Regioselective synthesis of 2′-O-modified nucleosides

Details Regioselective synthesis of 2′-O-modified nucleosides Regioselective synthesis of 2′-O-modified nucleosides

1999-01-08

2002-06-11

Wilson, James O. (Department: 1623)

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

Organic compounds

Carbohydrates or derivatives

C536S027100, C536S024300, C536S024500, C536S025300, C536S027210, C536S028100

Reexamination Certificate

active

06403779

ABSTRACT:

FIELD OF THE INVENTION
This invention is directed to methods of regioselective alkylation of pentosyl sugar moieties at the 2′-OH position. The present invention is further directed to reduction and derivatization of the 2′-O-alkylated compounds produced by these methods. The methods are useful for the preparation of 2′-O-alkyl nucleotides, nucleosides and nucleoside surrogates that are useful as precursors for the preparation of oligomeric compounds. Such oligomeric compounds are useful as therapeutics, diagnostics, and research reagents.
In certain embodiments of the invention, the inclusion of one or more 2′-O-aminooxyethyl moieties in an oligonucleotide provides, inter alia, improved binding of the oligonucleotide to a complementary strand. In further embodiments of the invention, the inclusion of one or more 2′-O-aminooxyethyl moieties in an oligomeric compound of the invention provides one or more conjugation sites useful for the conjugation of various useful ligands. Such ligands include, for example, reporter groups and groups for modifying uptake, distribution or other pharmacodynamic properties. The specific objectives and advantages of the present invention will become apparent to the art-skilled from the description of the preferred embodiments of the invention.
BACKGROUND OF THE INVENTION
It has been recognized that oligonucleotides and oligonucleotide analogs (oligomeric compounds) can be used to modulate mRNA expression by a mechanism that involves the complementary hybridization of relatively short oligonucleotides to mRNA such that the normal and essential functions of these intracellular nucleic acids are disrupted. Hybridization is the sequence-specific base pair hydrogen bonding of an oligonucleotide to a complementary RNA or DNA.
Oligonucleotides are used as diagnostics, therapeutics and as research reagents. For this, the ability of an oligonucleotide to bind to a specific DNA or RNA with fidelity is an important factor. The relative ability of an oligonucleotide to bind to complementary nucleic acids is compared by determining the melting temperature of a particular hybridization complex. The melting temperature (T
m
), a characteristic physical property of double helices, is the temperature (in ° C.) at which 50% helical versus coil (unhybridized) forms are present. T
m
is measured by using UV spectroscopy to determine the formation and breakdown (melting) of hybridization. Base stacking, which occurs during hybridization, is accompanied by a reduction in UV absorption (hypochromicity). Consequently, a reduction in UV absorption indicates a higher T
m
. The higher the T
m
, the greater the strength of the binding of the nucleic acid strands. Therefore, oligonucleotides modified to hybridize with appropriate strength and fidelity to targeted RNA (or DNA) are greatly desired for use as research reagents, diagnostic agents, and as oligonucleotide therapeutics.
Various modifications to the base, sugar and internucleoside linkage have been introduced into oligonucleotides at selected positions, and the resultant effect relative to the unmodified oligonucleotide compared. A number of modifications have been shown to improve one or more aspects of the oligonucleotide. Useful 2′-modifications that have been shown to improve aspects of oligonucleotides include halo, alkoxy and allyloxy groups. Many 2′-O-modified oligonucleotides having increased hybridization and nuclease resistance have been used in antisense research.
