4-desmethyl nucleoside analogs and oligomers thereof

Details 4-desmethyl nucleoside analogs and oligomers thereof 4-desmethyl nucleoside analogs and oligomers thereof

1999-10-07

2001-11-20

Wilson, James O. (Department: 1623)

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

Organic compounds

Carbohydrates or derivatives

C536S022100, C536S023100, C536S024300, C536S024500, C536S025320, C536S026600, C536S027100, C536S027210, C536S027600, C536S027810, C536S028100, C536S028400, C536S028500, C536S028530, C536S028540, C435S006120, C435S375000, C514S04400A

Reexamination Certificate

active

06320040

ABSTRACT:

FIELD OF THE INVENTION
This invention is directed to oligomeric compounds that are functionalized to include covalently bound groups. Specifically, the oligomeric compounds include a tetrahydrofuran moiety that is functionalizedwith a pendant conjugate group.
BACKGROUND OF THE INVENTION
Oligonucleotides and their analogs have been developed and used in molecular biology in certain procedures as probes, primers, linkers, adapters, and gene fragments. Modifications to oligonucleotides used in these procedures include labeling with non-isotopic 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. These modifications include use of methyl and other alkyl phosphonates, phosphorothioates, phosphorodithioate, phosphoamidate and phosphotriester linkages, and 2′-O-methyl ribose sugar units. Further modifications include modification made to modulate uptake and cellular distribution. Phosphorothioate oligonucleotides are presently being used in human clinical trials for various disease states, including use as antiviral agents. In view of the success of these oligonucleotides for both diagnostic and therapeutic uses, there exists an ongoing demand for improved oligonucleotide analogs.
Oligonucleotides and like molecules can interact with native DNA and RNA in several ways. One of these is duplex formation between an oligonucleotide and a single stranded nucleic acid. A further method is via triplex formation between an oligonucleotide and double stranded DNA to form a triplex structure.
Naturally occurring or synthetic oligonucleotides, together with hybrid species having both synthetic and natural components, can collectively be referenced as “oligomeric compounds.” Because of their properties, these oligomeric compounds are known to be useful in a number of different areas. They can be used as probes in cloning, blotting procedures, and in applications such as fluorescence in situ hybridization (FISH). Also, since local triplex formation inhibits gene transcription, such oligomeric compounds can be used to inhibit gene transcription. Labeled oligomers can also be used to directly map DNA molecules, such as by tagging an oligomer with a fluorescent label and effecting hybridization to complementary sequences in duplex DNA. Oligomers can also be used as identification tags in combinatorial chemical libraries as is disclosed in patent publication WO 94/08051 and Ohlmeyer et al.,
Proc. Natl. Acad. Sci. USA
, 1993, 90, 10922-10926.
Considerable research is being directed to the application of oligonucleotides and oligonucleotide analogs that bind complementary DNA and RNA strands for use as diagnostics, research reagents and potential therapeutics.
For most uses, it is desirable to append to oligomeric compounds groups that modulate or otherwise influence their activity or their membrane or cellular transport. One method of increasing such transport is by the attachment of a pendant lipophilic group. U.S. application Ser. No. 117,363, filed Sep. 3, 1993, entitled “Amine-Derivatized Nucleosides and Oligonucleosides”, describes several alkylamino functionalities and their use in the attachment of such pendant groups to oligonucleosides. Additionally, U.S. application Ser. No. 07/943,516, filed Sep. 11, 1992, entitled “Novel Amines and Methods of Making and Using the Same” and corresponding to published PCT application WO 94/06815 describe other novel amine-containing compounds, and their incorporation into oligonucleotides for, inter alia, the purposes of enhancing cellular uptake, increasing lipophilicity, causing greater cellular retention and increasing the distribution of the compound within the cell. U.S. application Ser. No. 08/116,801, filed Sep. 3, 1993, entitled “Thiol-Derivatized Nucleosides and Oligonucleosides”, describes nucleosides and oligonucleosides derivatized to include a thioalkyl functionality, through which pendant groups are attached.
