Process for the synthesis of 2′-O-substituted...

Details Process for the synthesis of 2′-O-substituted... Process for the synthesis of 2′-O-substituted...

1998-01-07

2001-04-24

Riley, Jezia (Department: 1656)

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

Organic compounds

Carbohydrates or derivatives

C435S006120, C536S022100, C536S024300, C536S025300, C536S025310, C536S025320, C536S025330, C536S026100, C536S026120, C536S026220

Reexamination Certificate

active

06222025

ABSTRACT:

FIELD OF THE INVENTION
This invention is directed to an improved process for the synthesis of 2′-O-substituted pyrimidine nucleotides and oligomeric compounds containing these nucleotides. The invention features treating a 2,2′-anhydropyrimidine nucleoside or a 2S,2′-anhydropyrimidine nucleoside with a weak nucleophile and a Lewis acid. The process is economically advantageous relative to processes currently in use and is applicable to large scale synthesis. The invention further features oligomeric compounds having at least one modified pyrimidine monomeric sub-unit with modifications at 2′-O-position of the sugar and the 5 position of the pyrimidine. Oligomeric compounds of the invention exhibit increased binding affinity to nucleic acids and increased nuclease resistance. acids and increased nuclease resistance.
BACKGROUND OF THE INVENTION
2′-O-Substituted pyrimidine nucleosides are useful per se in the preparation of oligonucleotides and related compounds. 2′-O-Substituted pyrimidine nucleosides are commercially available from companies such as, for example, Glen Research, Sterling, Va., and are considered to be items of commerce. The present invention is directed to new and useful processes for the preparation of 2′-O-substituted pyrimidine nucleosides.
Oligonucleotides and their analogs have been developed for various uses in molecular biology, including use as probes, primers, linkers, adapters, and gene fragments. Modifications to oligonucleotides used in these procedures include labeling with nonisotopic labels such as fluorescein, biotin, digoxigenin, alkaline phosphatase or other reporter molecules. Modifications also have been made to the ribose phosphate backbone to increase the nuclease stability of the resulting analog. These modifications include use of methyl phosphonates, phosphorothioates, phosphorodithioate linkages, and 2′-O-methyl ribose sugar units. Other modifications have been directed to the modulation of oligonucleotide uptake and cellular distribution. The success of these oligonucleotides for both diagnostic and therapeutic uses has created an ongoing demand for improved oligonucleotide analogs.
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 functions, contribute in major proportion to many diseases and regulatory functions in animals and man. 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 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 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 the action of transcription factors. 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 have also found use in the diagnostic testing of materials including, for example, biological fluids, tissues, intact cells or isolated cellular components. As with gene expression inhibition, diagnostic applications utilize the ability of oligonucleotides to hybridize with a complementary strand of nucleic acid. Hybridization is the sequence specific hydrogen bonding of oligonucleotides 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 are also widely used as research reagents. They are particularly useful in studies exploring the function of biological molecules, as well as in the preparation of biological molecules. For example, the use of both natural and synthetic oligonucleotides 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 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. Representative of such uses are as Synthetic Oligonucleotide Probes, Screening Expression Libraries with Antibodies and Oligonucleotides, DNA Sequencing, In Vitro Amplification of DNA by the Polymerase Chain Reaction and Site-directed Mutagenesis of Cloned DNA (see Book 2 of
Molecular Cloning, A Laboratory Manual,
supra) and DNA-Protein Interactions and The Polymerase Chain Reaction (see Vol. 2 of
Current Protocols In Molecular Biology,
supra).
Oligonucleotides can be synthesized to have custom properties that are tailored for a desired use. Thus a number of chemical modifications have been introduced into oligonucleotides 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, to provide a mode of disruption (a terminating event) once sequence-specifically bound to a target, and to improve the pharmacokinetic properties of the oligonucleotide.
Gibson, K. J., and Benkovic, S. J.,
Nucleic Acids Research,
1987, 15, 6455-6467, report a phthalimide-protected 5-(3-aminopropyl)-2′-deoxyuridine nucleoside probe, which is incorporated into oligonucleotides.
Haralambidis, J., et.al.,
Nucleic Acids Research,
1987, 15, 4857-4876, reports C-5 substituted deoxyuridines which are incorporated into oligonucleotides. The substituent has a masked primary aliphatic amino group which can be further substituted with various groups.
PCT Application WO 94/17094, filed Jan. 22 1993, published Aug. 4, 1994, reports 5-substituted pyrimidine (cytosine or uracil) bases wherein the 5-substituent is C
3-14
n-alkyl, C
2-8
(E)-n-1-alkenyl, ethynyl, or a C
4-12
n-1-alkyl group, and the synthesis of oligonucleotides having one or more of the modified 5-substituted pyrimidine bases.
PCT Application No. WO 93/10820, filed Nov. 24, 1992, published Jun. 10, 1993, reports 5-(1-propynyl)uracil and 5-(1-propynyl)cytosine or related analogs, and the synthesis of oligonucleotides having one or more of the modified 5-substituted pyrimidine bases.
PCT Application No. WO 93/10820, filed Nov. 24, 1992, reports 2′- and 5-substituted pyrimidine nucleotides which are incorporated into oligonucleotides having

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