title: The chemoenzymatic synthesis of clofarabine and related 2'-deoxyfluoroarabinosyl nucleosides: the electronic and stereochemical factors determining substrate recognition by E. coli nucleoside phosphorylases.
abstract: 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, (2F)Ara-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 ((2F)Ara-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.
authors.affiliation: Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya 16/10, 117997 GSP, Moscow B-437, Russia.
authors.forename: Ilja V
authors.initials: IV
authors.lastname: Fateev
authors.affiliation: Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya 16/10, 117997 GSP, Moscow B-437, Russia.
authors.forename: Konstantin V
authors.initials: KV
authors.lastname: Antonov
authors.affiliation: Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya 16/10, 117997 GSP, Moscow B-437, Russia.
authors.forename: Irina D
authors.initials: ID
authors.lastname: Konstantinova
authors.affiliation: Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya 16/10, 117997 GSP, Moscow B-437, Russia.
authors.forename: Tatyana I
authors.initials: TI
authors.lastname: Muravyova
authors.affiliation: Laboratory of Bioorganic Chemistry and Chemical Biology, Center for Nanotechnology, Heisenbergstraße 11, D-48149 Münster, Germany.
authors.forename: Frank
authors.initials: F
authors.lastname: Seela
authors.affiliation: Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya 16/10, 117997 GSP, Moscow B-437, Russia.
authors.forename: Roman S
authors.initials: RS
authors.lastname: Esipov
authors.affiliation: Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya 16/10, 117997 GSP, Moscow B-437, Russia.
authors.forename: Anatoly I
authors.initials: AI
authors.lastname: Miroshnikov
authors.affiliation: Institute of Bioorganic Chemistry, National Academy of Sciences, Acad. Kuprevicha 5/2, 220141 Minsk, Belarus.
authors.forename: Igor A
authors.initials: IA
authors.lastname: Mikhailopulo
keywords: chemoenzymatic synthesis; clofarabine; nucleoside phosphorylases; phosphopentomutase; recombinant E. coli ribokinase