catalysts Communication A Multi-Enzymatic Cascade Reaction for the 0 Synthesis of Vidarabine 5 -Monophosphate Marina Simona Robescu 1 , Immacolata Serra 2,*, Marco Terreni 1, Daniela Ubiali 1,* and Teodora Bavaro 1 1 Department of Drug Sciences, University of Pavia, viale Taramelli 12, I-27100 Pavia, Italy; [email protected] (M.S.R.); [email protected] (M.T.); [email protected] (T.B.) 2 Department of Food, Environmental and Nutritional Sciences, University of Milan, via Mangiagalli 25, I-20133 Milano, Italy * Correspondence: [email protected] (I.S.); [email protected] (D.U.); Tel.: +39-02-50319248 (I.S.); +39-0382-987889 (D.U.) Received: 4 December 2019; Accepted: 27 December 2019; Published: 1 January 2020 Abstract: We here described a three-step multi-enzymatic reaction for the one-pot synthesis of vidarabine 50-monophosphate (araA-MP), an antiviral drug, using arabinosyluracil (araU), adenine (Ade), and adenosine triphosphate (ATP) as precursors. To this aim, three enzymes involved in the biosynthesis of nucleosides and nucleotides were used in a cascade mode after immobilization: uridine phosphorylase from Clostridium perfringens (CpUP), a purine nucleoside phosphorylase from Aeromonas hydrophila (AhPNP), and deoxyadenosine kinase from Dictyostelium discoideum (DddAK). Specifically, CpUP catalyzes the phosphorolysis of araU thus generating uracil and α-d-arabinose-1-phosphate. AhPNP catalyzes the coupling between this latter compound and Ade to form araA (vidarabine). This nucleoside becomes the substrate of DddAK, which produces the 50-mononucleotide counterpart (araA-MP) using ATP as the phosphate donor. Reaction conditions (i.e., medium, temperature, immobilization carriers) and biocatalyst stability have been balanced to achieve the highest conversion of vidarabine 5 -monophosphate ( 95.5%). The combination of 0 ≥ the nucleoside phosphorylases twosome with deoxyadenosine kinase in a one-pot cascade allowed (i) a complete shift in the equilibrium-controlled synthesis of the nucleoside towards the product formation; and (ii) to overcome the solubility constraints of araA in aqueous medium, thus providing a new route to the highly productive synthesis of araA-MP. Keywords: enzyme cascade; enzyme immobilization; vidarabine 50-monophosphate; nucleoside phosphorylase; deoxyadenosine kinase; transglycosylation; phosphorylation; unnatural nucleosides 1. Introduction Nucleoside and nucleotide analogues are synthetic, chemically modified molecules that can mimic the natural building blocks of DNA/RNA synthesis; they can act, indeed, as antimetabolites, thus exerting anticancer/antiviral effects [1]. Upon cellular uptake, nucleosides are activated in vivo to 50-triphosphates by a sequential phosphorylation reaction catalyzed by host cell kinases or virus-encoded kinases. This means that nucleoside analogues must not only be substrates for polymerases, they also need to be recognized and phosphorylated by kinases [2]. Once phosphorylated, nucleotide analogues can elicit their effects by impairing nucleic acid synthesis [3]. Some modified nucleosides are frequently used as 50-monophosphate prodrugs. The use of 50-monophosphates allows both for circumventing the poor water solubility of the parent nucleosides and the first activation step through phosphorylation which is often a rate-limiting reaction [2]. This is the case of the antiviral drug vidarabine (Vira-A®, arabinosyladenine, araA) which was the first of Catalysts 2020, 10, 60; doi:10.3390/catal10010060 www.mdpi.com/journal/catalysts Catalysts 2020, 10, x FOR PEER REVIEW 2 of 12 the FDA-approved nucleoside analogues to be administered systemically in clinics [4]. Similarly, the 2-fluorinated counterpart of vidarabine (Fludara®, arabinosyl-2-fluoroadenine 5′-monophosphate, F- araA-MP)Catalysts is2020 used, 10, 60as 5′-monophosphate in the treatment of haematological malignancies [5].2 of 12 Chemical methods for nucleoside/nucleotide synthesis involve multi-step routes [6,7] which often require harsh reaction conditions and are plagued by moderate yields. the FDA-approved nucleoside analogues to be administered systemically in clinics [4]. Similarly, In recent decades, several biocatalytic approach®es have been described exploiting enzymes that the 2-fluorinated counterpart of vidarabine (Fludara , arabinosyl-2-fluoroadenine 50-monophosphate, are naturally involved in the biosynthesis of nucleosides and nucleotides such as nucleoside F-araA-MP) is used as 50-monophosphate in the