Immobilization of thermostable nucleoside phosphorylases on MagReSyn® epoxide microspheres and their application for the synthesis of 2,6-dihalogenated purine nucleosides

Zhou, Xinrui; Mikhailopulo, Igor A.; Bournazou, Mariano Nicolás Cruz; Neubauer, Peter

doi:10.1016/j.molcatb.2015.02.009

Cited by 31 publications

(20 citation statements)

References 33 publications

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“…The detailed immobilisation process and the evaluation are presented elsewhere. [26] Results and Discussion Scrutiny of the published methods for the chemo-enzymatic synthesis of base-and pentofuranose-modified purine nucleosides prompted us to formulate a new versatile approach to the synthesis of this class of biologically important compounds. Our approach consists in the enzymatic synthesis of 2,6-dihalogenated purine nucleosides, in particular 2,6-dichloropurine (26DCP) and 6-chloro-2-fluoropurine (6C2FP), as valuable scaffolds for the enzymatic and/or chemical preparation of a broad palette of modified nucleosides.…”

Section: Introductionmentioning

confidence: 99%

Synthesis of 2,6‐Dihalogenated Purine Nucleosides by Thermostable Nucleoside Phosphorylases

et al. 2015

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The enzymatic transglycosylation of 2,6-dichloropurine (26DCP) and 6-chloro-2-fluoropurine (6C2FP) with uridine, thymidine and 1-(b-d-arabinofuranosyl)-uracil as the pentofuranose donors and recombinant thermostable nucleoside phosphorylases from G. thermoglucosidasius or T. thermophilus as biocatalysts was studied. Selection of 26DCP and 6C2FP as substrates is determined by their higher solubility in aqueous buffer solutions compared to most natural and modified purines and, furthermore, synthesized nucleosides are valuable precursors for the preparation of a large number of biologically important nucleosides. The substrate activity of 26DCP and 6C2FP in the synthesis of their ribo-and 2'-deoxyA C H T U N G T R E N N U N G ribo-nucleosides was closely similar to that of related 2-amino-(DAP), 2-chloro-and 2-fluoroadenines; the efficiency of the synthesis of b-d-arabinofuranosides of 26DCP and 6C2FP was lower vs. that of DAP under similar reaction conditions. For a convenient and easier recovery of the biocatalysts, the thermostable enzymes were immobilized on MagReSyn epoxide beads and the biocatalyst showed high catalytic efficiency in a number of reactions. As an example, 6-chloro-2-fluoro-(b-d-ribofuranosyl)-purine (9), a precursor of various antiviral and antitumour drugs, was synthesized by the immobilized enzymes at 60 8C under high substrate concentrations (uriA C H T U N G T R E N N U N G dine:purine ratio of 2:1, mol). The synthesis was successfully scaled-up [uridine (2.5 mmol), base (1.25 mmol); reaction mixture 50 mL] to afford 9 in 60% yield. The reaction reveals the great practical potential of this enzymatic method for the efficient production of modified purine nucleosides of pharmaceutical interest.

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Section: Introductionmentioning

confidence: 99%

Synthesis of 2,6‐Dihalogenated Purine Nucleosides by Thermostable Nucleoside Phosphorylases

et al. 2015

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“…Small-and semi-preparative-scale experiments employing the improved conditions confirmed the calculated predictions. Hence, under optimized conditions, higher product yields were observed compared to previous studies [20,21].…”

Section: Discussionmentioning

confidence: 55%

“…A sugar donor-to-nucleobase ratio of 3.34 in the presence of 3.34 equivalents of phosphate (compared to the base concentration) was used. Using an immobilized PyNP of Thermus thermophilus and the same PNP, Zhou and colleagues later observed increasing product formation with reduced phosphate concentrations [21]. A negative impact on the product yield of phosphate equivalents above 0.3 in comparison to the nucleobase was recently confirmed by thermodynamic studies on transglycosylation reactions [13,22].…”

Section: Discussionmentioning

confidence: 93%

Efficient Biocatalytic Synthesis of Dihalogenated Purine Nucleoside Analogues Applying Thermodynamic Calculations

et al. 2020

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The enzymatic synthesis of nucleoside analogues has been shown to be a sustainable and efficient alternative to chemical synthesis routes. In this study, dihalogenated nucleoside analogues were produced by thermostable nucleoside phosphorylases in transglycosylation reactions using uridine or thymidine as sugar donors. Prior to the enzymatic process, ideal maximum product yields were calculated after the determination of equilibrium constants through monitoring the equilibrium conversion in analytical-scale reactions. Equilibrium constants for dihalogenated nucleosides were comparable to known purine nucleosides, ranging between 0.071 and 0.081. To achieve 90% product yield in the enzymatic process, an approximately five-fold excess of sugar donor was needed. Nucleoside analogues were purified by semi-preparative HPLC, and yields of purified product were approximately 50% for all target compounds. To evaluate the impact of halogen atoms in positions 2 and 6 on the antiproliferative activity in leukemic cell lines, the cytotoxic potential of dihalogenated nucleoside analogues was studied in the leukemic cell line HL-60. Interestingly, the inhibition of HL-60 cells with dihalogenated nucleoside analogues was substantially lower than with monohalogenated cladribine, which is known to show high antiproliferative activity. Taken together, we demonstrate that thermodynamic calculations and small-scale experiments can be used to produce nucleoside analogues with high yields and purity on larger scales. The procedure can be used for the generation of new libraries of nucleoside analogues for screening experiments or to replace the chemical synthesis routes of marketed nucleoside drugs by enzymatic processes.

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“…Recently, Rivero et al reported successful immobilization of E. coli ATCC 12407 in agarose matrix [16]. Moreover, immobilization on fused silica Open Tubular Capillary [22], aldehyde-agarose [23], and MagReSyn epoxide microspheres [24,25], in calcium alginate, agar, and kcarrageenan matrix [18], was described in the literature. Thus, we believe, that the immobilisation of our enzyme is possible.…”

Section: International Journal Of Chemical Engineeringmentioning

confidence: 99%

An Efficient Chemoenzymatic Process for Preparation of Ribavirin

Сахаров

Baykov

Konstantinova

et al. 2015

International Journal of Chemical Engineering

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Ribavirin is an important antiviral drug, which is used for treatment of many diseases. The pilot-scale chemoenzymatic process for synthesis of the active pharmaceutical ingredient Ribavirin was developed with 32% overall yield and more than 99.5% purity. The described method includes the chemical synthesis of 1,2,4-triazole-3-carboxamide, which is a key intermediate and enzyme-catalyzed transglycosylation reaction for preparation of the desired product. 1,2,4-Triazole-3-carboxamide was synthesized from 5-amino-1,2,4-triazole-3-carboxylic acid by classical Chipen-Grinshtein method. Isolated fromE. сoliBL21(DE3)/pERPUPHHO1 strain the purine nucleoside phosphorylase was used as a biocatalytical system. All steps of this process were optimized and scaled.

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Immobilization of thermostable nucleoside phosphorylases on MagReSyn® epoxide microspheres and their application for the synthesis of 2,6-dihalogenated purine nucleosides

Cited by 31 publications

References 33 publications

Synthesis of 2,6‐Dihalogenated Purine Nucleosides by Thermostable Nucleoside Phosphorylases

Synthesis of 2,6‐Dihalogenated Purine Nucleosides by Thermostable Nucleoside Phosphorylases

Efficient Biocatalytic Synthesis of Dihalogenated Purine Nucleoside Analogues Applying Thermodynamic Calculations

An Efficient Chemoenzymatic Process for Preparation of Ribavirin

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