TEMPLATED MOLECULES AND METHODS FOR USING SUCH MOLECULES

• US 20100016177A1
• Filed: 12/09/2008
• Published: 01/21/2010
• Est. Priority Date: 06/20/2001
• Status: Active Grant

• Current Assignee: Nuevolution A/S (Amgen Inc.)
• Original Assignee: Nuevolution A/S (Amgen Inc.)
• Inventors: Franch, Thomas, Slok, Frank Abilgaard, Sams, Christian Klarner, Olsen, Eva Kampmann, Hyldtoft, Lene, Freskgard, Per-Ola, Husemoen, Gitte Nystrup, Gouilaev, Alex Haahr, Glad, Sanne Schroder, Norregaard-Madsen, Mads, Pedersen, Henrik, Holtmann, Anette, Felding, Jakob, Godskesen, Michael Anders, Thisted, Thomas
• Granted Patent: US 8,932,992 B2
• Time in Patent Office: Days
• US Class Current: 506/17
• 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 19/06 Pyrimidine radicals
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 19/16 Purine radicals
• CPC Class Codes: C07H 19/20 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• 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 23/00 Compounds containing boron, silicon, or a metal, e.g. chelates, vitamin B12 esters with inorganic acids C07H11/00; metal salts, see parent compounds
• CPC Class Codes: C12N 15/1058 Directional evolution of libraries, e.g. evolution of libraries is achieved by mutagenesis and screening or selection of mixed population of organisms
• 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: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C40B 40/00 Libraries per se, e.g. arrays, mixtures

Abstract

The present invention relates to a method for synthesising templated molecules. In one aspect of the invention, the templated molecules are linked to the template which templated the synthesis thereof. The intion allows the generation of libraries which can be screened for e.g. therapeutic activity.

Claims

1. A method for synthesising a templated molecule comprising a plurality of functional groups, said method comprising the steps of

i) providing at least one template comprising a sequence of n coding elements,

wherein each coding element comprises at least one recognition group capable of recognising a predetermined complementing element, and

wherein n is an integer of more than 1,

ii) providing a plurality of building blocks, wherein each building block comprises

a) at least one complementing element comprising at least one recognition group capable of recognising a predetermined coding element, each complementing element consisting of 13 to 40 subunits,

b) at least one functional entity comprising at least one functional group and at least one reactive group, and

c) at least one linker separating the at least one functional entity from the at least one complementing element,

iii) contacting each of said coding elements with a complementing element capable of recognising said coding element,

iv) optionally, obtaining a complementing template, and

v) obtaining a templated molecule comprising covalently linked, functional groups by linking, by means of a reaction involving reactive groups a functional group of at least one functional entity to a functional group of another, functional entity,

wherein the templated molecule is linked by means of a covalent linker to the complementing template or template that templated the synthesis of the templated molecule, and

wherein the synthesis of the templated molecule does not involve ribosome mediated translation of a nucleic acid.

2. The method of claim 1, wherein the template comprising n coding elements is a linear sequence of coding elements or a branched template.

3. The method of claim 1, wherein n has a value of from 2 to 30.

4. The method of claim 1, wherein n has a value of from 3 to 20.

5. The method of claim 1, wherein n has a value of from 4 to 10.

6. The method of claim 1, wherein the template or complementing template is attached to a solid support.

7. The method of claim 1, wherein the template comprises nucleotides selected from the group consisting of deoxyribonucleic acids (DNA), ribonucleic acids (RNA), peptide nucleic acids (PNA), locked nucleic acids (LNA), and morpholinos sequences, and any analog or derivative thereof.

8. The method of claim 1, wherein the template or complementing template is amplifiable.

9. The method of claim 1, wherein each coding element is linked to a neighbouring coding element by a covalent chemical bond.

10. The method of claim 9, wherein said covalent chemical bond is selected from the group of covalent bonds consisting of phosphodiester bonds, phosphorothioate bonds, and peptide bonds.

11. The method of claim 1, wherein the coding elements are selected from the group consisting of nucleotides.

12. The method of claim 11, wherein the nucleotides are deoxyribonucleic acids comprising a base selected from adenine (A), thymine (T), guanine (G), and cytosine (C).

13. The method of claim 11, wherein the nucleotides are ribonucleic acids comprising a base selected from adenine (A), uracil (U), guanine (G), and cytosine (C).

14. The method of claim 1, wherein the coding elements are natural and non-natural nucleotides selected from the group consisting of deoxyribonucleic acids.

15. The method of claim 1, wherein the coding elements are natural and non-natural nucleotides selected from the group consisting of ribonucleic acids.

16. The method of claim 1, wherein the coding element subunits are selected from the group consisting of nucleotides, nucleotide derivatives and nucleotide analogs in which one or more of a base moiety and/or a phosphate moiety and/or a ribose moiety and/or a deoxyribose moiety have been substituted by an alternative molecular entity.

17. The method of claim 1, wherein a complementing template is obtained by complementing a plurality of predetermined coding elements by contacting each of said coding elements with a complementing element capable of recognising said coding element.

18. The method of claim 17, wherein the complementing template comprises nucleotides selected from the group consisting of deoxyribonucleic acids (DNA), ribonucleic acids (RNA), peptide nucleic acids (PNA), locked nucleic acids (LNA), and morpholinos sequences, and any analog or derivative thereof.

