Nucleoside H-Phosphonates. 18. Synthesis of Unprotected Nucleoside 5′-H-Phosphonates and Nucleoside 5′-H-Phosphonothioates and Their Conversion into the 5′-Phosphorothioate and 5′-Phosphorodithioate Monoesters Jadwiga Jankowska, Anna Sobkowska, Jacek Cies ´lak, Michal Sobkowski, and Adam Kraszewski* Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704 Poznan ´ , Poland Jacek Stawin ´ ski* Department of Organic Chemistry, Arrhenius Laboratory, Stockholm University, S-106 91 Stockholm, Sweden David Shugar Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawin ´ skiego 5A, 02-106 Warsaw, Poland Received March 16, 1998 A simple and efficient protocol for the preparation of unprotected nucleoside 5′-H-phosphonates and nucleoside 5′-H-phosphonothioates via a one-step deprotection of suitable precursors with methylamine has been developed. The synthetic utility of the unprotected nucleotide derivatives was demonstrated by converting them under mild conditions to the corresponding nucleoside 5′- phosphorothioate and nucleoside 5′-phosphorodithioate monoesters. Factors affecting oxidation of H-phosphonate, H-phosphonothioate, and phosphite derivatives with elemental sulfur are also discussed. Introduction In the last two decades phosphorothioate analogues of naturally occurring substances have become a firmly established tool in biochemical and biological studies directed toward unraveling of enzyme functions and mechanisms. 1 Due to their chirality at the phosphorus center and higher stability toward enzymatic hydrolysis, phosphorothioate derivatives have proven to be valuable as model constructs for designing enzyme inhibitors and transition state analogues, as stereochemical probes in elucidation of mechanisms of enzyme-catalyzed phospho- ryl transfer reactions, or for probing structures of ri- bozymes, etc. 1-3 Although nucleoside 5′-phosphate monoesters are cen- tral compounds in numerous biochemical and pharma- cological studies, 2,4,5 their phosphorodithio analogues, in contradistinction to those of phosphate diesters, have received relatively little attention. 6-8 In fact, nucleoside phosphorodithioates, bearing two sulfur atoms at the nonbridging positions of the phosphomonoester moiety, have been prepared only recently 6,7,9,10 and their synthe- ses highlighted some fundamental problems connected with their preparation. Due to the inherent instability of phosphorodithioate monoesters, most synthetic organic methods for thio- phosphorylation of nucleosides 1 have been found inap- plicable to the synthesis of these phosphate analogues. For example, an attempted synthesis of nucleoside phos- phorodithioates via S-alkyl and O-alkyl phosphorodithio- ate diesters or the corresponding phosphotriesters failed, due to problems encountered during the removal of phosphate protecting groups. 6 An approach involving nucleoside H-phosphonodithioates as intermediates has been more successful, but also this synthesis was ham- pered by a low yield in some crucial steps and formation of significant amounts of side products during the final deprotection. 6 The route via 2-thio-1,3,2-dithiaphos- pholane derivatives, as proposed by Okruszek et al., 7 produced nucleoside phosphorodithioates in acceptable yields (ca. 50%), but it required the inclusion of two extra steps (the introduction and removal of a 2-cyanoethyl group) into the synthetic protocol to prevent a severe decomposition of the products during the hydrolytic opening of the dithiaphospholane ring. The development of 9-fluorenemethyl H-phosphonothioate as a -(H)P(S)- (1) Eckstein, F. Annu. Rev. Biochem. 1985, 54, 367-402. (2) Eckstein, F.; Gish, G. TIBS 1989, 97-100. (3) Herschlag, D.; Piccirilli, J. A.; Cech, T. R. Biochemistry 1991, 30, 4844-4854. (4) Mitchell, A. G.; Thomson, W.; Nicholls, D.; Irwin, W. J.; Freeman, S. J. Chem. Soc., Perkin Trans. 1 1992, 2345-2353. (5) Perigaud, C.; Gosselin, G.; Lefebvre, I.; Girardet, J.-L.; Benzaria, S.; Barber, I.; Imbach, J.-L. Bioorg. Med. Chem. Lett. 1993, 3, 2521- 2526. (6) Seeberger, P. H.; Yau, E.; Caruthers, M. H. J. Am. Chem. Soc. 1995, 117, 1472-1478. (7) Okruszek, A.; Olesiak, M.; Krajewska, D.; Stec, W. J. J. Org. Chem. 1997, 62, 2269-2272. (8) Golos, B.; Dzik, J. M.; Rode, W.; Jankowska, J.; Kraszewski, A.; Stawin ´ ski, J.; Shugar, D. Interaction of thymidylate synthase with the 5′-thiophosphates and 5′-H-phosphonates of 2′-deoxyuridine, thymidine and 5-fluoro-2′-deoxyuridine; In Chemistry and Biology of Pteridines and Folates; Pfleiderer, W., Rokos, H., Eds.; Blackwell Science: Berlin, 1997; pp 423-426. (9) Seeberger, P. H.; Jorgensen, P. N.; Bankaitisdavis, D. M.; Beaton, G.; Caruthers, M. H. J. Am. Chem. Soc. 1996, 118, 9562-9566. (10) Jankowska, J.; Cieslak, J.; Kraszewski, A.; Stawin ´ ski, J. Tetrahedron Lett. 1997, 38, 2007-2010. 8150 J. Org. Chem. 1998, 63, 8150-8156 10.1021/jo980491u CCC: $15.00 © 1998 American Chemical Society Published on Web 10/28/1998