Biocatalytic nucleoside (trans-)glycosylations catalyzed by nucleoside phosphorylases have graduated to a practical and convenient approach to the preparation of modified nucleosides, which are important pharmaceuticals for the treatment of various cancers and viral infections. However, the obtained yields in these reactions are generally determined exclusively by the innate thermodynamic properties of the nucleosides involved, hampering the biocatalytic access to many sought-after target nucleosides. We herein report an orthogonal dimension for reaction engineering of these systems. We show how apparent equilibrium shifts in phosphorolysis and glycosylation reactions can be effected through entropically driven, biased esterification of nucleosides with inorganic borate. Our multifaceted analysis further describes the kinetic implications of this in situ reactant esterification for a model phosphorylase. Our results suggest an unusual pseudo-non-competitive inhibitory mechanism where reversible binding of the borate ester of the nucleoside substrate yields a non-productive inhibitor-enzyme complex. This complex exhibits constricted molecular dynamics and exists in a rapid equilibrium with the productive enzyme-substrate complex via hydrolytic interconversion. Collectively, this report presents a partial departure from the stringent thermodynamic constraints of nucleoside phosphorolysis reactions and shines light on the molecular processes regulating the activity of nucleoside-binding enzymes in the presence of borate.
Author contributions, accessibility statement, experimental procedures, additional discussions, and supplementary items, among others.