The use of antisense compounds as drug candidates with potential clinical applications requires that they form stable duplexes with target mRNA's, prevent translation of messages (most often via RNase H-mediated cleavage), and have resistance to nucleases. Phosphorothioate backbone modified oligonucleotides having 2′-O-modified monomers at selected positions have been reported to be effective antisense molecules. Cook, P. D., 1998, Second Generation 2′-Modified Antisense Oligonucleotides, J. A. Bristol (Ed.),
Annu. Rep. Med. Chem
., Vol. 33, pp. 313-325, Academic Press, New York. The phosphorothioate internucleoside linkage enhances nuclease resistance, while the 2′-O-modification increases hybridization. Superior antisense activity has been shown for 2′-O-modified oligomeric compounds. Martin,
Helv. Chim. Acta
., 1995, 78, 486-504. These oligonucleotides were prepared using “gapmer” technology. Monia et al.,
J. Biol. Chem
., 1993, 268, 14514-14522. Gapmer technology utilizes nuclease-resistant internucleoside linkages at selected positions while using native or other internucleoside linkages at internal positions. Generally, the 3′ and 5′ regions of the oligomeric compound will have contiguous internucleoside linkages providing superior nuclease resistance while the internal region may have native or other internucleoside linkages.
In addition to 2′-O-methoxyethyl modified oligomeric compounds, oligomeric compounds having pseudoisosteres of 2′-O-methoxyethyl modification have also shown superior hybridization qualities. Included in this group of 2′-O-modifications is the 2′-O-aminooxyethyl (AOE) modification. Kawasaki et al., 1998, Synthesis, Hybridization, and Nuclease Resistance Properties of 2′-O-Aminooxyethyl Modified Oligonucleotides, G. Gosselin and B. Rayner (Eds.),
XIII International Round Table, Nucleosides, Nucleotides, and their Biological Applications
, Montpellier, France. The hydroxylamino function present in this modification is observed in nature in the form of glycosylated antibiotics. Walker et al.,
J. Am. Chem
., 1994, 116, 3197-3206. The hydroxylamino function has also been synthetically incorporated into oligonucleotide backbones. Peoc'h et al.,
Nucleosides Nucleotides
, 1997, 16, 959-962. Among the unique properties of the hydroxylamino function are the unusual conformational preferences of the N—O bond and the surprisingly low pK
a
(MeONH
2
, 4.2, MeONHMe, 4.75, MeONHMe
2
, 3.65).
Ikehara et al. (
European J. Biochem
., 1984, 139, 447) have reported the synthesis of a mixed octamer containing one 2′-deoxy-2′-fluoroguanosine residue or one 2′-deoxy-2′-fluoroadenine residue. Guschlbauer and Jankowski (
Nucleic Acids Res
, 1980, 8, 1421) have shown that the contribution of the C3′-endo conformer increases with increasing electronegativity of the 2′-substituent. Thus, 2′-deoxy-2′-fluorouridine contains 85% of the C3′-endo conformer.
Furthermore, evidence has been presented which indicates that 2′-substituted-2′-deoxyadenosine polynucleotides resemble double-stranded RNA rather than DNA. Ikehara et al. (
Nucleic Acids Res
., 1978, 5, 3315) have shown that a 2′-fluoro substituent in poly A, poly I, or poly C duplexed to its complement is significantly more stable than the ribonucleotide or deoxyribonucleotide poly duplex as determined by standard melting assays. Ikehara et al. (
Nucleic Acids Res
., 1978, 4, 4249) have shown that a 2′-chloro or -bromo substituent in poly(2′-deoxyadenylic acid) provides nuclease resistance. Eckstein et al. (
Biochemistry
, 1972, 11, 4336) have reported that poly-(2′-chloro-2′-deoxy-uridylic acid) and poly(2′-chloro-2′-deoxycytidylic acid) are resistant to various nucleases. Inoue et al. (
Nucleic Acids Res
., 1987, 15, 6131) have described the synthesis of mixed oligonucleotide sequences containing 2′-OMe substituents on every nucleotide. The mixed 2′-OMe-substituted oligonucleotide hybridized to its RNA complement as strongly as the RNA-RNA duplex which is significantly stronger than the same sequence RNA-DNA hetero duplex (T
m
s, 49.0 and 50.1 versus 33.0 degrees for nonamers) Shibahara et al. (
Nucleic Acids Res
., 1987, 17, 239) have reported the synthesis of mixed oligonucleotides containing 2′-OMe substituents on every nucleotide. The mixed 2′-OMe-substituted oligonucleotides were designed to inhibit HIV replication.
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