C(R’)=N—N=, -C(R’)2-N=N-, and -C(R’)2-N(R’)-N(R’)-; and (iV) A is phenylene; B is a nt linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, -O-, kylene or substituted alkylene)-, -S-, -S(O)-, -, -NS(O)2-, 2-, -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -N(R’)-, -C(O)N(R’)-, -CON(R’)-(alkylene or substituted alkylene)-, -CSN(R’)-, -N(R’)CO-(alkylene or substituted alkylene)-, -N(R’)C(O)O-, -N(R’)C(S)-, -S(O)N(R’), -S(0)2N(R’), -N(R’)C(O)N(R’)-, -N(R’)C(S)N(R’)-, -N(R’)S(O)N(R’)-, -N(R’)S(O)2N(R’)-, -N(R’)-N=, -C(R’)=N—N(R’)-, -C(R’)=N—N=, -C(R’)2-N=N-, and -C(R’)2-N(R’)-N(R’)-; :le SR' O a. S / \ OR' R.

A: art/wL 7 7 ,or O\ /R la rr" each R’ is independently H, alkyl, or substituted alkyl, R1 is optional, and when present, is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and R2 is optional, and when present, is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; and each R3 and R4 is independently H, halogen, lower alkyl, or substituted lower alkyl, R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl, In addition, amino acids haVing the structure of Formula (XXXVHI) are included: 0 (XXXVHI), wherein: A is optional, and when present is lower alkylene, tuted lower alkylene, lower lkylene, substituted lower cycloalkylene, lower lene, substituted lower alkenylene, alkynylene, lower alkylene, substituted heteroalkylene, lower cycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene; B is optional, and when present is a linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, - where k is l, 2, or 3, -S(O)k(alkylene or substituted alkylene)-, -C(O)-, -NS(O)2-, -OS(O)2-, -C(O)-(alkylene or substituted alkylene)-, , -C(S)-(alkylene or substituted alkylene)-, -N(R’)-, -NR’-(alkylene or tuted alkylene)-, (R’)-, -CON(R’)-(alkylene or substituted ne)-, -CSN(R’)-, -CSN(R’)-(alkylene or substituted alkylene)-, -N(R’)CO-(alkylene or substituted ne)-, -N(R’)C(O)O-, -S(O)kN(R’)-, -N(R’)C(O)N(R’)-, -N(R’)C(S)N(R’)-, -N(R’)S(O)kN(R’)-, -N(R’)-N=, -C(R’)=N—, -C(R’)=N—N(R’)-, -C(R’)=N—N=, -C(R’)2-N=N-, and -C(R’)2-N(R’)-N(R’)-, where each R’ is independently H, alkyl, or substituted alkyl; R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R1 is H, an amino protecting group, resin, at least one amino acid, polypeptide, or polynucleotide; and R2 is OH, an ester protecting group, resin, at least one amino acid, polypeptide, or polynucleotide; with a proviso that when A is phenylene, B is present; and that when A is 4-, B is not — (CH2CH2)-; and that when A and B are , R is not methyl. Such non-natural amino acids may be in the form of a salt, or may be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.

In on, amino acids having the structure of Formula (XXXIX) are included: Ra BTR R1 \ R2 O (XXXIX), wherein: B is a linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O)k- where k is l, 2, or 3, -S(O)k(alkylene or substituted alkylene)-, -C(O)-, -NS(O)2-, -OS(O)2-, -C(O)-(alkylene or substituted ne)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R’)-, -NR’-(alkylene or substituted alkylene)-, -C(O)N(R’)-, -CON(R’)-(alkylene or substituted alkylene)-, -CSN(R’)-, -CSN(R’)-(alkylene or tuted alkylene)-, -N(R’)CO-(alkylene or substituted alkylene)-, -N(R’)C(O)O-, -S(O)kN(R’)-, -N(R’)C(O)N(R’)-, -N(R’)C(S)N(R’)-, -N(R’)S(O)kN(R’)-, -N=, =N—, -C(R’)=N—N(R’)-, -C(R’)=N—N=, -C(R’)2-N=N-, and -C(R’)2-N(R’)-N(R’)-, where each R’ is independently H, alkyl, or substituted alkyl; R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R1 is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and R2 is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; each Ra is independently selected from the group consisting of H, halogen, alkyl, substituted alkyl, - N(R’)2, -C(O)kR’ where k is l, 2, or 3, -C(O)N(R’)2, -OR’, and -S(O)kR’, where each R’ is independently H, alkyl, or substituted alkyl. Such non-natural amino acids may be in the form of a salt, or may be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.

In addition, the following amino acids are included: 0 O OQK H \N/NY \N OH H OH H2N H N H2N O H2N COOH 0 O 7 7 7 7 Qt O O 0 NJKH OH H OH H2N H2N H2N H2N COOH o o and , , , Such non-natural amino acids may be are optionally amino protected group, carboxyl protected and/or in the form of a salt, or may be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.

In addition, the following amino acids haVing the structure of Formula (XXXX) are included: (CR8 n)\B)J\R R1\N R2 (XXXX) wherein -NS(O)2-, -OS(O)2-, optional, and when t is a linker ed from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted ne)-, -S-, -S- (alkylene or tuted alkylene)-, -S(O)k- where k is l, 2, or 3, -S(O)k(alkylene or substituted alkylene)-, -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -, -NR’-(alkylene or substituted alkylene)-, -C(O)N(R’)-, -CON(R’)-(alkylene or substituted alkylene)-, -CSN(R’)-, ’)-(alkylene or substituted alkylene)-, -N(R’)CO-(alkylene or substituted alkylene)-, -N(R’)C(O)O-, -S(O)kN(R’)-, -N(R’)C(O)N(R’)-, -N(R’)C(S)N(R’)-, -N(R’)S(O)kN(R’)-, -N=, -C(R’)=N—, -C(R’)=N—N(R’)-, -C(R’)=N—N=, -C(R’)2-N=N-, and 2-N(R’)-N(R’)-, where each R’ is ndently H, alkyl, or substituted alkyl; R is H, alkyl, substituted alkyl, cycloalkyl, or tuted lkyl; R1 is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and R2 is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; each R5 is independently selected from the group consisting of H, halogen, alkyl, substituted alkyl, - N(R’)2, -C(O)kR’ where k is l, 2, or 3, -C(O)N(R’)2, -OR’, and -S(O)kR’, where each R’ is independently H, alkyl, or substituted alkyl; and n is O to 8; with a proviso that when A is —(CH2)4-, B is not —NHC(O)(CH2CH2)-. Such non-natural amino acids may be in the form of a salt, or may be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and ally post translationally modified.

In addition, the following amino acids are included: 5 55555555 5 5 5 w,herein such compounds are ally amino protected, optionally carboxyl protected, optionally amino protected and carboxyl ted, or a salt thereof, or may be incorporated into a non-natural amino acid ptide, polymer, ccharide, or a polynucleotide and optionally post translationally modified.

] In addition, the following amino acids haVing the structure of Formula (XXXXI) are included: 0 (XXXXI), wherein, A is optional, and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, tuted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene; B is optional, and when present is a linker selected from the group consisting of lower alkylene, tuted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O)k- where k is l, 2, or 3, -S(O)k(alkylene or substituted ne)-, -C(O)-, -NS(O)2-, -OS(O)2-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R’)-, -NR’-(alkylene or tuted alkylene)-, (R’)-, -CON(R’)-(alkylene or substituted alkylene)-, -CSN(R’)-, -CSN(R’)-(alkylene or tuted alkylene)-, -N(R’)CO-(alkylene or tuted alkylene)-, -N(R’)C(O)O-, -S(O)kN(R’)-, -N(R’)C(O)N(R’)-, -N(R’)C(S)N(R’)-, -N(R’)S(O)kN(R’)-, -N(R’)-N=, -C(R’)=N—, -C(R’)=N—N(R’)-, -C(R’)=N—N=, -C(R’)2-N=N-, and -C(R’)2-N(R’)-N(R’)-, where each R’ is independently H, alkyl, or substituted alkyl; R1 is H, an amino protecting group, resin, amino acid, ptide, or cleotide; and R2 is OH, an ester ting group, resin, amino acid, polypeptide, or polynucleotide.

Such tural amino acids may be in the form of a salt, or may be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.

In addition, the following amino acids haVing the structure of Formula (XXXXH) are included: Ra BYOOJ R1 \ R2 0 (XXXXII), wherein, B is optional, and when present is a linker selected from the group ting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, kylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O)k- where k is l, 2, or 3, -S(O)k(alkylene or substituted alkylene)-, , -NS(O)2-, -OS(O)2-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R’)-, -NR’-(alkylene or substituted alkylene)-, -C(O)N(R’)-, -CON(R’)-(alkylene or substituted alkylene)-, -CSN(R’)-, -CSN(R’)-(alkylene or substituted alkylene)-, -N(R’)CO-(alkylene or substituted alkylene)-, -N(R’)C(O)O-, -S(O)kN(R’)-, -N(R’)C(O)N(R’)-, -N(R’)C(S)N(R’)-, S(O)kN(R’)-, -N(R’)-N=, -C(R’)=N—, -C(R’)=N—N(R’)-, =N—N=, 2-N=N-, and -C(R’)2-N(R’)-N(R’)-, where each R’ is independently H, alkyl, or substituted alkyl; R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl, WO 66560 R1 is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and R2 is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; wherein each Ra is independently ed from the group consisting of H, n, alkyl, tuted alkyl, -N(R’)2, -C(O)kR’ where k is l, 2, or 3, -C(O)N(R’)2, -OR’, and -S(O)kR’, where each R’ is ndently H, alkyl, or substituted alkyl.

Such non-natural amino acids may be in the form of a salt, or may be incorporated into a non-natural amino acid polypeptide, polymer, ccharide, or a polynucleotide and optionally post translationally modified.

In addition, the following amino acids are included: Wfiww ”it?<53”? 1?” wherein such compounds are optionally amino protected, optionally carboxyl protected, optionally amino protected and carboxyl protected, or a salt thereof, or may be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and ally post translationally modified.

In addition, the following amino acids haVing the structure of Formula (XXXXIV) are included: (CRa)n\BA? R1\ R2 0 (XXXXIV), wherein, B is al, and when present is a linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower lene, substituted lower alkenylene, lower alkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O)k- where k is l, 2, or 3, -S(O)k(alkylene or substituted alkylene)-, -C(O)-, -NS(O)2-, -OS(O)2-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R’)-, -NR’-(alkylene or substituted ne)-, (R’)-, -CON(R’)-(alkylene or substituted alkylene)-, -CSN(R’)-, -CSN(R’)-(alkylene or substituted alkylene)-, -N(R’)CO-(alkylene or substituted alkylene)-, -N(R’)C(O)O-, -S(O)kN(R’)-, -N(R’)C(O)N(R’)-, -N(R’)C(S)N(R’)-, -N(R’)S(O)kN(R’)-, -N(R’)-N=, -C(R’)=N—, -C(R’)=N—N(R’)-, -C(R’)=N—N=, -C(R’)2-N=N-, and -C(R’)2-N(R’)-N(R’)-, where each R’ is independently H, alkyl, or substituted alkyl; R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R1 is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and R2 is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; each R21 is independently selected from the group consisting of H, n, alkyl, substituted alkyl, - , -C(O)kR’ where k is l, 2, or 3, -C(O)N(R’)2, -OR’, and -S(O)kR’, where each R’ is independently H, alkyl, or tuted alkyl; and n is O to 8.

Such non-natural amino acids may be in the form of a salt, or may be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.

In addition, the following amino acids are included: (E3 (E3 (E3 003 003 /—\ o o HzNJ:,(0'4 ’HzNLVfOH ’HzNji'fOH O 0“ 0“ H2N HZN HEN/gOH O O O and , , , , n such compounds are optionally amino protected, optionally carboxyl protected, optionally amino protected and carboxyl protected, or a salt thereof, or may be incorporated into a non-natural amino acid polypeptide, r, polysaccharide, or a polynucleotide and optionally post translationally modified.

In addition to monocarbonyl structures, the non-natural amino acids described herein may include groups such as dicarbonyl, dicarbonyl like, masked dicarbonyl and protected dicarbonyl groups.

For example, the following amino acids haVing the structure of Formula (XXXXV) are included: A\B)H(R R1\ R2 0 (XXXXV), wherein, A is optional, and when present is lower alkylene, substituted lower ne, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, tuted alkarylene, aralkylene, or substituted lene; B is optional, and when t is a linker selected from the group ting of lower alkylene, substituted lower alkylene, lower alkenylene, tuted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O)k- where k is l, 2, or 3, -S(O)k(alkylene or substituted alkylene)-, -C(O)-, -NS(O)2-, -OS(O)2-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R’)-, -NR’-(alkylene or substituted alkylene)-, -C(O)N(R’)-, -CON(R’)-(alkylene or substituted alkylene)-, ’)-, -CSN(R’)-(alkylene or substituted alkylene)-, -N(R’)CO-(alkylene or substituted alkylene)-, C(O)O-, N(R’)-, -N(R’)C(O)N(R’)-, -N(R’)C(S)N(R’)-, -N(R’)S(O)kN(R’)-, -N(R’)-N=, -C(R’)=N—, -C(R’)=N—N(R’)-, -C(R’)=N—N=, -C(R’)2-N=N-, and -C(R’)2-N(R’)-N(R’)-, where each R’ is independently H, alkyl, or substituted alkyl; R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R1 is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and R2 is OH, an ester ting group, resin, amino acid, polypeptide, or polynucleotide.

Such non-natural amino acids may be in the form of a salt, or may be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a cleotide and optionally post translationally modified.

In addition, the following amino acids haVing the structure of a I) are included: Ra B R1\ R2 0 (XXXXVI), wherein, B is al, and when present is a linker ed from the group ting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower alkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, - where k is l, 2, or 3, -S(O)k(alkylene or substituted alkylene)-, -C(O)-, -NS(O)2-, -OS(O)2-, (alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R’)-, -NR’-(alkylene or substituted alkylene)-, -C(O)N(R’)-, -CON(R’)-(alkylene or substituted alkylene)-, -CSN(R’)-, -CSN(R’)-(alkylene or substituted alkylene)-, -N(R’)CO-(alkylene or substituted alkylene)-, -N(R’)C(O)O-, -S(O)kN(R’)-, -N(R’)C(O)N(R’)-, -N(R’)C(S)N(R’)-, -N(R’)S(O)kN(R’)-, -N(R’)-N=, -C(R’)=N—, -C(R’)=N—N(R’)-, -C(R’)=N—N=, -C(R’)2-N=N-, and -C(R’)2-N(R’)-N(R’)-, where each R’ is ndently H, alkyl, or substituted alkyl; R is H, alkyl, substituted alkyl, cycloalkyl, or tuted cycloalkyl; R1 is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and R2 is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; wherein each R21 is independently ed from the group consisting of H, halogen, alkyl, substituted alkyl, -N(R’)2, -C(O)kR’ where k is l, 2, or 3, -C(O)N(R’)2, -OR’, and -S(O)kR’, where each R’ is ndently H, alkyl, or substituted alkyl.

Such non-natural amino acids may be in the form of a salt, or may be incorporated into a non-natural amino acid polypeptide, r, polysaccharide, or a polynucleotide and optionally post translationally modified.

In addition, the following amino acids are included: o o o (W m HWA (Cr 0 o +H3N 000- +H3N 000' +H3N 000- and+H3N coo- 7 7 7 wherein such compounds are optionally amino protected and carboxyl protected, or a salt thereof. Such non-natural amino acids may be in the form of a salt, or may be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.

] In addition, the following amino acids haVing the ure of Formula (XXXXVH) are included: (CRa)n\ R R1\ R2 0 O (XXXXVH), wherein, B is optional, and when present is a linker ed from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, kylene or substituted alkylene)-, -S(O)k- where k is l, 2, or 3, -S(O)k(alkylene or substituted alkylene)-, -C(O)-, -NS(O)2-, -OS(O)2-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted ne)-, -N(R’)-, -NR’-(alkylene or substituted alkylene)-, -C(O)N(R’)-, -CON(R’)-(alkylene or substituted ne)-, -CSN(R’)-, -CSN(R’)-(alkylene or substituted alkylene)-, -N(R’)CO-(alkylene or substituted alkylene)-, -N(R’)C(O)O-, -S(O)kN(R’)-, -N(R’)C(O)N(R’)-, -N(R’)C(S)N(R’)-, -N(R’)S(O)kN(R’)-, -N(R’)-N=, -C(R’)=N—, -C(R’)=N—N(R’)-, -C(R’)=N—N=, 2-N=N-, and -C(R’)2-N(R’)-N(R’)-, where each R’ is independently H, alkyl, or substituted alkyl; R is H, alkyl, substituted alkyl, cycloalkyl, or substituted lkyl; R1 is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and R2 is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; each R21 is independently selected from the group consisting of H, halogen, alkyl, substituted alkyl, -N(R’)2, -C(O)kR’ where k is l, 2, or 3, -C(O)N(R’)2, -OR’, and -S(O)kR’, where each R’ is independently H, alkyl, or substituted alkyl, and n is O to 8.

Such non-natural amino acids may be in the form of a salt, or may be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.

In addition, the following amino acids are included: 0 3 NH OH OH OH OH OH OH OH H2N H2N H2N H2N H2N H2N H2N o o o o o o o 7 7 7 7 7 7 7 and 0 wherein such compounds are optionally amino protected and carboxyl protected, or a salt thereof, or may be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.

In addition, the following amino acids haVing the structure of Formula HI) are included: 0 O A/ 1\L)KR R1HNXClolR2 (XXXXVIII); wherein: A is optional, and when present is lower alkylene, substituted lower ne, lower cycloalkylene, tuted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower alkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted e, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene, R is H, alkyl, substituted alkyl, lkyl, or substituted cycloalkyl, R1 is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and R2 is OH, an ester protecting group, resin, amino acid, polypeptide, or cleotide; X1 is C, S, or S(O); and L is alkylene, substituted alkylene, N(R’)(alkylene) or N(R’)(substituted alkylene), where R’ is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl.

Such non-natural amino acids may be in the form of a salt, or may be incorporated into a non-natural amino acid polypeptide, polymer, ccharide, or a polynucleotide and optionally post translationally In addition, the following amino acids haVing the structure of a (XXXXIX) are included: 0 O A/ \L)kR R1HNXClolR2 (XXXXIX) wherein: A is optional, and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower lene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, e, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted lene, R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl, R1 is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and R2 is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; L is alkylene, substituted alkylene, N(R’)(alkylene) or N(R’)(substituted alkylene), where R’ is H, alkyl, substituted alkyl, lkyl, or substituted cycloalkyl.

Such non-natural amino acids may be in the form of a salt, or may be incorporated into a non-natural amino acid ptide, polymer, polysaccharide, or a cleotide and optionally post translationally modified.

In addition, the following amino acids haVing the structure of Formula (XXXXX) are included: O 0 \S// A KA/ \ R R 1H N C (0 )R2 (XXXXX) wherein: A is optional, and when present is lower alkylene, substituted lower alkylene, lower lkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, lene, or substituted aralkylene, R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl, R1 is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and R2 is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; L is ne, substituted alkylene, N(R’)(alkylene) or N(R’)(substituted alkylene), where R’ is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl.

Such non-natural amino acids may be in the form of a salt, or may be incorporated into a non-natural amino acid polypeptide, polymer, ccharide, or a polynucleotide and optionally post translationally modified.

In addition, the following amino acids haVing the structure of Formula (XXXXXI) are included: 0 o l' A1 A/ \ R X (c R ER 9)n MN 0(0le wherein: A is optional, and when t is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene, R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl, R1 is H, an amino ting group, resin, amino acid, ptide, or polynucleotide; and R2 is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; X1 is C, S, or S(O); and n is O, l, 2, 3, 4, or 5; and each R8 and R9 on each CRgR9 group is independently selected from the group consisting of H, , alkylamine, halogen, alkyl, aryl, or any R8 and R9 can together form =0 or a cycloalkyl, or any to adjacent R8 groups can together form a cycloalkyl, Such non-natural amino acids may be in the form of a salt, or may be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally ] In addition, the following amino acids haVing the structure of Formula (XXXXXH) are included: O O é' A A/ R X \(CR5R9>n MN 0(0le (XXXXXII) wherein: A is optional, and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower lene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted lene, aralkylene, or substituted aralkylene, R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl, R1 is H, an amino protecting group, resin, amino acid, ptide, or polynucleotide; and R2 is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; n is O, l, 2, 3, 4, or 5; and each R8 and R9 on each CRgR9 group is independently selected from the group consisting of H, alkoxy, alkylamine, halogen, alkyl, aryl, or any R8 and R9 can together form =0 or a cycloalkyl, or any to adjacent R8 groups can together form a cycloalkyl, Such non-natural amino acids may be in the form of a salt, or may be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post ationally modified.

] In on, the following amino acids haVing the structure of Formula (XXXXXHI) are included: 0 O 0 \S/ A A/ \ R X (c R ER 9)“ MN 0(0le (XXXXXIII) wherein: A is optional, and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower lene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted alkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted lene, R is H, alkyl, tuted alkyl, cycloalkyl, or substituted cycloalkyl, R1 is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and R2 is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; n is O, l, 2, 3, 4, or 5; and each R8 and R9 on each CRgR9 group is independently selected from the group consisting of H, alkoxy, alkylamine, halogen, alkyl, aryl, or any R8 and R9 can together form =0 or a cycloalkyl, or any to adjacent R8 groups can together form a cycloalkyl.

Such non-natural amino acids may be in the form of a salt, or may be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post ationally modified.

In addition, the following amino acids haVing the ure of Formula (XXXXXIV) are included: (XXXXXIV); wherein: A is al, and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, lene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted e, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene, R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl, R1 is H, an amino protecting group, resin, amino acid, ptide, or polynucleotide; and R2 is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; X1 is C, S, or S(O); and L is ne, substituted ne, N(R’)(alkylene) or N(R’)(substituted alkylene), where R’ is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl.

Such tural amino acids may be in the form of a salt, or may be incorporated into a tural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.

In addition, the following amino acids haVing the structure of Formula (XXXXXV) are included: 0 0 /C \ A N —|_ R X R' MIN 0(0le (XXXXXV) wherein: A is optional, and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted e, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene, R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl, R1 is H, an amino ting group, resin, amino acid, polypeptide, or polynucleotide; and R2 is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; L is alkylene, substituted alkylene, N(R’)(alkylene) or substituted alkylene), where R’ is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl.

Such non-natural amino acids may be in the form of a salt, or may be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.

In addition, the following amino acids haVing the structure of Formula (XXXXXVI) are included: 0\S/ A0 o A/ \N —L R X R I MIN 0(0le (XXXXXVI) wherein: A is optional, and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, lene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, tuted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene, R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl, R1 is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and R2 is OH, an ester ting group, resin, amino acid, polypeptide, or polynucleotide; L is alkylene, substituted alkylene, N(R’)(alkylene) or N(R’)(substituted ne), where R’ is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl.

Such tural amino acids may be in the form of a salt, or may be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.

In addition, amino acids haVing the structure of Formula VII) are included: O (XXXXXVII), wherein: A is optional, and when present is lower alkylene, tuted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, e, tuted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene; (b) (b) (b) "T” /R3 “I" “T" “I" (b) (b) (b) M is -C(R3)-, (a)E/C\—C\—g (b)R4 R4 (3)E/C=C\—éR4 (a)E/R—O—éR4 (a) is/C\—s—§R4 , , , , (b) (b) (b) m R R film /R3 l ”i 0—C—E/ s_ /_3C (b) (b) _C_§ c=c—§ (b) g (b) / R /c\ R4 l I I R4 JJJ m m m (a) (a) (a) or (a) where (a) indicates a , , , bonding to the A group and (b) indicates bonding to respective carbonyl groups, R3 and R4 are independently chosen from H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl, or R3 and R4 or two R3 groups or two R4 groups optionally form a cycloalkyl or a heterocycloalkyl; R is H, halogen, alkyl, substituted alkyl, lkyl, or substituted cycloalkyl, T3 is a bond, C(R)(R), O, or S, and R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or tuted cycloalkyl, R1 is H, an amino protecting group, resin, amino acid, polypeptide, or cleotide; and R2 is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide.

Such non-natural amino acids may be in the form of a salt, or may be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and ally post translationally modified.

In addition, amino acids haVing the structure of a (XXXXXVIH) are included: Ra T3\ R1\N R2 O (XXXXXVIH), wherein: (b) (b) (b) WV‘ R3 m m m <|:—c/—§ (b) (b) (b) / \ 0” \ L—c—g é—o—g é—s—g \ / \ Mis -C(R3)-, (3) R4 R4 (a) 19/ — R4 (3) R4 (80 as/ \R4 , , , , (b) (b) (b) (b) m R m R3 J‘ri /R3 Pr{ C=C— (b) O—C— (b) S—C— (b) /C—C—3 <b> / I | I R3 \R4 J‘s-5‘ R4 m m WU" (a) (a) (a) or (a) Where (a) indicates , , , , bonding to the A group and (b) indicates bonding to respective carbonyl groups, R3 and R4 are independently chosen from H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl, or R3 and R4 or two R3 groups or two R4 groups optionally form a cycloalkyl or a heterocycloalkyl; R is H, halogen, alkyl, tuted alkyl, cycloalkyl, or substituted cycloalkyl, T3 is a bond, ), O, or S, and R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl, R1 is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and R2 is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; each Ra is independently ed from the group consisting of H, halogen, alkyl, substituted alkyl, -N(R’)2, R’ Where k is 1, 2, or 3, -C(O)N(R’)2, -OR’, and -S(O)kR’, Where each R’ is independently H, alkyl, or substituted alkyl.

Such non-natural amino acids may be in the form of a salt, or may be orated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.

] In addition, amino acids haVing the structure of Formula (XXXXXIX) are included: R 0 wherein: R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or tuted cycloalkyl; and T3 is O, or S.

Such non-natural amino acids may be in the form of a salt, or may be incorporated into a non-natural amino acid polypeptide, r, polysaccharide, or a polynucleotide and optionally post translationally modified.

In addition, amino acids having the structure of Formula (XXXXXX) are included: R O O (XXXXXX), wherein: R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted lkyl.

] In addition, the ing amino acids having structures of Formula (XXXXXX) are included: 0 O O O RPE R2 RDE R2 0 and 0 , .

Such non-natural amino acids may be in the form of a salt, or may be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.

The carbonyl or dicarbonyl functionality can be reacted selectively With a ylamine- containing reagent under mild conditions in aqueous solution to form the corresponding oxime linkage that is stable under physiological conditions. See, e.g., Jencks, W. P., J. Am. Chem Soc. 81, 1 (1959); Shao, J. and Tam, J. P., J. Am. Chem. Soc. 117(14):3893-3899 (1995). Moreover, the unique reactivity of the carbonyl or dicarbonyl group allows for selective modification in the presence of the other amino acid side . See, e.g., Cornish, V. W., et al., J. Am. Chem. Soc. 118:8150-8151 (1996); Geoghegan, K. F. & Stroh, J. G., Bioconjug. Chem. 3:138-146 (1992); Mahal, L. K., et al., e 276:1125-1128 (1997).

The synthesis of p-acetyl-(+/-)-phenylalanine and m—acetyl-(+/-)-phenylalanine is described in Zhang, Z., et al., Biochemistry 42: 6735-6746 (2003), incorporated by reference. Other carbonyl- or dicarbonyl-containing amino acids can be similarly ed.

In some embodiments, a polypeptide comprising a non-natural amino acid is chemically modified to generate a reactive carbonyl or dicarbonyl functional group. For instance, an aldehyde onality useful for conjugation ons can be generated from a functionality having adjacent amino and hydroxyl groups. Where the biologically active le is a polypeptide, for example, an N—terminal serine or threonine (which may be normally present or may be d Via chemical or enzymatic digestion) can be used to generate an aldehyde functionality under mild oxidative cleavage ions using periodate. See, e.g., Gaertner, et. al., Bioconjug. Chem. 3: 262-268 (1992); Geoghegan, K. & Stroh, J., Bioconjug. Chem. 3:138-146 (1992); Gaertner et al., J. Biol. Chem. 269:7224-7230 (1994). r, methods known in the art are restricted to the amino acid at the N—terminus of the peptide or protein.

Additionally, by way of example a non-natural amino acid bearing adjacent yl and amino groups can be incorporated into a polypeptide as a “masked” aldehyde functionality. For example, -hydroxylysine bears a hydroxyl group adjacent to the epsilon amine. Reaction conditions for generating the aldehyde typically involve addition of molar excess of sodium riodate under mild conditions to avoid oxidation at other sites within the polypeptide. The pH of the ion on is typically about 7.0. A typical reaction involves the addition of about 1.5 molar excess of sodium meta periodate to a buffered solution of the polypeptide, followed by incubation for about 10 minutes in the dark. See, e. g.

US. Patent No. 6,423,685.

B. ure and sis 0fN0n-Natural Amino Acids: Dicarbonyl, Dicarbonyl-like, Masked Dicarbonyl, and Protected Dicarbonyl Groups Amino acids with an ophilic reactive group allow for a variety of reactions to link molecules Via nucleophilic on reactions among others. Such ophilic reactive groups include a dicarbonyl group (including a diketone group, a ketoaldehyde group, a ketoacid group, a ketoester group, and a ketothioester group), a dicarbonyl-like group (which has reactiVity similar to a dicarbonyl group and is structurally similar to a dicarbonyl group), a masked dicarbonyl group (which can be readily converted into a onyl group), or a protected dicarbonyl group (which has reactiVity similar to a dicarbonyl group upon deprotection). Such amino acids include amino acids having the structure of Formula (xxxvn): 0 (XXXVH). wherein: A is optional, and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower lene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene, B is optional, and when present is a linker linked at one end to a diamine ning moiety, the linker ed from the group consisting of lower alkylene, tuted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-(alkylene or substituted alkylene)-, -S-(alkylene or substituted alkylene)-, -C(O)R”-, - alkylene or substituted alkylene)-, where k is l, 2, or 3, -C(O)-(alkylene or substituted alkylene)-, -C(S)-(alkylene or substituted alkylene)-, -NR”-(alkylene or substituted ne)-, -CON(R”)-(alkylene or substituted alkylene)-, -CSN(R”)-(alkylene or substituted alkylene)-, and -N(R”)CO-(alkylene or substituted alkylene)-, where each R” is independently H, alkyl, or substituted alkyl, 0 0 0 o o #2 Mkawk k /$71. 5,: Kisz' T1 \555 / 1 T2 \T \ T1 597 o 21 «duo Y 5&5\T3—T3—T2;?fi a , where, T1 is a bond, optionally substituted C1-C4 alkylene, ally substituted C1-C4 alkenylene, or optionally substituted heteroalkyl; wherein each optional substituents is independently selected from lower alkylene, substituted lower alkylene, lower lkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, tuted lower cycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene, T2, is selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, kylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O)k- where k is l, 2, or 3, -S(O)k(alkylene or substituted alkylene)-, , -C(O)-(alkylene or substituted alkylene)-, , -C(S)-(alkylene or substituted alkylene)-, -N(R’)-, -NR’-(alkylene or substituted alkylene)-, -C(O)N(R’)-, -CON(R’)-(alkylene or tuted alkylene)-, -CSN(R’)-, -CSN(R’)-(alkylene or substituted alkylene)-, -N(R’)CO-(alkylene or substituted ne)-, _N(R’)C(O)O_a -S(O)kN(R’)-, _N(R’)C(O)N(R’)_a -N(R’)C(S)N(R’)-, 'N(R’)S(O)kN(R’)'a -N(R’)- N=, -C(R’)=N—, -C(R’)=N—N(R’)-, -C(R’)=N—N=, -C(R’)2-N=N-, and -C(R’)2-N(R’)-N(R’)-, where each R’ is independently H, alkyl, or substituted alkyl; T ,5 ix): E “2352 , , or Where each X1 is independently selected from the group consisting of -O-, -S-, -N(H)-, -N(R)-, -N(Ac)-, and — N(OMe)-; X2 is —OR, -OAc, -SR, -N(R)2, -N(R)(Ac), -N(R)(OMe), or N3, and Where each R’ is independently H, alkyl, or substituted alkyl; R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted lkyl; R1 is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and R2 is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; or the —A—B-K-R groups together form a bicyclic or tricyclic cycloalkyl or heterocycloalkyl comprising at least one yl group, including a dicarbonyl group, protected carbonyl group, ing a protected onyl group, or masked carbonyl group, including a masked dicarbonyl group; or the —K-R group together forms a monocyclic or bicyclic cycloalkyl or cycloalkyl comprising at least one carbonyl group, including a dicarbonyl group, protected carbonyl group, including a protected dicarbonyl group, or masked carbonyl group, including a masked dicarbonyl group.

