Method for synthesising templated molecules

• US 8,808,984 B2
• Filed: 07/24/2008
• Issued: 08/19/2014
• Est. Priority Date: 03/15/2002
• Status: Expired due to Term

• Current Assignee: Nuevolution A/S (Amgen Inc.)
• Original Assignee: Neuvolution A/S
• Inventors: Pedersen, Henrik, Holtmann, Anette, Franch, Thomas, Gouliaev, Alex Haahr, Felding, Jakob
• Primary Examiner(s): Whisenant, Ethan C
• Application Number:
  US12/179,323
• Publication Number:
  US 20090143232A1
• Time in Patent Office: 2,217 Days
• Field of Search: 435/6, 435/6.1, 536/22.1, 536/23.1, 536/24.3, 506/23, 506/41
• US Class Current: 435/6.1
• 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 synthesizing 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 of different bifunctional complexes each bifunctional complex comprising a scaffolded molecule covalently linked by a linker to one or both strands of a double stranded identifier oligonucleotide comprising complementary nucleotide sequences, wherein the nucleotide sequence of each and both strands of the identifier oligonucleotide identifies the scaffolded molecule to which the identifier oligonucleotide is covalently linked.

2. The library according to claim 1, wherein the number of different bifunctional complexes in the library is at least 10³.

3. The library according to claim 1, wherein the number of different bifunctional complexes in the library is at least 10⁶.

4. The library according to claim 1, wherein each nucleotide monomer present in the identifier oligonucleotides of the library is composed of a nucleobase and a backbone moiety.

5. The library according to claim 4, wherein the nucleobases of the nucleotides of the identifier oligonucleotides of the library are naturally occurring nucleobases.

6. The library according to claim 4, wherein the nucleotides of the identifier oligonucleotides of the library contain naturally occurring nucleobases and non-natural nucleobases.

7. The library according to claim 6, wherein the nucleobases of the nucleotides of the identifier oligonucleotides of the library are purine and pyrimidine hetero-cycles, including heterocyclic analogues and tautomers thereof.

8. The library according to claim 6, wherein the nucleobases of the nucleotides of the identifier oligonucleotides of the library are 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.

9. The library according to claim 4, wherein the backbone moieties of the nucleotide monomers present in the identifier oligonucleotides of the library are selected from the group consisting of

10. The library according to claim 4, wherein the backbone moieties of the nucleotide monomers present in the identifier oligonucleotides of the library are made up of a pentose sugar moiety and an internucleoside linker.

11. The library according to claim 10, 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 (LNA), and wherein the nucleobase is attached to the 1′

position of the pentose sugar moiety.

12. The library according to claim 10, wherein the internucleoside linker is connecting the 3′

end of a preceding pentose monomer to a 5′

end of a succeeding pentose monomer.

13. The library according to claim 10, wherein the internucleoside linkers of the backbone moieties of the nucleotide monomers of the identifier oligonucleotides of the library are selected from the group of consisting of phosphodiester linkers, phosphorothioate linkers, methylphosphonate linkers, phosphoramidate linkers, phosphotriester linkers, and phosphodithioate linkers.

14. The library according to claim 4, wherein the identifier oligonucleotides of the library comprise nucleosides selected from the group of nucleosides consisting of deoxyadenosine, deoxyguanosine, deoxythymidine, and deoxycytidine, wherein said nucleosides are connected through phosphodiester linkages.

15. The library according to claim 4, wherein the identifier oligonucleotides of the library comprise nucleosides selected from the group of nucleosides consisting of adenosine, guanosine, uridine, cytidine, and inosine, wherein said nucleosides are connected through phosphodiester linkages.

16. The library according to claim 4, wherein the identifier oligonucleotides of the library comprise nucleosides selected from a first group of nucleosides consisting of deoxyadenosine, deoxyguanosine, deoxythymidine, and deoxycytidine, as well as nucleosides selected from a second group of nucleosides consisting of adenosine, guanosine, uridine, cytidine, and inosine, wherein said nucleosides are connected through phosphodiester linkages.

17. The library according to claim 1, wherein the linker is a polyethylene glycol (PEG) linker.

18. The library according to claim 1, wherein the scaffolded molecules of the library 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,

bridged polycyclic heterocycles; and

aliphatic polycycles, aromatic polycycles, and polyheterocycles.