EP1487850A2 - Bloc de construction formant une liaison atomique c-c ou c hetero apres reaction - Google Patents

Bloc de construction formant une liaison atomique c-c ou c hetero apres reaction

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Publication number
EP1487850A2
EP1487850A2 EP03711855A EP03711855A EP1487850A2 EP 1487850 A2 EP1487850 A2 EP 1487850A2 EP 03711855 A EP03711855 A EP 03711855A EP 03711855 A EP03711855 A EP 03711855A EP 1487850 A2 EP1487850 A2 EP 1487850A2
Authority
EP
European Patent Office
Prior art keywords
group
aryl
independently
alkylene
alkyl
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP03711855A
Other languages
German (de)
English (en)
Inventor
Alex Haahr Gouliaev
Henrik Pedersen
Kim Birkebaerd Jensen
Mikkel Dybro Lundorf
Christian Sams
Jakob Felding
Michael Anders Godskesen
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Nuevolution AS
Original Assignee
Nuevolution AS
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Filing date
Publication date
Application filed by Nuevolution AS filed Critical Nuevolution AS
Publication of EP1487850A2 publication Critical patent/EP1487850A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
    • C07D405/04—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1068—Template (nucleic acid) mediated chemical library synthesis, e.g. chemical and enzymatical DNA-templated organic molecule synthesis, libraries prepared by non ribosomal polypeptide synthesis [NRPS], DNA/RNA-polymerase mediated polypeptide synthesis
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures

Definitions

  • the present invention relates to a building block comprising a complementing element and a precursor for a functional entity.
  • the building block is designed to transfer the functional entity precursor with an adjustable efficiency to a recipient reactive group upon recognition between the complementing element and an encoding ele- ment associated with the reactive group.
  • the invention also relates to a method for transferring a functional entity precursor to recipient a reactive group.
  • the first oligonucleotide and a second oligonucleotide having a 3' amino group is aligned on a template such that the thioester group and the amino group are positioned in close proximity and a transfer is effected resulting in a coupling of the peptide to the second oligonucleotide through an amide bond.
  • the present invention relates to a building block of the general formula: Complementing Element - Linker - Carrier - C-F-connecting group - Functional entity precursor capable of transferring a Functional entity precursor to a recipient reactive group, wherein
  • Complementing Element is a group identifying the Functional entity precursor
  • Linker is a chemical moiety comprising a spacer and a S-C-connecting group, wherein the spacer is a valence bond or a group distancing the Functional entity precursor to be transferred from the complementing element and the S-C- connecting group connects the spacer with the Carrier
  • Carrier is arylene, heteroarylene, C C 6 alkylene, C-
  • C-F-connecting group is chosen from the group consisting of -SO 2 -O-, -O-SO 2 -O-, -C(O)-O-, -S + (R 3 RRrr)-, -C-U-C(V)-O-, -P + (W) 2 -O-, -P(W)-O- where U is
  • Functional entity precursor is -C(H)(R 3 )-R 4 or functional entity precursor is heteroaryl or aryl optionally substituted with one or more substituents belonging to the group comprising R 3 and R 4 .
  • C 3 -C 7 cycloheteroalkyl refers to a radical of totally saturated heterocycle like a cyclic hydrocarbon containing one or more heteroatoms selected from nitrogen, oxygen, phosphor, boron and sulphur independently in the cycle such as pyrrolidine (1- pyrrolidine; 2- pyrrolidine; 3- pyrrolidine; 4- pyr- rolidine; 5- pyrrolidine); pyrazolidine (1- pyrazolidine; 2- pyrazolidine; 3- pyra- zolidine; 4-pyrazolidine; 5-pyrazolidine); imidazolidine (1- imidazolidine; 2- imida- zolidine; 3- imidazolidine; 4- imidazolidine; 5- imidazolidine); thiazolidine (2- thia- zolidine; 3- thiazolidine; 4- thiazolidine; 5- thiazolidine); piperidine (1- piperidine; 2- piperidine; 3- piperidine; 4- piperidine;
  • Aryl is also intended to include the partially hydrogenated derivatives of the carbocyclic systems as well as up to four fused fused aromatic- or partially hydrogenated rings, each ring comprising 5-7 carbon atoms.
  • heteroaryl as used herein includes heterocyclic unsaturated ring systems containing, in addition to 2-18 carbon atoms, one or more heteroatoms selected from nitrogen, oxygen and sulphur such as furyl, thienyl, pyrrolyl, heteroaryl is also intended to include the partially hydrogenated derivatives of the heterocyclic systems enumerated below.
