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. 1995 Dec 11;23(23):4872–4877. doi: 10.1093/nar/23.23.4872

A new 2'-hydroxyl protecting group for the automated synthesis of oligoribonucleotides.

H Rastogi 1, D A Usher 1
PMCID: PMC307477  PMID: 8532531

Abstract

We have developed a new type of 2'-hydroxyl protecting group for the automated machine synthesis of RNA oligomers: a 2-hydroxyisophthalate formaldehyde acetal (HIFA). The unique feature of this protecting group is that, as the bis ester, it is relatively stable to the acidic conditions that are used for repeated removal of dimethoxytrityl groups during chain elongation, but the final deprotection step in alkali, which cleaves the chain from the support and removes the base and phosphate protecting groups, converts it to the bis carboxylate and this can be removed relatively rapidly by treatment with mild acid. Conversion of the bis ester to the bis carboxylic acid increases the rate of acid-catalyzed hydrolysis of the acetal by 42-fold at pH 1, and, possibly, by 1320-fold at pH 3. The bis ester is 112 times more stable than the 1-(2-fluorophenyl)-4-methoxypiperidin-4-yl group (Fpmp) towards hydrolysis at pH 1, while the bis acid is only 2.35 times more stable than Fpmp at pH 3. In synthesis of the dimers UpU and UpG, with a coupling time of 5 min, the dimethoxytrityl cation assay indicated coupling yields of > 98%.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bartel D. P., Szostak J. W. Isolation of new ribozymes from a large pool of random sequences [see comment]. Science. 1993 Sep 10;261(5127):1411–1418. doi: 10.1126/science.7690155. [DOI] [PubMed] [Google Scholar]
  2. Bock L. C., Griffin L. C., Latham J. A., Vermaas E. H., Toole J. J. Selection of single-stranded DNA molecules that bind and inhibit human thrombin. Nature. 1992 Feb 6;355(6360):564–566. doi: 10.1038/355564a0. [DOI] [PubMed] [Google Scholar]
  3. Cech T. R. The chemistry of self-splicing RNA and RNA enzymes. Science. 1987 Jun 19;236(4808):1532–1539. doi: 10.1126/science.2438771. [DOI] [PubMed] [Google Scholar]
  4. Crick F. H. The origin of the genetic code. J Mol Biol. 1968 Dec;38(3):367–379. doi: 10.1016/0022-2836(68)90392-6. [DOI] [PubMed] [Google Scholar]
  5. Dai X., De Mesmaeker A., Joyce G. F. Cleavage of an amide bond by a ribozyme. Science. 1995 Jan 13;267(5195):237–240. doi: 10.1126/science.7809628. [DOI] [PubMed] [Google Scholar]
  6. Doudna J. A., Usman N., Szostak J. W. Ribozyme-catalyzed primer extension by trinucleotides: a model for the RNA-catalyzed replication of RNA. Biochemistry. 1993 Mar 2;32(8):2111–2115. doi: 10.1021/bi00059a032. [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. Gerecke M., Borer R., Brossi A. Selective ther cleavage in the aporphine series. Conversion of (S)-bulbocapnine into (S)-corytuberine and (S)-corydine methyl ether. Helv Chim Acta. 1976 Nov 3;59(7):2551–2557. doi: 10.1002/hlca.19760590731. [DOI] [PubMed] [Google Scholar]
  9. Griffin B. E., Jarman M., Reese C. B. The synthesis of oligoribonucleotides. IV. Preparation of dinucleoside phosphates from 2',5'-protected ribonucleoside derivatives. Tetrahedron. 1968 Jan;24(2):639–662. doi: 10.1016/0040-4020(68)88015-9. [DOI] [PubMed] [Google Scholar]
  10. Guerrier-Takada C., Lumelsky N., Altman S. Specific interactions in RNA enzyme-substrate complexes. Science. 1989 Dec 22;246(4937):1578–1584. doi: 10.1126/science.2480641. [DOI] [PubMed] [Google Scholar]
