[a] Proteases have long been used as biocatalysts for organic transformations.[1] The greatest synthetic value of a protease lies perhaps with its ability to catalyze reverse hydrolysis and other nonhydrolytic reactions in which a nucleophile other than water is engaged in the formation of the desired product. For a serine or cysteine protease, the nucleophile acts as the acceptor of the acyl group from the acyl-enzyme intermediate in a reaction that is made under either thermodynamic or kinetic control.[2] Although a number of useful strategies have been developed and applied successfully to the synthesis of peptides [3][4][5][6][7] and peptide esters, [8] enzymatic synthesis of peptide thioesters has remained a challenge. Herein we demonstrate that subtiligase, a designer peptide ligase derived from the serine protease subtilisin, [6] is a viable catalyst for the thiolysis of peptide esters to form peptide thioesters, an important class of compounds that have seen increased use in the total synthesis of proteins. [9,10] Catalytic peptide ester-to-thioester transesterification by any serine or cysteine protease may appear straightforward, as thiolysis of the acyl-enzyme (acyl-E) intermediate would lead to direct formation of a peptide thioester product in a manner similar to aminolysis (Scheme 1). However, unlike aminolysis of a peptide ester in which a more stable peptide bond is formed, thiolysis forms a more labile thioester bond, which can be quickly hydrolyzed under normal enzyme catalysis conditions. Owing to this and the risk of cleavage of the susceptible peptide bonds notwithstanding, it would be difficult to use a natural serine or cysteine protease for such a transesterification reaction. In fact, our attempts to use subtilisin and papain for this purpose were unsuccessful. We therefore turned our attention to subtiligase. Subtiligase is an engineered double mutant of subtilisin, in which the active site residue Ser 221 is changed to Cys, and the Pro 225 residue to Ala. [6] It has all the structural features of a cysteine protease but lacks the normal amidase activity of the latter while retaining significant esterase activity to catalyze the aminolysis of a suitable peptide ester for peptide ligation.[6] With its decreased hydrolase activity, subtiligase generates a longer-lived acyl-E intermediate, which would make its capture possible by a thiol nucleophile. Because thiols are better nucleophiles than water, a kinetically controlled reaction would favor thiolysis over hydrolysis, and a less hydrolytically active subtiligase would make it possible to isolate the thioester product before it is hydrolyzed by the enzyme.To test our hypothesis, we produced a subtiligase variant that has a His 6 tag at its C terminus to facilitate purification. The presence of this His 6 tag did not affect the enzymatic activity, as tested in a model ligation reaction between the glycolate ester peptide, Ac-His-Ala-Ala-Pro-Phe-glc-Phe-Gly-NH 2 , and the peptide nucleophile, H-Ala-Phe-Ala-NH 2 , which respectively re...

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