Oligoribonucleotide and ribozyme analogs with terminal...

Details Oligoribonucleotide and ribozyme analogs with terminal... Oligoribonucleotide and ribozyme analogs with terminal...

1998-04-14

2002-07-16

Riley, Jezia (Department: 1656)

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

Organic compounds

Carbohydrates or derivatives

C536S023100, C536S024500, C536S025100, C536S025300, C536S026130, C536S026710, C435S006120, C514S04400A

Reexamination Certificate

active

06420546

ABSTRACT:

The invention relates to oligoribonucleotide analogs with terminal 3′—3′ and/or 5′—5′ internucleotide linkages. This modification stabilizes the molecules altered in this way, including ribozymes, without adversely altering their properties, including, where appropriate, catalytic activities.
Nucleic acid fragments whose sequence is complementary to the coding or sense sequence of a messenger RNA or to the codogenic strand of the DNA are called antisense oligonucleotides. Oligonucleotides of this type are increasingly being used for inhibiting gene expression, usually from the viewpoint of medical therapy, in vitro, in cell culture systems and in vivo (1. E. Uhlmann, A. Peyman, Chem. Rev. 90 (1990) 543-584; 2. J. Goodchild, Bioconjugate Chem. 1 (1990) 165-187; 3. L. Whitesell, A. Rosolen, L. Neckers, Antisense Research and Development 1 (1991) 343).
Variations of the antisense principle are:
I. Triple helix-forming oligonucleotides: nucleic acid fragments which are able to bind to the DNA double strand to form a triple helix and which modulate gene expression by inhibiting transcription (J. Chubb and M. Hogan, TIBTECE 10 (1992) 132-136).
II. Ribozymes: Ribonucleic acid fragments with enzymatic activity which comprises cleavage of the target RNA, for example an zRNA, after the specific binding of the ribozyme by the same (T. R. Cech, J. Am. Med. Assoc. 260 (1988) 3030).
For it to be possible to employ antisense oligonucleotides, triple helix-forming oligonucleotides and ribozymes in biological systems it is, however, necessary for the following conditions to be fulfilled (E. Uhlmann, A. Peyman, Chem. Rev. 90 (1990) 543-584):
1. on the one hand they must be readily soluble in water, but on the other hand easily pass through the lipophilic cell membrane,
2. they must be sufficiently stable to degradation inside the cell, i.e. stable to nucleases,
3. they must form stable hybrids with in intracellular nucleic acids at physiological temperatures,
4. the hybridization must be selective; the difference in the dissociation temperature to an oligonucleotide which results in a mispairing must be sufficiently large for it still to be possible for the latter to be specifically washed out,
5. in the case of ribozymes, the catalytic activity must be retained.
Unmodified oligonucleotides and, in particular, unmodified oligoribonucleotides are subject to extensive nucleolytic degradation. This is why at an early stage investigations were carried out into the structural modification of oligonucleotides so that they better meet the abovementioned requirements, in particular are better protected against nuclease degradation. For this purpose a large number of oligonucleotide analogs has been prepared, in some cases with enormous synthetic effort (1. E. Uhlmann, A. Peyman, Chem. Rev. 90 (1990) 543-584; 2. J. Goodchild, Bioconjugate Chem. 1 (1990) 165-187).
It was recently shown that 3′-3′- and/or 5′-51-terminally linked oligodeoxynucleotides and their analogs have distinctly increased stability against nucleolytic degradation (1. B. Seeliger, A. Fröhlich, M. Montenarh: Nucleosides+Nucleotides 10 (1991) 469-477; Z. H. Rösch, A. Fröhlich, J. Ramalho-Ortigao, J. Flavio, M. Montenarh, H. Seeliger: EP 0464638A2). Surprisingly, it has now been found that the same type of terminal linkage, which is easily accessible synthetically
a) is also able to stabilize the very much more labile oligoribonucleotides to nucleases,
