J.Org. Chem. 1994,59,7273-7283
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Photochemistry of Di(deoxyribonuc1eoside) Methylphosphonates Containing NS-Methyl-4-thiothymine Pascale Clivio and Jean-Louis Fourrey* Institut de Chimie des Substances Naturelles, CNRS,F-91198Gif sur Yvette Cedex, France Tomas Szab6 and Jacek Stawinski* Department of Organic Chemistry, University of Stockholm, S-106 91 Stockholm, Sweden Received June 15, 1994@ (Rp)- and (Sp)-5’-W-methyl-4-thiothymidine 3’4thymidinyl methylphosphonate) (Tpm3s4T) (lla and 1lb), respectively, have been synthesized in order to elucidate their photochemical behavior in comparison with that of their phosphate congener lb. The dinucleoside methylphosphonates proved to be less rapidly photolyzed than lb and gave more photoproducts. The structure and stereochemistry of each photoproduct were determined by NMR studies. A plausible mechanism to explain the formation of the major photoproducts has been proposed. It involves two pathways: either cycloaddition or radical coupling. The former one led to thietane derivatives, the formation of which is of relevance to the mechanism to give (6-4)bipyrimidine photoproducts in DNA. Most of the other photoproducts were probably formed from a biradical generated via hydrogen abstraction from the C5 methyl of the Tp- part of each dimer. Compounds 15 which represent a new type of photoproduct in the nucleic acid series are most likely formed by this route. Another significant finding of this study was t h a t the Rp dinucleoside methylphosphonate (lla)is a better mimic of the phosphate backbone, a s found in DNA, than its Sp diastereomers (llb).
Introduction The antisense approach for specific gene inhibition is currently receiving a great deal of attenti0n.l In this domain, the use of unmodified oligonucleotides has been limited by several factors such as susceptibility to nucleases digestion and poor cell penetration. To circumvent these problems methylphosphonate oligonucleotide analogs have been designed. Such nonionic constructions exhibit reasonable passive cell membrane penetration as well as strong nuclease resistance. Moreover, they form stable hybrids with a complementary DNA (or RNA) strand although the effects of the P-methyl group on helix formation have not yet been fully elucidated.2 It is generally accepted that the stereochemistry of the phosphorus atom might have an influence on several factors governing the geometry of the modified diester backbone such as the hydration pattern and electrostatic interaction^.^ Thus, oligonucleotide methylphosphonates with predominantly Rp configuration have been considered to afford more stable hybrids with complementary DNA than their Sp analog^.^ For a number of related investigations, di(deoxyribonuc1eotides)and their analogs have served as model systems either to predict conformational modifications produced by the incorporation of a modified deoxynucleoside in DNA or to study DNA Abstract published in Advance ACS Abstracts, October 15, 1994. ( l ) ( a ) Uhlmann, E.; Peyman, A. Chem. Rev. 1990,90,544-579. (b) Crooke, S.T.Annua1. Rev. Pharmacol. Toxicol. 1992,32,329-376. (c) Gene Regulation: Biology of Antisense RNA and DNA; Erickson, R. P., Izant, J. G., Eds.; Raven Press: New York, 1992. (d) Milligan, J. F.; Matteucci, M. D.;Martin, J. C. J . Med. Chem. 1993,36,19231937. (d) Beaucage, S. L.; Iyer, R. P. Tetrahedron 1993,49, 61236194. (e) Varma, R. S. Synthesis 1993,340-346. (2)(a) Miller, P. S BwlTechnology 1991,9,358-362. 6)Lesnikowski, 2. J. Bioorg. Chem. 1993,21,127-155. (3)(a)Hausheer, F. H.; Chandra Singh, U.;Palmer, T. C.; Saxe, J . D.J.Am. Chem. Soc. 1990,112,9468-9474.(b)Hausheer, F. H.; Rao, B. G.; Saxe, J. D.;Singh, U.C. Ibid. 1992,114,3201-3206. (4)(a) Lesnikowski, Z.J.; Jaworska, M.; Stec, W. J. Nucleic Acids Res. 1990, 18,2109-2115. (b) Samstag, W.;Engels, J. W. Angew. Chem., Int. Ed. Engl. 1992,31,1386-1388. @
0022-326319411959-7273$04.50/0
reactivity. In this respect, the conformation of several dinucleoside methylphosphonates has been studied to get a n insight into the conformational state of methylphosphonate-containing oligonucleotides. Rp configurated dinucleoside methylphosphonate isomers were found to better mimic dinucleoside phosphates than Sp i~omers.4~2~ Similarly, various dinucleoside phosphates have been used to study the mechanism of cyclobutane or (6-4) pyrimidine-pyrimidone photoproduct formation which are responsible for major photolesions in DNA.6 Recently, a model system based on the photochemistry of the dinucleoside phosphate la (Tps4T)has been designed to study the mechanism of formation of (6-4) lesions in DNA.’ Thymidylyl-(3’-5’)-4-thiothymidine (la) is a n analog of thymidylyl-(3’-5’)-thymidine (TpT) in which thymidine at the 3’-position is replaced by the selectively photoreactive 4-thiothymidine. In contrast to TpT, irradiation of la did not lead to cyclobutane dimers but provided in satisfactory yield (6-4)bipyrimidine photoproducts as well as another type of product arising from the generation of a radical a t the methyl position of thymidine a t the r - e n d . Altogether, these observations could be conveniently interpreted on the basis of earlier photochemical studies which demonstrated that the ( 5 ) (a) Miller, P. S.; Yano, J.; Yano, E.; Carroll, C.; Jayaraman, K.; Ts’o, P. 0. P. Biochemistry 1979,23,5134-5143.(b) Kan, L. 5.; Cheng, D.M.; Miller, P. S.; Yano, J.;Ts’o, P. 0. P.Zbid. 1980,19,2122-2132. (c) Swarna Latha, Y.; Yathindra, N. J . Bwmol. Struct. Dyn. 1991, 9, 613-631. (6)(a) Cadet, J.; Vigny, P. The Photochemistry of Nucleic Acids; Morrison, H., Ed.; Wiley and Sons: New York, 1990;Vol. 1, p 79. (b) Koning, T . M. G.; Van Soest, J. J. G.; Kaptein, R. Eur. J . Biochem. 1991,195,29-40. (c) Douki,T.; Voituriez, L.; Cadet, J. Photochem. Photobiol. 1991,53,293-297. (d) Kan, L.-S.; Voituriez, L.; Cadet, J. J . Photochem. Photobiol. B: Biol. 1992,12,339-357. (e) Douki,T.; Cadet, J. J . Photochem. Photobwl., B: Bwl. 1992,15, 199-213. (0 Kao, J . L.-F.; Nadji, S.; Taylor, J.-S. Chem. Res. Toxicol. 1993,6,561567. (g) Tabaczynski, W.A.; Lemaire, D. G. E.; Ruzsicska, B. P.; Alderfer, J. L. Biopolymers 1993,33,1365-1375. (h) Lemaire, D.G. E.; Ruzsicska, B. P. Photochem. Photobiol. 1993,57, 755-769. (7)Clivio, P.;Fourrey, J.-L.; Gasche, J.; Favre, A. J . Am. Chem. Soc. 1991,113,5481-5483.
0 1994 American Chemical Society
7274 J. Org. Chem., Vol. 59, No. 24, 1994
Clivio et al.
Scheme 1
Scheme 2
N
