Enzymatic Synthesis of Therapeutic Nucleosides using a Highly Versatile Purine Nucleoside 2’‐DeoxyribosylTransferase from Trypanosoma brucei

Abstract

The use of enzymes for the synthesis of nucleoside analogues offers several advantages over multistep chemical methods, including chemo-, regio-and stereoselectivity as well as milder reaction conditions. Herein, the production, characterization and utilization of a purine nucleoside 2'-deoxyribosyltransferase (PDT) from Trypanosoma brucei are reported. TbPDT is a dimer which displays not only excellent activity and stability over a broad range of temperatures (50-70 8C), pH (4-7) and ionic strength (0-500 mM NaCl) but also an unusual high stability under alkaline conditions (pH 8-10). TbPDT is shown to be proficient in the biosynthesis of numerous therapeutic nucleosides, including didanosine, vidarabine, cladribine, fludarabine and nelarabine. The structure-guided replacement of Val11 with either Ala or Ser resulted in variants with 2.8-fold greater activity. TbPDT was also covalently immobilized on glutaraldehyde-activated magnetic microspheres. MTbPDT3 was selected as the best derivative (4200 IU/g, activity recovery of 22 %), and could be easily recaptured and recycled for > 25 reactions with negligible loss of activity. Finally, MTbPDT3 was successfully employed in the expedient synthesis of several nucleoside analogues. Taken together, our results support the notion that TbPDT has good potential as an industrial biocatalyst for the synthesis of a wide range of therapeutic nucleosides through an efficient and environmentally friendly methodology.[a] Dr.

N-Ribosyltransferase From Archaeoglobus veneficus: A Novel Halotolerant and Thermostable Biocatalyst for the Synthesis of Purine Ribonucleoside Analogs

“…The heteroaromatic ring of the nucleobase would thus get sandwiched between the phenyl rings of Phe12 and Phe13, on one side, and the hydrophobic sidechains of Phe56 and Leu119# on the opposite side. The Met120# sidechain sulfur appears close to the H1' atom, which suggests that it likely plays a role, together with the carboxylate of Asp62, in stabilization of the oxocarbenium reaction intermediate, as proposed for other NDTs (Pérez et al, 2018;Del Arco et al, 2019a,b).…”

“…NDTs catalyze the interchange of the 2 ′ -deoxyribose moiety between a purine and/or pyrimidine 2 ′ -deoxynucleoside (donor) and a purine and/or pyrimidine base (acceptor) (Kaminski, 2002;Fernández-Lucas et al, 2010;Fresco-Taboada et al, 2013;Crespo et al, 2017;Del Arco et al, 2019a). According to their substrate specificity, NDTs are usually classified as: (i) type I NDTs (PDT), which perform this type of transglycosylation reaction between purine bases (Fresco-Taboada et al, 2013;Pérez et al, 2018) and (ii) type II NDTs (NDT), which do not discriminate between purines and pyrimidines (Kaminski, 2002;Fernández-Lucas et al, 2010;Fresco-Taboada et al, 2016). Interestingly, NDTs are highly regioselective for natural bases and a high number of nucleobase derivatives (Fresco-Taboada et al, 2013;Acosta et al, 2018).…”

8-Fluoro-N-2-isobutyryl-2′-deoxyguanosine: Synthesis and Reactivity

“…Syntheses of adenine nucleosides, such as 2-fluoro-2 -deoxyadenosine 2a [4][5][6][7] and 2-fluoroadenosine 2b [6,8] have been demonstrated through different approaches. Introduction of fluorine to the C-8 position of purine nucleosides is more challenging, likely due to the instability of the resulting nucleosides.…”

An Expedient Synthesis of Flexible Nucleosides through Enzymatic Glycosylation of Proximal and Distal Fleximer Bases

“…[5] Nucleoside phosphorylases (NPs) and N-deoxyribosyl-transferases (NDTs) are the predominant classes of enzymes used in the synthesis of nucleosides in which they mediate the transglycosylation of an ucleoside sugar to af ree heterocyclic base. [6][7][8][9][10] NPs (EC 2.4.2.1) catalyze the reversible phosphorolysis of ribo-or 2'-deoxyribonucleosides to generate af ree nucleobase and ribose-or 2-deoxyribose-1-phosphatei nt he presence of inorganic ortho-phosphate. Addition of as econd nucleobase to the reaction mixture can promote the formation of an ew nucleoside with the equilibrium in favor of nucleoside formation.…”

“…[11,12] NPs have been reportedt oa ccept modified bases as well as unnatural glycosyldonors. [6][7][8][9][13][14][15] NDTs( EC 2.4.2.6) catalyzet he transfer of 2-deoxyriboseb etween purine and pyrimidine bases. [16] TwoN DT types have been defined based on their substrate specificity:N DT-I (also named PTD), which is specific for purines, [17] and NDT-II, which accepts either purine or pyrimidine but has as trong preference for 2'-deoxyribosyl-pyrimidinea st he donor substrate.…”