WO2019104070A1 - O-nitrobenzyl photocleavable bifunctional linker - Google Patents

Abstract

Disclosed herein is an ortho-nitrobenzyl compound, or a stereoisomer or salt thereof, and a composition comprising the same, as well as processes for the preparation of the same. Also disclosed herein is a method of using the ortho-nitrobenzyl compound, or a stereoisomer thereof, in probes capable of hybridizing to, detecting or sequencing nucleic acid molecules.

Description

O-NITROBENZYL PHOTOCLEAVABLE BIF UNCTION AL LINKER

RELATED APPLICATIONS

[0001] This application claims priority to, and the benefit of, U.S.S.N. 62/589,067, filed on November 21, 2017, the entire contents of which is incorporated by reference in its entirety.

INCORPORATION OF SEQUENCE LISTING

[0002] The contents of the text file named "NATE-035001 wO_SeqList_ST25," which was created on November 14, 2018, and is 1 KB in size, is hereby incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

[0003] Photocleavable ("PC") compounds play an important role as protecting groups in blocking functional groups present in nucleosides, nucleotides, sugars and amino acids, which are used for the synthesis of biomolecules, e.g. nucleic acids and their derivatives, proteins, peptides and carbohydrates. Such compounds have the advantage that deprotection of the protected functional group can be performed simply via light exposure. Therefore, photocleavable compounds provide the basis for the photolithography based spatially resolved synthesis of oligonucleotides or peptides on solid supports, such as microarrays. The use of photolabile compounds for the synthesis of microarrays is well known (Pease, et al. Proc. Natl. Acad. Sci. USA 91 (1994) 5022-5026), Hasan, et al. Tetrahedron 53 (1997) 4247- 4264).

[0004] Photocleavable compounds can also be used as photocleavable linker molecules to link different elements, such as two biomolecules, a biomolecule and a solid phase, a biomolecule and streptavidin. For example, two different oligonucleotides may be connected via the photocleavable compound in order to deliver a single stranded molecule to a biochemical or biological assay, which upon irradiation with light releases the two individual oligonucleotides which then may form a double stranded DNA and exhibit biological function. The elements, which are photocleavably connected by the above mentioned compounds can be separated from each other simply due to light exposure. Such components are well known (Olejnik, et al. Proc. Natl. Acad. Sci. USA 92 (1995) 7590-7594; Olejnik, et al. Nucleic Acids Res. 26 (1998) 3572-3576; Olejnik, et al. Nucleic Acids Res. 27 (1999) 4626-4631). [0005] There is a continued need in the art for sensitive photocleavable linkers, as well as improved methods of detecting nucleic acid molecules using these linkers. The present disclosure addresses these needs.

SUMMARY OF THE INVENTION

[0006] The present disclosure provides a compound of the Formula (I):

or a stereoisomer or salt thereof, wherein: R₁ is hydrogen, halogen, C_1-6alkyl, C_2-6alkenyl, C_{2- 6}alkynyl, wherein said C_1-6alkyl, C_2-6alkenyl, C_2-6alkynl are each independently optionally substituted with at least one substituent R₁₀; R2 is O, NH, or N(C_1-6alkyl); R3 is cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each optionally substituted with at least one substituent R₁₀; each R₄ and R₇ are independently C_1-6alkylene, C_2-6alkenylene, C_2-6alkynylene, wherein said C_1-6alkylene, C_2-6alkenylene, C_2-6alkynlene are each independently optionally substituted with at least one substituent R₁₀; R₅ and R₉ are each independently cycloalkyl,

heterocycloalkyl, aryl, or heteroaryl, each optionally substituted with at least one substituent R₁₀; R₆ is O, NH or N( C_1-6alkyl); Rs is O, NH, or N(C_1-6alkyl); each R₁₀ is independently hydrogen, halogen, -C_1-6alkyl, -C_2-6alkenyl, -C_2-6alkynyl, haloC_1-6alkyl, haloC_2-6alkenyl, haloC_2-6alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, -CN, -NO2, oxo, -OR11, -SO2R11, - SO₃-, -COR11, -CO2R11, -CONR11R12, -C(=NR₁₁)NR₁2R₁3, -NR11R12, -NR12COR12, - NR11CONR12R13, -NR11CO2R12, -NR11SONR12R13, -NR11SO2NR12R13, or -NR11SO2R12; and R11, R12, and R13, which may be the same or different, are each independently hydrogen, - C_{1- 6}alkyl, -C_2-6alkenyl, -C_2-6alkynyl, haloC_1-6alkyl, haloC_2-6alkenyl, haloC_2-6alkynyl, C_{1- 6}alkyloxyC_1-6alkyl-, cycloalkyl, heterocyclyl, aryl, or heteroaryl.

[0007] In one aspect, R₁ is C_1-6alkyl, preferably C_1-3alkyl such as methyl, ethyl, propyl or isopropyl; R2 is NH or N(C_1-6alkyl); R3 is a 5- to 6-membered cycloalkyl, preferably cyclohexyl; R₄ is C_1-6alkylene, preferably C_1-3alkylene such as methylene, ethylene, propylene, or isopropylene; Rs is a 5- to 6- membered heterocyclyl comprising one nitrogen atom and 0 or 1 additional heteroatoms selected from N, O and S, wherein said heterocyclyl is optionally substituted with one or two R₁₀; R₆ is O; R₇ is C_1-6alkylene, preferably C_1- 3alkylene such as methylene, ethylene, propylene, or isopropylene; R₈ is O; R₉ is a 5- to 6- membered heterocyclyl comprising one nitrogen atom and 0 or 1 additional heteroatoms selected from N, O and S, wherein said heterocyclyl is optionally substituted with one or two R₁₀; and each R₁₀ is independently halogen, C_1-6alkyl, haloC_1-6alkyl, oxo, -SO₂H, or - SO₃ ^".

[0008] In one aspect, R₃ is cyclohexyl, R₄ is methylene, R₅ is lH-pyrrole-2,5-dione, and R₉ is pyrrolidine-2,5-dione, optionally substituted with SO₃ ^".

[0009] In a preferred aspect, the compound is

or a stereoisomer or salt thereof.

[0010] In a preferred aspect, the compound is

or a stereoisomer or salt thereof.

[0011] The present disclosure also provides a compound of the Formula (Π):

or a stereoisomer or salt thereof, wherein:

R21 is hydrogen, halogen, C_1-6alkyl, C_2-6alkenyl, C_2-6alkynyl, wherein said C_1-6alkyl, C_2- ealkenyl, C_2-6alkynl are each independently optionally substituted with at least one substituent R30;

R22 is O, NH, orN(C_1-6alkyl);

R23 is cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each optionally substituted with at least one substituent R30;

R24 is C_1-6alkylene, C_2-6alkenylene, C_2-6alkynylene, wherein said C_1-6alkylene, C_2- ealkenylene, C_2-6alkynlene are each independently optionally substituted with at least one substituent R30;

R26 is a bond, O, NH or N(C_1-6alkyl);

R27 is a bond, C_1-6alkylene, C_2-6alkenylene, C_2-6alkynylene, wherein said C_1-6alkylene, C_2- ealkenylene, C_2-6alkynlene are each independently optionally substituted with at least one substituent R30;

provided that when R26 is a bond, then R27 is also a bond;

R28 is OP(OR₁XNR31R32);

each R30 is independently hydrogen, halogen, -C_1-6alkyl, -C_2-6alkenyl, -C_2-6alkynyl, haloC_{1- 6}alkyl, halo C_2-6alkenyl, haloC_2-6alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, -CN, -

NO2, OXO, -OR31, -SO2R31, -SO₃ ^", -COR31, -CO2R31, -CONR31R32, -C(=NR3l)NR32R33, -

NR31R32, -NR32COR32, -NR31CONR32R33, -NR31C02R32, -NR31SONR32R33, -

NR31SO2NR32R33, or -NR31S02R32; and

R31, R32, and R33, which may be the same or different, are each independently hydrogen, -C_{1- 6}alkyl, -C_2-6alkenyl, -C_2-6alkynyl, haloC_1-6alkyl, haloC_2-6alkenyl, haloC_2-6alkynyl, C_{1- 6}alkyloxyC_1-6alkyl-, cycloalkyl, heterocyclyl, aryl, heteroaryl, or a protecting group.

[0012] The present disclosure also provides a probe comprising a first region comprising a first target-specific sequence and a second region which does not overlap with the first region; second region comprising a first label attachment region which is hybridized to a first DNA or RNA molecule, wherein the first DNA or RNA molecule is attached to one or more label monomers that emit light constituting a first signal; and a second label attachment region, which is non-overlapping to the first label attachment region, and which is hybridized to a second DNA or RNA molecule, wherein the second DNA or RNA molecule is attached to one or more label monomers that emit light constituting a second signal, and wherein the label attachment regions do not overlap; and a compound of Formula I disclosed herein linking the first region and the second region.

[0013] The present disclosure also provides a composition comprising a first probe and a second probe, the first probe comprising a first region comprising a first target-specific sequence; a second region which does not overlap with the first region, the second region comprising a first label attachment region which is hybridized to a first DNA or RNA molecule, wherein the first DNA or RNA molecule is attached to one or more label monomers that emit light constituting a first signal; and a second label attachment region, which is non-overlapping to the first label attachment region, and which is hybridized to a second DNA or RNA molecule, wherein the second DNA or RNA molecule is attached to one or more label monomers that emit light constituting a second signal, and wherein the label attachment regions do not overlap; and a compound of Formula I disclosed herein linking the first region and the second region; the second probe comprising a first region comprising a first target-specific sequence, wherein the target-specific sequence of the first probe and the target-specific sequence of the second probe are different; and a second region that does not overlap with the first region, does not bind to the target molecule and comprises at least one affinity moiety.

[0014] The present disclosure also provides methods of detecting nucleic acid molecules utilizing the probes and compositions disclosed herein.

[0015] Any of the above aspects can be combined with any other aspect.

[0016] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0017] As used herein, the singular forms of a word also include the plural form of the word, unless the context clearly dictates otherwise; as examples, the terms "a," "an," and "the" are understood to be singular or plural and the term "or" is understood to be inclusive. By way of example, "an element" means one or more element.

[0018] Throughout the specification the word "comprising," or variations such as

"comprises," will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps. Throughout the specification the word

"consisting of," or variations such as "consists of," will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, and the exclusion of any other element, integer or step, or group of elements, integers or steps. Throughout the specification the word "consisting essentially of," or variations such as "consists essentially of," will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, and any other element, integer or step, or group of elements, integers or steps that do not materially affect the basic and novel characteristics of the claimed invention.

