Solid phase native chemical ligation of unprotected or N-terminal Cysteine protected peptides in aqueous solution

Abstract

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) an aqueous solution comprising a solid phase support, said solid phase support having bound thereto a plurality of first peptides comprising partially or completely unprotected peptides having (i) an N-terminus that is bound to said solid phase support via a cleavable handle, and (ii) a C-terminus that is unprotected and capable of chemoselective chemical ligation; and    (b) a plurality of second peptides comprising partially or completely unprotected peptides having a C-terminus and an N-terminus that is (i) unprotected and (ii) capable of chemoselective ligation in said aqueous solution to said C-terminus of said first peptides.    
     
     
         2 . The apparatus of  claim 1 , wherein the C-terminus of said first peptides comprises a thioacid or a thioester.  
     
     
         3 . The apparatus of  claim 2 , wherein the C-terminus of said first peptides comprises a thioacid.  
     
     
         4 . The apparatus of  claim 2 , wherein the C-terminus of said first peptides comprises a thioester.  
     
     
         5 . The apparatus of  claim 2 , wherein the N-terminus of said second peptides comprises an N-terminal cysteine.  
     
     
         6 . The apparatus of  claim 5 , wherein the C-terminus of said second peptides comprises a C-terminal thioacid or thioester.  
     
     
         7 . The apparatus of  claim 5 , wherein the C-terminus of said second peptides comprises a C-terminal thioacid.  
     
     
         8 . The apparatus of  claim 5 , wherein the C-terminus of said second peptides comprises a C-terminal thioester.  
     
     
         9 . The apparatus of  claim 1 , wherein said solid phase support is a bead resin.  
     
     
         10 . The apparatus of  claim 1 , wherein said first peptides and said second peptides range in size from 5 to 99 amino acid residues.  
     
     
         11 . The apparatus of  claim 1 , wherein said first peptides and said second peptides are prepared by solid phase synthesis.  
     
     
         12 . 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.  
     
     
         13 . The apparatus of  claim 1 , wherein, said plurality of second peptides is comprised of peptides having the same length, but different amino acid sequences.  
     
     
         14 . The apparatus of  claim 1 , wherein said plurality of second peptides consists essentially of identical peptides.  
     
     
         15 . An apparatus for producing assembled polypeptides, said apparatus comprising a reaction vessel containing as components: 
 (a) an aqueous solution comprising a solid phase support, said solid phase support having bound thereto a plurality of first peptides comprising partially or completely unprotected peptides having (i) a C-terminus that is bound to said solid phase support via a cleavable handle, and (ii) an N-terminus that is unprotected and capable of chemoselective chemical ligation; and    (b) a plurality of second peptides comprising partially or completely unprotected peptides having an N-terminus and a C-terminus that is (i) unprotected and (ii) capable of chemoselective ligation in said aqueous solution to said N-terminus of said first peptides.    
     
     
         16 . The apparatus of  claim 15 , wherein the N-terminus of said first peptides comprises an N-terminal cysteine residue.  
     
     
         17 . The apparatus of  claim 15 , wherein the C-terminus of said second peptides comprises a thioacid or a thioester.  
     
     
         18 . The apparatus of  claim 15 , wherein the C-terminus of said second peptides comprises a thioacid.  
     
     
         19 . The apparatus of  claim 15 , wherein the C-terminus of said second peptides comprises a thioester.  
     
     
         20 . The apparatus of  claim 15 , wherein the N-terminus of said second peptides comprises an N-terminal cysteine residue.  
     
     
         21 . The apparatus of  claim 15 , wherein said plurality of second peptides is comprised of peptides having the same length, but different amino acid sequences.  
     
     
         22 . The apparatus of  claim 15 , wherein said plurality of second peptides consists essentially of identical peptides.  
     
     
         23 . The apparatus of  claim 15 , wherein the C-terminus of said first peptides comprises a thioacid.  
     
     
         24 . The apparatus of  claim 15 , wherein the C-terminus of said first peptides comprises a thioester.  
     
     
         25 . The apparatus of  claim 15 , wherein said solid phase support is a bead resin.  
     
     
         26 . The apparatus of  claim 15 , wherein said first peptides and said second peptides range in size from 5 to 99 amino acid residues.  
     
     
         27 . The apparatus of  claim 15 , wherein said first peptides and said second peptides are prepared by solid phase synthesis.  
     
     
         28 . The apparatus of  claim 15 , wherein at least one of said plurality of first peptides and said plurality of second peptides comprises peptides having an unnatural backbone structure.