  11. Inoue H., Hayase Y., Imura A., Iwai S., Miura K., Ohtsuka E. Synthesis and hybridization studies on two complementary nona(2'-O-methyl)ribonucleotides. Nucleic Acids Res. 1987 Aug 11;15(15):6131–6148. doi: 10.1093/nar/15.15.6131. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Milligan J. F., Groebe D. R., Witherell G. W., Uhlenbeck O. C. Oligoribonucleotide synthesis using T7 RNA polymerase and synthetic DNA templates. Nucleic Acids Res. 1987 Nov 11;15(21):8783–8798. doi: 10.1093/nar/15.21.8783. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Morris K. N., Tarasow T. M., Julin C. M., Simons S. L., Hilvert D., Gold L. Enrichment for RNA molecules that bind a Diels-Alder transition state analog. Proc Natl Acad Sci U S A. 1994 Dec 20;91(26):13028–13032. doi: 10.1073/pnas.91.26.13028. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Noller H. F., Hoffarth V., Zimniak L. Unusual resistance of peptidyl transferase to protein extraction procedures. Science. 1992 Jun 5;256(5062):1416–1419. doi: 10.1126/science.1604315. [DOI] [PubMed] [Google Scholar]
  15. Orgel L. E. Evolution of the genetic apparatus. J Mol Biol. 1968 Dec;38(3):381–393. doi: 10.1016/0022-2836(68)90393-8. [DOI] [PubMed] [Google Scholar]
  16. Prudent J. R., Uno T., Schultz P. G. Expanding the scope of RNA catalysis. Science. 1994 Jun 24;264(5167):1924–1927. doi: 10.1126/science.8009223. [DOI] [PubMed] [Google Scholar]
  17. Reese C. B., Saffhill R., Sulston J. E. 4-methoxytetrahydropyran-4-yl. A symmetrical alternative to the tetrahydropyranyl protecting group. Tetrahedron. 1970 Feb;26(4):1023–1030. doi: 10.1016/s0040-4020(01)98779-4. [DOI] [PubMed] [Google Scholar]
  18. Robertson D. L., Joyce G. F. Selection in vitro of an RNA enzyme that specifically cleaves single-stranded DNA. Nature. 1990 Mar 29;344(6265):467–468. doi: 10.1038/344467a0. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Sandström A., Kwiatkowski M., Chattopadhyaya J. Chemical synthesis of a pentaribonucleoside tetraphosphate constituting the 3'-acceptor stem sequence of E. coli tRNAIle using 2'-O-(3-methoxy-1,5-dicarbomethoxypentan-3-yl)-ribonucleoside building blocks. Application of a new achiral and acid-labile 2'-hydroxyl protecting group in tRNA synthesis. Acta Chem Scand B. 1985;39(4):273–290. doi: 10.3891/acta.chem.scand.39b-0273. [DOI] [PubMed] [Google Scholar]
  21. Sassanfar M., Szostak J. W. An RNA motif that binds ATP. Nature. 1993 Aug 5;364(6437):550–553. doi: 10.1038/364550a0. [DOI] [PubMed] [Google Scholar]
  22. Scaringe S. A., Francklyn C., Usman N. Chemical synthesis of biologically active oligoribonucleotides using beta-cyanoethyl protected ribonucleoside phosphoramidites. Nucleic Acids Res. 1990 Sep 25;18(18):5433–5441. doi: 10.1093/nar/18.18.5433. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Sinha N. D., Biernat J., McManus J., Köster H. Polymer support oligonucleotide synthesis XVIII: use of beta-cyanoethyl-N,N-dialkylamino-/N-morpholino phosphoramidite of deoxynucleosides for the synthesis of DNA fragments simplifying deprotection and isolation of the final product. Nucleic Acids Res. 1984 Jun 11;12(11):4539–4557. doi: 10.1093/nar/12.11.4539. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Tuerk C., Gold L. Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science. 1990 Aug 3;249(4968):505–510. doi: 10.1126/science.2200121. [DOI] [PubMed] [Google Scholar]
  25. Wincott F., DiRenzo A., Shaffer C., Grimm S., Tracz D., Workman C., Sweedler D., Gonzalez C., Scaringe S., Usman N. Synthesis, deprotection, analysis and purification of RNA and ribozymes. Nucleic Acids Res. 1995 Jul 25;23(14):2677–2684. doi: 10.1093/nar/23.14.2677. [DOI] [PMC free article] [PubMed] [Google Scholar]

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