  • aryl and “heteroaryl” as used herein refers to an aryl which can be op- tionally substituted or a heteroaryl which can be optionally substituted and includes phenyl, biphenyl, indenyl, naphthyl (1-naphthyl, 2-naphthyl), N- hydroxytetrazolyl, N-hydroxytriazolyl, N-hydroxyimidazolyl, anthracenyl (1- anthracenyl, 2-anthracenyl, 3-anthracenyl), thiophenyl (2-thienyl, 3-thienyl), furyl (2-furyl, 3-furyl), indolyl, oxadiazolyl, isoxazolyl, quinazolinyl, fluorenyl, xanthenyl, isoindanyl, benzhydryl, acridinyl, thiazolyl, pyrrolyl
  • the Functional Entity carries elements used to interact with host molecules and optionally reactive elements allowing further elaboration of an encoded molecule of a library. Interaction with host molecules like enzymes, receptors and polymers is typi- cally mediated through van der waal's interactions, polar- and ionic interactions and pi-stacking effects. Substituents mediating said effects may be masked by methods known to an individual skilled in the art (Greene, T. W.; Wuts, P. G. M. Protective Groups in Organic Synthesis; 3rd ed.; John Wiley & Sons: New York, 1999.) to avoid undesired interactions or reactions during the preparation of the individual building blocks and during library synthesis. Analogously, reactive elements may be masked by suitably selected protection groups. It is appreciated by one skilled in the art that by suitable protection, a functional entity may carry a wide range of substi- tutents.
  • the Functional Entity Precursor is a masked. Functional Entity that is incorporated into an encoded molecule. After incorporation, reactive elements of the Functional Entity may be revealed by un-masking allowing further synthetic operations. Finally, elements mediating recognition of host molecules may be un-masked.
  • Functional entity precursor is -C(H)(R 1 )-R 11 ' or functional entity precursor is heteroaryl or aryl substituted with 0-3 R 11 , 0-3 R 13 and 0-3 R 15 , wherein
  • R 11 and R 11 ' are independently H, or selected among the group consisting of a C C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, C 4 -C 8 alkadienyl, C 3 -C 7 cycloalkyl, C 3 -C 7 cyclo- heteroalkyl, aryl, and heteroaryl, said group being substituted with 0-3 R 12 , 0-3 R 13 and 0-3 R 15 , or R 11 and R 11 ' are C C 3 alkylene-NR 12 2 , C C 3 alkylene-NR 12 C(O)R 16 , d-C 3 al- kylene-NR 12 C(O)OR 16 , C C 2 alkylene-O-NR 12 2 , C C 2 alkylene-O-NR 12 C(O)R 16 ,
  • R 12 is H or selected independently among the group consisting of C C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, C 3 -C 7 cycloalkyl, C 3 -C 7 cycloheteroalkyl, aryl, heteroaryl, said group being substituted with 0-3 R 13 and 0-3 R 15 ,
  • R 13 is selected independently from -N 3 , -CNO, -C(NOH)NH 2 , -NHOH,
  • R 14 where R 14 is independently selected from -NO 2 , -C(O)OR 17 , -COR 17 , -CN,
  • R 16 is H, C r C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, C 3 -C 7 cycloalkyl, aryl or C ⁇ -C 6 alkylene-aryl substituted with 0-3 substituents independently selected from -F, -CI, -
  • R 17 is selected independently from H, C C 6 alkyl, C 3 -C 7 cycloalkyl, aryl, C C 6 al-
  • the function of the carrier is to ensure the transferability of the functional entity precursor.
  • a skilled chemist can design suitable substitutions of the carrier by evaluation of initial attempts.
  • the transferability may be adjusted in response to the chemical composition of the functional entity precursor, to the nature of the complementing element, to the conditions under which the transfer and recognition is performed, etc.
  • the carrier is selected from the group consisting of ary- lene, heteroarylene or -(CF 2 ) m . substituted with 0-3 R 1 wherein m is an integer between 1 and 10, and C-F-connecting group is -SO 2 -O-. Due to the high reactivity of such compounds a broad range of recipient reactive groups may be employed in the construction of carbon-carbon bonds or carbon-hetero atom bonds.
  • the carrier is -(CF 2 ) m - wherein m is an integer between 1 and 10, the C-F-connecting group is -SO 2 -O-; and the func- tional entity precursor is aryl or heteroaryl substituted with 0-3 R 11 , 0-3 R 13 and 0-3 R 15 .
  • the C-F-connecting group determines in concert with the carrier the transferability of the functional entity precursor.
  • the C-F-connecting group is -S + (R 11 )-,
  • the C-F-connecting group is chosen from the group consisting of -SO 2 -O-, and -S + (R 17 )-; wherein R 17 is selected independently from H, C C 6 alkyl, C 3 -C 7 cycloalkyl, aryl, C ⁇ -C 6 alkylene-aryl.
  • an aromatic moiety may be transferred from the C-F-connecting group to a recipient reactive group. Further, the transfer may be initiated by adding the catalyst, independently of the annealing of encoding - and complementing elements.
  • the S-C-connecting group provide a means for connecting the Spacer and the Carrier. As such it is primarily of synthetic convenience and does not influence the function of a building block.
