Template directed split and mix synthesis of small molecule libraries

Abstract

The invention combines the advantages of split and mix synthesis with the advantages of template directed synthesis. The method comprises the steps of: a) adding a linker molecule L to one or more reaction wells; b) adding a molecule fragment to each of said reaction wells; c) adding an oligonucleotide identifier to each of said reaction wells; d) subjecting said wells to conditions sufficient to allow said molecule fragments and said oligonucleotide identifiers to become attached to said linker molecule, or conditions sufficient for said molecule fragments to bind to other molecule fragments and sufficient for said oligonucleotide identifiers to bind to other oligonucleotide identifiers; e) combining the contents of said one or more reaction wells; and f) contacting the resulting bifunctional molecule(s) of step e) with one or more (oligonucleotide) templates each capable of hybridizing to at least one of the oligonucleotide identifiers added in step c).

1 . (canceled) 
     
     
         2 . A method for obtaining a bifunctional molecule comprising an encoded molecule part and a coding part, said method comprising:
 adding one or more molecule fragments to a nascent bifunctional molecule, wherein said nascent bifunctional molecule comprises a first reactive group suitable for coupling of said one or more molecule fragments and a second reactive group suitable for enzymatic coupling of one or more oligonucleotide identifiers; and   adding one or more oligonucleotide identifiers;   under conditions where said one or more molecule fragments become covalently attached to said first reactive group, and said one or more oligonucleotide identifiers become covalently attached to said second reactive group using one or more enzymes, to produce said bifunctional molecule; wherein the reactions involving said first and said second reactive groups may be performed in any order;   wherein the oligonucleotide identifiers comprise natural oligonucleotides;   wherein said first reactive group is chosen from the following list of reactive groups: —NH 2 , —COOH, —CHO, —OH, —NHR, —CSO 2 OH, phenylchloride, —SH, and —SS; and   wherein the encoded molecule part of the bifunctional molecule comprises one or more molecule fragments;   wherein the coding part of the bifunctional molecule comprises one or more oligonucleotide identifier(s); and   wherein the encoded molecule part comprises a polycyclic heterocycle.   
     
     
         3 . The method according to  claim 2 , wherein the bifunctional molecule obtained in  claim 2 , is reacted further one or more times with one or more molecule fragments and is provided with respective oligonucleotide identifiers to produce a bifunctional molecule comprising at least two molecule fragments and at least two oligonucleotide identifiers. 
     
     
         4 . The method of  claim 3 , wherein two or more bifunctional molecules are generated to produce a collection of bifunctional molecules each comprising an encoded molecule part and a coding part,
 wherein the encoded molecule part of each bifunctional molecule comprises two or more molecule fragments;   wherein the coding part of each bifunctional molecule comprises two or more oligonucleotide identifiers.   
     
     
         5 . A method for identifying a molecule with desired characteristics comprising:
 a) producing a collection of bifunctional molecules each comprising an encoded molecule part and a coding part, by using the method of  claim 4 ;   b) contacting the bifunctional molecules of step (a) with immobilized target molecules such that a subset of the collection of bifunctional molecule becomes bound to the immobilized target molecules;   c) washing the immobilized target molecules;   d) releasing bound bifunctional molecules from the immobilized target molecules by releasing the encoded molecule part from the immobilized target molecules;   e) contacting the released bifunctional molecules of step (d) with immobilized target molecules such that a subset of the collection of bifunctional molecules becomes bound to the immobilized target molecules;   f) washing the immobilized target molecules;   g) optionally releasing the bifunctional molecules from the immobilized target molecules;   h) identifying the bifunctional molecules resulting after step (b), (c), (d), (e), (f) or (g);   i) producing the encoded molecules of the identified bifunctional molecules of step (h) in their free form by organic chemistry; and   j) analyzing the characteristics of the encoded molecules in their free form; wherein the immobilized target molecules are immobilized on a solid support.   
     
     
         6 . The method of  claim 5  wherein a linker comprising polyethylene glycol connects the encoded molecule part and the coding part, and wherein the linker has a length of 1-50A. 
     
     
         7 . The method of  claim 6  wherein the linker has a length of 10-25 Å. 
     
     
         8 . The method of  claim 5  where at least one of said encoded molecule parts comprises a bridged polycyclic heterocycle. 
     
     
         9 . The method of  claim 5  where at least one of said encoded molecule parts comprises a cyclohexane-backbone modified beta-peptide, a cyclopentane-backbone modified beta-peptide, an azatide, a peptoid, or a trifunctional nonaromat carbocycle. 
     
     
         10 . The method of  claim 5  where at least one of said reactions between a reactive group and a molecule fragment leads to formation of a vinyl substituted aromatic compound, a substituted cycloalkane, an oxime, or a hydroxylamine ether. 
     
     
         11 . The method of  claim 5  involving the use of at least one accessory reagent chosen from the following list: nickel, rhodium, copper, cobalt, iron, osmium, titanium, and phosphine. 
     
     
         12 . The method of  claim 5  involving the use of at least one protecting group that is cleaved by LiOH, PdCl 2 , TCEP, or NaIO 4 . 
     
     
         13 . The method of  claim 5  wherein at least 100 of said two or more bifunctional molecules are generated. 
     
