Process for the preparation of oligonucleotides

Details Process for the preparation of oligonucleotides Process for the preparation of oligonucleotides

2000-08-16

2004-10-26

McGarry, Sean (Department: 1635)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C536S023100, C536S025310, C536S025320, C536S025330, C435S091100

Reexamination Certificate

active

06809195

ABSTRACT:

FIELD OF THE INVENTION
The present invention is directed to methods for synthesizing oligonucleotides and analogs thereof. In one aspect of the invention the methods combine oxidation and capping into a single step to improve the efficiency of synthesis. The overall synthesis preferably is completed in less time with a reduction in bulk reagents required. More specific objectives and advantages of the invention will hereinafter be made clear or become apparent to those skilled in the art during the course of explanation of preferred embodiments of the invention.
BACKGROUND OF THE INVENTION
Modified oligonucleotides are of great value in molecular biological research and in applications such as anti-viral therapy. Modified oligonucleotides which can block RNA translation, and are nuclease resistant, are useful as antisense reagents. In addition to oligonucleotides that have phosphodiester internucleotide linkages, sulfurized oligonucleotides which contain, for example, phosphorothioate linkages are also of interest in these areas. Because of their chirality (Rp and Sp) phosphorothioate containing oligonucleotides are useful in determining stereochemical pathways of certain enzymes which recognize nucleic acids.
It is well known that most of the bodily states in multicellular organisms, including most disease states, are effected by proteins. Such proteins, either acting directly or through their enzymatic or other function, contribute in major proportion to many diseases and regulatory functions in animals and humans. For disease states, classical therapeutics has generally focused upon interactions with such proteins in efforts to moderate their disease-causing or disease-potentiating functions. In newer therapeutic approaches, modulation of the actual production of such proteins is desired. By interfering with the production of proteins, the maximum therapeutic effect may be obtained with minimal side effects. It is therefore a general object of such therapeutic approaches to interfere with or otherwise modulate gene expression, which would lead to undesired protein formation.
One method for inhibiting specific gene expression is with the use of oligonucleotides, especially oligonucleotides which are complementary to a specific target messenger RNA (mRNA) sequence. Several oligonucleotides are currently undergoing clinical trials for such use. Phosphorothioate oligonucleotides are presently being used as such antisense agents in human clinical trials for various disease states, including use as antiviral agents.
Transcription factors interact with double-stranded DNA during regulation of transcription. Oligonucleotides can serve as competitive inhibitors of transcription factors to modulate their action. Several recent reports describe such interactions (see Bielinska, A., et. al.,
Science,
1990, 250, 997-1000; and Wu, H., et. al.,
Gene,
1990, 89, 203-209).
In addition to such use as both indirect and direct regulators of proteins, oligonucleotides and their analogs also have found use in diagnostic tests. Such diagnostic tests can be performed using biological fluids, tissues, intact cells or isolated cellular components. As with gene expression inhibition, diagnostic applications utilize the ability of oligonucleotides and their analogs to hybridize with a complementary strand of nucleic acid. Hybridization is the sequence specific hydrogen bonding of oligomeric compounds via Watson-Crick and/or Hoogsteen base pairs to RNA or DNA. The bases of such base pairs are said to be complementary to one another.
Oligonucleotides and their analogs are also widely used as research reagents. They are useful for understanding the function of many other biological molecules as well as in the preparation of other biological molecules. For example, the use of oligonucleotides and their analogs as primers in PCR reactions has given rise to an expanding commercial industry. PCR has become a mainstay of commercial and research laboratories, and applications of PCR have multiplied. For example, PCR technology now finds use in the fields of forensics, paleontology, evolutionary studies and genetic counseling. Commercialization has led to the development of kits which assist non-molecular biology-trained personnel in applying PCR. Oligonucleotides and their analogs, both natural and synthetic, are employed as primers in such PCR technology.
Oligonucleotides and their analogs are also used in other laboratory procedures. Several of these uses are described in common laboratory manuals such as
Molecular Cloning, A Laboratory Manual
, Second Ed., J. Sambrook, et al., Eds., Cold Spring Harbor Laboratory Press, 1989; and
Current Protocols In Molecular Biology
, F. M. Ausubel, et al., Eds., Current Publications, 1993. Such uses include as synthetic oligonucleotide probes, in screening expression libraries with antibodies and oligomeric compounds, DNA sequencing, in vitro amplification of DNA by the polymerase chain reaction, and in site-directed mutagenesis of cloned DNA. See Book 2 of
Molecular Cloning, A Laboratory Manual
, supra. See also “DNA-protein interactions and The Polymerase Chain Reaction” in Vol. 2 of
Current Protocols In Molecular Biology
, supra.
Oligonucleotides and their analogs can be synthesized to have customized properties that can be tailored for desired uses. Thus a number of chemical modifications have been introduced into oligomers to increase their usefulness in diagnostics, as research reagents and as therapeutic entities. Such modifications include those designed to increase binding to a target strand (i.e. increase their melting temperatures, Tm), to assist in identification of the oligonucleotide or an oligonucleotide-target complex, to increase cell penetration, to stabilize against nucleases and other enzymes that degrade or interfere with the structure or activity of the oligonucleotides and their analogs, to provide a mode of disruption (terminating event) once sequence-specifically bound to a target, and to improve the pharmacokinetic properties of the oligonucleotide.
The synthesis of oligonucleotides has classically involved obtaining a desired product on a small scale. The synthesis of oligonucleotides has more recently evolved to the point that routine syntheses are being performed on kilogram scale. Moving forward the next step is the synthesis of oligonucleotides and analogs on ton scale to supply large quantities to meet demands for ongoing pharmaceutical sales and clinical trials. The evolution of oligonucleotide synthetic techniques toward larger scale is requiring a reevaluation of each aspect of the synthetic process. There is an ongoing need in the art of oligomer synthesis to improve the efficiency of synthesis.
The chemical literature discloses numerous protocols for coupling nucleosides through phosphorous-containing covalent linkages to produce oligonucleotides of defined sequence. One of the most routinely used protocols is the phosphoramidite protocol (see, e.g, Advances in the Synthesis of Oligonucleotides by the Phosphoramidite Approach, Beaucage, S. L.; Iyer, R. P., Tetrahedron, 1992, 48, 2223-2311 and references cited therein; and The synthesis of Modified Oligonucleotides by the Phosphoramidite Approach and their applications, Beaucage, S. L.; Iyer, R. P.,
Tetrahedron,
1993, 49, 6123-6194 and references cited therein), wherein a nucleoside or oligonucleotide having a free hydroxyl group is reacted with a protected phosphoramidite monomer in the presence of a weak acid to form a phosphite-linked structure. Oxidation of the phosphite linkage with a suitable reagent effects conversion of a P
III
internucleoside linkage to a P
V
internucleoside linkage. For the purpose of this application, such reagents include oxygen transfer reagents and sulfur transfer reagents. Subsequent hydrolysis of the cyanoethyl group yields the desired phosphodiester or phosphorothioate linkage.
Phosphoramidites are commercially available from a variety of commercial sources (included are: Glen Research, Sterling, Va.; Amersham Pharmacia Biotech Inc., Piscata

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