[0019] About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from the context, all numerical values provided herein are modified by the term "about."

[0020] Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The references cited herein are not admitted to be prior art to the claimed disclosure. In the case of conflict, the present Specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting. Other features and advantages of the disclosure will be apparent from the following detailed description and claim.

BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 is a schematic illustration of an exemplary probe comprising a linker of the present disclosure.

[0022] Figure 2 is a schematic illustration of an exemplary nanreporter probe complex comprising a linker of the present disclosure.

[0023] Figure 3 is a schematic illustration of an exemplary sequencing method using a probe comprising a linker modification of the present disclosure.

[0024] Figure 4 is a schematic illustration of an exemplary probe complex and the locations within the complex that may comprise linkers of the present disclosure.

DETAILED DESCRIPTION OF THE INVENTION [0025] The present disclosure provides a compound of the Formula (I):

or a stereoisomer or salt thereof, wherein:

R₁ is hydrogen, halogen, C_1-6alkyl, C_2-6alkenyl, C_2-«alkynyl, wherein said C_1-6alkyl, C_2- ealkenyl, C_2-6alkynl are each independently optionally substituted with at least one substituent R₁₀;

R2 is O, NH, orN(C_1-6alkyl);

R3 is cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each optionally substituted with at least one substituent R₁₀;

each R» and R₇ are independently C_1-6alkylene, C_2-6alkenylene, C_2-6alkynylene, wherein said C_1-6alkylene, C_2-6alkenylene, C_2-6alkynlene are each independently optionally substituted with at least one substituent R₁₀;

R5 and R₉ are each independently cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each optionally substituted with at least one substituent R₁₀;

R₆ is O, NH or N(C_1-6alkyl);

R₈ is O, NH, orN(C_1-6alkyl);

each R₁₀ is independently hydrogen, halogen, -C_1-6alkyl, -C_2-6alkenyl, -C_2-6alkynyl, haloC_{1- 6}alkyl, halo C_2-6alkenyl, haloC_2-6alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, -CN, - NO2, oxo, -OR11, -SO2R11, -SO₃H, -COR11, -CO2R11, -CONR11R12, -C(=NR₁₁)NR₁2R₁3, - NR11R12, -NR12COR12, -NR11CONR12R13, -NR11CO2R12, -NR11SONR12R13, - NR11SO2NR12R13, or -NR11SO2R12; and

R11, R12, and R13, which may be the same or different, are each independently hydrogen, -C_{1- 6}alkyl, -C_2-6alkenyl, -C_2-6alkynyl, haloC_1-6alkyl, haloC_2-6alkenyl, haloC_2-6alkynyl, C_{1- 6}alkyloxyC_1-6alkyl-, cycloalkyl, heterocyclyl, aryl, or heteroaryl.

[0026] In an aspect, provided is a compound of Formula (I) wherein

R₁ is C_1-6alkyl, preferably C_1-3alkyl such as methyl, ethyl, propyl or isopropyl;

R2 is NH orN(C_1-6alkyl);

R3 is a 5- to 6-membered cycloalkyl, preferably cyclohexyl; R₄ is C_1-6alkylene, preferably C_1-3alkylene such as methylene, ethylene, propylene, or isopropylene;

R5 is a 5- to 6- membered heterocyclyl comprising one nitrogen atom and 0 or 1 additional heteroatoms selected from N, O and S, wherein said heterocyclyl is optionally substituted with one or two R₁₀;

R₆ is O;

R₇ is C_1-6alkylene, preferably C_1-3alkylene such as methylene, ethylene, propylene, or isopropylene;

R₈ is O;

R₉ is a 5- to 6- membered heterocyclyl comprising one nitrogen atom and 0 or 1 additional heteroatoms selected from N, O and S, wherein said heterocyclyl is optionally substituted with one or two R₁₀; and

each R₁₀ is independently halogen, C_1-6alkyl, haloC_1-6alkyl, oxo, -SO2H, or -SO₃H.

[0027] In an aspect, R3 is cyclohexyl and R» is methylene.

[0028] In an aspect, Rs is lH-pyrrole-2,5-dione.

[0029] In an aspect,R₉ is pyrrolidine-2,5-dione, optionally substituted with SO₃H.

[0030] In an aspect is provided a compound

or a stereoisomer or salt thereof.

[0031] In an aspect is provided a compound

or a stereoisomer or salt thereof.

[0032] In an aspect is provided a compound of the Formula (Π):

or a stereoisomer or salt thereof, wherein:

R21 is hydrogen, halogen, C_1-6alkyl, C_2-«alkenyl, C_2-6alkynyl, wherein said C_1-6alkyl, C_{2- 6}alkenyl, C_2-6alkynl are each independently optionally substituted with at least one substituent R30;

R22 is O, NH, orN(C_1-6alkyl);

R23 is cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each optionally substituted with at least one substituent R30;

R24 is C_1-6alkylene, C_2-«alkenylene, C_2-6alkynylene, wherein said C_1-6alkylene, C_{2- 6}alkenylene, C_2-6alkynlene are each independently optionally substituted with at least one substituent R30;

R26 is a bond, O, NH or N(C^alkyl);

R27 is a bond, C_1-6alkylene, C_2-6alkenylene, C_2-6alkynylene, wherein said C_1-6alkylene, C_{2- 6}alkenylene, C_2-6alkynlene are each independently optionally substituted with at least one substituent R30;

provided that when R26 is a bond, then R27 is also a bond;

R28 is OP(OR₁XNR31R32); each R30 is independently hydrogen, halogen, -C_1-6alkyl, -C_2-«alkenyl, -C_2-6alkynyl, haloC_{1- 6}alkyl, haloC_2-6alkenyl, haloC_2-6alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, -CN, -

NO2, OXO, -OR31, -SO2R31, -SO₃ , -COR31, -CO2R31, -CONR31R32, -C(=NR3l)NR32R33, -

NR31R32, -NR32COR32, -NR31CONR32R33, -NR31C02R32, -NR31SONR32R33, -

NR31SO2NR32R33, or-NR31S02R32; and

R31, R32, and R33, which may be the same or different, are each independently hydrogen, -C_{1- 6}alkyl, -C_2-«alkenyl, -C_2-6alkynyl, haloC_1-6alkyl, haloC_2-6alkenyl, haloC_2-6alkynyl, C_{1- 6}alkyloxyC_1-6alkyl-, cycloalkyl, heterocyclyl, aryl, heteroaryl, or a protecting group.

[0033] In an aspect, provided is a compound of Formula (Π) wherein

R21 is C_1-6alkyl, preferably C_1-3alkyl such as methyl, ethyl, propyl or isopropyl;

R22 is NH or N(C_1-6alkyl);

R23 is a 5- to 6-membered cycloalkyl, preferably cyclohexyl;

R24 is C_1-6alkylene, preferably C_1-3alkylene such as methylene, ethylene, propylene, or isopropylene, substituted with -OR31;

R26 is O;

R27 is C_1-6alkylene, preferably C_1-3alkylene such as methylene, ethylene, propylene, or isopropylene;

R28 is a phosphoramidite moiety; and

R31 is hydrogen or a hydroxyl protecting group, such as DMT.

[0034] In an aspect, R23 is cyclohexyl and R24 is methylene substituted with -OH, optionally protected by a protecting group.

[0035] In an aspect is provided a compound

wherein PG is hydrogen or a protecting group, and wherein n is 1-3, or a stereoisomer or salt thereof.

[0036] In an aspect is provided a compound

wherein PG is hydrogen or a protecting group, or a stereoisomer or salt thereof.

[0037] In an aspect is provided a compound

wherein PG is hydrogen or a protecting group, or a stereoisomer or salt thereof.

[0038] As used herein, including the appended claims, the singular forms of words such as "a", "an", and "the", include their corresponding plural references unless the context clearly dictates otherwise.

[0039] The term "or" is used to mean, and is used interchangeably with, the term "and/or" unless the context clearly dictates otherwise.

[0040] The term "alkyl" herein refers to a hydrocarbon group selected from linear and branched saturated hydrocarbon groups comprising from 1 to 18, such as from 1 to 12, further such as from 1 to 10, more further such as from 1 to 8, or from 1 to 6, or from 1 to 4, carbon atoms. Examples of alkyl groups comprising from 1 to 6 carbon atoms (i.e., C_1-6 alkyl) include, but not limited to methyl, ethyl, 1-propyl or n-propyl ("n-Pr"), 2-propyl or isopropyl ("i-Pr"), 1 -butyl or n-butyl ("n-Bu"), 2-methyl- 1-propyl or isobutyl ("i-Bu"), 1- methylpropyl or s-butyl ("s-Bu"), 1,1 -dimethyl ethyl or t-butyl ("t-Bu"), 1-pentyl, 2-pentyl, 3- pentyl, 2-methyl-2-butyl, 3-methyl-2-butyl, 3 -methyl- 1 -butyl, 2-methyl- 1 -butyl, 1-hexyl, 2- hexyl, 3-hexyl, 2-methyl -2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 3-methyl-3-pentyl, 2-methyl-3-pentyl, 2,3-dimethyl-2-butyl and 3,3-dimethyl-2-butyl groups. The term also encompasses saturated hydrocarbon groups present in bivalent form, also referred to herein as "alkylene" groups, with exemplary groups including methylene, ethylene, 1 -propylene, and the like.

[0041] The term "halogen" herein refers to fluoro (F), chloro (CI), bromo (Br) and iodo (I).

[0042] The term "haloalkyl" herein refers to an alkyl group in which one or more hydrogen is/are replaced by one or more halogen atoms such as fluoro (F), chloro (CI), bromo (Br), and iodo (I). Examples of the haloalkyl include haloC_1-6alkyl, haloC_1-6alkyl or halo C_1-4alkyl, but not limited to -CF₃, -CH2CI, -CH2 CF3, -CCI2, CF₃, and the like.

[0043] The term "alkenyl" herein refers to a hydrocarbon group selected from linear and branched hydrocarbon groups comprising at least one C=C double bond and from 2 to 18, such as from 2 to 8, further such as from 2 to 6, carbon atoms. Examples of the alkenyl group, e.g., C_2-6 alkenyl, include, but not limited to ethenyl or vinyl, prop-l-enyl, prop-2- enyl, 2-methylprop-l-enyl, but-l-enyl, but-2-enyl, but-3-enyl, buta-l,3-dienyl, 2-methylbuta- 1,3-dienyl, hex-l-enyl, hex-2-enyl, hex-3-enyl, hex-4-enyl, and hexa-l,3-dienyl groups. The term also encompasses the corresponding hydrocarbon groups present in bivalent form, also referred to herein as "alkenylene" groups, with exemplary groups including ethenylene, vinylene, and the like.

