| Abstract
| - 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‘-deoxyadenosinewith 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-butylalcohol 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 (Ki: 45 ± 0.5,16 ± 1, and 15 ± 1 μM, respectively). Compound 4 caused complete conversion of the enzymefrom its E-NAD+ to E-NADH form during the inactivation process. This indicates that 4 is asubstrate for the 3‘-oxidative activity of AdoHcy hydrolase (type I inhibitor). In contrast, theNAD+/NADH content of the enzyme was not affected during the inactivation process with 5and 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 activesite should lead to the formation of the corresponding thioester 7. This acylating-intermediateagent could then undergo nucleophilic attack by a protein residue, leading to a type IImechanism-based inactivation. ElectroSpray mass spectra analysis of the inactivated proteinby 5 supports this mechanistic proposal. Further studies (MALDI-TOF and ESI/MSn experiments) of the trypsin and endo-Lys-C proteolytic cleavage of the fragments of inactivatedAdoHcy hydrolase by 5 were carried out for localization of the labeling. The antiviral activityof 4, 5, and 6 against a large variety of viruses was determined. Significant activity (EC50: 1.9μM) was noted with 5 against vaccinia virus.
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