Processes for the synthesis of oligomeric compounds

Details Processes for the synthesis of oligomeric compounds Processes for the synthesis of oligomeric compounds

1998-11-06

2001-01-02

Geist, Gary (Department: 1623)

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

Organic compounds

Carbohydrates or derivatives

C536S025330, C536S025340

Utility Patent

active

06169177

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to methods for the preparation of oligomeric compounds, especially those having at least one phosphodiester or phosphorothioate linkage. The methods of the invention include the use of an allyl protecting group either at selected linkages or all linkages, followed by a novel deprotection strategy step using a mercapto compound in an aqueous amine or concentrated ammonium hydroxide to effect cleavage of the allyl protecting group.
BACKGROUND OF THE INVENTION
Oligonucleotides and their analogs have been developed and used in molecular biology in a variety of procedures as probes, primers, linkers, adapters, and gene fragments. Modifications to oligonucleotides used in these procedures include labeling with nonisotopic labels, e.g. fluorescein, biotin, digoxigenin, alkaline phosphatase, or other reporter molecules. Other modifications have been made to the ribose phosphate backbone to increase the nuclease stability of the resulting analog. Examples of such modifications include incorporation of methyl phosphonate, phosphorothioate, or phosphorodithioate linkages, and 2′-O-methyl ribose sugar units. Further modifications include those made to modulate uptake and cellular distribution. With the success of these compounds for both diagnostic and therapeutic uses, there exists an ongoing demand for improved oligonucleotides, their analogs and synthetic processes for their preparation.
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 on 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, maximum therapeutic effect may be obtained with minimal side effects. It is, therefore, a general object of such therapeutic approaches to interfere with or modulate gene expression which would otherwise lead to undesired protein formation.
One method for inhibiting specific gene expression involves using 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 et. al.,
Science,
1990, 250, 997-1000; and Wu et. al.,
Gene,
1990, 89, 203-209.
In addition to their use as both indirect and direct regulators of protein production, oligonucleotides and their analogs 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 inhibition of gene expression, 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 the preparation and study of many 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 individuals untrained in molecular biology to use PCR technology. 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., Sambrook et. al., Eds., Cold Spring Harbor Laboratory Press, 1989; and
Current Protocols In Molecular Biology,
Ausubel et. al., Eds., Current Publications, 1993. Such oligonucleotides may be used as synthetic oligonucleotide probes, in screening expression libraries with antibodies and oligomeric compounds, in DNA sequencing, for 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; and “DNA-Protein Interactions and The Polymerase Chain Reaction” in Vol. 2 of
Current Protocols In Molecular Biology
, supra.
Oligonucleotides and their analogs can be tailored for desired uses. Thus a number of chemical modifications have been introduced into oligomeric compounds 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 the melting temperature, 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 the oligonucleotide is sequence-specifically bound to the target, and to improve the pharmacokinetic properties of the oligonucleotide.
Several processes are known for the solid phase synthesis of oligonucleotide compounds. These are generally disclosed in the following United States Patents: U.S. Pat. Nos. 4,458,066, issued Jul. 3, 1984; No. 4,500,707, issued Feb. 19, 1985; and No. 5,132,418, issued Jul. 21, 1992. Additionally, a process for the preparation of oligonucleotides using 2-cyanoethyl phosphoramidite intermediates is disclosed in U.S. Pat. No. 4,973,679, issued Nov. 27, 1990.
A process for the preparation of oligonucleotides using an allylic phosphorus protecting group is disclosed in U.S. Pat. No. 5,026,838, issued Jun. 25, 1991.
The chemical literature discloses numerous processes for coupling nucleosides through phosphorous-containing covalent linkages to produce oligonucleotides of defined sequence. One of the most popular processes is the phosphoramidite technique. See, e.g., Advances in the Synthesis of Oligonucleotides by the Phosphoramidite Approach, Beaucage and Iyer,
Tetrahedron,
1992, 48, 2223-2311, and references cited therein. A nucleoside or oligonucleotide having a free hydroxyl group is reacted with a protected cyanoethyl phosphoramidite monomer in the presence of a weak acid to form a phosphite-linked structure. Id. Oxidation of the phosphite linkage, followed by hydrolysis of the cyanoethyl group, yields the desired phosphodiester or phosphorothioate linkage. Id.
The cyanoethyl phosphoramidite technique, however, has significant disadvantages. For example, cyanoethyl phosphoramidite monomers are quite expensive. Considerable quantities of monomer remain unreacted in a typical phosphoramidite coupling. Unreacted monomer can be recovered, if at all, only with great difficulty. Moreover, undesired cyanide ion results from these processes. Consequently, there remains a need in the art for synthetic methods that overcome these problems.
One protectin

Affiliated with

Also associated with

Rating

Processes for the synthesis of oligomeric compounds does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Processes for the synthesis of oligomeric compounds, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Processes for the synthesis of oligomeric compounds will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-2547780