  • the spacer serves to distance the functional entity precursor to be transferred from the bulky complementing element.
  • the identity of the spacer is not crucial for the function of the building block. It may be desired to have a spacer which can be cleaved by light. In this case, the spacer is provided with e.g. the group
  • the spacer may be provided with a polyethylene glycol part of the general formula:
  • the complementing element serves the function of trans- ferring genetic information e.g. by recognising a coding element.
  • the recognition implies that the two parts are capable of interacting in order to assemble a complementing element - coding element complex.
  • a variety of interacting molecular parts are known which can be used according to the invention. Examples include, but are not restricted to protein-protein interactions, protein- polysaccharide interactions, RNA-protein interactions, DNA-DNA interactions, DNA-
  • RNA interactions RNA-RNA interactions, biotin-streptavidin interactions, enzyme- ligand interactions, antibody-ligand interaction, protein-ligand interaction, etc.
  • the interaction between the complementing element and coding element may result in a strong or a weak bonding. If a covalent bond is formed between the parties of the affinity pair the binding between the parts can be regarded as strong, whereas the establishment of hydrogen bondings, interactions between hydrophobic do- mains, and metal chelation in general results in weaker bonding. In general relatively weak bonding is preferred.
  • the complementing element is capable of reversible interacting with the coding element so as to provide for an attachment or detachment of the parts in accordance with the chang- ing conditions of the media.
  • the interaction is based on nucleotides, i.e. the complementing element is a nucleic acid.
  • the complementing element is a sequence of nucleotides and the coding element is a sequence of nucleo- tides capable of hybridising to the complementing element.
  • the sequence of nucleotides carries a series of nucleobases on a backbone.
  • the nucleobases may be any chemical entity able to be specifically recognized by a complementing entity.
  • the nucleobases are usually selected from the natural nucleobases (adenine, guanine, uracil, thymine, and cytosine) but also the other nucleobases obeying the Watson- Crick hydrogen-bonding rules may be used, such as the synthetic nucleobases disclosed in US 6,037,120. Examples of natural and non-natural nucleobases able to perform a specific pairing are shown in figure 2.
  • the backbone of the sequence of nucleotides may be any backbone able to aggregate the nucleobases is a sequence. Examples of backbones are shown in figure 4. In some aspects of the in- vention the addition of non-specific nucleobases to the complementing element is advantegeous, figure 3
  • the coding element can be an oligonucleotide having nucleobases which complements and is specifically recognised by the complementing element, i.e. in the event the complementing element contains cytosine, the coding element part contains guanine and visa versa, and in the event the complementing element contains thymine or uracil the coding element contains adenine.
  • the complementing element may be a single nucleobase. In the generation of a library, this will allow for the incorporation of four different functional entities into the template-directed molecule. However, to obtain a higher diversity a complementing element preferably comprises at least two and more preferred at least three nucleotides. Theoretically, this will provide for 4 2 and 4 3 , respectively, different functional entities uniquely identified by the complementing element.
  • the complementing element will usually not comprise more than 100 nucleotides. It is preferred to have complementing elements with a sequence of 3 to 30 nucleotides.
  • the building blocks of the present invention can be used in a method for transferring a functional entity precursor to a recipient reactive group, said method comprising the steps of providing one or more building blocks as described above and contacting the one or more building blocks with a corresponding encoding element associated with a recipient reactive group under conditions which allow for a recognition between the one or more complementing elements and the encoding elements, said contacting being performed prior to, simultaneously with, or subse- quent to a transfer of the functional entity precursor to the recipient reactive group.
  • the encoding element may comprise one, two, three or more codons, i.e. sequences that may be specifically recognised by a complementing element.
  • Each of the codons may be separated by a suitable spacer group.
  • all or at least a majority of the codons of the template are arranged in sequence and each of the codons are separated from a neighbouring codon by a spacer group.
  • the number of codons of the encoding element is 2 to 100.
  • encoding elements comprising 3 to 10 codons.
  • a codon comprises 1 to 50 nucleotides and the complementing element comprises a sequence of nucleotides complementary to one or more of the encoding sequences.
  • the recipient reactive group may be associated with the encoding element in any appropriate way.
  • the reactive group may be associated covalently or non- covalently to the encoding element.
  • the recipient reactive group is linked covalently to the encoding element through a suitable linker which may be separately cleavable to release the reaction product.
  • the reactive group is coupled to a complementing element, which is capable of recognis- ing a sequence of nucleotides on the encoding element, whereby the recipient reactive group becomes attached to the encoding element by hybridisation.
  • the recipient reactive group may be part of a chemical scaffold, i.e. a chemical entity having one or more reactive groups available for receiving a functional entity precursor from a building block.
  • the recipient reactive group may be any group able to participate in cleaving the bond between the carrier and the functional entity precursor to release the functional entity precursor.
  • the recipient reactive group is a nucleophilic atom such as S, N, O, C or P.
