Method for cross-linking peptides

Patent metadata

Field	Value
Publication number	US-9290537-B2
Application number	US-201113996583-A
Country	US
Kind code	B2
Filing date	Dec 23, 2011
Priority date	Dec 23, 2010
Publication date	Mar 22, 2016
Grant date	Mar 22, 2016

Abstract

The present invention relates to a method for cross-linking peptides using an activated furan-moiety. In particular, the present invention provides a method for cross-linking peptides comprising the steps of: a) providing a composition comprising furan-peptides, said furan-peptides comprising at least one amino acid comprising a furan-moiety; b) contacting said composition comprising furan-peptides with second peptides, thereby obtaining a mixture comprising furan-peptides and second peptides; c) adding an activation signal to said mixture of step b), thereby activating said furan-peptides to activated furan-peptides, and d) reacting said activated furan-peptides with said second peptides, thereby cross-linking said activated furan-peptides with said second peptides.

First claim

The invention claimed is:

1. A method for cross-linking peptides comprising the steps of:
a) providing a composition comprising furan-peptides, said furan-peptides comprising at least one amino acid comprising a furan-moiety, wherein said furan-peptides are not coupled to a solid support selected from a polystyrene resin comprising an acid labile linker or a polystyrene-co-polyethyleneglycol resin comprising an acid labile linker;
b) contacting said composition comprising furan-peptides with second peptides, thereby obtaining a mixture comprising furan-peptides and second peptides;
c) adding an activation signal to said mixture of step b), thereby activating said furan-peptides to activated furan-peptides, and
d) reacting said activated furan-peptides with said second peptides, thereby cross-linking said activated furan-peptides with said second peptides.

2. The method for cross-linking peptides according to claim 1 , wherein said activation signal activates said furan-moiety to an enal-moiety.

3. The method for cross-linking peptides according to claim 1 , wherein said activated furan-peptides comprise an enal-moiety.

4. The method for cross-linking peptides according to claim 1 , wherein said second peptides comprise at least one amino acid comprising a sulfhydryl group, hydroxyl group, amine group, imidazole group and/or indole group.

5. The method for cross-linking peptides according to claim 4 , wherein said enal-moiety of said activated furan-peptides reacts with said sulfhydryl group, hydroxyl group, amine group, imidazole group and/or indole group of said amino acid of said second peptides.

6. The method for cross-linking peptides according to claim 1 , further comprising the step of identifying said second peptides cross-linked with said activated furan-peptides of step d).

7. The method for cross-linking peptides according to claim 5 , further comprising identifying said amino acid of said second peptides reacted with said enal-moiety of said activated furan-peptides.

8. The method for cross-linking peptides according to claim 7 , wherein said amino acid of said second peptide is Cys, Lys, Arg, Ser, Thr, Tyr, His, Trp and/or an N-terminal amino acid.

9. The method for cross-linking peptides according to claim 1 , wherein said activation signal is selected from the group consisting of chemical oxidants, enzymes and singlet oxygen.

10. The method for cross-linking peptides according to claim 9 , wherein said chemical oxidants are selected from the group consisting of NBS (N-bromo-succinimide), NaOCl, H 2 O 2 and peracids.

11. The method for cross-linking peptides according to claim 9 , wherein said enzymes belong to the class of cytochrome P450 enzymes.

12. The method for cross-linking peptides according to claim 1 , wherein said composition comprises at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% furan-peptides.

13. The method for cross-linking peptides according to claim 1 , wherein prior to step (a), furan-peptides are produced by incorporating at least one furan amino acid into a peptide during solid-phase peptide synthesis (SPPS) of said peptide.

14. The method for cross-linking peptides according to claim 1 , wherein prior to step (a), furan-peptides comprising an N-terminal furan-moiety are produced by a method comprising the steps of:
synthesizing peptides by coupling amino acids via amide bonds on a solid support, wherein at least the N-terminal amino acid comprises a furan-moiety, thereby obtaining furan-peptides coupled to a solid support comprising an N-terminal furan-moiety;
capping the N-terminal amino acid of said furan-peptides comprising an N-terminal furan-moiety with a capping moiety, thereby obtaining capped furan-peptides comprising an N-terminal furan-moiety; and
cleaving in solution said capped furan-peptides comprising an N-terminal furan-moiety from said solid support, thereby producing cleavage products in solution, wherein at least 60% of said cleavage products in solution are furan-peptides.