[0044] The term "alkynyl" herein refers to a hydrocarbon group selected from linear and branched hydrocarbon group, comprising at least one C≡C triple bond and from 2 to 18, such as 2 to 8, further such as from 2 to 6, carbon atoms. Examples of the alkynyl group, e.g., C_2-6 alkynyl, include, but not limited to ethynyl, 1-propynyl, 2-propynyl (propargyl), 1-butynyl, 2- butynyl, and 3-butynyl groups. The term also encompasses the corresponding hydrocarbon groups present in bivalent form, also referred to herein as "alkynylene" groups, with exemplary groups including ethynylene, 1-propynylene, and the like.

[0045] The term "alkyloxy" herein refers to an alkyl group as defined above bonded to oxygen, represented by -Oalkyl. Examples of an alkyloxy, e.g., C_1-6alkyloxy or CM alkyloxy includes, but not limited to, methoxy, ethoxy, isopropoxy, propoxy, n-butoxy, tert-butoxy, pentoxy and hexoxy and the like.

[0046] The term "cycloalkyl" herein refers to a hydrocarbon group selected from saturated and partially unsaturated cyclic hydrocarbon groups, comprising monocyclic and polycyclic (e.g., bicyclic and tricyclic) groups. For example, the cycloalkyl group may comprise from 3 to 12, such as from 3 to 10, further such as 3 to 8, further such as 3 to 6, 3 to 5, or 3 to 4 carbon atoms. Even further for example, the cycloalkyl group may be selected from monocyclic group comprising from 3 to 12, such as from 3 to 10, further such as 3 to 8, 3 to 6 carbon atoms. Examples of the monocyclic cycloalkyl group include cyclopropyl, cyclobutyl, cyclopentyl, 1-cyclopent-l-enyl, l-cyclopent-2-enyl, l-cyclopent-3-enyl, cyclohexyl, 1-cyclohex-l-enyl, l-cyclohex-2-enyl, l-cyclohex-3-enyl, cyclohexadienyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, cycloundecyl, and cyclododecyl groups. In particular, Examples of the saturated monocyclic cycloalkyl group, e.g., C3-8 cycloalkyl, include, but not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl groups. In a preferred aspect, the cycloalkyl is a monocyclic ring comprising 3 to 6 carbon atoms (abbreviated as C3-6 cycloalkyl), including but not limited to, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. Examples of the bicyclic cycloalkyl groups include those having from 7 to 12 ring atoms arranged as a bicyclic ring selected from [4,4], [4,5], [5,5], [5,6] and [6,6] ring systems, or as a bridged bicyclic ring selected from

bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane, and bicyclo[3.2.2]nonane. Further Examples of the bicyclic cycloalkyl groups include those arranged as a bicyclic ring selected from [5,6]

4. f and [6,6] ring systems, such as

^f^-^ and wherein the wavy lines indicate the points of attachment. The ring may be saturated or have at least one double bond (i.e.

partially unsaturated), but is not fully conjugated, and is not aromatic, as aromatic is defined herein.

[0047] The term "aryl" used alone or in combination with other terms refers to a group selected from: 5- and 6-membered carbocyclic aromatic rings, e.g., phenyl; bicyclic ring systems such as 7 to 12 membered bicyclic ring systems, wherein at least one ring is carbocyclic and aromatic, e.g., naphthyl and indanyl; and, tricyclic ring systems such as 10 to 15 membered tricyclic ring systems wherein at least one ring is carbocyclic and aromatic, e.g., fluorenyl. The terms "aromatic hydrocarbon ring" and "aryl" are used interchangeable throughout the disclosure herein. In some aspects, a monocyclic or bicyclic aromatic hydrocarbon ring has 5 to 10 ring-forming carbon atoms (i.e., C5-10 aryl). Examples of a monocyclic or bicyclic aromatic hydrocarbon ring includes, but not limited to, phenyl, naphth-l-yl, naphth-2-yl, anthracenyl, phenanthrenyl, and the like. In some aspects, the aromatic hydrocarbon ring is a naphthalene ring (naphth-l-yl or naphth-2-yl) or phenyl ring. In some aspects, the aromatic hydrocarbon ring is a phenyl ring.

[0048] The term "heteroaryl" herein refers to a group selected from: 5-, 6- or 7-membered aromatic, monocyclic rings comprising at least one heteroatom, for example, from 1 to 4, or, in some aspects, from 1 to 3, in some aspects, from 1 to 2, heteroatoms, selected from nitrogen (N), sulfur (S) and oxygen (O), with the remaining ring atoms being carbon; 8- to 12-membered bicyclic rings comprising at least one heteroatom, for example, from 1 to 4, or, in some aspects, from 1 to 3, or, in other aspects, 1 or 2, heteroatoms, selected from N, O, and S, with the remaining ring atoms being carbon and wherein at least one ring is aromatic and at least one heteroatom is present in the aromatic ring; and 11- to 14-membered tricyclic rings comprising at least one heteroatom, for example, from 1 to 4, or in some aspects, from 1 to 3, or, in other aspects, 1 or 2, heteroatoms, selected from N, O, and S, with the remaining ring atoms being carbon and wherein at least one ring is aromatic and at least one heteroatom is present in an aromatic ring. In a preferred aspect, heteroaryl is 5- to 6-membered heteroaryl comprising one nitrogen atom and 0 or 1 additional heteroatom selected from N, O and S, including but not limited to pyridinyl, isoxazolyl, and oxazolyl.

[0049] When the total number of S and O atoms in the heteroaryl group exceeds 1, those heteroatoms are not adjacent to one another. In some aspects, the total number of S and O atoms in the heteroaryl group is not more than 2. In some aspects, the total number of S and O atoms in the aromatic heterocycle is not more than 1. When the heteroaryl group contains more than one heteroatom ring member, the heteroatoms may be the same or different. The nitrogen atoms in the ring(s) of the heteroaryl group can be oxidized to form N-oxides.

[0050] The terms "aromatic heterocyclic ring" and "heteroaryl" are used interchangeable throughout the disclosure herein. In some aspects, a monocyclic or bicyclic aromatic heterocyclic ring has 5-, 6-, 7-, 8-, 9- or 10-ring forming members with 1, 2, 3, or 4 heteroatom ring members independently selected from nitrogen (N), sulfur (S) and oxygen (O) and the remaining ring members being carbon. In some aspects, the monocyclic or bicyclic aromatic heterocyclic ring is a monocyclic or bicyclic ring comprising 1 or 2 heteroatom ring members independently selected from nitrogen (N), sulfur (S) and oxygen (O). In some aspects, the monocyclic or bicyclic aromatic heterocyclic ring is a 5- to 6- membered heteroaryl ring, which is monocyclic and which has 1 or 2 heteroatom ring members independently selected from nitrogen (N), sulfur (S) and oxygen (O). In some aspects, the monocyclic or bicyclic aromatic heterocyclic ring is a 8- to 10-membered heteroaryl ring, which is bicyclic and which has 1 or 2 heteroatom ring members

independently selected from nitrogen, sulfur and oxygen.

[0051] Examples of the heteroaryl group or the monocyclic or bicyclic aromatic heterocyclic ring include, but are not limited to, (as numbered from the linkage position assigned priority 1) pyridyl (such as 2-pyridyl, 3-pyridyl, or 4-pyridyl), cinnolinyl, pyrazinyl, 2,4-pyrimidinyl, 3,5-pyrimidinyl, 2,4-imidazolyl, imidazopyridinyl, isoxazolyl, oxazolyl, thiazolyl, isothiazolyl, thiadiazolyl (such as 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, or 1,3,4-thiadiazolyl), tetrazolyl, thienyl (such as thien-2-yl, thien-3-yl), triazinyl, benzothienyl, furyl or furanyl, benzofuryl, benzoimidazolyl, indolyl, isoindolyl, indolinyl, oxadiazolyl (such as 1,2,3- oxadiazolyl, 1,2,4-oxadiazolyl, or 1,3,4-oxadiazolyl), phthalazinyl, pyrazinyl, pyridazinyl, pyrrolyl, triazolyl (such as 1,2,3-triazolyl, 1,2,4-triazolyl, or 1,3,4-triazolyl), quinolinyl, isoquinolinyl, pyrazolyl, pyrrolopyridinyl (such as lH-pyrrolo[2,3-b]pyridin-5-yl), pyrazolopyridinyl (such as lH-pyrazolo[3,4-b]pyridin-5-yl), benzoxazolyl (such as benzo[d]oxazol-6-yl), pteridinyl, purinyl, l-oxa-2,3-diazolyl, l-oxa-2,4-diazolyl, l-oxa-2,5- diazolyl, l-oxa-3,4-diazolyl, l-thia-2,3-diazolyl, l-thia-2,4-diazolyl, l-thia-2,5-diazolyl, 1- thia-3,4-diazolyl, furazanyl (such as furazan-2-yl, furazan-3-yl), benzofurazanyl,

benzothiophenyl, benzothiazolyl, benzoxazolyl, quinazolinyl, quinoxalinyl, naphthyridinyl, furopyridinyl, benzothiazolyl (such as benzo[d]thiazol-6-yl), indazolyl (such as lH-indazol- 5-yl) and 5,6,7,8-tetrahydroisoquinoline.

