DNA Synthesis Without Base Protection

Mitsuo Sekine1

1 Tokyo Institute of Technology, Yokohama
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 3.10
DOI:  10.1002/0471142700.nc0310s18
Online Posting Date:  October, 2004
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Abstract

DNA synthesis can be achieved by using O‐selective methods for internucleotide bond formation. This greatly simplifies the synthesis of oligodeoxyribonucleotides by eliminating the need for nucleobase protection and deprotection steps. This unit describes strategies that can be used for DNA synthesis without base protection. The discussion includes synthesis of phosphoramidite and H‐phosphonate monomers, solid‐phase assembly by the phosphoramidite and H‐phosphonate methods, and future prospects for DNA synthesis using N‐unprotected approaches.

Keywords: O‐selectivity; DNA synthesis; base protection; internucleotide bond

     
 
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Table of Contents

  • Liquid‐Phase Synthesis
  • Solid‐Phase Synthesis Using the Phosphoramidite Approach
  • Solid‐Phase Synthesis Using the H‐Phosphonate Approach
  • Advantages and Prospects Of the N‐Unprotected Approach
  • Literature Cited
  • Figures
     
 
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Materials

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Literature Cited

   Adamiak, R.W. and Stawinski, J. 1977. A highly effective route to N,N′‐disubstituted ureas under mild conditions. An application to the synthesis of tRNA anticodon loop fragments containing ureidonucleosides. Tetrahedron Lett. 18:1935‐1936.
   Adamiak, R.W., Biala, E., Grzeskowiak, K., Kierzek, R., Kraszewski, A., Markiewicz, W.T., Okupniak, J., Stawinski, J., and Wiewiorowski, M. 1978. The chemical synthesis of the anticodon loop of an eukaryotic initiator tRNA containing the hypermodified nucleoside N6‐/N‐threonylcarbonyl/‐adenosine/t6A/. Nucl. Acids Res. 5:1889‐1905.
   Alvarez, K., Vasseur, J.‐J., and Imbach, J.‐L. 1999. Use of photolabile amino‐protecting groups in the synthesis of base‐sensitive DNA SATE‐phosphotriesters. Nucleosides Nucleotides 18:1435‐1436.
   Baddiley, J. and Todd, A.R. 1947. Nucleotides. I. Muscle adenylic acid and adenosine diphosphate. J. Chem. Soc. 648‐651.
   Beaucage, S.L. and Iyer, R.P. 1992. Advances in the synthesis of oligonucleotides by the phosphoramidite approach. Tetrahedron 48:2223‐2311.
   Boal, J.H., Wilk, A., Harindranath, N., Max, E.E., Kempe, T., and Beaucage, S.L. 1996. Cleavage of oligodeoxyribonucleotides from controlled‐pore glass supports and their rapid deprotection by gaseous amines. Nucl. Acids Res. 24:3115‐3117.
   Butkus, V., Kayushin, A.L., Berlin, Y.A., Kolosov, M.N., and Smirnov, I.V. 1983. Cleavage of 5′‐O‐protecting trityl groups in oligodeoxynucleotide synthesis. Effect of substrate structure and reaction conditions on detritylation and depurination rates. Bioorg. Khim. 9:1518‐1530.
   Caruthers, M.H. 1991. Chemical synthesis of DNA and DNA analogues. Acc. Chem. Res. 24:278‐284.
   Claesen, C., Tesser, G.I., Dreef, C.E., Marugg, J.E., van der Marel, G.A., and van Boom, J.H. 1984. Use of 2‐methylsulfonylethyl as a phosphorus protecting group in oligonucleotide synthesis via a phosphite triester approach. Tetrahedron Lett. 25:1307‐1310.
