Application of various inverse substrates to thrombin-catalyzed peptide synthesis

Thrombin-catalyzed peptide synthesis has been studied using nine series of "inverse substrates:" i.e., p-amidinophenyl, p- and m-guanidinophenyl, p- a nd m-(guanidinomethyl)phenyl, and four position isomers of guanidinonaphthy l esters derived from N-alpha-(tert-butyloxycarbonyl)amino acid as acyl don or components. These substrates were classified into two types with respect to their response to thrombin, One group includes p-amidino- and p-guanidi nophenyl esters, which undergo less enantioselective coupling reaction. Sub strates classified into the other group are m-guanidinophenyl, p- and m-(gu anidinomethyl)phenyl, and four position isomers of guanidinonaphthyl esters which are involved in the enantioselective coupling reaction. Thus amino a cid residues of L-series (in the present case; N-alpha-Boc-L-Ala) are readi ly coupled to afford peptides by assigning them to either of the inverse su bstrates, The optimum condition for the coupling reaction was studied by ch anging organic solvent, pH, and acyl acceptor concentration, It was found t hat the enzymatic hydrolysis of the resulting product was negligible.