During our development of the PUB module for continuous high-throughput phosphate detection in biochemical assays, we serendipitously found that the presence of borate effects a series of inhibitory phenomena during pyrimidine nucleoside phosphorolysis as well as an apparent equilibrium shift caused by biased borate esterification of nucleosides over ribose 1-phosphate (Rib1P). While saccharides are well known to undergo complex equilibrium reactions with borate in aqueous solution, 28,29 comparably little is known about in situ competition of such processes. Similarly, although the literature offers some examples for biocatalytic applications of equilibrium shift phenomena through preferential esterification of one reactant with borate (namely, the enzymatic epimerization of fructose, lactose, galactose, and arabinose ) the exact species involved in these processes remain largely elusive, as do the kinetic implications of this esterification on the enzyme-level. Despite the precedents for borate inhibiting NAD + -dependent enzymes in a noncompetitive fashion, the molecular determinants and mechanisms of these phenomena have remained equally elusive. To shed light on the thermodynamic and kinetic implications of borate ester formation on nucleoside phosphorylase-catalyzed reactions, we herein report a multifaceted analysis of this reaction system with spectroscopic and computational approaches. Furthermore, we examined the synthetic utility of this biased borate esterification, as concentration-dependent apparent equilibrium shifts provide an orthogonal dimension for reaction engineering in biocatalytic glycosylation reactions.