Although each of the above-noted patent applications describe useful compounds, there remains a need in the art for additional stable compounds that bind complementary DNA and RNA. There further remains a need in the art for additional methods of attaching pendant groups to oligomeric compounds to further enhance or modulate their binding, cellular uptake, or other activity.
OBJECTS OF THE INVENTION
It is an object of the invention to provide novel nucleosides for use in attaching pendant conjugate groups to oligomeric compounds.
It is a further object of the invention to provide oligomeric compounds that have pendant intercalators, nucleic acid cleaving agents, cell surface phospholipids, diagnostic agents, fluorescent agents and other conjugate groups.
It is yet another object of the invention to provide improvements in research and diagnostic methods and materials for assaying bodily states in animals, especially disease states.
It is a further object of the present invention to provide methods of producing these novel compounds.
These and other objects will become apparent from the following description and accompanying claims.
SUMMARY OF THE INVENTION
The present invention provides compounds useful in diagnostic assays and as research reagents, as well as methods and intermediates for the preparation thereof.
In one aspect of the invention there are provided compounds of structure:
wherein:
R
L
is azido or a group of formula:
 R
C
—[Y]
e
—Z—
 in which
Z is O, S or HN;
Y is a bivalent linker;
e is 0 or 1; and
R
C
is alkyl, alkenyl, alkynyl, O-alkyl, O-alkenyl, O-alkynyl, aryl, alkaryl, phosphinyl, a polyamine, a polyether, polyglycol, a steroid molecule, a reporter molecule, an aromatic or nonaromatic ring system, an aromatic lipophilic molecule, a non-aromatic lipophilic molecule, a reporter enzyme, a peptide, a protein, a water soluble vitamin, a lipid soluble vitamin, a carbohydrate, a terpene molecule, a phospholipid, an intercalator, a cell receptor binding molecule, a crosslinking agent, or a porphyrin;
B
X
is a nucleobase;
X is H, OH, O-alkyl, O-alkoxyalkyl, O-alkylamino or F;
Q is O, S, CH
2
, CHF or CF
2
; and
R
D
is H, hydroxyl, an activated phosphorous group, a nucleoside, an activated nucleotide, a nucleotide, an oligonucleotide, an oligonucleoside or a protected derivative thereof.
Compounds are also provided according to the invention having structure:
wherein:
L is a group of formula:
 R
7
—[S—(CH
2
)
q
]
u
—[HN—(CH
2
)
n
]
t
—[O—(CH
2
)
m
]
v
—Z—
 in which
Z is O, S or HN;
t, u and v are each independently integers from 0 to 200;
m, n and q are each independently integers from 1 to 4; and
R
7
is R
C
, H or a protecting group; and
X, B
X
, R
C
and R
D
are defined as above.
In some preferred embodiments t and u are 0, or u and v are zero, or t and v are zero. In other preferred embodiments t and u are 0 and m is 2. In other preferred embodiments t and v are 0 and q is 2, or u and v are 0 and n is 2. In even other preferred embodiments u is 0 and each of t and v are 1, and m and n are each 2.
Also provided according to the invention are compounds of structure:
wherein:
R
C
, B
X
, X, Q, and R
D
are defined as above.
In the above structures, in some preferred compounds, R
C
is a polyether or a polyamine. In other preferred embodiments, R
C
is a steroid molecule, preferably cholic acid, deoxycholic acid, dehydrocholic acid, cortisone, digoxigenin, testosterone, cholesterol or 3-trimethylaminomethylhydrazido cortisone.
In some preferred embodiments R
C
is a water soluble vitamin, preferably thiamine, riboflavin, nicotinic acid, pyridoxal phosphate, pyridoxine, pyridoxamine, deoxypyridoxine, pantothenic acid, biotin, folic acid, 5′-deoxyadenosylcobalamin, inositol, choline or ascorbic acid.
In other preferred embodiments R
C
is a lipid soluble vitamin, preferably a retinal, a retinol, retinoic acid, &bgr;-carotene, vitamin D, cholecalciferol, a tocopherol, or a phytol.

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