Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2000-12-20
2004-07-27
McGarry, Sean (Department: 1635)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S023100, C536S025340, C435S091100
Reexamination Certificate
active
06768005
ABSTRACT:
The present invention concerns a method for the synthesis of phosphorothioate triesters, and particularly oligonucleotides.
In the past 15 years or so, enormous progress has been made in the development of the synthesis of oligodeoxyribonucleotides (DNA sequences), oligoribonucleotides (RNA sequences) and their analogues ‘Methods in Molecular Biology, Vol. 20, Protocol for Oligonucleotides and Analogs’, Agrawal, S. Ed., Humana Press, Totowa, 1993. Much of the work has been carried out on a micromolar or even smaller scale, and automated solid phase synthesis involving monomeric phosphoramidite building blocks Beaucage, S. L.; Caruthers, M. H.
Tetrahedron Lett.,
1981, 22, 1859-1862 has proved to be the most convenient approach. Indeed, high molecular weight DNA and relatively high molecular weight RNA sequences can now be prepared routinely with commercially available synthesisers. These synthetic oligonucleotides have met a number of crucial needs in biology and biotechnology.
Whereas milligram quantities have generally sufficed for molecular biological purposes, gram to greater than 100 gram quantities are required for clinical trials. Several oligonucleotide analogues that are potential antisense drugs are now in advanced clinical trials. If, as seems likely in the very near future, one of these sequences becomes approved, say, for the treatment of AIDS or a form of cancer, kilogram, multikilogram or even larger quantities of a specific sequence or sequences will be required.
Many of the oligonucleotides currently of interest in the phamaceutical industry are analogues of natural oligonucleotides which comprise phosphorothioated-internucleoside linkages. When phosphorothioate linkages are present, particularly when such linkages comprise a major proportion of the linkages, and especially when they comprise 100% of the internucleoside linkages, it is highly desirable that the concentration of impurity, non-phosphorothioated linkages in the final product is kept to a pharmacologically acceptable level.
A large number of protocols for the synthesis of oligonucleotides employ acetonitrile as a solvent for the reagents employed. Acetonitrile is attractive as a solvent because it is inert towards the reagents and oligonucleotide product, it has good solvation properties and is environmentally acceptable. Commonly, for large-scale syntheses, a high concentration of acetonitrile is present during the stage when the oligonucleotide product is cleaved from the solid support. Hitherto, this has been acceptable for large scale synthesis because of the perceived inert nature of acetonitrile. However, during the course of the studies resulting in the present invention, it has now been surprisingly found that higher purity oligonucleotides can be obtained by reducing the concentration of acetonitrile present during the cleavage stage.
According to one aspect of the present invention, there is provided a process for the large-scale synthesis of phosphorothioate oligonucleotides which comprises:
a) assembling an oligonucleotide bound to a solid support in the presence of acetonitrile; and
b) cleaving the oligonucleotide from the solid support;
characterised in that the concentration of acetonitrile is reduced to less than 10% by weight of the oligonucleotide plus solid support prior to the cleavage of the oligonucleotide from the solid support.
The phosphorothioate oligonucleotides can be assembled by known techniques for solid phase synthesis, for example using H-phosphonate or particularly phosphoramidite chemistry. For the phosphoramidite approach, commonly, the sequence employed is: deprotection of the nucleoside bound to solid support, preferably at the 5′-position; coupling of a, preferably 3′-, phosphoramidite nucleoside to form a supported oligonucleotide; sulphurisation of the supported oligonucleotide by reaction with a sulphurising agent to produce a supported phosphorothioate oligonucleotide; and capping of unreacted supported nucleoside with a capping reagent. This cycle is then repeated as often as is necessary to assemble the desired sequence of the oligonucleotide. When a mixed phosphate/phosphorothioate product is desired, the sulphurisation stage can be replaced with an oxidation step to produce a phosphate linkage at the desired location. On completion of the assembly, and prior to cleavage from the support, the supported oligonucleotide is commonly washed with acetonitrile in order to remove traces of unreacted reagents.
Acetonitrile can be removed by drying of the supported oligoncleotide, optionally under reduced pressure. The acetonitrile is commonly removed at ambient temperature, for example from 15 to 30° C., although elevated temperatures, such as from 30 to 80° C., for example from 40 to 60° C., may be employed.
The process according to the first aspect of the present invention is employed for large scale synthesis of oligonucleotides. Large scale synthesis of oligonucleotides is often regarded as being at or above a batch size of 10 mmol oligonucleotide, commonly at or above 15 mmol, often at or above 25 mmol, for example greater than 50 mmol, and especially greater than 75 mmol of oligonucleotide. In many embodiments, the process of the present invention is employed for oligonucleotide synthesis at a scale in the range of from 100-500 mmol.
On completion of the assembly of the desired product, the product may be cleaved from the solid support. Cleavage methods employed are those known in the art for the given solid support. When the product is bound to the solid support via a cleavable linker, cleavage methods appropriate for the linker are employed, for example, contact with methylamine, aqueous methylamine solution, liquified ammonia, gaseous ammonia and particularly contact with concentrated aqueous ammonia solution. Following cleavage, the product can be purified using techniques known in the art, such as one or more of ion-exchange chromatography, reverse phase chromatography, and precipitation from an appropriate solvent. Further processing of the product by for example ultrafiltration may also be employed.
Solid supports that are employed in the process according to the present invention are substantially insoluble in the solvent employed, and include those supports well known in the art for the solid phase synthesis of oligonucleotides. Examples include silica, controlled pore glass, polystyrene, copolymers comprising polystyrene such as polystyrene-poly(ethylene glycol) copolymers and polymers such as polyvinylacetate. Additionally, microporous or soft gel supports, especially poly(acrylamide) supports, such as those more commonly employed for the solid phase synthesis of peptides may be employed if desired. Preferred poly(acrylamide) supports are amine-functionalised supports, especially those derived from supports prepared by copolymerisation of acryloyl-sarcosine methyl ester, N,N-dimethylacrylamide and bis-acryloylethylenediamine, such as the commercially available (Polymer Laboratories) support sold under the catalogue name PL-DMA. The procedure for preparation of the supports has been described by Atherton, E.; Sheppard, R. C.; in
Solid Phase Synthesis: A Practical Approach
, Publ., IRL Press at Oxford University Press (1984). The functional group on such supports is a methyl ester and this is initially converted to a primary amine functionality by reaction with an alkyl diamine, such as ethylene diamine.
According to a second aspect of the present invention, there is provided a process for the synthesis of phosphorothioate oligonucleotides which comprises:
a) assembling an oligonucleotide bound to a solid support in the presence of acetonitrile;
b) prior to cleaving the oligonucleotide from the solid support washing the oligonucleotide bound to a solid support with a solvent other than acetonitrile; and
c) cleaving the oligonucleotide from the solid support.
Solvents which can be employed are preferably inert solvents which do not degrade the oligonucleotide under the conditions under which the solvent is employed. Examples of ine
Douglas Mark Edward
Mellor Ben James
Wellings Donald Alfred
Avecia Limited
Epps-Ford Janet L.
McGarry Sean
Pillsbury Winthorp LLP
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