S -Adenosyl- l -homocysteine Hydrolase Side Reactivity Enables Biocatalytic Access to 4′,5′-Dehydro Nucleosides as Nucleoside Drug Precursors

Abstract

S-Adenosyl-l-homocysteine hydrolase (SAHH) reversibly cleaves S-adenosyl-l-homocysteine (SAH) into adenosine and l-homocysteine, regulating the cellular potential to perform S-adenosyl-l-methionine (SAM)-dependent methylation of DNA, RNA, proteins, and small molecules. Another route of SAH metabolism involves the deamination to S-inosyl-l-homocysteine (SIH) followed by degradation to l-homocysteine and inosine catalyzed by S-inosyl-l-homocysteine hydrolase (SIHH). SAHHs have long been studied as regulators of cellular methylation potential, making them attractive targets for antimicrobial, antiviral, and anticancer drug development. In this study, we elucidate the often-reported co-detection of the nucleobases adenine and hypoxanthine in acid-quenched in vitro experiments testing SAHHs and SIHHs. This finding picks up on a reaction mechanism previously established for adenosine and demonstrates that it can also be applied to inosine. Intermediates formed during a dehydratase-like reaction catalyzed by SAHHs and SIHHs were detected by LC-MS/MS. Isolation of these intermediates enabled structural analysis by NMR spectroscopy and their identification as 4′,5′-dehydro nucleosides, as well as systematic assessment of their stability toward pH‑dependent depurination. The dehydratase-like reactivity of SAHHs and SIHHs can be repurposed and utilized in scaled-up reactions to efficiently produce 4′,5′-dehydro adenosine and 4′,5′-dehydro inosine. In addition, non-native nucleoside substrates were converted to the respective 4′,5′-dehydro nucleoside analogues. We propose that the SAHH- and SIHH-catalyzed dehydration reaction offers a straightforward strategy for the enzymatic synthesis of diverse 4′,5′-dehydro nucleosides as valuable precursors for chemical functionalization yielding 4′- or 5′-substituted nucleoside drug candidates.