15. The method for cross-linking peptides according to claim 14 , wherein said capping moiety is an aromatic moiety.

16. The method for cross-linking peptides according to claim 1 , wherein prior to step (a), furan-peptides are produced by incorporating at least one furan amino acid into a peptide during peptide translation in prokaryotes or in eukaryotes.

17. The method for cross-linking peptides according to claim 1 , wherein prior to step (a) furan-peptides are produced by a method comprising the steps of:
providing a translation system comprising: (i) a furan amino acid, (ii) an orthogonal
pyrrolysyl-tRNA synthetase of Methanosarcina mazei , or a functional fragment or variant thereof, (iii) an orthogonal tRNA-CUA of Methanosarcina mazei , wherein said orthogonal tRNA-CUA is specifically aminoacylated by said orthogonal pyrrolysyl-tRNA synthetase with the furan amino acid, and (iv) a nucleic acid encoding a peptide, wherein the nucleic acid comprises a codon that is recognized by said orthogonal tRNA-CUA; and
translating the nucleic acid, thereby incorporating the furan amino acid into the peptide.

18. The method according to claim 16 , wherein said furan amino acid is selected from a furan amino acid of Formula (XIa), (XIb) or (XIc), or a stereoisomeric form thereof, wherein X is selected from NH, O, S or P

19. A method for identifying a binding site of two interacting peptides comprising steps a) to d) of the method of claim 1 , wherein said amino acid comprising a furan-moiety is located at position n of said furan-peptides; said method further comprises the steps of:
e) determining the cross-link of said furan-peptides and said second peptides;
f) identifying said amino acid comprising a furan-moiety at position n as being a binding site of said two interacting peptides if cross-linking is detected; and
g) optionally repeating steps a) to f) with said furan-peptides, wherein said amino acid comprising a furan-moiety is located at position n+p of said furan-peptides; wherein position n may be any amino acid of said furan-peptides; and wherein p is a positive or negative integer (provided position n+p is located on said furan-peptides).

20. A method for identifying a binding site of two interacting peptides comprising the steps of:
a) providing a composition comprising furan-peptides, said furan-peptides comprising at least one amino acid comprising a furan-moiety, and wherein said amino acid comprising a furan-moiety is located at position n of said furan-peptides;
b) contacting said composition comprising furan-peptides with second peptides, thereby obtaining a mixture comprising furan-peptides and second peptides;
c) adding an activation signal to step b), thereby activating said furan-peptides to activated furan-peptides;
d) reacting said activated furan-peptides with said second peptides, thereby cross-linking said activated furan-peptides with said second peptides;
e) determining the cross-link of said interacting peptides;
f) identifying said amino acid comprising a furan-moiety as a binding site of two interacting peptides if cross-linking is detected; and
g) optionally repeating steps a) to f) with said furan-peptides, wherein said amino acid comprising a furan-moiety is located at position n+p of said furan-peptides; wherein position n may be any amino acid of said furan-peptides;
and wherein p is a positive or negative integer

Assignees

• Madder Annemieke
• Hoogewijs Kurt
• Deceuninck Annelies
• Carrette Lieselot
• Univ Gent

Inventors

• Madder Annemieke
• Hoogewijs Kurt
• Deceuninck Annelies
• Carrette Lieselot

Classifications

• C07K1/13

  Labelling of peptides · CPC title

• C12N9/0051

  acting on a sulfur group of donors (1.8) · CPC title

• C07K1/006 Primary

  of peptides containing derivatised side chain amino acids · CPC title

• C12Y108/01009

  Thioredoxin-disulfide reductase (1.8.1.9), i.e. thioredoxin-reductase · CPC title

• C07K1/1075 Primary

  by covalent attachment of amino acids or peptide residues · CPC title