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

Katja F Hellendahl,Anke Wagner,Albane Wetterwald,Peter Neubauer,Sarah Kamel

doi:10.3390/catal9120997

Katja F Hellendahl, Anke Wagner + Show 3 more

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https://doi.org/10.3390/catal9120997

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Abstract

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.

Highlights

Modified nucleosides and nucleotides are important small molecules in molecular, biological and pharmaceutical applications
While three were used biocatalysts, werewith usedfive forreplicas protein for elution estimate the amount ofreplicates bound enzyme and as immobilized biocatalysts, two replicas were used for protein elution to estimate the amount of to be later used as a soluble biocatalyst for the synthesis of nucleotide analogues
We studied the impact of guanosine triphosphate (GTP), adenosine triphosphate (ATP) and uridine triphosphate (UTP) on the conversion of fludarabine using heterologously expressed HsdCK

Summary

Introduction

Modified nucleosides and nucleotides are important small molecules in molecular, biological and pharmaceutical applications. Since they are involved in the same metabolic pathways as endogenous nucleosides and nucleotides, they act as antimetabolites, exerting useful pharmacological action [1]. In a number of approaches, bio-reversible protected ribonucleoside-5’-monophosphates (NMPs), such as the phosphoramidate prodrug sofosbuvir were administered to overcome the first activation step in vivo [3,4,5,6]. Nucleotides are mainly produced by chemical methods like the Yoshikawa protocol or the Ludwig-Eckstein method [7]. A common disadvantage of these multistep chemical synthesis reactions is a limited and stereo-selectivity leading leading to a need protection/deprotection steps. Several reactions is aregiolimited regioand stereo-selectivity to for a need for protection/deprotection steps

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