Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Patent
1997-09-15
1998-09-01
Jones, W. Gary
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
435 6, 536 2541, 536 253, 536 271, 536 2711, 536 2712, C07H 2100, C07H 1900, C07H 2102, C12Q 168
Patent
active
058012370
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL FIELD
Purification of synthetic oligonucleotides from mixtures in which the desired oligonucleotide contain a hydrophobic protecting group.
The number of nucleotide residues in a synthetic oligonucleotide may in principle be any integer >1. According to the synthetic routes employed today, the number is often <200 and usually <100.
Synthetic oligonucleotides comprise DNAs, RNAs and analogues containing modified bases, modified sugars and modified phosphate groups. A certain modification may be repeated in one, more or all of the nucleotide residues. A typical modification is thiolation in the backbone, e.g. the phosphate group may have one or two of its oxygen atoms replaced with sulphur (phosphorothioate and phosphorodithioate oligomers, respectively). Another typical modification is alkylation in certain bases, e.g. methylation.
TECHNICAL BACKGROUND AND PRIOR ART
The use of oligonucleotides as drugs will dramatically increase the demand for large scale production of highly purified synthetic oligonucleotides. Today oligonucleotides are typically synthesized by reacting, step-wise and in a predetermined order, 5'-protected nucleotides, activated at their respective phosphate group, with the deprotected 5-position in a terminal nucleotide residue of a growing oligonucleotide chain attached to a solid support. The most popular protecting groups for the 5'-position have been strongly hydrophobic with preference for 4,4'-dimethoxyphenylmethyl (dimethoxytrityl=DMTr). An alternative is 9-phenylxanthen-9-yl (pixyl=Px). The resulting 5-terminal protected oligonucleotides have typically been purified by combining chromatography on strongly hydrophobic matrixes (reverse phase chromatography=RPC) with anion exchange chromatography (IEX). In common for all RPC based purification procedures applied so far have been the use of water-soluble organic solvents (such as acetonitrile) to elute the adsorbed oligonucleotide.
A typical procedure for purification of oligonucleotides would, at minimum, include the following steps: synthesis. This is normally done by adding concentrated ammonia (25%) at an elevated temperature for several hours. 5-position in terminal nucleotide residues. still adsorbed onto the RPC resin or after elution from the resin. Deprotection is normally carried out by treatment with weak acids. increasing concentration of organic solvent, e.g. acetonitrile. column and subsequent elution by applying an increasing concentration of an inorganic salt.
For the synthesis and purification of oligonucleotides, including analogues thereof, see for instance Methods in Molecular Biology 20 (1993) (Ed. Agrawal S, Humana Press, Totowa. N.J., U.S.A.).
It has been suggested to run the RPC steps in a column in which particulate forms of a strongly hydrophobic matrix and an anion exchange matrix have been intimately mixed (U.S. Pat. No. 4,997,927). This approach demands the use of organic solvents such as acetonitrile and dichloromethane.
DRAWBACKS OF THE PRIOR ART METHODS
The earlier known methods have several drawbacks that make large scale processes difficult and expensive to design. The handling of large amounts of organic solvents demands explosive safe equipment and toxicity precautions. The production costs are further increased by the recovery and disposal of organic solvents and by extra costs related to labour and chromatography equipment for running multi-step processes.
OBJECTS OF THE INVENTION
The objects of the invention is to provide a simplified, safer and cheaper method for the purification of synthetic oligonucleotides.
THE INVENTION
These objects are achieved by applying a sample containing the desired protected oligonucleotide in water-soluble form onto a hydrophilic chromatography media (adsorbent) that is substituted with anion exchange groups and that binds the oligonucleotide under conditions of high as well as low ionic strength (for instance corresponding to a concentration of NaCl within the interval 0-3M) under conditions allowing adsorption of said protected ol
REFERENCES:
patent: 4997927 (1991-03-01), Blocker et al.
Yeung et al, "A general method of optimizing automated DNA synthesis to decrease chemical consumption to less than half", Anal. Biochem. 187:66-75, 1990.
Cubellis et al, "Use of fast protein liquid chromatography for the purification of synthetic oligonucleotides", J. Chromatography 329:406-414, 1985.
Gaur, "High performance liquid chromatography of synthetic oligonucleotides" J. Chromatography 549:207-215, 1991.
Fredman Jeffrey
Jones W. Gary
Pharmacia Biotech AB
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