General Principles for Yield Optimization of Nucleoside Phosphorylase‐Catalyzed Transglycosylations

Abstract

The biocatalytic synthesis of natural and modified nucleosides with nucleoside phosphorylases offers the protecting‐group‐free direct glycosylation of free nucleobases in transglycosylation reactions. This contribution presents guiding principles for nucleoside phosphorylase‐mediated transglycosylations alongside mathematical tools for straightforward yield optimization. We illustrate how product yields in these reactions can easily be estimated and optimized using the equilibrium constants of phosphorolysis of the nucleosides involved. Furthermore, the varying negative effects of phosphate on transglycosylation yields are demonstrated theoretically and experimentally with several examples. Practical considerations for these reactions from a synthetic perspective are presented, as well as freely available tools that serve to facilitate a reliable choice of reaction conditions to achieve maximum product yields in nucleoside transglycosylation reactions.

Efficient Biocatalytic Synthesis of Dihalogenated Purine Nucleoside Analogues Applying Thermodynamic Calculations

“…Transglycosylation reaction to produce dihalogenated purine nucleosides using thermostable nucleoside phosphorylases as biocatalysts and uridine (1a) and thymidine (1b) as sugar donors. To efficiently produce 2a-and 2b-containing nucleosides in a transglycosylation reaction, equilibrium state thermodynamic calculations were carried out [13]. We have recently shown that nucleoside phosphorolysis is a reversible endothermic reaction under tight thermodynamic control [14].…”

“…* Experiments were performed with 5 mM nucleobase, 5 mM sugar donor 1a or 1b, and 2 mM potassium phosphate buffer at 40 • C to determine the equilibrium constants. ** Equilibrium constants of phosphorolysis of the target compounds were calculated as described in [13]. Known equilibrium constants for these sugar donors at 40 • C were employed for the calculations [14].…”

“…To efficiently produce 2a-and 2b-containing nucleosides in a transglycosylation reaction, equilibrium state thermodynamic calculations were carried out [13]. We have recently shown that nucleoside phosphorolysis is a reversible endothermic reaction under tight thermodynamic control [14].…”

“…We have recently shown that nucleoside phosphorolysis is a reversible endothermic reaction under tight thermodynamic control [14]. Consequently, it is possible to predict the equilibrium states of transglycosylation reactions, if the equilibrium constants of phosphorolysis of both participating nucleosides are known [13]. However, for 3a-d, equilibrium constants of phosphorolysis have not been described before.…”

“…In the present study, the enzymatic synthesis of 2,6-dichloropurine riboside (3a), 2,6-dichloropurine deoxyriboside (3b), 6-chloro-2-fluoropurine riboside (3c), and 6-chloro-2-fluoropurine deoxyriboside (3d) with thermostable nucleoside phosphorylases was optimized based on thermodynamic equilibrium state calculations [13]. Equilibrium constants for dihalogenated nucleosides were calculated and used to determine optimum reactions to reach 90% or 95% product yields.…”