[0052] The term "heterocyclic" or "heterocycle" or "heterocyclyl" herein refers to a ring selected from 4-, 5-, 6-, 7-, 8-, 9-, 10-, 11- or 12-membered monocyclic, bicyclic and tricyclic, saturated and partially unsaturated rings comprising at least one carbon atoms in addition to at least one heteroatom, such as from 1-4 heteroatoms, further such as from 1-3, or further such as 1 or 2 heteroatoms, selected from nitrogen (N), sulfur (S) and oxygen (O). In some aspects, a heterocyclyl group is 4-, 5-, 6- or 7-membered monocyclic ring with at least one heteroatom selected from N, O and S. "Heterocycle" herein also refers to a 5- to 7- membered heterocyclic ring comprising at least one heteroatom selected from N, O, and S fused with 5-, 6-, and /or 7-membered cycloalkyl, carbocyclic aromatic or heteroaromatic ring, provided that the point of attachment is at the heterocyclic ring when the heterocyclic ring is fused with a carbocyclic aromatic or a heteroaromatic ring, and that the point of attachment can be at the cycloalkyl or heterocyclic ring when the heterocyclic ring is fused with cycloalkyl. "Heterocycle" herein also refers to an aliphatic spirocyclic ring comprising at least one heteroatom selected from N, O, and S, provided that the point of attachment is at the heterocyclic ring. The rings may be saturated or have at least one double bond (i.e.

partially unsaturated). The heterocycle may be substituted with oxo. The point of the attachment may be carbon or heteroatom in the heterocyclic ring. A heterocycle is not a heteroaryl as defined herein. In a preferred aspect, heterocyclyl is 5- to 6-membered heterocyclyl comprising one nitrogen atom and 0 or 1 additional heteroatom selected from N, O and S, including but not limited to pyrrolyl, dihydropyridine, morpholino, morpholinyl and tetraphydropyranyl. In other aspect, the substituted 5- to 6-membered heterocyclyl comprising one nitrogen atom and 0 or 1 additional heteroatom selected from N, O and S, includes, but not limited, γ-butyrolactam, δ-valerolactam, piperazin-2-one, pyrrolidine-2,5- dione, pyridin-2(lH)-one, l,5-dihydro-2H-pyrrol-2-one, pyrrolidin-2-one or lH-pyrrole-2,5- dione group.

[0053] Examples of the heterocycle include, but not limited to, (as numbered from the linkage position assigned priority 1) 1-pyrrolidinyl, 2-pyrrolidinyl, 2,4-imidazolidinyl, 2,3- pyrazolidinyl, 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-piperidinyl, 2,5-piperazinyl, pyranyl, morpholinyl, morpholino, 2-morpholinyl, 3-morpholinyl, oxiranyl, aziridinyl, thiiranyl, azetidinyl, oxetanyl, thietanyl, 1,2-dithietanyl, 1,3-dithietanyl, dihydropyridinyl, tetrahydropyridinyl, thiomorpholinyl, thioxanyl, piperazinyl, homopiperazinyl,

homopiperidinyl, azepanyl, oxepanyl, thiepanyl, 1,4-oxathianyl, 1,4-dioxepanyl, 1,4- oxathiepanyl, 1,4-oxaazepanyl, 1,4-dithiepanyl, 1,4-thiazepanyl and 1,4-diazepanyl, 1,4- dithianyl, 1,4-azathianyl, oxazepinyl, diazepinyl, thiazepinyl, dihydrothienyl, dihydropyranyl, dihydrofuranyl, tetrahydrofuranyl, tetrahydrothienyl, tetrahydropyranyl,

tetrahydrothiopyranyl, 1 -pyrrolinyl, 2-pyrrolinyl, 3-pyrrolinyl, indolinyl, 2H-pyranyl, 4H- pyranyl, 1,4-dioxanyl, 1,3-dioxolanyl, pyrazolinyl, pyrazolidinyl, dithianyl, dithiolanyl, pyrazolidinyl, imidazolinyl, pyrimidinonyl, l,l-dioxo-thiomoφholinyl, 3- azabicyco[3.1.0]hexanyl, 3-azabicyclo[4.1.0]heptanyl and azabicyclo[2.2.2]hexanyl. A substituted heterocycle also includes a ring system substituted with one or more oxo moieties, such as piperidinyl N-oxide, morpholinyl-N-oxide, 1 -oxo- 1 -thiomorpholinyl and 1,1-dioxo-l- thiomorpholinyl.

[0054] The term "protecting group" as used herein refers to a group used to mask a moiety which would be reactive or labile under conditions used in a chemical reaction. In some cases, the appropriate protecting group is subject to cleavage after the chemical reaction. In the case of hydroxyl (alcohol) protecting groups, exemplary groups include those applicable to oligonucleotide synthesis, such as dimethoxytrityl (DMT), methoxytrityl (MMT), and others known to those of skill in the art.

[0055] Compounds disclosed herein may contain an asymmetric center and may thus exist as enantiomers. "Enantiomers" refer to two stereoisomers of a compound which are non- superimposable mirror images of one another. Where the compounds disclosed herein possess two or more asymmetric centers, they may additionally exist as diastereomers.

Enantiomers and diastereomers fall within the broader class of stereoisomers. All such possible stereoisomers as substantially pure resolved enantiomers, racemic mixtures thereof, as well as mixtures of diastereomers are intended to be included. All stereoisomers of the compounds disclosed herein and /or pharmaceutically acceptable salts thereof are intended to be included. Unless specifically mentioned otherwise, reference to one isomer applies to any of the possible isomers. Whenever the isomeric composition is unspecified, all possible isomers are included.

[0056] The term "substantially pure" as used herein means that the target stereoisomer contains no more than 35%, such as no more than 30%, further such as no more than 25%, even further such as no more than 20%, by weight of any other stereoisomer(s). In some aspects, the term "substantially pure" means that the target stereoisomer contains no more than 10%, for example, no more than 5%, such as no more than 1%, by weight of any other stereoisomer(s).

[0057] When compounds disclosed herein contain olefinic double bonds, unless specified otherwise, such double bonds are meant to include both E and Z geometric isomers.

[0058] Some of the compounds disclosed herein may exist with different points of attachment of hydrogen, referred to as tautomers. For example, compounds including carbonyl

-CH2C(0)- groups (keto forms) may undergo tautomerism to form hydroxyl -CH=C(OH)- groups (enol forms). Both keto and enol forms, individually as well as mixtures thereof, are also intended to be included where applicable.

[0059] It may be advantageous to separate reaction products from one another and /or from starting materials. The desired products of each step or series of steps is separated and /or purified (hereinafter separated) to the desired degree of homogeneity by the techniques common in the art. Typically such separations involve multiphase extraction, crystallization from a solvent or solvent mixture, distillation, sublimation, or chromatography.

Chromatography can involve any number of methods including, for example: reverse-phase and normal phase; size exclusion; ion exchange; high, medium and low pressure liquid chromatography methods and apparatus; small scale analytical; simulated moving bed ("SMB") and preparative thin or thick layer chromatography, as well as techniques of small scale thin layer and flash chromatography. One skilled in the art will apply techniques most likely to achieve the desired separation. [0060] "Diastereomers" refers to stereoisomers of a compound with two or more chiral centers but which are not mirror images of one another. Di aster eom eric mixtures can be separated into their individual diastereomers on the basis of their physical chemical differences by methods well known to those skilled in the art, such as by chromatography and /or fractional crystallization. Enantiomers can be separated by converting the enantiomeric mixture into a diastereomeric mixture by reaction with an appropriate optically active compound (e.g., chiral auxiliary such as a chiral alcohol or Mosher's acid chloride), separating the diastereomers and converting (e.g., hydrolyzing) the individual

diastereoisomers to the corresponding pure enantiomers. Enantiomers can also be separated by use of a chiral HPLC column.

[0061] A single stereoisomer, e.g., a substantially pure enantiomer, may be obtained by resolution of the racemic mixture using a method such as formation of diastereomers using optically active resolving agents (Eliel, E. mid Wilen, S. Stereochemistry of Organic

Compounds. New York: John Wiley & Sons, Inc., 1994; Lochmuller, C. H., et al.

"Chromatographic resolution of enantiomers: Selective review. "J. Chromatogr., 113(3) (1975): pp. 283-302). Racemic mixtures of chiral compounds of the disclosure can be separated and isolated by any suitable method, including: (1) formation of ionic,

diastereomeric salts with chiral compounds and separation by fractional crystallization or other methods, (2) formation of diastereomeric compounds with chiral derivatizing reagents, separation of the diastereomers, and conversion to the pure stereoisomers, and (3) separation of the substantially pure or enriched stereoisomers directly under chiral conditions. See: Wainer, Irving W., Ed. Drug Stereochemistry: Analytical Methods mid Pharmacology. New York: Marcel Dekker, Inc., 1993.

[0062] Compounds described herein, including salts thereof, can be prepared using known organic synthesis techniques and can be synthesized according to any of numerous possible synthetic routes.

[0063] The reaction for preparing compounds described herein can be carried out in suitable solvents which can be readily selected by one of skill in the art of organic synthesis. Suitable solvents can be substantially non-reactive with the starting materials, the intermediates, or products at the temperatures at which the reactions are carried out, e.g., temperatures which can range from the solvent's boiling temperature. A given reaction can be carried out in one solvent or mixture of solvents. [0064] The selection of appropriate protecting group, can be readily determined by one skilled in the art.

[0065] Reactions can be monitored according to any suitable method known in the art, such as NMR, UV, HPLC, LC-MS and TLC. Compounds can be purified by a variety of methods, including HPLC and normal phase silica chromatography.

[0066] For example, compounds of Formula (I) can be formed as shown in Scheme I.

Nitration

Reduction

ffc ^R₃R4R₅

Scheme L 2J-I¾oxoDvrrolidin-l-vl 4-G4^'l-(4-ff2J^oxo-2.5-dihvdro-lH-pvrrol-l- vl)methvncvclohexane-l-carboxamido)ethvlM-nitrophenoxv)butanoa 1-1)

[0067] Bis(3-acetylphenyl) oxalate

[0068] 3 -Hydroxy acetophenone (10.0 g, 73.5 mmol) was dissolved with triethylamine (20.5 mL, 147 mmol) in ethyl acetate (80 mL). The solution was degassed by bubbling N2 and cooled to 0°C with an ice bath. A solution of oxalyl chloride (4.41 mL, 51.4 mmol) and ethyl acetate (20 mL) was added dropwise. The mixture was stirred overnight, the resultant precipitate was filtered and washed with water and ethyl acetate. The product was a white precipitate.

[0069] 1 -(5-Hydroxy-2-nitrophenyl)ethan- 1 -one

[0070] The solution of nitric acid (7 mL) and sulfuric acid (17 mL) was kept between -10 and -20 °C with an acetone/dry ice bath. Bis(3-acetylphenyl) oxalate (1.00 g, 3.06 mmol) was added slowly and allowed to react for 15 minutes, followed by a second addition of bis(3- acetylphenyl) oxalate (1.00 g, 3.06 mmol) and 15 minutes of stirring. To the reaction mixture water (100 mL) was added at 5 °C, stirred for 30 minutes, and filtered. The precipitate was washed with water and dissolved in methanol (60 mL) overnight. The solution was concentrated to dryness via rotary evaporation, and the resultant precipitate was stirred in water (15 mL) for 48 hours before filtering. The light yellow precipitate was collected as the desired product.

