Discovery of Type II (Covalent) Inactivation of S-Adenosyl-L-homocysteine Hydrolase Involving Its “Hydrolytic Activity”: Synthesis and Evaluation of Dihalohomovinyl Nucleoside Analogues Derived from Adenosine1

Treatment of the 5′-carboxaldehyde derived by Moffatt oxidation of 6-N-benzoyl-2′,3′-Oisopropylideneadenosine (1) with the “(bromofluoromethylene)triphenylphosphorane” reagent and deprotection gave 9-(6-bromo-5,6-dideoxy-6-fluoro-â-D-ribo-hex-5-enofuranosyl)adenine (4). Parallel treatment with a “dibromomethylene Wittig reagent” and deprotection gave 9-(6,6dibromo-5,6-dideoxy-â-D-ribo-hex-5-enofuranosyl)adenine (7), which also was prepared by successive bromination and dehydrobromination of the 6′-bromohomovinyl nucleoside 8. Bromination-dehydrobromination of the 5′-bromohomovinyl analogue 11 and deprotection gave (E)-9-(5,6-dibromo-5,6-dideoxy-â-D-ribo-hex-5-enofuranosyl)adenine (15). Compounds 4, 7, and 15 were designed as putative substrates of the “hydrolytic activity” of S-adenosyl-L-homocysteine (AdoHcy) hydrolase. Enzyme-mediated addition of water across the 5,6-double bond could generate electrophilic acyl halide or R-halo ketone species that could undergo nucleophilic attack by proximal groups on the enzyme. Such type II (covalent) mechanism-based inactivation is supported by protein labeling with 8-[3H]-4 and concomitant release of bromide and fluoride ions. Incubation of AdoHcy hydrolase with 7 or 15 resulted in irreversible inactivation and release of bromide ion. In contrast with type I mechanism-based inactivation, reduction of enzyme-bound NAD+ to NADH was not observed. Compounds 4, 7, and 15 were not inhibitory to a variety of viruses in cell culture, and weak cytotoxicity was observed only for CEM cells.