US 4,725,677 A · Process for the preparation of oligonucleotides

Abstract

The invention relates to a process for the preparation of oligonucleotides by the following steps: reaction of a nucleoside with a phosphine derivative, reaction of the nucleotide derivative thus obtained with a nucleoside bonded to a polymeric carrier, oxidation of the carrier-bound nucleoside-nucleotide thus obtained with formation of phosphotriester groups, blocking of free primary 5'-OH groups, elimination of a protective group from the terminal 5'-OH group, where appropriate single or multiple repetition of the abovementioned steps to introduce further nucleoside phosphate or oligonucleoside phosphate units, and cleavage of the nucleoside-carrier bond and, where appropriate, elimination of all protective groups present in the oligonucleoside phosphates. The phosphine derivative used is a compound of the general formula III ##STR1## in which X and L can react with OH groups of the sugar units in the oligonucleotides, and R.sup.3 is a protective group which can be liberated by .beta.-elimination.

A process for the preparation of oligonucleotides of the general formula I

in which B denotes a nucleoside base, R.sup.1 denotes hydrogen, hydroxyl or hydroxyl which is protected by a removable protective group and n denotes an integer from 1 to 200, comprising the steps of
(a) reacting a nucleoside of the general formula II ##STR14## in which R.sup.1 as defined as above, and R.sup.2 denotes a removable protective group and B' denotes the nucleoside base B protected by the protective groups which can be eliminated, with a phosphine derivative of the general formula III ##STR15## in which R.sup.3 is a protective group which can be eliminated, and X and L are groups which react with hydroxyl groups in the sugar moieties of the nucleotides or nucleosides, in the presence of a base to thereby form a nucleotide phosphite
(b) reacting the nucleotide phosphite obtained in step (a) and represented by the formula IV:
##STR16## in which B', R.sup.1, R.sup.2, R.sup.3 and L are as defined above, with a nucleoside, of the general formula V, bound to a polymeric carrier ##STR17## in which B' and R.sup.1 are as defined above and C denotes the polymeric carrier;
(c) oxidizing the carrier-bound nucleoside-nucleotides obtained in step (b) and represented by the formula:
##STR18## in which B', R.sup.1, R.sup.2, R.sup.3 and C are as defined above, with formation of phosphotriester groups,
(d) blocking free primary 5'-OH groups, which have not been reacted in the reaction according to step (b), with permanent protective groups;
(e) eliminating the protective group R.sup.2 ;
(f) optionally repeating steps (a) to (e) to introduce further nucleoside phosphate or oligonucleoside phosphate units;
and
(g) cleaving the nucleoside carrier bond and optionally eliminating the protective groups present in the oligonucleoside phosphates,
which process comprises using in step (a) as the phosphine derivative of the general formula III a compound in which R.sup.3 denotes a group of the formula VII ##STR19## in which the groups Y, which can be identical or different, represent hydrogen, methyl, and/or ethyl and Z represents an electron-attracting group, where, in the phosphine derivative of the formula III, X is chlorine, bromine, CN or SCN and L is CN or SCN, a secondary amino radical of the formula (VIII)
--NR.sub.2.sup.4
where the groups R.sup.4 are primary, secondary, or tertiary alkyl radicals having 1-10 carbon atoms, or together form a cycloalkyl radical having 5-7 carbon atoms, which can contain one or two nitrogen, oxygen, or sulfur atoms as hereoatoms, or are imidazole, triazole, tetrazole, 3-nitro-1,2,4-triazole, thiazole, pyrrole, benzotriazole, benzohydroxytriazole, imidazole substituted in the phenyl moiety, triazole substituted in the phenyl moiety, tetrazole substituted in the phenyl moiety, 3-nitro-1,2,4-triazole substituted in the phenyl moiety, thiazole substituted in the phenyl moiety, pyrrole substituted in the phenyl moiety, benzotriazole substituted in the phenyl moiety, or benzohydroxytrizole substituted in the phenyl moiety.

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Process for the preparation of oligonucleotides

Life of this patent

Process for the preparation of oligonucleotides

Life of this patent

Abstract

Claims19