Aminooxy-modified oligonucleotide synthetic intermediates

Details Aminooxy-modified oligonucleotide synthetic intermediates Aminooxy-modified oligonucleotide synthetic intermediates

2000-01-05

2001-02-27

Gitomer, Ralph (Department: 1623)

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

Organic compounds

Phosphorus esters

C564S300000, C536S025300

Reexamination Certificate

active

06194598

ABSTRACT:

FIELD OF THE INVENTION
This invention is directed to aminooxy modified oligonucleotides. Such oligonucleotides are useful as therapeutics, diagnostics, and research reagents. The invention is further directed to aminooxy nucleotides, nucleoside and nucleoside surrogates that are useful as precursors for the preparation of oligonucleotides. In certain embodiments of the invention, the inclusion of one or more aminooxy moieties of the invention in an oligonucleotide provides, inter alia, for improved binding of the oligonucleotides to a complementary strand. In further embodiments of the invention, the inclusion of one or more aminooxy moieties provides one or more conjugation sites useful for the conjugation of various useful ligands to the oligonucleotides. Such ligands include, for example, reporter groups and groups for modifying uptake, distribution or other pharmacodynamic properties.
BACKGROUND OF THE INVENTION
It has been recognized that oligonucleotides 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, 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.
For use in diagnostics and as research reagents and as well in therapeutics, 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 its targeted RNA (or DNA) are greatly desired for use as research reagents, diagnostic agents and as oligonucleotide therapeutics.
Various substitutions have been introduced in the base and sugar moieties of the nucleosides of oligonucleotides. The inclusion of certain of these substitutions has resulted in improvements in the resulting oligonucleotide. One such useful improvement is an increase in the nuclease resistance of the oligonucleotides by the introduction of 2′-substituents such as halo, alkoxy and allyloxy groups.
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 3′-endo 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 heteroduplex (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.
It is believed that the composite of the hydroxyl group's steric effect, its hydrogen bonding capabilities, and its electronegativity versus the properties of the hydrogen atom is responsible for the gross structural difference between RNA and DNA. Thermal melting studies indicate that the order of duplex stability (hybridization) of 2′-methoxy oligonucleotides is in the order of RNA-RNA>RNA-DNA>DNA-DNA.
U.S. Pat. No. 5,013,830, issued May 7, 1991, discloses mixed Oligonucleotides comprising an RNA portion, bearing 2′-O-alkyl substituents, conjugated to a DNA portion via a phosphodiester linkage. However, being phosphodiesters, these oligonucleotides are susceptible to nuclease cleavage.
European Patent application 339,842, filed Apr. 13, 1989, discloses 2′-O-substituted phosphorothioate oligonucleotides, including 2′-O-methylribooligonucleotide phosphorothioate derivatives. This application also discloses 2′-O-methyl phosphodiester oligonucleotides which lack nuclease resistance.
European Patent application 260,032, filed Aug. 27, 1987, discloses oligonucleotides having 2′-O-methyl substituents on the sugar moiety. This application also makes mention of other 2′-O-alkyl substituents, such as ethyl, propyl and butyl groups.
International Publication Number WO 91/06556, published May 16, 1991, and U.S. Pat. No. 5,466,786 discloses oligomers derivatized at the 2′ position with substituents, which are stable to nuclease activity. Specific 2′-O-substituents which were incorporated into oligonucleotides include ethoxycarbonylmethyl (ester form), and its acid, amide and substituted amide forms.
European Patent application 399,330, filed May 15, 1990, discloses nucleotides having 2′-O-alkyl substituents.
International Publication Number WO 91/15499, published Oct. 17, 1991, discloses oligonucleotides bearing 2′-O-alkyl, -alkenyl and -alkynyl substituents.
Martin, P.,
Helvetica Chimica Acta,
78, 1995, 486-504, discloses certain nucleosides and oligonucleotides prepared therefrom that include 2′-methoxyethoxy, 2′-methoxy(tris-ethoxy) and other substituents. Oligonucleotides containing nucleoside substituted with either the 2′-methoxyethoxy and 2′-methoxy(tris-ethoxy)substituents exhibited improved hybridization as judged by increase in Tm.
It has been recognized that oligonucleotides having improved fidelity hybridization are of great importance in the development of oligonucleotide useful as research reagents, diagnostic agents and therapeutic agents.
BRIEF DESCRIPTION OF THE INVENTION
In accordance with the present invention, aminooxy containing compositions that modulate the activity of DNA and RNA are provided. Preferred compositions include compounds of the structure:
wherein:
B
X
is purine or pyrimidine heterocyclic base;
T
1
and T
2
, independently, are OH, a hydroxyl protecting group, an activated phosphate group, a nucleotide, a nucleoside, or an oligonucleotide; and
L has one of the structures:
wherein
m is from 0 to 10;
y is from 1 to 10;
x is 1;
E is N(R
1
) (R
2
) or N=C(R
1
) (R
2
); and
each R
1
and R
2
is, independently, H, C
1
-C
10
alkyl, a

Affiliated with

Also associated with

Rating

Aminooxy-modified oligonucleotide synthetic intermediates does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Aminooxy-modified oligonucleotide synthetic intermediates, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Aminooxy-modified oligonucleotide synthetic intermediates will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-2583572