Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Patent
1985-06-18
1988-02-16
Griffin, Ronald W.
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
536 28, 536 29, C07H 1512, C07H 1700
Patent
active
047256770
DESCRIPTION:
BRIEF SUMMARY
DESCRIPTION
The invention relates to a process for the preparation of oligonucleotides of the general formula I indicated in claim 1. The oligonucleotides prepared according to the invention have defined sequences and can be used as specific primers and probes and are of great importance for the synthesis of complete genes (Arzneimittelforschung 30, 3a, 548, (1980)).
According to the most recent state of the art, oligonucleotides are prepared by either the phosphate or phosphite triester method using polymeric carriers (Nachr. Chem. Tech. Lab. 29, 230 (1981)). In order to be able to construct defined sequences, it is necessary for the individual units (nucleosides or nucleotides) to be provided with suitable protective groups. In this context, base-labile acyl groups are generally used for the protection of the exocyclic amino groups on the heterocyclic nucleobases, and a base-labile ester bond is used to attach the oligonucleotide chain to the polymeric carrier in a customary manner, and acid-labile trityl ether groups are used to protect the primary 5'-OH group. The phosphate protective group used in the phosphate triester method is customarily either the 2-chlorophenyl or the 4-chlorophenyl group, with an ester-type bond, which can only be removed by attack of a base or a nucleophile on the phosphorus atom. This type of step is inherently undesirable since it involves the risk of cleavage of the internucleotide phosphate ester bond. This risk has been greatly reduced by the use of oximate anions (Tetrahedron Lett. 19, 2727 (1978)), although these also attack the phosphorus atom in an undesired manner in the crucial step and, moreover, have the disadvantage that a relatively small amount of desired oligonucleotide is contaminated with every large amounts of involatile salts which are difficult to extract. This not only makes the working up and subsequent purification of the synthesized oligonucleotide difficult but also leads to considerable material losses.
In the phosphite triester method, the methyl group with an ester-type bond is customarily used as the phosphate protective group which can be removed by attack of a nucleophile on the methyl C atom (J. Amer. Chem. Soc. 99, 3526 (1977)). Since attack on the P atom is avoided, there is likewise avoidance of the risk of cleavage of the internucleotide bond. The nucleophile customarily used is thiophenol/triethylamine, which are unpleasant to manipulate and, moreover, lead to involatile compounds which are difficult to extract and which, as mentioned above, both make work-up difficult and lead to considerable material losses.
Although the actual synthesis of oligonucleotides by the solid phase/phosphite or phosphate triester method takes place very efficiently and rapidly, the preparation of oligonucleotides of defined sequence remains very time-consuming. This is primarily due to the problems of the subsequent work-up and purification which take up a multiple of the actual synthesis time. The process of the invention operates at this point and provides in this connection a crucial technical improvement.
In order to obtain compounds of the formula I indicated in claim 1, ##STR2## in which B denotes a nucleobase, for example adenine (A), guanine (G), cytosine (C), thymine (T) or uracil (U) or their analogs, and R.sup.1 denotes hydrogen, hydroxyl or hydroxyl which is protected by the protective groups customary in nucleotide chemistry, and n denotes an integer from 1 to 200, according to the invention a variety of defined reaction steps are carried out, as follows: ##STR3##
R.sup.1 of the general formula II can be hydrogen; in this case the compounds of the formula I are oligodeoxynucleotides. The group R.sup.1 can also be hydroxyl or hydroxyl which is, where appropriate, protected by the protective groups customary in nucleotide chemistry. Examples of protective groups of this type are trityl, monomethoxytrityl and dimethoxytrityl, acyl, for example acetyl, benzoyl; tetrahydropyranyl, methoxytetrahydropyranyl, o-nitrobenzyl and silyl ethers, such as, for
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Koster Hubert
Sinha Nanda D.
Biosyntech GmbH
Griffin Ronald W.
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