  • Scheme 1a shows the transfer of an alkyl group and scheme 1b shows the transfer of an vinyl group.
  • the recipient reactive group is a organometallic compound as shown in scheme 2.
  • the building blocks are used for the formation of a library of compounds.
  • the complementing element of the building block is used to identify the functional entity. Due to the enhanced proximity between reactive groups when the complementing entity and the encoding element are contacted, the functional entity precursor together with the identity programmed in the complementing element is transferred to the encoding element associated with recipient reactive group.
  • the sequence of the complementing element is unique in the sense that the same sequence is not used for another functional entity.
  • the unique identification of the functional entity enable the possibility of decoding the encoding element in order to determine the synthetic history of the molecule formed. In the event two or more functional entities have been transferred to a scaffold, not only the identity of the transferred functional entities can be determined.
  • each different member of a library comprises a complementing element having a unique sequence of nucleotides, which identifies the functional entity.
  • FIG. 1 Two setups for Functional Entity Transfer Figure 2. Examples of specific base pairing Figure 3. Example of non-specific base-pairing Figure 4. Backbone examples Figure 5 Three examples of building blocks
  • a building block of the present invention is characterized by its ability to transfer its functional entity precursor to a recipient reactive group. This is done by forming a new covalent bond between the recipient reactive group and cleaving the bond between the carrier moiety and the functional entity precursor of the building block.
  • FIG. 1 Two setups for generalized functional entity precursor transfer from a building block are depicted in figure 1.
  • one complementing element of a build- ing block recognizes a coding element carrying another functional entity precursor, hence bringing the functional entities in close proximity. This results in a reaction between functional entity precursor 1 and 2 forming a covalent bond between these concurrent with the cleavage of the bond between functional entity precursor 2 and its linker.
  • a template brings together two building blocks re- suiting in functional entity precursor transfer from one building block to the other.
  • Figure 5 illustrates three specific compounds according to the invention. For illustrative purposes the individual features used in the claims are indicated.
  • the upper compound is an example of a building block wherein the linker is backbone attached at the 3'-position.
  • the first part of the linker i.e. the spacer, is an aliphatic chain ending in a nitrogen atom.
  • the nitrogen atom bridges to the S-C-connecting group, which is an N-acylated arylmethyleamine.
  • the carrier attached to the left hand side carbonyl group of the S-C-connecting group is a benzene ring holding the C-F Connecting group in the para position.
  • the C-F Connecting group is a positively charged sulfur atom which is attached to the Functional Entity Precursor, in this case a benzyl group.
  • the middle compound illustrates a 5' attachment of a linker.
  • the linker is linked through a phosphate group and extends into a three membered aliphatic chain. Through another phosphate group and a PEG linker the complementing element is linked via an amide bond to the Carrier.
  • the Functional Entity Precursor is transferred resulting in an alkylation of the nucleophile.
  • the lower compound illustrates a nucleobase attachment of the linker.
  • the linker attaches to the 5 position of a pyrimidine type nucleobase and extents through an ⁇ - ⁇ unsaturated N-methylated amide to the S-C-connecting group, which is a 4- amino methyl benzoic acid derivative.
  • the functional entity precursor can be transferred to a nucleophilic recipient reactive group e.g. an amine or a thiol forming an allylic amine or thiol.
  • the functional entity precursor is of the formula -C(H)(R 3 )-R 4 or functional entity precursor is heteroaryl or aryl optionally substituted with one or more substituents belonging to the group comprising R 3 and R 4 .
  • the functional entity precursor is of the formula -C(H)(R 3 )-R 4 or functional entity precursor is heteroaryl or aryl optionally substituted with one or more substituents belonging to the group comprising R 3 and R 4 .
