Models and mechanism-based inhibitors for thiamin diphosphate-dependent enzymes.

摘要

Novel pyruvic acid analogs were synthesized and tested as potential mechanism-based inhibitors of several thiamin diphosphate-dependent enzymes. These compounds, 2-oxo-3-butynoic acid, 2-oxo-3-pentynoic acid and 2-oxo-3-heptynoic acid, incorporated a triple-bond in place of the methyl group of pyruvic acid. The synthesis of these compounds was achieved by reacting ethyl (N-methoxy-N-methylcarbamoyl)formate ("Weinreb amide") with the lithium salt or the Grignard of the respective1I-alkyne. The ethyl esters were hydrolyzed under careful pH control to yield the free acids, which were tested as potential mechanism-based inhibitors of Escherichia coli pyruvate dehydrogenase multienzyme complex and its E1 subunit, pyruvate decarboxylase from Saccharomyces cerevisiae and pyruvate oxidase from Lactobacillus plantarum. 2-Oxo-3-butynoic acid is the most potent inhibitor to date of the pyruvate dehydrogenase multenzyme complex. This compound was also found to be the most potent inhibitor for pyruvate oxidase and 2-oxo-3-heptynoic acid was the most potent one for pyruvate decarboxylase.;The first step in reactions catalyzed by thiamin diphosphate-dependent enzymes is the dissociation of the proton from the C-2 position on the thiazolium ring. In order to study this species, termed the ylide/carbene, several N-substitued-4,5-dimethylthiazolium salts were synthesized. The 13C-2-labeled compound was synthesized and the carbene was generated. Its reactivity with benzaldehyde-[alpha- 13C] was studied by NMR. The results of this study present the first direct demonstration that the carbene acts as a nucleophile and attacks the carbonyl carbon of benzaldehyde to form an enolate species resembling the enamine formed with thiamin diphosphate on the enzyme.;Acetoin dehydrogenase multienzyme complex, which catalyzes the oxidative cleavage of acetoin to acetaldehyde and acetylCoA, consists of three loosely bound subunits, of which the E1 subunit, acetoin dehydrogenase, is a thiamin diphosphate-dependent enzyme. It is well known that acetoin is a by-product of the pyruvate decarboxylase reaction and also that thiamin can catalyze the benzoin condensation. A synthetic model has been made, incorporating a thiazolium moiety and isoalloxazine, an oxidizing agent, hoping to effectively mimic, acetoin dehydrogenase and cleave alpha-hydroxyketones into their corresponding aldehyde and acid derivative fragments. Using this synthetic biscoenzyme model, alpha-hydroxyketones were successfully cleaved into their corresponding aldehyde and acid derivative via an oxidative retro-benzoin condensation.