To probe if borate esters like 1a* would be feasible species to effect apparent equilibrium shifts under dilute aqueous reaction conditions, we turned to density functional theory (DFT) calculations and NMR spectroscopy. DFT calculations (using B3LYP/TZP in combination with a COSMO solvent model for water, see the SI for details) suggested that the formation of the 2′-or 3′-borate monoesters of 1a would be highly disfavored processes, while the formation of the proposed five-membered cyclic diester 1a* offers around 40 kJ mol -1 net gain in Gibbs free energy, indicating that this esterification could feasibly proceed against the concentration gradient of water (Fig. ). Indeed, direct evidence for the presence of 1a* could be obtained by NMR spectroscopy. When 1a (10 mM) was incubated with 20 mM borate in glycine buffer at pH 9, a dominant borate-containing species in equilibrium with free borate could be observed by B NMR (Fig. ), while 1 H NMR showed additional signals indicative of a modification of the ribosyl moiety of 1a (Fig. ). Furthermore, slight changes in the coupling constants across the sugar ring implied the introduction of ring torsion, consistent with the configurational changes required in 1a* (Table ). In addition to this ester, we observed two distinct minor components in the mixture, which exhibited identical coupling constants to 1a* and existed in similar concentrations (ca. 1:1.2 ratio). Based on the well-characterized diastereomeric borate ester dimers of methyl apiose described by Ishii and Ono and the report of purine nucleoside solubilization as 2:1 complexes with borate by Tsuji and colleagues, we tentatively ascribe these species as the cis-and trans-isomers 1a** (Figs. and). Overall, the Gibbs free energies obtained experimentally by NMR spectroscopy or equilibrium state calculations based on UV data (Δ ca. -32 kJ mol -1 , see the SI for details) are comparable to those obtained by DFT calculation. Although an analogous six-membered ester (and potentially its dimers) between the 3′-and 5′-hydroxyl groups would introduce similar configurational changes to those ascribed to 1a*, DFT calculations revealed that its formation is much less exothermic and consequently disfavored. Similarly, the analogous five-membered borate ester of Rib1P is less favored than 1a* (which is requisite for the observed equilibrium shift), as supported by DFT and NMR data (Figs. and). Additionally, we observed no trace of dimers of Rib1P* by NMR, which is likely a result of the highly disfavored formal -5 charge of these species. Consequently, the formation of the borate ester 1a* should be expected to dominate in direct competition during a phosphorolysis reaction, which we could confirm by subjecting phosphorolysis reaction mixtures with increasing borate concentrations to NMR analysis (Fig. ). Further thermodynamic experiments indicated that this biased esterification is primarily driven by entropic effects. Arrhenius plots of the 1a-1a* equilibrium obtained by H NMR revealed a drastic temperaturedependence, favoring the presence of the free nucleoside at higher temperatures (Fig. ). In support of these observations, Arrhenius plots for the borate esterification derived from equilibrium shifts in the phosphorolysis reaction indicated an approach to an energetic balance between 1a* and Rib1P* at higher temperatures (Fig. ). Collectively, these results describe 1a* as the dominant borate ester in this reaction system, responsible for depleting the pool of free nucleoside in dilute aqueous solution at room temperature. Consistent with this conclusion, the 2′-deoxy nucleoside deoxy-1a, incapable of forming 1a* or 1a**, did not show an equilibrium shift during its phosphorolysis in the presence of borate or any reaction with borate discernible by NMR (Fig. ).