Ilja V. Fateev

The chemoenzymatic synthesis of clofarabine and related 2′-deoxyfluoroarabinosyl nucleosides: the electronic and stereochemical factors determining substrate recognition by E. coli nucleoside phosphorylases

Summary Two approaches to the synthesis of 2-chloro-9-(2-deoxy-2-fluoro-β-D-arabinofuranosyl)adenine (1, clofarabine) were studied. The first approach consists in the chemical synthesis of 2-deoxy-2-fluoro-α-D-arabinofuranose-1-phosphate (12a, 2FAra-1P) via three step conversion of 1,3,5-tri-O-benzoyl-2-deoxy-2-fluoro-α-D-arabinofuranose (9) into the phosphate 12a without isolation of intermediary products. Condensation of 12a with 2-chloroadenine catalyzed by the recombinant E. coli purine nucleoside phosphorylase (PNP) resulted in the formation of clofarabine in 67% yield. The reaction was also studied with a number of purine bases (2-aminoadenine and hypoxanthine), their analogues (5-aza-7-deazaguanine and 8-aza-7-deazahypoxanthine) and thymine. The results were compared with those of a similar reaction with α-D-arabinofuranose-1-phosphate (13a, Ara-1P). Differences of the reactivity of various substrates were analyzed by ab initio calculations in terms of the electronic structure (natural purines vs analogues) and stereochemical features (2FAra-1P vs Ara-1P) of the studied compounds to determine the substrate recognition by E. coli nucleoside phosphorylases. The second approach starts with the cascade one-pot enzymatic transformation of 2-deoxy-2-fluoro-D-arabinose into the phosphate 12a, followed by its condensation with 2-chloroadenine thereby affording clofarabine in ca. 48% yield in 24 h. The following recombinant E. coli enzymes catalyze the sequential conversion of 2-deoxy-2-fluoro-D-arabinose into the phosphate 12a: ribokinase (2-deoxy-2-fluoro-D-arabinofuranose-5-phosphate), phosphopentomutase (PPN; no 1,6-diphosphates of D-hexoses as co-factors required) (12a), and finally PNP. The substrate activities of D-arabinose, D-ribose and D-xylose in the similar cascade syntheses of the relevant 2-chloroadenine nucleosides were studied and compared with the activities of 2-deoxy-2-fluoro-D-arabinose. As expected, D-ribose exhibited the best substrate activity [90% yield of 2-chloroadenosine (8) in 30 min], D-arabinose reached an equilibrium at a concentration of ca. 1:1 of a starting base and the formed 2-chloro-9-(β-D-arabinofuranosyl)adenine (6) in 45 min, the formation of 2-chloro-9-(β-D-xylofuranosyl)adenine (7) proceeded very slowly attaining ca. 8% yield in 48 h.

A New Strategy for the Synthesis of Nucleosides: One-Pot Enzymatic Transformation of D-Pentoses into Nucleosides

A possibility of the one-pot synthesis of purine and pyrimidine nucleosides employing pure recombinant ribokinase, phosphopentomutase and nucleoside phosphorylases in a caskade transformation of D-pentoses into nucleosides is demonstrated. Preliminary results of this study point to reliability to develop practical methods for the preparation of a number of biologically important nucleosides.

Chemoenzymatic method of 1,2,4-triazole nucleoside synthesis: Possibilities and limitations

Possibilities and limitations of chemoenzymatic synthesis of novel structural analogues of an antiviral preparation of Ribavirin (1-β-D-ribofuranosyl-1,2,4-triazole-3-carboxamide) were established. A synthesis of various amides of 1H-1,2,4-triazole-3-carboxylic acid and its 5-substituted analogues—potential substrates of purine nucleoside phosphorylase—has been described. Comparative efficiency of preparation methods of these amides, as well as the methods of introduction of functional groups to the C5 position of heterocyclic system, were investigated. Novel analogues of Ribavirin containing various substitutes in the carboxamide group were synthesized. A biotechnological method was developed for the preparation of 1-β-D-ribofuranozyl-1,2,4-triazole-3-carbonitryl, an intermediate in the synthesis of Viramidine, the modern analogue of Ribavirin.

The arsenolysis reaction in the biotechnological synthesis of ribavirin. The in vitro and in vivo inhibition of influenza A virus replication with a combination of ribavirin and ozeltamivir

The biotechnological method of synthesis of the antiviral drug ribavirin based on the transglycosylation reaction was improved due to the addition of catalytic amounts of sodium arsenate. This approach allows us to hydrolyze the excess natural nucleoside guanosine, a ribose donor, and, hence, made the composition of the reaction mixture less complicated, thus facilitating the process of ribavirin isolation. It was shown that in cell cultures the combination of ribavirin and oseltamivir carboxylate inhibited the replication of the influenza A virus more effectively than each of them alone. Similar results were obtained in experiments on laboratory animals (mouse Balb/c) infected with the influenza A virus H3N2/Aichi/68 strain.

A New Strategy for the Synthesis of Nucleosides: One-Pot Enzymatic Transformation of D-Pentoses into Nucleosides~!2010-01-23~!2010-07-23~!2010-08-23~!

A possibility of the one-pot synthesis of purine and pyrimidine nucleosides employing pure recombinant ribokinase, phosphopentomutase and nucleoside phosphorylases in a caskade transformation of D-pentoses into nucleosides is demonstrated. Preliminary results of this study point to reliability to develop practical methods for the preparation of a number of biologically important nucleosides.