Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1997-08-05
2002-09-10
Geist, Gary (Department: 1623)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C536S023100, C536S024300, C536S028100, C536S028500, C536S028530, C536S029200
Reexamination Certificate
active
06447998
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention is related to enhanced triplex binding and compositions useful in achieving enhanced triplex binding. In some embodiments, the invention provides modified nucleosides that provide enhanced triplex binding of single stranded oligonucleotides containing one or more of the modified nucleosides to target duplex oligonucleotides. Also provided by the invention are methods of using the modified nucleosides to prepare oligonucleotides containing them and methods of using the oligonucleotides containing the modified nucleosides in triplex binding assays and methods of separation.
2. Brief Description of Related Art
Hsieh, H.-P. and McLaughlin, L. W.,
J. Org. Chem
. (1995), 60:5356-5359, discloses the syntheses of two pyridine C-nucleosides as “deletion-modified” analogues of dT and dC.
OBJECTS OF THE INVENTION
Selected embodiments of the present invention accomplish one or more of the following objects:
A principal object of the invention is sequence specific triplex binding of single stranded oligonucleotides to duplex oligonucleotides. In particular an object is diagnosis through triplex binding.
An additional object of the invention is to provide 2-aminopyridine and 2-pyridone C-nucleosides.
An additional object of the invention is to provide oligonucleotides containing 2-aminopyridine and 2-pyridone C-nucleosides.
An additional object of the invention is to provide 2-aminopyridine and 2-pyridone C-nucleosides as intermediates in the preparation of oligonucleotides containing 2-aminopyridine and 2-pyridone C-nucleosides.
SUMMARY OF THE INVENTION
Compounds or compositions having formula (I) or (II) are provided herein:
wherein:
each R
1
is independently H or a hydroxy protecting group, or both R
1
groups are taken together to form a cyclic hydroxy protecting group;
R
2
is H, F, —OR
1
, or —OR
6
;
R
3
is H or —CH
3
;
each R
4
of formula I and II is independently H or an amine protecting group, or both R
4
groups of formula I are taken together to form a cyclic amine protecting group;
R
5
is H
, —CH
3
or —C≡C—CH
3
; and
R
6
is
and salts, solvates, resolved enantiomers and purified diastereomers thereof.
Another aspect of the invention is directed to oligomers capable of triple helix formation comprising a multiplicity of nucleosides linked by internucleoside linkages wherein at least one nucleoside is a modified nucleoside comprising a nucleoside composition of the invention.
Another aspect of the invention is directed to methods of detecting the presence, absence or amount of a particular DNA duplex in a sample suspected of containing DNA comprising contacting the sample with an oligomer of the invention under conditions wherein a triple helix is formed between the oligomer and the particular DNA duplex.
DETAILED DESCRIPTION
The present invention is directed to 2-aminopyridine and 2-pyridone C-nucleosides. In particular the invention is directed to compositions comprising a compound of the formula:
Each R
1
is independently H or a hydroxy protecting group, or both R
1
groups are taken together to form a cyclic hydroxy protecting group. Typically, each R
1
is H.
Typical R
1
hydroxy protecting groups described in Greene (pages 14-118) include Ethers (Methyl); Substituted Methyl Ethers (Methoxymethyl, Methylthiomethyl, t-Butylthiomethyl, (Phenyldimethylsilyl)methoxymethyl, Benzyloxymethyl, p-Methoxybenzyloxymethyl, (4-Methoxyphenoxy)methyl, Guaiacolmethyl, t-Butoxymethyl, 4-Pentenyloxymethyl, Siloxymethyl, 2-Methoxyethoxymethyl, 2,2,2-Trichloroethoxymethyl, Bis(2-chloroethoxy)methyl, 2-(Trimethylsilyl)ethoxymethyl, Tetrahydropyranyl, 3-Bromotetrahydropyranyl, Tetrahydropthiopyranyl, 1-Methoxycyclohexyl, 4-Methoxytetrahydropyranyl, 4-Methoxytetrahydrothiopyranyl, 4-Methoxytetrahydropthiopyranyl S,S-Dioxido, 1-[(2-Chloro-4-methyl)phenyl]-4-methoxypiperidin-4-yl, 35, 1,4-Dioxan-2-yl, Tetrahydrofuranyl, Tetrahydrothiofuranyl, 