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

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.

Key advances in biocatalytic phosphorylations in the last two decades: Biocatalytic syntheses in vitro and biotransformations in vivo (in humans)

“…[103] Recombinant human deoxycytidine kinase has been demonstrated to catalyze the phosphorylation of natural and modified nucleoside analogues to their 5′-monophosphates. [104] For the phosphorylation of fludarabine a conversion of 60% was achieved when the soluble enzyme was used and a slightly lower conversion of 55% in the case of the immobilized enzyme. [104] Several nucleoside analogues that inhibit reverse transcriptases have been approved for treating viral infections since the approval of zidovudine.…”

“…[ 104 ] For the phosphorylation of fludarabine a conversion of 60% was achieved when the soluble enzyme was used and a slightly lower conversion of 55% in the case of the immobilized enzyme. [ 104 ] Several nucleoside analogues that inhibit reverse transcriptases have been approved for treating viral infections since the approval of zidovudine. [ 105 ] These nucleoside reverse transcriptase inhibitors act as chain terminators for viral DNA polymerization by their corresponding triphosphates, which are formed in the host cells by kinase‐catalyzed phosphorylations.…”

A Multi-Enzymatic Cascade Reaction for the Synthesis of Vidarabine 5′-Monophosphate

“…Uridine phosphorylase from Clostridium perfringens (CpUP) and a purine nucleoside phosphorylase from Aeromonas hydrophila (AhPNP) were used in the preparative synthesis of araA yielding 3.5 g/L of the desired product (purity 98.7%) [18]. Moreover, an enzymatic approach for the phosphorylation of araA to araA-MP and of F-araA to F-araA-MP using either a deoxyribonucleoside kinase from Drosophila melanogaster (DmdNK) [11], a deoxyadenosine kinase from Dictyostelium discoideum (DddAK) [12], or human deoxycytidine kinase (HsdCK) [17] has been developed.…”

“…dNKs catalyze the transfer of the γ-phosphate group from a nucleotide (generally ATP) to the 5 -hydroxyl group of nucleosides thus forming the corresponding 5 -mononucleotide [14]. The potential of NPs and dNKs in the synthesis of nucleoside/nucleotide analogues has been already demonstrated by several papers published in recent years [9][10][11][12][15][16][17]. Uridine phosphorylase from Clostridium perfringens (CpUP) and a purine nucleoside phosphorylase from Aeromonas hydrophila (AhPNP) were used in the preparative synthesis of araA yielding 3.5 g/L of the desired product (purity 98.7%) [18].…”

“…Uridine phosphorylase from Clostridium perfringens (CpUP) and a purine nucleoside phosphorylase from Aeromonas hydrophila (AhPNP) were used in the preparative synthesis of araA yielding 3.5 g/L of the desired product (purity 98.7%) [18]. Moreover, an enzymatic approach for the phosphorylation of araA to araA-MP and of F-araA to F-araA-MP using either a deoxyribonucleoside kinase from Drosophila melanogaster (DmdNK) [11], a deoxyadenosine kinase from Dictyostelium discoideum (DddAK) [12], or human deoxycytidine kinase (HsdCK) [17] has been developed.Biocatalysts are not only environmentally benign reagents and highly selective, but they are compatible with each other within certain ranges of operating conditions [19,20]. This feature enables integration of sequential biocatalytic transformations in one-pot cascades for the synthesis of complex molecules [21].…”