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.

The increased interest in (enzymatic) transformations between nucleosides and nucleobases has demanded the development of efficient analytical tools. In this report, we present an update and extension of our recently described method for monitoring these reactions by spectral unmixing. The presented method uses differences in the UV absorption spectra of nucleosides and nucleobases after alkaline quenching to derive their ratio based on spectral shape by fitting normalized reference spectra. It is applicable to a broad compound spectrum comprising more than 35 examples, offers HPLC‐like accuracy, ease of handling and significant reductions in both cost and data acquisition time compared to other methods. This contribution details the principle of monitoring reactions by spectral unmixing, gives recommendations regarding solutions to common problems and applications that necessitate special sample treatment. We provide software, workflows and reference spectra that facilitate the straightforward and versatile application of the method.

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.

Natural ribonucleoside-5’-monophosphates are building blocks for nucleic acids which are used for a number of purposes, including food additives. Their analogues, additionally, are used in pharmaceutical applications. Fludarabine-5´-monophosphate, for example, is effective in treating hematological malignancies. To date, ribonucleoside-5’-monophosphates are mainly produced by chemical synthesis, but the inherent drawbacks of this approach have led to the development of enzymatic synthesis routes. In this study, we evaluated the potential of human deoxycytidine kinase (HsdCK) as suitable biocatalyst for the synthesis of natural and modified ribonucleoside-5’-monophosphates from their corresponding nucleosides. Human dCK was heterologously expressed in E. coli and immobilized onto Nickel-nitrilotriacetic acid (Ni-NTA) superflow. A screening of the substrate spectrum of soluble and immobilized biocatalyst revealed that HsdCK accepts a wide range of natural and modified nucleosides, except for thymidine and uridine derivatives. Upon optimization of the reaction conditions, HsdCK was used for the synthesis of fludarabine-5´-monophosphate using increasing substrate concentrations. While the soluble biocatalyst revealed highest product formation with the lowest substrate concentration of 0.3 mM, the product yield increased with increasing substrate concentrations in the presence of the immobilized HsdCK. Hence, the application of immobilized HsdCK is advantageous upon using high substrate concentration which is relevant in industrial applications.

This publication introduces a thermal segment microwell plate control device, integrating smart sensor technology and standardized well-plate geometry for automated biolab setups. It is tested in a high-throughput enzymatic activity assay.

Nucleoside kinases (NKs) are key enzymes involved in the in vivo phosphorylation of nucleoside analogues used as drugs to treat cancer or viral infections. Having different specificities, the characterization of NKs is essential for drug design and the production of nucleotide analogues in an in vitro enzymatic process. Therefore, a fast and reliable substrate screening assay for NKs is of great importance. Here, we report the validation of a well-known luciferase-based assay for the detection of NK activity in 96-well plate format. The assay was semi-automated using a liquid handling robot. A good linearity was demonstrated (r² >0.98) in the range of 0 to 500 µM ATP, and it was shown that also alternative phosphate donors like dATP or CTP were accepted by the luciferase. The developed high-throughput assay revealed comparable results to HPLC analysis. The assay was exemplary used for the comparison of the substrate spectra of four nucleoside kinases using 20 (8 natural and 12 modified) substrates. The screening results correlated well with literature data and, additionally, previously unknown substrates were identified for three of the NKs studied. Our results demonstrate that the developed semi-automated high-throughput assay is suitable to identify best performing NKs for a wide range of substrates.