2,3,3a,4,5,6,7,7a-Octahydro-7,8,8-trimethyl-4,7-methanobenzofuran-2-yl)); Substituted Ethyl Ethers (1-Ethoxyethyl, 1-(2-Chloroethoxy)ethyl, 1-Methyl-1-methoxyethyl, 1-Methyl-1-benzyloxyethyl, 1-Methyl-1-benzyloxy-2-fluoroethyl, 2,2,2-Trichloroethyl, 2-Trimethylsilylethyl, 2-(Phenylselenyl)ethyl, t-Butyl, Allyl, p-Chlorophenyl, p-Methoxyphenyl, 2,4-Dinitrophenyl, Benzyl); Substituted Benzyl Ethers (p-Methoxybenzyl, 3,4-Dimethoxybenzyl, o-Nitrobenzyl, p-Nitrobenzyl, p-Halobenzyl, 2,6-Dichlorobenzyl, p-Cyanobenzyl, p-Phenylbenzyl, 2- and 4-Picolyl, 3-Methyl-2-picolyl N-Oxido, Diphenylmethyl, p,p′-Dinitrobenzhydryl, 5-Dibenzosuberyl, Triphenylmethyl, &agr;-Naphthyldiphenylmethyl, p-methoxyphenyldiphenylmethyl, Di(p-methoxyphenyl)phenylmethyl, Tri(p-methoxyphenyl)methyl, 4-(4′-Bromophenacyloxy)phenyldiphenylmethyl, 4,4′,4″-Tris(4,5-dichlorophthalimidophenyl)methyl, 4,4′,4″-Tris(levulinoyloxyphenyl)methyl, 4,4′,4″-Tris(benzoyloxyphenyl)methyl, 3-(Imidazol-1-ylmethyl)bis(4′,4″-dimethoxyphenyl)methyl, 1,1-Bis(4-methoxyphenyl)-1′-pyrenylmethyl, 9-Anthryl, 9-(9-Phenyl)xanthenyl, 9-(9-Phenyl-10-oxo)anthryl, 1,3-Benzodithiolan-2-yl, Benzisothiazolyl S,S-Dioxido); Silyl Ethers (Trimethylsilyl, Triethylsilyl, Triisopropylsilyl, Dimethylisopropylsilyl, Diethylisopropylsily, Dimethylthexylsilyl, t-Butyldimethylsilyl, t-Butyldiphenylsilyl, Tribenzylsilyl, Tri-p-xylylsilyl, Triphenylsilyl, Diphenylmethylsilyl, t-Butylmethoxyphenylsilyl); Esters (Formate, Benzoylformate, Acetate, Choroacetate, Dichloroacetate, Trichloroacetate, Trifluoroacetate, Methoxyacetate, Triphenylmethoxyacetate, Phenoxyacetate, p-Chlorophenoxyacetate, p-poly-Phenylacetate, 3-Phenylpropionate, 4-Oxopentanoate (Levulinate), 4,4-(Ethylenedithio)pentanoate, Pivaloate, Adamantoate, Crotonate, 4-Methoxycrotonate, Benzoate, p-Phenylbenzoate, 2,4,6-Trimethylbenzoate (Mesitoate)); Carbonates (Methyl, 9-Fluorenylmethyl, Ethyl, 2,2,2-Trichloroethyl, 2-(Trimethylsilyl)ethyl, 2-(Phenylsulfonyl)ethyl, 2-(Triphenylphosphonio)ethyl, Isobutyl, Vinyl, Allyl, p-Nitrophenyl, Benzyl, p-Methoxybenzyl, 3,4-Dimethoxybenzyl, o-Nitrobenzyl, p-Nitrobenzyl, S-Benzyl Thiocarbonate, 4-Ethoxy-1-naphthyl, Methyl Dithiocarbonate); Groups With Assisted Cleavage (2-Iodobenzoate, 4-Azidobutyrate, 4-Niotro-4-methylpentanoate, o-(Dibromomethyl)benzoate, 2-Formylbenzenesulfonate, 2-(Methylthiomethoxy)ethyl Carbonate, 4-(Methylthiomethoxy)butyrate, 2-(Methylthiomethoxymethyl)benzoate); Miscellaneous Esters (2,6-Dichloro-4-methylphenoxyacetate, 2,6-Dichloro-4-(1,1,3,3 tetramethylbutyl)phenoxyacetate, 2,4-Bis(1,1-dimethylpropyl)phenoxyacetate, Chorodiphenylacetate, Isobutyrate, Monosuccinoate, (E)-2-Methyl-2-butenoate (Tigloate), o-(Methoxycarbonyl)benzoate, p-poly-Benzoate, &agr;-Naphthoate, Nitrate, Alkyl N,N,N′,N′-Tetramethylphosphorodiamidate, N-Phenylcarbamate, Borate, Dimethylphosphinothioyl, 2,4-Dinitrophenylsulfenate); and Sulfonates (Sulfate, Methanesulfonate (Mesylate), Benzylsulfonate, Tosylate).
More typically, R
1
hydroxy protecting groups include substituted methyl ethers, substituted benzyl ethers, silyl ethers, and esters including sulfonic acid esters, still more typically, trialkylsilyl ethers, tosylates and acetates.
Typical 1,2-diol protecting groups (thus, generally where two OH groups are taken together with the R
1
protecting functionality) are described in Greene at pages 118-142 and include Cyclic Acetals and Ketals (Methylene, Ethylidene, 1-t-Butylethylidene, 1-Phenylethylidene, (4-Methoxyphenyl)ethylidene, 2,2,2-Trichloroethylidene, Acetonide (Isopropylidene), Cyclopentylidene, Cyclohexylidene, Cycloheptylidene, Benzylidene, p-Methoxybenzylidene, 2,4-Dimethoxybenzylidene, 3,4-Dimethoxybenzylidene, 2-Nitrobenzylidene); Cyclic Ortho Esters (Methoxymethylene, Ethoxymethylene, Dimethoxymethylene, 1-Methoxyethylidene, 1-Ethoxyethylidine, 1,2-Dimethoxyethylidene, &agr;-Methoxybenzylidene, 1-(N,N-Dimethylamino)ethylidene Derivative, &agr;-(N,N-Dimethylamino)benzylidene Derivative, 2-Oxacyclopentylidene); Silyl Derivatives (Di-t-butylsilylene Group,
Froehler Brian C.
Gutierrez Arnold J.
Matteucci Mark D.
Crane L. Eric
Geist Gary
Isis Pharmaceuticals , Inc.
Woodcock Washburn Kurtz Mackiewicz & Norris LLP
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