[0071] Methyl 4-(3-acetyl-4-nitrophenoxy)butanoate

[0072] 6-Hydroxy-2-nitroacetophenone (2.56 g, 14.1 mmol), ethyl -4-bromobutyrate (2.72 g, 13.9 mmol), and potassium carbonate (3.89 g, 28.1 mmol) were added to DMF (15 mL) and stirred overnight at 75 °C. The solution was poured into water (300 mL), stirred for 1 hour at room temperature, and for 2 hours at 5 °C. The resultant precipitate was filtered and washed with water. The product was dried and collected as a yellow solid.

[0073] 4-(3-Acetyl-4-nitrophenoxy)butanoic acid

[0074] Ethyl (4-(3-acetyl-4-nitrophenoxy)butanoate (2.00 g, 8.26 mmol) was stirred in the solution of water (25 mL) and trifluoroacetic acid (2.5 mL) at 90 °C for 2 hours. The solution was allowed to cool to room temperature resulting in the formation of pale yellow precipitate. The precipitate was filtered and recrystallized in water to yield pale yellow crystals. [0075] 4-(3-(l-Hydroxyethyl)-4-nitrophenoxy)butanoic acid

OH

0₂N

O^' CO2H

[0076] (4-(3-Acetyl-4-nitrophenoxy) butanoic acid (2.33 g, 8.70 mmol) was dissolved in ethanol (50 mL) and cooled with an ice bath. To this solution sodium borohydride (0.494 g, 13.1 mmol) was added slowly. The reaction was stirred overnight and the solvent was removed under vacuum. The resulting precipitate was dissolved in 15 mL of water, acidified with 2N HC1, and kept at 5 °C overnight. The resulting precipitate was recrystallized with water, resulting in the desired pale yellow precipitate.

[0077] 4-(3-(l-Bromoethyl)-4-nitrophenoxy)butanoic acid

Br

[0078] 4-(3-(l-Hydroxyethyl)-4-nitrophenoxy)butanoic acid (1.00 g, 3.71 mmol) was dissolved in DCM (5 mL) and cooled with an ice bath. The solution of phosphorous tribromide (1.24 mL, 13.0 mmol) was added dropwise and allowed to react for 3.5 hours. Water (2 mL) was then added dropwise to react remaining phosphorous tribromide, and the organic layer was washed with water and sodium bicarbonate (3x eq. vol.). The solution was then dried with magnesium sulfate and dried under vacuum to give 4-(3-(l-bromoethyl)-4- nitrophenoxy)butanoic acid. [0079] 4-(3-(l-Aminoethyl)-4-nitrophenoxy)butanoic acid

\ .NH₂

0₂N

[0080] The brominated product 4-(3-(l-bromoethyl)-4-nitrophenoxy)butanoic acid was then dissolved in THF (5 mL) and added to a solution of ammonium hydroxide (45 mL). The reaction mixture was stirred overnight, followed by removal of THF and ammonia under vacuum, and acidified with 2N HC1. The aqueous layer was washed with ethyl acetate (3 x eq. vol.)- The product was concentrated to dryness via rotary evaporation and recrystallized with MeOH to yield 4-(3-(l-aminoethyl)-4-nitrophenoxy)butanoic acid as a pale yellow solid.

[0081] 4-(3-( 1 -(4-((2,5-Dioxo-2,5-dihydro- lH-pyrrol- 1 -yl)methyl)cyclohexane-l - carboxamido)ethyl)-4-nitrophenoxy)butanoi c acid

[0082] 4-(3-(l-Aminoethyl)-4-nitrophenoxy)butanoic acid (268 mg, 10 mmol) was dissolved in dry DMF (10 ml). To the solution was added succinimidyl 4-[N- maleimidomethyl]cyclohexane-l-carboxylate (334 mg, 10 mmol) and triethylamine (1.0 m). The reaction mixture was stirred at room temperature for 2 hours, and DMF and

triethylamine were removed under vacuum. The residue was recrystallized with EtOAc and hexanes to yield 4-(3-(l-(4-((2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)methyl)cyclohexane-l- carboxamido)ethyl)-4-nitrophenoxy)butanoic acid as a white solid. [0083] 2,5-Dioxopyrrolidin-l-yl 4<3-(l-(4<(2,5-d^^

yl)metoyl)cyclohexane-l-carboxamido)eAy

[0084] 4-(3-(l -(4-((2,5-Dioxo-2,5-dihydro- 1 H-pyrrol- 1 -yl)methyl)cyclohexane- 1 - carboxamido)ethyl)-4-nitrophenoxy)butanoic acid (245 mg, 0.5 mmol) was dissolved in dry dimethylformamide (10 mL) and dry pyridine (0. 5 mL). To the solution was added N- hydroxysuccinimide (85 mg) and l^thyl-3^3-dimemylaminopropyl)carbodiirnide (150 mg). The reaction mixture was stirred at room temperature overnight, and DMF and pyridine were removed under vacuum. The residue was dissolved in ethyl acetate and washed with water. The crude product was concentrated to dryness via rotary evaporation and recrystallized with EtOAc and hexanes to yield 2,5-dioxopyrrolidin-l-yl 4-(3-(l-(4-((2,5-dioxo-2,5-dihydro-lH- pyrrol-l-yl)methyl)cyclohexane-l^arboxamido)ethyl)-4-nitrophenoxy)butanoate as a white solid.

[0085] The present disclosure also provides probes comprising two regions, a first region comprising a target-specific sequence, and a second region that does not bind to the target containing at least one label attachment region, wherein the interface between the first region and the second region comprises a linker of the present disclosure. The first region and the second region can be linked by a compound of Formula I. The probes of the present disclosure can be referred to as "nanoreporter probes".

[0086] The first region and the second region do not overlap. The at least one label attachment region is capable of hybridizing to at least one DNA or RNA molecule, wherein the DNA or RNA molecule is labeled or attached to one or more label monomers that emit a signal that contributes to a code for detecting and identifying the target molecule. This probe is referred to herein as a reporter probe or a nanoreporter, these terms are used

interchangeably herein. Nanoreporters are preferably synthetic, i.e., non-naturally-occurring, nucleic acid molecules. The first region with the target-specific sequence is also referred to herein as the target binding domain. The second region containing at least one label attachment region is also referred to herein as the barcode domain.

[0087] The target binding domain of a nanoreporter probe can comprise one more nucleotide sequences that can be hybridized to a target nucleic acid that is to be detected or sequenced, or to a barcode domain of another nanoreporter probe.

[0088] The target binding domain can comprise a series of nucleotides (e.g. is a

polynucleotide). The target binding domain can comprise DNA, RNA, or a combination thereof In the case when the target binding domain is a polynucleotide, the target binding domain binds to a target nucleic acid by hybridizing to a portion of the target nucleic acid that is complementary to the target binding domain of the sequencing probe. The target binding domain can be any amount or number of nucleotides in length. The target binding domain can comprise 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, at least 10, at least 11, at least 12, at least 13, at least 14, at least IS, at least 16, at least 17, at least 18, at least 19, or at least 20 nucleotides.

[0089] The target binding domain can comprise at least one natural base. The target binding domain can comprise no natural bases. The target binding domain can comprise at least one modified nucleotide or nucleic acid analog. The target binding domain can comprise no modified nucleotides or nucleic acid analogs. The target binding domain can comprise at least one universal base. The target binding domain can comprise no universal bases. The target binding domain can comprise at least one degenerate base. The target binding domain can comprise no degenerate bases.

[0090] The target domain can comprise any combination natural bases (e.g. 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more natural bases), modified nucleotides or nucleic acid analogs (e.g. 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more modified or analog nucleotides), universal bases (e.g. 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more universal bases), or degenerate bases (e.g. 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more degenerative bases). When present in a combination, the natural bases, modified nucleotides or nucleic acid analogs, universal bases and degenerate bases of a particular target binding domain can be arranged in any order. [0091] The terms "modified nucleotides" or "nucleic acid analogues" include, but are not limited to, locked nucleic acids (LNA), bridged nucleic acids (BNA), propyne-modified nucleic acids, zip nucleic acids (ZNA^®), isoguanine and isocytosine. The target binding domain can include zero to six (e.g. 0, 1, 2, 3, 4, 5 or 6) modified nucleotides or nucleic acid analogues. Preferably, the modified nucleotides or nucleic acid analogues are locked nucleic acids (LNAs).

[0092] The term "locked nucleic acids (LNA)" as used herein includes, but is not limited to, a modified RNA nucleotide in which the ribose moiety comprises a methylene bridge connecting the 2' oxygen and the 4' carbon. This methylene bridge locks the ribose in the 3'- endo confirmation, also known as the north confirmation, that is found in A-form RNA duplexes. The term inaccessible RNA can be used interchangeably with LNA. The term "bridged nucleic acids (BNA)" as used herein includes, but is not limited to, modified RNA molecules that comprise a five-membered or six-membered bridged structure with a fixed 3'- endo confirmation, also known as the north confirmation. The bridged structure connects the 2' oxygen of the ribose to the 4' carbon of the ribose. Various different bridge structures are possible containing carbon, nitrogen, and hydrogen atoms. The term "propyne-modified nucleic acids" as used herein includes, but is not limited to, pyrimidines, namely cytosine and thymine/uracil, that comprise a propyne modification at the C5 position of the nucleic acid base. The term "zip nucleic acids (ZNA^®)" as used herein includes, but is not limited to, oligonucleotides that are conjugated with cationic spermine moieties.

[0093] The term "universal base" as used herein includes, but is not limited to, a nucleotide base does not follow Watson-Crick base pair rules but rather can bind to any of the four canonical bases (A, T/U, C, G) located on the target nucleic acid. The term "degenerate base" as used herein includes, but is not limited to, a nucleotide base that does not follow Watson- Crick base pair rules but rather can bind to at least two of the four canonical bases A, T/U, C, G), but not all four. A degenerate base can also be termed a Wobble base; these terms are used interchangeably herein.

[0094] The target binding domain can also comprise a minor-groove binder moiety. A minor- groove binder moiety is a chemical modification of an oligonucleotide that adds a chemical moiety that can bind to the minor groove of the target nucleotide to which the oligonucleotide is hybridized. Without being bound by theory, the inclusion of a minor-groove binder moiety increases the affinity of a target binding domain for a target nucleic acid, increasing the melting temperature of the target binding domain-target nucleic acid duplex. The higher binding affinity can allow for use of a smaller target binding domain.

