Process for the synthesis of ribonucleic acid (RNA) using a nove

Details Process for the synthesis of ribonucleic acid (RNA) using a nove Process for the synthesis of ribonucleic acid (RNA) using a nove

1994-07-26

1996-09-03

Kunz, Gary L.

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

536 253, C07H 102, C07H 2102

Patent

active

055525393

DESCRIPTION:

BRIEF SUMMARY
The present invention relates to a process for the synthesis of ribonucleic acid (RNA) using a deprotection agent making it possible to rapidly obtain a total deprotection of the hydroxyl groups in the 2'-position of the RNA.
For a number of years, processes for the synthesis of ribonucleic acid fragments have developed greatly as a result of the increasing interest in RNA, which plays an important part in numerous cellular processes.
These synthesis processes are similar to those used in the synthesis of deoxyribonucleic acid (DNA) and are e.g. described by Ogilvie et al in Proc. Natl. Acad. Sci., Vol. 85, pp. 5764-5768, August 1988 and by Gasparutto et al in Nucleosides & Nucleotides 9(8), pp. 1087-1098, 1990.
The starting product for this synthesis consists of ribonucleosides, whose hydroxyl in the 2'-position is protected by an appropriate group and which also has groups suitable for nucleotide synthesis and successive condensation takes place of these nucleoside derivatives in order to form ribonucleic acid.
Thus, in the aforementioned document Ogilvie et al used ribonucleoside derivatives in which the hydroxyl groups in the 2'-position are protected by the t-butyl-dimethyl silyl radical. After coupling all the nucleosides, a ribonucleic acid is consequently obtained, whose hydroxyls in the 2'-position are protected. Therefore, it is appropriate to carry out at the end of the operation a deprotection treatment in order to eliminate these 2'-hydroxyl protecting groups.
Hitherto, said deprotection has been performed by using as the deprotection reagent tetrabutyl ammonium fluoride in tetrahydrofuran as described in Glen Research Report, Vol. 4, No. 1, March 1991, RNA Synthesis--Problems in Deprotection. The deprotection of the hydroxyl function in the 2'-position of a nucleoside or nucleotide in free form carrying in the 2'-position a tert.-butyl dimethyl silyl (TBDMS) function takes place within a few minutes in a tetrabutyl ammonium fluoride. It is much more difficult in the case of an oligonucleotide carrying t-butyl dimethyl silyl in the 2'-position and takes hours (cf. FIG. 3, curve 5). It has been found that in the case of very long RNA fragments, e.g. more than 25 bases, the deprotection of the hydroxyl groups is not complete with this reagent, because there are at least 1 to 2% non-deprotected hydroxyl groups per base unit, i.e. each RNA molecule has at least one non-deprotected hydroxyl, which can be prejudicial to the biological activity of RNA, as has been indicated by Ogilvie.
The present invention relates to a process for the synthesis of ribonucleic acid (RNA), which makes it possible to totally and rapidly obtain the total deprotection of the hydroxyl groups in the 2'-position of the synthesized ribonucleic acid.
This process consists of ##STR1## in which R.sup.1 is a radical derived from a pyrimidic or puric base chosen from among the radicals of formula: ##STR2## in which R.sup.5 is H or CH.sub.3 and R.sup.6, R.sup.7 and R.sup.8 are hydrogen atoms or an acyl group protecting an exocyclic NH.sub.2 group,
R.sup.2 is an unstable radical in an acid medium appropriate for nucleotide synthesis,
R.sup.3 is a solid support and
R.sup.4 is a trialkyl silyl radical, (I), in which R.sup.1, R.sup.2 and R.sup.4 have the meanings given hereinbefore and R.sup.3 is a phosphorus radical appropriate for nucleotide synthesis, in order to form a synthetic ribonucleic acid, whereof all the hydroxyl groups in the 2'-position are protected by R.sup.4 and synthesized ribonucleic acid with a deprotection agent complying with the formula: ##STR3## in which R.sup.9 represents a straight or branched C.sub.1 to C.sub.10 alkyl radical, R.sup.10 and R.sup.11, which can be same or different represent H or a straight or branched C.sub.1 to C.sub.10 alkyl radical, which can be the same or different to R.sup.9 and n is a number between 1 and 3, which may or may not be an integer.
In this process, successive condensation cycles are performed in order to couple the different nucleosides to one another using conventional met

REFERENCES:
Derwent Publications Ltd., London, GB; Section Ch, Week 9119, 26 Jun. 1991, Class B, p. 21; JP,A,3 074 398 (Yuki Gosei Yakuhin) Mar. 28, 1991.

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