We have previously reported a general synthetic approach to analogues of coenzyme A (CoA) and CoA esters using a combination of enzymatic and nonenzymatic reactions (Martin et al. J. Am. Chem. Sec. 1994, 116, 4660). We report here the extension of this method to a CoA ester analogue 1c in which the orientation of the thioester is reversed. A key to this synthesis is the use of a trithioortho ester as a protected thioester. The reversed thioester analogue Ic is a time-dependent inhibitor of thiolase, apparently forming an acyl enzyme in which the CoA moiety rather than an acetyl moiety is covalently attached to an active site nucleophile. This analogue also serves as a general synthon for analogues having other functionality at the site of the thioester group. This has been applied to the synthesis of a reversed thioester analogue of succinyl-CoA 6 and hydroxamate 7 and hydrazide 8 analogues of acetyl-CoA, analogues which are not available by the previously described methodology. The hydroxamate and hydrazide analogues are potent inhibitors of the enzyme citrate synthase. The reversed thioester analogue of acetyl-CoA may have useful applications in enzymology and permits the ready access to a range of additional CoA analogues modified in the thioester moiety.