Optimized Biocatalytic Synthesis of 2-Selenopyrimidine Nucleosides by Transglycosylation*.

Katja F Hellendahl,Felix Kaspar,Anke Kurreck,Xinrui Zhou,Peter Neubauer,Zhen Huang,Zhaoyi Yang

doi:10.1002/cbic.202100067

Katja F Hellendahl, Felix Kaspar + Show 5 more

Open Access

https://doi.org/10.1002/cbic.202100067

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Abstract

Selenium‐modified nucleosides are powerful tools to study the structure and function of nucleic acids and their protein interactions. The widespread application of 2‐selenopyrimidine nucleosides is currently limited by low yields in established synthetic routes. Herein, we describe the optimization of the synthesis of 2‐Se‐uridine and 2‐Se‐thymidine derivatives by thermostable nucleoside phosphorylases in transglycosylation reactions using natural uridine or thymidine as sugar donors. Reactions were performed at 60 or 80 °C and at pH 9 under hypoxic conditions to improve the solubility and stability of the 2‐Se‐nucleobases in aqueous media. To optimize the conversion, the reaction equilibria in analytical transglycosylation reactions were studied. The equilibrium constants of phosphorolysis of the 2‐Se‐pyrimidines were between 5 and 10, and therefore differ by an order of magnitude from the equilibrium constants of any other known case. Hence, the thermodynamic properties of the target nucleosides are inherently unfavorable, and this complicates their synthesis significantly. A tenfold excess of sugar donor was needed to achieve 40−48 % conversion to the target nucleoside. Scale‐up of the optimized conditions provided four Se‐containing nucleosides in 6–40 % isolated yield, which compares favorably to established chemical routes.

Highlights

IntroductionBy coupling the enzymatic phosphorolysis of a sugar donor with the glycosylation of a free nucleobase in situ, nucleosides can formally be synthesized directly from their corresponding nucleobases
Selenium derivatization is a powerful tool for structure and function studies of nucleic acids
We demonstrated on an analytical scale that 2- and 4-Se-substituted pyrimidine nucleosides can be prepared via transglycosylation reactions catalyzed by thermostable nucleoside phosphorylases (NPs)

Summary

Introduction

By coupling the enzymatic phosphorolysis of a sugar donor with the glycosylation of a free nucleobase in situ, nucleosides can formally be synthesized directly from their corresponding nucleobases The application of these enzymes resolves the need for protecting groups, laborious preparation as well as isolation of sugar synthons,[22] and greatly reduces the use of toxic organic solvents by employing aqueous systems[23,24] Our work aims for biocatalytic direct glycosylation of a selenium-containing nucleobase This proof-of-concept study, revealed that several challenges remained to be addressed for the biocatalytic synthesis of Se-containing nucleosides using NPs to transition from our previous report to a synthetically useful procedure. This report presents improved yields compared to established procedures and describes the first biocatalytic synthesis of 2-Sepyrimidine nucleosides

Results and Discussion

Equilibrium constant of phosphorolysis

Enzymatic synthesis and purification in semipreparative scale

Experimental Section