Process for the synthesis of oligonucleotides incorporating...

Details Process for the synthesis of oligonucleotides incorporating... Process for the synthesis of oligonucleotides incorporating...

1998-09-01

2001-01-16

Wilson, James O. (Department: 1623)

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

Organic compounds

Carbohydrates or derivatives

C536S025310, C536S025320, C536S025330, C536S025340

Reexamination Certificate

active

06175004

ABSTRACT:

FIELD OF THE INVENTION
This invention is related to synthesis of oligonucleotides. In accordance with preferred embodiments this invention provides improved processes for the synthesis of oligonucleotides incorporating 2-aminoadenosine and similar moieties.
BACKGROUND OF THE INVENTION
Oligonucleotides and their analogs have been developed for various uses in molecular biology, including use as probes, primers, linkers, adapters, and gene fragments. In a number of these applications, the oligonucleotides specifically hybridize to a target nucleic acid sequence. 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.
One method of use for oligonucleotides is the inhibition of specific gene expression, where the oligonucleotides are complementary to specific target messenger RNA (mRNA) or other sequences. This mode of action is commonly known as “antisense”. The specific binding of an antisense oligonucleotide to its target mRNA or other sequence can inhibit gene expression by at least two major mechanisms. The binding of the oligonucleotide to its target may hinder protein binding for translation and/or regulation. Moreover, the oligonucleotides may act through RNase-mediated degradation of a DNA/RNA duplex. Antisense technology is used in research applications to study the functions of certain genes. Antisense oligonucleotides can also be therapeutic agents, with one antisense drug having been approved for use and several oligonucleotides currently undergoing clinical trials.
Phosphorothioate oligonucleotides, which incorporate a phosphorothioate linkage (P═S), as opposed to a phosphodiester linkage (P═O), are presently being used as antisense agents in human clinical trials for various disease states, including use as antiviral, anticancer and anti-autoimmune agents.
Oligonucleotides have also found use in the diagnostic testing of materials including, for example, biological fluids, tissues, intact cells and isolated cellular components. As with gene expression inhibition, diagnostic applications can utilize the ability of oligonucleotides to hybridize with a complementary strand of nucleic acid. There are numerous examples of commercially available kits using probe technologies that hybridize to a target sequence for diagnostic purposes.
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 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
, supra, and DNA-Protein Interactions and The Polymerase Chain Reaction, Vol. 2 of
Current Protocols In Molecular Biology
, supra.
It is greatly desired that oligonucleotides be able to be synthesized to have customized properties which are tailored for desired uses. 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.
Oligonucleotides bind in a sequence specific manner to their target, a stretch of nucleotides from either DNA or RNA, especially messenger RNA (mRNA). The base-pairing of such interactions is the well known; A-T (or A-U) and G-(C base pairing. The G-C base pair provides three hydrogen bonds, while the A-T and A-U base pairs provide only two. Thus, the A-T base pair is energetically less favorable. By increasing the A-T base pair to have three hyrogen bonds, through chemical modification of these bases, it has been shown that a more stable duplex structure can be formed.
This increased hybridization is expected to have wide-reaching application in molecular biology. Diagnostic probes and therapeutic agents are expected to be more effective.
The incorporation of 2-aminoadenosine (2,6-diamino-purine) and similar moieties into oligonucleotides in place of adenosine provides an additional site for hydrogenbonding to uridine or thymidine. This modification has been shown to increase the binding affinity of oligonucleotides to their target RNA sequences and, to a lesser extent, DNA sequences (Gryaznov, S., et al.,
Tetrahedron Lett.
1994, 35, 2489-2492).
The use of oligonucleotides containing 2-aminoadenosine has been described in Lamm, G. M., et al. (
Nucleic Acids Res.
1991, 19, 3193-3198) and Barabino, S. M., et al., (
Nucleic Acids Res.
1992, 20, 4457-4464) wherein 2′-O-allyl antisense probes containing 2-aminoadenine were used to deplete U5 snRNP from cell extracts and to inhibit exon ligation. Oligonucleotides without 2-aminoadenosine were less effective.
Despite the benefit of enhanced hybridization, from such modification, oligonucleotides incorporating such substitutions have not been widely used due to the difficulty of incorporating 2-modified aminoadenosines into oligonucleotide chains. It has been particularly difficult to devise a protection scheme which is stable to chemical and oligonucleotide synthesis, yet can be cleanly removed during a normal deprotection cycle on a DNA synthesizer. The two exocyclic amines on the moieties have very different electronic properties and therefore different protection/deprotection properties; the N2 is electron rich and the N6 is electron poor. From an empirical point of view, when they are both protected as amides (e.g. isobutyryl), the N6 comes off very easily such that dilute basic solutions will cause partial deprotection and lower the isolated yield of the bis protected nucleotide. Once the N6 is deprotected, the N2 is extremely stable and is therefore difficult to remove.
A wide variety of methods have been used to produce oligonucleotides containing 2-aminoadenosine. Gaffney, B. L., et al. (
Tetrahedron
1984, 40, 3-13) prepared short hexamer and octomer sequences incorporating 2-aminoadenine by protecting with N-acyl. However, this synthesis method did not allow the incorporation of multiple 2-aminoadenine residues into an oligonucleotide of defined sequence.
Chollet, A., et al. (
Chemica Scripta
1986, 26, 37-40) prepared 2-aminoadenosine phosphoroamidites and phosphotriesters by protecting the N2 position of adenine with isobutyryl and the N6 position with 1-methyl-2,2-diethoxy pyrrolidine. The monomers were incorporated in an oligonucleotide chain using standard synthesis methods. How

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