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A series of ribo‐ and deoxyribonucleosides bearing 2‐aminopurine as a nucleobase with 7,8‐difluoro‐ 3,4‐dihydro‐3‐methyl‐2H‐[1,4]benzoxazine (conjugated directly or through an aminohexanoyl spacer) was synthesized using an enzymatic transglycosylation reaction. Nucleosides 3‐6 were resistant to deamination under action of adenosine deaminase (ADA) Escherichia coli and ADA from calf intestine. The antiviral activity of the modified nucleosides was evaluated against herpes simplex virus type 1 (HSV‐1, strain L2). It has been shown that at sub‐toxic concentrations, nucleoside (S)‐4‐[2‐amino‐9‐(β‐D‐ribofuranosyl)‐purin‐6‐yl]‐7,8‐difluoro‐3,4‐dihydro‐3‐methyl‐2H‐[1,4]benzoxazine exhibit significant antiviral activity (SI > 32) on the model of HSV‐1 in vitro, including an acyclovir‐resistant virus strain (HSV‐1, strain L2/R).

A comparative study of the possibilities of using ribokinase → phosphopentomutase → nucleoside phosphorylase cascades in the synthesis of modified nucleosides was carried out. Recombinant phosphopentomutase from Thermus thermophilus HB27 was obtained for the first time: a strain producing a soluble form of the enzyme was created, and a method for its isolation and chromatographic purification was developed. It was shown that cascade syntheses of modified nucleosides can be carried out both by the mesophilic and thermophilic routes from D-pentoses: ribose, 2-deoxyribose, arabinose, xylose, and 2-deoxy-2-fluoroarabinose. The efficiency of 2-chloradenine nucleoside synthesis decreases in the following order: Rib (92), dRib (74), Ara (66), F-Ara (8), and Xyl (2%) in 30 min for mesophilic enzymes. For thermophilic enzymes: Rib (76), dRib (62), Ara (32), F-Ara (<1), and Xyl (2%) in 30 min. Upon incubation of the reaction mixtures for a day, the amounts of 2-chloroadenine riboside (thermophilic cascade), 2-deoxyribosides (both cascades), and arabinoside (mesophilic cascade) decreased roughly by half. The conversion of the base to 2-fluoroarabinosides and xylosides continued to increase in both cases and reached 20-40%. Four nucleosides were quantitatively produced by a cascade of enzymes from D-ribose and D-arabinose. The ribosides of 8-azaguanine (thermophilic cascade) and allopurinol (mesophilic cascade) were synthesized. For the first time, D-arabinosides of 2-chloro-6-methoxypurine and 2-fluoro-6-methoxypurine were synthesized using the mesophilic cascade. Despite the relatively small difference in temperatures when performing the cascade reactions (50 and 80 °C), the rate of product formation in the reactions with Escherichia coli enzymes was significantly higher. E. coli enzymes also provided a higher content of the target products in the reaction mixture. Therefore, they are more appropriate for use in the polyenzymatic synthesis of modified nucleosides.

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