From batch to flow synthesis of purine ribonucleosides by enzymatic transglycosylation

Purine nucleoside phosphorylases (PNPs, EC 2.4.2.1) catalyze the reversible phosphorolysis of the glycosydic bond of purine nucleosides; upon addition of a second nucleobase, these enzymes may transfer the glycosyl moiety to it, resulting in the chemo-, regio- and stereoselective formation of a new nucleoside (transglycosylation, Scheme 1). This chemoenzymatic process represents an advantageous alternative to conventional chemical strategies which are frequently hampered by several drawbacks such as multistep processes, need for protecting groups, low chemo-, regio- and stereoselectivity. A PNP from Aeromonas hydrophila (AhPNP) has been recently characterized in terms of substrate specificity [1], also upon immobilization on the inner surface of a silica capillary coupled on-line with a chromatographic column [2]. Because of its wide substrate specificity, AhPNP was then exploited to catalyze the “one-pot, one-enzyme” transglycosylation of 7-methylguanosine iodide with a series of 6-substituted purines, resulting in a moderate to high conversion (18-65%) of the bases into a 23-compound library of 6-substituted purine-9-ribosides. Moreover, AhPNP was covalently immobilized (25 mg immobilized enzyme, 50% yield) in a pre-packed stainless steel column containing aminopropyl silica particles. The resulting AhPNP-IMER (Immobilized Enzyme Reactor) was coupled to a HPLC apparatus containing an analytical or a semi-preparative chromatographic column associated with a UV-visible detector. This system was used to synthesize five 6-substituted purine ribonucleosides at a mg scale by transglycosylation through a “flow-based” approach. Coupling of transglycosylation reaction and product separation resulted in a fast and efficient process (52-89% conversion) with minimized sample handling. To date, AhPNP-IMER completely retained its activity upon 50 reactions in 10 months. [1] D. Ubiali, C. D. Serra, I. Serra, C. F. Morelli, M. Terreni, A. M. Albertini, P. Manitto, G. Speranza Adv. Synth. Catal. 2012, 354, 96-104 [2] E. Calleri, D. Ubiali, I. Serra, C. Temporini, G. Cattaneo, G. Speranza, C. F. Morelli, G. Massolini J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2014, 968, 79-86