Modified oligonucleotides, their preparation and their use

Details Modified oligonucleotides, their preparation and their use Modified oligonucleotides, their preparation and their use

2000-08-22

2002-11-12

Riley, Jezia (Department: 1656)

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

Organic compounds

Carbohydrates or derivatives

C435S006120, C536S023100, C536S024300, C536S025300

Reexamination Certificate

active

06479651

ABSTRACT:

BACKGROUND OF THE INVENTION
The present invention relates to novel oligonucleotides which contain modified bases and which possess valuable physical, biological and pharmacological properties, to a process for their preparation and to their use as inhibitors of gene expression (antisense oligonucleotides, ribozymes, sense oligonucleotides and triplex-forming oligonucleotides), as probes for detecting nucleic acids, as aids in molecular biology and as pharmaceuticals or diagnostic agents.
Numerous chemical modifications of oligonucleotides are known from the literature. These modifications can affect the sugar-phosphate skeleton or the nucleotide bases. Reviews of the state of the art are provided, for example, by Uhlmann & Peyman, Chem. Rev. 1990, 90, 543 and Milligan et al., J. Med. Chem. 1993, 36, 1923.
As a rule, it is necessary to modify oligonucleotides chemically since unmodified oligonucleotides are very rapidly degraded by nucleolytic activities both in cells and in the cell culture medium. Stabilization against nucleolytic degradation can be achieved by replacing the sugar-phosphate backbone or by modifying the phosphate bridge, the sugar moiety or the nucleotide base [Milligan et al., above and Uhlmann & Peyman, above].
In addition to modifications which lead to oligonucleotides which possess increased stability towards nucleolytic degradation, those modifications are also of interest which alter the hybridization behavior of the modified oligonucleotides such that the latter are able, for example, to form more stable hybridization complexes (duplexes) with intracellular nucleic acids (so-called target nucleic acids). It is possible to alter the hybridization properties of oligonucleotides by, for example, modifying the bases. The altered hybridization properties of oligonucleotides which have been modified in this way can be recognized, for example, from the melting temperatures (T
m
values) of the duplexes, which temperatures are different from those obtained with the unmodified oligonucleotides.
Thus, oligonucleotides which contain 5-bromouracil, for example, form more stable hybridization complexes with the complementary nucleic acids than do oligonucleotides which contain the corresponding, unsubstituted bases (uracil) [G. D. Fasman, CRC Handbook of Biochemistry and Molecular Biology, 3rd edition, 1975, Nucleic Acids, Vol. I, 58-585].
In addition, PCT Application WO 93/10820 discloses oligonucleotides which contain modified uracil or cytosine bases and which are able to form duplex or triplex structures with the target nucleic acids which are more stable than those formed by the unmodified oligonucleotides. Oligonucleotides which contain the base analog 2-aminoadenine have also been reported to exhibit improved hybridization properties [Chollet et al., (1988), Nucleic Acid Research, 16, 305-317]. German Patent Application P4415370.8 discloses that incorporating 8-azapurine bases into oligonucleotides increases the stability of the corresponding hybridization complexes with the target nucleic acids. In addition WO 93/09127 discloses oligonucleotides which contain substituted or unsubstituted 7-deazapurine bases and which, as a result, are more readily able to form triplex structures with the target molecules (double-stranded DNA). Oligonucleotides which contain 7-deazapurine bases which are substituted in the 7 position are also disclosed in WO 94/22892 and WO 94/24144.
