Method for synthesising templated molecules

• US 10,731,151 B2
• Filed: 07/24/2014
• Issued: 08/04/2020
• Est. Priority Date: 03/15/2002
• Status: Active Grant

• Current Assignee: Nuevolution A/S (Amgen Inc.)
• Original Assignee: Nuevolution A/S (Amgen Inc.)
• Inventors: Pedersen, Henrik, Holtmann, Anette, Franch, Thomas, Gouliaev, Alex Haahr, Felding, Jakob
• Primary Examiner(s): Gross, Christopher M
• Application Number:
  US14/340,123
• Publication Number:
  US 20150252360A1
• Time in Patent Office: 2,203 Days
• Field of Search: None
• CPC Class Codes: C07B 2200/11 Compounds covalently bound to a solid support
• CPC Class Codes: C07D 405/04 directly linked by a ring-member-to-ring-member bond
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical
• CPC Class Codes: C12N 15/1068 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
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C40B 40/06 Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 40/14 Libraries containing macromolecular compounds and not covered by groups C40B40/06 - C40B40/12

Abstract

The invention relates to a method for synthesising templated molecules attached to the templated which directed the synthesis thereof. The method involves a template, a scaffold functional entity and a functional entity attached to a building block, which, in turn, is attached the template. The scaffold functional entity and the functional entity of the building block are both provided with complementary dimerization domains allowing the functional entities to come into close proximity when the complementary domains interact with to each other. The method may be used for generating libraries of templated molecules which may be selected for biological activity.

Claims

1. A library comprising a plurality of different scaffolded molecules comprising

a scaffold connected to a plurality of functional entities, wherein the scaffolded molecule is covalently linked to an identifier oligonucleotide comprising

a first plurality of oligonucleotide sequences and a second plurality oligonucleotide sequences comprising oligonucleotide sequences complementary to the first plurality of oligonucleotide sequences,

wherein the first plurality is covalently linked to the second plurality and each of the first and second plurality identifies the scaffolded molecule and further wherein the library comprises at least 10³ different scaffolded molecules.

2. The library according to claim 1, wherein the identifier oligonucleotide comprises nucleotide monomers composed of a nucleobase and a backbone moiety.

3. The library according to claim 2, wherein the nucleobases of the nucleotide monomers are naturally occurring nucleobases.

4. The library according to claim 2, wherein the identifier oligonucleotide comprises nucleotide monomers comprising naturally occurring nucleobases and nucleotide monomers comprising non-natural nucleobases.

5. The library according to claim 4, wherein the nucleotide monomers comprise nucleobases selected from the group consisting of purine and pyrimidine hetero cycles and heterocyclic analogues and tautomers thereof.

6. The library according to claim 4, wherein the nucleotide monomers comprise nucleobases selected from the group consisting of

adenine, 8-oxo-N⁶-methyladenine;

guanine, isoguanine, 7-deazaguanine;

cytosine, isocytosine, pseudoisocytosine, N⁴,N⁴-ethanocytosine,

5-methylcytosine, 5-(C³-C⁶)-alkynylcytosine;

thymine;

uracil, 5-bromouracil, 5-fluorouracil;

inosine;

purine, diaminopurine, N⁶,N⁶-ethano-2,6-diamino-purine;

xanthine, 7-deazaxanthine;

pyrimidine and 2-hydroxy-5-methyl-4-triazolopyridine; and

heterocyclic analogues and tautomers thereof.

7. The library according to claim 2, wherein the nucleotide monomers comprise backbone moieties selected from the group consisting of

8. The library according to claim 2, wherein the nucleotide monomers comprise backbone moieties comprising a pentose sugar moiety and an internucleoside linker.

9. The library according to claim 8, wherein the pentose sugar moiety is selected from the group consisting of ribose, 2′

-deoxyribose, 2′

-O-methyl-ribose, 2′

-flour-ribose, and 2′

-4′

-O-methylene-ribose, and wherein the nucleobase is attached to the 1′

position of the pentose sugar moiety.

10. The library according to claim 8, wherein the internucleoside linker connects the 3′

end of a preceding pentose monomer to the 5′

end of a succeeding pentose monomer.

