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. 1996 Oct 1;24(19):3811–3820. doi: 10.1093/nar/24.19.3811

Solid support synthesis of all-Rp-oligo(ribonucleoside phosphorothioate)s.

H Almer 1, J Stawinski 1, R Strömberg 1
PMCID: PMC146170  PMID: 8871563

Abstract

The first method for solid support synthesis of all-Rp-oligo(ribonucleoside phosphorothioate)s is presented as well as attempts to increase the stereoselectivity of the key step in this approach. The synthetic strategy consists of (i) a solid support synthesis procedure, using 5'-O-(4-methoxytriphenylmethyl)-2'-O-tert-butyldimethylsilyl-ri bon ucleoside 3'-H- phosphonates, that due to stereoselectivity in the condensation step, gives oligomers with mostly Sp-H-phosphonate diesters (72-89% under standard conditions), (ii) stereospecific sulfurization with S8 in pyridine to produce oligo(ribonucleoside phosphorothioate)s enriched with internucleosidic linkages of Rp configuration, (iii) treatment of the deprotected oligonucleotides with the enzyme Nuclease P1 from Penicillium citrinum, that specifically catalyses cleavage of Sp-phosphorothioate diester linkages, which leaves a mixture of oligomers having all internucleosidic linkages as Rp-phosphorothioates, and finally (iv) isolation and HPLC purification of the full length all-Rp oligomer. Mixed sequences containing the four common nucleosidic residues up to the chain length of a heptamer were synthesized. Change of N-4-protection on the cytidine building block from propionyl to N-methylpyrrolidin-2-ylidene gave a slightly improved diastereoselectivity in H-phosphonate diester formation. Increased selectivity up to 99+% was obtained with the guanosine building block when the amount of pyridine in the coupling step was reduced.