] Non-limiting example of dicarbonyl amino acids haVing the structure of Formula (X X XVII) include: o o 0 O OH OH HZN HZN HZN O O O 7 7 7 O 0 SM 0 O O O HZNgM HZNWo’\ OH O O O 7 7 7 O / O O HZN iOH HzN OH OH HZN HZN O O O O 7 ' 7 7 O O O O O O O O CF3 CHFZ OH OH OH OH HzN HZN HZN HZN O 0 O O OH OH OH HZN H2N H2N o o and 0 Such non-natural amino acids may be in the form of a salt, or may be incorporated into a non-natural amino acid polypeptide, polymer, ccharide, or a polynucleotide and optionally post translationally modified.

C. Structure and Synthesis of Non-Natural Amino Acids: Ketoalkyne, Ketoalkyne - like, Masked Ketoalkyne, Protected Ketoalkyne , Alkyne, and Cycloalkyne Groups Amino acids containing reactive groups with dicarbonyl-like vity allow for the linking of molecules Via philic addition reactions. Such electrophilic reactive groups include a ketoalkyne group, a ketoalkyne-like group (which has reactivity r to a ketoalkyne group and is structurally r to a kyne group), a masked kyne group (which can be readily converted into a ketoalkyne group), or a protected ketoalkyne group (which has vity similar to a ketoalkyne group upon deprotection). In some embodiments, amino acids containing reactive groups with a al alkyne, al alkyne or cycloalkyne allow for linking of molecules Via cycloaddition reactions (e.g., 1,3-dipolar cycloadditions, azide-alkyne Huisgen cycloaddition, etc.) Such amino acids include amino acids haVing the structure of Formula (XXXXXXI-A) or (XXXXXXI-B): R l H 3 A G—CEC—R \B/ R3 A\ Q R3 B R (R19)q R1\N 2 R1\N R2 H R4 H R O (XXXXXXI-A), 4o (XXXXXXI-B), wherein: A is optional, and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, arylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene, B is optional, and when present is a linker linked at one end to a diamine containing moiety, the linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, tuted lower heteroalkylene, -O-(alkylene or substituted alkylene)-, -S-(alkylene or substituted alkylene)-, -C(O)R”-, - S(O)k(alkylene or substituted alkylene)-, where k is l, 2, or 3, -C(O)-(alkylene or tuted alkylene)-, -C(S)-(alkylene or substituted ne)-, -NR”-(alkylene or substituted alkylene)-, -CON(R”)-(alkylene or substituted alkylene)-, -CSN(R”)-(alkylene or substituted alkylene)-, and -N(R”)CO-(alkylene or substituted alkylene)-, where each R” is ndently H, alkyl, or substituted alkyl, )K T G is optional, and when present is Sonora/4);; 555% ix?“ T4 is a carbonyl protecting group including, but not limited to, \_/ 55:2le Wm “Mn XUCI cQ‘Z‘XZ 921/le2 ; ; or where each X1 is independently selected from the group ting of -O-, -S-, -N(H)-, -N(R)-, -N(Ac)-, and —N(OMe)-; X2 is —OR, -OAc, -SR, -N(R)2, - N(R)(Ac), -N(R)(OMe), or N3, and where each R’ is independently H, alkyl, or substituted alkyl; R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R1 is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; R2 is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; each of R3 and R4 is independently H, halogen, lower alkyl, or substituted lower alkyl, or R3 and R4 or two R3 groups optionally form a cycloalkyl or a heterocycloalkyl; each R19 is ndently selected from the group ting of C1-C6 alkyl, C1-C6 alkoxy, ester, ether, thioether, aminoalkyl, n, alkyl ester, aryl ester, amide, aryl amide, alkyl halide, alkyl amine, alkyl sulfonic acid, alkyl nitro, thioester, sulfonyl ester, halosulfonyl, nitrile, alkyl nitrile, and nitro; and qis 0,1, 2, 3, 4, 5, 6, 7, 8, 9,10 or 11.

D. Structure and Synthesis ofNon-Natural Amino Acids: Ketoamine, Ketoamine-like, Masked Ketoamine, and Protected Ketoamine Groups ]Amino acids containing reactive groups with dicarbonyl-like reactiVity allow for the linking of molecules Via nucleophilic addition ons. Such reactive groups include a ine group, a ketoamine-like group (which has reactiVity similar to a ketoamine group and is structurally r to a ketoamine group), a masked ketoamine group (which can be readily converted into a ketoamine group), or a protected ketoamine group (which has reactiVity similar to a ketoamine group upon deprotection). Such amino acids include amino acids haVing the structure of Formula (XXXXXXH): R3 A\B/G_T1\ N—R' R1\N R2 R H R4 0 (XXXXXXII) wherein: A is optional, and when present is lower alkylene, tuted lower alkylene, lower cycloalkylene, tuted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted alkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted lene, aralkylene, or substituted aralkylene; B is optional, and when present is a linker linked at one end to a diamine containing moiety, the linker selected from the group consisting of lower ne, substituted lower alkylene, lower lene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-(alkylene or substituted alkylene)-, -S-(alkylene or substituted alkylene)-, -C(O)R”-, - S(O)k(alkylene or substituted ne)-, where k is l, 2, or 3, -C(O)-(alkylene or substituted alkylene)-, -C(S)-(alkylene or substituted alkylene)-, -NR”-(alkylene or tuted alkylene)-, -CON(R”)-(alkylene or substituted alkylene)-, -CSN(R”)-(alkylene or substituted alkylene)-, and -N(R”)CO-(alkylene or substituted alkylene)-, where each R” is ndently H, alkyl, or substituted alkyl; 2* 5' T " 2/ 4\ 5'S, G is or ; T1 is an optionally substituted C1-C4 alkylene, an optionally substituted C1-C4 alkenylene, or an optionally substituted heteroalkyl; 555><9LL fir T4 is a yl protecting group including, but not limited to, R'O OR' \_/ fyk “w X X1 v 09-239 ‘12“11: ; ; or where each X1 is independently selected from the group consisting of -O-, -S-, -N(H)-, -N(R’)-, -N(Ac)-, and —N(OMe)-; X2 is —OR, -OAc, -SR’, -N(R’)2, -N(R’)(Ac), -N(R’)(OMe), or N3, and where each R’ is ndently H, alkyl, or substituted alkyl; R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R1 is H, an amino protecting group, resin, amino acid, ptide, or polynucleotide; and R2 is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; each of R3 and R4 is independently H, halogen, lower alkyl, or substituted lower alkyl, or R3 and R4 or two R3 groups optionally form a cycloalkyl or a heterocycloalkyl.

Amino acids haVing the structure of a (XXXXXXH) include amino acids haVing the structure of Formula XHI) and Formula (XXXXXXIV): Ra /Gfl B /RV Ra F O ’R’ R' N\ RE,1 a R1\ R2 R1\ R2 N N H H O (XXXXXXIII), O (XXXXXXIV) wherein each Ra is independently selected from the group consisting of H, halogen, alkyl, substituted alkyl, -N(R’)2, -C(O)kR’ where k is 1, 2, or 3, -C(O)N(R’)2, -OR’, and R’, where each R’ is ndently H, alkyl, or substituted alkyl.

E. Structure and sis ofNon-Natural Amino Acids: Diamine, Diamine-like, Masked Diamine, Protected Amines and Azides Amino acids with a nucleophilic reactive group allow for a variety of reactions to link les Via electrophilic addition reactions among others. Such nucleophilic ve groups include a diamine group (including a hydrazine group, an e group, an imine group, a l,l-diamine group, a 1,2-diamine group, a 1,3-diamine group, and a l,4-diamine group), a diamine-like group (which has reactiVity similar to a diamine group and is structurally similar to a e group), a masked e group (which can be readily ted into a diamine group), or a protected diamine group (which has reactiVity similar to a diamine group upon deprotection). In some embodiments, amino acids containing reactive groups with azides allow for linking of les Via cycloaddition reactions (e.g., 1,3-dipolar cycloadditions, azide-alkyne Huisgen cycloaddition, etc.).

In another aspect are methods for the chemical synthesis of hydrazine-substituted molecules for the derivatization of carbonyl-substituted dolastatin derivatives. In one embodiment, the hydrazine- substituted molecule can dolastatin linked derivatives. In one embodiment are methods for the preparation of hydrazine-substituted molecules suitable for the derivatization of carbonyl-containing non-natural amino acid polypeptides, including by way of example only, ketone-, or aldehyde-containing non-natural amino acid polypeptides. In a further or additional embodiment, the non-natural amino acids are incorporated site-specifically during the in Vivo translation of proteins. In a further or additional embodiment, the hydrazine-substituted dolastatin derivatives allow for the site-specific derivatization of carbonyl-containing tural amino acids Via philic attack of each carbonyl group to form a heterocycle-derivatized polypeptide, including a nitrogen-containing heterocycle-derivatized polypeptide in a site-specific fashion. In a further or additional embodiment, the method for the preparation of hydrazine-substituted dolastatin tives provides access to a wide variety of site-specifically derivatized polypeptides. In a further or additional embodiment are s for synthesizing ine- functionalized polyethyleneglycol (PEG) linked dolastatin derivatives.

Such amino acids include amino acids having the structure of Formula (XXXVII—A) or (XXXVII-B): R3 R3 A\ /K\ A\ /N 3 R3 B R R3 B R1 \ R2 R \1 R2 N N H R4 H R4 0 (XXXVII), o (XXXVII-B), wherein: A is optional, and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, tuted lower cycloalkylene, lower alkenylene, substituted lower lene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted e, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene; B is optional, and when present is a linker linked at one end to a diamine ning moiety, the linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower alkylene, -O-(alkylene or substituted ne)-, -S-(alkylene or substituted alkylene)-, ”-, -C(O)R”- or substituted alkylene)-, where k is , -S(O)k(alkylene l, 2, or 3, -C(O)-(alkylene or substituted ne)-, -C(S)-(alkylene or substituted alkylene)-, -NR”-(alkylene or substituted alkylene)-, -CON(R”)-(alkylene or tuted alkylene)-, -CSN(R”)-(alkylene or substituted alkylene)-, and -N(R”)CO-(alkylene or substituted alkylene)-, where each R” is independently H, alkyl, or substituted alkyl, R R R2 H R8 $9 $8 |9 H\N/8 Ill H‘ l; | N\ /N\ \ \ / N\ /N / T1 H T2 \R9 [.111 / \ T2 T1 \ \ / \ 55y K IS ”LL!/ 555‘ J‘p’ “L21 ‘11.}. 555 01‘ a ’ ’ a H\I\I\ I 1L1 ;where R8 and R9 are independently selected from H, alkyl, substituted alkyl, cycloalkyl, substituted lkyl, or amine protecting group; T1 is a bond, optionally substituted C1-C4 ne, optionally substituted C1-C4 alkenylene, or optionally substituted heteroalkyl, T2 is optionally substituted C1-C4 ne, optionally substituted C1-C4 alkenylene, optionally substituted heteroalkyl, optionally substituted aryl, or optionally substituted heteroaryl; wherein each optional substituents is independently selected from lower alkyl, substituted lower alkyl, lower cycloalkyl, substituted lower cycloalkyl, lower l, substituted lower alkenyl, alkynyl, lower heteroalkyl, substituted heteroalkyl, lower heterocycloalkyl, substituted lower heterocycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkaryl, substituted alkaryl, aralkyl, or substituted aralkyl, R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R1 is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and R2 is OH, an ester protecting group, resin, amino acid, ptide, or polynucleotide; each of R3 and R4 is independently H, n, lower alkyl, or substituted lower alkyl, or R3 and R4 or two R3 groups optionally form a cycloalkyl or a heterocycloalkyl; or the —A—B-K-R groups together form a bicyclic or tricyclic cycloalkyl or heterocycloalkyl comprising at least one diamine group, protected diamine group or masked diamine group; or the —B-K-R groups together form a bicyclic or tricyclic cycloalkyl or cycloaryl or cycloalkyl comprising at least one diamine group, protected diamine group or masked diamine group; or the —K-R group er forms a monocyclic or bicyclic cycloalkyl or heterocycloalkyl sing at least one diamine group, protected diamine group or masked diamine group; wherein at least one amine group on —A—B-K-R is optionally a protected amine.

In one aspect are compounds comprising the structures 1 or 2: NH 2 R3 R3A, 1% NH 3' \TI 2 R3 3A f ‘B’ 2NH2 Rl‘N R2 R1. R2 R N H 4O H R40 1 2 wherein: A is optional, and when t is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, tuted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, arylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or tuted aralkylene, B is al, and when present is a linker linked at one end to a diamine containing moiety, the linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower lene, lower heteroalkylene, substituted lower alkylene, -O-(alkylene or substituted alkylene)-, -S-(alkylene or substituted alkylene)-, -C(O)R”-,-S(O)k(alkylene or substituted alkylene)-, where k is l, 2, or 3, -C(O)-(alkylene or substituted alkylene)-, -C(S)-(alkylene or substituted alkylene)-, -NR”-(alkylene or substituted alkylene)-, -CON(R”)-(alkylene or substituted alkylene)-, -CSN(R”)-(alkylene or substituted alkylene)-, and -N(R”)CO-(alkylene or substituted alkylene)-, where each R” is independently H, alkyl, or substituted alkyl; T1 is a bond or CH2; and T2 is CH; wherein each optional substituents is independently selected from lower alkyl, substituted lower alkyl, lower cycloalkyl, substituted lower cycloalkyl, lower alkenyl, substituted lower alkenyl, alkynyl, lower heteroalkyl, substituted heteroalkyl, lower heterocycloalkyl, substituted lower heterocycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkaryl, substituted alkaryl, aralkyl, or substituted aralkyl; R1 is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and R2 is OH, an ester protecting group, resin, amino acid, ptide, or polynucleotide; each of R3 and R4 is independently H, halogen, lower alkyl, or substituted lower alkyl, or R3 and R4 or two R3 groups optionally form a cycloalkyl or a cycloalkyl; or the —A—B-diamine ning moiety together form a ic cycloalkyl or heterocycloalkyl comprising at least one diamine group, ted diamine group or masked diamine group; or the —B-diamine containing moiety groups together form a bicyclic or tricyclic cycloalkyl or ryl or heterocycloalkyl comprising at least one diamine group, protected diamine group or masked diamine group; n at least one amine group on —A—B-diamine containing moiety is optionally a protected amine; or an active metabolite, salt, or a pharmaceutically acceptable prodrug or solvate thereof.

The following non-limiting es of amino acids having the structure of Formula (XXXVII) are included: qfiflfif NH2 NH2 7 7 If»?Ni WO 66560 NH NH NH2 NHz OH OH OH OH HZN HZN HZN HZN NH2 OW NH2 NH2 OH OH OH HZN HZN HZN O O and O 7 , Such non-natural amino acids may also be in the form of a salt or may be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and/or optionally post translationally modified.

In certain embodiments, compounds of Formula (XXXVII) are stable in aqueous solution for at least 1 month under mildly acidic conditions. In certain embodiments, compounds of Formula (XXXVII) are stable for at least 2 weeks under mildly acidic conditions. In n embodiments, compound of Formula (XXXVII) are stable for at least 5 days under mildly acidic conditions. In n embodiments, such acidic conditions are pH about 2 to about 8.

In certain embodiments of compounds of Formula (XXXVII), B is lower alkylene, substituted lower alkylene, O-(alkylene or substituted alkylene)-, C(R’)=NN(R’)-, -N(R’)CO-, C(O)-, =N—, C(O)-(alkylene or substituted alkylene)-, CON(R’)(alkylene or substituted alkylene)-, -S(alkylene or substituted alkylene)-, -S(O)(alkylene or substituted alkylene)-, or -S(O)2(alkylene or substituted alkylene)-. In certain embodiments of compounds of Formula (XXXVII), B is —O(CH2)-, -CH=N—, CH=NNH-, -NHCH2-, -NHCO-, C(O)-, C(O)(CH2)-, CONH(CH2)-, -SCH2-, CH2-, or -S(O)2CH2-.

In certain embodiments of compounds of a (XXXVII), R is C1-6 alkyl or cycloalkyl. In n embodiments of compounds of Formula (XXXVII) R is —CH3, -CH(CH3)2, or ropyl. In certain embodiments of compounds of Formula (XXXVII), R1 is H, tert-butyloxycarbonyl (Boc), 9- Fluorenylmethoxycarbonyl (Fmoc), N—acetyl, tetrafluoroacetyl (TFA), or benzyloxycarbonyl (Cbz). In certain embodiments of compounds of Formula (XXXVII), R1 is a resin, amino acid, polypeptide, or cleotide. In certain embodiments of compounds of Formula (XXXVII), R1 is an antibody, antibody fragment or monoclonal antibody. In certain embodiments of compounds of Formula (XXXVII), R2 is OH, O-methyl, O-ethyl, or O-t-butyl. In n embodiments of compounds of Formula (XXXVII), R2 is a resin, at least one amino acid, ptide, or polynucleotide. In certain embodiments of compounds of Formula (XXXVII), R2 is an antibody, antibody fragment or monoclonal antibody.

] The ing non-limiting examples of amino acids haVing the structure of Formula (XXXVII) are also included: \NHZ N 0vilk E‘NHz 02:21E H 5? NH2 oE OH HZN Ez Ez O O O NH2 NH2 OH OH OH HZN HZN HZN o O zE NH2 Om o: OH H2N Ez H2N O O 0 7 7 7 HzN NHZ NH2 NH2 / NH2 NH2 OH OH H2N HzN H2N O O O 7 7 7 NHz H N H2N HzN 0 and 0%O , ‘ Non—limiting examples of protected amino acids having the structure of Formula (XXXVII) include: ’N= H H N \ ,CH3 CH3 H CH3 N\ 0vN N=c\ \N=C: CH3 CH3 OH OH H2N HZN HZN O O O and 7 7 7 N\ ,CHs N—C\_ F. ure and Synthesis 0fN0n-Natural Amino Acids: Aromatic Amines Non-natural amino acids with nucleophilic reactive groups, such as, by way of example only, an aromatic amine group (including ary and tertiary amine ), a masked aromatic amine group (Which can be readily converted into a aromatic amine group), or a protected aromatic amine group (Which has reactiVity similar to an aromatic amine group upon deprotection) allow for a variety of reactions to link molecules via various reactions, including but not limited to, reductive alkylation reactions with aldehyde containing dolastatin linker derivatives. Such ic amine containing non- natural amino acids include amino acids having the structure of Formula (XXXXXXV): (Ra>n\/A NH R3 B Rpg R2 o (XXXXXXV) wherein: is selected from the group consisting of a monocyclic aryl ring, a bicyclic aryl ring, a multicyclic aryl ring, a clic heteroaryl ring, a bicyclic heteroaryl ring, and a yclic heteroaryl ring; A is independently CR,, or N; B is independently CRa, N, O, or S; each R2, is independently selected from the group consisting of H, halogen, alkyl, -N02, -CN, substituted alkyl, -N(R’)2, -C(O)kR’, -C(O)N(R’)2, -OR’, and -S(O)kR’, where k is l, 2, or 3; andn is O, l, 2, 3, 4, , or 6; R1 is H, an amino protecting group, resin, at least one amino acid, polypeptide, or polynucleotide; and R2 is OH, an ester ting group, resin, at least one amino acid, polypeptide, or polynucleotide; each of R3 and R4 is independently H, halogen, lower alkyl, or substituted lower alkyl, or R3 and R4 or two R3 groups optionally form a cycloalkyl or a heterocycloalkyl; M is H or —CH2R5; or the M-N—C(R5) moiety may form a 4 to 7 ed ring structure; R5 is alkyl, substituted alkyl, alkenyl, substituted l, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide, tuted polyalkylene oxide, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycle, substituted heterocycle, alkaryl, tuted alkaryl, aralkyl, substituted aralkyl, -C(O)R”, -C(O)OR”, -C(O)N(R”)2, -C(O)NHCH(R”)2, lene or substituted alkylene)-N(R”)2, -(alkenylene or substituted alkenylene)-N(R”)2, -(alkylene or substituted alkylene)-(aryl or substituted aryl), -(alkenylene or substituted alkenylene)-(aryl or substituted aryl), -(alkylene or substituted alkylene)- ON(R”)2, -(alkylene or substituted alkylene)-C(O)SR”, -(alkylene or substituted alkylene)-S-S—(aryl or substituted aryl), wherein each R” is independently hydrogen, alkyl, tuted alkyl, alkenyl, substituted alkenyl, alkoxy, tuted alkoxy, aryl, substituted aryl, aryl, substituted heteroaryl, heterocycle, tuted heterocycle, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, or -C(O)OR’; or two R5 groups optionally form a cycloalkyl or a heterocycloalkyl; l 14 or R5 and any Ra optionally form a cycloalkyl or a heterocycloalkyl; and each R’ is independently H, alkyl, or tuted alkyl.

Such non-natural amino acids may also be in the form of a salt, or may be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally reductively ted.

(Ron\/AQNH The structure (as presented in all examples herein) does not present the relative orientations of “A,” “B,” “NH-M” and “Ra”; rather these four features of this structure may be oriented in any chemically-sound manner (along With other features of this structure), as illustrated by example herein.

Non-natural amino acids containing an aromatic amine moiety haVing the structure of Formula (A) include non-natural amino acids having the structures: /A B-A1 \ v A A/, B\‘ \A/ /:j::,'AAV R3 I \ 2A R3 A R R 1\N 2 H R4 0 and , , A‘=A‘ B \A‘ R3 A R‘N R2 R4 M 0 ; Wherein, each A’ is independently selected from CRa, N, or C_NH, and up to two A’ may be C_NH With the remaining A’ selected from CR,, or N.

Such non-natural amino acids may also be in the form of a salt, or may be incorporated into a non-natural amino acid polypeptide, r, ccharide, or a polynucleotide and optionally ively alkylated.

Non-limiting examples of non-natural amino acids containing an aromatic amine moiety haVing the structure of a (XXXXXXV) include non-natural amino acids haVing the structure of Formula (XXXXXXVI), and Formula (XXXXXXVII), (XXXXXXVI), (XXXXXXVH), wherein; G is an amine protecting group, including, but not limited to, l 15 95.)" OVCC13 ,s‘“ 1/ \fl/0\/ 0,5 OQ . fYoQ ; ; ‘g’ o “1:3;O 555 CH CH 0 3 ’ 3 if $5_ ,5: 0 SiMe —C Or it W 3 Such non-natural amino acids may be in the form of a salt, or may be orated into a tural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally reductively alkylated.

] Non-natural amino acids containing an aromatic amine moiety have the following structures: Wherein each Ra is independently selected from the group consisting of H, halogen, alkyl, -N02, -CN, substituted alkyl, -N(R’)2, -C(O)kR’, -C(O)N(R’)2, -OR’, and -S(O)kR’, Where k is l, 2, or 3; M is H or —CH2R5; or the M-N—C(R5) moiety may form a 4 to 7 membered ring structure; R1 is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; R2 is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; R5 is alkyl, substituted alkyl, alkenyl, substituted alkenyl, l, substituted alkynyl, alkoxy, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide, substituted polyalkylene oxide, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycle, substituted heterocycle, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, -C(O)R”, -C(O)OR”, -C(O)N(R”)2, HCH(R”)2, -(alkylene or substituted ne)-N(R”)2, -(alkenylene or substituted alkenylene)-N(R”)2, -(alkylene or substituted alkylene)-(aryl or substituted aryl), -(alkenylene or substituted alkenylene)-(aryl or substituted aryl), -(alkylene or substituted alkylene)- 2, -(alkylene or substituted alkylene)-C(O)SR”, -(alkylene or substituted alkylene)-S-S-(aryl or substituted aryl), Wherein each R” is independently hydrogen, alkyl, tuted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycle, substituted heterocycle, alkaryl, substituted l, aralkyl, tuted aralkyl, or -C(O)OR’; or R5 and any Ra optionally form a cycloalkyl or a heterocycloalkyl; and each R’ is independently H, alkyl, or substituted alkyl. Such non-natural amino acids may also be in the form of a salt, or may be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide.

Such tural amino acids of Formula (XXXXXXV) may be formed by reduction of protected or masked amine moieties on the aromatic moiety of a non-natural amino acid. Such protected or masked amine moieties include, but are not limited to, , hydrazines, nitro, or azide substituents.

The reducing agents used to reduce such protected or masked amine moieties include, but are not limited to, TCEP, NaZS, Na2S204, LiAlH4, NaBH4 or NaBCNHg.

V. Non-Natural Amino Acid Linked Dolastatin Derivatives In another aspect bed herein are methods, strategies and techniques for incorporating at least one such atin linker derivatives into a non-natural amino acid. Also included With this aspect are methods for ing, purifying, characterizing and using such dolastatin linker derivatives containing at least one such non-natural amino acid. Also included With this aspect are compositions of and methods for producing, purifying, characterizing and using oligonucleotides (including DNA and RNA) that can be used to produce, at least in part, a dolastatin linker derivative containing at least one tural amino acid. Also included With this aspect are compositions of and methods for producing, purifying, characterizing and using cells that can express such oligonucleotides that can be used to produce, at least in part, a atin linker derivative containing at least one non-natural amino acid.

Thus, dolastatin linker derivatives comprising at least one non-natural amino acid or modified non-natural amino acid With a carbonyl, dicarbonyl, alkyne, cycloalkyne, azide, oxime or hydroxylamine group are provided and described herein. In certain embodiments, dolastatin linker derivatives with at least one non-natural amino acid or modified tural amino acid With a carbonyl, onyl, alkyne, cycloalkyne, azide, oxime or hydroxylamine group include at least one post-translational modification at some position on the polypeptide. In some embodiments the co-translational or post-translational modification occurs via the cellular machinery (e. g., glycosylation, ation, acylation, lipid- modification, palmitoylation, palmitate addition, orylation, ipid-linkage ation, and the like), in many instances, such cellular-machinery-based co-translational or post-translational modifications occur at the naturally occurring amino acid sites on the polypeptide, however, in certain embodiments, the cellular-machinery—based co-translational or post-translational modifications occur on the tural amino acid site(s) on the polypeptide.

In other ments, the post-translational modification does not utilize the cellular machinery, but the onality is instead provided by attachment of a molecule (a polymer; a water-soluble polymer; a derivative of polyethylene glycol; a second protein or ptide or polypeptide analog; an antibody or antibody fragment; and any combination thereof) comprising a second reactive group to the at least one non-natural amino acid comprising a first reactive group (including but not limited to, non-natural amino acid containing a ketone, de, acetal, hemiacetal, alkyne, cycloalkyne, azide, oxime, or hydroxylamine functional group) utilizing chemistry methodology described herein, or others suitable for the ular reactive groups. In certain ments, the co-translational or post-translational ation is made in vivo in a eukaryotic cell or in a karyotic cell. In certain embodiments, the post-translational modification is made in vitro not utilizing the ar machinery. Also included With this aspect are methods for ing, purifying, characterizing and using such dolastatin linker derivatives containing at least one such co-translationally or post-translationally modified non-natural amino acids.

Also included Within the scope of the methods, compositions, strategies and techniques described herein are reagents capable of ng with a dolastatin linker derivative (containing a carbonyl or dicarbonyl group, oxime group, alkyne, cycloalkyne, azide, hydroxylamine group, or masked or protected forms thereof) that is part of a polypeptide so as to produce any of the aforementioned post-translational modifications. In certain embodiments, the resulting post-translationally d dolastatin linker derivative Will contain at least one oxime group; the ing modified oxime-containing dolastatin linker derivative may undergo subsequent modification reactions. Also included with this aspect are methods for producing, purifying, characterizing and using such reagents that are capable of any such post- ational modifications of such dolastatin linker derivative(s).

In certain embodiments, the polypeptide or non-natural amino acid linked dolastatin derivative includes at least one nslational or post-translational modification that is made in vivo by one host cell, Where the post-translational modification is not normally made by another host cell type. In n embodiments, the polypeptide includes at least one co-translational or post-translational modification that is made in vivo by a eukaryotic cell, Where the co-translational or ranslational modification is not normally made by a non-eukaryotic cell. Examples of such co-translational or ranslational modifications include, but are not limited to, glycosylation, acetylation, acylation, lipid-modification, palmitoylation, palmitate addition, orylation, glycolipid-linkage modification, and the like. In one embodiment, the co-translational or ranslational modification comprises attachment of an oligosaccharide to an asparagine by a GlcNAc-asparagine linkage (including but not limited to, Where the oligosaccharide comprises (GlcNAc-Man)Z-Man-GlcNAc-GlcNAc, and the like). In another embodiment, the co-translational or post-translational modification comprises attachment of an oligosaccharide ding but not limited to, Gal-GalNAc, Gal-GlcNAc, etc.) to a serine or threonine by a GalNAc- serine, a GalNAc-threonine, a GlcNAc-serine, or a GlcNAc-threonine linkage. In certain embodiments, a protein or polypeptide can comprise a secretion or zation sequence, an epitope tag, a FLAG tag, a polyhistidine tag, a GST fusion, and/or the like. Also included with this aspect are methods for producing, purifying, characterizing and using such polypeptides containing at least one such co-translational or post- translational ation. In other embodiments, the glycosylated non-natural amino acid polypeptide is produced in a ycosylated form. Such a non-glycosylated form of a glycosylated non-natural amino acid may be produced by methods that e chemical or enzymatic removal of oligosaccharide groups from an isolated or substantially purified or unpurified glycosylated non-natural amino acid polypeptide; production of the non-natural amino acid in a host that does not glycosylate such a non-natural amino acid polypeptide (such a host including, prokaryotes or eukaryotes engineered or d to not glycosylate such a polypeptide), the introduction of a glycosylation inhibitor into the cell culture medium in which such a non-natural amino acid polypeptide is being produced by a eukaryote that normally would glycosylate such a polypeptide, or a combination of any such methods. Also described herein are such non-glycosylated forms of normally-glycosylated non-natural amino acid polypeptides (by normally- glycosylated is meant a polypeptide that would be glycosylated when produced under conditions in which naturally-occurring polypeptides are glycosylated). Of course, such non-glycosylated forms of normallyglycosylated non-natural amino acid polypeptides (or indeed any polypeptide described herein) may be in an unpurified form, a substantially purified form, or in an isolated form.

In certain embodiments, the non-natural amino acid polypeptide includes at least one post- translational ation that is made in the ce of an accelerant, wherein the post-translational modification is stoichiometric, stoichiometric-like, or near-stoichiometric. In other embodiments the ptide is ted with a reagent of Formula (XIX) in the presence of an accelerant. In other embodiments the accelerant is selected from the group consisting of: ::NH2 C: OH 3:SH 0H [I \ NH2 NH2 H CE N \(I NSH OH NH2 NH2 NH2 0H N NH2 N NH /\/NH2 NOH [53 2 H2N H2N H2N H N 7 7 7 7 7 7 7 7 7 7 7 and N.

A. Chemical Synthesis 0fN0n-Nataral Amino Acid Linked Dolastatin tives: Oxime- Containing Linked atin Derivatives Non-natural amino acid dolastatin linked derivatives containing an oxime group allow for reaction with a variety of reagents that contain n reactive yl- or dicarbonyl- groups ding but not limited to, s, aldehydes, or other groups with similar reactivity) to form new non-natural amino acids comprising a new oxime group. Such an oxime exchange reaction allows for the further functionalization of dolastatin linked tives. Further, the original dolastatin linked derivative containing an oxime group may be useful in their own right as long as the oxime linkage is stable under conditions necessary to incorporate the amino acid into a polypeptide (e.g., the in vivo, in vitro and chemical synthetic methods described herein).

Thus, in certain ments described herein are non-natural amino acid dolastatin linked derivatives with sidechains comprising an oxime group, an oxime-like group (which has reactivity similar to an oxime group and is urally similar to an oxime group), a masked oxime group (which can be readily converted into an oxime group), or a protected oxime group (which has reactivity similar to an oxime group upon deprotection).