  • R 5 , R 6 , R 7 and R 8 independently is H, C C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, C 4 -C 8 alkadienyl, C 3 -C 7 cycloalkyl, C 3 -C 7 cycloheteroalkyl, aryl or heteroaryl and wherein R 5 and R 6 may together form a 3-8 membered heterocyclic ring or R 5 and R 7 may together form a 3-8 membered heterocyclic ring or R 6 and R 7 may together form a 3- 8 membered heterocyclic ring,
  • R 5 , R 6 , R 7 and R 8 independently is H, C C 6 alkyl, C 3 -C 7 cycloalkyl, C 3 -C 7 cyclohet- eroalkyl, aryl or heteroaryl and wherein R 5 and R 6 may together form a 3-8 membered heterocyclic ring or R 5 and R 7 may together form a 3-8 membered heterocyclic ring or R 6 and R 7 may together form a 3-8 membered heterocyclic ring,
  • R 5 , R 6 , R 7 and R 8 independently is H, C C 6 alkyl, C 3 -C 7 cycloalkyl, C 3 -C 7 cyclohet- eroalkyl, aryl or heteroaryl and wherein R 5 and R 6 may together form a 3-8 membered heterocyclic ring or R 5 and R 7 may together form a 3-8 membered heterocyclic ring or R 6 and R 7 may together form a 3-8 membered heterocyclic ring,
  • R 5 , R 6 , R 7 and R 8 independently is H, d-C 6 alkyl, C 3 -C 7 cycloalkyl, C 3 -C 7 cycloheteroalkyl, aryl or heteroaryl and wherein R 5 and R 6 may together form a 3-8 membered heterocyclic ring or R 5 and R 7 may together form a 3-8 membered heterocyclic ring or R 6 and R 7 may together form a 3-8 membered heterocyclic ring,
  • R 5 , R 6 , R 7 and R 8 independently is H, C C 6 alkyl, C 3 -C 7 cycloalkyl, C 3 -C 7 cycloheteroalkyl, aryl or heteroaryl and wherein R 5 and R 6 may together form a 3-8 membered heterocyclic ring or R 5 and R 7 may together form a 3-8 membered heterocyc- lie ring or R 6 and R 7 may together form a 3-8 membered heterocyclic ring,
  • R 5 , R 6 , R 7 and R 8 independently is H, C C 6 alkyl, C 3 -C 7 cycloalkyl, C 3 -C 7 cycloheteroalkyl, aryl or heteroaryl and wherein R 5 and R 6 may together form a 3-8 membered heterocyclic ring or R 5 and R 7 may together form a 3-8 membered heterocyclic ring or R 6 and R 7 may together form a 3-8 membered heterocyclic ring, in still another prefered embodiment,
  • R 5 , R 6 , R 7 and R 8 independently is H, C C 6 alkyl, C 3 -C 7 cycloalkyl, C 3 -C 7 cycloheteroalkyl, aryl or heteroaryl and wherein R 5 and R 6 may together form a 3-8 mem- bered heterocyclic ring or R 5 and R 7 may together form a 3-8 membered heterocyclic ring or R 6 and R 7 may together form a 3-8 membered heterocyclic ring,
  • R 5 , R 6 , R 7 and R 8 independently is H, methyl, ethyl, propyl, butyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, phenyl, naphthyl, thienyl, furyl, pyridinyl, quinolinyl or isoquinolinyl and wherein R 5 and R 6 may together form a 3-8 membered het- erocyclic ring or R 5 and R 7 may together form a 3-8 membered heterocyclic ring or R 6 and R 7 may together form a 3-8 membered heterocyclic ring,
  • R 5 , R 6 , R 7 and R 8 independently is H, methyl, ethyl, propyl, butyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, phenyl, naphthyl, thienyl, furyl, pyridinyl, quinolinyl or isoquinolinyl and wherein R 5 and R 6 may together form a 3-8 membered heterocyclic ring or R 5 and R 7 may together form a 3-8 membered heterocyclic ring or R 6 and R 7 may together form a 3-8 membered heterocyclic ring,
  • R 5 , R 6 , R 7 and R 8 independently is H, methyl, ethyl, propyl, butyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, phenyl, naphthyl, thienyl, furyl, pyridinyl, quinolinyl or isoquinolinyl and wherein R 5 and R 6 may together form a 3-8 membered heterocyclic ring or R 5 and R 7 may together form a 3-8 membered heterocyclic ring or R
  • R 5 , R 6 , R 7 and R a independently is H, methyl, ethyl, propyl, butyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, phenyl, naphthyl, thienyl, furyl, pyridinyl, quinolinyl or isoquinolinyl and wherein R 5 and R 6 may together form a 3-8 membered het- erocyclic ring or R 5 and R 7 may together form a 3-8 membered heterocyclic ring or R 6 and R 7 may together form a 3-8 membered heterocyclic ring,
  • R 5 , R 6 , R 7 and R 8 independently is H, methyl, ethyl, propyl or butyl and wherein R 5 and R 6 may together form a 3-8 membered heterocyclic ring or R 5 and R 7 may to- gether form a 3-8 membered heterocyclic ring or R 6 and R 7 may together form a 3-8 membered heterocyclic ring,