   Dreef‐Tromp, C.M., Van Dam, E.M.A., van den Elst, H., van den Boogaart, J.E., van der Marel, G.A., and van Boom, J.H. 1991. Solid‐phase synthesis of RNA via a silyl‐protecting‐group strategy. Recl. Trav. Chim. Pays Bas 110:378‐383.
   Finnan, J.L., Varshney, A., and Letsinger, R.L. 1980. Developments in the phosphite‐triester method of synthesis of oligonucleotides. Nucl. Acids Res. Symp. Ser. 7:133‐145.
   Fourrey, J.‐L. and Varenne, J. 1985. Preparation and phosphorylation reactivity of N‐nonacylated nucleoside phosphoramidites. Tetrahedron Lett. 26:2663‐2666.
   Froehler, B.C. and Matteucci, M.D. 1983a. Dialkylformamidines: Depurination resistant N6‐protecting group for deoxyadenosine. Nucl. Acids Res. 11:8031‐8036.
   Froehler, B.C. and Matteucci, M.D. 1983b. Substituted 5‐phenyltetrazoles: Improved activators of deoxynucleoside phosphoramidites in deoxyoligonucleotide synthesis. Tetrahedron Lett. 24:3171‐3174.
   Gryaznov, S.M. and Letsinger, R.L. 1991. Synthesis of oligonucleotides via monomers with unprotected bases. J. Am. Chem. Soc. 113:5876‐5877.
   Gryaznov, S.M. and Letsinger, R.L. 1992. Selective O‐phosphitylation with nucleoside phosphoramidite reagents. Nucl. Acids Res. 20:1879‐1882.
   Hall, R.H. 1971. The Modified Nucleosides in Nucleic Acids. Columbia University Press, New York.
   Hata, T. and Kurihara, T. 1973. Preparation of intermediates in the synthesis of polynucleotides. II. N4‐Benzoylation of deoxycytidylic and cytidylic acids by 2‐chloromethyl‐4‐nitrophenyl benzoate. Chem. Lett. 8:859‐862.
   Hayakawa, Y. 2001. Toward an ideal synthesis of oligonucleotides: Development of a novel phosphoramidite method with high capability. Bull. Chem. Soc. Jpn. 74:1547‐1565.
   Hayakawa, Y. and Kataoka, M. 1998. Facile synthesis of oligodeoxyribonucleotides via the phosphoramidite method without nucleoside base protection. J. Am. Chem. Soc. 120:12395‐12401.
   Hayakawa, Y., Aso, Y., Uchiyama, M., and Noyori, R. 1983a. Facile nucleoside phosphorylation via hydroxyl activation. Tetrahedron Lett. 24:1165‐1168.
   Hayakawa, Y., Aso, Y., Uchiyama, M., and Noyori, R. 1983b. Chemoselective phosphorylation of N‐unprotected nucleosides via aluminum alkoxides. Tetrahedron Lett. 24:5641‐5644.
   Hayakawa, Y., Uchiyama, M., and Noyori, R. 1984. A convenient method for the formation of internucleotide linkage. Tetrahedron Lett. 25:4003‐4006.
   Hayakawa, Y., Uchiyama, M., Nobori, T., and Noyori, R. 1985. A convenient synthesis of 2′‐5′‐linked oligoribonucleotides. Tetrahedron Lett. 26:761‐764.
   Hayakawa, Y., Nobori, T., Noyori, R., and Imai, J., 1987. Synthesis of 2′‐5′,3′‐5′ linked triadenylates. Tetrahedron Lett. 28:2623‐2626.
   Hayakawa, Y., Kataoka, M., and Noyori, R. 1996. Benzimidazolium triflate as an efficient promoter for nucleotide synthesis via the phosphoramidite method. J. Org. Chem. 61:7996‐7997.
   Hayakawa, Y., Kawai, R., and Kataoka, M. 2001. Nucleotide synthesis via methods without nucleoside‐base protection. Eur. J. Pharm. Sci. 13:5‐16.
   Ikemoto, T., Haze, A., Hatano, H., Kitamoto, Y., Ishida, M., and Nara, K. 1995. Phosphorylation of nucleosides with phosphorus oxychloride in trialkyl phosphate. Chem. Pharm. Bull. 43:210‐215.
