Thermodynamic Reaction Control of Nucleoside Phosphorolysis

Abstract

Nucleoside analogs represent a class of important drugs for cancer and antiviral treatments. Nucleoside phosphorylases (NPases) catalyze the phosphorolysis of nucleosides and are widely employed for the synthesis of pentose‐1‐phosphates and nucleoside analogs, which are difficult to access via conventional synthetic methods. However, for the vast majority of nucleosides, it has been observed that either no or incomplete conversion of the starting materials is achieved in NPase‐catalyzed reactions. For some substrates, it has been shown that these reactions are reversible equilibrium reactions that adhere to the law of mass action. In this contribution, we broadly demonstrate that nucleoside phosphorolysis is a thermodynamically controlled endothermic reaction that proceeds to a reaction equilibrium dictated by the substrate‐specific equilibrium constant of phosphorolysis, irrespective of the type or amount of NPase used, as shown by several examples. Furthermore, we explored the temperature‐dependency of nucleoside phosphorolysis equilibrium states and provide the apparent transformed reaction enthalpy and apparent transformed reaction entropy for 24 nucleosides, confirming that these conversions are thermodynamically controlled endothermic reactions. This data allows calculation of the Gibbs free energy and, consequently, the equilibrium constant of phosphorolysis at any given reaction temperature. Overall, our investigations revealed that pyrimidine nucleosides are generally more susceptible to phosphorolysis than purine nucleosides. The data disclosed in this work allow the accurate prediction of phosphorolysis or transglycosylation yields for a range of pyrimidine and purine nucleosides and thus serve to empower further research in the field of nucleoside biocatalysis.

General Principles for Yield Optimization of Nucleoside Phosphorylase-Catalyzed Transglycosylations

“…[16] To validate thesep redictionse xperimentally and demonstrate the varying impact of phosphate on the product yield, we prepared as eries of natural and base-modified ribosyl nucleosides from their respective nucleobases, using uridine as a sugar donor. Fitting of the experimental data to the equilibrium constraints [15,16] yielded equilibrium constants K 1 and K 2 very similar to those reported previously [17] and revealed a great range of apparent equilibrium constants K 2 (0.01 to 0.35 at 60 8C, pH 9) and K N (0.4 to 16.0). In all cases,t he experimental yields determined by HPLC agreedw ell with the predictions obtained for different phosphate concentrations ( Figure 2).…”

“…[16] Numerical solutions of this system allowed theoretical examination of the effect of phosphate and sugar donor excess on the product yield, considering ar easonable range of equilibriumc onstants. [17] Approaching zero phosphate concentration, the maximum (ideal) product yield can be obtained, but at higher phosphate concentrationsa na pparent loss of yield can be observed due to phosphorolysis (or non-synthesis) of the product nucleoside (Figure 1). Whereas the K 1 /K 2 ratio (equal to K N )d ictates the maximumy ield with minimal phosphate, K 2 determines the extent of yield loss in the presence of phosphate.…”

“…Realistic K 1 and K 2 valuesw ere assumed basedo nr ecently reportede quilibrium constants. [17] The graphsf or maximum yield (max. ;b lack), 0.1 equiv (green), 1equiv (blue)a nd 10 equiv (red) of phosphate were plotted using numericalsolutionso ft he system of equilibrium constraints (1) and(2) calculatedw ith the Pythonc ode describedint he external SupportingI nformation.…”

“…Thus, the maximum yield (at zero phosphate) can be calculated easily from Equation (4) to reflect ar ealistic estimate of the experimentaly ield if < 0.3 equiv of phosphate are used. Ideal yields for conversions employing ar ange of pyrimidine and purine ribosyl and 2'-deoxyribosyl nucleosides [17] with different reaction conditions including sugar donor excess and temperature, can be calculated with an Excel sheet freelya vailable from the externally hosted Supporting Information. [18] These considerations andp revious findings [15,16] bear several practical implications for NPase-catalyzed nucleoside transglycosylations.…”

“…Second,p yrimidine nucleosides serve better as sugar donors than purine nucleosides. [17] From ap ractical point of view,u ridine and thymidine recommend themselves as ribosyl and 2'-deoxyribosyl donor,r espectively,d ue to their simple commerciala vailability and high K value. Third, phosphate concentration in the transglycosylation reactionshould generally be kept as low as possible to prevent loss of product yield.…”