Such non-natural amino acid dolastatin linked tives include dolastatin linked derivatives having the structure of Formula (VIII) or (IX): (VH1) (1X) wherein: A is optional, and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower lene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted alkylene, lower heterocycloalkylene, tuted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, tuted alkarylene, aralkylene, or substituted aralkylene; B is optional, and when present is a linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower lene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, - S-, -S-(alkylene or substituted alkylene)-, -S(O)k- where k is l, 2, or 3, -S(O)k(alkylene or substituted alkylene)-, -C(O)—, -C(O)-(alkylene or substituted alkylene)-, , -C(S)- (alkylene or substituted alkylene)-, -N(R’)-, alkylene or substituted alkylene)-, -C(O)N(R’)-, -CON(R’)-(alkylene or substituted alkylene)-, -CSN(R’)-, -CSN(R’)-(alkylene or substituted alkylene)-, -N(R’)CO-(alkylene or substituted alkylene)-, -N(R’)C(O)O-, -S(0)kN(R’)-, C(0)N(R’)-, _N(R’)C(S)N(R’)_a -N(R’)S(O)kN(R’)-, -N(R’)-N=, - C(R’)=N—, -C(R’)=N—N(R’)-, -C(R’)=N—N=, -C(R’)2-N=N-, and -C(R’)2-N(R’)-N(R’)-, where each R’ is independently H, alkyl, or substituted alkyl, R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R1 is H, an amino ting group, resin, at least one amino acid, ptide, or polynucleotide; R2 is OH, an ester protecting group, resin, at least one amino acid, polypeptide, or polynucleotide; R3 and R4 are each independently H, halogen, lower alkyl, or substituted lower alkyl, or R3 and R4 or two R3 groups optionally form a cycloalkyl or a heterocycloalkyl; Z has the structure of: ‘7;\KKAr R5 ; R5 is H, CORg, C1-C6alkyl, or le; R8 is OH R6 is OH or H; Ar is phenyl or pyridine; R7 is C1-C6alkyl or hydrogen; L is a linker selected from the group ting of —alkylene—, —alkylene—C(O)—, —(alkylene—O)n— alkylene—, —(alkylene—O)n—alkylene—C(O)—, —(alkylene—O)n—(CH2)n~NHC(O)—(CH2)nv~ C(Me)2—S—S—(CH2)nu~NHC(O)—(alkylene—O)nmv—alkylene—, —(alkylene—O)n—alkylene—W—, — alkylene—C(O)—W—, —(alkylene—O)n—alkylene—U—alkylene—C(O)—, and —(alkylene—O)n— alkylene—U—alkylene—; W has the structure of: KTJK” U has the ure of: COZH Mimi each n, n', n", n'" and nH" are independently integers greater than or equal to one; or an active metabolite, or a pharmaceutically acceptable prodrug or solvate thereof.

In certain embodiments of compounds of Formula (VIII) and (IX), R5 is thiazole. In certain embodiments of compounds of Formula (VIII) and (IX), R6 is H. In n embodiments of compounds of Formula (VIII) and (IX), Ar is phenyl. In certain ments of compounds of Formula (VIII) and (IX), R7 is methyl. In certain embodiments of compounds of Formula (VIII) and (IX), n is an integer from O to 20. In certain embodiments of compounds of Formula (VIII) and (IX), n is an integer from O to 10.

In certain embodiments of compounds of Formula (VIII) and (IX), n is an integer from O to 5.

In n embodiments of compounds of Formula (VIII) and (IX), R5 is thiazole. In n embodiments of compounds of Formula (VIII) and (IX), R5 is hydrogen. In certain embodiments of WO 66560 compounds of Formula (VIII) and (IX), R5 is methyl, ethyl, propyl, iso-propyl, butyl, iso-butyl, sec-butyl, tert-butyl, pentyl, or hexyl. In certain embodiments of compounds of Formula (VIII) and (IX), R5 is — NH—(alkylene—O)n—NH2, wherein alkylene is —CH2—, —CH2CH2—, —CH2CH2CH2—, —CH2CH2CH2CH2—, — CH2CH2CH2CH2CH2—, —CH2CH2CH2CH2CH2CH2—, —CH2CH2CH2CH2CH2CH2CH2—, — CH2CH2CH2CH2CH2CH2CH2CH2—, 2CHZCHZCHZCHZCHZCHZCH2—, — CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2—, —CH2CHZCH2CHZCHZCHZCHQCHZCHZCHZCH2—, or — CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2—. In certain embodiments of Formula (VIII) and (IX), ne is methylene, ethylene, propylene, butylenes, pentylene, hexylene, or heptylene.

In certain ments of compounds of Formula (VIII) and (IX), R5 is —NH—(alkylene—O)n— NHZ, wherein n is 0,1, 2, 3, 4, 5, 6, 7, 8, 9,10,11,12,13,14,15,16,17,18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100.

In certain embodiments of compounds of Formula (VIII) and (IX), R6 is H.

In certain embodiments of compounds of Formula (VIII) and (IX), Ar is .

In certain embodiments of compounds of a (VIII) and (IX), R7 is methyl, ethyl, propyl, iso-propyl, butyl, sec-butyl iso-butyl, tert-butyl, pentyl, or hexyl. In certain embodiments of compounds of Formula (VIII) and (IX), R7 is hydrogen.

In certain embodiments of compounds of Formula (VIII) and (IX), each L is independently a cleavable linker or eavable linker. In n embodiments of compounds of Formula (VIII) and (IX), each L is independently a oligo(ethylene glycol) derivatized linker.

In certain embodiments of compounds of Formula (VIII) and (IX), each alkylene, alkylene', alkylene", and alkylene'" independently is —CH2—, —CH2CH2—, —CH2CH2CH2—, —CH2CH2CH2CH2—, — CH2CH2CH2CH2CH2—, —CH2CH2CH2CH2CH2CH2—, —CH2CH2CH2CH2CH2CH2CH2—, — CH2CH2CH2CH2CH2CH2CH2CH2—, —CH2CH2CHZCHZCHZCHZCHZCHZCH2—, — CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2—, —CH2CHZCH2CHZCHZCHZCHQCHZCHZCHZCH2—, or — CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2—. In certain embodiments of compounds of Formula (VIII) and (IX), ne is ene, ethylene, propylene, nes, pentylene, hexylene, or heptylene.

In n embodiments of compounds of Formula (VIII) and (IX), each n, n', n", n'", and n"" independently is 0,1, 2, 3, 4, 5, 6, 7, 8, 9,10,11,12,13,14,15,16,17,18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100.

In certain embodiments of compounds of Formula (VIII) or (IX), R1 is a polypeptide. In certain embodiments of compounds of Formula (VIII) or (IX), R2 is a polypeptide. In certain embodiments of compounds of Formula (VIII) or (IX), the polypeptide is an dy. In certain embodiments of compounds of Formula (VIII) or (IX), the antibody is herceptin.

WO 66560 Such non-natural amino acid dolastatin linked derivatives include dolastatin linked derivatives having the structure of Formula (X), (XI), (XII) 0r (XIII): Me Lfl HMJ1: R7 0 Me/_\MeMe OMeOM R1” —N “j“ 0%; B\A R7 0 M/KMeMe OMeO R3 RO4 R3 R2 Me O Me/\MeMe OMeO OMeOA“”85:er R2 31 L1 (XI) Me MNJL: \ R3 Me\ N 3 \ 3 N Y Me o Me/KMeMe OMeO OMeOA R IWNJL_ fiw R7 0 Me/_\MeMe OMeOM N\H ,L1 Z 0 Me R /N Y N (X11) L3—M meHJN Me B\A 0 R7 0 Me/_\MeMe OMeoM Me Me MeO NH WO 66560 M321;£3;qu R7 OMe/\MeMe OMeO OMe OA R2 81 J\N \ R3 C? ’i R3 R7 OMe/\MeMe OMeO OMeOA R R7 OMe/:\MeR12 OMeO OMeOA A is optional, and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower lkylene, lower alkenylene, substituted lower lene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene; B is optional, and when present is a linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, - S-, -S-(alkylene or substituted alkylene)-, - where k is l, 2, or 3, -S(O)k(alkylene or substituted alkylene)-, -C(O)—, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)- (alkylene or substituted alkylene)-, -, -NR’-(alkylene or substituted alkylene)-, -C(O)N(R’)-, -CON(R’)-(alkylene or substituted alkylene)-, -CSN(R’)-, -CSN(R’)-(alkylene or substituted alkylene)-, -N(R’)CO-(alkylene or tuted alkylene)-, -N(R’)C(O)O-, -S(0)kN(R’)-, _N(R’)C(O)N(R’)_a -N(R’)C(S)N(R’)-, 'N(R’)S(O)kN(R’)'a -N(R’)-N=, - C(R’)=N-, -C(R’)=N—N(R’)-, =N-N=, -C(R’)2-N=N-, and 2-N(R’)-N(R’)-, where each R’ is independently H, alkyl, or substituted alkyl, R is H, alkyl, substituted alkyl, cycloalkyl, or substituted lkyl; R1 is H, an amino protecting group, resin, at least one amino acid, polypeptide, or polynucleotide; R2 is OH, an ester protecting group, resin, at least one amino acid, polypeptide, or polynucleotide; R3 and R4 are each independently H, halogen, lower alkyl, or substituted lower alkyl, or R3 and R4 or two R3 groups optionally form a cycloalkyl or a heterocycloalkyl; Z has the structure of: €Yé\(KAr R5 ; R5 is H, COZH, C1-C6alkyl, or thiazole; R6 is OH or H; Ar is phenyl or pyridine; R7 is C1-C6alkyl or en; L1, L2, L3, and L4 are each linkers independently selected from the group consisting of a bond, — alkylene—, —(alkylene—O)n—alkylene—J—, —alkylene'—J—(alkylene—O)n—alkylene—, —J— (alkylene—O)n—alkylene—, —(alkylene—O)n—alkylene—J—(alkylene—O)n'—alkylene—J'—, — (alkylene—O)n—alkylene—J—alkylene'—, —W—, —alkylene—W—, alkylene'—J—(alkylene—NMe)n— alkylene—W—, —J—(alkylene—NMe)n—alkylene—W—, ylene—NMe—alkylene'—NMe— alkylene"—W—, and —alkylene—J—alkylene'—NMe—alkylene"—NMe—alkylene'"—W—; W has the structure of: ILWJUCVJK; o NH2 each I and J' independently have the structure of: aim/I51 \O/ii or J’S\Nj\e§ H H , , ; and each n and n' are independently integers greater than or equal to one.

In certain embodiments of compounds of Formula (X), (XI), (XII) or (XIII), R5 is thiazole or ylic acid. In certain embodiments of compounds of Formula (X), (XI), (XII) or (XIII), R6 is H. In certain embodiments of compounds of a (X), (XI), (XII) or (XIII), Ar is phenyl. In certain embodiments of compounds of Formula (X), (XI), (XII) or (XIII), R7 is methyl. In certain embodiments of compounds of Formula (X), (XI), (XII) or (XIII), n and n' are integers from O to 20. In certain embodiments of compounds of Formula (X), (XI), (XII) or (XIII), n and n' are integers from O to 10. In certain embodiments of compounds of Formula (X), (XI), (XII) or (XIII), n and n' are rs from O to 5.

In certain ments of compounds of Formula (X), (XI), (XII) or (XIII), R5 is thiazole. In certain ments of compounds of Formula (X), (XI), (XII) or (XIII), R5 is hydrogen. In certain embodiments of compounds of Formula (X), (XI), (XII) or (XIII), R5 is methyl, ethyl, propyl, iso-propyl, butyl, iso-butyl, sec-butyl, tert-butyl, pentyl, or hexyl. In certain embodiments of compounds of Formula (X), (XI), (XII) or (XIII), R5 is —NH—(alkylene—O)n—NH2, wherein alkylene is —CH2—, —CH2CH2—, — CH2CH2CH2—, —CH2CH2CH2CH2—, —CH2CH2CH2CH2CH2—, —CH2CH2CH2CH2CH2CH2—, — CH2CH2CH2CH2CH2CH2CH2—, —CH2CH2CH2CH2CH2CH2CH2CH2—, — CH2CH2CH2CH2CH2CH2CH2CH2CH2—, —CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2—, — CH2CHZCH2CH2CHZCHZCHZCHZCHZCHZCH2—, or —CH2CH2CHZCH2CHZCHZCHZCHZCHgCHZCHZCH2—.

In certain embodiments of Formula (X), (XI), (XII) or (XIII), alkylene is methylene, ethylene, propylene, butylenes, pentylene, hexylene, or heptylene.

In certain ments of compounds of Formula (X), (XI), (XII) or , R5 is —NH— ene—O)n—NH2, wherein n is O, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100.

In certain embodiments of compounds of Formula (X), (XI), (XII) or (XIII), R6 is H. In some ments of nds of Formula (X), (XI), (XII) or , R6 is hydroxy.

] In certain embodiments of compounds of Formula (X), (XI), (XII) or (XIII), Ar is phenyl.

In certain embodiments of compounds of a (X), (XI), (XII) or , R7 is methyl, ethyl, propyl, opyl, butyl, sec-butyl iso-butyl, tert-butyl, pentyl, or hexyl. In certain embodiments of compounds of Formula (X), (XI), (XII) or (XIII), R7 is en.

In n embodiments of compounds of Formula (X), (XI), (XII) or (XIII), each L1, L2, L3, and L4 is independently a cleavable linker or non-cleavable linker. In certain embodiments of compounds of Formula (X), (XI), (XII) or (XIII), each L1, L2, L3, and L4 is independently a oligo(ethylene glycol) derivatized linker.

In certain embodiments of compounds of Formula (X), (XI), (XII) or (XIII), each alkylene, alkylene', alkylene", and alkylene'" independently is —CH2—, —CH2CH2—, —CH2CH2CH2—, — CHZCHZCHgCH2—, —CH2CH2CH2CH2CH2—, —CH2CH2CH2CH2CH2CH2—, —CH2CH2CH2CH2CH2CH2CH2— , —CHQCH2CH2CH2CH2CH2CH2CH2—, —CH2CH2CHZCHZCHZCHZCHZCHZCH2—, CH2CH2CHZCHZCHZCHZCHZCHZCHZCH2—, —CH2CH2CH2CHZCHZCHZCHZCHZCHZCHZCH2—, or — CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2—. In n embodiments of compounds of Formula (X), (XI), (XII) or (XIII), alkylene is methylene, ethylene, propylene, butylenes, pentylene, hexylene, or heptylene.

In certain embodiments of compounds of Formula (X), (XI), (XII) or (XIII), each n and n' ndently is 0,1, 2, 3, 4, 5, 6, 7, 8, 9,10,11,12,13,14,15,16,17,18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100.

In certain embodiments of compounds of Formula (X), (XI), (XII) or (XIII), R1 is a polypeptide.

In certain embodiments of compounds of Formula (X), (XI), (XII) or (XIII), R2 is a polypeptide. In certain embodiments of compounds of Formula (X), (XI), (XII) or (XIII), the polypeptide is an antibody.

In certain embodiments of compounds of Formula (X), (XI), (XII) or (XIII), the antibody is herceptin.

In certain embodiments, compounds of Formula (X), (XI), (XII) or (XIII) are stable in aqueous solution for at least 1 month under mildly acidic conditions. In certain embodiments, compounds of Formula (X), (XI), (XII) or (XIII) are stable for at least 2 weeks under mildly acidic conditions. In certain embodiments, compound of Formula (X), (XI), (XII) or (XIII) are stable for at least 5 days under mildly acidic conditions. In certain embodiments, such acidic conditions are pH 2 to 8. Such non-natural amino acids may be in the form of a salt, or may be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.

Oxime-based non-natural amino acids may be synthesized by methods already described in the art, or by s described herein, ing: (a) reaction of a hydroxylamine-containing non-natural amino acid with a carbonyl- or onyl-containing reagent; (b) reaction of a yl- or dicarbonyl- containing non—natural amino acid with a hydroxylamine-containing reagent; or (c) reaction of an oxime- ning non-natural amino acid with n carbonyl- or dicarbonyl-containing reagents.

B. al Structure and Synthesis of Non-Natural Amino Acid Linked Dolastatin Derivatives: tea' Aromatic Amine Linked Dolastatin tives ] In one aspect are dolastatin linker derivatives for the chemical derivatization of non-natural amino acids based upon the reactivity of an aromatic amine group. In further or additional embodiments, at least one of the entioned non-natural amino acids is orated into a dolastatin linker tive, that is, such embodiments are non-natural amino acid linked dolastatin derivatives. In further or additional embodiments, the dolastatin linker derivatives are functionalized on their sidechains such that their reaction with a derivatizing non-natural amino acid generates an amine linkage. In further or additional embodiments, the dolastatin linker tives are selected from atin linker derivatives having aromatic amine sidechains. In further or additional embodiments, the dolastatin linker tives comprise a masked sidechain, including a masked ic amine group. In further or additional embodiments, the non-natural amino acids are selected from amino acids having aromatic amine sidechains. In further or additional embodiments, the non-natural amino acids comprise a masked sidechain, including a masked aromatic amine group.

In another aspect are carbonyl-substituted dolastatin linker derivatives such as, by way of example, aldehydes, and ketones, for the production of tized non-natural amino acid polypeptides based upon an amine linkage. In a further embodiment are aldehyde-substituted dolastatin linker derivatives used to derivatize aromatic amine-containing non-natural amino acid polypeptides via the formation of an amine linkage between the derivatizing dolastatin linker and the aromatic amine- containing non-natural amino acid polypeptide.

In r or additional ments, the non-natural amino acids comprise aromatic amine sidechains where the aromatic amine is ed from an aryl amine or a heteroaryl amine. In a further or additional embodiment, the non-natural amino acids resemble a natural amino acid in structure but contain aromatic amine groups. In another or further embodiment the non-natural amino acids resemble alanine or tyrosine (aromatic amino acids). In one embodiment, the non-natural amino acids have properties that are distinct from those of the natural amino acids. In one embodiment, such ct properties are the chemical reactivity of the sidechain; in a further embodiment this distinct chemical reactiVity permits the sidechain of the non-natural amino acid to o a reaction while being a unit of a polypeptide even though the sidechains of the naturally-occurring amino acid units in the same ptide do not undergo the aforementioned reaction. In a further embodiment, the sidechain of the non-natural amino acid has a chemistry orthogonal to those of the naturally-occurring amino acids. In a r embodiment, the sidechain of the non-natural amino acid comprises a nucleophile-containing moiety; in a further embodiment, the nucleophile-containing moiety on the sidechain of the non-natural amino acid can undergo a reaction to te an amine-linked derivatized dolastatin. In a further embodiment, the sidechain of the non-natural amino acid comprises an electrophile-containing moiety; in a further embodiment, the electrophile-containing moiety on the sidechain of the non-natural amino acid can undergo nucleophilic attack to generate an linked derivatized dolastatin. In any of the aforementioned embodiments in this paragraph, the non-natural amino acid may exist as a separate molecule or may be incorporated into a ptide of any length; if the latter, then the polypeptide may further incorporate lly-occurring or non-natural amino acids.

Modification of non-natural amino acids described herein using reductive alkylation or reductive amination reactions have any or all of the following advantages. First, aromatic amines can be ively alkylated with carbonyl-containing compounds, including aldehydes, and ketones, in a pH range of about 4 to about 10 (and in certain embodiments in a pH range of about 4 to about 7) to generate substituted amine, including secondary and tertiary amine, linkages. Second, under these reaction conditions the chemistry is selective for non-natural amino acids as the sidechains of naturally occurring amino acids are unreactive. This allows for site-specific derivatization of polypeptides which have incorporated non-natural amino acids containing aromatic amine moieties or protected aldehyde es, including, by way of example, recombinant proteins. Such derivatized polypeptides and proteins can thereby be prepared as defined neous products. Third, the mild conditions needed to effect the reaction of an aromatic amine moiety on an amino acid, which has been incorporated into a polypeptide, with an aldehyde-containing reagent generally do not rsibly destroy the ry structure of the polypeptide ting, of , where the purpose of the reaction is to destroy such tertiary structure).

Similarly, the mild ions needed to effect the reaction of an aldehyde moiety on an amino acid, which has been incorporated into a polypeptide and deprotected, with an aromatic amine-containing reagent generally do not irreversibly destroy the tertiary structure of the polypeptide (excepting, of course, where the purpose of the reaction is to destroy such tertiary structure). Fourth, the reaction occurs rapidly at room temperature, which allows the use of many types of polypeptides or reagents that would otherwise be unstable at higher temperatures. Fifth, the reaction occurs readily is aqueous conditions, again allowing use of polypeptides and reagents incompatible (to any extent) with non-aqueous solutions. Six, the reaction occurs y even when the ratio of polypeptide or amino acid to reagent is stoichiometric, iometric-like, or near-stoichiometric, so that it is unnecessary to add excess reagent or polypeptide to obufin a usefifl annnnn of reacfion product n the remflfing anfine can be produced regioselectively and/or regiospecifically, ing upon the design of the amine and carbonyl portions of the reactants. Finally, the reductive alkylation of aromatic amines with aldehyde-containing reagents, and the reductive amination of aldehydes with aromatic amine containing reagents, generates amine, ing secondary and tertiary amine, linkages which are stable under ical conditions.

Non-natural amino acids with nucleophilic reactive groups, such as, by way of example only, an aromatic amine group (including secondary and tertiary amine groups), a masked aromatic amine group (which can be readily converted into a aromatic amine group), or a protected aromatic amine group (which has vity similar to a aromatic amine group upon deprotection) allow for a y of reactions to link molecules via various reactions, including but not limited to, reductive alkylation reactions with aldehyde containing dolastatin linked tives. Such alkylated non-natural amino acid linked atin derivatives include amino acids having the structure of Formula (XXV), (XXVI), , (XXVHI), (XXIX), or (XXX): (R16)n—/TqNJ/L\NNZNILTUM;%Ne%I R7 OMe/\MeMe OMeOM (XXV) 0 R2 Me Me Me\NIN(N\)J\Ne Me H Me NH R6 N N I : I Ar HN R2 (XXVI) R7 o A Me OMeO OM90 Me Me o N—L \_H —M H N \I—// R O4 (R16)n L_NIMNenJ1jprOYkN R7 OMe/\MeMe OMeO OMeO \ NVL1 I Me (R16)n|—\/, H (xxvrr) Ri ‘R 1 Ls—w MHJDLNN 0 R2 R7 OMe/\MeMe OMeO OMeO M3;viKNOWH R7 OMe/\MeMe OMeO OMeOA R6 ,R1 H R2 H _ (XXVIH) Me M eH Me \_ 0 Me NAG R40 Me \JL'?‘ H H Me \ N N N N N—L3 (R16)n 1 H R7 0Me/\MeMe OMeO 0M6 OAr gift?KWR7 OMe/\MeMe OMeOM N\H \ N\/L1 Z (R16) —|“ K7 H NtR (XXIX) R4 1L3—N Me 0 R2 R7 OMe/\MeMe OMe OM 0 Z R7 OMe/\MeMe OMeOM NH 0 Z R7 OMe/\MeMe OMeO OMeOA“MI—Jim R , 1 HN R2 H —M Nikki—L3o LBJ/Q R40 (R16)n (XXX) R7 OMe/\MeMe OMeO OMeOAAr 6 Mka—L‘;O H R7 OMe/\MeMe OMeO r R5 wherein: Z has the structure of: R5 is H, COZH, C1-C6alkyl, or thiazole; R6 is OH or H; Ar is phenyl or pyridine; R1 is H, an amino protecting group, resin, at least one amino acid, polypeptide, or polynucleotide; R2 is OH, an ester protecting group, resin, at least one amino acid, ptide, or polynucleotide; R4 is H, halogen, lower alkyl, or tuted lower alkyl; R7 is C1-C6alkyl or hydrogen; L, L1, L2, L3, and L4 are each linkers selected from the group consisting of a bond, —alkylene—, — alkylene—C(O)—, lene—O)n—alkylene—, lene—O)n—alkylene—C(O)—, —(alkylene— O)n—(CH2)n~NHC(O) (CH2)nu C(Me)2 S S (CH2)nm NHC(O)—(alkylene—O)nmv—alkylene—, — (alkylene—O)n—alkylene—W—, —alkylene—C(O)—W—, lene—O)n—alkylene—J—, —alkylene'— J—(alkylene—O)n—alkylene—, —(alkylene—O)n—alkylene—J—alkylene', —J—(alkylene—O)n— alkylene—, —(alkylene—O)n—alkylene—J—(alkylene—O)n'—alkylene—J'—, —W—, —alkylene—W—, alkylene'—J— (alkylene—NMe)n—alkylene—W—, and J— (alkylene—NMe)n—alkylene—W—, — (alkylene—O)n—alkylene—U—alkylene—C(O)—, —(alkylene—O)n—alkylene—U—alkylene—; —J— alkylene—NMe—alkylene'—NMe—alkylene"—W—, and —alkylene—J—alkylene'—NMe—alkylene"— NMe—alkylene'"—W—; W has the structure of: Lwflfifk“; U has the structure of: COZH mink each J and J' independently have the structure of: zit/iiN/‘Zi fiNiO/‘Zi or Jimi‘g each n and n' are independently integers greater than or equal to one; and each R16 is independently selected from the group consisting of hydrogen, halogen, alkyl, N02, CN, and substituted alkyl.

Such alkylated non-natural amino acid linked dolastatin derivatives may also be in the form of a salt, or may be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally reductively alkylated.

In certain embodiments of compounds of Formula (XXV), (XXVI), (XXVII), (XXVIII), (XXIX), or (XXX), R5 is thiazole or ylic acid. In certain ments of compounds of Formula (XXV), (XXVI), (XXVII), (XXVIII), (XXIX), or (XXX), R6 is H. In n embodiments of compounds of Formula (XXV), (XXVI), (XXVII), (XXVIII), (XXIX), or (XXX), Ar is phenyl. In certain embodiments of compounds of a (XXV), (XXVI), (XXVII), (XXVIII), (XXIX), or (XXX), R7 is methyl. In certain embodiments of compounds of Formula (XXV), (XXVI), (XXVII), (XXVIII), (XXIX), or (XXX), n is an r from O to 20. In certain embodiments of compounds of a (XXV), (XXVI), (XXVII), (XXVIII), (XXIX), or (XXX), n is an r from O to 10. In certain embodiments of compounds of Formula (XXV), (XXVI), (XXVII), (XXVIII), (XXIX), or r (XXIV), n is an integer from O to 5.

In n embodiments of nds of Formula (XXV), (XXVI), (XXVII), (XXVIII), , or (XXX), R5 is thiazole or carboxylic acid. In certain embodiments of compounds of Formula (XXV), (XXVI), (XXVII), (XXVIII), (XXIX), or (XXX), R5 is hydrogen. In certain embodiments of compounds of Formula (XXV), (XXVI), ), I), (XXIX), or (XXX), R5 is methyl, ethyl, propyl, iso- propyl, butyl, iso-butyl, sec-butyl, tert-butyl, pentyl, or hexyl. In certain embodiments of compounds of Formula ((XXV), (XXVI), (XXVII), (XXVIII), (XXIX), or (XXX), R5 is —NH—(alkylene—O)n—NH2, Wherein alkylene is —CH2—, —CH2CH2—, —CH2CH2CH2—, —CH2CH2CH2CH2—, —CH2CH2CH2CH2CH2—, — CH2CH2CH2CH2CH2CH2—, —CH2CH2CH2CH2CH2CH2CH2—, —CH2CHZCH2CH2CH2CH2CH2CH2—, — CH2CH2CH2CH2CH2CH2CH2—, —CH2CH2CHZCHgCHZCHQCHZCHQCHZCH2—, — CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2—, or —CH2CHZCHZCHZCHZCHZCHZCHZCHZCHZCHQCH2—.

In certain embodiments of Formula (XXV), (XXVI), (XXVII), (XXVIII), (XXIX), or (XXX), alkylene is methylene, ethylene, propylene, butylenes, pentylene, hexylene, or heptylene.

In certain embodiments of nds of Formula (XXV), (XXVI), (XXVII), (XXVIII), (XXIX), or (XXX), R5 is —NH—(alkylene—O)n—NH2, Wherein n is O, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100.

In certain embodiments of compounds of Formula (XXV), (XXVI), (XXVII), (XXVIII), , or (XXX), R6 is H. In some embodiments of compounds of Formula (XXV), (XXVI), (XXVII), (XXVIII), (XXIX), or (XXX), R6 is hydroxy.

In certain embodiments of compounds of Formula (XXV), , (XXVII), (XXVIII), (XXIX), or (XXX), Ar is phenyl.

In certain ments of compounds of Formula (XXV), (XXVI), (XXVII), (XXVIII), (XXIX), or (XXX), R7 is methyl, ethyl, propyl, iso-propyl, butyl, sec-butyl iso-butyl, tert-butyl, pentyl, or hexyl.

In certain ments of compounds of Formula (XXV), (XXVI), ), (XXVIII), , or (XXX), R7 is hydrogen.

In certain embodiments of compounds of Formula (XXV), (XXVI), (XXVII), (XXVIII), (XXIX), or (XXX), each L, L1, L2, L3, and L4 is independently a cleavable linker or non-cleavable linker. In certain embodiments of compounds of a (XXV), (XXVI), (XXVII), (XXVIII), (XXIX), or (XXX), each L, L1, L2, L3, and L4 is independently a oligo(ethylene glycol) derivatized linker.

In certain embodiments of compounds of Formula (XXV), (XXVI), (XXVII), (XXVIII), (XXIX), or (XXX), each ne, alkylene', ne", and alkylene'" independently is —CH2—, H2—, -— CH2CH2CH2—, —CH2CH2CH2CH2—, —CH2CH2CH2CH2CH2—, —CH2CH2CH2CH2CH2CH2—, — CH2CH2CH2CH2CH2CH2CH2—, —CH2CH2CH2CH2CH2CH2CH2CH2—, — CH2CH2CH2CH2CH2CH2CH2CH2CH2—, —CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2—, — CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2—, or —CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2—.

In certain embodiments of compounds of Formula (XIX), (XX), (XXI), (XXII), (XXIII) or , alkylene is methylene, ethylene, ene, butylenes, pentylene, hexylene, or heptylene.

In certain embodiments of compounds of Formula (XXV), (XXVI), (XXVII), (XXVIII), (XXIX), or (XXX), each n, n', n", n'", and n"" independently is O, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100.

In certain embodiments of compounds of Formula (XXV), (XXVI), (XXVII), (XXVIII), (XXIX), or (XXX), R1 is a ptide. In certain embodiments of compounds of a (XXV), (XXVI), (XXVII), (XXVIII), (XXIX), or (XXX), R2 is a polypeptide. In certain embodiments of compounds of Formula (XXV), (XXVI), (XXVII), (XXVIII), (XXIX), or (XXX), the polypeptide is an dy. In certain embodiments of compounds of Formula (XXV), (XXVI), (XXVII), (XXVIII), (XXIX), or (XXX), the antibody is herceptin.

Compounds of Formula (XXV), (XXVI), ), (XXVIII), (XXIX), or (XXX) may be formed by the reductive alkylation of aromatic amine nds With carbonyl containing reagents such as, by way of example, ketones, esters, thioesters, and aldehydes.

In some embodiments, the masked amine moieties of non-natural amino acids contained in polypeptides are lly reduced to give non-natural amino acids containing aromatic amine es incorporated into non-natural amino acid polypeptides. Such aromatic amine moieties are then reductive alkylated With carbonyl-containing reagents described above to give polypeptides containing non-natural amino acids of Formula (XXV), (XXVI), (XXVII), (XXVIII), (XXIX), or (XXX). Such ons may also be applied to non-natural amino acids incorporated into synthetic polymers, polysaccharides, or polynucleotides. Additionally, such reactions may be applied to corporated tural amino acids. By way of example the reducing agent used to reduce masked amine moieties includes, but is not limited to, TCEP, NaZS, NaZSZO4, LiAlH4, B2H6, and NaBH4. By way of example only, reductive tion may occur in aqueous buffers with a pH of about 4 to about 7 and using a mild reducing agent, such as, by way of example only, sodium cyanoborohydride (NaBCNHg). In addition, other reducing agents may be used for reductive alkylation including, but not limited to, TCEP, NaZS, NaZSZO4, , B2H6, and NaBH4.

A non-limiting exemplary syntheses of non-natural amino acid polypeptides containing amino acids of Formula (XXV), (XXVI), (XXVII), (XXVIII), , or (XXX) by reductive alkylation of secondary aromatic amine moieties, contained in non-natural amino acids, with carbonyl-containing reagents described above. Such reductive alkylations give polypeptides containing non-natural amino acids with tertiary aromatic amine es. Such reactions may also be applied to non-natural amino acids orated into tic rs, polysaccharides, or polynucleotides. Additionally, such reactions may be applied to non-incorporated non-natural amino acids. By way of example only, reductive alkylation may occur in aqueous buffers with a pH of about 4 to about 7 and using a mild reducing agent, such as, by way of example only, sodium cyanoborohydride (NaBCNHg). In addition, other reducing agents may be used for reductive alkylation including, but not limited to, TCEP, NaZS, 4, LiAlH4, B2H6, and NaBH4.

