Linker nucleoside, and production and use of the same

Details Linker nucleoside, and production and use of the same Linker nucleoside, and production and use of the same

2000-07-11

2004-03-02

Wilson, James O. (Department: 1623)

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

Organic compounds

Carbohydrates or derivatives

C536S023100, C536S026700, C536S026800, C536S027130, C536S027210, C536S027600, C536S027800, C536S028530, C536S028540, C536S028550

Reexamination Certificate

active

06699978

ABSTRACT:

The present invention relates to a linker nucleoside, its preparation and use for the covalent bonding of biomolecules to oligonucleotides, in particular p-RNA oligonucleotides.
Pyranosylnucleic acids (p-NAs) are in general structures isomeric to the natural RNA, in which the pentose units are present in the pyranose form and are repetitively linked between the positions C-2′ and C-4′ by phosphodiester groups (FIG.
1
). “Nucleobase” is understood here as meaning the canonical nucleobases A, T, U, C, G, but also the pairs isoguanine/isocytosine and 2,6-diaminopurine/xanthine and, within the meaning of the present invention, also other purines and pyrimidines. p-NAs, namely the p-RNAs derived from ribose, were described for the first time by Eschenmoser et al. (Pitsch, S. et al. Helv. Chim. Acta 1993, 76, 2161; Pitsch, S. et al. Helv. Chim Acta 1995, 78, 1621; Angew. Chem. 1996, 108, 1619-1623). They exclusively form so-called Watson-Crick-paired, i.e. purine-pyrimidine- and purine-purine-paired, antiparallel, reversibly “melting”, quasi-linear and stable duplexes. Homochiral p-RNA strands of the opposite sense of chirality likewise pair controllably and are strictly non-helical in the duplex formed. This specificity, which is valuable for the synthesis of supramolecular units, is connected with the relatively low flexibility of the ribopyranose phosphate backbone and also with the strong inclination of the base plane to the strand axis and the tendency resulting from this for intercatenary base stacking in the resulting duplex and can finally be attributed to the participation of a 2′,4′-cis-disubstituted ribopyranose ring in the synthesis of the backbone. These significantly better pairing properties make p-NAs preferred pairing systems, compared with DNA and RNA, for use in the synthesis of supramolecular units. They form a pairing system which is orthogonal to natural nucleic acids, i.e. they do not pair with DNAs and RNAs occurring in the natural form, which is particularly of importance in the diagnostic field.
Eschenmoser et al. (1993, supra) has for the first time prepared a p-RNA, as shown in FIG.
2
and explained below.
In this connection, a suitable protected nucleobase was reacted with the anomer mixture of the tetrabenzoylribopyranose by action of bis(trimethylsilyl)acetamide and a Lewis acid such as, for example, trimethylsilyl trifluormethanesulphonate (analogously to H. Vorbrüggen, K. Krolikiewicz, B. Bennua, Chem. Ber. 1981, 114, 1234.). Under the action of base (NaOH in THF/methanol/water in the case of the purines; saturated ammonia in MeOH in the case of the pyrimidines), the acyl protective groups were removed from the sugar, and the product was protected in the 3′,4′-position under acidic catalysis with p-anisaldehyde dimethyl acetal. The diastereomer mixture was isolated in the 2′-position, the 3′, 4′-methoxybenzylidene-protected 2′-benzoate was deacetalized by acidic treatment, e.g. with trifluoroacetic acid in methanol, and reacted with dimethoxytrityl chloride. The 2′→3′ migration of the benzoate was initiated by treatment with p-nitrophenol/4-(dimethylamino)pyridine/triethylamine/pyridine
-propanol. Almost all reactions were worked up by column chromatography. The key structural unit synthesized in this way, the 4′-DMT-3′-benzoyl-1′-nucleobase derivative of the ribopyranose, was then partly phosphitylated or bonded to a solid phase via a linker.
