Pyranosyl nucleic acid conjugates

Details Pyranosyl nucleic acid conjugates Pyranosyl nucleic acid conjugates

2000-07-27

2003-08-19

Wilson, James O. (Department: 1623)

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

Organic compounds

Carbohydrates or derivatives

C536S024300, C536S024500

Reexamination Certificate

active

06608186

ABSTRACT:

This is a national stage application of international application PCT/EP98/05997, filed Sep. 21, 1998, which in turn claims priority to German application serial no. 197 41 715.9, filed Sep. 22, 1997.
The present invention relates to a pentopyranosylnucleoside of the formula (I) or of the formula (II)
its preparation and use for the production of a therapeutic, diagnostic and/or electronic component.
Pyranosylnucleic acids (p-NAs) are in general structural types which are isomeric to the natural RNA, in which the pentose units are present in the pyranose form and are repetitively linked by phosphodiester groups between the positions C-2′ and C-4′ (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. (see 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 construction of supramolecular units, is associated with the relatively low flexibility of the ribopyranose phosphate backbone and 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 construction of the backbone. These significantly better pairing properties make p-NAs pairing systems to be preferred compared with DNA and RNA for use in the construction of supramolecular units. They form a pairing system which is orthogonal to natural nucleic acids, i.e. they do not pair with the DNAs and RNAs occurring in the natural form, which is of importance, in particular, in the diagnostic field.
Eschenmoser et al. (1993, supra) has for the first time prepared a p-RNA, as shown in FIG.
2
and illustrated below.
In this context, a suitable protected nucleobase was reacted with the anomer mixture of the tetrabenzoylribopyranose by action of bis(trimethylsilyl)acetamide and of a Lewis acid such as, for example, trimethylsilyl trifluoromethanesulphonate (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 protected 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 acylated in the 2′-position, and 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 unit synthesized in this way, the 4′-DMT-3′-benzoyl-1′-nucleobase derivative of the ribopyranose, was then partly phosphitylated and bonded to a solid phase via a linker.
In the following 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 residual protective groups were then removed, and the product was purified and desalted by means of HPLC.
The described process of Eschenmoser et al. (1993, supra), however, shows the following disadvantages:
1. The use of non-anomerically pure tetrabenzoylpentopyranoses (H. G. Fletcher, J. Am. Chem. Soc. 1955, 77, 5337) for the nucleosidation reaction with nucleobases reduces the yields of the final product owing to the necessity of rigorous chromatographic cuts in the following working steps.
2. With five reaction stages, starting from ribopyranoses which have a nucleobase in the 1′-position, up to the protected 3′-benzoates, the synthesis is very protracted and carrying-out on the industrial scale is barely possible. In addition to the high time outlay, the yields of monomer units obtained are low: 29% in the case of the purine unit adenine, 24% in the case of the pyrimidine unit uracil.
3. In the synthesis of the oligonucleotides, 5-(4-nitrophenyl)-1H-tetrazole is employed as a coupling reagent in the automated p-RNA synthesis. The concentration of this reagent in the solution of tetrazole in acetonitrile is in this case so high that the 5-(4-nitrophenyl)-1H-tetrazole regularly crystallizes out in the thin tubing of the synthesizer and the synthesis thus comes to a premature end. Moreover, it was observed that the oligomers were contaminated with 5-(4-nitrophenyl)-1H-tetrazole.
4. The described work-up of p-RNA oligonucleotides, especially the removal of the base-labile protective groups with hydrazine solution, is not always possible if there is a high thymidine fraction in the oligomers.
It was therefore the object of the present invention to make available novel pentopyranosylnucleosides and a process for their preparation in which the preparation of the pentopyranosylnucleosides on a larger scale than by known processes is to be made possible and the disadvantages described above are avoided.
One subject of the present invention is therefore a pentopyranosylnucleoside of the formula (I)
in which
R
1
is equal to H, OH, Hal where Hal is equal to Br or Cl, or a radical selected from
 or —O—P[N(i-Pr)
2
]—(OCH
2
CH
2
CN)
where i-Pr is equal to isopropyl, R
2
, R
3
and R
4
independently of one another, identically or differently, are in each case H, Hal where Hal is equal to Br or Cl, NR
5
R
6
, OR
7
, SR
8
, ═O, C
n
H
2n+1
where n is an integer from 1-12, preferably 1-8, in particular 1-9, a &bgr;-eliminable group, preferably a group of the formula —OCH
2
CH
2
R
18
where R
18
is equal to a cyano or p-nitrophenyl radical or a fluorenylmethyloxycarbonyl (Fmoc) radical, or (C
n
H
2n
)NR
10
R
11
where R
10
R
11
is equal to H, C
n
H
2n+1
or R
10
R
11
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, are in each case H, OR
7
where R
7
has the abovementioned meaning, or C
n
H
2n+1
, or C
n
H
2n−1
, where n has the abovementioned meaning, and
R
5
, R
6
, R
7
and R
8
independently of one another, identically or differently, is in each case H, C
n
H
2n+1
, or C
n
H
2n−1
, where n has the abovementioned meaning, —C(O)R
9
where R
9
is equal to a linear or branched, optionally substituted alkyl or aryl radical, preferably a phenyl radical,
X, Y and Z independently of one another, identically or differently, is in each case ═N—, ═C(R
16
)— or —N(R
17
)— where R
16
and R
17
independently of one another, identically or differently, is in each case H or C
n
H
2n+1
or (C
n
H
2n
)NR
10
R
11
having the above-mentioned meanings, the dotted lines represent optional unsaturation, and S
c1
and S
c2
independently of one another, identically or differently, is in each case H or a protective group selected from an acyl, trityl or allyloxycarbonyl group, preferably a benzoyl or 4,4′-dimethoxytrityl (DMT) group, or of

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