C. al Synthesis of Non-Natural Amino Acid Linked atin Derivatives: Heteroaryl-Containing Linked Dolastatin Derivatives In one aspect are non-natural amino acids for the chemical derivatization of dolastatin linked derivatives based upon the reactivity of a dicarbonyl group, including a group containing at least one ketone group, and/or at least one aldehyde groups, and/or at least one ester group, and/or at least one carboxylic acid, and/or at least one thioester group, and wherein the dicarbonyl group can be a 1,2- dicarbonyl group, a 1,3-dicarbonyl group, or a l,4-dicarbonyl group. In further or additional aspects are non-natural amino acids for the chemical derivatization of dolastatin linked derivatives based upon the vity of a diamine group, including a hydrazine group, an amidine group, an imine group, a 1,1- diamine group, a 1,2-diamine group, a amine group, and a l,4-diamine group. In further or additional embodiments, at least one of the aforementioned non-natural amino acids is incorporated into a dolastatin linked derivative, that is, such embodiments are non-natural amino acid linked dolastatin tives. In further or additional ments, the non-natural amino acids are functionalized on their sidechains such that their reaction with a derivatizing molecule tes a linkage, ing a cyclic-based linkage, including a nitrogen-containing heterocycle, and/or an aldol-based linkage. In further or additional embodiments are non-natural amino acid polypeptides that can react with a derivatizing dolastatin linker to generate a non-natural amino acid linked dolastatin derivatives containing a linkage, including a heterocyclic-based linkage, including a nitrogen-containing heterocycle, and/or an aldol-based linkage. In further or additional embodiments, the non-natural amino acids are selected from amino acids having dicarbonyl and/or diamine sidechains. In r or additional embodiments, the non- natural amino acids comprise a masked sidechain, including a masked diamine group and/or a masked dicarbonyl group. In further or additional embodiments, the non-natural amino acids comprise a group selected from: keto-amine (i.e., a group containing both a ketone and an amine); keto-alkyne (i.e., a group ning both a ketone and an alkyne); and an one (i.e., a group containing a dicarbonyl group and an alkene).

In r or additional ments, the non-natural amino acids comprise dicarbonyl sidechains Where the carbonyl is selected from a ketone, an de, a carboxylic acid, or an ester, including a thioester. In another embodiment are non-natural amino acids containing a onal group that is capable of forming a heterocycle, ing a en-containing cycle, upon treatment With an appropriately functionalized reagent. In a further or additional embodiment, the non-natural amino acids resemble a natural amino acid in structure but contain one of the aforementioned functional groups.

In another or further embodiment the non-natural amino acids resemble phenylalanine or tyrosine (aromatic amino acids); While in a separate embodiment, the tural amino acids resemble alanine and leucine (hydrophobic amino acids). In one embodiment, the non-natural amino acids have properties that are distinct from those of the natural amino acids. In one embodiment, such distinct properties are the chemical reactivity of the sidechain, in a further ment this distinct al reactivity permits the sidechain of the non-natural amino acid to undergo a reaction While being a unit of a polypeptide even though the sidechains of the naturally-occurring amino acid units in the same polypeptide do not undergo the aforementioned reaction. In a further embodiment, the sidechain of the non-natural amino acid has a try orthogonal to those of the naturally-occurring amino acids. In a further embodiment, the sidechain of the non-natural amino acid comprises an electrophile-containing moiety; in a further ment, the electrophile-containing moiety on the sidechain of the non-natural amino acid can undergo nucleophilic attack to generate a heterocycle-derivatized protein, including a nitrogen-containing cycle-derivatized protein. In any of the aforementioned embodiments in this paragraph, the non- natural amino acid may exist as a separate molecule or may be incorporated into a polypeptide of any length; if the latter, then the polypeptide may further incorporate naturally-occurring or non-natural amino acids.

In another aspect are e-substituted molecules, wherein the diamine group is selected from a hydrazine, an amidine, an imine, a l,l-diamine, a l,2-diamine, a 1,3-diamine and a l,4-diamine group, for the production of tized non-natural amino acid linked dolastatin derivatives based upon a heterocycle, including a nitrogen-containing heterocycle, e. In a further embodiment are diamine- substituted atin derivatives used to derivatize dicarbonyl-containing tural amino acid polypeptides Via the formation of a heterocycle, including a nitrogen-containing heterocycle, linkage between the derivatizing molecule and the dicarbonyl-containing non-natural amino acid polypeptide. In further embodiments the aforementioned dicarbonyl-containing non-natural amino acid polypeptides are diketone-containing non-natural amino acid polypeptides. In further or additional embodiments, the dicarbonyl-containing non-natural amino acids comprise sidechains where the yl is selected from a ketone, an aldehyde, a carboxylic acid, or an ester, including a thioester. In further or additional embodiments, the diamine-substituted molecules comprise a group selected from a desired functionality.

In a further embodiment, the sidechain of the non-natural amino acid has a try orthogonal to those of the lly-occurring amino acids that allows the non-natural amino acid to react selectively with the diamine-substituted molecules. In a r embodiment, the sidechain of the non-natural amino acid comprises an ophile-containing moiety that reacts selectively with the diamine-containing molecule; in a further embodiment, the ophile-containing moiety on the sidechain of the non-natural amino acid can undergo nucleophilic attack to generate a cycle-derivatized protein, including a nitrogen- containing heterocycle-derivatized protein. In a further aspect related to the embodiments described in this paragraph are the modified non-natural amino acid polypeptides that result from the reaction of the derivatizing molecule with the non-natural amino acid ptides. Further embodiments include any further modifications of the already modified non-natural amino acid polypeptides.

In another aspect are dicarbonyl-substituted molecules for the production of derivatized non- natural amino acid polypeptides based upon a heterocycle, including a nitrogen-containing heterocycle, linkage. In a further embodiment are dicarbonyl-substituted molecules used to derivatize diamine- containing tural amino acid polypeptides via the formation of a cycle, including a nitrogen- containing heterocycle group. In a further embodiment are dicarbonyl-substituted molecules that can form such heterocycle, including a nitrogen-containing heterocycle groups with a diamine-containing non- natural amino acid polypeptide in a pH range between about 4 and about 8. In a r embodiment are dicarbonyl-substituted molecules used to tize diamine-containing non-natural amino acid polypeptides via the formation of a heterocycle, including a nitrogen-containing heterocycle, linkage between the derivatizing molecule and the diamine-containing non-natural amino acid polypeptides. In a further ment the dicarbonyl-substituted molecules are diketone-substitued molecules, in other aspects dehyde-substituted molecules, in other aspects id—substituted molecules, in other aspects ketoester-substituted molecules, including ketothioester-substituted les. In further embodiments, the dicarbonyl-substituted molecules comprise a group selected from a desired functionality. In further or additional embodiments, the aldehyde-substituted molecules are aldehyde- substituted polyethylene glycol (PEG) les. In a r embodiment, the sidechain of the non- natural amino acid has a chemistry onal to those of the naturally-occurring amino acids that allows the non-natural amino acid to react selectively with the carbonyl-substituted molecules. In a further embodiment, the sidechain of the non-natural amino acid comprises a moiety (e. g., diamine group) that reacts selectively with the dicarbonyl-containing molecule; in a further embodiment, the nucleophilic moiety on the sidechain of the non-natural amino acid can undergo electrophilic attack to generate a heterocyclic-derivatized protein, including a nitrogen-containing heterocycle-derivatized protein. In a further aspect d to the embodiments described in this paragraph are the modified non-natural amino acid polypeptides that result from the reaction of the derivatizing molecule with the non-natural amino acid polypeptides. Further embodiments include any further modifications of the already modified non- natural amino acid polypeptides.

In one aspect are methods to derivatize proteins Via the reaction of carbonyl and hydrazine reactants to generate a heterocycle-derivatized protein, including a nitrogen-containing heterocycle-derivatized dolastatin. ed Within this aspect are methods for the derivatization of dolastatin linker derivatives based upon the condensation of carbonyl- and hydrazine-containing reactants to generate a heterocycle-derivatized dolastatin, ing a nitrogen-containing heterocycle-derivatized dolastatin. In additional or r embodiments are methods to derivatize ketone-containing dolastatin derivatives or aldehyde-containing dolastatin derivatives With hydrazine-fiJnctionalized non-natural amino acids. In yet additional or further aspects, the hydrazine-substituted molecule can include ns, other rs, and small molecules.

In another aspect are methods for the chemical synthesis of hydrazine-substituted molecules for the derivatization of carbonyl-substituted dolastatin derivatives. In one embodiment, the hydrazine-substituted molecule is a atin linked derivative suitable for the tization of carbonyl- containing non-natural amino acid polypeptides, including by way of example only, ketone-, or aldehyde- ning non-natural amino acid polypeptides.

In one aspect are non-natural amino acids for the chemical derivatization of dolastatin analogs based upon a quinoxaline or phenazine linkage. In further or additional embodiments, the non-natural amino acids are onalized on their sidechains such that their on With a derivatizing dolastatin linker generates a quinoxaline or phenazine linkage. In further or additional embodiments, the non-natural amino acids are selected from amino acids haVing l,2-dicarbonyl or l,2-aryldiamine sidechains. In further or additional embodiments, the non-natural amino acids are selected from amino acids haVing protected or masked l,2-dicarbonyl or yldiamine sidechains. Further included are equivalents to l,2-dicarbonyl sidechains, or protected or masked equivalents to l,2-dicarbonyl sidechains.

In r aspect are derivatizing molecules for the production of tized non-natural amino acid polypeptides based upon quinoxaline or ine linkages. In one embodiment are l,2- dicarbonyl substituted dolastatin linker tives used to derivatize yldiamine containing non- natural amino acid polypeptides to form quinoxaline or phenazine es. In another embodiment are yldiamine substituted dolastatin linker derivatives used to derivatize l,2-dicarbonyl containing non- natural amino acid polypeptides to form quinoxaline or phenazine linkages. In a further aspect related to the above embodiments are the modified non-natural amino acid polypeptides that result from the reaction of the derivatizing dolastatin linker With the non-natural amino acid polypeptides. In one ment are l,2-aryldiamine containing non-natural amino acid polypeptides derivatized With l,2-dicarbonyl substituted dolastatin linker derivative to form quinoxaline or ine linkages. In another embodiment are l,2-dicarbonyl containing non-natural amino acid polypeptides derivatized With l,2-aryldiamine substituted dolastatin linker derivatives to form aline or phenazine linkages.

Provided herein in certain embodiments are tizing molecules for the production of toxic compounds comprising non-natural amino acid polypeptides based upon triazole linkages. In some embodiments, the reaction n the first and second reactive groups can proceed via a dipolarophile reaction. In certain embodiments, the first reactive group can be an azide and the second reactive group can be an alkyne. In further or alternative embodiments, the first reactive group can be an alkyne and the second reactive group can be an azide. In some embodiments, the Huisgen cycloaddition reaction (see, e.g., Huisgen, in l,3-DIPOLAR CYCLOADDITION CHEMISTRY, (ed. Padwa, A., 1984), p. 1-176) provides for the incorporation of turally encoded amino acids bearing azide and alkyne-containing side chains permits the resultant polypeptides to be modified with extremely high selectivity. In n embodiments, both the azide and the alkyne functional groups are inert toward the twenty common amino acids found in naturally-occurring polypeptides. When brought into close proximity, however, the ”spring-loaded” nature of the azide and alkyne groups is revealed and they react selectively and efficiently Via Huisgen [3 2] ddition reaction to generate the corresponding triazole. See, e.g., Chin et al., Science 301:964-7 (2003); Wang et al., J. Am. Chem. Soc., 125, 193 (2003); Chin et al., J. Am.

Chem. Soc., 26-9027 (2002). Cycloaddition reaction involving azide or alkyne-containing polypeptides can be carried out at room temperature under aqueous conditions by the addition of Cu(II) (e. g., in the form of a tic amount of CuSO4) in the presence of a ng agent for reducing Cu(II) to Cu(I), in situ, in tic amount. See, e.g., Wang et al., J. Am. Chem. Soc. 125, 193 (2003); Tornoe et al., J. Org. Chem. 67:3057-3064 (2002); RostovtseV, Angew. Chem. Int. Ed. 41:2596-2599 (2002). Preferred reducing agents include ascorbate, metallic copper, quinine, hydroquinone, Vitamin K, glutathione, cysteine, Fez, C02, and an applied electric potential.

Such tural amino acid heteroaryl-linked dolastatin derivatives include amino acids haVing the structure of Formula (XXXI), (XXXII), (XXXIII), (XXXIV), (XXXV), or (XXXVI): RNf,B\/L\:gf\)J\RRH 33 R7 O /\ Me OMeO R2 MeO NH (XXXI) HNIKAr (XXXII) OMe O L2—'\Il N\Z R7 OMe/\MeMe OMeO OMeO H R3R3 N /B\ R/ /L1 1 fr:A D (XXXIII) R2 0 H L3—'\Il \Z R7 OMe/\MeMe OMeO OMeO New NMHJ1lfimN“ANIFEWLZ R7 OMe/\MeMe OMeO OMeOA R3 R3N L1\ DA/B\ Me AR4 NR1 (XXXIII) R7 OMe/\MeMe OMeO OMeOA Lz—Mngn-vfl% R7 OMe/\MeMe OMeO—I\N3O§MER2 H R3 R3 NN; R1/ EA/ELD/M Me R2 0 L—Nlmn-fl“:pr0 (XXXV) R7 eMe OMeOM bfiw“TEEN:Me Ne R7 OMe/\MeMe OMeOM NH “AeINIJN .\ NfiNH R7 OMe/\MeMe OMeO OMeO r R5 R3 R3 H L1\ /B\ N\ Me 0 R4 N 0 R2 u—Ls OM6 0 Ar R5 (XXXVI) j Me O .~ H R7 OMe/\MeMe OMe 0 OMe OAr R6 - wherein: Z has the structure of: ‘7;\KKAr R5 ; R5 is H, COZH, C1-C6alkyl, or thiazole; R6 is OH or H; Ar is phenyl or pyridine; R1 is H, an amino protecting group, resin, at least one amino acid, polypeptide, or polynucleotide; R2 is OH, an ester protecting group, resin, at least one amino acid, polypeptide, or polynucleotide; R4 is H, halogen, lower alkyl, or substituted lower alkyl; R7 is C1-C6alkyl or en; L, L1, L2, L3, and L4 are each linkers selected from the group ting of a bond, —alkylene—, — alkylene—C(O)—, —alkylene—J—, —(alkylene—O)n—alkylene—, —(alkylene—O)n—alkylene—C(O)—, — (alkylene—O)n—J—, —(alkylene—O)n—J—alkylene—, —(alkylene—O)n—(CH2)n~NHC(O)—(CH2)nu— C(Me)2—S—S—(CH2)nu~NHC(O)—(alkylene—O)nmv—alkylene—, —(alkylene—O)n—alkylene—W—, — alkylene—C(O)—W—, lene—O)n—alkylene—J—, —alkylene'—J—(alkylene—O)n—alkylene—, — (alkylene—O)n—alkylene—J—alkylene', —J—(alkylene—O)n—alkylene—, —(alkylene—O)n—alkylene— J—(alkylene—O)n'—alkylene—J'—, —W—, ene—W—, alkylene'—J—(alkylene—NMe)n—alkylene— W—, —J—(alkylene—NMe)n—alkylene—W—, —(alkylene—O)n—alkylene—U—alkylene—C(O)—, — (alkylene—O)n—alkylene—U—alkylene—; —J—alkylene—NMe—alkylene'—NMe—alkylene"—W—, and —alkylene—J—alkylene'—NMe—alkylene"—NMe—alkylene'"—W—; W has the structure of: LWJLKTJK“; U has the structure of: COZH ,nmiwr each J and J' independently have the ure of: zit/iiN/‘Zi fiNiO/‘Zi or H {fimi‘i' a a each n and n' are independently integers greater than or equal to one; D has the structure of: each R17 is independently selected from the group consisting of H, alkyl, substituted alkyl, l, tuted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide, substituted polyalkylene oxide, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkaryl, substituted alkaryl, aralkyl, tuted aralkyl, -(alkylene or substituted alkylene)-ON(R”)2, -(alkylene or substituted alkylene)-C(O)SR”, -(alkylene or substituted alkylene)-S-S-(aryl or substituted aryl), -C(O)R”, -C(O)2R”, or -C(O)N(R”)2, n each R” is independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, , substituted alkoxy, aryl, substituted aryl, aryl, l, substituted alkaryl, aralkyl, or substituted each 21 is a bond, CR17R17, O, S, NR’, CR17R17-CR17R17, CR17R17-O, O-CR17R17, CR17R17-S, S-CR17R17, CR17R17-NR’, or NR’-CR17R17; each R’ is H, alkyl, or substituted alkyl; each 22 is selected from the group consisting of a bond, -C(O)-, -C(S)-, optionally substituted C1-C3 alkylene, optionally substituted C1-C3 alkenylene, and optionally tuted heteroalkyl; each 23 are independently selected from the group ting of a bond, optionally substituted C1-C4 alkylene, optionally substituted C1-C4 alkenylene, optionally substituted heteroalkyl, -O-, -S-, -C(O)-, -C(S)-, and -N(R’)-; each T3 is a bond, C(R”)(R”), O, or S; With the o that When T3 is O or S, R” cannot be halogen; each R” is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted lkyl; m and p are 0, l, 2, or 3, provided that at least one ofm or p is not 0; (b) (b) | ‘A'I'V‘ /R3 if; nlnr /C\— E03) /C—C\(b—E) (577R4 /C=C\—E(b) /C—O—Emlnn (b) /C\—5—§ (b) M2 is (a) R3 R4 (a5; R4 M3 (201K: (b) (b) (b) (b) M J‘ri R fi R 3 3 m R3 I / / R3/i—d—é /C=C—E (b) O—T—E (b) s—T—E (b) (b) \R4 >44“ R4 mIJu‘ w m (a) (a) (a) or (a) Where (a) indicates a , , , bonding to the B group and (b) indicates bonding to respective positions Within the heterocycle group; (b) (b) (b) (b) “if” /R3 mi: 3(1)) —c/R3—§(b) b)§ c—s—E (b) (f—ng (b) / / R M3 is (3)3? (a)$??/||— CR\R4 (352% (3)3/ 4 Where (a) indicates bonding to the B group and (b) indicates bonding to respective positions Within the heterocycle group; (b) (b) ”$3“ (b) (b) “I: “l” R—c'2—c=§ I— —g '— :31) /C\_q=g (b) 3 (b) 0 1|?4 >5: C}; (b) S /C—§(b) CE‘Nm) R3 M41s (3)19 (a) R3 (a) (a) or (a) , , a , 7 Where (a) indicates bonding to the B group and (b) indicates bonding to respective positions Within the heterocycle group; each R19 is independently selected from the group ting of C1-C6 alkyl, C1-C6 , ester, ether, thioether, lkyl, halogen, alkyl ester, aryl ester, amide, aryl amide, alkyl halide, alkyl amine, alkyl sulfonic acid, alkyl nitro, thioester, sulfonyl ester, halosulfonyl, nitrile, alkyl nitrile, and nitro; qis 0,1, 2, 3, 4, 5, 6, 7, 8, 9,10 or 11; and each R16 is independently selected from the group consisting of hydrogen, halogen, alkyl, N02, CN, and substituted alkyl.

In some embodiments, the compound of Formula (XXXI) include compounds having the structure of a (XXXI-A): R7 OMe/\M eMe OMeOM (XXXI—A) R3 3R40 “'1“ R1 R2 In n embodiments of compounds of Formula (XXXI), (XXXII), (XXXIII), ), , or (XXXVI), R5 is thiazole or carboxylic acid. In certain embodiments of compounds of Formula (XXXI), (XXXII), (XXXIII), (XXXIV), (XXXV), or (XXXVI), R6 is H. In certain embodiments of nds of Formula (XXXI), (XXXII), (XXXIII), (XXXIV), (XXXV), or (XXXVI), Ar is phenyl.

In certain embodiments of compounds of a (XXXI), (XXXII), (XXXIII), (XXXIV), (XXXV), or (XXXVI), R7 is methyl. In certain embodiments of compounds of Formula (XXXI), (XXXII), (XXXIII), (XXXIV), (XXXV), or (XXXVI), n is an integer from O to 20. In certain embodiments of compounds of Formula (XXXI), (XXXII), (XXXIII), (XXXIV), (XXXV), or (XXXVI), n is an integer from O to 10. In certain embodiments of compounds of Formula (XXXI), (XXXII), (XXXIII), (XXXIV), (XXXV), or (XXXVI), n is an integer from O to 5.

In certain embodiments of nds of Formula (XXXI), (XXXII), (XXXIII), (XXXIV), (XXXV), or (XXXVI), R5 is thiazole or carboxylic acid. In certain embodiments of compounds of Formula (XXXI), (XXXII), (XXXIII), (XXXIV), (XXXV), or (XXXVI), R5 is hydrogen. In certain embodiments of compounds of Formula (XXXI), (XXXII), (XXXIII), (XXXIV), (XXXV), or (XXXVI), R5 is methyl, ethyl, propyl, iso-propyl, butyl, iso-butyl, sec-butyl, utyl, pentyl, or hexyl. In certain ments of compounds of a (XXXI), (XXXII), (XXXIII), (XXXIV), (XXXV), or (XXXVI), R5 is —NH—(alkylene—O)n—NH2, wherein alkylene is —CH2—, —CH2CH2—, —CH2CH2CH2—, — CHZCHZCHZCH2—, —CH2CH2CH2CH2CH2—, 2CH2CH2CH2CH2—, —CH2CH2CH2CH2CH2CH2CH2— , ZCHZCHZCHZCHZCHZCH2—, —CHZCH2CHZCHZCHZCHZCHZCHZCH2—, CHZCH2CH2CHgCHgCHgCHZCHZCHZCH2—, —CH2CH2CH2CH2CHZCHZCHZCHZCHZCHZCH2—, or — CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2—. In certain embodiments of a (XXXI), (XXXII), (XXXIII), (XXXIV), (XXXV), or (XXXVI), alkylene is methylene, ethylene, propylene, butylenes, pentylene, hexylene, or heptylene.

In certain embodiments of compounds of Formula (XXXI), (XXXII), (XXXIII), (XXXIV), (XXXV), or (XXXVI), R5 is —NH—(alkylene—O)n—NH2, n n is O, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15,16,17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100.

In certain embodiments of compounds of Formula (XXXI), (XXXII), (XXXIII), (XXXIV), (XXXV), or ), R6 is H. In some embodiments of compounds of a (XXXI), (XXXII), (XXXIII), (XXXIV), (XXXV), or (XXXVI), R6 is hydroxy.

In certain embodiments of compounds of Formula (XXXI), (XXXII), (XXXIII), (XXXIV), (XXXV), or (XXXVI), Ar is phenyl.

] In certain embodiments of compounds of Formula (XXXI), (XXXII), (XXXIII), ), (XXXV), or (XXXVI), R7 is methyl, ethyl, propyl, iso-propyl, butyl, sec-butyl iso-butyl, tert-butyl, pentyl, or hexyl. In n embodiments of compounds of Formula (XXXI), (XXXII), (XXXIII), (XXXIV), (XXXV), or (XXXVI), R7 is hydrogen.

In certain embodiments of compounds of Formula , (XXXII), I), ), (XXXV), or (XXXVI), each L, L1, L2, L3, and L4 is independently a cleavable linker or non-cleavable linker. In certain embodiments of compounds of Formula (XXXI), (XXXII), (XXXIII), (XXXIV), (XXXV), or (XXXVI), each L, L1, L2, L3, and L4 is independently a oligo(ethylene glycol) derivatized linker.

] In certain embodiments of compounds of Formula (XXXI), (XXXII), (XXXIII), (XXXIV), (XXXV), or (XXXVI), each alkylene, alkylene', alkylene", and alkylene'" independently is —CH2—, -— CH2CH2—, H2CH2—, —CH2CH2CH2CH2—, —CH2CH2CH2CH2CH2—, —CH2CH2CH2CH2CH2CH2—, — CH2CH2CH2CH2CH2CH2CH2—, —CH2CH2CH2CH2CH2CH2CH2CH2—, — CH2CH2CH2CH2CH2CH2CH2CH2CH2—, 2CH2CH2CH2CH2CH2CH2CH2CH2—, — CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2—, or —CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2—.

In certain embodiments of compounds of Formula (XXXI), (XXXII), (XXXIII), (XXXIV), , or (XXXVI), alkylene is methylene, ethylene, propylene, butylenes, pentylene, ne, or heptylene.

In certain embodiments of nds of a (XXXI), (XXXII), (XXXIII), (XXXIV), (XXXV), or (XXXVI), each n, n', n", n'", and n"" independently is O, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100.

WO 66560 ] In n embodiments of compounds of Formula (XXXI), (XXXII), (XXXIII), (XXXIV), (XXXV), or (XXXVI), R1 is a polypeptide. In n embodiments of compounds of Formula , (XXXII), (XXXIII), (XXXIV), (XXXV), or ), R2 is a polypeptide. In certain embodiments of compounds of Formula (XXXI), (XXXII), (XXXIII), (XXXIV), (XXXV), or (XXXVI), the polypeptide is an antibody. In certain embodiments of compounds of a (XXXI), ), (XXXIII), (XXXIV), (XXXV), or (XXXVI), the antibody is herceptin. nds of Formula (XXXI), (XXXII), (XXXIII), (XXXIV), (XXXV), or (XXXVI) may be formed by the reductive alkylation of aromatic amine compounds with carbonyl containing reagents such as, by way of example, s, esters, thioesters, and des.

The formation of such non-natural amino acid heterocycle-linked dolastatin derivatives haVing the structure of Formula (XXXI), ), (XXXIII), (XXXIV), , or (XXXVI) includes, but is not limited to, (i) reactions of diamine-containing non-natural amino acids with dicarbonyl-containing dolastatin linked derivatives or reactions of diamine-containing non-natural amino acids with ketoalkyne- containing dolastatin linked derivatives, (ii) reactions of dicarbonyl-containing non-natural amino acids with either e-containing dolastatin linked tives or reactions of onyl-containing non- natural amino acids with ketoamine-containing dolastatin linked derivatives, (iii) reactions of ketoalkyne- containing non-natural amino acids with diamine-containing dolastatin linked derivatives, or (iV) reactions of ketoamine-containing non-natural amino acids with dicarbonyl-containing V.

Modification of dolastatin linked derivatives described herein with such reactions have any or all of the following advantages. First, diamines undergo condensation with dicarbonyl-containing compounds in a pH range of about 5 to about 8 (and in further embodiments in a pH range of about 4 to about 10, in other embodiments in a pH range of about 3 to about 8, in other embodiments in a pH range of about 4 to about 9, and in further embodiments a pH range of about 4 to about 9, in other embodiments a pH of about 4, and in yet another embodiment a pH of about 8) to generate cycle, including a nitrogen- containing heterocycle, es. Under these conditions, the sidechains of the naturally occurring amino acids are unreactive. Second, such selective chemistry makes possible the site-specific derivatization of recombinant proteins: derivatized proteins can now be prepared as defined homogeneous products. Third, the mild conditions needed to effect the reaction of the diamines described herein with the dicarbonyl- containing polypeptides described herein generally do not irreversibly destroy the tertiary structure of the polypeptide (excepting, of course, where the e of the reaction is to destroy such tertiary structure).

Fourth, the reaction occurs rapidly at room temperature, which allows the use of many types of polypeptides or reagents that would be unstable at higher temperatures. Fifth, the reaction occurs readily is aqueous ions, again allowing use of polypeptides and ts incompatible (to any extent) with non-aqueous solutions. Six, the reaction occurs readily even when the ratio of polypeptide or amino acid to reagent is stoichiometric, near stoichiometric, or stoichiometric-like, so that it is unnecessary to add excess reagent or polypeptide to obtain a useful amount of reaction product. Seventh, the resulting heterocycle can be produced regioselectively and/or regiospecif1cally, depending upon the design of the diamine and onyl portions of the reactants. Finally, the condensation of diamines with dicarbonyl- containing molecules tes heterocycle, including a nitrogen-containing heterocycle, linkages which are stable under biological conditions.

V1 Location 0fn0n-nataral amino acids in dolastatin linker derivatives The methods and compositions described herein include incorporation of one or more non-natural amino acids into a dolastatin linker derivative. One or more non-natural amino acids may be incorporated at one or more particular positions which do not disrupt activity of the dolastatin linker derivative. This can be achieved by making “conservative” substitutions, including but not limited to, substituting hydrophobic amino acids with non-natural or l hydrophobic amino acids, bulky amino acids with tural or natural bulky amino acids, hydrophilic amino acids with non-natural or natural hilic amino acids) and/or inserting the non-natural amino acid in a location that is not required for activity.

A variety of biochemical and ural approaches can be employed to select the desired sites for substitution with a non-natural amino acid within the dolastatin linker derivative. In some embodiments, the non-natural amino acid is linked at the inus of the dolastatin derivative. In other embodiments, the tural amino acid is linked at the N—terminus of the dolastatin derivative. Any position of the dolastatin linker derivative is suitable for selection to incorporate a non-natural amino acid, and selection may be based on rational design or by random selection for any or no particular desired purpose. Selection of d sites may be based on producing a non-natural amino acid polypeptide (which may be r modified or remain unmodified) having any desired property or activity, including but not limited to a receptor binding modulators, receptor activity modulators, modulators of binding to binder partners, binding r activity modulators, binding partner conformation modulators, dimer or multimer formation, no change to ty or property ed to the native molecule, or manipulating any physical or chemical property of the ptide such as solubility, aggregation, or ity.

Alternatively, the sites identified as critical to biological activity may also be good ates for substitution with a non-natural amino acid, again depending on the desired activity sought for the polypeptide. Another alternative would be to simply make serial substitutions in each position on the polypeptide chain with a non-natural amino acid and observe the effect on the activities of the polypeptide. Any means, technique, or method for selecting a position for substitution with a non-natural amino acid into any polypeptide is suitable for use in the s, techniques and compositions described herein.

The structure and activity of naturally-occurring mutants of a polypeptide that contain deletions can also be examined to determine regions of the protein that are likely to be tolerant of substitution with a non-natural amino acid. Once residues that are likely to be intolerant to substitution with non-natural amino acids have been eliminated, the impact of proposed tutions at each of the remaining positions can be examined using methods including, but not limited to, the three-dimensional structure of the relevant polypeptide, and any associated ligands or binding proteins. X-ray crystallographic and NMR structures of many polypeptides are available in the Protein Data Bank (PDB, WV}? centralized database containing three-dimensional structural data of large molecules of proteins and nucleic acids, one can be used to identify amino acid positions that can be substituted with non-natural amino acids. . In addition, models may be made investigating the secondary and tertiary structure of polypeptides, if three-dimensional structural data is not available. Thus, the identity of amino acid positions that can be substituted with non-natural amino acids can be readily obtained.

Exemplary sites of incorporation of a non-natural amino acid include, but are not limited to, those that are ed from potential or g regions, or regions for binding to binding proteins or ligands may be fully or partially t exposed, have minimal or no hydrogen-bonding interactions with nearby residues, may be minimally exposed to nearby reactive residues, and/or may be in regions that are highly flexible as ted by the three-dimensional crystal structure of a particular polypeptide with its associated receptor, ligand or binding ns.

A wide variety of tural amino acids can be substituted for, or incorporated into, a given position in a polypeptide. By way of example, a particular non-natural amino acid may be selected for incorporation based on an examination of the three dimensional crystal structure of a polypeptide with its associated , receptor and/or binding proteins, a preference for vative substitutions In one ment, the methods described herein include incorporating into the dolastatin linker derivative, where the dolastatin linker derivative comprises a first reactive group; and contacting the dolastatin linker derivative with a molecule (including but not d to a second protein or polypeptide or polypeptide analog; an antibody or antibody fragment; and any combination thereof) that comprises a second reactive group. In certain embodiments, the first reactive group is a hydroxylamine moiety and the second reactive group is a carbonyl or onyl moiety, whereby an oxime linkage is formed. In certain ments, the first reactive group is a carbonyl or onyl moiety and the second reactive group is a hydroxylamine moiety, whereby an oxime linkage is formed. In certain ments, the first reactive group is a carbonyl or dicarbonyl moiety and the second reactive group is an oxime moiety, whereby an oxime exchange reaction occurs. In certain embodiments, the first reactive group is an oxime moiety and the second reactive group is carbonyl or dicarbonyl moiety, whereby an oxime exchange reaction .

In some cases, the dolastatin linker derivative incorporation(s) will be combined with other additions, substitutions, or deletions within the polypeptide to affect other chemical, physical, pharmacologic and/or biological traits. In some cases, the other additions, substitutions or deletions may increase the stability (including but not limited to, resistance to proteolytic degradation) of the polypeptide or se affinity of the polypeptide for its appropriate receptor, ligand and/or binding proteins. In some cases, the other additions, substitutions or deletions may increase the solubility (including but not limited to, when expressed in i cili or other host cells) of the polypeptide. In some embodiments sites are selected for substitution with a naturally encoded or non-natural amino acid in addition to r site for oration of a non-natural amino acid for the purpose of sing the polypeptide solubility following expression in E coli, or other recombinant host cells. In some embodiments, the polypeptides comprise another addition, substitution, or deletion that modulates y for the associated ligand, 2012/039472 binding proteins, and/or receptor, modulates (including but not limited to, increases or decreases) receptor dimerization, stabilizes receptor dimers, modulates circulating half-life, tes release or bio- bility, facilitates purification, or improves or alters a particular route of administration. Similarly, the non-natural amino acid polypeptide can comprise chemical or enzyme cleavage sequences, protease cleavage ces, reactive groups, antibody-binding domains (including but not limited to, FLAG or poly-His) or other affinity based sequences (including but not limited to, FLAG, poly-His, GST, etc.) or linked molecules (including but not limited to, biotin) that improve detection (including but not d to, GFP), purification, transport thru tissues or cell membranes, prodrug release or activation, size reduction, or other traits of the polypeptide.

