Solid phase native chemical ligation of unprotected or N-terminal cysteine protected peptides in aqueous solution
View Patent ↗The present invention provides methods, apparatus and kits for synthesizing assembled peptides and proteins on a solid phase with sequential ligation of three or more unprotected peptide segments using chemoselective and mild ligation chemistries in aqueous solution. Also provided are methods of monitoring solid phase sequential ligation reactions using MALDI or electrospray ionization mass spectrometry of reaction products.
1. An apparatus for producing assembled polypeptides, said apparatus comprising a reaction vessel containing as components:
(a) a solid phase support having bound thereto a plurality of first peptides, said first peptides comprising partially or completely unprotected peptides having an N-terminus that is bound to said solid phase support via a cleavable handle, and a C-terminus that comprises a thioacid or a thioester;
(b) a plurality of second peptides comprising partially or completely unprotected peptides having an N-terminal cysteine and a C-terminal thioester or thioacid.
2. The apparatus of claim 1 , wherein the C-terminus of said first peptides comprises a thioacid.
3. The apparatus of claim 1 , wherein the C-terminus of said first peptides comprises a thioester.
4. The apparatus of claim 1 , wherein the C-terminus of said second peptides comprises a thioacid.
5. The apparatus of claim 1 , wherein the C-terminus of said second peptides comprises a thioester.
6. The apparatus of claim 1 , wherein said solid phase support is a bead resin.
7. The apparatus of claim 1 , wherein said first peptides and said second peptides range in size from 5 to 99 amino acid residues.
8. The apparatus of claim 1 , wherein said first peptides and said second peptides are prepared by solid phase synthesis.
9. The apparatus of claim 1 , wherein at least one of said plurality of first peptides and said plurality of second peptides comprises peptides having an unnatural backbone structure.
10. An apparatus for preparing assembled polypeptides, said apparatus comprising a reaction vessel containing as components:
a) a solid phase support having bound thereto a plurality of first peptides, said first peptides comprising partially or completely unprotected peptides having a C-terminus that is bound to said solid phase support via a cleavable linkage and an N-terminus that comprises a cysteine residue;
b) a plurality of second peptides comprising partially or completely unprotected peptides having an N-terminal cysteine and a C-terminal thioacid or thioester.
11. The apparatus of claim 10 , wherein said plurality of second peptides is comprised of peptides having the same length, but different amino acid sequences.
12. The apparatus of claim 10 , wherein said plurality of second peptides consists essentially of identical peptides.
13. The apparatus of claim 10 , wherein the C-terminus of said first peptides comprises a thioacid.
14. The apparatus of claim 10 , wherein the C-terminus of said first peptides comprises a thioester.
15. The apparatus of claim 10 , wherein the C-terminus of said second peptides comprises a thioacid.
16. The apparatus of claim 10 , wherein the C-terminus of said second peptides comprises a thioester.
17. The apparatus of claim 10 , wherein said solid phase support is a bead resin.
18. The apparatus of claim 10 , wherein said first peptides and said second peptides range in size from 5 to 99 amino acid residues.
19. The apparatus of claim 10 , wherein said first peptides and said second peptides are prepared by solid phase synthesis.
20. The apparatus of claim 10 , wherein at least one of said plurality of first peptides and said plurality of second peptides comprises peptides having an unnatural backbone structure.