Method for the synthesis of a bifunctional complex
Disclosed is a method for obtaining a bifunctional complex comprising a display molecule part and a coding part, wherein a nascent bifunctional complex comprising a chemical reaction site and a priming site for enzymatic addition of a tag is reacted at the chemical reaction site with one or more reactants, and provided with respective tag(s) identifying the reactant(s) at the priming site is using one or more enzymes.
1. A method for alternating parallel synthesis of a combinatorial library of different small molecules, said method comprising the steps of
i) providing a plurality of the same or different nascent bifunctional molecules;
ii) dividing said same or different nascent bifunctional molecules into a plurality of compartments;
iii) reacting one or more chemical reaction sites of a nascent bifunctional molecule in each of said compartments with the same or different reactants, wherein the reactions are conducted in organic media; and
iv) adding a nucleic acid tag to a nascent bifunctional molecule in each of said compartments, wherein each tag identifies one or more of said reactants, and wherein the addition is conducted in aqueous media;
v) repeating steps iii) and iv) one or more times;
vi) complementing the nucleic acid tags by hybridization to at least partly complementary nucleic acid anti-tags; and
vii) obtaining the combinatorial library of different small molecules.
2. The method of claim 1 , wherein nucleic acid tags are added by enzymatic or chemical ligation.
3. The method of claim 2 , wherein nucleic acid tags are added by enzymatic ligation.
4. The method of claim 1 , wherein the organic media is changed to an aqueous media prior to adding the nucleic acid tags to the nascent bifunctional molecules.
5. The method of claim 1 , wherein the aqueous media is changed to an organic media prior to reacting the reactants with the one or more chemical reaction sites of the nascent bifunctional molecules.
6. The method of claim 3 , wherein enzymatic ligation is conducted in aqueous media, followed by lyophilisation of the nascent bifunctional molecules, wherein the lyophilized nascent bifunctional molecules are dissolved or dispersed in an organic media, and wherein the one or more chemical reaction sites are reacted with the reactants in the organic media.
7. The method of claim 1 , wherein an organic solvent is added to the aqueous media after tag addition and prior to reacting the reactants with the one or more chemical reaction sites.
8. The method of claim 7 , wherein the organic solvent is miscible with the aqueous media.
9. The method of claim 8 , wherein the organic solvent and the aqueous media form a homogeneous reaction media.
10. The method of claim 7 , wherein the organic solvent and the aqueous media form a bi-phasic reaction media.
11. The method of claim 1 , wherein the tags are added enzymatically in aqueous media, and wherein the aqueous media is mixed with an organic solvent prior to reacting the reactants with the one or more chemical reaction sites.
12. The method of claim 1 , wherein one or more of the different small molecules have a molecular weight of less than 1000 Da.
13. The method of claim 1 , wherein one or more of the different small molecules are scaffolded small molecules.
14. The method of claim 13 , wherein the scaffolds are selected from the group consisting of benzodiazepines, steroids, hydantiones, piperasines, diketopiperasines, morpholines, tropanes, cumarines, qinolines, indoles, furans, pyrroles, oxazoles, amino acid precursors, and thiazoles.
15. The method of claim 2 , wherein nucleic acid tags are added by chemical ligation.
16. The method of claim 15 , wherein a first oligonucleotide tag comprises a 3′-OH group, and a second oligonucleotide tag comprises a 5′-phosphor-2-imidazole group, and wherein a reaction of said first and second oligonucleotides forms a phosphodiester internucleoside linkage.
17. The method of claim 15 , wherein a first oligonucleotide tag comprises a phosphoimidazolide group at the 3′-end, and a second oligonucleotide comprises a phosphoimidazolide group at the 5′-end, and wherein a reaction of said 5′ and 3′ oligonucleotides forms a phosphodiester internucleoside linkage.
18. The method of claim 15 , wherein a first oligonucleotide comprises a 3′-phosphorothioate group, and a second oligonucleotide comprises a 5′-iodine, and wherein a reaction of said 5′ and 3′ oligonucleotides forms 3′-O—P(═O)(OH)—S-5′ internucleoside linkage.
19. The method of claim 15 , wherein a first oligonucleotide comprises a 3′-phosphorothioate group, and a second oligonucleotide comprises a 5′-tosylate, and wherein a reaction of said 5′ and 3′ oligonucleotides forms a 3′-O—P(═O)(OH)—S-5′ internucleoside linkage.
20. The method of claim 1 , wherein step vi) comprises a polymerase extending a primer using the nucleic acid tags as a template for the extension.
21. The method of claim 20 , wherein the polymerase is a DNA polymerase or a RNA polymerase.
22. The method of claim 21 , wherein the polymerase is selected from the group consisting of Reverse Transcriptase, Taq DNA polymerase, Pfu polymerase, a Vent polymerase, Klenow fragment, DNA polymerase η and DNA polymerase t.
23. The method of claim 1 , wherein each nucleotide monomer of an oligonucleotide tag comprises
a nucleobase moiety, and
a backbone unit composed of a sugar moiety and an inter-nucleoside linker.
24. The method of claim 23 , wherein one or more of the nucleobase moieties is a natural nucleobase moiety.
25. The method of claim 23 , wherein one or more of the nucleobase moieties is independently selected from the group consisting of adenine, 8-oxo-N 6 -methyladenine; guanine, isoguanine, 7-deazaguanine; cytosine, isocytosine, pseudoisocytosine, N 4 ,N 4 -ethanocytosine, 5-methylcytosine, 5-(C 3 -C 6 )-alkynylcytosine; thymine; uracil, 5-bromouracil, 5-fluorouracil; inosine; purine, diaminopurine, N 6 ,N 6 -ethano-2,6-diamino-purine; xanthine, 7-deazaxanthine; pyrimidine and 2-hydroxy-5-methyl-4-triazolopyridine, and heterocyclic analogues and tautomers thereof.
26. The method of claim 23 , wherein one or more of the sugar moieties is a pentose moiety.
27. The method of claim 26 , wherein the pentose moiety is selected from the group consisting of ribose, 2′-deoxyribose, 2′-O-methyl-ribose, 2′-fluoro-ribose, and 2′-4′-O-methylene-ribose.
28. The method of claim 23 , wherein one or more of the inter-nucleoside linkers is a phosphodiester linker.
29. The method of claim 23 , wherein one or more of the inter-nucleoside linkers is a non-natural phosphodiester linker.
30. The method of claim 29 wherein the non-natural phosphodiester linker is independently selected from the group consisting of phosphorothioate inter-nucleoside linkers, methylphosphonate inter-nucleoside linkers, phosphoramidate inter-nucleoside linkers, phosphotriester inter-nucleoside linkers, and phosphodithioate inter-nucleoside linkers.
31. The method of claim 23 , wherein one or more of the inter-nucleoside linkers is a non-phosphorous-containing linker.
32. The method of claim 23 , wherein one or more of the backbone units is independently selected from the group consisting of
wherein B denotes a nucleobase.