A Multi-Enzymatic Cascade Reaction for the Synthesis of Vidarabine 5′-Monophosphate (original) (raw)
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An Enzymatic Flow-Based Preparative Route to Vidarabine
Molecules
The bi-enzymatic synthesis of the antiviral drug vidarabine (arabinosyladenine, ara-A), catalyzed by uridine phosphorylase from Clostridium perfringens (CpUP) and a purine nucleoside phosphorylase from Aeromonas hydrophila (AhPNP), was re-designed under continuous-flow conditions. Glyoxyl–agarose and EziGTM1 (Opal) were used as immobilization carriers for carrying out this preparative biotransformation. Upon setting-up reaction parameters (substrate concentration and molar ratio, temperature, pressure, residence time), 1 g of vidarabine was obtained in 55% isolated yield and >99% purity by simply running the flow reactor for 1 week and then collecting (by filtration) the nucleoside precipitated out of the exiting flow. Taking into account the substrate specificity of CpUP and AhPNP, the results obtained pave the way to the use of the CpUP/AhPNP-based bioreactor for the preparation of other purine nucleosides.
Journal of Molecular Catalysis B: Enzymatic, 2015
Since the preparation of nucleoside 5-diphosphates by classical methodologies is complex, multistep enzymatic systems were explored to synthesize pyrimidine nucleoside 5-diphosphates starting from readily available reagents. Different strategies were combined to prepare uridine-and thymidine 5-diphosphates as ribo-and deoxyribonucleoside models, respectively. For uridine 5-diphosphate synthesis, conversions between 38 and 66% were achieved, using a simple methodology that involves commercial yeast extract as biocatalyst and biocatalytically in situ prepared uridine 5-monophosphate. Corynebacterium ammoniagenes ATCC 19350 was used for the first time as biocatalyst to synthesize uridine 5-monophosphate from uracil and orotic acid while Raoultella planticola was the selected biocatalyst for uridine 5-monophosphate synthesis from uridine. The overall performances of all the tested approaches were similar but the use of uracil leads to a more suitable and cheaper process. Alternatively, for thymidine 5-diphosphate synthesis two consecutive one pot multistep enzyme systems were assayed. In the first biotransformation, 2-deoxyribose 5-phosphate was formed from glucose by Erwinia carotovora whole cells followed by the action of phosphopentomutase and thymidine phosphorylase affording thymidine in 85% conversion relative to 2-deoxyribose 5-phosphate. Finally, in the second one pot reaction, the nucleoside was converted to thymidine 5-diphosphate by the combined action of Escherichia coli BL21 pET22b-phoRp and Saccharomyces cerevisiae.
Processes catalyzed by enzymes offer numerous advantages over chemical methods although in many occasions the stability of the biocatalysts becomes a serious concern. Traditionally, synthesis of nucleosides using poorly water-soluble purine bases, such as guanine, xanthine, or hypoxanthine, requires alkaline pH and/or high temperatures in order to solubilize the substrate. In this work, we demonstrate that the 2′-deoxyribosyltransferase from Leishmania mexicana (LmPDT) exhibits an unusually high activity and stability under alkaline conditions (pH 8–10) across a broad range of temperatures (30–70 °C) and ionic strengths (0–500 mM NaCl). Conversely, analysis of the crystal structure of LmPDT together with comparisons with hexameric, bacterial homologues revealed the importance of the relationships between the oligomeric state and the active site architecture within this family of enzymes. Moreover, molecular dynamics and docking approaches provided structural insights into the substrate-binding mode. Biochemical characterization of LmPDT identifies the enzyme as a type I NDT (PDT), exhibiting excellent activity, with specific activity values 100- and 4000-fold higher than the ones reported for other PDTs. Interestingly, LmPDT remained stable during 36 h at different pH values at 40 °C. In order to explore the potential of LmPDT as an industrial biocatalyst, enzymatic production of several natural and non-natural therapeutic nucleosides, such as vidarabine (ara A), didanosine (ddI), ddG, or 2′-fluoro-2′-deoxyguanosine, was carried out using poorly water-soluble purines. Noteworthy, this is the first time that the enzymatic synthesis of 2′-fluoro-2′-deoxyguanosine, ara G, and ara H by a 2′-deoxyribosyltransferase is reported.