[0095] The target binding domain can also comprise one or more twisted intercalating nucleic acids (ITNAs). A TINA is a nucleic acid molecule that stabilizes the formation of Hoogsteen triplex DNA from double-stranded oligonucleotides and triplex-forming oligonucleotides. TINAs can be used to stabilize a double-stranded oligonucleotides, thereby improving the specificity and sensitivity of an oligonucleotide probe to a target nucleic acid.

[0096] The target binding domain can also comprise nucleic acid molecules comprising a 2'- O-methyl-modified base. A 2'-0-methyl-modified base is a nucleoside modification of RNA in which a methyl group is added to the 2' hydroxyl group of the ribose to produce a 2' methoxy group. A 2'-0-methyl-modified base offers superior protection against base hydrolysis and digestion by nucleases. Without being bound by theory, the addition of a 2'- O-m ethyl -modified base also increases the melting temperature of a nucleic acid duplex.

[0097] The target binding domain can also comprise a covalently linked stilbene

modification. A stilbene modification can increase the stability of a nucleic acid duplex.

[0098] Table 1 provides several other configurations of target binding domains of the present disclosure.

Table 1.

[0099] The target binding domain can comprise an antibody. The term "antibody" herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bi specific antibodies), and antibody fragments so long as they exhibit the desired antigen- binding activity. An antibody that binds to a target refers to an antibody that is capable of binding the target with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting the target. In one embodiment, the extent of binding of an anti-target antibody to an unrelated, non-target protein is less than about 10% of the binding of the antibody to target as measured, e.g., by a radioimmunoassay (RIA) or biacore assay. In certain embodiments, an antibody that binds to a target has a dissociation constant (Kd) of < 1 uM, < 100 nM, < 10 nM, < 1 nM, < 0.1 nM, < 0.01 nM, or < 0.001 nM (e.g. 10⁸ M or less, e.g. from 10* M to 10¹³ M, e.g., from 10⁹ M to 10¹³ M). In certain

embodiments, an anti-target antibody binds to an epitope of a target that is conserved among different species. The target binding domain can comprise an antibody fragment. An

"antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds.

Examples of antibody fragments include but are not limited to Fv, Fab, Fab¹, Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g. scFv); and multispecific antibodies formed from antibody fragments.

[00100] The barcode domain of a nanoreporter probe comprises a unique, designed nanoreporter. The nanoreporter comprises one or more, e.g., one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty or more, unique, designed nucleotide sequences that can be hybridized to other nanoreporter probes or to molecules bound by or comprising at least one detectable label. The nucleotide sequences can comprise at least 4, or at least 5, at least 6, or at least 7, or at least 8, or at least 9, or at least 10, or at least 11, or at least 12, or at least 13, or at least 14, or at least 15, or at least 16 or at least, 17 , or at least 18, or at least 19, or at least 20, or at least 21, or at least 22, or at least 23, or at least 24, or at least 25, or at least 26, or at least 27, or at least 28, or at least 29, or at least 30 nucleotides.

[00101] The nucleotide sequences of the nanoreporter of a single nanoreporter probe can be designed such that: (a) the nucleotide sequences specifically correspond to the nucleotide sequences present in the target binding domain of that same nanoreporter probe; and (b) the nucleotide sequences can be hybridized to one or more specific, predetermined nanoreporter probes or to one or more molecules bound by or comprising a specific, predetermined label. Thus, when the nanoreporter is hybridized to one or more molecules bound by or comprising a predetermined detectable label, the nanoreporter probe will emit a detectable single that distinguishes it from other nanoreporter probes and identifies the sequence present in the target binding domain of that nanoreporter probe.

[00102] The barcode domain can comprise at least one natural base. The barcode domain can comprise no natural bases. The barcode domain can comprise at least one modified nucleotide or nucleic acid analog. The barcode domain can comprise no modified nucleotides or nucleic acid analogs. The barcode domain can comprise at least one universal base. The barcode domain can comprise no universal bases. The barcode domain can comprise at least one degenerate base. The barcode domain can comprise no degenerate bases. The target domain can comprise any combination natural bases (e.g. 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more natural bases), modified nucleotides or nucleic acid analogs (e.g. 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more modified or analog nucleotides), universal bases (e.g. 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more universal bases), or degenerate bases (e.g. 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more degenerative bases). When present in a combination, the natural bases, modified nucleotides or nucleic acid analogs, universal bases and degenerate bases of a particular barcode domain can be arranged in any order.

[00103] A nanoreporter can comprise at least one detectable label. A nanoreporter can comprise at least 2 , at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100 detectable labels. [00104] A nanoreporter can comprise a plurality of a first detectable label. A nanoreporter can comprise a plurality of a first detectable label and a plurality of an at least second detectable label. A nanoreporter can comprise a plurality of a first detectable label and a plurality of an at least second detectable label, wherein the number of first detectable labels is equal to the number of detectable labels. A nanoreporter can comprise a plurality of a first detectable label and a plurality of an at least second detectable label, wherein the number of first detectable labels is not equal to the number of detectable labels.

[00105] The terms "detectable moiety", "detectable label" and "detectable reporter" are used herein interchangeably. A detectable moiety, label or reporter can be bound to a nanoreporter probe in a variety of ways, including the direct or indirect attachment of a detectable moiety such as a fluorescent moiety, colorimetric moiety and the like. One of skill in the art can consult references directed to labeling nucleic acids. Examples of fluorescent moieties include, but are not limited to, yellow fluorescent protein (YFP), green fluorescent protein (GFP), cyan fluorescent protein (CFP), red fluorescent protein (RFP), umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, cyanines, dansyl chloride, phycocyanin, phycoerythrin and the like.

[00106] Fluorescent labels and their attachment to nucleotides and/or oligonucleotides are described in many reviews, including Haugland, Handbook of Fluorescent Probes and Research Chemicals, Ninth Edition (Molecular Probes, Inc., Eugene, 2002); Keller and Manak, DNA Probes, 2nd Edition (Stockton Press, New York, 1993); Eckstein, editor, Oligonucleotides and Analogues: A Practical Approach (IRL Press, Oxford, 1991); and Wetmur, Critical Reviews in Biochemistry and Molecular Biology, 26:227-259 (1991). Particular methodologies applicable to the disclosure are disclosed in the following sample of references: U.S. Patent Nos. 4,757,141; 5,151,507; and 5,091,519. One or more fluorescent dyes can be used as labels for labeled target sequences, e.g., as disclosed by U.S. Patent Nos. 5,188,934 (4,7-dichlorofluorescein dyes); 5,366,860 (spectrally resolvable rhodamine dyes); 5,847,162 (4,7-dichlororhodamine dyes); 4,318,846 (ether-substituted fluorescein dyes); 5,800,996 (energy transfer dyes); Lee etal. 5,066,580 (xanthine dyes); 5,688,648 (energy transfer dyes); and the like. Labelling can also be carried out with quantum dots, as disclosed in the following patents and patent publications: U.S. Patent Nos. 6,322,901; 6,576,291; 6,423,551; 6,251,303; 6,319,426; 6,426,513; 6,444,143; 5,990,479; 6,207,392;

2002/0045045; and 2003/0017264. As used herein, the term "fluorescent label" comprises a signaling moiety that conveys information through the fluorescent absorption and/or emission properties of one or more molecules. Such fluorescent properties include fluorescence intensity, fluorescence lifetime, emission spectrum characteristics, energy transfer, and the like.

[00107] Commercially available fluorescent nucleotide analogues readily incorporated into nucleotide and/or oligonucleotide sequences include, but are not limited to, Cy3-dCTP, Cy3- dUTP, Cy5-dCTP, Cy5-dUTP (Amersham Biosciences, Piscataway, NJ), fluorescein- 12- dUTP, tetramethylrhodamine-6-dUTP, TEXAS RED™-5-dUTP, CASCADE BLUE™-7- dUTP, BODIPY TMFL-14-dUTP, BODIPY TMR-14-dUTP, BODIPY TMTR-14-dUTP, RHOD AMINE GREEN™-5-dUTP, OREGON GREENR™ 488-5-dUTP, TEXAS RED™- 12-dUTP, BODIPY TM 630/650- 14-dUTP, BODIPY TM 650/665- 14-dUTP, ALEXA FLUOR™ 488-5-dUTP, ALEXA FLUOR™ 532-5-dUTP, ALEXA FLUOR™ 568-5-dUTP, ALEXA FLUOR™ 594-5-dUTP, ALEXA FLUOR™ 546- 14-dUTP, fluorescein- 12-UTP, tetramethylrhodamine-6-UTP, TEXAS RED™-5-UTP, mCherry, CASCADE BLUE™-7- UTP, BODIPY TM FL-14-UTP, BODIPY TMR-14-UTP, BODIPY TM TR-14-UTP, RHODAMINE GREEN™-5-UTP, ALEXA FLUOR™ 488-5-UTP, LEXA FLUOR™ 546- 14-UTP (Molecular Probes, Inc. Eugene, OR) and the like. Alternatively, the above fluorophores and those mentioned herein can be added during oligonucleotide synthesis using for example phosphoroamidite or NHS chemistry. Protocols are known in the art for custom synthesis of nucleotides having other fluorophores (See, Henegariu et al. (2000) Nature Biotechnol. 18:345). 2-Aminopurine is a fluorescent base that can be incorporated directly in the oligonucleotide sequence during its synthesis. Nucleic acid could also be stained, a priori, with an intercalating dye such as DAPI, YOYO- 1 , ethidium bromide, cyanine dyes (e.g., SYBR Green) and the like.

[00108] Other fluorophores available for post-synthetic attachment include, but are not limited to, ALEXA FLUOR™ 350, ALEXA FLUOR™ 405, ALEXA FLUOR™ 430, ALEXA FLUOR™ 532, ALEXA FLUOR™ 546, ALEXA FLUOR™ 568, ALEXA

FLUOR™ 594, ALEXA FLUOR™ 647, BODIPY 493/503, BODIPY FL, BODIPY R6G, BODIPY 530/550, BODIPY TMR, BODIPY 558/568, BODIPY 558/568, BODIPY 564/570, BODIPY 576/589, BODIPY 581/591, BODIPY TR, BODIPY 630/650, BODIPY 650/665, Cascade Blue, Cascade Yellow, Dansyl, lissamine rhodamine B, Marina Blue, Oregon Green 488, Oregon Green 514, Pacific Blue, Pacific Orange, rhodamine 6G, rhodamine green, rhodamine red, tetramethyl rhodamine, Texas Red (available from Molecular Probes, Inc., Eugene, OR), Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7 (Amersham Biosciences, Piscataway, NJ) and the like. FRET tandem fluorophores can also be used, including, but not limited to, PerCP-Cy5.5, PE-Cy5, PE-Cy5.5, PE-Cy7, PE-Texas Red, APC-Cy7, PE-Alexa dyes (610, 647, and 680), APC-Alexa dyes and the like.