     
         14 . A method for identifying molecules with desired characteristics comprising:
 a) Adding linker molecules L to at least 100 reaction wells;   b) Contacting the linker molecules L of said reaction wells with a molecule fragment different from the molecule fragment added to the other wells, under conditions sufficient to allow the molecule fragment to attach to the linker molecules of the same well;   c) Contacting the linker molecules L of said reaction wells with an oligonucleotide identifier different from the oligonucleotide identifier added to any of the other wells, under conditions sufficient to allow the oligonucleotide identifier to attach to the linker molecules of the same well;   wherein steps b) and c) may be performed in any order to form bi-functional molecules comprising a molecule fragment, a linker molecule L, and an oligonucleotide identifier;   d) Combining the resulting bifunctional molecules of steps a) to c) into an admixture, and aliquoting said admixture into at least 100 different reaction wells;   e) Contacting the bi-functional molecules of each of said reaction wells with a molecule fragment different from the molecule fragment added to any of the other wells in this step e), under conditions sufficient to allow said molecule fragment to attach to said bi-functional molecules;   f) Contacting the bi-functional molecules of each of said reaction wells with an oligonucleotide identifier different from the oligonucleotide identifier added to any of the other wells in this step f), under conditions sufficient to allow said oligonucleotide identifier to attach to said bi-functional molecules;   wherein steps e) and f) may be performed in any order to form bi-functional molecules each comprising an encoded molecule comprising at least two molecule fragments, a linker molecule L, and at least two oligonucleotide identifiers;   g) Combining the resulting bi-functional molecules of steps d) to f) into an admixture;   wherein in step b) at least one reactive group of the linker molecule L reacts with a reactive group in the molecule fragment;   wherein in step c) at least one reactive group of the linker molecule L reacts with a reactive group in the oligonucleotide identifier;   wherein in step e) at least one reactive group of the molecule fragment reacts with a reactive group in a molecule fragment attached to Linker L,   wherein in step f) at least one reactive group of the oligonucleotide identifier reacts with a reactive group in an oligonucleotide identifier attached to Linker L;   wherein at least one of said encoded molecules comprise a tricyclic heterocycle, or at least two of said encoded molecules comprise a polycyclic heterocycle, of which one is a bridged polycyclic heterocycle, or at least one of said encoded molecules comprise a trifunctional aromatic heterocycle;   h) Contacting the admixture of bifunctional molecules of step g) with target molecules, under conditions allowing the binding of some of the bifunctional molecules to said target molecules, under conditions where the target molecules are immobilized on a solid support, during or after the binding of some of the bifunctional molecules to said target molecules;   i) Washing the solid support;   j) Releasing the bifunctional molecules from the solid support;   k) Contacting the released bifunctional molecules of step j) with target molecules, under conditions allowing the binding of some of the bifunctional molecules to said target molecules, under conditions where the target molecules are immobilized on a solid support, during or after the binding of some of the bifunctional molecules to said target molecules;   l) Washing the solid support;   m) Optionally releasing the bifunctional molecules from the solid support;   n) Sequencing the oligonucleotide identifiers of the bifunctional molecules that were recovered in step k), l) or m) to identify the encoded molecules that bound the target molecules;   o) Producing said encoded molecules in their free form by organic chemistry; and   p) Analysing the characteristics of said encoded molecules in their free form.   
     
     
         15 . The method of  claim 14 , wherein at least one of said encoded molecules comprise a tricyclic heterocycle, and at least two of said encoded molecules comprise a polycyclic heterocycle, of which one is a bridged polycyclic heterocycle, and at least one of said encoded molecules comprise a trifunctional aromatic heterocycle. 
     
     
         16 . The method of  claim 14 , wherein the characteristics of said encoded molecules in their free form, and thus without being attached to the oligonucleotide identifier, are analysed by an assay selected from the group consisting of: an enzyme inhibition assay, a cell-based receptor binding assay, a cell-based activity assay, CaCo2-cell-based analysis of membrane permeability, in vivo determination of animal toxicity, solubility, water-octanol partitioning measurements, and metabolic stability measurements. 
     
     
         17 . The method of  claim 14 , wherein the release of the bi-functional molecules from the solid support is effectuated by a reagent, pH change or light, wherein the reagent is a ligand that binds the target molecule, and wherein the ligand is selected from the group consisting of: a small molecule, a peptide, a DNA aptamer, and a protein. 
     
     
         18 . The method of  claim 14 , wherein the sequencing is by mass spectrometry-based sequencing, single molecule sequencing, or sequencing by hybridisation to oligonucleotide arrays. 
     
     
         19 . The method of  claim 14 , wherein the reaction between at least two reactive groups involves catalysis, where the catalysis is chosen from the list of homogenous, heterogenous, phase transfer and asymmetric catalysis. 
     
     
         20 . The method of  claim 14 , where the reaction between at least two reactive groups is a Wittig Olefination, a 1,3 Dipolar Cycloaddition, a nitro-Michael addition, a carbon-carbon bond forming reaction, or an organometallic coupling reaction. 
     
     
         21 . The method of  claim 14 , where at least one of said reactions between a reactive group and a molecule fragment leads to formation of a substituted cyclodiene, and where at least one of said reactions between a reactive group and a molecule fragment leads to formation of a beta-hydroxy ketone.