Chemical preparation of ubiquitin thioesters and modifications thereof

Abstract

The present invention discloses latent thioester functionalities attached to the C-terminus of a first polypeptide, or a first fragment thereof having a Cys residue at its N-terminus, and a process using this functionality for the preparation of polypeptide thioesters, in particular of ubiquitin thioesters, this process comprising preparing a polypeptide or a fragment thereof, being attached to a latent thioester functionality, which can then be ligated with a second polypeptide fragment, followed by selective activation of the latent thioester functionality group, to provide the requested polypeptide thioester. There are also provided the polypeptides obtained by this method, specific unnatural amino acids useful to be incorporated within the polypeptide thioesters, and kits for preparing them.

1 . A process for the preparation of ubiquitin thioesters, said process comprising:
 a. Chemically synthesizing a first modified ubiquitin polypeptide fragment selected from CKIQDKEGIPPDQQRLIF (Ub28-45) (SEQ ID NO: 10), CGKQLEDGRTLSDYNIQKESTLHLVLRLRGG (Ub46-76) (SEQ ID NO: 8) and CKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGG (UB28-76) (SEQ ID NO: 9), being attached to an N-S acyl trnsfer device on the C-terminal of said ubiquitin fragment, further wherein said ubiquitin fragment contains an unprotected Cysteine amino acid on the N-terminal side thereof;   b. chemically synthesizing a second ubiquitin fragment being complimentary to said first ubiquitin fragment obtained in step a, wherein said second ubiquitin fragment is in a thioester form;   c. ligating said first ubiquitin fragment with said second ubiquitin fragment, to obtain an modified unprotected ubiquitin polypeptide attached to said N-S acyl transfer device;   d. reacting said modified ubiquitin polypeptide attached to said N-S acyl transfer device with an external thiol under acidic conditions to obtain said ubiquitin thioester;   wherein said N-S acyl transfer device has the general Formula I:   
       
         
           
           
               
               
           
         
         wherein R, R 1 , R 2  and R 3  are selected from: 
         i. R=hydrogen or 2-nitrobenzyl; R 1 =hydrogen or methyl; R 2 =CONH 2 ; R 3 =hydrogen; 
         ii. R=hydrogen or 2-nitrobenzyl; R 1 =hydrogen or methyl; R 2 =CO—N-pyrroline; R 3 =hydrogen; 
         iii. R=hydrogen or 2-nitrobenzyl; R 1 =methyl, ethyl or benzyl; R 2 =hydrogen; R 3 =hydrogen; 
         iv. R=hydrogen or 2-nitrobenzyl; R 1 =C1 alkyl-CONH 2  or C1 alkyl-COOH; R 2 =hydrogen; R 3 =hydrogen. 
       
     
     
         2 . The process of  claim 1 , further comprising desulfurization of said ubiquitin thioester to turn said Cys amino acid into an Ala amino acid, either before or after step (d). 
     
     
         3 . The process of  claim 1 , wherein said N-S acyl transfer device is a residue of N-methyl cysteine. 
     
     
         4 . The process of  claim 1 , wherein said ubiquitin thioester contains at least one protected δ-mercaptolysine. 
     
     
         5 . The process of  claim 4 , wherein said protected δ-mercaptolysine is a thiazolidine (Thz)-protected mercaptolysine. 
     
     
         6 . The process of  claim 1 , wherein said ubiquitin thioester is prepared of two ubiquitin fragments by native chemical ligation (NCL), wherein said fragment attached to said N-S acyl transfer device is: CGKQLEDGRTLSDYNIQKESTLHLVLRLRGG (Ub46-76) (SEQ ID NO: 8) and said second fragment being in its thioester form is LQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIF (Ubl-45) (SEQ ID NO: 4). 
     
     
         7 . The process of  claim 1 , wherein said ubiquitin thioester is prepared of two ubiquitin fragments by native chemical ligation (NCL), wherein said fragment attached to said N-S acyl transfer device is: CKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGG (Ub28-76) (SEQ ID NO: 9) and said second fragment in a thioester form is LQIFVKTLTGKTITLEVEPSDTIENVK (Ub1-27) (SEQ ID NO: 6). 
     
     
         8 . The process of  claim 1 , wherein said ubiquitin polypeptide is prepared of three ubiquitin fragments by native chemical ligation (NCL), wherein said first fragment attached to said N-S acyl transfer device is CGKQLEDGRTLSDYNIQKESTLHLVLRLRGG (Ub46-76) (SEQ ID NO: 8), said second fragment attached to said LTF is CKIQDKEGIPPDQQRLIF (Ub28- 45) (SEQ ID NO: 10) and said third fragment in its thioester form is LQIFVKTLTGKTITLEVEPSDTIENVK (Ub1-27) (SEQ ID NO: 6). 
     
     
         9 . The process of  claim 1  wherein in at least one of said fragments, the amino acid K stands for a modified Lysine amino acid. 
     
     
         10 . The process of  claim 9 , wherein said modified Lysine amino acid is a protected δ-mercaptolysine. 
     
     
         11 . The process of  claim 10 , wherein said protected δ-mercaptolysine is thiazolidine (Thz)-protected mercaptolysine.