treatment of haematological malignancies [5]. phosphorylasesChemical (NPs, methods EC 2.4.2.x) for nucleoside and deoxyribonucleoside/nucleotide synthesis involve kinases multi-step (dNKs, EC routes 2.7.1.x) [6,7] which[8–13]. often NPs act in therequire salvage harsh reactionpathway conditions of andnucleobases are plagued and by moderate catalyze yields. the reversible conversion of (deoxy)ribonucleosidesIn recent decades, to their several corresponding biocatalytic approaches free base haveand α been-D-(deoxy)ribose-1-phosphate described exploiting enzymes in the presencethat areof inorganic naturally involvedorthophosphate in the biosynthesis (phosphorolysi of nucleosidess). The reversibility and nucleotides of phosphorolysis such as nucleoside reaction can bephosphorylases exploited for (NPs, synthetic EC 2.4.2.x) applications and deoxyribonucleoside: if a second nucleobase kinases (dNKs, reacts EC with 2.7.1.x) α-D [8-(deoxy)ribose-1-–13]. NPs act phosphate,in the salvage the formation pathway ofof nucleobasesa new nucleoside and catalyze can re thesult reversible (transglycosylation) conversion of (deoxy)ribonucleosides [10]. dNKs catalyze the to their corresponding free base and α-d-(deoxy)ribose-1-phosphate in the presence of inorganic transfer of the γ-phosphate group from a nucleotide (generally ATP) to the 5′-hydroxyl group of orthophosphate (phosphorolysis). The reversibility of phosphorolysis reaction can be exploited for nucleosides thus forming the corresponding 5′-mononucleotide [14]. The potential of NPs and dNKs synthetic applications: if a second nucleobase reacts with α-d-(deoxy)ribose-1-phosphate, the formation in theof synthesis a new nucleoside of nucleoside/nucleotide can result (transglycosylation) analogues [ 10has]. dNKsbeen already catalyze thedemonstrated transfer of the byγ several-phosphate papers published in recent years [9–12,15–17]. Uridine phosphorylase from Clostridium perfringens (CpUP) group from a nucleotide (generally ATP) to the 50-hydroxyl group of nucleosides thus forming and thea purine corresponding nucleoside 50-mononucleotide phosphorylase [ 14from]. The Aeromonas potential ofhydrophila NPs and ( dNKsAhPNP) in thewere synthesis used in of the preparativenucleoside synthesis/nucleotide of araA analogues yielding has been 3.5 alreadyg/L of the demonstrated desired product by several (purity papers 98.7%) published [18]. in Moreover, recent an enzymaticyears [9–12 approach,15–17]. Uridine for the phosphorylase phosphorylation from Clostridium of araA perfringensto araA-MP(Cp UP)and and of F-araA a purine to nucleoside F-araA-MP usingphosphorylase either a deoxyribonucleoside from Aeromonas hydrophila kinase(Ah PNP)from were Drosophila used in the melanogaster preparative synthesis(DmdNK) of araA[11], a deoxyadenosineyielding 3.5 g kinase/L of the from desired Dictyostelium product (purity discoideum 98.7%) [(18Dd].dAK) Moreover, [12], anor enzymatichuman deoxycytidine approach for thekinase (HsdCK)phosphorylation [17] has been of araAdeveloped. to araA-MP and of F-araA to F-araA-MP using either a deoxyribonucleoside Drosophila melanogaster Dm Dictyostelium kinaseBiocatalysts from are not only environmentally( dNK) benign [11], a deoxyadenosinereagents and highly kinase selective, from but they are discoideum (DddAK) [12], or human deoxycytidine kinase (HsdCK) [17] has been developed. compatible with each other within certain ranges of operating conditions [19,20]. This feature enables Biocatalysts are not only environmentally benign reagents and highly selective, but they are integrationcompatible of sequential with each biocatalytic other within transformation certain rangess in of one-pot operating cascades conditions for the [19, 20synthesis]. This of feature complex moleculesenables [21]. integration The design of sequential of enzymatic biocatalytic networks transformations in one-pot systems, in one-pot as cascadesoccurring for in the living synthesis cells, has evidentof complex advantages molecules allowing [21]. Thethe designincrease of enzymaticof reaction networks rate, higher in one-pot yields systems, and asalso occurring overcoming in substrate/productliving cells, has inhibition evident advantages [22]. Moreover, allowing therunning increase ofartificial reaction cascades rate, higher using yields immobilized and also biocatalystsovercoming can substrateprovide/ producta spatial inhibition compartmentalizati [22]. 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