  • R 5 , R 6 , R 7 and R 8 independently is H, methyl, ethyl, propyl or butyl and wherein R 5 and R 6 may together form a 3-8 membered heterocyclic ring or R 5 and R 7 may together form a 3-8 membered heterocyclic ring or R 6 and R 7 may together form a 3-8 membered heterocyclic ring,
  • R 5 , R 6 , R 7 and R 8 independently is H, methyl, ethyl, propyl or butyl and wherein R 5 and R 6 may together form a 3-8 membered heterocyclic ring or R 5 and R 7 may together form a 3-8 membered heterocyclic ring or R 6 and R 7 may together form a 3-8 membered heterocyclic ring,
  • R 5 , R 6 , R 7 and R 8 independently is H, methyl, ethyl, propyl or butyl and wherein R 5 and R 6 may together form a 3-8 membered heterocyclic ring or R 5 and R 7 may together form a 3-8 membered heterocyclic ring or R 6 and R 7 may together form a 3-8 membered heterocyclic ring,
  • R 5 , R 6 , R 7 and R 8 independently is H, cyclopropyl, cyclobutyl, cyclopentyl or cyclo- hexyl,
  • R 5 , R 6 , R 7 and R 8 independently is H, cyclopropyl, cyclobutyl, cyclopentyl or cyclo- hexyl,
  • R 5 , R 6 , R 7 and R 8 independently is H, cyclopropyl, cyclobutyl, cyclopentyl or cyclo- hexyl,
  • R 5 , R 6 , R 7 and R 8 independently is H, cyclopropyl, cyclobutyl, cyclopentyl or cyclo- hexyl,
  • R 5 , R 6 , R 7 and R 8 independently is H, cyclopropyl, cyclobutyl, cyclopentyl or cyclo- hexyl,
  • R 5 , R 6 , R 7 and R 8 independently is H, phenyl, naphthyl, thienyl, furyl, pyridinyl, qui- nolinyl or isoquinolinyl, in still another prefered embodiment,
  • R 5 , R 6 , R 7 and R 8 independently is H, phenyl, naphthyl, thienyl, furyl, pyridinyl, qui- nolinyl or isoquinolinyl,
  • R 5 , R 6 , R 7 and R 8 independently is H, phenyl, naphthyl, thienyl, furyl, pyridinyl, qui- nolinyl or isoquinolinyl,
  • R 5 , R 6 , R 7 and R 8 independently is H, phenyl, naphthyl, thienyl, furyl, pyridinyl, qui- nolinyl or isoquinolinyl,
  • R 5 , R 6 , R 7 and R 8 independently is H, phenyl, naphthyl, thienyl, furyl, pyridinyl, qui- nolinyl or isoquinolinyl,
  • R 3 and R 4 independently is H, C C 6 alkyl, C 3 -C 7 cycloalkyl, C 3 -C 7 cycloheteroalkyl, aryl or heteroaryl
  • R 3 and R 4 independently is H
  • R 3 and R 4 independently is C C 6 alkyl, C 3 -C 7 cycloalkyl or C 3 -C 7 cycloheteroalkyl,
  • R 3 and R 4 independently is methyl, ethyl, propyl or butyl
  • R 3 and R 4 independently is cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl
  • R 3 and R 4 independently is aziridinyl, pyrrolidinyl, piperidinyl or morpholinyl
  • R 3 and R 4 independently is aryl or heteroaryl
  • R 3 and R 4 independently is phenyl or naphthyl
  • R 3 and R 4 independently is thienyl, furyl, pyridyl, quinolinyl or isoquinolyl
  • the 4-halobenzoic acid (25 mmol) is added to a ice cooled solution of chloro sulfo- nic acid (140 mmol). The mixture is slowly heated to reflux and left at reflux for 2-3 hours. The mixture is added to 100 mL ice and the precipitate collected by filtration. The filtrate is washed with water (2 x 50 mL) and the dried in vacuo affording the corresponding sulfonoyl chloride in 60-80% yield.
  • the 3-chlorosulfonyl-4-halo- benzoic acid derivate (5 mmol) is dissolved in EtOH (5 mL) and added to a ice cooled mixture of NaOEt (10 mL, 2M).
  • Ps Polystyrene resin. Alternatively other acid labile linkers may be employed.
  • Step l A polystyrene resin with a wang linker (4-hydroxymethylphenol linker) (50 mg ⁇ 50 umol), a bi-functional carrier (200 umol, 4 equiv) in a solvent such as THF, DCM, DCE, DMF, NMP or a mixture thereof (500 uL) and a base such as TEA, DIEA, pyri- dine (400 umol, 8 equiv), optionally in the presence of DMAP (100 umol), are allowed to react at temperatures between -20 °C and 60 °C, preferably between 0 °C and 25 °C, for 1-24 h, preferably 1-4 h. The resin is washed with the solvent compo- sition used during the reaction (5x1 mL) and used in the following step.
  • a solvent such as THF, DCM, DCE, DMF, NMP or a mixture thereof (500 uL) and a base such as TEA, DIEA
  • a functional entity precursor carrying a hydroxy group in the position of the intended attachment to the C-F-connecting group (200 umol, 4 equiv) in a solvent such as THF, DCM, DCE, DMF, NMP or a mixture thereof (500 uL) and a base such as TEA,
  • DIEA, pyridine (400 umol, 8 equiv), optionally in the presence of DMAP, are added to the resin bound carrier isolated in step 1 and allowed to react at temperatures between 0 °C and 100 °C, preferably between 25 °C and 80 °C, for 2-48 h, preferably 4-16 h.
  • the resin is washed with the solvent composition used during the reac- tion (5x1 mL).