VII. HER2 Gene as Exemplar The methods, compositions, strategies and ques described herein are not limited to a particular type, class or family of polypeptides or proteins. Indeed, Virtually any polypeptides may be designed or modified to include at least one “modified or unmodified” non-natural amino acids containing dolastatin linker derivative bed . By way of example only, the polypeptide can be homologous to a therapeutic protein ed from the group consisting of: alpha-l antitrypsin, angiostatin, antihemolytic factor, antibody, antibody fragment, monoclonal antibody (e. g., bevacizumab, cetuximab, panitumumab, infliximab, adalimumab, basiliximab, daclizumab, omalizumab, ustekinumab, etanercept, gemtuzumab, alemtuzumab, rituximab, trastuzumab, nimotuzumab, palivizumab, and abciximab), apolipoprotein, apoprotein, atrial natriuretic factor, atrial natriuretic polypeptide, atrial peptide, C-X-C chemokine, T39765, NAP-2, ENA—78, gro-a, gro-b, gro-c, IP-10, GCP-2, NAP-4, SDF-l, PF4, MIG, calcitonin, c-kit ligand, cytokine, CC ine, monocyte chemoattractant n-l, monocyte chemoattractant protein-2, monocyte chemoattractant protein-3, monocyte inflammatory protein-l alpha, monocyte inflammatory n-i beta, , 1309, R83915, R91733, HCCl, T58847, , T64262, CD40, CD40 ligand, c-kit ligand, collagen, colony stimulating factor (CSF), complement factor 5a, complement inhibitor, complement receptor 1, cytokine, epithelial neutrophil activating peptide-78, MIP-l6, MCP-l, epidermal grth factor (EGF), lial neutrophil activating peptide, erythropoietin (EPO), exfoliating toxin, Factor IX, Factor VII, Factor VIII, Factor X, fibroblast grth factor (FGF), fibrinogen, fibronectin, four-helical bundle protein, G-CSF, glp-l, GM-CSF, erebrosidase, gonadotropin, grth factor, grth factor receptor, grf, hedgehog n, hemoglobin, hepatocyte grth factor (hGF), hirudin, human grth hormone (hGH), human serum albumin, ICAM-l, ICAM-l receptor, LFA—l, LFA—l receptor, insulin, insulin-like grth factor (IGF), IGF-I, IGF-II, interferon (IFN), IFN—alpha, IFN—beta, IFN—gamma, interleukin (IL), IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, keratinocyte grth factor (KGF), lactoferrin, leukemia inhibitory factor, luciferase, neurturin, neutrophil inhibitory factor (NIF), oncostatin M, osteogenic protein, oncogene product, tonin, parathyroid hormone, F, PDGF, peptide hormone, pleiotropin, protein A, protein G, pth, pyrogenic exotoxin A, pyrogenic in B, nic exotoxin C, pyy, n, renin, SCF, small biosynthetic protein, soluble complement receptor I, soluble I-CAM l, soluble interleukin receptor, e TNF receptor, medin, somatostatin, somatotropin, streptokinase, superantigens, lococcal enterotoxin, SEA, SEB, SECl, SEC2, SEC3, SED, SEE, steroid hormone receptor, superoxide dismutase, toxic shock syndrome toxin, thymosin alpha 1, tissue plasminogen tor, tumor growth factor (TGF), tumor necrosis factor, tumor necrosis factor alpha, tumor necrosis factor beta, tumor necrosis factor or (TNFR), VLA—4 protein, VCAM-l protein, vascular endothelial growth factor (VEGF), urokinase, mos, ras, raf, met, p53, tat, fos, myc, jun, myb, rel, estrogen receptor, progesterone receptor, testosterone receptor, aldosterone receptor, LDL receptor, and corticosterone.

In one embodiment is a method for treating solid tumor which overexpresses HER-2 selected from the group consisting of breast cancer, small cell lung oma, ovarian cancer, prostate cancer, gastric carcinoma, cervical cancer, esophageal carcinoma, and colon cancer. In another embodiment, the solid tumor is breast cancer. In a further embodiment the solid tumor is ovarian cancer.

Thus, the following description of trastuzumab is provided for rative purposes and by way of example only, and not as a limit on the scope of the methods, compositions, strategies and techniques described herein. Further, reference to trastuzumab in this application is intended to use the generic term as an example of any antibody. Thus, it is understood that the modifications and tries described herein with reference to trastuzumab can be equally applied to any antibody or monoclonal antibody, including those ically listed herein.

Trastuzumab is a zed monoclonal dy that binds to the domain IV of the extracellular t of the HER2/neu receptor. The HER2 gene (also known as HER2/neu and ErbB2 gene) is amplified in 20-30% of early-stage breast cancers, which makes it overexpressed. Also, in , HER2 may send s without mitogens arriving and binding to any receptor, making it overactive.

HER2 extends through the cell membrane, and s signals from outside the cell to the inside.

In healthy people, signaling compounds called mitogens arrive at the cell membrane, and bind to the outside part of other members of the HER family of receptors. Those bound receptors then link (dimerize) with HER2, activating it. HER2 then sends a signal to the inside of the cell. The signal passes through different biochemical pathways. This includes the PI3K/Akt y and the MAPK pathway. These signals promote invasion, survival and growth of blood vessels (angiogenesis) of cells.

Cells treated with zumab undergo arrest during the G1 phase of the cell cycle so there is reduced proliferation. It has been suggested that zumab induces some of its effect by downregulation of HER2/neu leading to disruption of receptor dimerization and signaling h the downstream PI3K cascade. P27Kipl is then not phosphorylated and is able to enter the nucleus and inhibit cdk2 activity, causing cell cycle arrest. Also, trastuzumab suppresses angiogenesis by both induction of antiangiogenic factors and repression of proangiogenic factors. It is thought that a contribution to the unregulated growth ed in cancer could be due to proteolytic cleavage of HER2/neu that results in the release of the extracellular domain. Trastuzumab has been shown to inhibit HER2/neu main cleavage in breast cancer cells.

VIII. Cellular uptake 0fn0n-natural amino acids Non-natural amino acid uptake by a eukaryotic cell is one issue that is typically ered when designing and selecting non-natural amino acids, including but not limited to, for incorporation into a protein. For example, the high charge density of oc-amino acids suggests that these compounds are unlikely to be cell ble. l amino acids are taken up into the eukaryotic cell via a collection of protein-based transport systems. A rapid screen can be done which assesses which non-natural amino acids, if any, are taken up by cells (examples 15 & l6 herein illustrate non-limiting examples of tests which can be done on tural amino acids). See, e. g., the toxicity assays in, e. g., the US. Patent Publication No. 2004/198637 entitled “Protein Arrays,” which is herein orated by reference in its entirety, and Liu, D.R. & Schultz, PG. (1999) Progress toward the evolution of an organism with an expanded genetic code. PNAS United States 96:4780-4785. Although uptake is easily analyzed with s assays, an alternative to designing non-natural amino acids that are amenable to cellular uptake pathways is to provide biosynthetic pathways to create amino acids in vivo.

Typically, the non-natural amino acid produced via cellular uptake as described herein is produced in a concentration sufficient for efficient protein biosynthesis, ing but not limited to, a natural cellular amount, but not to such a degree as to affect the concentration of the other amino acids or exhaust cellular resources. Typical concentrations produced in this manner are about 10 mM to about 0.05 IX. Biosynthesis 0fN0n-Natural Amino Acids Many biosynthetic pathways already exist in cells for the production of amino acids and other compounds. While a biosynthetic method for a particular non-natural amino acid may not exist in nature, including but not limited to, in a cell, the methods and compositions described herein provide such methods. For e, thetic pathways for non-natural amino acids can be generated in host cell by adding new enzymes or modifying existing host cell pathways. Additional new enzymes include naturally occurring enzymes or ially d s. For example, the biosynthesis of p- aminophenylalanine (as presented in an example in ed “In vivo incorporation of unnatural amino acids”) relies on the addition of a combination of known enzymes from other organisms.

The genes for these enzymes can be introduced into a eukaryotic cell by transforming the cell with a plasmid comprising the genes. The genes, when expressed in the cell, provide an enzymatic pathway to size the desired nd. Examples of the types of enzymes that are optionally added are provided herein. Additional s sequences are found, for example, in Genbank. Artificially evolved enzymes can be added into a cell in the same manner. In this manner, the ar machinery and resources of a cell are manipulated to produce non-natural amino acids.

] A variety of methods are available for producing novel enzymes for use in biosynthetic pathways or for evolution of existing ys. For example, recursive ination, including but not limited to, as developed by Maxygen, Inc. (available on the world wide web at www.maxygen.com), can be used to develop novel enzymes and pathways. See, e. g., Stemmer (1994), Rapid evolution of a protein in vitro by DNA Shuflling, ):389-391; and, Stemmer, (1994), DNA Shuflling by random fragmentation and reassembly: In vitro recombination for molecular evolution, Proc. Natl. Acad. Sci.

M, 91:10747-10751. Similarly DesignPathTM, developed by Genencor (available on the world wide web at genencor.com) is optionally used for metabolic pathway engineering, ing but not d to, to engineer a pathway to create a tural amino acid in a cell. This technology reconstructs existing ys in host organisms using a combination of new genes, including but not limited to those identified through functional genomics, molecular evolution and design. Diversa Corporation (available on the world wide web at diversa.com) also provides technology for rapidly screening libraries of genes and gene pathways, including but not d to, to create new pathways for biosynthetically producing non-natural amino acids. lly, the non-natural amino acid produced with an engineered biosynthetic pathway as bed herein is produced in a concentration sufficient for efficient protein biosynthesis, including but not limited to, a natural cellular amount, but not to such a degree as to affect the concentration of the other amino acids or exhaust cellular resources. Typical concentrations produced in vivo in this manner are about 10 mM to about 0.05 mM. Once a cell is transformed with a plasmid sing the genes used to produce enzymes desired for a specific pathway and a tural amino acid is generated, in vivo selections are optionally used to further ze the production of the non-natural amino acid for both mal protein synthesis and cell growth.

X. Additional tic Methodology The non-natural amino acids described herein may be synthesized using methodologies described in the art or using the techniques described herein or by a combination thereof. As an aid, the following table provides s starting electrophiles and nucleophiles which may be combined to create a desired functional group. The information provided is meant to be illustrative and not limiting to the synthetic techniques described herein.

Table 1: Examples of Covalent Linkages and Precursors Thereof Oximes aldehydes or ketones Hydroxylamines Alkyl amines alkyl halides amines/anilines Esters alkyl halides carboxylic acids Thioethers alkyl halides Thiols Ethers alkyl halides alcohols/phenols hers alkyl sulfonates Thiols Esters alkyl sulfonates carboxylic acids Ethers alkyl sulfonates alcohols/phenols Esters Anhydrides alcohols/phenols Carboxamides Anhydrides amines/anilines Thiophenols aryl halides Thiols Aryl amines aryl halides Amines Thioethers Azindines Thiols te esters Boronates Glycols amides carboxylic acids amines/anilines Esters carboxylic acids Alcohols hydrazines Hydrazides carboxylic acids N—acylureas or Anhydrides carbodiimides carboxylic acids Esters diazoalkanes carboxylic acids Thioethers es Thiols Thioethers haloacetamides Thiols Ammotriazines halotriazines amines/anilines Triazinyl ethers iazines alcohols/phenols Amidines imido esters /anilines Ureas lsocyanates amines/anilines Urethanes lsocyanates alcohols/phenols Thioureas isothiocyanates amines/anilines Thioethers Maleimides Thiols Phosphite esters phosphoramidites Alcohols Silyl ethers silyl halides ls Alkyl amines sulfonate esters amines/anilines Thioethers ate esters Thiols Esters ate esters carboxylic acids Ethers sulfonate esters Alcohols Sulfonamides sulfonyl halides amines/anilines Sulfonate esters sulfonyl halides phenols/alcohols ] In general, carbon electrophiles are susceptible to attack by complementary nucleophiles, including carbon nucleophiles, wherein an attacking nucleophile brings an electron pair to the carbon electrophile in order to form a new bond between the nucleophile and the carbon electrophile.

Non-limiting examples of carbon nucleophiles include, but are not limited to alkyl, l, aryl and alkynyl Grignard, organolithium, organozinc, , alkenyl , aryl- and alkynyl-tin reagents (organostannanes), alkyl-, alkenyl-, aryl- and alkynyl-borane reagents (organoboranes and organoboronates); these carbon nucleophiles have the advantage of being kinetically stable in water or polar organic solvents. Other non-limiting es of carbon nucleophiles include phosphorus ylids, enol and enolate reagents; these carbon nucleophiles have the advantage of being relatively easy to generate from precursors well known to those skilled in the art of synthetic organic chemistry. Carbon nucleophiles, when used in conjunction with carbon electrophiles, engender new carbon-carbon bonds between the carbon nucleophile and carbon electrophile.

Non-limiting examples of non-carbon nucleophiles suitable for coupling to carbon electrophiles include but are not limited to y and secondary amines, thiols, thiolates, and hers, alcohols, alkoxides, azides, semicarbazides, and the like. These non-carbon nucleophiles, when used in conjunction with carbon electrophiles, typically generate heteroatom linkages (C-X-C), wherein X is a hetereoatom, including, but not limited to, oxygen, sulfur, or nitrogen.

EXAMPLES Example 1: Synthesis ofCompound 1 Scheme 1 /\/0\/\0/\/0H 1_2 1-1 :9:NoN0\/\O/\/0\/\O/\,0H DIAD PPh;., HCI “\H H N0 o 0 /\ OMe O OMe O / l1: 1-5 N\—/_S 1 NaBH3CN eamwnwxgtf0 OMe o OMe o NH2NH2 xfla?OMeO OMe O Compound 1-3: Tetra (ethylene glycol ) 1-1 (10g, 51.5mmol), N—hydroxyphthalimide 1-2 ( 8.4g, 51.15mmol) and triphenylphosphine ( 17.6g, 67mmol) were dissolved in 300 mL of tetrahydrofiJran followed by addition of DIAD (12.8 mL, 61.78 mol) at 0°C. The ing solution was d at room temperature overnight, and then trated to dryness. The residue was purified by flash column chromatography to give 5.47g (31%) of compound 1-3.

Compound 1-4: To a on of compound 1-3 (200mg, 0.59mmol) in 15mL dichloromethane was added Dess-Martin Periodinane , 0.71 mmol). The reaction mixture was stirred at ambient temperature overnight. The reaction was ed with the solution of sodium bisulfite in 15 mL of saturated sodium bicarbonate. The e was separated. The organic layer was washed with saturated sodium bicarbonate, brine, dried over sodium sulfate, filtered and concentrated in vacuo. The residue was purified by flash column chromatography to give 150mg (75%) of compound 1-4.

Compound 1-6: To a on of monomethyldolastatin hydrochloride salt 1-5 (50mg, 0.062 mmol) in lmL of DMF was added compound 1-4 (63mg, 0.186mmol) and 70uL of acetic acid, followed by addition of 8 mg of sodium cyanoborohydride. The resulting mixture was d at ambient temperature for 2 hours. The reaction mixture was diluted with water and purified by HPLC to give 60mg (80%) of nd 1-6. MS (ESI) m/z 547 , 1092 [M+H].

Compound 1: Compound 1-6 (60mg, 0.05mmol) was dissolved in 1 mL of DMF. 32 uL of hydrazine was added. The resulting solution was stirred at ambient temperature for 1 hour. The reaction was quenched with 1N hydrochloride solution. The reaction mixture was purified by HPLC to give 33 mg (55%) of compound 1. MS (ESI) m/z 482 [M+2H], 962 [M+H].

Example 2: sis ofCompound 2 Scheme 2 HO\/\o/\/0\/\OH 6:10 03>:Noo\/\ /\/o\/\0 OH 2-1 DIAD PPh3 HCIHEN/{kRM N’o\/\o/\/o\/§o OMe O OMeO 21:" \ o 2-3 flewwwnummmfNaBH3CN OMe O OMe 0 Compound 2 was synthesized via a similar synthetic route as described in e 1. MS (ESI) m/z 460 [M+2H], 918 [M+H].

Example 3: Synthesis ofCompound 3 Scheme 3 NOH o o 6:10 o/\/°H HO/\/1\/\OH \ DIAD PPh3 0 3-2 OMe O OMe ON Qmwwmfw?NaBH3CNOMe O OMe O ‘ NHZNHZ “wing:$494M.5OMeO OMe 0 Compound 3 was synthesized Via similar synthetic route to Example 1. MS (ESI) m/z 438 [M+2H], 974 [M+H].

Example 4: Synthesis ofCompound 4 Compound 4-2: To a on of Val -OH.HCl 4-1 (1 g, 4.77 mmol) and bromoethanol (304.7 uL, 4.3 mmol) in 10 mL of DMF was added 1.68 ml of DIEA. The reaction mixture was stirred at room temperature for 2 days. 4.8 mmol of BocZO was added to the reaction mixture, followed by 0.84 mL of DIEA. The reaction mixture was stirred at room temperature for 2 days. The reaction e was concentrated in vacuo and extracted with ethyl acetate, and washed with water, brine, dried over sodium sulfate and concentrated in vacuo. The residue was purified by flash column chromatography to give 0.66 g of compound 4-2.

Scheme 4 0 Bco O Bco O °J< JED—0’ HO’VNEAO —>/\/Br I J< / “VA/“$0| J< :15.“ ; I ocz /\ /=\ O 4'3 /\ 4-1 i HATU o H“EJL'TI H H N/o\/\|}l N N \ Bee 0 /\ OMe O OMeO o OtBu o /\ OMeO HCI OMeoo OH HCHO o HNgkN H H N N \ N/O\/\lil g o I A OMe o OMe o O OH o NHZNHZl 4-8 .mwfiflfifiwofléoH H H o /\ OMeO OMeO 0 OH Compound 4-3: To a solution of compound 4-2 (500 mg, 1.58 mmol), N-hydroxyphthalimide (261 mg, 1.6 mmol) and triphenylphosphine (538 mg, 2.05 mmol) in 15 mL THF was added DIAD (394 uL, 1.9 mol) at at 0°C. The resulting solution was stirred at room temperature ght, and then concentrated in vacuo. The residue was purified by flash column chromatography to give 0.68 g of compound 4-3. nd 4-4: Compound 4-3 was dissolved in 15 mL 4N HCl/Dioxane. The reaction mixture was stirred at room temperature for 2 days and concentrated in vacuo. The residue was dissolved in DMF and treated with BocZO (230 uL, 1 mmol) and DIEA (352 uL, 2 mmol). The reaction mixture was stirred at room temperature for 2 days. The reaction mixture was d by HPLC to give 100 mg of compound 4-4.

] Compound 4-5: To a solution of nd Boc-Val-Dil-methleap-OH in DMF is added phe(OtBu)-OH.HCl, HATU and N—methylmorpholine. The reaction mixture is d at room temperature for 4 hours. The reaction e is concentrated in vacuo and extracted with ethyl acetate (100 mLXl, 50 mL X2). The organic layer is ed and washed with brine, dried over sodium sulfate and concentrated in vacuo. The residue is purified by flash chromatography. The resulting compound is treated with HCl/EtOAC to give compound 4-5.

Compound 4-6: To a solution of compound 4-5 in DMF is added compound 4-4, HATU and DIEA. The reaction mixture is stirred at room temperature for 4 hours. The reaction e is concentrated in vacuo and extracted with ethyl acetate (100 mLXl, 50 mL X2). The organic layer is combined and washed with brine, dried over sodium sulfate and concentrated in vacuo. The residue is d by flash chromatography to give compound 4-6.

] Compound 4-7: Compound 4-6 is dissolved in 15 mL 4N HCl/Dioxane. The reaction mixture is stirred at room temperature for 2 hours and concentrated in vacuo to give compound 4-7.

Compound 4-8: To a solution of compound 4-7 in 1 mL of DMF is added formylaldehyde and acetic acid, followed by addition of sodium cyanoborohydride. The resulting mixture is stirred at t temperature for 2 hours. The reaction mixture is d with water and purified by HPLC to give compound 4-8.

Compound 4: Compound 4-8 is dissolved in 1 mL of DMF. Hydrazine is added. The resulting solution is stirred at ambient temperature for 1 hour. The reaction is quenched with lN hydrochloride solution. The reaction mixture is purified by HPLC to give Compound 4.

Example 5: sis ofCompound 5 Compound 4-7 is dissolved in 1 mL of DMF. Hydrazine is added. The resulting solution is stirred at ambient temperature for 1 hour. The reaction is quenched with lN hydrochloride solution. The reaction mixture is purified by HPLC to give Compound 5.

Example 6: Synthesis ofCompound 6 Scheme 6 ”‘3' e.HC| X0YNJLT; OH —. ()qu —. HZNJKN OMe O OMe O OMe o OMe 00 OMe O OMe O COOMe Boc-N--methyl--Va| 3°C 0 /'\ OMeO OMeO COOMe Boc o /\ OMeO OMe 0 COOH CW\/\0 Ir“:RQN“‘61?6° o A OMeO OMeO COOH 6-6 I NaBH3CN N~M\/O\/\o/\/0\/\ (DH/:31,“ ZNHZ OMe o OMe oo 0-090909%99%0995 OMe O OMe O Compound 6-2: To a solution of compound 6-1 (500 mg, 0.875 mmol) in 3 mL of DMF was added 283 mg of phenylalanine hydrochloride, 433mg of HATU and 581 uL of N—methylmorpholine. The reaction mixture was stirred at room temperature for 4 hours. The reaction e was concentrated in vacuo and extracted with ethyl acetate (100 mLXl, 50 mL X2). The organic layer was combined and washed with brine, dried over sodium sulfate and trated in vacuo. The residue was purified by flash chromatography to give 560 mg (76%) of compound 6-2.

Compound 6-3: Compound 6-2 was dissolved in 15 mL 4N HCl/Dioxane. The reaction e was stirred at room temperature for 2 hours and concentrated in vacuo to give 511 mg of compound 6-3.

Compound 6-4: To a solution of compound 6-3 (368 mg, 0.55 mmol) in 3 mL of DMF was added 255 mg of Boc-N—methyl valine, 314mg of HATU and 303 uL of ylmorpholine. The reaction mixture was stirred at room ature for 4 hours. The reaction mixture was concentrated in vacuo and extracted with ethyl acetate (100 mLXl, 50 mL X2). The organic layer was combined and washed with brine, dried over sodium sulfate and trated in vacuo. The residue was purified by flash chromatography to give 370 mg (79%) of compound 6-4.

Compound 6-5: To a solution of compound 6-4 (170mg) in 10 mL MeOH was added 5eq of 1N LiOH. The reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was ed by lNHCl and extracted with ethyl acetate washed with brine, dried over sodium sulfate and concentrated in vacuo to give 150 mg (90%) of compound 6-5.

Compound 6-6: Compound 6-5 was dissolved in 4N HCl/Dioxane. The reaction mixture was stirred at room temperature for 2 hours and concentrated in vacuo and purified by HPLC to give 150mg of compound 6-6.

Compound 6-7: To a solution of compound 6-6 (50mg, 0.062 mmol) in lmL of DMF was added compound 1-4 (63mg, 0.186mmol) and 70uL of acetic acid, followed by addition of 8 mg of sodium cyanoborohydride. The resulting mixture was stirred at ambient temperature for 2 hours. The reaction mixture was diluted with water and purified by HPLC to give 60mg (80%) of compound 6-7.

Compound 6: Compound 6-7 (60mg, 0.05mmol) was dissolved in 1 mL of DMF. 32 [LL of ine was added. The resulting solution was stirred at ambient ature for 1 hour. The reaction was quenched with 1N hydrochloride solution. The reaction mixture was purified by HPLC to give 33 mg (55%) of nd 6.

Example 7: Synthesis ofCompound 7 Scheme 7 urifOMe O OMe O 6x13?“ l NaBH3CN CgowwwiwfifwOMeO OMe O l NHZNHZ “Wm;fifiwOMeo OM 00e nd 7 was synthesized via similar synthetic route to Compound 1. MS (ESI) m/z 440 [M+2H], 879 [M+H].

Example 8: Synthesis ofCompound 8 Scheme8 HCI “\Jx .nH N’°\/\o/\7o HN N ; \ l o/‘\‘ OMeO + 0 3'3 OMeoo N‘lTlO/Vo\/\ oniN/8-1\I\HNHZNHZOMeO OMeoo ”2”°N°$“In“dlfflrofidw OMeO OMeoo Compound 8 was synthesized Via similar synthetic route to Compound 1. MS (ESI) m/z 418 [M+2H], 835 [M+H]. 2012/039472 Example 9: Synthesis ofCompound 9 Scheme 9 drag/1W MM:0.o 0” /\ OMe o OMe o NV\O+ 9-1 /5\ HATUl »wj§fi$w%Boc O A OMe O OMe O /\ OMe O OMe O /N NH2NH2 HzN/ \/\N N \ I .

AI l OMe o OMe o /N Compound 9-1: To a solution of compound Boc-Val-Dil-methleap-OH in DMF is added 4- (2—Aminoethyl) pyridine, HATU and ylmorpholine. The reaction mixture is stirred at room temperature for 4 hours. The reaction mixture is trated in vacuo and extracted With ethyl acetate (100 mLXl, 50 mL X2). The organic layer is combined and washed With brine, dried over sodium sulfate and concentrated in vacuo. The residue is purified by flash chromatography. The ing compound is treated with HCl/EtOAC to give compound 9-1.

Compound 9-2: To a solution of compound 9-1 in DMF is added compound 4-4, HATU and DIEA. The reaction mixture is stirred at room temperature for 4 hours. The reaction mixture is concentrated in vacuo and extracted With ethyl acetate (100 mLXl, 50 mL X2). The organic layer is combined and washed With brine, dried over sodium sulfate and concentrated in vacuo. The residue is purified by flash chromatography to give compound 9-2.

Compound 9-3: Compound 9-2 is dissolved in 15 mL 4N HCl/Dioxane. The reaction mixture is d at room temperature for 2 hours and concentrated in vacuo to give compound 9-3.

Compound 9-4: To a solution of compound 9-3 in lmL of DMF is added formylaldehyde and acetic acid, followed by addition of sodium cyanoborohydride. The resulting mixture is stirred at ambient temperature for 2 hours. The reaction mixture is diluted With water and purified by HPLC to give compound 9-4.

] Compound 9: Compound 9-4 is ved in 1 mL of DMF. ine is added. The resulting solution is d at ambient temperature for 1 hour. The reaction is quenched With lN hydrochloride solution. The reaction mixture is purified by HPLC to give compound 9.

Example 10: Synthesis ound 10 Scheme 10 o NIOWHIl/NWJLN H “\H H N N \ I | o /=\ OMeO OMeO IN NH2NH2 é)12%Z:3:0 Z Z: IN2\ —Z Compound 10: Compound 9-3 is dissolved in 1 mL of DMF. Hydrazine is added. The resulting solution is stirred at ambient temperature for 1 hour. The reaction is quenched With lN hydrochloride solution. The on mixture is purified by HPLC to give Example 10.

Example 11: Synthesis ofCompound 11 Scheme 11 O N Vk \ OtBu 11-2 0 H0\/\o/\/0\/\o/\/OH H°\/\o/\/O\/\o/\/0\/\[rOtBu—>11 -4 Na! THF DIAD PPh3 THF 11-1 11-3 0 N’°\/\o/\/°\/\o/\/° OtBu Dioxane 0 11-5 0 N’O\/\ /\/O\/\o o/\/ \/\n/OH0 + o OMe O OMe 0 O o'q/lijiN 11-6 117 N\/:/s PyBroP No/\/ \/\o/\/ weO O /\/U\N o”\)kN OMe o OMe o N S NH2NH2 ~°wo~°on*~119*XQN CF3COOH OMe O OMe O N/N\:/S Compound 11-3: To a solution of tetra (ethylene glycol) 11-1 (40.6 mL, 235 mmol) in 100 mL of tetrahedrofuran was added 47 mg of . 12 mL of tert-butylacrylate was added after sodium was ved. The reaction mixture was stirred at room temperature for 24 hours. The reaction mixture was concentrated in vacuo and quenched with 2 mL of 1 N HCl. The e was suspended in brine and extracted with ethyl acetate (100 mLXl, 50 mL X2). The organic layer was combined and washed with brine, dried over sodium sulfate and concentrated in vacuo to give 6.4 g (23%) of compound 11-3.

Compound 11-5: Compound 11-3 (1.0 g, 3.12 mmol), N—hydroxyphthalimide 11-4 (611 mg, 3.744 mmol) and triphenylphosphine (1.23 g, 4.68 mmol) were dissolved in 20 mL of tetrahydrofuran followed by addition of DIAD (0.84 mL, 4.06 mol) at 0°C. The resulting solution was stirred at room temperature overnight, and then concentrated to dryness. The residue was purified by flash column chromatography using ep Cartridges (80g), g with 0-100% ethyl acetate/hexanes, to give 1.0 g (100%) of compound 11-5.

Compound 11-6: Compound 11-5 was dissolved in 15 mL 4N HCl/Dioxane. The reaction mixture was stirred at room temperature for 2 hours and concentrated in vacuo to give 1.0 g of nd 11-6.

Compound 11-8: To a solution of 30 mg (0.0372 mmol) of monomethyldolastatin hydrochloride, 31 mg 4 mmol) of compound 11-6 and 38.2 mg (0.082 mmol) of PyBroP in 1 mL of DMF was added 33 [LL (0.186 mmol) of diisopropylethylamine. The reaction mixture was stirred at room temperature for 5 hours. The reaction mixture was purified by HPLC to give 28mg (65%) of compound 11-8. MS (ESI) m/z 785 [M+2H], 1164[M+H].

Compound 11: Compound 11-8 (28 mg, 0.024 mmol) was dissolved in 1 mL of DMF. 23 [LL (0.72mmol) of ous hydrazine was added. The resulting solution was stirred at room temperature for 1 hour. The on was quenched with 1N hloride solution. The reaction e was purified by preparative HPLC, eluting with 20-70%CH3CN/H2O in 20 min at 254 nm, to give 20 mg (66%) of Compound 11. MS (ESI) m/z 518 [M+2H], 1034[M+H].

Example 12: Synthesis ofCompound 12 Scheme 12 mwmfiwwMmQaE—wmla?OMeO OMeO OMeO OMeO OMeO OMeO COOMe 121 12-2 Bee o A OMeO OMeO COOMe Bee o A OMeO OMeO NZ/No COOH 12-5 No/\/ \/\NH2o I;51%ng 31%ng OMe o OMe 0 COOH OMe 0 COOH \ITNfimQArN O \ NHzNHz \ENdeYQRWT OMe o OMe oo N/Vo\/\ON OMe o OMe 00 ”WeNH2 12-11 @111—CH12-9 < 2:0l 0 Bee How \/\u No/\/0\/\NH2 1. DIAD PPhalTHF 12-8 12'1° 2. 4N HClIDioxane Compound 12-2: To a solution of compound 12-1 (500 mg, 0.875 mmol) in 3 mL of DMF was added 283 mg of alanine hydrochloride, 433mg of HATU and 581 uL of ylmorpholine.

The reaction mixture was stirred at room temperature for 4 hours. The reaction mixture was concentrated in vacuo and extracted with ethyl acetate (100 mLXl, 50 mL X2). The organic layer was ed and washed with brine, dried over sodium sulfate and concentrated in vacuo. The residue was purified by flash chromatography to give 560 mg (76%) of compound 12-2.

Compound 12-3: Compound 12-2 was dissolved in 15 mL 4N oxane. The reaction mixture was stirred at room temperature for 2 hours and concentrated in vacuo to give 511 mg of compound 12-3.

Compound 12-4: To a solution of compound 12-3 (368 mg, 0.55 mmol) in 3 mL of DMF was added 255 mg of Boc-N—methyl valine, 314mg of HATU and 303 uL of N—methylmorpholine. The reaction mixture was stirred at room temperature for 4 hours. The reaction mixture was concentrated in vacuo and extracted with ethyl e (100 mLXl, 50 mL X2). The organic layer was combined and washed with brine, dried over sodium sulfate and concentrated in vacuo. The residue was d by flash chromatography to give 370 mg (79%) of compound 12-4.

Compound 12-5: To a solution of compound 12-4 (170mg) in 10 mL MeOH was added Seq of 1N LiOH. The reaction mixture was stirred at room temperature for 2 hours. The on mixture was acidified by 1NHCl and extracted with ethyl acetate washed with brine, dried over sodium sulfate and concentrated in vacuo to give 150 mg (90%) of compound 12-5.

