Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Patent
1989-06-19
1993-03-30
Brown, Johnnie R.
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
536 2531, 536 2533, 560 1, C07H 2100, C07C 6974
Patent
active
051985403
DESCRIPTION:
BRIEF SUMMARY
The invention relates to a process for the preparation of oligonucleotides by successive linking of individual or several nucleotide units in a homogeneous phase system, each linking step being followed by working up of the reaction mixture by chromatography.
Various processes have been disclosed as a result of the importance of synthetic oligonucleotides for isolating genetic material. According to the most recent state of the art, these processes are carried out either by the phosphate triester method or by the phosphite triester method, the use of polymeric carriers as the heterogeneous phase having proved to be particularly advantageous. The use of polymeric carriers serves, in particular, to simplify the working up of the reaction mixtures after the condensation steps by replacing expensive chromatographic separation processes with filtrations. Moreover, automation of the individual reaction steps is also possible. However, these processes have the disadvantage that oligonucleotides can thereby be isolated only in relatively small amounts.
The invention relates to a process of the abovementioned type, with which relatively large amounts of oligonucleotides having a defined sequence can be obtained. It is possible to prepare not only the naturally occurring oligo-diesters, but also analogous oligomers, such as oligo-triesters or oligophosphonates.
The process according to the invention is characterized by the following reaction steps:
a) reaction of a polyfunctional carrier molecule, which is soluble in the particular reaction mixture, of the general formula I compatible from the point of view of nucleotide chemistry and Sp is an optionally branched alkylene polyalkylene group with 1-30 carbon atoms, an arylene or polyarylene group with 1-10 aryl groups, a polyaralkylene group with 1-10 aryl groups, a polyaralkylene group with 2-100 aralkylene units, polyesters, polysiloxanes or polyamides with a molecular weight of up to 40,000, optionally branched alkylenedioxy compounds with 2-6 carbon atoms or optionally partially alkylated polyalkyleneoxy compounds with a molecular weight of up to 40,000, with at least an equimolar amount, based on --(X).sub.n, of a first nucleoside or nucleotide N.sup.1,
b) separation and purification and, if appropriate, phosphorylation of the compound obtained in stage a, of the general formula II
c) reaction of the compound II obtained in stage b with at least an equimolar amount, based on --(X--N.sup.1).sub.n, of a second nucleoside or nucleotide N.sup.2,
d) separation and purification and, if appropriate, phosphorylation of the compound obtained in stage c, of the general formula III
e) if appropriate, further successive linking and purification and, if appropriate, phosphorylation steps to form the oligonucleotide compound of the general formula IV
f) subsequent release of the oligonucleotides of the general formula V
According to a preferred embodiment of the process of the invention, carrier molecules of the general formula I in which the groups X can form a silyl ether, trityl ether or ester bond with the 3'- or 5'-hydroxyl groups of a nucleoside, nucleotide or oligonucleotide are used.
According to another advantageous embodiment of the invention, a compound in which the group Sp is an alkylene or polyalkylene group with 2-10 carbon atoms is used as the carrier molecule.
Examples of the reactive group X which are customary in nucleotide chemistry are described in Liebigs Ann. Chem. 1978, 839-853 and in Nucleic Acids Research, Symposium Series No. 7, 1980, 39-59. Typical examples are, inter alia, the following:
1. Acid halides, in particular acid chlorides and acid bromides.
2. Carboxylic acid groups, which can react with 5'-OH groups, for example in the presence of condensing agents; they can also be converted into activated trityl chloride derivatives according to the following equation:
3. Activated ester functions of the general formula --COOR'
4. OH, SH and NH.sub.2 groups.
Carrier molecules of the general formula Sp(OH).sub.n and the corresponding thio and amino de
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Brown Johnnie R.
Crane L. Eric
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