11. The library according to claim 10, wherein the internucleoside linker is selected from the group of consisting of phosphodiester linkers, phosphorothioate linkers, methylphosphonate linkers, phosphoramidate linkers, phosphotriester linkers, phosphodithioate linkers, and internucleoside linkers not containing phosphorous.

12. The library according to claim 2, wherein the identifier oligonucleotide comprises nucleosides selected from the group consisting of deoxyadenosine, deoxyguanosine, deoxythymidine, and deoxycytidine, and wherein said nucleosides are connected through phosphodiester linkers.

13. The library according to claim 2, wherein the identifier oligonucleotide comprises nucleosides selected from the group consisting of adenosine, guanosine, uridine, cytidine, and inosine, and wherein said nucleosides are connected through phosphodiester linkers.

14. The library according to claim 2, wherein the identifier oligonucleotide comprises nucleosides selected from a first group consisting of deoxyadenosine, deoxyguanosine, deoxythymidine, and deoxycytidine, and nucleosides selected from a second group consisting of adenosine, guanosine, uridine, cytidine, and inosine, wherein said nucleosides are connected through through a phosphodiester linker.

15. The library of claim 1, wherein the library comprises from about 10³ to about 10¹² different scaffolded molecules.

16. The library of claim 15, wherein the scaffolded molecules are small molecules.

17. The library of claim 15, wherein the oligonucleotide sequences in the second plurality of oligonucleotide sequences are covalently linked.

18. The library of claim 15, wherein the oligonucleotide identifier comprises one or more single stranded oligonucleotide sequences.

19. The library of claim 15, wherein the oligonucleotide identifier comprises double stranded oligonucleotide sequences.

20. The library of claim 15 wherein each of the first plurality and the second plurality of oligonucleotide sequences contain an oligonucleotide sequence identifying the scaffold.

21. The library of claim 15 wherein the scaffold is connected to at least two of the plurality of functional entities through a different reactive group on the scaffold or at a different position on the scaffold previously occupied by a reactive group.

40. The library according to claim 15, wherein the number of different scaffolded molecules in the library is at least 10⁶.

41. The library according to claim 15, wherein one or more scaffolded molecules are covalently linked to an identifier oligonucleotide through a linker comprising polyethylene glycol (PEG).

42. The library according to claim 15, wherein the scaffolded molecules are not polymeric molecules.

43. The library according to claim 15, wherein the scaffolded molecules are not polynucleotides.

44. The library according to claim 15, wherein the scaffolded molecules are selected from the group consisting of

monofunctional, difunctional, trifunctional and oligofunctional, open-chain hydrocarbons;

monocyclic, bicyclic, tricyclic and polycyclic hydrocarbons,

bridged polycyclic hydrocarbons;

monofunctional, difunctional trifunctional and oligofunctional, non-aromatic carbocycles;

monofunctional, difunctional, trifunctional and oligofunctional, aromatic carbocycles;

monocyclic, bicyclic, tricyclic and polycyclic, aromatic carbocycles;

monofunctional, difunctional, trifunctional and oligofunctional, non-aromatic heterocycles;

monofunctional, difunctional, trifunctional and oligofunctional, aromatic heterocycles;

monocyclic, bicyclic, tricyclic and polycyclic heterocycles; and

bridged polycyclic heterocycles.

45. The library of claim 15, wherein one or more of the different scaffolded molecules comprise one or more core structures covalently linked to a plurality of functional entities.

46. A method for selecting scaffolded molecules from the library of different scaffolded molecules of claim 15, the method comprising:

(1) exposing the library to conditions enriching the library with complexes having a predetermined activity toward a biological target;

(2) amplifying the complexes of the enriched library;

(3) optionally repeating steps (1) and (2); and

(4) obtaining an enriched library having a higher ratio of scaffolded molecules with the predetermined activity.

47. The library of claim 45 wherein the core structure is connected at least two of the plurality of functional entities through a different reactive group on the core structure or at a different position on the core structure previously occupied by a reactive group.

22. A library comprising a plurality of different scaffolded molecules comprising

a scaffold connected to two or more functional entities, wherein the scaffolded molecule is covalently linked to an identifier oligonucleotide comprising

a plurality of codons and a plurality of anticodons comprising oligonucleotide sequences complementary to the plurality of codons, wherein the plurality of codons is covalently linked to the plurality of anticodons, and

each of the plurality of codons and the plurality of anti-codons identifies the scaffolded molecule, and wherein at least two of the functional entities are connected to different reactive groups on the scaffold or different positions on the scaffold previously occupied by reactive groups.