Compound 12-6: Compound 12-5 was ved in 4N HCl/Dioxane. The reaction mixture was stirred at room temperature for 2 hours and concentrated in vacuo and purified by HPLC to give 150mg of compound 12-6.

Compound 12-7: To a solution of compound 12-6 in DMF was added formylaldehyde (3eq) and 20 eq of acetic acid, followed by addition of 2 eq of sodium cyanoborohydride. The resulting mixture was stirred at ambient temperature for 2 hours. The reaction mixture was diluted with water and purified by HPLC to give compound 12-7. nd 12-10: tert—Butyl 2-(2-hydroxyethoxy)ethylcarbamate (2.05 g, 10 mmol), N- hydroxyphthalimide (1.8 g, 11 mmol) and triphenylphosphine (3.67 g, 14 mmol) were dissolved in 100 mL of tetrahydrofuran followed by on of DIAD (2.48 mL, 12 mol) at 0°C. The resulting solution was stirred at room temperature overnight, and then concentrated to dryness. The residue was d with 50 mL of 4N HCl/dioxane. The mixture was stirred at room temperature for 2 hours. The solvent was removed in vacuo. The residue was d with ether, filtered, washed with ether and dried in vacuo to get 2.6 g (91%) of compound 12-10. MS (ESI) m/z 251 [M+H].

Compound 12-11: To a solution of compound 12-10 (20 mg, 0.026 mmol) in 1 mL of DMF was added 11.2 mg of compound 12-10, 15 mg of HATU and 23 [LL of DIEA. The reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was purified by HPLC to give 20 mg (70%) of compound 12-4. MS (ESI) m/z 490 , 978[M+H].

Compound 12: Compound 12-11 (20 mg, 0.0183 mmol) was ved in 1 mL of DMF. 18 ML (0.56mmol) of anhydrous hydrazine was added. The resulting solution was stirred at room temperature for 1 hour. The reaction was quenched with 1N hydrochloride solution. The reaction mixture was purified by preparative HPLC, eluting with 20-70%CH3CN/H2O in 20 min at 254 nm, to give 14 mg (72%) of nd 12. MS (ESI) m/z 425 , H].

Example 13: Synthesis ofCompound 13 ° [1°kaOtBu NOWOH HOMOtBu—. 4N HCIIDioxane 13-1 DIAD PPh3 13-3 HOSu DCCCine $QAOH ValCItPABOH 13.5 :NoOM:N 36- HNJ/ 1lBNP,DIEA02“NH2 0A HCI o M nJ1 Q”° N’0 11”9 N N OMeO OMeO \ H : H 0 13-8 13-7 A HOBt,D|EA o NH2 0 0 OH JK H \H H N -‘ N N.0\/\/\)J\N o _ /\ 0M9 O 0M9 ° \ H N\/U\H: 0 13-9 O NH2 NH2NH2 o 0 OH H “H H o o N\)LNH OJKNjng$Nj/\I:W(%N| 5 ‘ /\ OMe o OMe o H N’o\/\/\/U\N2 H H of 13 0%NH2 Compound 13-2: Tert-butyl 6-hydroxyhexanoate 13-1 (1.5 g, ol), N- hydroxyphthalimide ( 1.42g, 8.76mmol) and triphenylphosphine ( 2.82g, 10.76 mmol) were dissolved in 50 mL of tetrahydrofuran ed by addition of DIAD (2 mL, 9.564 mol) at 0°C. The resulting solution was stirred at room temperature overnight, and then concentrated to dryness. The residue was purified by flash column chromatography to give 2.5g (95%) of compound 13-2.

Compound 13-3: The compound 13-2 was treated with 15 mL 4N HCl in dioxane. The reaction mixture was stirred at t temperature for 12 hours and concentrated to s in vacuo to give 900mg (100%) of compound 13-3.

Compound 13-4: To a solution of compound 13-3 (900mg, 3.0mmol) in 10 mL of THF was added 397 mg of N—hydroxysuccinimide, followed by adding 669 mg of DCC. The reaction mixture was stirred at ambient ature overnight and filtered. The filtration was concentrated and treated with 10 mL of DCM. The DCM solution was stayed at ambient temperature for 1 hour and filtered. The filtration was concentrated and purified by flash column chromatography to give 800mg (71%) of compound 13-4.

] Compound 13-6: The mixture of compound 13-4 (435 mg, 1.16 mmol) and Val-Cit-PABOH 13-51 (400mg, 1.054 mmol) in 12 mL of DMF was stirred at ambient temperature for 24 hours. The solvent was removed in vacuo. The residue was treated with ether, filtered and washed with ether. The solid was dried in vacuo to give 660mg (98%) of nd 13-6.

Compound 13-7: To the solution of compound 13-6 (200mg, 0.313 mmol) in 6 mL of DMF was added bis (p-nitrophenyl) carbonate (286 mg, 0.94 mmol), followed by addition of 110.2 uL of DIEA. The reaction mixture was stirred at ambient temperature for 5 hours and concentrated. The residue was treated with ether and filtered. The collected solid was washed with ether, 5% citric acid, water, ether and dried in vacuo to give 210mg (83%) compound 13-7. nd 13-9: To a solution of thylauristatin hydrochloride salt 13-8 (100mg, 0.1325 mmol) in 2mL of DMF was added compound 13-7 (159mg, 0.2mmol) and 10 mg of HOBt, followed by addition of 35.2 uL of DIEA. The ing mixture was stirred at ambient temperature for 2 days. The reaction mixture was diluted with water and purified by HPLC to give 93mg (51%) of nd 13-9. MS (ESI) m/z 692 [M+2H], 1382 [M+H].

Compound 13: The compound 13-9 (50mg, 0.036mmol) was dissolved in 1 mL of DMF. 23 [LL of hydrazine was added. The resulting solution was stirred at ambient temperature for 3 hours. The reaction was quenched with 1N hydrochloride solution. The reaction mixture was purified by HPLC to give 32mg (65%) of Compound 13. MS (ESI) m/z 638.5 [M+Na+2H], 1253.3 [M+H], 1275.8 [M+Na].

Example 14: Synthesis ofCompound 14 Scheme 14 0 &°N \Aogaufld \ 14.4 o H0\/\o/\/ \/\o/\/ —.0 0H HO\/\°/\/ /°\/\n/013u —.0 N—°\/\°/\/°\/\o/\/° OtBu Na, THF DIAD, PPh3, THF \ W 0 ° 14.1 23% 100% 0 14-5 14-3 Dioxane \ 100% “\iN N--Hydroxysuccinimide No 0 DCC, THF N \/\°/\/ / We)”O 0 N— \/\°/\/°\/\o/\/°WOH 100% \ of 0 0 14-6 ogNNH2 92% DMF / N:/\/°\/\o/\/°\/\o/\iN 0N?”go” BNP, DIEAIDMF / N:/\/°\/\°/\,o\/\°/\iN OHEiN/©/\°N HNJ/ 14-10 HNJ/ 14-9 0%NH2 02W”: DIEA wwwmmwaemf\=/ HNJ/ 14-11 o NH2 NHZNHZ 81/0 1{(13%WE HZNo/Vowo/VowoA/Lkujggrj/3i”mo OMeO OMeO CF3COOH N\_/s— HNJ/ 14 o NH2 Compound 14-3: To a solution of tetra (ethylene glycol) 14-1 (40.6 mL, 235 mmol) in 100 mL of tetrahedrofuran was added 47 mg of . 12 mL of tert-butylacrylate was added after sodium was dissolved. The reaction mixture was stirred at room temperature for 24 hours. The reaction mixture was concentrated in vacuo and quenched with 2 mL of 1 N HCl. The residue was ded in brine and extracted with ethyl acetate (100 mLXl, 50 mL X2). The organic layer was combined and washed with brine, dried over sodium sulfate and concentrated in vacuop to give 6.4 g (23%) of compound 14-3.

Compound 14-5: Compound 14-3 (1.0 g, 3.12 mmol), N—hydroxyphthalimide 14-4 (611 mg, 3.744 mmol) and triphenylphosphine (1.23 g, 4.68 mmol) were dissolved in 20 mL of ydrofuran followed by addition of DIAD (0.84 mL, 4.06 mol) at 0°C. The resulting solution was stirred at room temperature overnight, and then concentrated to dryness. The residue was purified by flash column chromatography using ep Cartridges (80g), eluting with 0-100% ethyl acetate/hexanes, to give 1.0 g (100%) of compound 14-5.

Compound 14-6: Compound 14-5 was dissolved in 15 mL 4N HCl/Dioxane. The reaction mixture was stirred at room temperature for 2 hours and concentrated in vacuo to give 1.0 g of compound 14-6.

Compound 14-7: To a solution of compound 6 (1.93 g, 4.68 mmol) and N- hydroxysuccinimide (646 mg, 5.616 mmol) in 20 mL of tetrahedrofuran was added 1.062 g (5.148 mmol) of DCC. The reaction mixture was stirred at room temperature overnight and filtered. The filtration was trated and purified by flash column chromatography using SiliaSep Cartridges (80g), g with 0-100% ethyl acetate/hexanes to give 2.37g (100%) of compound 14-7.

Compound 14-8: Compound 14- 8 was made ing to the literature (Bioconjugat Chem. 2002, 13 (4), 855-869.) Compound 14-9: To a solution of compound 14-8 (200 mg, 0.527 mmol) in 2 mL of DMF was added 295 mg (0.58 mmol) of compound 14-7. The reaction mixture was stirred at room temperature overnight and trated in vacuo. The residue was treated with ether, filtered, washed with ether and dried in vacuo to give 402 mg (98%) of compound 14-9.

Compound 14-10: To a solution of compound 14-9 (406 mg, 0.527 mmol) and bis(p- henol) carbonate (481 mg, 1.58 mmol) in 10 mL of DMF was added 0.186 mL(1.054 mmol) of diisopropylethylamine. The reaction e was stirred at room temperature for 5 hours. The solvent was d in vacuo. The residue was treated with ether, filtered, washed with ether, 5% citic acid, water, ether and dried in vacuo to give 350 mg (72%) of compound 14-10.

] Compound 14-11: To a solution of 50 mg (0.062 mmol) of monomethyldolastatin hydrochloride, 87.2 mg (0.093 mmol) of compound 14-10 and 4.7 mg (0.031 mmol) of HOBt in 1 mL of DMF was added 22 [LL (0.124 mmol) of diisopropylethylamine. The reaction mixture was stirred at room temperature for 16 hours. The reaction mixture was purified by HPLC to give 41mg (42%) of compound 14-11. MS (ESI) m/z 785 [M+2H].

Compound 14: Compound 14-11 (41 mg, 0.026 mmol) was dissolved in 1 mL of DMF. 17 ML (0.52mmol) of ous ine was added. The resulting solution was stirred at room temperature for 1 hour. The reaction was quenched with 1N hydrochloride solution. The reaction mixture was purified by preparative HPLC, eluting with 20-70%CH3CN/H2O in 20 min at 254 nm, to give 22 mg (58%) of compound 14. MS (ESI) m/z 720 [M+2H]. 2012/039472 Example 15: Synthesis ofCompound 15 Scheme 15 Jk o o HCI \AJLN \H O N N N’°\/\/\)LN HN O /=\ I OMe O OMeO \ H N’ S o f \=/ '1 13'7 DIEAIHOBt o NH2 O O H “H H o o OAOATIfVLaQ/VWN NEJkNH O OMeO Compound 15-2: To a on of 50 mg (0.062 mmol) of monomethyldolastatin hydrochloride, 75 mg (0.093 mmol) of compound 13-7 and 4.7 mg (0.031 mmol) of HOBt in 1 mL of DMF was added 22 uL (0.124 mmol) of diisopropylethylamine. The reaction e was stirred at room temperature for 16 hours. The reaction mixture was purified by HPLC to give 41mg (42%) of compound -2. MS (ES1)m/z 718 [M+2H], 1435 [M+H].

Compound 15-2: Compound 15-2 (41 mg, 0.026 mmol) was dissolved in 1 mL of DMF. 17 uL (0.52mmol) of anhydrous hydrazine was added. The resulting solution was stirred at room temperature for 1 hour. The reaction was quenched with 1N hydrochloride solution. The reaction mixture was purified by preparative HPLC, g with 20-70%CH3CN/H20 in 20 min at 254 nm, to give 22 mg (58%) of example 15. MS (ESI) m/z 653 [M+2H], 1305 [M+H].

Example 16: Synthesis ofCompound 16 Scheme 16 A 16-8 0 NH2 HO\/\OH 16-1 DMF BNP,DIEA VLOtBu NO Na THF 0 16-2 Jk o o o HO W/\/O OtBu Fmoc\N N \)J\NDA H : H 163. o o o f @ 16-9 N—OH DIAD, PPh3, THF OANHZ 16-40 MMD, HOBt DIEAIDMF N’owo/Qkomu < \i ‘0 16-5 (3)111fo$RQJWT§>N \ 4NHC|IDioxane Fmoc~b‘j;(“\:/(l)J\N/©/\o OMe O OMe O 16-10 o 0 N )J\OH HN 16-6 0 ogNNH2 l DiethylamineTHF 0 o O o/\iop + HZEH¢kN/©/\o11:1:1:12:15OMeO OMeO 16-7 I 16-11 1 NHZ HNJ/ 16-12 Compound 16-3: To a solution of ne glycol 16-1 (13.1 mL, 235 mmol) in 100 mL of tetrahedrofuran was added 47 mg of sodium. 12 mL of tert-butylacrylate was added after sodium was dissolved. The reaction mixture was stirred at room temperature for 24 hours. The reaction mixture was concentrated in vacuo and quenched with 2 mL of 1 N HCl. The residue was suspended in brine and extracted with ethyl acetate (100 mLXl, 50 mL X2). The organic layer was combined and washed with brine, dried over sodium sulfate and concentrated in vacuo. The residue was purified by flash column chromatography to give 5.2 g (24%) of compound 16—3.

Compound 16—5: Compound 16-3 (2.0 g, 10.5 mmol), oxyphthalimide (2.05 g, 12.6 mmol) and triphenylphosphine (3.58 g, 13.65 mmol) were dissolved in 50 mL of tetrahydrofuran followed by addition of DIAD (3.26 mL, 15.75 mol) at 0°C. The resulting solution was stirred at room temperature overnight, and then trated to dryness. The residue was d by flash column chromatography to give compound 16—5.

] Compound 16—6: Compound 16—5 was dissolved in 15 mL 4N HCl/Dioxane. The reaction mixture was stirred at room temperature for 2 hours and concentrated in vacuo to give compound 16—6.

Compound 16—7: To a on of compound 16—6 (5.16 mmol) and N—hydroxysuccinimide (722 mg, 6.7 mmol) in 20 mL of tetrahedrofuran was added 1.28 g (6.2 mmol) of DCC. The reaction mixture was stirred at room temperature overnight and filtered. The filtration was concentrated and purified by flash column chromatography to give 500mg of compound 16—7. nd 16—8: Compound 16— 8 was made according to the literature (Bioconjugat Chem. 2002, 13 (4), 9.) Compound 16—9: To a solution of compound 16—8 (5.0 g, 8.3 mmol) and bis(p-nitrophenol) carbonate (7.6 g, 25 mmol) in 100 mL of DMF was added 2.92 mL(16.6 mmol) of diisopropylethylamine.

The reaction mixture was stirred at room temperature for 16 hours. The solvent was removed in vacuo.

The residue was d with ether, filtered, washed with ether, 5% citic acid, water, ether and dried in vacuo to give 5.0 g (81%) of compound 16—9.

Compound 16—10: To a solution of 1.0 g (1.24 mmol) of monomethyldolastatin hydrochloride, 1.42 g (1.8575 mmol) of compound 16-9 and 95 mg (0.62 mmol) of HOBt in 10 mL of DMF was added 437 uL (2.48 mmol) of diisopropylethylamine. The reaction mixture was stirred at room temperature for 16 hours. The reaction mixture was purified by HPLC to give 1.0 g (58%) of compound 16-10. MS (ESI) m/z 700 [M+2H], 1398 [M+H].

Compound 16—11: To a solution of compound 16—10 (1.0 g, 0.715 mmol) in 15 mL of tetrahedrofuran was added 5 mL (48 mmol) of diethylamine. The reaction mixture was stirred at room temperature for 1.5 hours and concentrated in vacuo. The residue was dissolved in 20 mL of DCM, treated with 200 mL of ether and filtered, wshed with ether and dried in vacuo to give 860 mg of nd 16- 11. MS (ESI) m/z 589 [M+2H], 1176 [M+H].

Compound 16: To a on of 50 mg (0.0425 mmol) of compound 16-11 in 1 mL of DMF was added 32 mg (0.085 mmol) of compound 16—7. The on mixture was stirred at room temperature for 16 hours. The HPLC and MS showed reaction done. 27.2 uL (0.85 mmol) of anhydrous hydrazine was added to the reaction mixture. The on was done in 2 hours. The reaction mixture was acidified with 1N HCl and purified by HPLC to give 40mg (66%) of nd 16. MS (ESI) m/z 654 [M+2H], 1307[M+H].

Example I 7: Synthesis ofCompound I 7 Scheme 17 o O 0 17-1 ‘HOSu OLNIrVgfiYWYNo o H “H H ° o 0%" HQK lo 5 I OMeO OMeO N’ o‘ + H2" N A N/ S \ H 0 or \:l 16-11 17-2 l 06km2 O O °1NOJLIW1J1©A° l O g1 OMe O OMe O \ H a H 0 HNJ/ 17-3 o NH2 1 o o HZNDJLEH$N©AO O 1‘ . 0 AT OMe o OMe o N ’s H E H CF3COOH 0 06km2 ] Compound 17-2: To a solution of compound 17-1 (1.0 g, 4.52 mmol) and N- hydroxysuccinimide (572 mg, 4.97 mmol) in 20 mL of tetrahedrofiiran was added 1.12 g (5.424 mmol) of DCC. The reaction e was stirred at room temperature overnight and filtered. The filtration was trated to give compound 17-2.

Compound 17: To a solution of 50 mg (0.0425 mmol) of compound 16-11 in 1 mL of DMF was added 41 mg (0.1275 mmol) of compound 17-2. The reaction mixture was stirred at room temperature for 16 hours. The HPLC and MS showed reaction done. 20 uL (0.625 mmol) of anhydrous hydrazine was added to the reaction mixture. The reaction was done in 2 hours. The reaction mixture was acidified with 1N HCl and purified by HPLC to give 35mg (60%) of compound 17. MS (ESI) m/z 625 [M+2H], 1249[M+H].

Example 18: Synthesis ofCompound 18 Scheme 18 Wwwwwfir*0mg1.19m HNJ/ 0 OH 14—10 A o NH2 a H J/ 18-1 0 NH2 1 NHZNHZDMF H2N\ONO\A0~O\Ao/\)kr\I(N\-ANO H gxxmxOMeO OMe O H 5 Of 18 o¢L\NH2 Compound 18-1: To a solution of compound 6-6, mg (0.062 mmol) of compound 14-10 and HOBt in 1 mL of DMF was added diisopropylethylamine. The reaction mixture was stirred at room temperature for 16 hours. The reaction e was purified by HPLC to give compound 18-1.

] Compound 18: Compound 18-1 was ved in 1 mL of DMF. Anhydrous ine was added. The resulting solution was stirred at room temperature for 1 hour. The reaction was quenched with 1N hydrochloride solution. The reaction mixture was purified by preparative HPLC, eluting with 20- 70%CH3CN/H20 in 20 min at 254 nm, to give compound 18.

Example 19: Synthesis ofCompound 19 Scheme 19 iokflo/VOWONOWOHO Dess-Martin i DC—TM OMONOWONO\/§o ~o\/\N,Boc 14-3 19-5 19-1 1.NaBH;CN,DMF MMD 1.D|AD,PPhg/THF 1 N—0H 2. 4N HCI/Dioxane ofNHZ >LOMONO\AONO\/\MEN/:51mm‘rn /_/ OMe o OMe o é:/ l N—o N\_/s_ 19-6 3°01 NH2 Lys-(Boc)2-OH Hc. 4N HCI/Dioxane EDC, HOBt o o 4N HCI/Dioxane .3: o —. >—\ H ofNH NH ofNH 0\/\ /\/°\/\N N + o o Boc o NH2 /—’o H HCI ONEiN OMe o OMe o N / s l N-o l N-0 \—/ 19-3 194 19-7 0 O HATU/DIEA 75% wow°$“Id/{LI\ 1%; Z”; OMe o OMe o CF3c00H N, o /—/o—/— Lowowowow11:ng“\iRQAH‘‘CF3c00H o /'\ OMe o OMe o l N-o N S 19-8 NHZNHZIDMF 71% HNNONOWO/Vo‘AMEN/figmmrH—s OMe o OMe o CF3c00H N/ HII‘NJJ\/\o/\/°\/\o/\/°\/\MEN?RQAVHS CF3COOHO OMe o OMe o HZN—O/—/ CF3COOH 19 Compound 19-2: tert—Butyl 2-(2-hydroxyethoxy)ethylcarbamate 13 (2.05 g, 10 mmol), N- hydroxyphthalimide (1.8 g, 11 mmol) and triphenylphosphine (3.67 g, 14 mmol) were dissolved in 100 mL of tetrahydrofuran followed by addition of DIAD (2.48 mL, 12 mol) at 0°C. The resulting solution was d at room temperature overnight, and then concentrated to dryness. The residue was treated with 50 mL of 4N HCl/dioxane. The e was stirred at room temperature for 2 hours. The solvent was removed in vacuo. The residue was d with ether, filtered, washed with ether and dried in vacuo to get 2.6 g (91%) of compound 19-2. MS (ESI) m/z 251 [M+H].

] Compound 19-3: To the mixture of compound 19-2 (315 mg, 1.1 mmol), Boc-Lys(Boc)-OH (365 mg, 1 mmol), EDC (382 mg, 2 mmol) and HOBt (306 mg, 2 mmol) in 10 mL of DCM was added 2012/039472 1.056 mL (6 mmol) of diisopropylethylamine. The on mixture was stirred at room temperature for 3 hours and extracted with ethyl acetate, washed with 5% citric acid, saturate sodium bicarbonate, brine, dried over sodium sulfate, filtered and concentrated in vacuo. The residue was purified flash column tography using SiliaSep Cartridges (40g), eluting with 0-100% ethyl acetate/hexanes, to give 405 mg (70%) of compound 19-3.

] Compound 19-4: Compound 19-3 was dissolved in 15 mL 4N HCl/Dioxane. The reaction e was stirred at room temperature for 2 hours and concentrated in vacuo to give 315 mg (98%) of compound 19-4. MS (ESI) m/z 379 [M+H].

Compound 19-5: To a solution of compound 14-3 (322 mg, 1 mmol) in 20 mL dichloromethane was added artin Periodinane (636 mg, 1.5 mmol). The reaction mixture was d at room temperature for 3 hours. The reaction was quenched with a solution of sodium thiosulfate (1.4 g, 8.85 mmol) in 15 mL of saturated sodium bicarbonate. The mixture was separated. The organic layer was washed with saturated sodium bicarbonate, brine, dried over sodium sulfate, filtered and concentrated in vacuo. The residue was purified by flash column chromatography using SiliaSep Cartridges (40g), eluting with 0-100% ethyl e/hexanes to give 170mg (53%) of compound 19-5.

Compound 19-6: To a solution of thyldolastatin hydrochloride 1.0 g (1.24 mmol) in mL of DMF was added 1.19 g (3.72 mmol) of compound 17 followed by 1.4 mL (24.8 mmol) of acetic acid and 156 mg (2.48 mmol) of sodium cyanoborohydride. The resulting mixture was stirred at room temperature for 2 hours. The solvent was removed in vacuo. The residue was adjusted to pH 8 by sodium bicarbonate and extracted with DCM, washed with brine, dried over sodium e, filtered and concentrated in vacuo. The residue was purified by flash column chromatography using SiliaSep Cartridges (40g), eluting with 0-5% methanol/DCM to give 680mg (51%) of compound 19-6. MS (ESI) m/z 538 [M+2H], 1075 [M+H].

Compound 19-7: To a solution of compound 19-6 (680 mg, 0.632 mmol) in 5 mL of DCM was added 20 mL of 4N HCl/dioxane. The reaction mixture was d at room temperature for 2 hours and concentrated in vacuo. The e was treated with ether, filtered, washed with ether and dried in vacuo to give 660 mg (98%) of compound 19-7. MS (ESI) m/z 510 [M+2H], 1019 [M+H].

Compound 19-8: To a solution of compound 19-7 (280 mg, 0.257 mmol), compound 19-4 (38 mg, 0.0857 mmol) and ylmorpholine (0.283 mL, 2.57 mmol) in 5 mL of N- methylmpyrrolidinone was added 98 mg (0.257 mmol) of HATU. The reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was purified by HPLC to give 160mg (71%) of nd 19-8. MS (ESI) m/z 596 [M+4H], 794[M+3H], 1191 [M+2H].

Compound 19: Compound 19-8 (160 mg, 0.0613 mmol) was dissolved in 1.5 mL of DMF.

ML (0.613mmol) of anhydrous hydrazine was added. The resulting solution was stirred at room temperature for 1 hour. The reaction was quenched with 1N hloride solution. The reaction mixture was purified by preparative HPLC, eluting with 20-70% CH3CN/H2O in 20 min at 254 nm, to give 120 mg (75%) of compound 19. MS (ESI) m/z 451[M+5H], 563[M+4H], 751 [M+3H], 1126 [M+2H].

Example 20: Synthesis ofCompound 20 Scheme 20 NaH,THF .

Ho/\/o\/\o/\/o\/\OH T T35~o/\/°\/\o/\/°\/\0H m. \/O\/\o/\,O\/§o -1 20.2 20-3 1. NaBH3CN, MMD, DMF 2. HF. pyridine \ \/\o/\/o\/\NENEiRQI/Rrh‘ OMe O OMe O 204 NS\=/ HCI cool2 o o_/—NH NHZ Clio/\/O\/\o/\/O\/\PINK/N?RQArN /_/ HCI OMe O OMe O l N-0 NS\:/ -5 o l DCM, DIEA 0wo~0w0~~3§~w9%%~0 0 CM 0 A CMe e /_S CFchOH N\_/ 0 2H /—/°f °=<°wo~0wo~~ @_0 ”j:EYOYLH:5CMe 0 0M9 o 38 CF3c00Ho NHZNHZ H .\H H O\/\o/\/ \/\o/\/‘o NEN$$QHWNi 1 O 0 /\ OMeO OMeON,s NH CFchOH \=, MEI—{NH O O=< /_/ “\jL °\/\o/\/°\/\o/\/N H2N‘0 a CFSCOOHO OMe O OMe ON CFchOH 39 N\=/s Compound 20-2: To a solution of tetra (ethylene glycol) 20-1 (8.0 g, 41.2 mmol) in 100 mL of tetrahedrofuran was added 1.65 g of sodium hydride at 0°C. The on mixture was stirred at room temperature for 30min. 6.21 g of TBS-Cl was added to this solution. The reaction e was stirred at room temperature overnight. The reaction mixture was trated in vacuo and quenched with 2 mL of 1 N HCl. The residue was suspended in brine and extracted with ethyl acetate (100 mLXl, 50 mL X2).

The organic layer was combined and washed with brine, dried over sodium sulfate and concentrated in vacuo. The residue was purified by flash column chromatography to give 5.7g of nd 20-2.

Compound 20-3: To a solution of compound 20-2 (500 mg, 1.62 mmol) in 30 mL dichloromethane was added artin Periodinane (1.03 g, 2.43 mmol). The reaction mixture was stirred at room temperature for 3 hours. The reaction was quenched with a solution of sodium thiosulfate (1.4 g, 8.85 mmol) in 15 mL of saturated sodium bicarbonate. The mixture was separated. The organic layer was washed with ted sodium onate, brine, dried over sodium sulfate, filtered and concentrated in vacuo. The residue was purified by flash column chromatography to give 400mg of compound 20-3.

Compound 20-4: To a on of monomethyldolastatin hydrochloride 213 mg (0.263 mmol) in 4 mL of DMF was added 245 mg (0.75 mmol) of compound 20-3 followed by 0.303 mL (5 mmol) of acetic acid and 34 mg (0.5 mmol) of sodium cyanoborohydride. The resulting mixture was stirred at room temperature for 2 hours. The solvent was removed in vacuo. 3 mL of 60% acetonitrile was added, followed by 0.2 mL of HF.Pyridine at 00C. The resulting solution was stirred at room temperature for 2 hours. The organic solvent was removed in vacuo. The residue was adjusted to pH 8 by sodium onate and extracted with DCM, washed with brine, dried over sodium sulfate, filtered and concentrated in vacuo. The residue was purified by flash column chromatography to give 160mg of compound 20-4. MS (ESI) m/z 474 [M+2H], 947[M+H].

] Compound 20-5: To a on of compound 20-4 (50mg, 0.062 mmol) in 4 mL ofDCM was added 0.3 mL of phosgene/toluene at 0°C. The reaction mixture was stirred at 0°C for 3 hours and concentrated in vacuo for next step t purification.

Compound 20-6: To a solution of compound 19-4 (7.6 mg, 0.017 mmol) and compound 20-5 (0.062 mmol) was added 25 [LL of diisopropylethylamine. The reaction mixture was stirred at room temperature for 1 hour. The reaction e was purified by HPLC, eluting with 20-70% CH3CN/H2O in 20 min at 254 nm, to give 33 mg of compound 20-6. MS (ESI) m/Z 582[M+4H], 775[M+3H], 1 163 [M+2H].

Compound 20: Compound 20-6 (33 mg, 0.014 mmol) was dissolved in 1 mL of DMF. 14 uL (0.43mmol) of anhydrous hydrazine was added. The resulting solution was d at room temperature for 1 hour. The reaction was quenched with 1N hydrochloride solution. The reaction mixture was d by preparative HPLC, eluting with 20-70%CH3CN/H2O in 20 min at 254 nm, to give 10 mg of compound 20. MS (ESI) m/z 549[M+4H], 732[M+3H], 1098[M+2H].

Example 21: sis ofCompound 21 Scheme 21 \i J< o | \ 1 HN\/\1|1H 2 Xor/Nwwmkok 21-1 21 2 4NHCIIDioxane I 1"Ir“fiNfiWNQK'W“ ”°1,NH“CY\1“*HCIOOHDECPK+H2jgf/Nr-H\ijLNDAD OMeO OMeO HN/r 16-11 21-3 NHZ OANHz 2H0 NMP OH; DH HOWNwN/Jngfj/\ijLN OMeO OMeO N,_s o of"” NH2 + o Efi‘N-O/—/ Hc' HNJ/ 21-4 19.4 OANHz HATU NMP \ 1I.grow5 ,N\/\rf/\/U\Nj;r-o1 H\jLrm) OMeO OMeO NHZNHZ ..“rsiMIMrWIMROflOMeo OMe o , CFchOH ° o2NNH2 ofNH | CFchOH Ni orig“ /_/ HNY\/N\/\NNAN MED/w“ OMS O OMe O HzN-O 0 CF3COOH CF COOH of 3 21 0 NH2 Compound 21-2: The mixture of N, N’-Dimethy1ene diamine 21-1 (5 mL, 46.5 mmol) and tert-butyl acrylate 13 mL (116 mmol) was heated at 85 0C for 1 hour. Another 13 mL (116 mmol) of tertbutyl acrylate was added. The reaction mixture was continuely heated at 85°C for 1 hour and stirred at room temperature overnight. The reaction mixture was concentrated in vacuo. The residue wasdiluted with hexanes and purified by flash column tography using SiliaSep Cartridges (120g), eluting with 0-5% ol/DCM to give 10.1 g (62%) of compound 21-2. MS (ESI) m/z 345 [M+H].

] Compound 21-3: To a solution of compound 21-2 (5.0 g, 14.5 mmol) in 50 mL of DCM was added 40 mL of 4N HCl/dioxane. The reaction mixture was stirred at room temperature for 2 days and concentrated in vacuo. The e was treated with ether, d, washed with ether and dried in vacuo to give 4.3 g (97%) of compound 21-3.

] Compound 21-4: To a solution of 166 mg(0.544 mmol) of compound 21-3 and 0.15 mL of N—methylmorpholine in 10 mL of N—methylpyrrolidinone was added 160 mg of compound 16—11, followed by 0.068 mL (0.408 mmol) of DECP. The on mixture was stirred at room temperature for 1 hour. The reaction mixture was purified by preparative HPLC, eluting with 35-70% CH3CN/H2O in 20 min at 254 nm, to give 100 mg (50%) of compound 21-4. MS (ESI) m/z 464[M+3H], 696 [M+2H], 1391 [M+H].

