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.

Keywords: chemoenzymatic synthesis; clofarabine; nucleoside phosphorylases; phosphopentomutase; recombinant E. coli ribokinase.

Figure 1

The structures of purine nucleosides studied in the chemoenzymatic synthesis and in a cascade one-pot transformation of D-pentoses into nucleosides of 2-chloroadenine catalyzed by the recombinant E. coli ribokinase (RK), phosphopentomutase (PPM) and purine nucleoside phosphorylase (PNP) (purine numbering was used throughout of the manuscript).

Scheme 1

Chemical synthesis of 2-deoxy-2-fluoro-α/β-D-arabinofuranose-1-phosphates (12a,b). Reagents and conditions: (a) 9/AcBr/H₃PO₄, 50 °C, 5 h; (b) intermediate 10/dioxane/n-Bu₃N, rt, 12–18 h; (c) intermediate 11/water/LiOH, rt, 1 h.

Figure 2

The structures of 1-phosphates of α-D-arabinofuranose (13a; Ara^Fur-1P) and β-D-arabinopyranose (13b; Ara^Pyr-1P).

Figure 3

Geometry optimization of 1-phosphates of 2-deoxy-2-fluoro-α-D-arabinofuranose (12a) and the β-anomer 12b [dilithium salts; HyperChem 8.1; AMBER Force Field starting approximation, then the ab initio calculations (3-21G/total charge equal to zero; Polak–Ribiere conjugate gradient)].

Figure 4

Progress of the formation of 9-(2-deoxy-2-fluoro-β-D-arabinofuranosyl)-2-chloroadenine (1), 2-amino-9-(2-deoxy-2-fluoro-β-D-arabinofuranosyl)adenine (2a) and 9-(2-deoxy-2-fluoro-β-D-arabinofuranosyl)hypoxanthine (3a) (Reaction conditions are shown in Table 1).

Figure 5

Clofarabine content in the reaction mixture vs time (hours) of the reaction.

Scheme 2

Suggested mechanism of purine nucleoside synthesis catalyzed by E. coli purine nucleoside phosphorylase.

Figure 6

Progress of the formation of β-D-arabinofuranosides and 2-deoxy-2-fluoro-β-D-arabinofuranosides of 5-aza-7-deazaguanine 4b and 4a, and 8-aza-7-deazahypoxanthine 5b and 5a. Reaction conditions: reactions (1 mL) were performed in the presence of 2 μmol heterocyclic base and 4 μmol phosphate 12a (as a mixture of two anomers, so that the real quantity of the substrate 12a is half of the indicated quantity, i.e., 2 μmol) or 10 μmol arabinose-1-phosphate (real quantity of α-D-arabinofuranose-1-phosphate is 2 μmol) in water at 50 °C for 24 h with addition of 3.9 units PNP; the following preparations of E. coli enzymes have been employed: PNP (52 units/mg; 15 mg/mL).

Figure 7

Tautomeric structures of 5-aza-7-deazaguanine (17).

Figure 8

Progress of the formation of clofarabine (1), 9-(β-D-arabinofuranosyl)-2-chloroadenine (6), 9-(β-D-xylofuranosyl)-2-chloroadenine (7) and 2-cloroadenosine (8).