19. The method of claim 1, wherein the complementing elements are selected from nucleotides, and the complementing elements are linked enzymatically.

20. The method of claim 1, wherein the templated molecule is selected from the group consisting of a linear sequence of functional groups, a branched sequence of functional groups and a cyclic sequence of functional groups.

21. The method of claim 1, wherein the templated molecule is an oligomer or a polymer comprising at least one repetitive sequence of functional groups.

22. The method of claim 1, wherein the templated molecule comprises molecules or molecular entities selected from the group consisting of α

-peptides, β

-peptides, γ

-peptides, ω

-peptides, mono-, di- and tri-substituted α

-peptides, β

-peptides, γ

-peptides, ω

-peptides, peptides, wherein the amino acid residues are in the L-form or in the D-form, vinylogous polypeptides, glycopoly-peptides, polyamides, vinylogous sulfonamide peptide, polysulfonamide, conjugated peptides comprising prosthetic groups, polycyclic compounds comprising aliphatic or aromatic cycles, polyheterocyclic compounds, proteoglycans, and polysiloxanes, and any combination thereof.

23. The method of claim 1, wherein neighbouring residues of the templated molecule is linked by a chemical bond selected from the group of chemical bonds consisting of peptide bonds, sulfonamide bonds, ester bonds, saccharide bonds, carbamate bonds, carbonate bonds, urea bonds, phosphonate bonds, urethane bonds, azatide bonds, peptoid bonds, ether bonds, ethoxy bonds, thioether bonds, single carbon bonds, double carbon bonds, triple carbon bonds, disulfide bonds, sulfide bonds, phosphodiester bonds, oxime bonds, imine bonds, imide bonds, and any combination thereof.

24. The method of claim 1, wherein the backbone structure of said templated molecule comprises a molecular group selected from —

NHN(R)CO—

;

—

NHB(R)CO—

;

—

NHC(RR′

)CO—

;

—

NHC(═

CHR)CO—

;

—

NHC₆H₄CO—

;

—

NHCH₂CHRCO—

;

—

NHCHRCH₂CO—

;

—

COCH₂—

;

—

COS—

;

—

CONR—

;

—

COO—

;

—

CSNH—

;

—

CH₂NH—

;

—

CH₂CH₂—

;

—

CH₂S—

;

—

CH₂ SO—

;

—

CH₂SO₂—

;

—

CH(CH₃)S—

;

—

CH═

CH—

;

—

NHCO—

;

—

NHCONH—

;

—

CONHO—

;

—

C(═

CH₂)CH₂—

;

—

PO₂⁻

NH—

;

—

PO₂⁻

CH₂—

;

—

PO₂⁻

CH₂N⁺—

;

—

SO₂NH⁻

—

; and

lactams.

25. A method for synthesising a library of different templated molecules each comprising a plurality of functional groups, said method comprising the steps of performing the method of claim 1 and using a plurality of templates having different coding elements and/or a different order of coding elements.

26. The method of claim 25, wherein more than 10⁵ different templates are used.

27. The method of claim 25, wherein more than 10⁷ different templates are used.

28. The method of claim 25, wherein the plurality of different templates results in the formation of a library of different templated molecules, wherein each of said templated molecules is linked to the specific template or complementing template that templated the molecule.

29. The method of claim 28 comprising the further step of releasing the template or complementing template from the templated molecule, and obtaining a templated molecule that is not linked to the complementing template or template that templated the synthesis of the templated molecule.

30. A library of different templated molecules obtained by or obtainable by the method of claim 25, said molecules optionally being linked to the complementing template or template that templated the synthesis of the templated molecule, wherein said different templated molecules comprises a plurality of residues.

31. The library of different templated molecules according to claim 30, said different templated molecules being independently selected from the group consisting of different α

-peptides, different β

-peptides, different γ

-peptides, different ω

-peptides, different mono-, di- and tri-substituted α

-peptides, different β

-peptides, different γ

-peptides, and different ω

-peptides, different peptides, wherein the amino acid residues are in either the L-form and/or in the D-form, different vinylogous polypeptides, different glycopoly-peptides, different polyamides, different vinylogous sulfonamide peptides, different polysulfonamides, and different conjugated peptides comprising prosthetic groups.

32. The library of different templated molecules according to claim 30, wherein said library comprises a plurality of more than or about 10³ different templated molecules.

33. The library of different templated molecules according to claim 30, wherein said library comprises a plurality of more than or about 10⁷ different templated molecules.

34. The library of different templated molecules according to claim 30, wherein said library further comprises the templates which templated the synthesis of each templated molecule, or a subset thereof.

35. The library of different templated molecules according to claim 34, wherein the templated molecules are linked to the templates that templated the synthesis of the templated molecule.

36. The library of different templated molecules according to claim 35, wherein the templated molecules are covalently linked to the templates.

37. The library of different templated molecules according to claim 35, wherein the templated molecules are further linked to the complementing templates.

38. The library of different templated molecules according to claim 37, wherein the templated molecules are covalently linked to the complementing templates.

39. The library of different templated molecules according to claim 30, wherein the templates do not comprise or consist of natural nucleotides when the templated molecule is an α

-peptide.

40. The library of different templated molecules according to claim 30, wherein the templated molecules are not optionally substituted α

-peptides.