Compound 21-5: To a solution of compound 19-4 (11 mg, 0.025 mmol), compound 21-4 (115 mg, 0.077 mmol) and N—methylmorpholine (0.028 mL, 0.25 mmol) in 1.5 mL thylmpyrrolidinone was added 29.3 mg (0.077 mmol) of HATU. The reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was purified by HPLC, eluting with 20-70% CH3CN/H2O in 20 min at 254 nm, to give 60 mg (67%) of compound 21-5. MS (ESI) m/z 625 [M+5H], 781 [M+4H], 1041[M+3H].

] Compound 21: Compound 21-5 (60 mg, 0.014 mmol) was dissolved in 1 mL of DMF. 7 uL (0.21mmol) of anhydrous hydrazine was added. The resulting solution was stirred at room temperature for 1 hour. The on was quenched with 1N hydrochloride solution. The reaction mixture was purified by preparative HPLC, eluting with 20-70% CH3CN/H2O in 20 min at 254 nm, to give 29 mg (58%) of compound 21. MS (ESI) m/z 599[M+5H], 749[M+4H], 998 [M+3H].

Example 22: sis ofCompound 22 Scheme 22 H H H o \N NJN N N + O HICI o /i\ I OMeO OMeO c00H NNOV‘O’N H 0 12-7 19-4 :3“ HATU i \thrNdLNoH H H N N I 3 o I A OMe0 OMeO N” CFacOOH 0 JENIROVHo o H H H \ ~~°w°'“ I I H o /\ OMeO OMeO 0 CF3COOH H NHZNHZl ‘INNJIoH H I MNH o/'\I OMeO 0Meoo NH CFacOOH \T 2 i 'i‘ ”N \/\or o A OMeO OMeO N CFacOOH 0 Compound 22-1: To a on of nd 19-4 (7.6 mg, 0.017 mmol), compound 12-7 (40 mg, 0.051 mmol) and DIEA (0.030 mL, 0.17 mmol) in 2 mL of DMF was added 32 mg (0.085 mmol) of HATU. The reaction mixture was stirred at room temperature for 2 hour. The reaction e was purified by HPLC, eluting with 20-70% CH3CN/H2O in 20 min at 254 nm, to give 24 mg (68%) of compound 22-1. MS (ESI) m/z 612 [M+3H], 917 [M+2H], 1834[M+H].

Compound 22: Compound 22-1 (24 mg, 0.012 mmol) was dissolved in 1 mL of DMF. 12 uL (0.36mmol) of anhydrous hydrazine was added. The resulting solution was stirred at room temperature for 1 hour. The on was quenched with 1N hydrochloride solution. The reaction mixture was purified by preparative HPLC, eluting with 20-70% CH3CN/H2O in 20 min at 254 nm, to give 15 mg (58%) of Example 21. MS (ESI) m/z 569[M+3H], 852[M+2H], 1726[M+2H].

Example 23: Synthesis ofCompound 23 Scheme 23 o O >LOMONTA’TONOWOH fl» NOWO/VOW(:5; 23-1 Naml \IKO/Nme“15> HZNNOWONOwo/\j\oJ<‘_H2/Pd-c >LChe/:owo/VOWNS OMe o OMe 0 COOH 23_3 HCI l 3%217%wi o CF3COOHO OMe o OMe o o o ”WOVEN \/\o/\)koJ< H H H “ \N NdLN N N I O I I I + O /\ OMe O OMe O o N’VOV‘O’VOwo’QkOH 23_ H ”(:NOworN HATU CF3COOHO OMe OWE OMe O N/VOw Now/QK° ° $Y%N CF3COOHO OMe O OMe O O M/VowoA/Owo“)1”:in«AK/m}? 23-6 QAE; [ NHZNHZ CF3COOHO OMeO OMeO N~o% ~O\/\ VkNH° ° H/tioLN O CF3COOH N/\/O\/\O,NH2 0M9 0 0MB 00 ”NOWONOWO H CF3COOH Compound 23-1: To a solution of compound 14-3 (4.0 g, 12.4 mmol) and 6.6 mL (37.2 mmol) of DIEA in 50 mL of DCM was added 3.31 g of tolunenesulfonyl chloride at 0°C. The on mixture was stirred at room temperature for 2 days. The reaction mixture was extracted with ethyl acetate.

The organic layer was combined and washed with 5% citric acid, water, brine, dried over sodium sulfate and concentrated in vacuo. The residue was purified by flash column chromatography to give 3.5g of compound 23-1.

Compound 23-2: To a solution of compound 23-1 (3.5 g, 7.34 mmol) in 20 mL DMF was added solium azide (1.44 g, 22.02 mmol). The reaction mixture was stirred at 50°C for 2 days. The reaction mixture was extracted with ethyl acetate. The organic layer was washed with water, brine, dried over sodium sulfate, filtered and concentrated in vacuo. The residue was purified by flash column chromatography to give 2.1 g of compound 23-2.

Compound 23-3: To a solution of compound 23-2 (2.1 g, 6.05 mmol) in 50 mL MeOH was added 400 mg (10%) of Pd—C. The resulting mixture was stirred at room temperature under 1 atm Hg for 24 hours. The reaction mixture was filtered and concentrated in vacuo to give 2.1 g of compound 23-3.

MS (ESI) m/z 322[M+H].

Compound 23-4: To a solution of compound 23-3 (33mg, 0.102 mmol), compound 12-7 (40 mg, 0.051 mmol) and 54 [LL of diisopropylethylamine in 1 mL of DMF was added 38 mg of HATU. The reaction mixture was stirred at room temperature for 4 hours. The reaction mixture was d by HPLC to give 52 mg of compound 23-4. MS (ESI) m/z 525[M+2H], 1049[M+H].

Compound 23-5: Compound 23-4 (52 mg, 0.045 mmol) was ved in 5 mL 4N HCl/Dioxane. The reaction mixture was stirred at room ature for 2 hours and concentrated in vacuo to give 52 mg (100%) of compound 23-5. MS (ESI) m/z 497[M+2H], 993[M+H].

Compound 23-6: To a solution of compound 19-4 (7.6 mg, 0.017 mmol), nd 23-6 (52 mg, 0.051 mmol) and DIEA (0.030 mL, 0.17 mmol) in 2 mL of DMF was added 32 mg (0.085 mmol) of HATU. The reaction mixture was stirred at room temperature for 2 hour. The reaction mixture was purified by HPLC, eluting with 20-70% CH3CN/H2O in 20 min at 254 nm, to give 26 mg (61%) of compound 23-6. MS (ESI) m/z 583[M+4H], 777[M+3H], 1165[M+2H].

Compound 23: Compound 23-6 (26 mg, 0.01 mmol) was dissolved in 1 mL of DMF. 10 [LL mol) of anhydrous hydrazine was added. The resulting solution was d at room temperature for 1 hour. The reaction was quenched with 1N hydrochloride solution. The reaction mixture was purified by preparative HPLC, eluting with 20-70% H2O in 20 min at 254 nm, to give 10 mg (40%) of Example 23. MS (ESI) m/z 550[M+4H], 733[M+3H], +2H].

Table 1. Structures of Compounds 1-23 Structure Example "'2No/\/0\/\o/\/0\/\NIH-IL\ijkRQI/‘WVN OMe O OMe O N/N\=/S 2 HZN’O\/\o/\/O\/\NIrH-jLN OMe o W158OMe 0 N\=IS WO 66560 2012/039472 0\/\ H “\H N H HZN’ H \ g Ii] I /'\ OMeO OMeO /N 11 H2N\o/\/O\/\o/\/O\/\o/\)LN N\_)LNH H H \\\ N N | i | /\ OMeO OMe O N/ S H H H 12 \\ ‘ \N NgLN N N I o A I OMeO OMeoo N/\/0\/\O,NH2 (AMENo 0 OH H ”\H H o o _ mMH/kfi: “WON“ N\/U\NH | 5 I o /\ OMeO OMeO H i H 13 of o)\NH2 o o H H H H2N\o/\/O\/\o/\/0V\O/\)LN NEJL” I I 14 O A OMeO OMeO H a HNJ/O O NH i H\i H H N N N O O m N N H I I O /i\ OMeO OMeO H [WOMEN. N 2 N/ S H of: H \=/ O NH i H\i H H N N N O O m H T i T 16 H2N’o\/\0/\)LN N o OMeO OMeO . N N/ s H of: H \=/ O NH WO 66560 17 HZN’OdLN 0 NH 0 o OJKN HN$N “H H N N o O /\)L “\JL I i I 18 HN2 ‘oNOV‘o’VOV‘o o OMeO OM o /\ e I u 0 CH 0 NH O O H H H HNMONOV\ONOV\T¥NEJL%MN: O I A OMeO OMeO \3 N I S 19 \:l o o H \H H ofNH HNJVONOWONO$NITN9LNW$YMN o /=\ I OMeO OMeO HzN-O N,’_ S, U »“ H ~ ~ oV‘o/VowO/Vl 5 'i‘ O O /\ OMeO OMeO N’ S NH \=I °>—N pfNH=<4H o H N N¢LH N n OWONOWO’VI ! Iii H H H " " I AIM N 9*? W HN OMeO OMeO ‘g’\’N\/\ A 'l H H ’_S 0 if l o NH2 0 o H H H o O/Nokv N%%%N \ o ofNjH /_, ”NYVNWN’QL”l N; °/\_ OMeO H owneoN’s HZN-O | _ o of o NH2 NQLNH H H \N N N I O/‘\|i OMeO OMeO NH O :2 H 12 Example 24: Analysis 0fHER-t0x binding to HER2 receptor Her2-Fc was immobilized on CMS chip to a density of N 280 RU. Flow rate was adjusted to 50 ul/min with HBS-EP as buffer. HerTox ts were injected for 3 min with 15 dissociation phase. 30sec pulse of 20mM HCl was used for regeneration. The Bivalent Analyte model was utilize to fit the data (Figure 1). Analysis of the data indicates that HerTox HA121-NC2D: Kd N 60 pM (chi2=4) and HerTox HA121-NC1D: Kd N 300 pM (chi2=l4).

Example 25: ent Transfection WO 66560 2012/039472 CHO-S culture is seeded at 0.75x10A6/mL approximately 16 hours pre-transfection in FreeStyle Cho medium. Cells are ready to transfect the next day when the cell count has d 1.4 — 1.6x10A6/mL. When cells reach target count, 400 mM pAF stock is added to a 1.4mM final culture concentration. PEI/DNA complex is prepared as described: DNA (1.42 ug/1x10A6 cells) is dissolved in RPMI (5% (v/v) of total culture volume), DNA/RPMI mixture is incubated at room temperature for 2 minutes, PEI stock (1 mg/mL) is added to DNA solution at a 3:1 ratio (mL PEI/ug DNA), and the mixture is incubated at room ature for 5 min. The culture is gently added to the mixture and swirled. The flasks are transferred to a 32°C incubator. At day 6 ransfection, a western blot analysis is performed.

At day 7 post-transfection, the atant is harvested.

Example 26: Anti-Her2 variant expression test 30 ml shaker cultures, CHO-S in yle medium; 56 ug DNA in PEI reagent were used. 1.5 mM pAF was also used. At day 6, the supernatant was harvested. Titer was determined by Fc ELISA. (Figures 2 and 3) Example 27: In vitro Inhibition ofProliferation Assay At day 1, the cells were seeded. The media was aspirated and the T-225 culture flasks of cells was rinsed with 30mL PBS -/. PBS was aspirated and 6 mL of 0.25% Trypsin—EDTA was added to each flask. The flasks were incubated at 37C, 5%CO2 for 2-5 minutes. Adherent cells were dislodged by hitting flask and trypsin was neutralized by adding 14mL culture medium. The cell suspension was mixed and transered to a 50 mL conical tube. The cells were spun down at 1200 rpm, 5 min, room temperature.

The resultant cell pellet was resuspended in 12 mL of e medium. The cells were counted in a hemacytometer. Cells were seeded at appropriate cell densities into 96-well flat-bottom, clear plates and incubated overnight at 37°C, 5% C02 to allow cells to attach. Plating volume was 80uL/well. 80uL/well of culture medium was utilized as the “no cell” control.

Cell plating density es include: BT474 (high Her2) — 20,000 cells/well in Fl2k/DMEM (50/50), 10% FBS, P/S MDA—MB-468 (Her2 ve) — 6,000 cells/well in Fl2k/DMEM (50/50), 10% FBS, P/S HCC1954 — 5,000 cells/well in RPMI 1640, 10% FBS, P/S SKOV-3 — 6,000 cells/well in RPMI 1640, 10% FBS, P/S HT29 — 20,000 cells/well in McCoy’ s 5A, 10% FBS, P/S At day 2, the test samples were added to the cells. The test samples were diluted in culture medium to a 9x stock concentration in Column 2 of a round-bottom 96-well plate. 3x serial dilutions were made from Column 2 to Column 11 with a channel or. 10uL/well of the above samples was added in duplicate to the appropriate wells of the seeded plate. The total volume in the wells was about 90uL/well. 10uL/well of culture medium was added to avoid edge s for sample wells. Media Control wells were ed in the inner wells where no sample was added (just 10uL/well of culture medium) to be used in proliferation calculations. The plates were incubated at 37°C, 5%C02 for 72 hrs.

WO 66560 At day 5, proliferation readout occurred. To detect cell proliferation, lOuL/well of WST-8 reagent was added (Cell Counting Kit-8, cat# CKO4-20, o Labs) to all wells. The plate was incubated for 4 hrs at 37°C, 5%C02. The plates were measured for absorbance at OD 450nm. Calculation of proliferation inhibition of test samples: subtract the “no cell control” OD450 value from all wells’ OD450 ; calculate average of the Media Control (untreated) wells; calculate each sample’s % Media Control value using the formula: (OD450 sample/OD450 avg % Media Control) *100; calculate the average, standard deviation, and %CV of each ’s duplicate % Media Control values; plot each samples average % Media l value against sample concentration; calculate ICSO values using 4- parameter logistic fit regression analysis to determine potency of test samples.

Figure 4 and Figure 5 illustrate results from the in Vitro proliferation assay with dolastatin linker derivatives and breast cancer line HCCl954, HER2 +++. Figures 6 and 7 illustrate results from the in Vitro proliferation assay with dolastatin linker derivatives and ovarian cancer line SKOV-3, HER2 +++. s 8 and 9 illustrate results from the in Vitro proliferation assay with dolastatin linker derivatives and breast cancer line MDS-MB-468, HER2 negative.

TABLE 2. x Proliferation Assay Summary: IC50 Values [nM] after 72hr drug treatment Data Set I Experiment Date 9/4/10 9/4/10 9/4/10 9/4/10 9/4/10 9/4/10 9/13/10 0 9/13/10 HER2 exp. (literature/in house) +++/+++ +++/+++ ?/+++ +++/++++ + +++/++++ +++/+++ +++/+++ ? In vivo sensitivity to Herceptin + + - ? - ? + - ? HCC195 4 NCI-N87 ZR30 Dolastatin 0.1 0.06 0.2 >30 0.2 >30 0.1 0.1 no fit NC-Dl 2.7 0.9 11 >100 3.7 >100 2.9 2.1 no fit NC-D2 2.4 1.5 5.2 >100 3.4 >100 2.8 2.3 no fit PHC-D2 Herceptin >300 >300 >300 >300 >300 >300 >300 >300 >300 Mab HA121- NC-Dl 0.2 0.1 >10 >10 (0.04)* >10 0.2 no fit 0.3 Mab HA121- NC-D2 1.0 0.3 >10 >10 0.3 >10 0.4 no fit 0.7 Mab HA121- PHC-D2 -——-——-==—- Fab K136-NC- m III-I...- Data Set 11 ment Date 9/24/10 9/24/10 10/8/10 10/8/10 10/8/10 HER2 exp. (literature/in house) ++/? H/H: H/H: :H/H: -/? In vivo sensitivity to Herceptin unlikely -- -- - ? Sa_mpl_e HT29 BT474 HCC1954 SKOV-3 MDA-MB-468 NC-Dl 4 (45)* <0.01 <0.1 <0.1 NC-D2 4 3 <0.01 (0.1)* 0.3 PHC-D2 12 7 2 8 2 Herceptin >300 >300 >300 >300 >300 Mab HA121- NC-Dl >10 1 0.2 1.3 >30 Mab HA121- NC-D2 >10 0.8 0.03 (1.3)* >30 Mab HA121- PHC-D2 5* 2 0.1 0.3 (5.8)* Fab K136pAF Fab K136-NC-D1 TABLE 3. In vitro Cellular Data -----SmallMolecule, IC50, nM ADC, IC50, nM EGFR C225—HC- C225—NC- Cancer Cell Line KRAS BRAF MMD NC-D-l C-D-l NC-D-2 C-D-1 expression D-1 D-2 WO 66560 HCT-116 mut wt 0.13 2.14 24.86 5.7 51.73 >100 62.8 HT-29 wt mut 0.1 4.3 1.7 36.9 16.7 SW620 mut wt - 0.14 5 3.2 121 56.8 HCT-15 mut wt ++ 2.65 31.03 >100 >100 >300 >300 A549* mut wt --+ 0.19 6.44 39.82 39.82 >100 >100 Lung H2122 mut wt -- 0.11 12.71 31.76 31.76 >100 >100 H460 mut wt + 0.48 10.4 95.1 95.1 >300 >300 Prostate DU145 wt wt --+ 0.24 4.8 20.51 20.51 >100 >100 Example 28: In vivo anti-tumor efficacy 0fHer2-ADCs in HCCI954 (human breast carcinoma) xenograft animal model 4 (human breast carcinoma) cells were obtained from American Type Culture Collection (Manassas, VA) and cultured in RPMI + 10% FBS, 37°C, 5% C02 until 80% confluent. Cells were harvested by trypsinization and suspended in PBS at 1 X 108 cells/mL.

Female, SClD—beige mice, 5—8 weeks old, were obtained from Charles River Laboratories.

HCCl954 cells (human, breast carcinoma, ATCC, # 38) were mixed l:l with Matrigel (BD Biosciences, Bedford MA) and injected subcutaneously into the mice. When tumors reached an average size of 100—200 mm3, mice were sorted into groups of 9—10 mice each. r measurements were taken twice weekly until the end of the study. To estimate tumor volume, two orthogonal diameters were measured with calipers and the values entered into the formula, (L X W X W)./2=V, (Where W=the shortest diameter, L=the longest diameter and V=volume), to obtain an estimated volume. The tumor volume was ted to tumor weight in the Excel data file by assuming 1 mm3 = l mg. Endpoint was based on a study design of tumor growth inhibition (TGl). When the mean tumor volume of the control group reached . 1,000 mm3 all mice were ized or day 28, whichever came first.

Mice were given a single IV injection (tail vein) on day 1 of dosing. Test article was ved at 4 mg/mL, 2 mg/mL and 0.66 mg/mL and administered at a dose volume of 5 mL/kg to deliver 20, 10 and 3.3 mg/kg. Test articles were: Herceptin® al grade (Trastuzumab), Her2-HS122- NCD1 (Ab:Drug ratio = 1:2, non-cleavable linker), and Her2-HS122/LK145-HCD1 (Ab:Drug ratio = 1:4, cleavable linker) See Figure 16.

Example 29: In vivo Studies 0fHer2-Dolastatin linked Derivative HCCI954 cells were utilized for this study with 107 cells/mouse in Matrigel, SC in the right flank. Mice were SCID-bg female 4-8 weeks. Grouping was performed at day 5 after cell implantation (tumors ~100 mm3): sorted into 11 groups of 10 mice each. A single IV dose was given on day 1 of dosing with each compound at 3 dose levels, 20 mg/kg, 10 mg/kg and 3.3 mg/kg. Tumor volume was monitored until the endpoint was reached (1,000 mm3 or 60 days). (Figure 10) Paclitaxel 25 mg/kg, 1V, qod X 5 was employed as the l herapy. The vehicle = 50 mM histamine, 0.1 M NaCl, 5% trehalose, pH 6. Herceptin® clinical grade (Trastuzumab), Her2-HS122-NCD1 (non-cleavable linker) and Her2-HS122/LK145-HCD1 (cleavable linker) were tested. (Figures lland 12) Table 4. ation of T/C (Treated / Control) for the HCC1954 study at day 28 Median Tumor olume Group 11 Schedule (mm3) T/C 1# 10 vehicle - iv qd x 1 486 -- 2 10 trastuzumab 3.3 iv qd x 1 405 0.833 3 10 trastuzumab 10 iv qd x 1 446 0.918 4 10 trastuzumab 20 iv qd x 1 385 0.792 10 Her-HS122-NC1D-002 3.3 iv qd x 1 40 0.082 6 10 122-NC1D-002 10 iv qd x 1 14 0.029 7 10 Her-HS122-NC1D-002 20 iv qd x 1 18 0.037 8 10 Her-HS122/LK145-HC1D-001 3.3 iv qd x 1 40 0.082 9 10 Her-HS122/LK145-HC1D-001 10 iv qd x 1 25 0.051 10 Her-HS122/LK145-HC1D-001 20 iv qd x 1 18 0.037 11 10 paclitaxel 25 iv qod x 5 18 0.037 Example 30: Pharmacokinetic studies Assay was performed that detected antibody binding to ErbB2 receptor. (Figure 13) Assay was performed that detected at least two dolastatins linked to an antibody (Figure 14).

Figure 15.

Example 31: entfor Breast Cancer Human Clinical Trial of the Safety and/0r Efficacy of Trastuzumab-Linked Dolastatin Derivative for Breast Cancer y Objective: To compare the safety and pharmacokinetics of administered composition comprising trastuzumab-linked dolastatin derivative.

Study Design: This study will be a Phase I, -center, open-label, randomized dose escalation study followed by a Phase II study in breast cancer patients. Patients should not have had exposure to trastuzumab-linked dolastatin derivative prior to the study entry. Patients must not have ed treatment for their cancer within 2 weeks of beginning the trial. Treatments include the use of chemotherapy, hematopoietic growth s, and ic therapy such as monoclonal antibodies.

Patients must have recovered from all ties (to grade 0 or 1) associated with previous treatment. All subjects are evaluated for safety and all blood collections for pharmacokinetic analysis are collected as led. All studies are performed with institutional ethics committee approval and patient consent.

Phase 1: Patients receive i.v. zumab-linked dolastatin derivative on days 1, 8, and 15 of each 28-day cycle. Doses of trastuzumab-linked dolastatin derivative may be held or modified for toxicity based on assessments as outlined below. Treatment repeats every 28 days in the absence of unacceptable toxicity. Cohorts of 3-6 patients receive escalating doses of trastuzumab-linked dolastatin derivative until the maximum tolerated dose (MTD) for zumab-linked dolastatin derivative is determined. The MTD is defined as the dose preceding that at which 2 of 3 or 2 of 6 patients experience dose-limiting toxicity.

Dose limiting toxicities are determined according to the definitions and standards set by the National Cancer Institute (NCI) Common ology for Adverse Events (CTCAE) Version 3.0 (August 9, 2006).

Phase 11: Patients e trastuzumab-linked dolastatin tive as in phase I at the MTD determined in phase 1. Treatment repeats every 4 weeks for 2-6 courses in the absence of disease progression or unacceptable toxicity. After completion of 2 courses of study therapy, patients who achieve a complete or partial response may receive an additional 4 courses. ts who maintain stable disease for more than 2 months after completion of 6 courses of study therapy may receive an additional 6 courses at the time of disease ssion, provided they meet al eligibility criteria.

Blood Sampling Serial blood is drawn by direct vein puncture before and after administration of trastuzumab-linked dolastatin derivative. Venous blood samples (5 mL) for determination of serum concentrations are obtained at about 10 minutes prior to dosing and at imately the ing times after dosing: days 1, 8, and 15. Each serum sample is divided into two aliquots. All serum samples are stored at -20°C. Serum samples are shipped on dry ice.

Pharmacokinetics: Patients undergo plasma/serum sample tion for pharmacokinetic evaluation before beginning treatment and at days 1, 8, and 15 . Pharmacokinetic parameters are calculated by model independent methods on a Digital Equipment Corporation VAX 8600 computer system using the latest version of the BIOAVL software. The following pharmacokinetics parameters are determined: peak serum concentration (Cmax); time to peak serum concentration (tmax); area under the concentration-time curve (AUC) from time zero to the last blood sampling time (AUC 042) calculated with the use of the linear oidal rule; and terminal ation half-life (t1/2), computed from the elimination rate constant. The elimination rate constant is ted by linear sion of consecutive data points in the al linear region of the log-linear concentration-time plot. The mean, standard deviation (SD), and coefficient of variation (CV) of the pharmacokinetic parameters are calculated for each treatment. The ratio of the parameter means (preserved ation/non—preserved formulation) is calculated.

Patient se to combination therapy: Patient response is assessed via imaging with X- ray, CT scans, and MRI, and imaging is performed prior to beginning the study and at the end of the first cycle, with additional imaging performed every four weeks or at the end of subsequent cycles. Imaging modalities are chosen based upon the cancer type and feasibility/availability, and the same imaging modality is ed for similar cancer types as well as throughout each patient’s study course. Response rates are determined using the RECIST criteria. (Therasse et al, J. Natl. Cancer Inst. 2000 Feb 2; 92(3):205-l6; http://ctep.cancer.gov/forms/TherasseRECISTJNCIpdf). ts also undergo /tumor biopsy to assess changes in progenitor cancer cell phenotype and clonogenic growth by flow cytometry, Western blotting, and IHC, and for changes in cytogenetics by FISH. After tion of study treatment, patients are followed periodically for 4 weeks.

Example 32: Treatmentfor Breast Cancer Human Clinical Trial of the Safety and Efficacy of Trastuzumab-Linked Dolastatin Derivative for Breast Cancer Therapy Objective: Compare the efficacy and toxicity of trastuzumab-linked atin derivative alone followed at e progression by combination trastuzumab and paclitaxel vs first-line combination trastuzumab and paclitaxel in women with verexpressing metastatic breast cancer.

Study Design: This study is a randomized, multicenter study. Patients are stratified according to degree of HER2/neu-overexpression (2+ vs 3+), prior anthracycline-containing adjuvant treatment (no prior treatment vs prior treatment without radiotherapy to left chest wall vs prior treatment with radiotherapy to left chest wall), estrogen-receptor status (positive vs negative vs unknown), prior therapy (first-line vs second/third-line), and center. Patients are randomized to one of two treatment arms. Arm I: Patients receive zumab-linked dolastatin derivative IV over 30-90 minutes weekly. At time of disease progression, ts receive combination trastuzumab-linked dolastatin derivative IV and paclitaxel IV as in arm II. Arm II: Patients receive trastuzumab-linked dolastatin derivative IV over 30-90 minutes . Paclitaxel is administered IV over 1 hour weekly for 3 weeks followed by 1 week of rest.

Treatment continues in both arms in the absence of disease progression or unacceptable toxicity.

Quality of life is assessed at baseline and day l of s 2, 3, 4, 5, 6, 8, 10, and 12. Patients are followed at l, 3, and 6 months and then every 6 months thereafter. e 33: Treatmentfor Bladder Cancer Objective: Determine the acute toxicity of paclitaxel and radiotherapy with or without a atin derivative bed herein in patients who have undergone prior transurethral bladder resection for muscle-invasive transitional cell carcinoma of the bladder.

Disease Characteristics: Histologically or cytologically is confirmed y tional cell carcinoma (TCC) of the bladder; histologic ce of muscularis propria invasion; meets l of the following stage criteria: stage T2-4a; NX, NO, or N1; and M0 disease or clinical stage Tl, grade 3/3 disease AND requires definitive local therapy; tumor involvement of the prostatic urethra allowed ed the following ia are met: tumor is visibly completely resected; no evidence of stromal invasion of the prostate, no evidence of distant metastases by chest x-ray or CT scan AND abdominal/pelvic CT scan; has undergone transurethral bladder resection (as thorough as is judged safely possible) within the past 3-8 weeks, including bimanual examination with tumor mapping; sufficient tumor tissue available for HER2/neu analysis; not a candidate for radical cystectomy.

Study Design: This study is a ndomized, multicenter study. Patients are assigned to 1 of 2 treatment groups according to HER2/neu status (HER2/neu 2+ or 3+ staining [group 1] vs HER2/neu O or 1+ ng [group 2]).

Group 1: Patients receive paclitaxel IV over 1 hour on days 1, 8, 15, 22, 29, 36, and 43 and a dolastatin tive described herein Via IV over 90 minutes on day 1 and then over 30 minutes on days 8, 15, 22, 29, 36, and 43. Patients also undergo radiotherapy once daily on days 1-5, 8-12, 15-19, 22-26, 29-33, 36-40, 43-47, and 50. Treatment continues in the absence of disease progression or unacceptable toxicity.

Group 2: Patients receive axel and undergo radiotherapy as in group 1. After completion of study treatment, patients are followed at 4-5 weeks, every 3 months for 1 year, every 4 months for 1 year, every 6 months for 3 years, and then annually thereafter.

Example 34: Treatmentfor Ovarian Cancer Human Clinical Trial of the Safety and y of aDolastatin Derivative described herein for Ovarian Cancer Therapy Objective: Evaluate the safety and efficacy of a four week once weekly IV dosage of ition sing a dolastatin derivative described herein in women with HER2-overexpressing ovarian cancer.

Study Design: This study is a non-randomized, open-label, 11 week, enter study. This study will evaluate the safety profile of four once weekly IV dosage, the MTD, PK and immunogenicity of trastuzumab-linked dolastatin derivative. Patients are assigned to a single group. Patients receive one dose of trastuzumab-linked dolastatin derivative once a week for 4 weeks. Trastuzumab-linked dolastatin derivative will be administered by IV infusion on Study Days 1, 8, 15, and 22. Urine s will be taken on days 1 and 22.

Blood Sampling Serial blood is drawn by direct vein puncture before and after administration of the atin derivative. Venous blood samples (5 mL) for determination of serum concentrations are obtained at about 10 minutes prior to dosing and at approximately the following times after dosing: days 1, 2, 4, 5, 8, 15, 22, 36, 43 and 50. Each serum sample is diVided into two aliquots. All serum samples are stored at -20°C. Serum samples are shipped on dry ice.

Treatment continues in the absence of disease progression or unacceptable toxicity. Quality of life is ed at baseline and day 1 of courses 2, 3, 4, 5, 6, 8, 10, and 12. Patients are followed on days 29. 36, 43, and 50. Patients will be asked about adverse events. Patients will have an imaging scan and ECG to evaluate tumor siz and heart function (day 43). At the termination of the study ts will have a physical exam day 50). ts with evidence of disease regression may receive continued therapy until evidence of ssion of disease is documented.

Claims (53)

The claims defining the ion are as follows:

1. A compound, of Formula (I) or salt thereof: Me Me Me O Me H H L N N Me (I) Y N N R7 O Me OMe O Me Me MeO NH O Z ; 5 wherein: Z has the structure of: R5 ; R5 is H, COR8, C1-C6alkyl, or thiazole; R8 is OH or –NH–(alkylene–O)n–NH2; 10 R6 is OH or H; Ar is phenyl or pyridine; R7 is C1-C6alkyl or hydrogen; Y are each selected from the group consisting of an hydroxylamine, methyl, aldehyde, protected aldehyde, ketone, protected ketone, thioester, ester, dicarbonyl, 15 ine, amidine, imine, diamine, azide, keto-amine, keto-alkyne, alkyne, cycloalkyne, and ene-dione; L is a linker selected from the group consisting of ene–, –alkylene–C(O)–, – (alkylene–O)n–alkylene–, –(alkylene–O)n–alkylene–C(O)–, –(alkylene–O)n– '–NHC(O)–(CH2)n''–C(Me)2–S–S–(CH2)n'''–NHC(O)–(alkylene–O)n''''– 20 alkylene–, –(alkylene–O)n–alkylene–W–, –alkylene–C(O)–W–, –(alkylene–O)n– alkylene–U–alkylene–C(O)–, and –(alkylene–O)n–alkylene–U–alkylene–; provided that when R5 is H, C1-C6alkyl or le or R8 is OH, L is not alkylene or – (alkylene-O)n-alkylene; W has the structure of: Me Me O O O N N H H O NH2 ; U has the structure of: CO2H O ; or L is , Y is , R5 is COR8, and R8 is –NH–(alkylene–O)n–NH2; 5 and each n, n', n'', n''' and n'''' are independently integers greater than or equal to one.

2. The compound of claim 1, wherein R5 is thiazole.

3. The compound of claim 1, wherein R6 is H.

4. The compound of claim 1, wherein Ar is phenyl. 10

5. The compound of claim 1, wherein R7 is methyl.

6. The compound of claim 1, where n is an integer from 0 to 20.

7. The compound of claim 1, where n is an integer from 0 to 10.

8. The compound of claim 1, where n is an integer from 0 to 5.

9. The compound of claim 1, wherein Y is cyclooctyne. 15

10. The nd of claim 9, wherein the cyclooctyne has a structure of: (R19)q ; each R19 is independently selected from the group ting of C1-C6 alkyl, C1-C6 alkoxy, ester, ether, thioether, aminoalkyl, halogen, alkyl ester, aryl ester, amide, aryl amide, alkyl halide, alkyl amine, alkyl sulfonic acid, alkyl nitro, thioester, 20 sulfonyl ester, halosulfonyl, nitrile, alkyl nitrile, and nitro; and q is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11.