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Selected References

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  1. Altona C., Sundaralingam M. Conformational analysis of the sugar ring in nucleosides and nucleotides. A new description using the concept of pseudorotation. J Am Chem Soc. 1972 Nov 15;94(23):8205–8212. doi: 10.1021/ja00778a043. [DOI] [PubMed] [Google Scholar]
  2. Bryant F. R., Benkovic S. J. Stereochemical course of the reaction catalyzed by 5'-nucleotide phosphodiesterase from snake venom. Biochemistry. 1979 Jun 26;18(13):2825–2828. doi: 10.1021/bi00580a022. [DOI] [PubMed] [Google Scholar]
  3. Burgers P. M., Eckstein F., Hunneman D. H. Stereochemistry of hydrolysis by snake venom phosphodiesterase. J Biol Chem. 1979 Aug 25;254(16):7476–7478. [PubMed] [Google Scholar]
  4. Damha M. J., Ogilvie K. K. Oligoribonucleotide synthesis. The silyl-phosphoramidite method. Methods Mol Biol. 1993;20:81–114. doi: 10.1385/0-89603-281-7:81. [DOI] [PubMed] [Google Scholar]
  5. Eckstein F. Nucleoside phosphorothioates. Annu Rev Biochem. 1985;54:367–402. doi: 10.1146/annurev.bi.54.070185.002055. [DOI] [PubMed] [Google Scholar]
  6. Frey P. A. Chiral phosphorothioates: stereochemical analysis of enzymatic substitution at phosphorus. Adv Enzymol Relat Areas Mol Biol. 1989;62:119–201. doi: 10.1002/9780470123089.ch4. [DOI] [PubMed] [Google Scholar]
  7. Gasparutto D., Livache T., Bazin H., Duplaa A. M., Guy A., Khorlin A., Molko D., Roget A., Téoule R. Chemical synthesis of a biologically active natural tRNA with its minor bases. Nucleic Acids Res. 1992 Oct 11;20(19):5159–5166. doi: 10.1093/nar/20.19.5159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Griffiths A. D., Potter B. V., Eperon I. C. Stereospecificity of nucleases towards phosphorothioate-substituted RNA: stereochemistry of transcription by T7 RNA polymerase. Nucleic Acids Res. 1987 May 26;15(10):4145–4162. doi: 10.1093/nar/15.10.4145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Herschlag D., Piccirilli J. A., Cech T. R. Ribozyme-catalyzed and nonenzymatic reactions of phosphate diesters: rate effects upon substitution of sulfur for a nonbridging phosphoryl oxygen atom. Biochemistry. 1991 May 21;30(20):4844–4854. doi: 10.1021/bi00234a003. [DOI] [PubMed] [Google Scholar]
  10. Knowles J. R. Enzyme-catalyzed phosphoryl transfer reactions. Annu Rev Biochem. 1980;49:877–919. doi: 10.1146/annurev.bi.49.070180.004305. [DOI] [PubMed] [Google Scholar]
  11. McSwiggen J. A., Cech T. R. Stereochemistry of RNA cleavage by the Tetrahymena ribozyme and evidence that the chemical step is not rate-limiting. Science. 1989 May 12;244(4905):679–683. doi: 10.1126/science.2470150. [DOI] [PubMed] [Google Scholar]
  12. Mizrahi V., Henrie R. N., Marlier J. F., Johnson K. A., Benkovic S. J. Rate-limiting steps in the DNA polymerase I reaction pathway. Biochemistry. 1985 Jul 16;24(15):4010–4018. doi: 10.1021/bi00336a031. [DOI] [PubMed] [Google Scholar]
  13. Pon R. T., Usman N., Ogilvie K. K. Derivatization of controlled pore glass beads for solid phase oligonucleotide synthesis. Biotechniques. 1988 Sep;6(8):768–775. [PubMed] [Google Scholar]
  14. Potter B. V., Connolly B. A., Eckstein F. Synthesis and configurational analysis of a dinucleoside phosphate isotopically chiral at phosphorus. Stereochemical course of Penicillium citrum nuclease P1 reaction. Biochemistry. 1983 Mar 15;22(6):1369–1377. doi: 10.1021/bi00275a008. [DOI] [PubMed] [Google Scholar]
  15. Rozners E., Westman E., Strömberg R. Evaluation of 2'-hydroxyl protection in RNA-synthesis using the H-phosphonate approach. Nucleic Acids Res. 1994 Jan 11;22(1):94–99. doi: 10.1093/nar/22.1.94. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Sarkar M., Sigurdsson S., Tomac S., Sen S., Rozners E., Sjöberg B. M., Strömberg R., Gräslund A. A synthetic model for triple-helical domains in self-splicing group I introns studied by ultraviolet and circular dichroism spectroscopy. Biochemistry. 1996 Apr 16;35(15):4678–4688. doi: 10.1021/bi9523466. [DOI] [PubMed] [Google Scholar]
  17. Stawinski J., Strömberg R., Thelin M., Westman E. Studies on the t-butyldimethylsilyl group as 2'-O-protection in oligoribonucleotide synthesis via the H-phosphonate approach. Nucleic Acids Res. 1988 Oct 11;16(19):9285–9298. doi: 10.1093/nar/16.19.9285. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Stec W. J., Grajkowski A., Koziolkiewicz M., Uznanski B. Novel route to oligo(deoxyribonucleoside phosphorothioates). Stereocontrolled synthesis of P-chiral oligo(deoxyribonucleoside phosphorothioates). Nucleic Acids Res. 1991 Nov 11;19(21):5883–5888. doi: 10.1093/nar/19.21.5883. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Wagner R. W. Gene inhibition using antisense oligodeoxynucleotides. Nature. 1994 Nov 24;372(6504):333–335. doi: 10.1038/372333a0. [DOI] [PubMed] [Google Scholar]
  20. Westman E., Strömberg R. Removal of t-butyldimethylsilyl protection in RNA-synthesis. Triethylamine trihydrofluoride (TEA, 3HF) is a more reliable alternative to tetrabutylammonium fluoride (TBAF). Nucleic Acids Res. 1994 Jun 25;22(12):2430–2431. doi: 10.1093/nar/22.12.2430. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Wilk A., Stec W. J. Analysis of oligo(deoxynucleoside phosphorothioate)s and their diastereomeric composition. Nucleic Acids Res. 1995 Feb 11;23(3):530–534. doi: 10.1093/nar/23.3.530. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Wu T., Ogilvie K. K., Pon R. T. Prevention of chain cleavage in the chemical synthesis of 2'-silylated oligoribonucleotides. Nucleic Acids Res. 1989 May 11;17(9):3501–3517. doi: 10.1093/nar/17.9.3501. [DOI] [PMC free article] [PubMed] [Google Scholar]

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