Coenzyme A (CoA) analogues containing alpha,beta-unsaturated ester, ketone, and sulfone moieties were prepared by chemo-enzymatic synthesis as inhibitors of coenzyme A disulfide reductase (CoADR), a proven and as yet unexploited drug target in Staphylococcus aureus. Among these Michael acceptor-containing CoA analogues, which were designed to target CoADR's single essential active site cysteine for conjugate addition, a phenyl vinyl sulfone-containing analogue showed the most potent inhibition with a competitive K-i of similar to 40 nM, and time-dependent inactivation with a second-order rate of inactivation constant of similar to 40 000 s(-1).M-1. Our results suggest that electrophilic substrate analogues should be considered as potential inhibitors of other medicinally relevant disulfide reductase enzymes.