11. The compound of claim 1, wherein Y is azide.

12. The compound of claim 1, comprising Formula (II): Me Me Me O Me H H H2N L N N Me (II) O N N R7 O Me OMe O Me Me MeO NH N Ph 5

13. The compound of claim 12, wherein L is –(alkylene–O) n–alkylene–C(O)–.

14. The compound of claim 13, wherein each alkylene is –CH2CH2–, n is equal to 4, and R7 is methyl.

15. The compound of claim 12, wherein L is lene–O) n–(CH2)n'–NHC(O)–(CH2)n''– C(Me)2–S–S–(CH2)n'''–NHC(O)–(alkylene–O)n''''–alkylene–. 10

16. The compound of claim 15, n each alkylene is –CH2CH2–, n is equal to 1, n' is equal to 2, n'' is equal to 1, n ''' is equal to 2, n'''' is equal to 4, and R7 is methyl.

17. A compound of Formula (III), (IV), (V) or (VI), or salt thereof,: Me Me Me O Me H H N N L2 N N Me R7 O Me OMe O Me Me MeO NH L1 O Z Me Me Me (III) Y O Me H H N N L3 N N Me R7 O Me OMe O Me Me MeO NH O Z ; Me Me Me O Me H H Me N N N N Me Me O Me OMe O Me Me MeO NH O Ar HN L2 Me Me Me (IV) O Me R6 H H Me N N Me L1 N N Me O Me OMe O V Me Me MeO NH HN L3 Ar O R6 ; Me Me Me O Me H H N N L2 N N Me R7 O Me OMe O Me Me MeO NH L1 O Z Y Me Me Me O Me H H (V) N N L3 N N Me R7 O Me OMe O Me Me MeO NH J O Z Me Me Me O Me H H N N L4 N N Me R7 O Me OMe O Me Me MeO NH O Z ; Me Me Me O Me H H Me N N N N Me Me O Me OMe O Me Me MeO NH O Ar HN L2 Me Me Me O Me R6 H H Me N N L1 N N Me Me O Me OMe O Me Me MeO NH HN L3 (VI) Ar O Me Me Me O Me R6 H H J Me N N N N Me Me O Me OMe O Me Me MeO NH HN L4 Ar O wherein: Z has the structure of: R5 ; 5 R5 is H, COR8, C1-C6alkyl, or le; R8 is OH; R6 is OH or H; Ar is phenyl or pyridine; R7 is C1-C6alkyl or hydrogen; 10 Y and V are each selected from the group consisting of an hydroxylamine, methyl, aldehyde, protected de, ketone, protected ketone, thioester, ester, dicarbonyl, hydrazine, amidine, imine, diamine, azide, keto-amine, lkyne, alkyne, cycloalkyne, and ene-dione; L1, L2, L3, and L4 are each linkers independently selected from the group consisting of 15 a bond, –alkylene–, –(alkylene–O)n–alkylene–J–, –alkylene'–J–(alkylene–O)n– alkylene–, –J–(alkylene–O)n–alkylene–, –(alkylene–O)n–alkylene–J–(alkylene– O)n'–alkylene–J'–, –(alkylene–O)n–alkylene–J–alkylene'–, –W–, –alkylene–W–, alkylene'–J–(alkylene–NMe)n–alkylene–W–, –J–(alkylene–NMe)n–alkylene–W–, – J–alkylene–NMe–alkylene'–NMe–alkylene''–W–, and –alkylene–J–alkylene'– NMe–alkylene''–NMe–alkylene'''–W–; 5 W has the structure of: Me Me O O O N N H H O NH2 ; each J and J' independently have the structure of: O O O N N O or N H , H , H ; and each n and n' are independently integers greater than or equal to one. 10

18. The compound of claim 17, wherein the compound is of a (VII): Me Me Me O Me H H Me N N N N Me Me O Me OMe O Me Me MeO NH O Ar HN L2 Me Me Me (VII) O Me R6 H H Me N N L1 N N Me Me O Me OMe O V Me Me MeO NH HN L3 Ar O R6 .

19. The nd of claim 18, wherein L1 is –(alkylene–O)n–alkylene–J–, L2 is –alkylene'–J'– (alkylene–O)n'–alkylene–, L3 is –J''–(alkylene–O)n''–alkylene–, alkylene is –CH2CH2–, alkylene' is –(CH2)4–, n is 1, n' and n'' are 3, J has the structure of H , J' and J'' have N O the structure of H , and R7 is methyl.

20. The nd of claim 18, wherein the compound is of Formula (IV) and wherein L1 is – J–(alkylene–O)n–alkylene–, L2 is –(alkylene–O)n'–alkylene–J'–alkylene'–, L3 is –(alkylene– 5 O)n''–alkylene–J''–, alkylene is –CH2CH2–, alkylene' is –(CH2)4–, n is 1, n' and n'' are 4, and J, J' and J'' have the structure of H .

21. A compound of Formula (VIII) or (IX): Me Me Me O Me H H L N N O N N Me R N R7 O Me OMe O Me Me (VIII) MeO NH B O Z A O R3 R4 R3 R2 R1 ; Me Me Me O Me H H Me R6 Me N N N N N (IX) Me O Me OMe O Me Me OMe O O NH R3 R3 R1 L NH O A N B O R2 R ; 10 wherein: A is al, and when present is lower alkylene, lower lkylene, lower alkenylene, lene, lower heteroalkylene, lower heterocycloalkylene, arylene, heteroarylene, alkarylene, aralkylene; B is optional, and when present is a linker selected from the group consisting of lower 15 alkylene, lower alkenylene, lower heteroalkylene, -O-, -O-(alkylene)-, -S-, -S- (alkylene)-, -S(O)k- where k is 1, 2, or 3, -S(O)k(alkylene)-, -C(O)-, -C(O)- (alkylene)-, -C(S)-, -C(S)-(alkylene)-, -N(R’)-, -NR’-(alkylene)-, -C(O)N(R’)- , -CON(R’)-(alkylene)-, -CSN(R’)-, -CSN(R’)-(alkylene)-, -N(R’)CO-(alkylene)- , -N(R’)C(O)O-, -S(O)kN(R’)-, -N(R’)C(O)N(R’)-, -N(R’)C(S)N(R’)- , S(O)kN(R’)-, -N(R’)-N=, -C(R’)=N-, -C(R’)=N-N(R’)-, -C(R’)=N- 5 N=, -C(R’)2-N=N-, and -C(R’)2-N(R’)-N(R’)-, where each R’ is independently H,or alkyl; R is H, alkyl, or cycloalkyl; R1 is H, an amino ting group, resin, at least one amino acid, polypeptide, or polynucleotide; 10 R2 is OH, an ester protecting group, resin, at least one amino acid, polypeptide, or polynucleotide; R3 and R4 are each independently H, halogen, lower alkyl, or R 3 and R4 or two R3 groups optionally form a cycloalkyl or a heterocycloalkyl; Z has the structure of: 15 R5 ; R5 is H, COR8, lkyl, or thiazole; R8 is OH; R6 is OH or H; Ar is phenyl or ne; 20 R7 is C1-C6alkyl or en; L is a linker selected from the group consisting of–alkylene–C(O)–, –(alkylene–O)n– alkylene–C(O)–, –(alkylene–O)n–(CH2)n'–NHC(O)–(CH2)n''–C(Me)2–S–S–(CH2)n'''– NHC(O)–(alkylene–O)n''''–alkylene–, –(alkylene–O)n–alkylene–W–, –alkylene– C(O)–W–, –(alkylene–O)n–alkylene–U–alkylene–C(O)–, and –(alkylene–O)n– 25 alkylene–U–alkylene–; W has the structure of: Me Me O O O N N H H O NH2 ; U has the structure of: CO2H O ; and each n, n', n'', n''' and n'''' are independently integers greater than or equal to one; 5 or solvate thereof.

22. The compound of claim 21, wherein R1 is a polypeptide.

23. The compound of claims 22, wherein the polypeptide is an antibody.

24. The nd of claim 23, wherein the antibody is zumab.

25. The compound of claim 21, wherein R2 is a polypeptide. 10

26. The compound of claims 25, wherein the polypeptide is an dy.

27. The compound of claim 26, wherein the antibody is trastuzumab.

28. A compoundof Formula (X), (XI), (XII) or (XIII) or salt thereof: Me Me Me O Me H H N N L2 N N Me R7 O Me OMe O Me Me MeO NH L1 O Z Me Me Me O O Me (X) R N H H N N L3 N N Me A O R7 O Me OMe O Me Me MeO NH R3 R4 R2 O Z R1 ; Me Me Me O Me Me O H H H Me N N N N N N L2 Me O Me OMe O OMe O Me Me Ar R6 R2 R1 L1 (XI) Me Me Me R3 O Me Me O A H O H H R3 Me N N N N B N N N L3 Me O Me OMe O OMe O R Me Me Ar R6 ; Me Me Me O Me H H N N L2 N N Me R7 O Me OMe O Me Me MeO NH L1 O Z Me Me Me O O Me R N H H N N (XII) L3 N N Me A O R7 O Me OMe O R3 R4 Me Me MeO NH R2 J Me O Z R3 Me Me HN O Me R1 H N N L4 N N Me R7 O Me OMe O Me Me MeO NH O Z ; Me Me Me O Me Me O H H H Me N N N N N N L2 R7 O Me OMe O OMe O Me Me Ar R6 R2 R1 L1 (XIII) Me Me Me R3 O Me Me O O A H H H R3 Me N N N N B N N N L3 R7 O Me OMe O H OMe O R Me Me Ar R6 Me Me Me O Me Me O H H H Me N N N N N N L4 R7 O Me OMe O H OMe O Me Me Ar R6 5 n: A is optional, and when present is lower alkylene, lower cycloalkylene, lower alkenylene, alkynylene, lower heteroalkylene, lower heterocycloalkylene, arylene, heteroarylene, lene, or aralkylene; B is optional, and when present is a linker selected from the group consisting of lower 5 alkylene, lower alkenylene, lower heteroalkylene, -O-, -O-(alkylene)-, -S-, -S- (alkylene)-, -S(O)k- where k is 1, 2, or 3, -S(O)k(alkylene)-, -C(O)-, -C(O)- (alkylene)-, -C(S)-, -C(S)-(alkylene)-, -N(R’)-, -NR’-(alkylene)-, -C(O)N(R’)- , -CON(R’)-(alkylene)-, -CSN(R’)-, -CSN(R’)-(alkylene)-, -N(R’)CO-(alkylene)- , -N(R’)C(O)O-, -S(O)kN(R’)-, -N(R’)C(O)N(R’)-, -N(R’)C(S)N(R’)- 10 , -N(R’)S(O)kN(R’)-, -N(R’)-N=, =N-, -C(R’)=N-N(R’)-, -C(R’)=NN =, -C(R’)2-N=N-, and -C(R’)2-N(R’)-N(R’)-, where each R’ is independently H, or alkyl; R is H, alkylor cycloalkyl; R1 is H, an amino protecting group, resin, at least one amino acid, polypeptide, or 15 polynucleotide; R2 is OH, an ester ting group, resin, at least one amino acid, polypeptide, or polynucleotide; R3 and R4 are each independently H, halogen, lower alkyl, or R 3 and R4 or two R3 groups optionally form a cycloalkyl or a heterocycloalkyl; 20 Z has the structure of: R5 ; R5 is H, CO2H, C1-C6alkyl, or thiazole; R6 is OH or H; Ar is phenyl or ne; 25 R7 is C1-C6alkyl or hydrogen; L1, L2, L3, and L4 are each s ndently ed from the group consisting of a bond, –alkylene–, –(alkylene–O)n–alkylene–J–, –alkylene'–J–(alkylene–O)n– alkylene–, –J–(alkylene–O)n–alkylene–, –(alkylene–O)n–alkylene–J–(alkylene– O)n'–alkylene–J'–, –(alkylene–O)n–alkylene–J–alkylene'–, –W–, –alkylene–W–, ne'–J–(alkylene–NMe)n–alkylene–W–, –J–(alkylene–NMe)n–alkylene–W–, – J–alkylene–NMe–alkylene'–NMe–alkylene''–W–, and –alkylene–J–alkylene'– NMe–alkylene''–NMe–alkylene'''–W–; W has the structure of: Me Me O O O N N H H 5 O NH2 ; each J and J' independently have the structure of: O O O N N O or N H , H , H ; and each n and n' are independently integers r than or equal to one. 10

29. The compound of claim 28, n R1 is a polypeptide.

30. The compound of claims 29, wherein the polypeptide is an dy.

31. The compound of claim 30, wherein the antibody is trastuzumab.

32. The compound of claim 28, wherein R2 is a polypeptide.

33. The compound of claims 32, wherein the polypeptide is an antibody. 15

34. The compound of claim 33, wherein the antibody is trastuzumab.

35. A method for derivatizing a dolastatin analog of Formula (I), (III), (IV), (V), or (VI) so as to produce a derivative of said dolastatin analog, the method comprising contacting the dolastatin analog with a reagent of Formula (XXXVII), wherein Formula (I), (III), (IV), (V), or (VI) correspond to: Me Me Me O Me H H L N N Me (I) Y N N R7 O Me OMe O Me Me MeO NH 20 O Z ; Me Me Me O Me H H N N L2 N N Me R7 O Me OMe O Me Me MeO NH L1 O Z Me (III) Y Me Me O Me H H N N L3 N N Me R7 O Me OMe O Me Me MeO NH O Z ; Me Me Me O Me H H Me N N N N Me Me O Me OMe O Me Me MeO NH O Ar HN L2 Me Me Me (IV) O Me R6 H H Me N N N N Me L1 Me O Me OMe O V Me Me MeO NH HN L3 Ar O R6 ; Me Me Me O Me H H N N L2 N N Me R7 O Me OMe O Me Me MeO NH L1 O Z Y Me Me Me O Me H H (V) N N L3 N N Me R7 O Me OMe O Me Me MeO NH J O Z Me Me Me O Me H H N N L4 N N Me R7 O Me OMe O Me Me MeO NH O Z ; Me Me Me O Me H H Me N N N N Me Me O Me OMe O Me Me MeO NH O Ar HN L2 Me Me Me O Me R6 H H Me N N L1 N N Me Me O Me OMe O Me Me MeO NH HN L3 (VI) Ar O Me Me Me O Me R6 H H J Me N N N N Me Me O Me OMe O Me Me MeO NH HN L4 Ar O R6 ; Z has the structure of: R5 ; 5 R5 is H, COR8, C1-C6alkyl, or thiazole; R8 is OH or –NH–(alkylene–O)n–NH2; R6 is OH or H; Ar is phenyl or pyridine; R7 is C1-C6 alkyl or hydrogen; 10 Y is NH2–O– or methyl; L, L1, L2, L3, and L4 are each linkers selected from the group consisting of a bond, – alkylene–, –alkylene–C(O)–, –(alkylene–O)n–alkylene–, –(alkylene–O)n–alkylene– C(O)–, –(alkylene–O)n–(CH2)n'–NHC(O)–(CH2)n''–C(Me)2–S–S–(CH2)n'''– NHC(O)–(alkylene–O)n''''–alkylene–, –(alkylene–O)n–alkylene–W–, –alkylene– 15 C(O)–W–, –(alkylene–O)n–alkylene–J–, –alkylene'–J–(alkylene–O)n–alkylene–, – (alkylene–O)n–alkylene–J–alkylene', kylene–O)n–alkylene–, –(alkylene–O)n– alkylene–J–(alkylene–O)n'–alkylene–J'–, –W–, –alkylene–W–, alkylene'–J– (alkylene–NMe)n–alkylene–W–, and J– (alkylene–NMe)n–alkylene–W–, – (alkylene–O)n–alkylene–U–alkylene–C(O)–, –(alkylene–O)n–alkylene–U– alkylene–; –J–alkylene–NMe–alkylene'–NMe–alkylene''–W–, and –alkylene–J– 5 alkylene'–NMe–alkylene''–NMe–alkylene'''–W–; W has the structure of: Me Me O O O N N H H O NH2 ; U has the structure of: CO2H 10 O each J and J' independently have the ure of: O O O N N O or N H , H , H or L is absent, Y is methyl, V is selected from the group consisting of an hydroxylamine, methyl, aldehyde, protected aldehyde, , ted 15 ketone, thioester, ester, dicarbonyl, hydrazine, amidine, imine, diamine, azide, keto-amine, lkyne, alkyne, cycloalkyne, and ene-dione; R5 is COR8, and R8 is –NH–(alkylene–O)n–NH2; and each n, n', n'', n''' and n'''' are independently integers greater than or equal to one; 20 wherein Formula (XXXVII) corresponds to: A K R3 B R (XXXVII) R1 R2 H R4 wherein: A is optional, and when present is lower alkylene, tuted lower alkylene, lower alkenylene, substituted lower alkenylene, arylene, substituted arylene, 5 heteroarylene, substituted heteroarylene, lene, substituted alkarylene, aralkylene, or substituted aralkylene; B is optional, and when present is a linker selected from the group consisting of lower alkylene, lower alkenylene, -O-, -O-()-, -S-, -S-(alkylene)-, - where k is 1, 2, or 3, -S(O)k(alkylene)-, -C(O)-, -C(O)-(alkylene)-, -C(S)-, -C(S)-(alkylene)-, - 10 N(R’)-, -NR’-(alkylene)-, -C(O)N(R’)-, -CON(R’)-(alkylene)-, -CSN(R’)- , -CSN(R’)-(alkylene)-, -N(R’)CO-(alkylene)-, -N(R’)C(O)O-, -S(O)kN(R’)- , C(O)N(R’)-, C(S)N(R’)-, -N(R’)S(O)kN(R’)-, -N(R’)-N=, - C(R’)=N-, -C(R’)=N-N(R’)-, -C(R’)=N-N=, 2-N=N-, and -C(R’)2-N(R’)-N(R’)-, where each R’ is independently H, or alkyl; 15 each R’ is independently H or alkyl; O O S OR' SR' R' O O K is , O , , , , , O , or O R' R is H, alkyl or cycloalkyl; R1 is H, an amino protecting group, resin, at least one amino acid, or polynucleotide; 20 R2 is OH, an ester protecting group, resin, at least one amino acid, or polynucleotide; R3 and R4 are each independently H, halogen, lower alkyl, or R 3 and R4 or two R3 groups optionally form a cycloalkyl or a heterocycloalkyl.

36. The method of claim 35, wherein the derivative of said dolastatin analog is at least one oxime containing amino acid having the ure of Formula (VIII), (IX), (X), (XI), (XII), or (XIII): Me Me Me O Me H H L N N O N N Me R N R7 O Me OMe O Me Me (VIII) MeO NH B O Z A O R3 R4 R3 R2 Me Me Me O Me H H Me R6 Me N N N N N (IX) Me O Me OMe O Me Me OMe O O NH R3 R3 R1 L NH O A N B O R2 5 R Me Me Me O Me H H N N L2 N N Me R7 O Me OMe O Me Me MeO NH L1 O Z O Me Me O Me (X) R N H H N N L3 N N Me A O R7 O Me OMe O Me Me MeO NH R3 R4 R2 O Z Me Me Me O Me Me O H H H Me N N N N N N L2 Me O Me OMe O OMe O Me Me Ar R6 R2 R1 L1 (XI) Me Me Me R3 O Me Me O A H O H H R3 Me N N N N B N N N L3 Me O Me OMe O OMe O R Me Me Ar R6 ; Me Me Me O Me H H N N L2 N N Me R7 O Me OMe O Me Me MeO NH L1 O Z O Me Me O Me R N H H N N (XII) L3 N N Me A O R7 O Me OMe O Me Me R3 R4 MeO NH J O Z R3 R2 Me Me Me HN O R1 H N N L4 N N Me R7 O Me OMe O Me Me MeO NH O Z ; Me Me Me O Me Me O H H H Me N N N N N N L2 R7 O Me OMe O OMe O Me Me Ar R6 R2 R1 L1 (XIII) Me Me Me R3 O Me Me O O A H H H R3 Me N N N N B N N N L3 R7 O Me OMe O H OMe O R Me Me Ar R6 Me J Me Me O Me Me O H H H Me N N N N N N L4 R7 O Me OMe O H OMe O Me Me Ar R6 5

37. The method of claim 35, wherein the dolastatin analog is contacted with the t of Formula (XXXVII) in aqueous solution under mildly acidic conditions.

38. A nd of Formula (XXV), (XXVI), (XXVII), (XXVIII), (XXIX), or (XXX): Me Me Me O Me H H H N L N N N N Me (R16)n H N R7 O Me OMe O R1 Me Me MeO NH (XXV) R4 O Z O R2 ; Me Me Me O Me H H Me R6 Me N N N N N R1 R7 O Me OMe O HN R2 (XXVI) Me Me OMe O O N L H H N R4 O (R16)n Me Me Me O Me Me H H H N N N L2 N N Z R7 O Me OMe O OMe O Me Me N L1 (R16)n Me Me Me H O Me (XXVII) H Me N H H R1 N N N R4 L3 N N Z O R2 R7 O Me OMe O 5 OMe O Me Me Me Me Me O Me Me O H H H Me N N N N N N L2 R7 O Me OMe O OMe O Me Me Ar R6 R1 HN R2 L1 (XXVIII) Me Me Me H O Me R4 O Me O N H H H Me N N N N N N L3 (R16)n R7 O Me OMe O H OMe O Me Me Ar R6 Me Me Me O Me H H N N L2 N N Me R7 O Me OMe O Me Me MeO NH N L1 O Z (R16)n Me Me Me H O Me N H H (XXIX) R1 N N R4 L3 N N Me O R2 R7 O Me OMe O Me Me MeO NH J O Z Me Me Me O Me H H N N L4 N N Me R7 O Me OMe O Me Me MeO NH O Z Me Me Me O Me Me O H H H Me N N N N N N L2 R7 O Me OMe O OMe O Me Me Ar R6 R1 HN R2 Me Me Me H O Me Me O N R4 O H H H Me N N N N N N L3 (R16)n (XXX) R7 O Me OMe O OMe O Me Me Ar R6 Me Me Me O Me Me O H H H Me N N N N N N L4 R7 O Me OMe O OMe O Me Me Ar R6 5 n: Z has the structure of: R5 ; R5 is H, CO2H, C1-C6alkyl, or thiazole; R6 is OH or H; 10 Ar is phenyl or pyridine; R1 is H, an amino protecting group, resin, at least one amino acid, polypeptide, or polynucleotide; R2 is OH, an ester protecting group, resin, at least one amino acid, polypeptide, or polynucleotide; 5 R4 is H, halogen, or lower alkyl; R7 is C1-C6alkyl or hydrogen; L, L1, L2, L3, and L4 are each linkers selected from the group consisting of a bond, – alkylene–, –alkylene–C(O)–, ene–J–, –(alkylene–O)n–alkylene–, –(alkylene– O)n–alkylene–C(O)–, –(alkylene–O)n–J–, lene–O)n–J–alkylene–, lene– 10 O)n–(CH2)n'–NHC(O)–(CH2)n''–C(Me)2–S–S–(CH2)n'''–NHC(O)–(alkylene–O)n''''– alkylene–, –(alkylene–O)n–alkylene–W–, –alkylene–C(O)–W–, –(alkylene–O)n– alkylene–J–, –alkylene'–J–(alkylene–O)n–alkylene–, –(alkylene–O)n–alkylene–J– alkylene', –J–(alkylene–O)n–alkylene–, –(alkylene–O)n–alkylene–J–(alkylene– lkylene–J'–, –W–, –alkylene–W–, alkylene'–J–(alkylene–NMe)n–alkylene– 15 W–, –J–(alkylene–NMe)n–alkylene–W–, –(alkylene–O)n–alkylene–U–alkylene– C(O)–, –(alkylene–O)n–alkylene–U–alkylene–; –J–alkylene–NMe–alkylene'– NMe–alkylene''–W–, and –alkylene–J–alkylene'–NMe–alkylene''–NMe– alkylene'''–W–; 20 W has the structure of: Me Me O O O N N H H O NH2 ; U has the structure of: CO2H each J and J' independently have the structure of: O O O N N O or N H , H , H ; each n and n' are independently integers greater than or equal to one; and each R16 is ndently selected from the group ting of hydrogen, halogen, alkyl, NO2, and CN. 5

39. The compound of claim 38, wherein R1 is a polypeptide.

40. The compound of claims 39, wherein the polypeptide is an antibody.

41. The compound of claim 40, wherein the antibody is trastuzumab.

42. The compound of claim 38, wherein R2 is a polypeptide.

43. The compound of claims 42, wherein the polypeptide is an antibody. 10

44. The compound of claim 43, wherein the dy is trastuzumab.

45. A compound of Formula (XXXI), (XXXII), (XXXIII), (XXXIV), (XXXV), or (XXXVI): Me Me Me O Me H R3 R3 H H N B L N N R1 A D N N Me R7 O Me OMe O R2 O Me Me MeO NH (XXXI) O Z ; Me Me Me O Me H H Me R6 Me N N N N N Ar ) R7 O Me OMe O Me Me OMe O O NH R3 R3 H L B N D A R1 O R2 ; Me Me Me O Me Me H H H N N N L2 N N Z R7 O Me OMe O OMe O R3 R3 Me Me N B L1 R1 A D R4 Me Me Me (XXXIII) O Me H Me R2 O H N N N L3 N N Z R7 O Me OMe O OMe O Me Me 15 ; Me Me Me O Me Me O H H H Me N N N N N N L2 R7 O Me OMe O OMe O Me Me Ar R6 R3 R3 L1 H B N Me Me Me D A R1 (XXXIII) O Me Me O R4 H H H Me N N N O R2 N N N L3 R7 O Me OMe O OMe O Me Me Ar R6 Me Me Me O Me H H N N L2 N N Me R7 O Me OMe O H R3 R3 Me Me MeO NH N B L1 O Z R1 A D Me Me Me R4 O Me H H R2 O N N (XXXV) L3 N N Me R7 O Me OMe O Me Me MeO NH J O Z Me Me Me O Me H H N N L4 N N Me R7 O Me OMe O Me Me MeO NH O Z Me Me Me O Me Me O H H H Me N N N N N N L2 R7 O Me OMe O OMe O Me Me Ar R6 R3 R3 H L1 B N Me Me Me D A R1 O Me Me O R4 H H H Me N N N O R2 N N N L3 R7 O Me OMe O OMe O Me Me Ar R6 ) Me Me Me O Me Me O H H H Me N N N N N N L4 R7 O Me OMe O OMe O Me Me Ar R6 5 ; wherein: Z has the structure of: R5 ; R5 is H, CO2H, C1-C6alkyl, or thiazole; R6 is OH or H; 5 Ar is phenyl or pyridine; R1 is H, an amino protecting group, resin, at least one amino acid, polypeptide, or polynucleotide; R2 is OH, an ester protecting group, resin, at least one amino acid, polypeptide, or polynucleotide; 10 R4 is H, halogen or lower alkyl; R7 is C1-C6alkyl or hydrogen; L, L1, L2, L3, and L4 are each linkers ed from the group consisting of a bond, – alkylene–, –alkylene–C(O)–, –alkylene–J–, –(alkylene–O)n–alkylene–, –(alkylene– O)n–alkylene–C(O)–, –(alkylene–O)n–J–, –(alkylene–O)n–J–alkylene–, –(alkylene– 15 O)n–(CH2)n'–NHC(O)–(CH2)n''–C(Me)2–S–S–(CH2)n'''–NHC(O)–(alkylene–O)n''''– alkylene–, –(alkylene–O)n–alkylene–W–, –alkylene–C(O)–W–, –(alkylene–O)n– alkylene–J–, ene'–J–(alkylene–O)n–alkylene–, –(alkylene–O)n–alkylene–J– alkylene', –J–(alkylene–O)n–alkylene–, –(alkylene–O)n–alkylene–J–(alkylene– O)n'–alkylene–J'–, –W–, –alkylene–W–, ne'–J–(alkylene–NMe)n–alkylene– 20 W–, –J–(alkylene–NMe)n–alkylene–W–, –(alkylene–O)n–alkylene–U–alkylene– C(O)–, –(alkylene–O)n–alkylene–U–alkylene–; –J–alkylene–NMe–alkylene'– NMe–alkylene''–W–, and –alkylene–J–alkylene'–NMe–alkylene''–NMe– alkylene'''–W–; 25 W has the structure of: Me Me O O O N N H H O NH2 ; U has the structure of: CO2H each J and J' independently have the structure of: O O O N N O or N 5 H , H , H ; each n and n' are independently integers greater than or equal to one; D has the structure of: R17 R17 R17 Z1 Z1 Z1 Z1 N N N Z1 R17 N N N N N Z1 N M3 NH Z2 N Z2 T , Z2 N , , Z2 T , Z3 T , R17 R17 R17 R17 R17 R17 R17 T3 R17 N R17 N R18 H Z1 Z3 R18 H N (R18)p M4 N M2 N N T3 , T3 , , R17 R17 m(R18) , H H H N N N N N or N , , N N H H , H (R19)q each R17 is independently ed from the group consisting of H, alkyl, alkenyl, 10 alkynyl, alkoxy, alkylalkoxy, polyalkylene oxide, aryl, heteroaryl, alkaryl, aralkyl, -(alkylene)-ON(R”)2, -(alkylene)-C(O)SR”, -(alkylene)-S-S- (aryl), -C(O)R”, -C(O)2R”, or -C(O)N(R”)2, wherein each R” is ndently hydrogen, alkyl, l, alkoxy, aryl, heteroaryl, alkaryl, or aralkyl; each Z1 is a bond, CR17R17, O, S, NR’, CR17R17-CR17R17, 7-O, O-CR17R17, CR17R17-S, S-CR17R17, CR17R17-NR’, or NR’-CR17R17; each R’ is H or alkyl; each Z2 is selected from the group consisting of a bond, -C(O)-, , C1-C3 5 alkylene, C1-C3 alkenylene, and heteroalkyl; each Z3 are independently selected from the group consisting of a bond, C1-C4 alkylene, C1-C4 lene, heteroalkyl, -O-, -S-, -C(O)-, -C(S)-, and -N(R’)-; each T3 is a bond, C(R’’)(R’’), O, or S; with the proviso that when T3 is O or S, R’’ cannot be halogen; 10 each R’’ is H, halogen, alkyl or cycloalkyl; m and p are 0, 1, 2, or 3, provided that at least one of m or p is not 0; (b) (b) C (b) C C (b) C C (b) C O (b) M2 is (a) R3 , (a) R4 R4 , (a) R4 , (a) R4 , (b) (b) (b) R3 C C (b) O C (b) C C (b) C S (b) R3 R4 (a) R4 , (a) , (a) , (a) , or S C (b) (a) , where (a) tes bonding to the B group and (b) indicates 15 bonding to respective positions within the heterocycle group; (b) (b) (b) (b) C (b) C C (b) C O (b) C S (b) M3 is (a) , (a) R4 , (a) , (a) , or C C (b) (a) , where (a) indicates bonding to the B group and (b) indicates bonding to respective positions within the heterocycle group; (b) (b) (b) R3 C C (b) O C (b) C (b) C C (b) M4 is (a) , (a) R3 R3 , (a) , (a) , or S C (b) (a) , where (a) indicates bonding to the B group and (b) indicates g to tive positions within the heterocycle group; each R19 is independently ed from the group ting of C1-C6 alkyl, C1-C6 5 alkoxy, ester, ether, thioether, aminoalkyl, halogen, alkyl ester, aryl ester, amide, aryl amide, alkyl halide, alkyl amine, alkyl sulfonic acid, alkyl nitro, thioester, sulfonyl ester, halosulfonyl, nitrile, alkyl nitrile, and nitro; q is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11; and each R18 is independently selected from the group consisting of hydrogen, n, 10 alkyl, NO2, CN, and alkyl.

46. The compound of claim 45, wherein R1 is a polypeptide.

47. The compound of claims 46, wherein the polypeptide is an antibody.

48. The compound of claim 47, wherein the antibody is zumab.

49. The compound of claim 45, wherein R2 is a polypeptide. 15

50. The compound of claims 49, wherein the polypeptide is an antibody.

51. The compound of claim 50, wherein the antibody is trastuzumab.

52. The compound of claim 45, wherein the compound is of Formula (XXXI-A): Me Me Me O Me H H L N N Me B N N N R3 A N N R7 O Me OMe O Me Me MeO NH (XXXI-A) R3 R4 O Z R1 R2 .

53. A pharmaceutical composition comprising a compound of any of claims 1-34 and 38-52 20 and a pharmaceutically acceptable carrier, excipient, or binder.