3'-Desoxypentopyranosyl nucleic acid, its production...

Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives

Reexamination Certificate

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C536S024300, C536S024500, C536S025340, C536S027100, C536S027110, C536S028100, C435S091100

Reexamination Certificate

active

06696555

ABSTRACT:

This application is a national stage application filed under 35 U.S.C. §371 of PCT International Application No. PCT/EP99/06036, filed on Aug. 18, 1999 and which claims priority from German Application No. 198 37 387.2, filed Aug. 18, 1998.
The present invention relates to a 3′-deoxypentopyranosylnucleic acid consisting essentially of 3′-deoxypentopyranosylnucleosides of the formula (I) or of the formula (II)
their preparation and use for the production of a therapeutic, diagnostic and/or electronic component.
Pyranosylnucleic acids (p-NAs) are structural types which in general are isomeric to the natural RNA and in which the pentose units are present in the pyranose form and are repetitively linked between positions C-2′ and C-4′ by phosphodiester groups. “Nucleobase” is understood here as meaning the canonical nucleobases A, T, U, C, G, but also the pairs isoguanine/isocytosine and 2,6-diaminopurinelxanthine and, within the meaning of the present invention, also other purines and pyrimidines. p-NAs, to be precise the p-RNAs derived from ribose, were described for the first time by Eschenmoser et al. (Helv. Chim. Acta 1993, 76, 2161; 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 duplices. Homochiral p-RNA strands of opposite chiral sense likewise pair controllably and are strictly non-helical in the duplex formed. This specificity, which is valuable for the synthesis of supramolecular units, is associated with the relatively low flexibility of the ribopyranose phosphate backbone and with the high 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 essentially better pairing properties make p-NAs preferred pairing systems compared with DNA and RNA for application 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 the DNAs and RNAs occurring in the natural form, which is particularly of importance in the diagnostic field.
p-RNA, however, has the following disadvantages which are to be attributed to the presence of the 3′-hydroxyl function:
1. The necessary protection of the 3′-hydroxyl group with a benzoyl protective group complicates and prolongs the synthetic route to the monomeric units considerably.
2. On account of the use of the allyl radical as a base and phosphate protective group, the deprotection and the removal of the oligonucleotide must be carried out by two successively connected steps. First, the allyl radicals are removed by the Noyori method (R. Noyori, J. Am. Chem. Soc. 1990, 112, 1691-6). Then the base-labile acyl groups must be cleaved and the oligonucleotide removed from the carrier.
3. After oligonucleotide synthesis is complete, the cleavage of the 3′-benzoyl radicals from the oligonucleotide causes difficulties. In order to remove these radicals effectively the use of hydrazine is necessary, which can lead to ring-opening of the pyrimidine bases, especially uracil and thymine.
4. 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 so high here that in general the 5-(4-nitrophenyl)-1H-tetrazole 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. The benzimidazolium triflate alternatively used also has negative points: it crystallizes out, even though rarely, in the tubing, is expensive and must moreover be recrystallized before its use.
The object of the present invention was therefore to provide and to oligomerize novel pentopyranosylnucleosides for orthogonal pairing systems, whereby the disadvantages described above can be circumvented.
Surprisingly, it has now been found that 3′-deoxypentopyranosylnucleic acids (p-DNA) do not have the disadvantages described and still have the advantageous orthogonal pairing properties (see FIG.
3
).
A subject of the present invention is therefore a 3′-deoxypentopyranosylnucleic acid consisting essentially of 3′-deoxypentopyranosylnucleosides of the formula (I),
in which
R
1
is equal to H, OH, Hal where Hal is equal to Br or Cl, a radical selected from
or —O—P[N(i-Pr)
2
] (OCH
2
CH
2
CN) where i-Pr is equal to isopropyl, or —O—PH—(═O)(—O),
R
2
, R
3
and R
4
independently of one another, identically or differently, are in each case H, 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 1-4, 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
is bonded 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
2+1
, or C
n
H
2−1
, where n has the abovementioned meaning, and
R
5
, R
6
, R
7
and R
8
independently of one another, identically or differently, are in each case H, C
n
H
2n+1
, or C
n
H
2n−1
, where n has the above-mentioned 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, are in each case ═N-, ═C(R
16
)- or —N(R
17
)- where R
16
and R
17
independently of one another, identically or differently, are in each case H or C
n
H
2n+1
or (C
n
H
2n
)NR
10
R
11
with the abovementioned meanings, the dotted lines represent optional unsaturation, and
S
c1
is hydrogen or a protective group selected from an acyl, trityl, allyloxycarbonyl, a photolabile or &bgr;-eliminable protective group, preferably a fluorenymethyloxycarbonyl (Fmoc) or 4, 4′-dimethoxytrityl (DMT) group,
or of the formula (II)
in which R
1′
is equal to H, OH, Hal where Hal is equal to Br or Cl, a radical selected from
or —O—P[N(i-Pr)
2
] (OCH
2
CH
2
CN) where i-Pr is equal to isopropyl, or —O—PH—(═O)(—O)
R
2′
, R
3′
, and R
4′
independently of one another, identically or differently, are in each case H, ═O, C
n
H
2n+1
or OC
n
H
2n−1
, 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′
, independently of one another has the abovementioned meaning of R
10
or R
11
, and
X′, Y′, and Z′ independently of one another, identically or differently, are in each case ═N—, ═C(R
16′
)— or —N(R
17′
), where R
16′
and R
17′
independently of one another have the abovementioned meaning of R
16
and R
17
, the dotted lines represent optional unsaturation, and S
c1′
, has the abovementioned meaning of S
c1
.
According to the present invention, the nucleic acid according to the invention is synthesized from 3′-deoxypentopyranosylnucleosides, further modifications, such as the conjugates described in greater detail below, likewise being included by the invention.
The 3′-deoxypentopyranosylnucleosides are in general 3′-deoxyribo-, 3′-deoxyarabino-, 3′-deoxylyxo- and/or 3′-deoxyxylopyranosylnucleos

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