Synthesis, mechanism of action, and antiviral activity of a new series of covalent mechanism-based inhibitors of S-adenosyl-L-homocysteine hydrolase

A direct method for the preparation of 5 ' -S-alkynyl-5 ' -thioadenosine an d 5 ' -S-allenyl-5 ' -thioadenosine has been developed. Treatment of a prot ected 5 ' -acetylthio-5 ' -deoxyadenosine with sodium methoxide and proparg yl bromide followed by deprotection gave the 5 ' -S-propargyl-5 ' -thioaden osine 4. Under controlled base-catalysis with sodium tert-butoxide in tert- butyl alcohol 4 was quantitatively converted into 5 ' -S-allenyl-5 ' -thioa denosine 5 or 5 ' -S-propynyl-5 ' -thioadenosine 6. Incubation of recombina nt human placental AdoHcy hydrolase with 4, 5, or 6 resulted in time- and c oncentration-dependent inactivation of the enzyme (K-i: 45 +/- 0.5, 16 +/- 1, and 15 +/- 1 muM, respectively). Compound 4 caused complete conversion o f the enzyme from its E-NAD(+) to E-NADH form during the inactivation proce ss. This indicates that 4 is a substrate for the 3 ' -oxidative activity of AdoHcy hydrolase (type I inhibitor). In contrast, the NAD(+)/NADH content of the enzyme was not affected during the inactivation process with 5 and 6 , and their mechanism of inactivation was further investigated. Addition of enzyme-sequestered water on the S-allenylthio group of 5 or S-propynylthio group of 6 within the active site should lead to the formation of the corr esponding thioester 7. This acylating-intermediate agent could then undergo nucleophilic attack by a protein residue, leading to a type II mechanism-b ased inactivation. ElectroSpray mass spectra analysis of the inactivated pr otein by 5 supports this mechanistic proposal. Further studies (MALDI-TOF a nd ESI/MSn experiments) of the trypsin and endo-Lys-C proteolytic cleavage of the fragments of inactivated AdoHcy hydrolase by 5 were carried out for localization of the labeling. The antiviral activity of 4, 5, and 6 against a large variety of viruses was determined. Significant activity (EC50: 1.9 muM) was noted with 5 against vaccinia virus.