We report on the first synthesis, kinetic evaluation and application of novel substrate-derived inhibitors against the Staphylococcus aureus cysteine protease-transpeptidase, sortase (staphylococcal surface protein sorting A, SrtA). The peptidyl-diazomethane and peptidyl-chloromethane analogues, Cbz (benzyloxycarbonyl)-Leu-Pro-Ala-Thr-CHN2 (I) and Cbz-Leu-Pro-Ala-Thr-CH2Cl (II) respectively were found to act as time-dependent irreversible inhibitors of recombinant sortase (SrtAΔN). The peptidyl-chloromethane analogue (II) was the most powerful with an inhibitor specificity constant (ki/Ki) of 5.3×104M-1·min-1, approx. 2-fold greater than that determined for the peptidyl-diazomethane (I). Additionally, using Western-blot analysis, we have been able to demonstrate that a biotinylated version of the peptidyl-diazomethane analogue, biotin-Ahx (aminohexanoyl)-Leu-Pro-Ala-Thr-CHN2 (III), can be used as an affinity label to detect the presence of wild-type SrtA in crude cell lysates prepared from S. aureus.

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