Bioresource Technology, 2020
In this work, a mono-and a bi-enzymatic analytical immobilized enzyme reactors (IMERs) were developed as prototypes for biosynthetic purposes and their performances in the inflow synthesis of nucleoside analogues of pharmaceutical interest were evaluated. Two biocatalytic routes based on nucleoside 2′-deoxyribosyltransferase from Lactobacillus reuteri (LrNDT) and uridine phosphorylase from Clostridium perfrigens (CpUP)/purine nu-cleoside phosphorylase from Aeromonas hydrophila (AhPNP) were investigated in the synthesis of 2′-deoxy, 2′,3′-dideoxy and arabinonucleoside derivatives. LrNDT-IMER catalyzed the synthesis of 5-fluoro-2′-deoxyuridine and 5-iodo-2′-deoxyuridine in 65-59% conversion yield, while CpUP/AhPNP-IMER provided the best results for the preparation of arabinosyladenine (60% conversion yield).
2021
Herein we report the conversion of cytidine 2 to N-hydroxycytidine 7 catalysed by cytidine deaminase (CD). The wild-type enzyme operates efficiently at high sustrate loadings and hydroxylamine concentrations to favor N-hydroxy-cytidine formation over uridine. Although the wild-type enzyme demonstrated good activity, we were able to further enhance the ratio of N-hydroxycytidine to uridine produced through directed evolution of CD. In particular, a T123G mutation close to the active site dramatically reduces cytidine hydrolysis activity whilst preserving desired amination activty. The approach reported provides a new route to a key intermediate for the COVID-19 experimental drug Molnupiravir 1.
Tetrahedron Letters, 2010
Bis(dichlorophosphino)methane was converted to a β,γ-methylenetriphosphitylating reagent. The reagent was immobilized on aminomethyl polystyrene resin-bound linker of 4-acetoxy-3phenylbenzyl alcohol to afford a polymer-bound β,γ-methylenetriphosphitylating reagent, which was reacted with unprotected nucleosides followed by oxidation with tert-butyl hydroperoxide, deprotection of cyanoethoxy groups with DBU, and acidic cleavage, to produce 5′-O-β,γ-methylene triphosphate nucleosides in 53-82% overall yields. Among all the compounds, cytidine 5′-O-β,γmethylenetriphosphate inhibited completely RNase H activity of HIV-1 reverse transcriptase at 700 μM. Phosphate transfer is involved in several enzymatic catalyzed reactions 1-3 and therefore is a subject of considerable interest in biological systems. Triphosphate mimics, such as methylenetriphosphates, halogenated methylenetriphosphates, and imidotriphosphates, have been used to probe the mechanism of phosphoryl transfer in enzyme-catalyzed processes 2-4 and to target specific receptors or enzymes that bind or hydrolyze triphosphates. 5-8 Replacement of labile P-O-P bond in nucleoside triphosphates with a stable isosteric P-CH 2-P bond in nucleotide analogs results in enhanced metabolic stability. Synthesis of nonhydrolyzable triphosphate analogs of nucleosides is considered a challenge. A number of solution phase strategies have been previously reported for the synthesis of nucleoside 5′-O-β,γ-methylenetriphosphates including some of the compounds described here by the coupling reactions of nucleoside 5′-monophosphate salt forms or activated nucleoside monophosphates with diphosphonates (methylene diphosphonic acids). Some examples Correspondence to: Keykavous Parang. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Supplementary data: Supplementary data including experimental procedures and characterization of resins with IR and final compounds with NMR, high-resolution mass spectrometry, and quantitative phosphorus analysis, enzyme assay procedures can be found in the online version of this article.
BBA: Proteins and proteomics, 2020
Nowadays enzymatic synthesis of nucleic acid derivatives is gaining momentum over traditional chemical synthetic processes. Biotransformations catalyzed by whole cells or enzymes offer an ecofriendly and efficient alternative to the traditional multistep chemical methods, avoiding the use of chemical reagents and organic solvents that are expensive and environmentally harmful. Herein we report for the first time the covalent im-mobilization a uracil phosphoribosyltransferase (UPRT). In this sense, UPRT from Thermus thermophilus HB8 was immobilized onto glutaraldehyde-activated MagReSyn®Amine magnetic iron oxide porous microparticles (MTtUPRT). According to the catalyst load experiments, MTtUPRT3 was selected as optimal biocatalyst for further studies. MTtUPRT3 was active and stable in a broad range of temperature (70-100 °C) and in the pH interval 6-8, displaying maximum activity at 100 °C and pH 7 (activity 968 IU/g support , retained activity 100%). In addition, MTtUPRT3 could be reused up to 8 times in the synthesis of uridine-5′-monophosphate (UMP). Finally, MTtUPRT3 was successfully applied in the sustainable synthesis of different 5-modified uridine-5′-monophosphates at short times. Taking into account these results, MTtUPRT3 would emerge as a valuable biocatalyst for the synthesis of nucleoside monophosphates through an efficient and environmentally friendly methodology.