  • the desired Carrier-Functional entity reagent is cleaved from the resin obtained in step 2 by treatment with an acid like TFA, HF or HCI in a solvent such as THF, DCM, DCE or a mixture thereof (1 mL) at temperatures between -20 °C and 60 °C, preferably between 0 °C and 25 °C, for 1-4 h, preferably 1-2 h.
  • a solvent such as THF, DCM, DCE or a mixture thereof
  • the Carrier-Functional entity reagent may be bound to the Spacer by several different reactions as illustrated below. Formation of an amide bond between a carboxylic acid of the Carrier and an amine group of a Spacer
  • 15 ⁇ L of a 150 mM building block solution of FE 1 -Carrier-COOH is mixed with 15 ⁇ L of a 150 mM solution of EDC and 15 ⁇ L of a 150 mM solution of N-hydroxy- succinimide (NHS) using solvents like DMF, DMSO, water, acetonitril, THF, DCM, methanol, ethanol or a mixture thereof.
  • the mixture is left for 15 min at 25°C.
  • 45 ⁇ L of an aminooligo (10 nmol) in 100 mM buffer at a pH between 5 and 10, preferably 6.0-7.5 is added and the reaction mixture is left for 2 hours at 25°C. Excess building block and organic by-products were removed by extraction with EtOAc (400 ⁇ L).
  • EtOAc Remaining EtOAc is evaporated in vacuo using a speedvac.
  • the building block is purified following elution through a BioRad micro-spin chromatography column, and analyzed by electron spray mass spectrometry (ES-MS).
  • Oligo A 5'-YACGATGGATGCTCCAGGTCGC
  • Funtional Entity-OH is a phenol, n is an integer between 3 and 6.
  • the reaction mixture from step 1 is added to a solution of an aminooligo (10 nmol) in 100 mM buffer at a pH between 5 and 10, preferably 6.0-7.5 optionally in the presence of NHS.
  • the reaction mixture is left for 2 hours at 25°C. Excess building block and organic by-products were removed by extraction with EtOAc (400 ⁇ L). Remaining EtOAc is evaporated in vacuo using a speedvac.
  • the building aminooligo is purified following elution through a BioRad micro-spin chromatography column, and analyzed by electron spray mass spectrometry (ES-MS). Use of building blocks
  • An oligonucleotide building block carrying functional entity FE 1 is combined at 2 ⁇ M final concentration with one equivalent of a complementary building block displaying a nucleophilic recipient group. Reaction proceeds at temperatures between 0 °C and 100 °C preferably between 15 °C-50 °C for 1-48 hours, preferably 10-20 hours in DMF, DMSO, water, acetonitril, THF, DCM, methanol, ethanol or a mixture thereof, pH buffered to 4-10, preferably 6-8. Organic by-products are removed by extraction with EtOAc, followed by evaporation of residual organic solvent for 10 min in vacuo. Pd catalyst is removed and oligonucleotides are isolated by eluting sample through a BioRad micro-spin chromatography column. Coupling efficiency is quantified by ES-MS analysis.
  • a carrier coupled functional entity oligo (Example 1) (250 pmol) was added to a scaffold oligo B (200 pmol) in 50 ⁇ l 100 mM MES, pH 6. The mixture was incubated overnight at 25 °C. Subsequently, the mixture was purified by gel filtration using a microspin column equilibrated with H 2 O and transfer of the functional entity was veri- fied by electron spray mass spectrometry (ES-MS). Transfer efficiency is expressed in percent and were calculated by dividing the abundance of scaffold oligo carrying transferred functional entities to total abundance of scaffold oligos (with and without transferred functional entities).
  • An oligonucleotide building block carrying functional entity FE 1 is combined at 2 ⁇ M final concentration with one equivalent of a complementary building block displaying a nucleophilic recipient group.
  • the reaction pro- ceeds at temperatures between 0 °C and 100 °C preferably between 15 °C-50 °C for 1-48 hours, preferably 10-20 hours in DMF, DMSO, water, acetonitrile, THF, DCM, methanol, ethanol or a mixture thereof, pH buffered to 4-10, preferably 6-8.
  • Organic by-products are removed by extraction with EtOAc, followed by evaporation of residual organic solvent for 10 min in vacuo.
  • Pd catalyst is removed and oligonu- cleotides are isolated by eluting sample through a BioRad micro-spin chromatogra- phy column. Coupling efficiency is quantified by ES-MS analysis.
  • R 1 H, Me, Et, iPr, CI, N0 2
  • R 2 H, Me, Et, iPr, CI, N0 2
  • R 1 and R 2 may be used to tune the reactivity of the sulphate to allow appropriate reactivity. Chloro and nitro substitution will increase reactivity. Alkyl groups will decrease reactivity. Ortho substituents to the sulphate will due to steric reasons direct incoming nucleophiles to attack the R-group selectively and avoid attack on sulphur. E.g.