   Ishido, R. 1989. Protection of 5′‐hydroxy groups of cytidine derivatives. Jpn. Kokai Tokkyo Koho Jp Pat. 01308294; Chem. Abstr. 1990. 112:700.
   Iyer, R.P., Yu, D., Ho, N.‐H., Devlin, T., and Agrawal, S. 1995. O‐ and S‐Methyl phosphotriester oligonucleotides: Facile synthesis using N‐pent‐4‐enoyl nucleoside phosphoramidites. J. Org. Chem. 60:8132‐8133.
   Iyer, R.P., Yu, D., Devlin, T., Ho, N.‐H., Johnson, S., and Agrawal, S. 1996. Synthesis, biophysical properties, and stability studies of mixed backbone oligonucleotides containing novel non‐ionic linkages. Nucleosides Nucleotides 16:1491‐1495.
   Kataoka, M. and Hayakawa, Y. 1999. A convenient method for the synthesis of N‐free 5′‐O‐(p,p′‐dimethoxytrityl)‐2′‐deoxyribonucleosides via 5′‐O‐selective tritylation of the parent substances. J. Org. Chem. 64:6087‐6089.
   Kobori, A., Miyata, K., Ushioda, M., Seio, K., and Sekine, M. 2002a. A new silyl ether‐type linker useful for the automated synthesis of oligonucleotides having base‐labile protective groups. Chem. Lett. 31:16‐17.
   Kobori, A., Miyata, K., Ushioda, M., Seio, K., and Sekine, M. 2002b. A new method for the synthesis of oligodeoxyribonucleotides containing 4‐N‐alkoxycarbonyldeoxycytidine derivatives and their hybridization properties. J. Org. Chem. 67:476‐485.
   Koole, L.H., Buck, H.M., Kanters, J.A., and Schouten, A. 1987. Molecular conformation of 2′‐deoxy‐3′,5′‐di‐O‐acetyladenosine. Crystal structure and high resolution proton nuclear magnetic resonance investigations. Can. J. Chem. 65:326‐331.
   Köster, H., Stumpe, A., and Wolter, A. 1983. Polymer support oligonucleotide synthesis. 13. Rapid and efficient synthesis of oligodeoxynucleotides on porous glass support using triester approach. Tetrahedron Lett. 24:747‐750.
   Krotz, A.H., McElroy, B., Scozzari, A.N. Cole, D.L., and Ravikumar, V.T. 2003. Controlled detritylation of antisense oligonucleotides. Org. Process Res. Dev. 7:47‐52.
   Kume, A., Iwase, R., Sekine, M., and Hata, T. 1984. Cyclic diacyl groups for protection of the N6‐amino group of deoxyadenosine in oligodeoxynucleotide synthesis. Nucl. Acids Res. 12:8525‐8538.
   Kung, P.‐P. and Jones, R.A. 1992. H‐Phosphonate DNA synthesis without amino protection. Tetrahedron Lett. 33:5869‐5872.
   Letsinger, R.L., Finnan, J.L., Jacobs, S.A., Juodka, B.A., and Varshney, A.K. 1976. Exploration of new procedures for the synthesis of polynucleotides. In Proceedings of the International Conference on Synthesis, Structure and Chemistry of Transfer Ribonucleic Acids and Their Components, Dymaczewo, Poland, September 13‐17, 1976. Institute of Organic Chemistry, Polish Academy of Sciences.
   Levin, A.S., Tabatadze, E.R., and Komarova, N.I. 1991. Synthesis of oligodeoxyribonucleotides by H‐phosphonate method using N‐unblocked synthons. Sibirskii Khim. Z. 6:142‐144
   Marugg, J.E., Tromp, M., Kuyl‐Yeheskiely, E., van der Marel, G.A., and van Boom, J.H. 1986. A convenient and general approach to the synthesis of properly protected d‐nucleoside 3′‐hydrogen phosphonates via phosphite intermediates. Tetrahedron Lett. 27:2661‐2664.
