Selective enzymatic amidation of c-terminal esters or acids of peptides

Abstract

The present invention relates to a process for the amidation of C-terminal esters or acids of peptide substrates in solution-phase synthesis of peptides, comprising amidating one or more peptide substrates comprising C-terminal esters or acids using the protease subtilisin in any suitable form in the presence of an ammonium salt derived from an acid having a pKa above 0. This process is useful in the production of protected or unprotected peptides.

1 . A process for the amidation of C-terminal esters or acids of peptide substrates in solution-phase synthesis of peptides, comprising amidating one or more peptide substrates comprising C-terminal esters or acids using the protease subtilisin in any suitable form in the presence of one or more ammonium salts derived from an acid having a pKa above 0. 
     
     
         2 . A process according to  claim 1 , wherein C-terminal esters of peptide substrates are amidated. 
     
     
         3 . A process according to  claim 2 , wherein the esters of the C-terminal esters of the peptide substrate are selected from the group of C 1-12  (ar)alkyl esters. 
     
     
         4 . A process according to  claim 3 , wherein the esters of the C-terminal esters of the peptide substrate are selected from the group of primary C 1-12  (ar)alkyl esters. 
     
     
         5 . A process according to  claim 4 , wherein the esters of the C-terminal esters of the peptide substrate are selected from the group of primary C 1-4  alkyl esters. 
     
     
         6 . A process according to  claim 5 , wherein the ester of the C-terminal ester of the peptide substrate is the C-terminal methyl ester. 
     
     
         7 . A process according to  claim 1 , wherein the ammonium salt is derived from an acid having a pKa above 3.5. 
     
     
         8 . A process according to  claim 7 , wherein the ammonium salt has the following chemical structure (I): 
       
         
           
           
               
               
           
         
         wherein 
         R1 is selected from the group of hydrogen, C 1-12  (ar)alkyl, C 6-12  aryl, —N(R2) 2 , —OH, and R3-O − NH 4   + , 
         R2 is selected from the group of hydrogen and/or C 1-4  alkyl, and 
         R3 is a bond, a carbonyl group, or a C 1-4  alkyl carbonyl group, optionally substituted with one or more hydroxyl groups and/or —COO − NH 4   + , optionally in hydrated form. 
       
     
     
         9 . A process according to  claim 8 , wherein R1 is selected from the group of C 1-12  (ar)alkyl, C 6-12  aryl, —NH 2 , —OH, and —O − NH 4   + . 
     
     
         10 . A process according to  claim 9 , wherein the ammonium salt is selected from ammonium carbamate, ammonium carbonate, ammonium bicarbonate, ammonium acetate, ammonium benzoate, and mixtures thereof. 
     
     
         11 . A process according to  claim 10 , wherein the ammonium salt is ammonium carbamate. 
     
     
         12 . A process according to  claim 1 , wherein the molar ratio of ammonium salt to peptide substrate ranges from 2:1 to 20:1. 
     
     
         13 . The process according to  claim 1 , wherein the peptide substrate comprising the C-terminal ester or acid comprises a C-terminal acyl residue which is an α-amino acyl residue from natural or synthetic origin. 
     
     
         14 . The process according to  claim 13 , wherein the α-amino acyl residue is selected from Ala, protected Cys, protected Asp, protected Glu, Phe, Gly, His, (protected) Lys, Leu, Met, Asn, Gln, (protected) Arg, (protected) Ser, Thr, Val, (protected) Trp and (protected) Tyr. 
     
     
         15 . The process according to  claim 1 , wherein the peptide substrate is prepared according to a process for rapid solution synthesis of a peptide in an organic solvent or a mixture of organic solvents, the process comprising repetitive cycles of steps (a)-(d):
 a) a coupling step, using an excess of an activated carboxylic component to acylate an amino component,   b) a quenching step in which a scavenger is used to remove residual activated carboxylic functions, wherein the scavenger may also be used for deprotection of the growing peptide,   c) one or more aqueous extractions and optionally, (d) a separate deprotection step, followed by one or more aqueous extractions,   whereby in at least one cycle in process step b an amine comprising a free anion or a latent anion is used as a scavenger of residual activated carboxylic functions.   
     
     
         16 . The process according to  claim 1 , wherein the protease subtilisin is of the family EC 3.4.21.62. 
     
     
         17 . The process according to  claim 1 , wherein the protease subtilisin is free subtilisin. 
     
     
         18 . The process according to  claim 1 , wherein the protease subtilisin is cross-linked enzyme aggregate (CLEA) subtilisin. 
     
     
         19 . The process according to  claim 1 , wherein an organic solvent is used. 
     
     
         20 . The process according to  claim 19 , wherein the organic solvent is selected from N,N-dimethylformamide (DMF), N-methyl-2-pyrrolidone (NMP), dioxane, N,N-dimethylacetamide (DMA), dichloromethane (DCM), tetrahydrofuran (THF), acetonitrile, tert-butanol, tert-amyl alcohol, dichloroethane (DCE), tert-butyl methyl ether (MTBE), and mixtures thereof. 
     
     
         21 . The process according to  claim 20 , wherein the organic solvent is a mixture of tert-butanol and DMF, tert-butanol and NMP, tert-amyl alcohol and DMF, or tert-amyl alcohol and NMP. 
     
     
         22 . The process according to  claim 19 , wherein water is present in the organic solvent ranging from 0.0001 to 5% (v/v). 
     
     
         23 . The process according to  claim 1 , wherein pH at which the reaction is performed is selected from the range of 5.5-10. 
     
     
         24 . The process according to  claim 1 , wherein the reaction temperature for the amidation is 15-60° C. 
     
     
         25 . The process according to  claim 1 , wherein the amount of protease subtilisin ranges from 1 to 50 wt. % related to the peptide substrate. 
     
     
         26 . The process according to  claim 1 , wherein the amidation is performed by stepwise adding portions of the protease subtilisin (in any suitable form) into the reaction mixture comprising one or more peptide substrates comprising C-terminal esters or acids. 
     
     
         27 . The process according to  claim 1 , wherein the amidation is performed by stepwise adding portions of the ammonium salt into the reaction mixture comprising one or more peptide substrates comprising C-terminal esters or acids.