[00109] Metallic silver or gold particles can be used to enhance signal from fluorescently labeled nucleotide and/or oligonucleotide sequences (Lakowicz elal. (2003) BioTechniques 34:62).

[00110] Other suitable labels for an oligonucleotide sequence can include fluorescein (FAM, FITC), digoxigenin, dinitrophenol (DNP), dansyl, biotin, bromodeoxyuridine (BrdU), hexahistidine (6xHis), phosphor-amino acids (e.g., P-tyr, P-ser, P-thr) and the like. The following hapten/antibody pairs can be used for detection, in which each of the antibodies is derivatized with a detectable label: biotin/a-biotin, digoxigenin/a-digoxigenin, dinitrophenol (DNPya-DNP, 5-Carboxyfluorescein (FAM)/a-FAM.

[00111] Figure 1 shows an illustrative schematic of an exemplary nanoreporter probe comprising a target binding domain and a barcode domain in which the interface between the target binding domain and the barcode domain comprises the photocleavable linker of the present disclosure.

[00112] Figure 2 shows an exemplary nanoreporter probe complex comprising the photocleavable linker of the present disclosure. The nanoreporter probe complex comprises seven nanoreporter probes. One nanoreporter probe, in this example referred to as the primary nanoreporter probe, comprises a target binding domain and a barcode domain, in which the interface between the target binding domain and the barcode domain comprises the photocleavable linker of the present disclosure. Six nanoreporter probes, in this example referred to as secondary nanoreporter probes, are hybridized to the barcode domain of the primary nanoreporter. Each secondary nanoreporter probe is hybridized to 5 molecules that are bound to a detectable label. In this example, the positioning of the cleavable linker of the present disclosure allows for the controllable and rapid separation of the target binding and barcode domains of the primary nanoreporter probe. This controllable and rapid separation can be beneficial in various applications, including the detection and sequencing of target nucleic acids, as it allows a user to remove the detectable signal from specific nanoreporter probe to allow for easier detection of other nanoreporter probes.

[00113] Figure 3 is a schematic illustration of an exemplary sequencing method using a nanoreporter probe comprising the cleavable linker modification of the present disclosure. In the first step (far left panel of Figure 3), a nanoreporter probe, referred to in this example as a sequencing probe, is hybridized via the target binding domain to a target nucleic acid that is to be sequenced. Additionally, a secondary nanoreporter probe is hybridized to a position on the barcode domain of the sequencing probe. This position of the barcode domain of the sequencing probe corresponds to the identity of the first two nucleotides of the target binding domain of the sequencing probe. Thus, the secondary nanoreporter probe that hybridizes to this position also corresponds to the identity of these first two nucleotides. In the next step, the barcode domain of the secondary nanoreporter probe is hybridized to oligos labeled with fluorescent dyes. The secondary nanoreporter probe comprises a sequence such that three oligos that are labeled with a green dye and three oligos that are labeled with a red dye are hybridized, creating a green-red color combination that corresponds to the identity of the first two nucleotides of the target binding domain. Thus, by detecting the green-red color combination using a fluorescence microscope, the user can identify the first two nucleotides of the target binding domain of the sequencing probes and thereby, the identity of the complementary nucleotides in the target nucleic acid. In the next step, the linker of the present disclosure is cleaved using UV light to separate the labeled barcode domain and target binding domain of the secondary nanoreporter probe, thereby ensuring that the sequencing probe no longer emits a detectable signal associated with the identity of the first two nucleotides of the target binding domain. In the next step, a different secondary nanoreporter probe is hybridized to a position of the barcode domain of the sequencing probe. This position corresponds to the identity of the third and fourth nucleotides of the target binding domain of the sequencing probe. The secondary nanoreporter probe that hybridizes in this position is therefore specific to the identity of the third and fourth nucleotides of the target binding domain. The barcode domain of this secondary nanoreporter probe is hybridized to labeled oligos such that the secondary nanoreporter probe emits a detectable signal that is specific to the identity of the third and fourth nucleotides of the target binding domain.

[00114] In the example depicted in Figure 3, the photocleavable linker of the present disclosure is necessary to remove the labeled barcode domain that corresponds to the identity of the first two nucleotides to allow for the detection of the labeled barcode domain that corresponds to the identity of the third and fourth nucleotides. If the labeled barcode domain that corresponds to the identity of the first two nucleotides was not removed, the fluorescent signals from the barcode domains of the two secondary nanoreporter probes would not be distinguishable using standard fluorescence microscopy techniques. [00115] Figure 4 shows an exemplary nanoreporter probe complex and the locations within the complex that could comprise the photocleavable linker of the present disclosure. The structure of the nanoreporter complex is the same as that depicted in Figure 2.

[00116] In some aspects, the reporter probe may comprise two label attachment regions, wherein the first and second label attachment regions are capable of hybridizing to a first and second DNA or RNA molecule respectively, and each first and second DNA or RNA molecule is attached to one or more label monomers that emit light that constitute a first and second signal, respectively. In some aspects, the second probe may comprise three or more label attachment regions, wherein at each label attachment region, an DNA or RNA molecule attached to one or more label monomers that emit light that constitute a signal can bind. The signals emitted from the labeled monomers contribute to the code that identifies the specific target molecule.

[00117] The present disclosure also provides a method of preparing probes that include a linker of the present disclosure. The method comprises: 1) Dissolving 32.7 nmol of 3'-amino modified 84mer oligo, with the nucleotide sequence

AGGCTGCACACCTTAGGCTGCACACCTTAGGCTGCACACCTTCGGCAAATGGCGT TCGGCAAATGGCGTTCGGCAAATGGCGTT (SEQ ID NO: 1) in 32.7 μΐ. of water tube; 2) To the 1.0 mM amino modified oligo solution of step 1, adding 27.5 μL of saturated NaHCCb (approximately a 10-fold molar excess in addition to factoring for oligo

length/number of phosphate backbones), vortexing briefly to mix, and subsequently drying in vacuo via Savant to facilitate and ensure sodium counterion exchange (and removal of ammonium or triethylammonium counterion) of the amino-modified starting material prior to reaction with the NHS-ester moiety of the custom bifunctional cross-linker reagent; 3) Dissolving the dried-down pellet from step 2 in 84.8 μL of 0.1 M Borate buffer pH 8.5. 3.0 μί, can be optionally retained for HPLC analysis of starting material; 4) To the remaining 81.8 uL of Borate buffer solution, adding 14.7 μί_^ of a 55.5 mM solution of cross-linker reagent that contains an NHS-ester reactive group on one terminus and maleimide group on other terminus and that is dissolved in anhydrous DMF (25-fold molar excess); 5) Vortexing the reaction mixture well for 10 seconds, briefly spinning down, and allowing to react at room temperature (RT) in the dark; 6) After 30 minutes, vortexing the reaction mixture again for 10 seconds, briefly spinning down, and allowing to react at RT in the dark overnight.

[00118] The method further comprising: 7) After approximately 18 hours of reaction, quenching the excess NHS-ester in the solution of step 6 by reacting the NHS-ester with a 1.1 molar excess of Tris-HCl (2.04 uL of 1 M Tris-HCl solution) for 15 minutes at RT; 8) To the resulting reaction mixture, adding 3 M NaOAc (10.7 μL to render 10% by volume), followed by 321.6 μί, of ice cold EtOH to precipitate desired DNA-conjugate intermediate; 9) Vortexing the suspension for about 10 seconds, spining briefly via benchtop centrifuge, and incubating in -80°C freezer for 1 hour; 10) Centrifuging the desired intermediate product at 4°C for 30 minutes at full speed (13,500 RPM), and washing the resulting pellet twice with 200 μL of ice cold 70% aq. EtOH. After each wash and prior to decanting or pipette removal of supernatants, spinning briefly via benchtop centrifuge at 4°C for 10 minutes at full speed; 11) Drying the final pellet in vacuo via Savant for 15 minutes.

[00119] The method further comprising: 12) Dissolving 195.9 nmol of reporter molecule (5'- ThioMC6-D/CGAGAATCCTAGAC, SEQ ID NO: 2) in 195.9 μL· of PBS pH 7.4, resulting in a reporter molecule concentration of 1.0 mM concentration; 13) To the 1.0 mM reporter solution, adding a 100-fold excess of TCEP reagent (19.6 umol; 39.2 μL of 0.5 M solution of TCEP) to reduce any disulfide linkages to free thiol groups. Vortexing the reaction mixture well for 10 seconds, briefly spinning down, and allowing to react at RT in the dark for 2 hours; 14) Diluting the reaction mixture from step 13 with water to total volume of 750 μί, loading onto a pre-equilibrated NAP- 10 column (also in water), and washing the column with 250 μL of water.; 15) Eluting and collecting the reduced thiol product and concentrating the product to near-dryness via Savant; 16) Adding 39.2 uL of 0.1 M NaHC03 pH 8.3 to the reduced 14mer thiol, vortexing for 10 seconds to mix, and then briefly spinning down; 17) Adding the mixture from step 16 to a solution of the above dried pellet of step 11 dissolved in 81.75 μL of 0.1 M NaHC03 pH 8.3. This reaction corresponds to a 6-fold excess of the reduced 14mer thiol relative to the maleimide reactive group of the 84mer conjugate; 18) Vortexing the combined mixture for 10 seconds to mix, briefly spinning down, and allowing to react at RT for at least 2 hours in the dark (or overnight); 19) Purifying the resulting product via RP-HPLC under standard nucleic acid purification conditions to afford final desired bifunctional 98mer product.

[00120] The reporter probes of the present disclosure can be labeled with any of a variety of label monomers, such as a fluorochrome, dye, enzyme, nanoparticle, chemiluminescent marker, biotin, or other monomer known in the art that can be detected directly (e.g., by light emission) or indirectly (e.g., by binding of a fluorescently-labeled antibody). Generally, one or more of the label attachments regions in the reporter probe is labeled with one or more label monomers, and the signals emitted by the label monomers attached to the label attachment regions of a reporter probe or capture probe constitute a code that identifies the target. In certain aspects, the lack of a given signal from the label attachment region (i.e., a "dark" spot) can also constitute part of the code. In certain preferred aspects, the label monomers are fluorophores or quantum dots.

