Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Patent
1995-02-22
1999-06-22
Kunz, Gary L.
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
536 245, 536 271, 536 281, C07H 19067, C07H 19167, C07H 2102, C07H 1910
Patent
active
059143967
DESCRIPTION:
BRIEF SUMMARY
FIELD OF INVENTION
This invention is directed to processes for the preparation of 2'-O-alkyl uridine and cytidine phosphoramidites. This invention is also directed to processes for the preparation of 2'-O-alkyl, 3'-O-alkyl and 2',3'-di-O-alkyl 2,6-diaminopurine riboside, 2'-O-alkyl guanosine and 2'-O-alkyl guanosine analogs, phosphoramidites of these compounds and methods of use thereof.
BACKGROUND OF THE INVENTION
A number of oligonucleotide analogs have been made. One class of oligonucleotides that have been synthesized are the 2'-O-substituted oligonucleotides. Such oligonucleotides have certain unique and useful properties. In U.S. patent application Ser. No. 814,961, filed Dec. 24, 1991, now abandoned, entitled Gapped 2' Modified Phosphorothioate Oligonucleotides, assigned to the same assignee as this application, the entire contents of which are herein incorporated by reference, 2' substituted nucleotides are introduced within an oligonucleotide to induce increased binding of the oligonucleotide to a complementary target strand while allowing expression of RNase H activity to destroy the targeted strand.
In a recent article, Sproat, B. S., Beijer, B. and Iribarren, A., Nucleic Acids Research, 1990, 18:41, the authors noted further use of 2'-O-methyl substituted oligonucleotides as "valuable antisense probes for studying pre-mRNA splicing and the structure of spliceosomes".
2'-O-methyl and ethyl nucleotides and methods of making the same have been reported by a number of authors.
Robins, M. J., Naik, S. R. and Lee, A. S. K., J. Org. Chem., 39:1891 (1974) reported a low yield synthesis of 2'-O- and 3'-O-methyl guanosine via a stannous chloride catalyzed monomethylation by diazomethane. As was later reported by Robins, M. J., Hansske, F. and Bernier, S. E., Can. J. Chem., 59:3360 (1981), "convenient and high yield methods have been devised for synthesis of the 2'--O-- and 3'-O-methyl ethers of adenosine, cytidine, and uridine . . . However, guanosine has presented significant difficulties." In the foregoing paper, the authors reported an improved synthesis of 2'-O and 3'-O-methyl guanosine. The synthesis was improved by effecting the stannous chloride catalyzed diazomethane methylation of 2,6-diamino-9-(.beta.-D-ribofuranosyl)purine (2-aminoadenosine) in place of guanosine. The diamino purine moiety was then reduced to the corresponding guanine moiety with adenosine deaminase. In a further diazoation reaction described by Singer and Kusmierek, Biochemistry 15:5052 (1976), a mixture of 2' and 3'-O-ethyl guanosine was reported to result from the treatment of guanosine with diazoethane. The alkylation also resulted in alkylation of the heterocyclic base. The alkylated product was treated with base to remove the ethyl group from the heterocyclic base. The resulting product was identified by virtue of having the same UV spectrum as that of guanosine, but a Rf differing from the Rf of guanosine.
A further improvement in the synthesis of 2'-O-methyl nucleosides was reported by Inoue, H., Hayase, Y. Imura, A., Iwai, S., Miura, K. and Ohtsuka, E., Nucleic Acids Research, 15:6131 (1987). This method of synthesis was effected utilizing CH3I in the presence of Ag.sub.2 O. This method proved useful for all of the common nucleotides with the exception of guanosine. As reported by these authors, guanosine proved refractory to this synthetic method. Thus these authors again had to effect the 2'-O-methylation of guanosine with diazomethane. In order to avoid methylation of the amino functionality of the guanine base moiety, the guanine base moiety was blocked with an isobutyryl group. Additionally, to avoid methyl esterification of the 3'-O functionality of the sugar moiety, a TIPDS (tetraisopropyldisiloxane) blocking group was used to block both the 3' and the 5' hydroxyls of the sugar moiety.
Sproat et al., supra and Sproat, B. S., Iribarren, A. M., Garcia, R. G. and Beijer, B., Nucleic Acids Research, 19:733 (1991) addressed the synthesis of 2'-O-methyl guanosine (and 2'-O-allyl guanosine). In both of these Sproa
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Cook Phillip Dan
Guinosso Charles John
ISIS Pharmaceuticals Inc.
Kunz Gary L.
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