   McCollum, C. and Andrus, A. 1991. An optimized polystyrene support for rapid, efficient oligonucleotide synthesis. Tetrahedron Lett. 32:4069‐4072.
   Michelson, M. and Todd, A.D. 1954. Nucleotides. Part XXIII. Mononucleotides derived from deoxycytidine. Note on the structure of cytidylic acids a and b. J. Chem. Soc. 34‐40.
   Nikolenko, L.N., Nezavibat'ko, V.N., and Tolmacheva, N.S. 1967. Selective N‐benzoylation of deoxycytidine and cytidine. Khim. Prir. Soedin. 3:359.
   Nishino, S., Nagato, Y., Hasegawa, Y., Yamamoto, H., Kamaike, K., and Ishido, Y. 1991. Partial protection of carbohydrate derivatives. Part 27. Efficient deanilidation of phosphoranilidates by the use of nitrites and acetic anhydride. Heteroatom. Chem. 2:187‐196.
   Ogilvie, K.K. 1973. The tert‐butyldimethylsilyl group as a protecting group in deoxynucleosides. Can. J. Chem. 51:3799‐3807.
   Ogilvie, K.K. and Nemer, M. 1981. Nonaqueous oxidation of phosphites to phophates in nucleotide synthesis. Tetrahedron Lett. 22:2531‐2532.
   Ohkubo, A., Ezawa, Y., Seio, K., and Sekine, M. 2002. A new strategy for the synthesis of oligodeoxynucleotides in the phosphoramidite method without base protection via phosphite intermediates. Nucl. Acids Res. Suppl. 2:29‐30.
   Okupniak, J., Adamiak R.W., and Wiewiorowski, M. 1981. New conditions for a selective introduction of trityl type protective group into adenosine and cytidine 3′‐phosphates. Pol. J. Chem. 55:679‐682.
   Pearson, R.G. 1987. Recent advances in the concept of hard and soft acids and bases. J. Chem. Educ. 64:561‐567.
   Pearson, R.G. 1990. Hard and soft acids and bases—the evolution of a chemical concept. Coord. Chem. Rev. 100:403‐425.
   Schaller, H., Weimann, G., Lerch, B., and Khorana, H.G. 1963. Studies on polynucleotides. XXIV. The stepwise synthesis of specific deoxyribopolynucleotides. Protected derivatives of deoxyribonucleosides and new syntheses of deoxyribonucleoside 3′‐phosphates. J. Am. Chem. Soc. 85:3821‐3827.
   Schulhof, J.C., Molko, D., and Teoule, R. 1987. Facile removal of new base protecting groups useful in oligonucleotide synthesis. Tetrahedron Lett. 28:51‐54.
   Sekine, M., Matsuzaki, J., Satoh, M., and Hata, T. 1982. Improved 3′‐O‐phosphorylation of guanosine derivatives by O6‐oxygen protection. J. Org. Chem. 47:571‐573.
   Sekine, M., Masuda, N., and Hata, T. 1985. Introduction of the 4,4′,4′′‐tris(benzoyloxy)trityl group into the exo amino groups of deoxyribonucleosides and its properties. Tetrahedron 41:5445‐5453.
   Sekine, M., Ohkubo, A., and Seio, K. 2003. Proton‐block strategy for the synthesis of oligodeoxynucleotides without base protection, capping reaction, and P‐N bond cleavage reaction. J. Org. Chem. 68:5478‐5492.
   Septak, M. 1996. Kinetic studies on depurination and detritylation of CPG‐bound intermediates during oligonucleotide synthesis. Nucl. Acids Res. 24:3053‐3058.
   Shena, M. 1999. DNA Microarrays: A practical approach. Oxford University Press, London.
   Spinelli, N., Meyer, A., Hayakawa, Y., Imbach, J.‐L., and Vasseur, J.‐J. 2002. Use of allylic protecting groups for the synthesis of base‐sensitive prooligonucleotides. Eur. J. Org. Chem. 49‐56.
   Tanaka, T. and Letsinger, R.L. 1982. Syringe method for stepwise chemical synthesis of oligonucleotides. Nucl. Acids Res. 10:3249‐3260.