[00121] The present disclosure also provides a probe pair which includes a reporter probe as described herein and a capture probe. A capture probe comprises a first region containing a target-specific sequence, and a second non-overlapping region containing one or more affinity moieties. In the probe pair, the reporter probe and capture probe bind to the same target molecule at non-overlapping locations. The affinity moieties can be utilized for purification and/or for immobilization. The affinity moieties may be attached to the capture probe by covalent or non-covalent means. Various affinity moieties appropriate for purification and/or for immobilization are known in the art. Preferably, the affinity moiety is biotin. In some aspects, the capture probe may contain a second affinity moiety, such as repeat sequences, which are used for affinity purification through hybridization to an oligonucleotide column, in which the column contains oligonucleotides that are

complementary to the repeat sequences of the capture probe. In some aspects, the capture probe comprises at least one label attachment region that is hybridized to at least one RNA or DNA molecule, wherein the RNA or DNA molecule is labeled or attached to one or more label monomers that emit a signal that contributes to the code.

[00122] The present disclosure also provides methods of detecting or quantifying an individual or plurality of target molecules in a biomolecular sample, using the compositions described herein.

[00123] In certain aspects, the methods of detection are performed in multiplex assays, whereby a plurality of target molecules are detected in the same assay (a single reaction mixture). In a preferred aspect, the assay is a hybridization assay in which the plurality of target molecules are detected simultaneously. In certain aspects, the plurality of target molecules detected in the same assay is at least 5 different target molecules, at least 10 different target molecules, at least 20 different target molecules, at least SO different target molecules, at least 75 different target molecules, at least 100 different target molecules, at least 200 different target molecules, at least 500 different target molecules, or at least 750 different target molecules, or at least 1000 different target molecules. In other aspects, the plurality of target molecules detected in the same assay is up to 50 different target molecules, up to 100 different target molecules, up to 150 different target molecules, up to 200 different target molecules, up to 300 different target molecules, up to 500 different target molecules, up to 750 different target molecules, up to 1000 different target molecules, up to 2000 different target molecules, or up to 5000 different target molecules. In yet other aspects, the plurality of target molecules detected is any range in between the foregoing numbers of different target molecules, such as, but not limited to, from 20 to 50 different target molecules, from 50 to 200 different target molecules, from 100 to 1000 different target molecules, from 500 to 5000 different target molecules, and so on and so forth. Preferably, the target molecule is DNA (including cDNA) or RNA (including mRNA and cRNA). The present disclosure may be particularly useful for multiplex assays to detect a plurality of target molecules in a sample, for example, a set of genes. Each target molecule, or gene of interest, in a multiplex assay is associated with a unique reporter code as designated by the unique linear arrangement of label monomers associated with the reporter probe.

[00124] Nanoreporters can bind to target molecules directly or indirectly via an

oligonucleotide molecule (also referred to as a tag). Nanoreporter systems that utilize capture and reporter probes that contain target-specific sequences for binding directly to the target molecule are described in U.S. Patent Publications US2010/0015607 and US2010/0047924. Nanoreporter systems that indirectly bind to the target molecule are described in U.S. Patent Publication US2014/0371088.

Claims

This listing of claims will replace all prior versions, and listings, of claims in the application:

1. A compound of the Formula (I) :

or a stereoisomer or salt thereof, wherein:

R₁ is hydrogen, halogen, C_1-6alkyl, C_2-6alkenyl, C_2-6alkynyl, wherein said C_1-6alkyl, C₂- ₆alkenyl, C_2-6alkynl are each independently optionally substituted with at least one substituent R₁₀;

R2 is O, NH, orN(C_1-6alkyl);

R3 is cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each optionally substituted with at least one substituent R₁₀;

each R₄ and R₇ are independently C_1-6alkylene, C_2-6alkenylene, C_2-6alkynylene, wherein said C_1-6alkylene, C_2-6alkenylene, C_2-6alkynlene are each independently optionally substituted with at least one substituent R₁₀;

R5 and R₉ are each independently cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each optionally substituted with at least one substituent R₁₀;

R₆ is O, NH orN(Cwalkyl);

R₈ is O, NH, orN(C_1-6alkyl);

each R₁₀ is independently hydrogen, halogen, -C_1-6alkyl, -C_2-6alkenyl, -C_2-6alkynyl, haloC_1-6alkyl, halo C_2-6alkenyl, haloC_2-6alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, - CN, -NO2, oxo, -OR₁₁, -SO2R11, - SO₃ , -COR11, -CO2R11, -CONR11R12, -C(=NR₁₁)NR₁₂R₁₃, - NR11R12, -NR12COR12, -NR11CONR12R13, -NR11CO2R12, -NR11SONR12R13, - NR11SO2NR12R13, or -NR11SO2R12; and

R11, R12, and R13, which may be the same or different, are each independently hydrogen, -C_1-6alkyl, -C_2-6alkenyl, -C_2-6alkynyl, haloC_1-salkyl, halo C_2-6alkenyl, haloC_2- ealkynyl, C_1-6alkyloxyC_1-6alkyl-, cycloalkyl, heterocyclyl, aryl, or heteroaryl.

2. The compound of claim 1, wherein R₁ is C_1-6alkyl, preferably C_1-3alkyl such as methyl, ethyl, propyl or isopropyl; Ra is NH orNCC_1-6alkyl);

R3 is a 5- to 6-membered cycloalkyl, preferably cyclohexyl;

R₄ is C_1-6alkylene, preferably C_1-3alkylene such as methylene, ethylene, propylene, isopropylene;

Rs is a S- to 6- membered heterocyclyl comprising one nitrogen atom and 0 or 1 additional heteroatoms selected from N, O and S, wherein said heterocyclyl is optionally substituted with one or two R₁₀;

R₆ is O;

R₇ is C_1-6alkylene, preferably C_1-3alkylene such as methylene, ethylene, propylene, isopropylene;

R₈ is O;

R₉ is a 5- to 6- membered heterocyclyl comprising one nitrogen atom and 0 or 1 additional heteroatoms selected from N, O and S, wherein said heterocyclyl is optionally substituted with one or two R₁₀; and

each R₁₀ is independently halogen, C_1-6alkyl, haloC_1-6alkyl, oxo, -SO2H, or -SO₃ ^".

3. The compound of claim 1 , wherein R₃ is cyclohexyl, R₄ is methylene, Rs is 1H- pyrrole-2,5-dione, and R₉ is pyrrolidine-2,5-dione, optionally substituted with SCh".

4. The compound of claim 1, which is

or a stereoisomer or salt thereof; or

or a stereoisomer or salt thereof.

5. A compound of the Formula (II):

or a stereoisomer or salt thereof, wherein:

R21 is hydrogen, halogen, C_1-6alkyl, C_2-6alkenyl, C_2-6alkynyl, wherein said C_1-6alkyl, C_2-6alkenyl, C_2-6alkynl are each independently optionally substituted with at least one substituent R30;

R22 is O, NH, or N(C walkyl);

R23 is cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each optionally substituted with at least one substituent R₁₀;

R24 is C_1-6alkylene, C₂-salkenylene, C_2-6alkynylene, wherein said C_1-6alkylene, C_2- ealkenylene, C_2-6alkynlene are each independently optionally substituted with at least one substituent R₁₀;

R26 is a bond, O, NH or N(C_1-6alkyl);

R27 is a bond, C_1-6alkylene, C_2-6alkenylene, C_2-6alkynylene, wherein said C_1-6alkylene, C_2-6alkenylene, C_2-6alkynlene are each independently optionally substituted with at least one substituent R₁₀;

provided that when R26 is a bond, then R27 is also a bond; R28 is OP(OR₁XNRnR₁2);

each R30 is independently hydrogen, halogen, -C_1-6alkyl, -C_2-6alkenyl, - C_2-6alkynyl, haloC_1-6alkyl, haloC_2-6alkenyl, haloC_2-6alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, - CN, -NO2, oxo, -OR11, -SO2R31, -S03-, -COR31, -CO2R31, -CONR31R32, -C(=NR31)NR32R33, - NR31R32, -NR32COR32, -NR31CONR32R33, -NR31CO2R32, -NR31SONR32R33, - NR31SO2NR32R33, or -NR31S02R32; and

R31, R32, and R33, which may be the same or different, are each independently hydrogen, -C_1-6alkyl, - C_2-6alkenyl, -C_2-6alkynyl, haloC_1-salkyl, haloC_2-6alkenyl, haloC_2- ealkynyl, C_1-6alkyloxyC_1-6alkyl-, cycloalkyl, heterocyclyl, aryl, heteroaryl, or a protecting group.

6. A probe comprising

A. a first region comprising a first target-specific sequence;

B. a second region which does not overlap with the first region, the second region comprising

i. a first label attachment region which is hybridized to a first DNA or RNA molecule, wherein the first DNA or RNA molecule is attached to one or more label monomers that emit light constituting a first signal; and

ii. a second label attachment region, which is non-overlapping to the first label attachment region, and which is hybridized to a second DNA or RNA molecule, wherein the second DNA or RNA molecule is attached to one or more label monomers that emit light constituting a second signal, and wherein the label attachment regions do not overlap; and

C. a compound of any one of claims 1-4 linking the first region and the second region.

7. A composition comprising a first probe and a second probe,

A. the first probe comprising

i. a first region comprising a first target-specific sequence;

ii. a second region which does not overlap with the first region, the second region comprising

a. a first label attachment region which is hybridized to a first DNA or RNA molecule, wherein the first DNA or RNA molecule is attached to one or more label monomers that emit light constituting a first signal; and b. a second label attachment region, which is non-overlapping to the first label attachment region, and which is hybridized to a second DNA or RNA molecule, wherein the second DNA or RNA molecule is attached to one or more label monomers that emit light constituting a second signal, and wherein the label attachment regions do not overlap; and

iii. a compound of any one of claims 1-4 linking the first region and the second region;

B. the second probe comprising

i. a first region comprising a first target-specific sequence, wherein the target- specific sequence of the first probe and the target-specific sequence of the second probe are different; and

ii. a second region that does not overlap with the first region, does not bind to the target molecule and comprises at least one affinity moiety.