23. The library of claim 22, wherein the library comprises from about 10³ to about 10¹² different scaffolded molecules.

24. The library according to claim 23, wherein the scaffolded molecules are small molecules.

25. The library according to claim 23, wherein the scaffolded molecules are selected from the group consisting of

monofunctional, difunctional, trifunctional and oligofunctional, open-chain hydrocarbons;

monocyclic, bicyclic, tricyclic and polycyclic hydrocarbons,

bridged polycyclic hydrocarbons;

monofunctional, difunctional trifunctional and oligofunctional, non-aromatic carbocycles;

monofunctional, difunctional, trifunctional and oligofunctional, aromatic carbocycles;

monocyclic, bicyclic, tricyclic and polycyclic, aromatic carbocycles;

monofunctional, difunctional, trifunctional and oligofunctional, non-aromatic heterocycles;

monofunctional, difunctional, trifunctional and oligofunctional, aromatic heterocycles;

monocyclic, bicyclic, tricyclic and polycyclic heterocycles; and

bridged polycyclic heterocycles.

26. The library of claim 23, wherein anti-codons in the plurality of anti-codons are covalently linked.

27. The library of claim 22, wherein the oligonucleotide identifier comprises one or more single stranded oligonucleotide sequences.

28. The library of claim 22 wherein the oligonucleotide identifier comprises double stranded oligonucleotide sequences.

29. The library of claim 22, wherein one or more of the different scaffolded molecules comprises a core structure connected to the two or more functional entities, wherein at least two of the functional entities are connected to different reactive groups on the core structure or different positions on the core structure previously occupied by reactive groups.

30. The library of claim 22 wherein each of the plurality of codons and the plurality of anti-codons contains an oligonucleotide sequence identifying the scaffold.

31. The library of claim 22 wherein the scaffold is connected to each of the functional entities through a different reactive group on the scaffold or a different position on the scaffold previously occupied by a reactive group.

32. The library according to claim 22, wherein one or more scaffolded molecules are covalently linked to an identifier oligonucleotide through a linker comprising polyethylene glycol (PEG).

33. The library according to claim 22, wherein the identifier oligonucleotide comprises nucleotide monomers comprising naturally occurring nucleobases and nucleotide monomers comprising non-natural nucleobases.

34. The library according to claim 33, wherein the nucleotide monomers comprise nucleobases selected from the group consisting of

adenine, 8-oxo-N⁶-methyladenine;

guanine, isoguanine, 7-deazaguanine;

cytosine, isocytosine, pseudoisocytosine, N⁴,N⁴-ethanocytosine,

5-methylcytosine, 5-(C³-C⁶)-alkynylcytosine;

thymine;

uracil, 5-bromouracil, 5-fluorouracil;

inosine;

purine, diaminopurine, N⁶,N⁶-ethano-2,6-diamino-purine;

xanthine, 7-deazaxanthine;

pyrimidine and 2-hydroxy-5-methyl-4-triazolopyridine; and

heterocyclic analogues and tautomers thereof.

35. The library according to claim 33, wherein the nucleotide monomers comprise backbone moieties selected from the group consisting of

36. The library according to claim 33, wherein the nucleotide monomers comprise backbone moieties comprising a pentose sugar moiety and an internucleoside linker.

37. The library according to claim 36, wherein the internucleoside linker is selected from the group of consisting of phosphodiester linkers, phosphorothioate linkers, methylphosphonate linkers, phosphoramidate linkers, phosphotriester linkers, phosphodithioate linkers, and internucleoside linkers not containing phosphorous.

38. A method for selecting scaffolded molecules from the library of different scaffolded molecules of claim 22, the method comprising:

(1) exposing the library to conditions enriching the library with complexes having a predetermined activity toward a biological target;

(2) amplifying the complexes of the enriched library;

(3) optionally repeating steps (1) and (2); and

(4) obtaining an enriched library having a higher ratio of scaffolded molecules with the predetermined activity.

39. The library according to claim 22, wherein the scaffolded molecules are not polynucleotides.