Human Deoxycytidine Kinase Is a Valuable Biocatalyst for the Synthesis of Nucleotide Analogues

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.DFG, 392246628, Chemo-enzymatische Synthese von Selen-modifizierten Nukleosiden, Nukleotiden und OligonukleotidenTU Berlin, Open-Access-Mittel - 201

Semi-automated high-throughput substrate screening assay for nucleoside kinases

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 nucleotide analogue production in an in vitro enzymatic process. Therefore, a fast and reliable substrate screening method for NKs is of great importance. Here, we report on the validation of a well-known luciferase-based assay for the detection of NK activity in a 96-well plate format. The assay was semi-automated using a liquid handling robot. Good linearity was demonstrated (r² > 0.98) in the range of 0–500 µM ATP, and it was shown that alternative phosphate donors like dATP or CTP were also accepted by the luciferase. The developed high-throughput assay revealed comparable results to HPLC analysis. The assay was exemplarily used for the comparison of the substrate spectra of four NKs using 20 (8 natural, 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.DFG, 414044773, Open Access Publizieren 2021 - 2022 / Technische Universität BerlinDFG, 392246628, Chemo-enzymatische Synthese von Selen-modifizierten Nukleosiden, Nukleotiden und Oligonukleotide

Presence and Diversity of Different Enteric Viruses in Wild Norway Rats (Rattus norvegicus)

Abstract: Rodents are common reservoirs for numerous zoonotic pathogens, but knowledge about diversity of pathogens in rodents is still limited. Here, we investigated the occurrence and genetic diversity of enteric viruses in 51 Norway rats collected in three different countries in Europe. RNA of at least one virus was detected in the intestine of 49 of 51 animals. Astrovirus RNA was detected in 46 animals, mostly of rat astroviruses. Human astrovirus (HAstV-8) RNA was detected in one, rotavirus group A (RVA) RNA was identified in eleven animals. One RVA RNA could be typed as rat G3 type. Rat hepatitis E virus (HEV) RNA was detected in five animals. Two entire genome sequences of ratHEV were determined. Human norovirus RNA was detected in four animals with the genotypes GI.P4-GI.4, GII.P33-GII.1, and GII.P21. In one animal, a replication competent coxsackievirus A20 strain was detected. Additionally, RNA of an enterovirus species A strain was detected in the same animal, albeit in a different tissue. The results show a high detection rate and diversity of enteric viruses in Norway rats in Europe and indicate their significance as vectors for zoonotic transmission of enteric viruses. The detailed role of Norway rats and transmission pathways of enteric viruses needs to be investigated in further studies.Peer Reviewe

Modular Enzymatic Cascade Synthesis of Nucleotides Using a (d)ATP Regeneration System

A correction to this article has been published in Front. Bioeng. Biotechnol., 26 October 2020 | https://doi.org/10.3389/fbioe.2020.606584Eine Korrektur zu diesem Artikel wurde publiziert in Front. Bioeng. Biotechnol., 26 October 2020 | https://doi.org/10.3389/fbioe.2020.606584Nucleoside-5’-triphosphates (NTPs) and their analogs are building blocks of DNA and are important compounds in both pharmaceutical and molecular biology applications. Currently, commercially available base or sugar modified NTPs are mainly synthesized chemically. Since the chemical production of NTPs is time-consuming and generally inefficient, alternative approaches are under development. Here we present a simple, efficient and generalizable enzymatic synthesis method for the conversion of nucleosides to NTPs. Our one-pot method is modular, applicable to a wide range of natural and modified nucleotide products and accesses NTPs directly from cheap nucleoside precursors. Nucleoside kinases, nucleoside monophosphate (NMP) kinases and a nucleoside diphosphate (NDP) kinase were applied as biocatalysts. Enzymes with different substrate specificities were combined to produce derivatives of adenosine and cytidine triphosphate with conversions of 4 to 26%. The implementation of a (deoxy)ATP recycling system resulted in a significant increase in the conversion to all NTP products, furnishing 4 different NTPs in quantitative conversion. Natural (deoxy)NTPs were synthesized with 60 to >99% conversion and sugar- and base-modified NTPs were produced with 69 to >99% and 27 to 75% conversion, respectively. The presented method is suitable for the efficient synthesis of a wide range of natural and modified NTPs in a sustainable one-pot process.TU Berlin, Open-Access-Mittel – 2020DFG, 392246628, Chemo-enzymatische Synthese von Selen-modifizierten Nukleosiden, Nukleotiden und Oligonukleotide

Spectral Unmixing‐Based Reaction Monitoring of Transformations between Nucleosides and Nucleobases†

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.DFG, 390540038, EXC 2008: Unifying Systems in Catalysis "UniSysCat"TU Berlin, Open-Access-Mittel – 2020DFG, 392246628, Chemo-enzymatische Synthese von Selen-modifizierten Nukleosiden, Nukleotiden und Oligonukleotide

Human Deoxycytidine Kinase Is a Valuable Biocatalyst for the Synthesis of Nucleotide Analogues

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

Semi-Automated High-Throughput Substrate Screening Assay for Nucleoside Kinases

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 nucleotide analogue production in an in vitro enzymatic process. Therefore, a fast and reliable substrate screening method for NKs is of great importance. Here, we report on the validation of a well-known luciferase-based assay for the detection of NK activity in a 96-well plate format. The assay was semi-automated using a liquid handling robot. Good linearity was demonstrated (r² > 0.98) in the range of 0–500 µM ATP, and it was shown that alternative phosphate donors like dATP or CTP were also accepted by the luciferase. The developed high-throughput assay revealed comparable results to HPLC analysis. The assay was exemplarily used for the comparison of the substrate spectra of four NKs using 20 (8 natural, 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

Semi-automated high-throughput substrate screening assay for nucleoside kinases

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

General Principles for Yield Optimization of Nucleoside Phosphorylase-Catalyzed Transglycosylations

This is our publication "General Principles for Yield Optimization of Nucleoside Phosphorylase-Catalyzed Transglycosylations" that described the prediction and optimization of yields in nucleoside transglycosylations mediated by nucleoside phosphorylases. As a continuation to previous reports, we highlight the varying effect of excess phosphate on product yield both theoretically and experimentally, as this is a crucially important feature of the reaction system previous undecribed. Furthermore, we provide a simplified equation for the estimation of product yield that allows for straightforward analytical solutions instead of the numerical solutions previously required. Herein, we provide the full text draft of our work along with the supplementary information. Externally hosted supplementary files can be accessed via the links cited in the main document

Biocatalytic Synthesis of 2-Seleno Pyrimidine Nucleosides via Transglycosylation

Selenium-modified nucleosides are powerful tools to study the structure and function of nucleic acids and their protein interactions. The wide-spread application of 2-seleno pyrimidine nucleosides is currently limited by low yields in established synthetic routes. Here, 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 thus belong to the highest described so far. Thus, a ten-fold 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.<br /