Full Papers Novel Chemoenzymatic Protocol for the Synthesis of 3′-O-Dimethoxytrityl-2′-deoxynucleoside Derivatives as Building Blocks for Oligonucleotide Synthesis Alba Dı ´az-Rodrı ´guez, † Susana Ferna ´ndez, † Yogesh S. Sanghvi, ‡ Miguel Ferrero, † and Vicente Gotor* ,† Departamento de Quı ´mica Orga ´ nica e Inorga ´ nica and Instituto de Biotecnologı ´a de Asturias, UniVersidad de OViedo, 33071-OViedo (Asturias), Spain, and Rasayan Inc., 2802 Crystal Ridge Road, Encinitas, California 92024-6615, U.S.A. Abstract: An easy, efficient, and scalable chemoenzymatic strategy for the synthesis of 3′-O-dimethoxytrityl-2′-deoxynucleosides has been developed. A key feature of this approach is the regiose- lective synthesis of 5′-O-levulinyl-2′-deoxynucleosides through enzymatic acylation in the presence of Candida antarctica lipase B. In addition, it was observed that the deblocking of levulinyl group from the 5′-position is perfectly compatible with con- ventional base protecting groups. To demonstrate the scalability of this method, 3′-O-dimethoxytritylthymidine (4a) was syn- thesized on 25-g scale. These monomers (4a-d) are useful building blocks for the synthesis of oligonucleotides. Introduction Synthetic oligonucleotides are an emerging class of chemotherapeutic agents with tremendous potential for treatment of a wide range of cardiovascular, inflammatory, metabolic and infectious diseases, and a variety of cancers. 1 Vitravene and Macugen are two shining examples of FDA approved oligonucleotide drugs along with 40 others that are advancing through human clinical trials at a fast pace. As a result, a number of pharmaceutical companies are actively engaged in the discovery and development of oligonucleotide drugs. 2 Assuming successful human clinical trials with these products and the possibility of their commercial launch, it is anticipated that soon very large quantities of therapeutically useful oligonucleotides may be required. During the past decade enormous efforts have been made in the development of synthetic methodologies for oligonucleotides, particularly for their large-scale synthesis. 3,4 As modified oligonucleotides have become a major field of investigation for chemists, methods for their suitable protection/deprotection for the synthesis of nucleoside mono- mers have become equally important. Selective protection of a multifunctional compound is a challenging problem in organic synthesis. 5 Among the plethora of synthetic tools available to chemists, application of biocatalysts in organic chemistry has become one of the most attractive alternatives to the conventional chemical methods for a variety of reasons. 6 For example, enzymes are environmentally accept- able, work under mild conditions, are compatible in organic solvents, and demonstrate high chemo- and regio-selectivity during chemical transformations with recycling possibilities. In nucleoside chemistry, selective manipulation of the hydroxyl groups of carbohydrate moiety over amino groups of the bases is synthetically challenging and requires a multistep protocol. 7 Recently, we reported the use of enzymes for efficient synthesis of 3′- and 5′-O-levulinyl nucleosides avoiding several tedious chemical protection/deprotection steps. 8 We further demonstrated that enzymes are capable * To whom correspondence should be addressed. E-mail: VGS@fq.uniovi.es. † Universidad de Oviedo. ‡ Rasayan Inc. (1) (a) Crooke, S. T., Ed. Antisense Drug Technology Principles, Stategies and Applications; Marcel Dekker: New York, 2001. (b) Sanghvi, Y. S. In ComprehensiVe Natural Products Chemistry; Kool, E. T., Ed.; Elsevier: Amsterdam, 1999; Vol. 7, Chapter 7, pp 285-311 and references therein. (2) ISIS Pharmaceuticals (www.isispharm.com) has the largest portfolio of antisense oligonucleotide products currently in clinical trials. (3) (a) For a recent review of oligo- and poly-nucleotide synthesis, see: Reese, C. B. Org. Biomol. Chem. 2005, 3, 3851-3868. (b) Sanghvi, Y. S.; Andrade, M.; Deshmukh, R. R.; Holmberg, L.; Scozzari, A. N.; Cole, D. L. In Manual of Antisense Methodology; Hartmann, G.; Endres, S., Eds.; Kluwer Academic Publishers: Hingham, 1999; pp 3-23. (4) (a) Gukathasan, R.; Massoudipour, M.; Gupta, I.; Chowdhury, A.; Pulst, S.; Sivakumar, R.; Sanghvi, Y. S.; Laneman, S. J. Organomet. Chem. 2005, 690, 2603-2607. (b) Wang, Z.; Cedillo, I.; Cole, D. L.; Sanghvi, Y. S.; Hinz, M.; Prukala, W.; Sobkowski, M.; Seliger, H.; Rimmler, M.; Ditz, R.; Hoffmeyer, J. In InnoVation and PerspectiVes in Solid-Phase Synthesis & Combinatorial Libraries; Epton, R., Ed.; Mayflower Worldwide Ltd.: England, 2004; pp 118-122. (c) Sanghvi, Y. S.; Guo, Z.; Pfundheller, H. M.; Converso, A. Org. Process Res. Dev. 2000, 4, 175-181. (d) Pon, R. T.; Yu, S.; Guo, Z.; Sanghvi, Y. S. Nucleic Acid Res. 1999, 27, 1531- 1538. (5) Greene, T. W.; Wuts, P. G. M. ProtectiVe Groups in Organic Synthesis; 3rd ed.; Wiley: New York, 1999. (6) (a) Klibanov, A. M. Nature 2001, 409, 241-246. (b) Koeller, K. M.; Wong, C.-H. Nature 2001, 409, 232-240. (c) Patel, R. N. StereoselectiVe Biocatalysis; Marcel Dekker: New York, 2000. (d) Carrea, G.; Riva, S. Angew. Chem. Int. Ed. 2000, 39, 2226-2254. (e) Bornscheuer, U. T.; Kazlauskas, R. J. Hydrolases in Organic Synthesis. Regio- and Stereose- lectiVe Biotransformations; Wiley-VCH: Weinheim, 1999. (7) For reviews on enzymatic transformations in nucleosides, see: (a) Ferrero, M.; Gotor, V. Chem. ReV. 2000, 100, 4319-4347. (b) Ferrero, M.; Gotor, V. Monatsh. Chem. 2000, 131, 585-616. (8) (a) Lavandera, I.; Garcı ´a, J.; Ferna ´ndez, S.; Ferrero, M.; Gotor, V.; Sanghvi, Y. S. In Current Protocols in Nucleic Acid Chemistry; Beaucage, S. L., Bergstrom, D. E., Glick, G. D., Jones, R. A., Eds.; John Wiley and Sons: New York, 2005; Chapter 2.11. (b) Garcı ´a, J.; Ferna ´ndez, S.; Ferrero, M.; Sanghvi, Y. S.; Gotor, V. Org. Lett. 2004, 6, 3759-3762. (c) Garcı ´a, J.; Ferna ´ndez, S.; Ferrero, M.; Sanghvi, Y. S.; Gotor, V. Tetrahedron: Asymmetry 2003, 14, 3533-3540. (d) Garcı ´a, J.; Ferna ´ndez, S.; Ferrero, M.; Sanghvi, Y. S.; Gotor, V. J. Org. Chem. 2002, 67, 4513-4519. Organic Process Research & Development 2006, 10, 581-587 10.1021/op050253i CCC: $33.50 © 2006 American Chemical Society Vol. 10, No. 3, 2006 / Organic Process Research & Development • 581 Published on Web 02/04/2006