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

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Human Deoxycytidine Kinase Is a Valuable Biocatalyst for the Synthesis of Nucleotide Analogues catalysts Article Human Deoxycytidine Kinase Is a Valuable Biocatalyst for the Synthesis of Nucleotide Analogues Katja F. Hellendahl 1, Sarah Kamel 1,2, Albane Wetterwald 2,3, Peter Neubauer 1 and Anke Wagner 1,2,* 1 Faculty III Process Sciences, Institute of Biotechnology, Technische Universität Berlin, Ackerstr. 76, 13355 Berlin, Germany; [email protected] (K.F.H.); [email protected] (S.K.); [email protected] (P.N.) 2 BioNukleo GmbH, Ackerstr. 76, 13355 Berlin, Germany; [email protected] 3 Ecole Nationale Supérieure d’Agronomie et des Industries Alimentaires, 2 Avenue de la Forêt de Haye, 54505 Vandœuvre-lès-Nancy, France * Correspondence: [email protected]; Tel.: +49-30-314-72183 Received: 30 September 2019; Accepted: 25 November 2019; Published: 27 November 2019 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. Keywords: human deoxycytidine kinase; Ni-NTA sepharose; activity screening; nucleoside analogue; cladribine; clofarabine; fludarabine 1. 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, thus, exerting useful pharmacological action [1]. Most of the known nucleoside analogue drugs are activated in vivo to their respective active forms (nucleoside-5’-diphosphate or nucleoside-5’-triphosphate), nevertheless, in vivo activation is often insufficient [2]. Therefore, 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 [3–6]. Catalysts 2019, 9, 997; doi:10.3390/catal9120997 www.mdpi.com/journal/catalysts Catalysts 2019, 9, 997 2 of 14 To date, nucleotides are mainly produced by chemical methods like the Yoshikawa protocol or the Catalysts 2019, 9, x FOR PEER REVIEW 2 of 14 Ludwig-Eckstein method [7]. A common disadvantage of these multistep chemical synthesis reactions reactionsis a limited is a regio-limited and regio stereo-selectivity- and stereo-selectivity leading leading to a need to fora need protection for protection/deprotection/deprotection steps.Several steps. Ssideeveral products side products are produced, are produced which, inwhich return in complicatesreturn complicates the purification the purification process. process. The overall The overall process processis laborious, is laborious and often, and only often moderate only moderate product pr yieldsoduct are yields achieved. are achieved. Furthermore, Furthermore, nucleotide nucleotide products productsharboring harboring sensitive sensitive functional functional groups, such groups as a, such triazide as a ring triazide cannot ring be cannot synthesized be synthesized at all in a chemicalat all in aapproach, chemical approach, as they cannot as they withstand cannot withstand the harsh conditionsthe harsh conditions [8]. [8]. DespiteDespite the the progress progress made made in in the the optimization optimization of of chemical chemical synthesis synthesis routes, routes, enzymatic enzymatic or or chemochemo-enzymatic-enzymatic methods methods are are often often a agood good alternative alternative [9] [9.]. Compared Compared to to chemical chemical synthesis synthesis routes, routes, biotransformationbiotransformation usually usually does does not not require require protection protection or or deprotection deprotection steps steps and and lead lead to to high high final final productproduct yields yields,, due due to to the the high high regio regio-- and and stereo stereo-selectivity-selectivity of of the the enzymes. enzymes. Deoxyribonucleoside Deoxyribonucleoside kinaseskinases were were described described as assuitable suitable biocatalysts biocatalysts for forthe thesynthesis synthesis of nucleotide of nucleotide analogues analogues [10,11] [10. As,11 ]. anAs example, an example, the deoxynucleoside the deoxynucleoside kinase kinase (dNK) (dNK)from the from fruit the-fly fruit-fly DrosophilaDrosophila melanogaster melanogaster was shownwas toshown have toa havewide a widesubstrate substrate spectrum spectrum and andto touse use 2’ 2’-deoxy-ribonucleosides,-deoxy-ribonucleosides, arabinosyl arabinosyl-uracil,-uracil, arabinosylarabinosyl-adenine-adenine and and fludarabine fludarabine as as substrates substrates [11] [11.]