A direct method for the preparation of 5'-S-alkynyl-5'-thioadenosine and 5'-S-allenyl-5'-thioadenosine has been developed. Treatment of a protected 5'-acetylthio-5'-deoxyadenosine with sodium methoxide and propargyl bromide followed by deprotection gave the 5'-S-propargyl-5'-thioadenosine 4. Under controlled base-catalysis with sodium tert-butoxide in tert-butyl alcohol 4 was quantitatively converted into 5'-S-allenyl-5'-thioadenosine 5 or 5'-S-propynyl-5'-thioadenosine 6. Incubation of recombinant human placental AdoHcy hydrolase with 4, 5, or 6 resulted in time- and concentration-dependent inactivation of the enzyme (K(i): 45 +/- 0.5, 16 +/- 1, and 15 +/- 1 microM, respectively). Compound 4 caused complete conversion of the enzyme from its E-NAD(+) to E-NADH form during the inactivation process. 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 corresponding thioester 7. This acylating-intermediate agent could then undergo nucleophilic attack by a protein residue, leading to a type II mechanism-based inactivation. ElectroSpray mass spectra analysis of the inactivated protein by 5 supports this mechanistic proposal. Further studies (MALDI-TOF and ESI/MS(n) 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 (EC(50): 1.9 microM) was noted with 5 against vaccinia virus.