DOI: 10.1021/jo101368z Published on Web 09/09/2010 J. Org. Chem. 2010, 75, 6605–6613 6605 r 2010 American Chemical Society pubs.acs.org/joc Enzymatic Parallel Kinetic Resolution of Mixtures of D/L 2 0 -Deoxy and Ribonucleosides: An Approach for the Isolation of β-L-Nucleosides † Sa ul Martı´nez-Montero, ‡ Susana Fern andez, ‡ Yogesh S. Sanghvi, § Vicente Gotor,* ,‡ and Miguel Ferrero* ,‡ ‡ Departamento de Quı´mica Org  anica e Inorg  anica and Instituto Universitario de Biotecnologı´a de Asturias, Universidad de Oviedo, 33006-Oviedo (Asturias), Spain, and § Rasayan Inc., 2802 Crystal Ridge Road, Encinitas, California 92024-6615 MFerrero@uniovi.es; VGS@uniovi.es Received July 12, 2010 We have developed a lipase-catalyzed parallel kinetic resolution of mixtures of β-D/L-nucleosides. The opposite selectivity during acylation exhibited by Pseudomonas cepacia lipase (PSL-C) with β-D- and β-L-nucleosides furnished acylated compounds that have different R f values. As a consequence, isolation of both products was achieved by simple column chromatography. Computer modeling of the transition-state analogues during acylation of β-D- and β-L-2 0 -deoxycytidine with PSL-C was carried out to explain the high selectivity. PSL-C favored the 3 0 -O-levulination of the β-D enantiomer, whereas the 5 0 -OH group was acylated in 2 0 -deoxy-β-L-cytidine. In both cases, the cytosine base was placed in the alternate hydrophobic pocket of PSL’s substrate-binding site, where it can form extra hydrogen bonds (in addition to the five essential catalytically relevant hydrogen bonds) that stabilize these intermediates catalyzing the selective acylation of β-D/L-nucleosides. Introduction In recent years, unnatural β-L-nucleoside analogues have received much attention as a new class of antiviral and anticancer agents. 1 Since the discovery of lamivudine 2 (L-2 0 ,3 0 -dideoxy-3 0 - thiacytidine, 3TC; 1, Chart 1) for the treatment of human immunodeficiency virus (HIV) and hepatitis B virus (HBV) infections, various L-nucleoside derivatives have been synthe- sized and evaluated for their potential therapeutic applications. Recently, telbivudine (LdT; 4), the L-isomer of thymidine, was approved for the treatment of HBV infection. Clinical trials have shown LdT to be significantly more effective than lamivudine or adefovir and less likely to cause resistance. 3 Another anti-HBV L-nucleoside is valtorcitabine 4 (val-LdC; 5), the 3 0 -O-valinyl ester of L-deoxycytidine, which has been developed as a fixed dose combination with LdT. In clinical trials, the combination of the two agents had demonstrated greater antiviral activity than either drug alone. One promising candidate for the treatment of chronic HBV is β-L-2 0 ,3 0 - dideoxy-3 0 -thia-5-fluorocytidine (emtricitabine, FTC; 2), the 5-fluorinated analogue of 3TC, available in combination with other antiretroviral agents as an anti-HIV therapeutic agent. This drug is currently being evaluated as a potential treatment for chronic HBV. 5 † In memoriam to Professor Jos e Manuel Concell on. (1) See a mini-review: Math e, C.; Gosselin, G. Antiviral Res. 2006, 71, 276–281. (2) (a) Jarvis, B.; Faulds, D. Drugs 1999, 58, 101–141. (b) Cameron, J. M.; Collins, P.; Daniel, M.; Storer, R. Drugs Future 1993, 18, 319–323. (c) Chang, C.-N.; Doong, S.-L.; Zhou, J. H.; Beach, J. W.; Jeong, L. S.; Chu, C. K.; Tsai, C.-H.; Cheng, Y.-C. J. Biol. Chem. 1992, 267, 13938–13942. (d) Doong, S. L.; Tsai, C.-H.; Schinazi, R. F.; Liotta, D. C.; Cheng, Y.-C. Proc. Natl. Acad. Sci. U. S. A. 1991, 88, 8495–8499. (3) Charuworn, P.; Keeffe, E. B. Clin. Med. Ther. 2009, 1, 157–166. (4) (a) Gosselin, G.; Pierra, C.; Benzaria, S.; Dukhan, D.; Imbach, J.-L.; Loi, A. G.; La Colla, P.; Cretton-Scott, E.; Bridges, E. G.; Standring, D. N.; Sommadossi, J. P. β-L-2 0 -Deoxythymidine (L-dT) and β-L-2 0 -Deoxycytidine (LdC): How Simple Structures can be Potent, Selective and Specific Anti- HBV Drugs. In Frontiers in Nucleosides and Nucleic Acids; Schinazi, R. F., Liotta, D. C., Eds.; IHL Press: Tucker, GA, 2004; pp 309-317. (b) Licensed by Novartis. It has been discontinued in 2008 for reasons that were not disclosed: Ryder, N. S. Expert Opin. Invest. Drugs 2010, 19, 1–21. (5) (a) Furman, P. A.; Davis, M.; Liotta, D. C.; Paff, M.; Frick, L. W.; Nelson, D. J.; Dornsife, R. E.; Wurster, J. A.; Wilson, L. J.; Fyfe, J. A.; Tuttle, J. V.; Miller, W. H.; Condreay, L.; Averett, D. R.; Schinazi, R. F.; Painter, G. R. Antimicrob. Agents Chemother. 1992, 36, 2686–2692. (b) Schinazi, R. F.; McMillan, A.; Cannon, D.; Mathis, R.; Lloyd, R. M.; Peck, A.; Sommadossi, J.-P.; Clair, M. St.; Wilson, J.; Furman, P. A.; Painter, G. R.; Choi, W. B.; Liotta, D. C. Antimicrob. Agents Chemother. 1992, 36, 2423–2431. (c) Jenh, A. M.; Thio, C. L.; Pham, P. A. Pharmacotherapy 2009, 29, 1212–1227. (d) Saag, M. S. Clin. Infect. Dis. 2006, 42, 126–131.