  • 3-Aminophenol (6) is treated with maleic anhydride, followed by treatment with an acid e.g. H 2 SO 4 or P 2 O 5 and heat to yield the maleimide (7).
  • the ring closure to the maleimide may also be achieved when an acid stable O-protection group is used by treatment with or Ac 2 O with or without heating, followed by O-deprotection. Alternatively reflux in Ac 2 O, followed by O-deacetylation in hot water/dioxane to yield (7).
  • a thiol carrying oligonucleotide in buffer 50 mM MOPS or hepes or phosphate pH 7.5 is treated with a 1-100 mM solution and preferably 7.5 mM solution of the organic building block (9) in DMSO or alternatively DMF, such that the DMSO/DMF concentration is 5-50%, and preferably 10%.
  • the mixture is left for 1-16 h and preferably 2-4 h at 25 °C.
  • methylating monomer building block (10) To give the alkylating in this case methylating monomer building block (10).
  • reaction of the alkylating monomer building block (10) with an amine carrying monomer building block may be conducted as follows:
  • the oligonucleotides are annealed to the template by heating to 50 °C and cooled (2 °C/ second) to 30 °C. The mixture is then left o/n at a fluctuating temperature (10 °C for
  • a vinylating monomer building block may be prepared and used similarily as described above for an alkylating monomer building block.
  • the intermediate chlorosulphate is isolated and treated with an enolate or O-trialkylsilylenolate with or without the presence of fluoride.
  • the thiol carrying oligonucleotide in buffer 50 mM MOPS or hepes or phosphate pH 7.5 is treated with a 1-100 mM solution and preferably 7.5 mM solution of the organic building block (12) in DMSO or alternatively DMF, such that the DMSO/DMF concentration is 5-50%, and preferably 10%.
  • the mixture is left for 1-16 h and preferably 2-4 h at 25 °C.
  • the sulfonylenolate (13) may be used to react with amine carrying monomer building block to give an enamine (14a and/or 14b) or e.g. react with an carbanion to yield (15a and/or 15b).
  • enamine 14a and/or 14b
  • carbanion 15a and/or 15b
  • the reaction of the vinylating monomer building block (13) and an amine or nitroal- kyl carrying monomer building block may be conducted as follows:
  • the oligonucleotides are annealed to the template by heating to 50 °C and cooled (2 °C/ second) to 30 °C.
  • the mixture is then left o/n at a fluctuating temperature (10 °C for 1 second then 35 °C for 1 second), to yield template bound (14a/b or 15a/b).

Abstract

L'invention concerne un bloc de construction apte au transfert d'informations génétiques et d'un précurseur d'entité fonctionnel vers un groupe réactif de cellules réceptrices. Le bloc de construction peut servir à générer un complexe simple ou des pharmacothèques de différents complexes, le complexe comprenant une molécule codée liée à un élément codant. Les pharmacothèques de complexes sont utilisées lors de demandes de composés pharmaceutiquement actifs.
EP03711855A 2002-03-15 2003-03-14 Bloc de construction formant une liaison atomique c-c ou c hetero apres reaction Withdrawn EP1487850A2 (fr)

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US43442302P 2002-12-19 2002-12-19
DKPA200201950 2002-12-19
DK200201950 2002-12-19
US434423P 2002-12-19
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WO2002102820A1 (fr) 2001-06-20 2002-12-27 Nuevolution A/S Derives nucleosidiques pour elaboration de bibliotheque combinatoire
US7413854B2 (en) 2002-03-15 2008-08-19 Nuevolution A/S Method for synthesising templated molecules
US10730906B2 (en) 2002-08-01 2020-08-04 Nuevolutions A/S Multi-step synthesis of templated molecules
EP3299463B1 (fr) 2002-10-30 2020-10-21 Nuevolution A/S Codage enzymatique
WO2004056994A2 (fr) 2002-12-19 2004-07-08 Nuevolution A/S Procedes de synthese guidee a fonction et structure quasi-selectives
WO2004074429A2 (fr) 2003-02-21 2004-09-02 Nuevolution A/S Procede de production d'une banque de deuxieme generation
DE602004019764D1 (de) 2003-03-20 2009-04-16 Nuevolution As Ligationsvermittelnde codierung von kleinen molekülen
WO2005026387A1 (fr) 2003-09-18 2005-03-24 Nuevolution A/S Procede permettant d'obtenir des informations structurelles concernant une molecule codee et procede permettant de selectionner des composes
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EP1940755A2 (fr) 2005-10-28 2008-07-09 Praecis Pharmaceuticals Inc. Procedes d'identification de composes etudies, par recours a des bibliotheques codees
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KR20170119729A (ko) 2009-02-13 2017-10-27 엑스-켐, 인크. Ｄｎａ―코딩된 라이브러리의 생성 및 스크리닝 방법
US11225655B2 (en) 2010-04-16 2022-01-18 Nuevolution A/S Bi-functional complexes and methods for making and using such complexes
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