Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1998-06-11
2002-01-01
Geist, Gary (Department: 1623)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S025300
Reexamination Certificate
active
06335439
ABSTRACT:
FIELD OF THE INVENTION
This invention is directed to methods for the preparation of phosphoramidite compounds that are useful, for example, in the solid phase synthesis of oligonucleotides. The oligonucleotides are useful as diagnostic reagents, research reagents and therapeutics agents.
BACKGROUND OF THE INVENTION
It is well known proteins are significantly involved in many of the bodily states in multicellular organisms, including most disease states. 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 production of such proteins is desired. By interfering with the production of proteins, the maximum therapeutic effect might be obtained with minimal side effects. It is the 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.
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 functioning as both indirect and direct regulators of proteins, oligonucleotides have also 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 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 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 as primers in polymerase chain reactions (PCR) 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 is used 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, both natural and synthetic, are employed as primers in PCR technology.
Laboratory uses of oligonucleotides are described generally in 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 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
, ibid.) and DNA-Protein Interactions and The Polymerase Chain Reaction (see Vol. 2 of
Current Protocols In Molecular Biology
, ibid).
Oligonucleotides can be custom-synthesized 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 (terminating event) once sequence-specifically bound to a target; and to improve the pharmacokinetic properties of the oligonucleotides.
Thus, it is of increasing value to prepare oligonucleotides and other phosphorus-linked oligomers for use in basic research or for diagnostic or therapeutic applications. Consequently, and in view of the considerable expense and time required for synthesis of specific oligonucleotides, there has been a longstanding effort to develop successful methodologies for the preparation of specific oligonucleotides with increased efficiency and product purity.
Synthesis of oligonucleotides can be accomplished using both solution phase and solid phase methods. Oligonucleotide synthesis via solution phase in turn can be accomplished with several coupling mechanisms. However, solution phase chemistry requires purification after each internucleotide coupling, which is labor intensive and time consuming.
The current method of choice for the preparation of naturally occurring oligonucleotides, as well as modified oligonucleotides such as phosphorothioate and phosphoro-dithioate oligonucleotides, is via solid-phase synthesis wherein an oligonucleotide is prepared on a polymer support (a solid support) such as controlled pore glass (CPG); oxalyl-controlled pore glass (see, e.g., Alul, et al.,
Nucleic Acids Research
1991, 19, 1527); TENTAGEL Support, (see, e.g., Wright, et al.,
Tetrahedron Letters
1993, 34, 3373); or POROS, a polystyrene resin available from Perceptive Biosystems. Solid-phase synthesis relies on sequential addition of nucleotides to one end of a growing oligonucleotide chain. Typically, a first nucleoside (having protecting groups on any exocyclic amine functionalities present) is attached to an appropriate glass bead support and activated phosphite compounds (typically nucleotide phosphoramidites, also bearing appropriate protecting groups) are added stepwise to elongate the growing oligonucleotide. The nucleotide phosphoramidites are reacted with the growing oligonucleotide using “fluidized bed” technology to mix the reagents. The known silica supports suitable for anchoring the oligonucleotide are very fragile and thus cannot be exposed to aggressive mixing.
Additional methods for solid-phase synthesis may be found in Caruthers U.S. Pat. Nos. 4,415,732; 4,458,066; 4,500,707; 4,668,777; 4,973,679; and 5,132,418; and Koster U.S. Pat. Nos. 4,725,677 and Re. 34,069.
Phosphoramidites typically have been prepared by one of three routes. In the first, a suitably protected nucleobase is reacted with a protected bis-dialkylamino phosphite compound in the presence of 1H-tetrazole or a tetrazole salt. See Nielsen, J. et al., Nucleic Acids Res. 1986, 14, 7391; Nielsen, J. et al., J. Chem. Res.(S) 1986, 26; Hamamoto, S. et al., Chem. Lett. 1986, 1401; and Nielsen, J. et al., Nucleic Acids Res. 1987, 15, 3626. This method is disadvantageous because, inter alia, tetrazole is a health hazard, and poses disposal problems due to its explosive nature.
A second method for the preparation of phosphoramidites involves reacting the 3′-hydroxyl of a nucleoside with a protected dialklyamino chloro phosphitylting reagent. See Hering, G. et al., Nucleosides Nucleotides 1985, 4, 169; and Ugi, I. et al., J. Chem. Soc. Chem. Commun. 1997, 877. This method also is disadvantageous because of the explosive nature of the phosphitylting reagent.
A third method for the synthesis of phosphoramidites involves reacting the 3′-hydroxyl of a nucleoside with a dialklyamino
Capaldi Daniel C.
Eleuteri Alessandra
Ravikumar Vasulinga T.
Geist Gary
ISIS Pharmaceuticals Inc.
Owens Howard V.
Woodcock Washburn Kurtz Mackiewicz & Norris
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