Broadening of the substrate tolerance of α-chymotrypsin by using the carbamoylmethyl ester as an acyl donor in kinetically controlled peptide synthesis

摘要

Although enzymic methods for peptide synthesis 1 have severalnadvantages over conventional chemical methodologies, annarrow substrate specificity is often regarded as a major drawbacknfrom a synthetic standpoint: only limited amino acidnresidues are acceptable as substrates. Accordingly, developingna means to broaden the applicability of proteases for peptidensynthesis remains a challenging task. In the preceding paper,2nwe have reported that the coupling efficiency in the kineticallyncontrolled peptide-bond formation catalysed by α-chymotrypsinnis profoundly improved by the use of activated estersnsuch as the 2,2,2-trifluoroethyl ester as an acyl donor insteadnof the conventional methyl ester in an organic solvent such asnacetonitrile with low water content and that this approach isnuseful for the incorporation of non-protein amino acids suchnas halogenophenylalanines into peptides. The success of thisnapproach prompted us to attempt to overcome the narrow substratentolerance of the protease by modulating the ester moietynof an acyl donor. Thus, we have examined a series of activatednesters for the couplings of α-chymotrypsin’s inherently poornsubstrates such as Ala lacking a large hydrophobic side chain.nWe have at last found that the carbamoylmethyl ester is superiorneven to the halogenated alkyl esters,3 and that this particularnester is able to broaden the protease’s substrate tolerance. Thisnpaper describes the details of the relevant works.