. HumanHuman deoxycytidine deoxycytidine kinase kinase (Hs (HsdCKdCK)) is is another another interesting interesting biocatalyst biocatalyst for for the the synthesis synthesis of of nucleonucleotidetide analogues. analogues. InIn vivo vivo, ,it itplays plays an an important important role role in in maintaining maintaining the the homeostasis homeostasis of of the the 2’2’-deoxy-ribonucleotide-deoxy-ribonucleotide pool pool within within the the cell. cell. Using Using adenosine adenosine triphosphate triphosphate or or uridine uridine triphosphate triphosphate asas aa phosphate phosphate donor, donor, it it catalyzes catalyzes the the 5′ 5-0-phosphorylationphosphorylation of of deoxycytidine, deoxycytidine, deoxyadenosine, deoxyadenosine, and and deoxyguanosinedeoxyguanosine [12 [12––15]15.]. Furthermore, Furthermore, dCK dCK activity activity is is essential essential for for the the activation activation of of a awide wide variety variety of of importantimportant therapeutic therapeutic prodrugs, prodrugs, suchsuch asas thethe antiviral antiviral agent agent lamivudine lamivudine (3TC), (3TC), for for the the treatment treatment of HIV of HIVinfection infection [16], [16] or, antineoplastic or antineoplastic agents agents like like cladribine cladribine [17] [17] (Scheme (Scheme1). 1). Scheme 1. Nucleoside analogues accepted as substrates of human deoxycytidine kinase (HsdCK). Scheme 1. Nucleoside analogues accepted as substrates of human deoxycytidine kinase (HsdCK). Both whole immobilized cells and purified enzymes have been used for the synthesis of NMPBoth analogues whole immobilized [18,19]. Immobilization cells and purified techniques enzymes help have to been stabilize used for biocatalysts the synthesis under of NMP harsh analoguesconditions [18,19] (e.g.,. highImmobilization temperature, techniques extreme help pH, to solvents) stabilize [ 19biocatalysts]. As theimmobilized under harsh biocatalystsconditions (e.g.can, behigh used temperature, repeatedly, theextreme processes pH, getsolvents) more cost-e[19]. ffAsective, the immobilized which is an important biocatalysts consideration can be used for repeatedly,industrial applications.the processes Using get more dNK cost and-effective, deoxyadenosine which is kinase an important (dAK) of considerationDrosophila melanogaster for industrialand applications.Dictyostelium Using discoideum dNK, respectively, and deoxyadenosine fludarabine-5’-monophosphate kinase (dAK) of (2F-AraAMP)Drosophila melanogaster was synthesized and Dictyosteliumfrom fludarabine discoideum (2F-AraA), respectively, with a high fludarabine conversion-5’-monophosphate percentage (> 90%) (2F [-10AraAMP),11]. was synthesized from fludarabineIn this study, (2F the-AraA) chemo-enzymatic with a high conversion synthesis of percentage NMP analogues (> 90%) from [10,11] nucleosides. using soluble andIn immobilized this study, theHs chemodCK is-enzymatic demonstrated synthesis (Scheme of NMP2). HsanaloguesdCK was from chosen nucleosides as it is ausing well-studied soluble and immobilized HsdCK is demonstrated (Scheme 2). HsdCK was chosen as it is a well-studied enzyme showing a wide substrate spectrum. However, it is potential for industrial application was not studied so far. Nickel-nitrilotriacetic acid (Ni-NTA) superflow was applied as the support, since the applied enzymes bear an N-terminal His-tag and it is described as a gentle way of immobilization. Catalysts 2019, 9, 997 3 of 14 enzyme showing a wide substrate spectrum. However, it is potential for industrial application was not studiedCatalysts
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