   Ti, G.S., Gaffney, B.L., and Jones, R.A. 1982. Transient protection: Efficient one‐flask syntheses of protected deoxynucleosides. J. Am. Chem. Soc. 104:1316‐1319.
   Ushioda, M., Kadokura, M., Moriguchi, T., Kobori, A., Aoyagi, M., Seio, K., and Sekine, M. 2002. Unique participation of unprotected internucleotidic phosphodiester residues on unexpected cleavage reaction of the Si‐O bond of the diisopropylsilandiyl group used as a linker for the solid‐phase synthesis of 5′‐terminal guanylated oligodeoxynucleotides. Helv. Chim. Acta 85:2930‐2945.
   Venner, H. 1964. Studies on nucleic acids. IX. Stability of the N‐glycosidic linkage in nucleosides. Z. Physiol. Chem. 339:14‐27.
   Venner, H. 1966. Research on nucleic acids. XII. Stability of the N‐glycoside bond of nucleotides. Z. Physiol. Chem. 344:189‐196.
   Ven'yaminova, A.G., Komarova, N.I., Levin, A.S., and Repkova, M.N. 1988. Synthesis of ribonucleoside‐3′‐ and ‐5′‐H‐phosphonates via salicylchlorophosphine. Bioorg. Khim. 14:484‐489.
   Wada, T. and Sekine, M. 1995. Chemical synthesis of oligothymidylate having hydroxymethylphosphonate internucleotidic linkages. Tetrahedron Lett. 36:845‐848.
   Wada, T., Sato, Y., Honda, F., Kawahara, S., and Sekine, M. 1997. Chemical synthesis of oligodeoxyribonucleotides using N‐unprotected H‐phosphonate monomers and carbonium and phosphonium condensing reagents: O‐Selective phosphonylation and condensation. J. Am. Chem. Soc. 119:12710‐12721.
   Wada, T., Kobori, A., Kawahara, S., and Sekine, M. 1998a. Synthesis and properties of oligodeoxyribonucleotides containing 4‐N‐acetylcytosine bases. Tetrahedron Lett. 39:6907‐6910.
   Wada, T., Mochizuki, A., Sato, Y., and Sekine, M. 1998b. Functionalization of solid supports with N‐unprotected deoxyribonucleosides. Tetrahedron Lett. 39:5593‐5596.
   Wada, T., Honda, F., Sato, Y., and Sekine, M. 1999. First synthesis of H‐phosphonate oligonucleotides bearing N‐unmodified bases. Tetrahedron Lett. 40:915‐918.
   Wada, T., Kobori, A., Kawahara, S., and Sekine, M. 2001a. Synthesis and hybridization ability of oligodeoxyribonucleotides incorporating N‐acyldeoxycytidine derivatives. Eur. J. Org. Chem. 4583‐4593.
   Wada, T., Mochizuki, A., Higashiya, S., Tsuruoka, H., Kawahara, S., Ishikawa, M., and Sekine, M. 2001b. Synthesis and properties of 2‐azidodeoxyadenosine and its incorporation into oligodeoxynucleotides. Tetrahedron Lett. 42:9215‐9219.
   Watanabe, K.A. and Fox, J.J. 1966. Simple method for selective acylation of cytidine on the 4‐amino group. Angew. Chem. Int. Ed. Engl. 5:579‐580.
   Yamana, K., Nishijima, Y., Negishi, K., Yoshiki, T., Nishio, K., and Nakano, H. 1991. Deoxyribonucleoside 3′‐phosphorobisamidites in the synthesis of isopropyl phosphotriester oligodeoxyribonucleotide analogs. Tetrahedron Lett. 32:4721‐4724.
   Yoshikawa, M. and Kato, T. 1967. Studies of phosphorylation. I. Phosphorylation of 2′,3′‐O‐isopropylidene nucleoside by phosphoryl chloride. Bull. Chem. Soc. Jpn. 40:2849‐2853.
   Yuodka, Y., Lunsford, W.B., and Letsinger, R.L. 1976. Oligonucleotides and nucleotide‐peptides. XXV. New method for the synthesis of oligonucleotides. Bioorg. Chem. 2:1218‐1324.
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