Mar 21, 2013 - via the phosphoramidite method without nucleoside base protection. Masanori Kataoka, Rie Kawai and Yoshihiro Hayakawa. Graduate School ...
© 1999 Oxford University Press
Nucleic Acids Symposium Series No. 42 105—106
A facile synthesis of DNA oligomers with modified backbones via the phosphoramidite method without nucleoside base protection Masanori Kataoka, Rie Kawai and Yoshihiro Hayakawa
Graduate School of Human Informatics, Nagoya University, Chikusa, Nagoya 464-8601, Japan
INTRODUCTION Oligodeoxyribonucleotides with modified backbones are attractive as antisense molecules and accordingly the convenient synthesis of the nucleotides have been strongly required. The synthesis is achieved via a variety of approaches including phosphoramidite method and Hphosphonate method, but they, except for some ones(l), are not absolutely satisfactory, because they require problematic protection of the adenine, cytosine, and guanine bases. Introduction of a protecting group to the base generally requires multi steps. In some cases, use of expensive reagents is necessary. N-Protected, particularly, /V-acylated deoxyadenosine and deoxyguanosine derivatives easily suffer depurination. Removal of the protectors, which is usually carried out under rather harsh conditions, sometimes causes decomposition of the target products. Thus, the method without base-protection (Nunprotected method) is ideal for the synthesis. We recently disclosed synthesis of DNA oligomers via such a N-unprotected phosphoramidite method(2). This paper
reports the application of this new strategy to the synthesis of oligodeoxyribonucleotides with some modified backbones. RESULTS AND DISCUSSION In the N-unprotected approach, an important subject is invention of a convenient preparation of the N-free-5'-0dimethoxytrityl-2'-deoxyribonuc!eosides, 1-4. Among these, the derivatives 1(3), 2(4), and 4(5), except for the guanosine derivative 3, can be provided via the direct dimethoxytritylation of the parent nucleosides by the previous methods. In contrast, preparation of 3 by the existing method is troublesome. It requires the following three steps: (i) protection of the nucleoside base by an amidine group, (ii) dimethoxytritylation, and (iii) removal of the transient amidine protector. It is also problematic that this process requires expensive dimethoxydimethylaminomethane(6) in the second step. Accordingly, we first examined the invention of a convenient approach to 3 and succeeded in the single-step preparation via the direct dimethoxytritylation of 2'deoxyguanosine(7). Thus, treatment of 2'-deoxyguanosine with /;,//-dimethoxytrityl chloride in the presence of a 1:1:1 mixture of methanesulfonic acid, imidazole, and diisopropylethylamine (2 equiv each toward the nucleoside) in DMF (25 °C, 2 h) afforded 3 in 78% isolated yield. This new method could be applied to the preparation of 1, 2, and 4. The yields of these products were 85-89%. The 5'-O-protected compounds, 1-4, were converted in >95% yields to their 3'-phosphoramidites, 5-9, through the DMTrO.
DMTrC
1,B = Ade 2, B = Cyt 3, B = Gua 4, B = Thy
5, B = Ade; R1 = NCCH2CH2O; R2 = /-C3H7 6, B = Cyt; R1 = NCCH2CH2O; R2 = /-C3H7 7, B = Gua; R1 = NCCH2CH2O; R2 = /-C3H7 8, B = Thy; R1 = NCCH2CH2O; R2 = /-C3H7 9, B = Thy; R1 = C 6 H 5 ; R2 = C 2 H 5
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ABSTRACT A convenient synthesis of DNA oligomers with modified back bones including phosphorothioates and phosphotriesters has been developed on the basis of the phosphoramidite strategy without nucleoside base protection. In this synthesis, a new, convenient method for the preparation of N-free-5'-0-dimethoxytrityl-2'deoxyribonucleoside by the direct O-selective dimethoxytritylation of the parent nucleosides has also been disclosed.
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Figure 1. Capillary gel electrophoresis profiles of crude products synthesized by the N-unprotected method: (A) 10; (B) 11. condensation of them and CNCH2CH2OP[N(z-C3H7)2]Cl or C 6 H 5 P [ N ( C 2 H 5 ) 2 ] C l by the assistance of diisopropylethylamine in THF. Two types of backbone-modified oligomers, GpsCpsCpsCpsApsApsGpsCpsTpsGpsGpsCpsApsTpsCps CpsGpsTpsCpsA (ps = phosphorothioate) (10) and Tp(C6H5)GpTpCpGpApCpApCpCpCpApApTp(C6H5)T [p(C6H5) = phenylphosphonate] (11), were prepared on controlled pore glass on an Applied Biosystems 381A DNA synthesizer. In the synthesis of 10, the chain elongation was performed via (i) the phosphitylation with a suitable phosphoramidite monomer, 5,6, 7, or 8, and imidazolium triflate as the activator, (ii) conversion of the undesirably formed /V-phosphitylated adenosine and cytidine to the N-free materials by brief treatment with benzimidazolium triflate in methanol, and (iii) thioation using Beaucage reagent(8). While, internucleotide linkage of 11 was constructed via phosphitylation using 5-8 or phenylphosphinylation using 9, followed by oxidation with ferf-butyl hydroperoxide. The target nucleotides were obtained by treatment with ammonia which removes cyanoethyl protectors and detaches the products from the solid supports. As shown in Figure 1, the products, 10 and 11, have sufficiently high purity in their crude forms. The TOF-MS analysis supported the structures of them. CONCLUSION We have demonstrated that the N-unprotected phosphoramidite method is useful for the synthesis of oligodeoxyribonucleotides with modified back bones such as phosphorothioates and phenylphosphonates. The present N-unprotected approach is superior to existing Nprotected methods because the target products can be prepared at lower cost; the synthetic process is shorter; and a risk of depurination is reduced.
REFERENCES 1. Representative methods without nucleoside base, see (a) Gryaznov, S.M. and Letsinger, R.L. (1991)/ Am. Chem. Soc, 113, 5876-5877 (the phosphoramidite method); (b) Gryaznov, S.M. and Letsinger, R.L. (1992) Nucleic Acids Res., 20, 1879-1882 (the phosphoramidite method) Wada, T., Sato, Y., Honda, F., Kawahara, S., Sekine, M. (1997) J. Am. Chem. Soc. 119, 12710-12721 (the W-phosphonate method). 2. Hayakawa, Y. and Kataoka, M. (1998) J. Am. Chem. Soc. 120. 12395-I24OI. 3. Ti, G.S., Gaffiney, B.L. and Jones, A.R. (1982) J. Am. Chem. Soc. 104, 1316-1319. 4. Ishido, Y. (1990) Chem. Abstr., 112, 700. 5. Schaller, H., Weimann, G., Lerch, B. and Khorana, H.G. (1963) J. Am. Chem. Soc, 85, 3821-3827. 6. Vu, H., McColumn, C , Jacobson, K., Theisen, P., Vinayak, R., Spiess, E. and Andrus, A. (1990) Tetrahedron Lett., 31, 7269-7272. 7. Kataoka, M. and Hayakawa, Y. (1999) J. Org. Chem., in press. 8. Iyer, R.P., Phillips, L.R., Egan, W., Regan, J.B. and Beaucage, S.L. (1990) J. Am. Chem. Soc, 112, 46934699.
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