In the subsequent automated oligonucleotide synthesis, the carrier-bonded component in the 4′-position was repeatedly acidically deprotected, a phosphoramidite was coupled on under the action of a coupling reagent, e.g. a tetrazole derivative, still-free 4′-oxygen atoms were acetylated and the phosphorus atom was oxidized in order thus to obtain the oligomeric product. The remaining protective groups were then removed, and the product was purified and desalted by means of HPLC.
The disadvantage of the already known p-RNA oligonucleotides is that no methods are known to covalently bond other biomolecules to these oligonucleotides.
It was therefore the object of the present invention to make available suitable constructs which make possible covalent bonding of other biomolecules to oligonucleotides, in particular to p-RNA oligonucleotides.
A subject of the present invention is therefore a linker nucleoside of the formula (I) or (II),
in which R
1
is equal to H, OH, phosphoramidite, Hal where Hal is preferably equal to Br or Cl,
R
2
, R
3
and R
4
independently of one another, identically or differently, in each case are OC
n
H
2n−1
for formula (I) or (C
n
H
2n
)NR
10
R
11
for formula (I) and (II) where
R
10
R
11
is linked via a radical of the formula
in which R
12
, R
13
, R
14
and R
15
independently of one another, identically or differently, in each case are H, C
n
H
2n+1
or C
n
H
2n−1
or OR
7
, where R
7
is equal to H, C
n
H
2n−1
or C
n
H
2n−1
, —C(O)R where R
8
is equal to a linear or branched, optionally substituted alkyl or aryl radical, preferably a phenyl radical, where n is equal to an integer from 1-12, preferably 1-8, in particular 1-4, X, Y and Z independently of one another, identically or differently, in each case are ═N—, ═C(R
9
)— or —N(R
9
)— where R
9
and R
9′
independently of one another, identically or differently, in each case are H or C
n
H
2n+1
or (C
n
H
2n
)NR
10
R
11
having the abovementioned meanings, in a particular embodiment the radicals R
2
, R
3
and R
4
and the atoms X, Y and Z taken together have the meaning assigned to them by the structure of the linker nucleoside of the formula (I) or (II) as a pentopyranosyl- or pentofuranosylpurine, -2,6-diaminopurine, -6-purinthiol, -adenosine, -guanosine, -isoguanosine, -6-thioguanosine, -xanthine, -hypoxanthine, -indole, -tryptamine, -N-phtaloyl-tryptamine, -caffeine, -theobromine, -theophylline or benzotriazole, and S
c1
and S
c2
independently of one another, identically or differently, in each case are H or a protective group selected from an acyl, trityl or allyloxycarbonyl group, preferably a benzoyl or 4, 4′-dimethoxytrityl (DMT) group, or a phosphoester(III), phosphoester(V), thiophosphate(V), phosphonate or phosphoramidite,
or of the formula (III) or (IV)
in which R
1′
is equal to H, OH, phosphoramidite or Hal where Hal is preferably equal to Br or Cl, R
2′
,R
3′
and R
4′
independently of one another, identically or differently, in each case for formula (III) OC
n
H
2n−1
where n is equal to an integer from 1-12, preferably 1-8, in particular 1-4, or for formula (III) and (IV) (C
n
H
2n
)NR
10′
R
11′
, where R
11′
, independently of one another has the abovementioned meaning of R
10
or R
11
, and X′ in each case is ═N—, ═C(R
9′
)— or —N(R
9″
)—, where R
9′
and R
9″
independently of one another have the above-mentioned meaning of R
9
and R
9′
, in a particular embodiment the radicals R
2′
, R
3′
and R
4′
and the atom X taken together have the meaning assigned to it by the structure of the linker nucleoside of the formula (III) or (IV) as a pentopyranosyl- or pentofuranosylpyridine, -pyrimidine, -thymidine, -cytosine, -isocytosine, -uracil, and S
c1′
and S
c2′
have the abovementioned meaning of S
c1
and S
c2
.
The pentose according to the invention is in general a ribose, arabinose, lyxose and/or xylose, preferably a ribopyranose, where the pentopyranosyl moiety can have the D configuration, but also the L configuration.
Customarily, the linker nucleoside according to the invention is a pentopyranosyl- or pentofuranosylpurine, -2,6-diaminopurine, -6-purinthiol, -pyridine, -pyrimidine, -adenosine, -guanosine, -isoguanosine, -6-thioguanosine, -xanthine, -hypoxanthine, -thymidine, -cytosine, -isocytosine, -indole, -tryptamine, -N-phthaloyltryptamine, -uracil, -caffeine, -theobromine, -theophylline, -benzotriazol or -ac

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