However, it is not possible to predict the base modifications which will lead to an increase in duplex stability. Thus, numerous examples are known of base modifications which diminish duplex stability. Thus, PCT Application WO 92/002258 describes pyrimidine-modified oligonucleotides which exhibit decreased binding affinity for the target nucleic acids. Methyl or bromine substituents which are introduced at the 8 position of the purine ring also decrease the stability of the corresponding duplexes [E. N. Kanaya et al., Biochemistry (1987) 26 7159, and Biochemistry, 1984, 23, 4219]. Oligonucleotides which contain 7-deazaadenine form significantly fewer stable duplexes with complementary oligonucleotides than do oligonucleotides which contain adenine [Seela et al., Nucleic Acid Research (1982) 10, 1389].
SUMMARY OF THE INVENTION
The object of the present invention is, therefore, to make available novel oligonucleotides which possess advantageous properties.
It has now been found, surprisingly, that oligonucleotides which possess at least one substituted 7-deazapurine base form hybridization complexes with the target nucleic acids which are significantly more stable than those formed by comparable oligonucleotides which possess unsubstituted 7-deazapurine bases.
The invention consequently relates to oligonucleotides of the formula I
and the physiologically tolerated salts thereof, in which
B are, independently of each other, a base which is customary in nucleotide chemistry, and at least one B is a base of the formula II
 in which
R
15
and R
16
are, independently of each other,
1. hydrogen,
2. halogen,
3. (C
1
-C
10
)-alkyl,
4. (C
2
-C
13
)-alkenyl,
5. (C
2
-C
10
)-alkynyl,
6. NO
2
,
7. NH
2
,
8. cyano,
9. —S—(C
1
-C
6
)-alkyl,
10. (C
1
-C
6
)-alkoxy,
11. (C
6
-C
20
)-aryloxy,
12. SiH
3
,
13.
14. a radical as described under 3., 4. or 5. which is substituted by one or more radicals from the group SH, S—(C
1
-C
6
)-alkyl, (C
1
-C
6
)-alkoxy, OH, —NR(c)R(d), —CO—R(b), —NH—CO—NR(c)R(d), —NR(c)R(g), —NR(e)R(f) or —NR(e)R(g), or by a polyalkyleneglycol radical of the formula —[O—(CH
2
)
r
]
s
—NR(c)R(d), where r and s are, independently of each other, an integer between 1 and 18, preferably between 1 and 6, with it being possible for functional groups such as OH, SH, —CO—R(b), —NH—CO—NR(c)R(d), —NR(c)R(d), —NR(e)R(f), —NR(e)R(g) or —NR(c)R(g) additionally to be linked to one or more groups, where appropriate via a further linker, which favor intracellular uptake or serve for labeling a DNA or RNA probe, or, when the oligonucleotide analog hybridizes to the target nucleic acid, attack the latter while binding, cross-linking or cleaving, or
15. a radical as defined under 3., 4. or 5. in which from one to all the H atoms are substituted by halogen, preferably fluorine;
R(a) is OH, (C
1
-C
6
)-alkoxy, (C
6
-C
20
)-aryloxy, NH
2
or NH—T, where T is an alkylcarboxyl group or alkylamino group which is linked to one or more groups, where appropriate via a further linker, which favor intracellular uptake or serve for labeling a DNA or RNA probe or, when the oligonucleotide analog hybridizes to the target nucleic acid, attack the latter while binding, cross-linking or cleaving, R(b) is hydroxyl, (C
1
-C
6
)-alkoxy or —NR(c)R(d), R(c) and R(d) are, independently of each other, H or (C
1
-C
6
)-alkyl which is unsubstituted or substituted by —NR(e)R(f) or —NR(e)R(g), R(e) and R(f) are, independently of each other, H or (C
1
-C
6
)-alkyl, R(g) is (C
1
-C
6
)-alkyl-COOH; with the proviso that R
15
and R
16
cannot each simultaneously be hydrogen, NO
2
, NH2, cyano or SiH
3
;
E and F are, independently of each other, H, OH or NH
2
,
R
1
is hydrogen, C
1
-C
18
-alkyl, C
2
-C
18
-alkenyl, C
2
-C
18
-alkynyl, C
2
-C
18
-alkylcarbonyl, C
3
-C
19
-alkenylcarbonyl, C
3
-C
19
-alkynylcarbonyl, (C
6
-C
14
)-aryl (C
1
-C
8
)-alkyl, a protective group which is customary in nucleotide chemistry, or a radical of the formula IIIa
R
1a
is hydrogen, C
1
-C
8
-alkyl, C
2
-C
18
-alkenyl, C
2
-C
18
-alkynyl, C
2
-C
18
-alkylcarbonyl, C
3
-C
19
-alkenylcarbonyl, C
3
-C
19
-alkynylcarbonyl, (C
6
-C
14
)-aryl-(C
1
-C
8
)-alkyl, or a radical of the formula IIIb
R
2
is hydrogen, hydroxyl, C
1
-C
18
-alkoxy , C
1
-C
6
-alkenyloxy, in particular allyloxy, halogen, in particular F, azido or NH
2
;
a is oxy, sulfanediyl or methylene;
n is an integer ≧1;
W is oxo, thioxo or selenoxo;
V is oxy, sulfanediyl or imino;
Y is oxy, sulfanediyl, imino or methylene;
Y′ is oxy, sulfanediyl, imino, (CH
2
)
m
or V(CH
2
)
m
, in which
m is an integer from 1 to 18;
X is hydr

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