b) is able to stabilize ribozymes (oligoribonucleotides with particular sequence requirements) to nucleases without impairing the catalytic activity,
c) is additionally able to stabilize oligoribonucleotides and ribozymes which have been protected from nucleases by chemical modification.
The invention therefore relates to oligoribonucleotides of the formula I
in which
R
1
is hydrogen or a radical of the formula II
R
2
is hydrogen or a radical of the formula III
 but where at least one of the radicals R
2
or R
2
is a radical of the formula II or III;
B is a base such as, for example, natural bases such as adenine, thymine, cytosine, guanine or unnatural bases such as, for example, purine, 2,6-diaminopurine, 7-deazaadenine, 7-deazaguanine, N
4
,N
4
-ethanocytosine or their prodrug forms;
R
3
is, independently of one another, OH, hydrogen, O(C
1
-C
18
)alkyl, O(C
2
-C
18
)alkenyl, F, NH
2
or its prodrug forms and N
3
, but at least one R
3
radical is different from H, and R
1
is preferably OH, hydrogen, O(C
1
-C
6
) alkyl, O(C
2
-C
6
)alkenyl, F, NH
23
.
W and W′ are, independently of one another, oxygen or sulfur;
Z and Z′ are, independently of one another, O
−
; S
−
; C
1
-C
18
,-alkoxy, preferably C
12
-C
8
-alkoxy, particularly preferably C
1
-C
3
-alkoxy, especially methoxy; C
1
-C
18
-alkyl, preferably C
1
-C
8
-alkyl, particularly preferably C
1
-C
3
-alkyl, especially methyl; NER
4
with R
4
=preferably C
1
-C
18
-alkyl, particularly preferably C
1
-C
8
-alkyl, especially C
1
-C
4
-alkyl or C
1
-C
4
-alkoxy-C
1
-C
6
-alkyl, preferably methoxyethyl; NR
4
R
5
, in which R
4
is as defined above and R
5
is preferably C
1
-C
18
,-alkyl, particularly preferably C
1
-C
8
-alkyl, especially C
1
-C
4
-alkyl, or in which R
4
and R
5
are, together with the nitrogen atom carrying them, a 5-6-membered heterocyclic ring which may additionally contain another hetero atom from the series comprising O, S and N, such as, for example, morpholine;
where X is OH, H, F, Cl, Br, NH
2
, N
3
, O—C(O)—(C
1
-C
18
)-alkyl, O—C(O)—(C
2
-C
18
)-alkenyl, O—C(O)—(C
2
-C
18
)alkynyl, O—C(O)—(C
6
-C
18
)aryl, O—(C
1
-C
18
)-alkyl, O—(C
2
-C
18
)-alkenyl, O—(C
2
C
18
)alkynyl, O—(C
6
-C
18
)aryl, P(O)YY′, where Y and Y′ are defined as Z and Z′. R
3
and X in formula II can together′form a cyclic phosphoric diester.
X is preferably OR, H, F, particularly preferably OH, and
n is an integer from 5-60, preferably 10-40 and especially preferably 15-25,
and their physiologically tolerated salts.
Aryl is to be understood to mean in this connection, for example, phenyl, phenyl substituted (1-3 times) by C
1
-C
6
-alkyl, C
1
-C
6
-alkoxy and/or halogen.
The oligoribonucleotides of the formula I are preferred. Furthermore preferred are oligoribonucleotides of the formula I in which R
2
is a radical of the formula III and R
1
is hydrogen; R
1
or R
2
is a radical of the formulae II and III respectively; or R
2
is hydrogen and R
1
is a radical of the formula II, where either W or Z in the latter case is not oxygen.
Furthermore, particular mention may be made of oligoribonucleotides of the formula I in which W is oxygen, or Z and W are both oxygen.
Furthermore, particular mention may be made of oligoribonucleotides of the formula I whose base sequence B
1
, B
2
, . . . B
n
corresponds to the sequence requirements for ribozymes.
Emphasis should be placed in this connection on hammerhead ribozymes (for example Uhlenbeck, Nature 328 (1987) 596; Haseloff, Gerlach, Nature 334 (1988) 585), the hairpin ribozymes (for example Hampel et al., Nucl. Acids. Res. 18 (1990) 299), the human hepatitis &agr;-virus ribozyme (for example Branch, Robertson, Proc. Natl. Acad. Sci. USA 88 (1991) 10163) and the external guide sequence for RNase P (for example Forster, Altman, Science 249 (1990) 783), but very especially the hammerhead ribozymes.
Very particularly preferred oligoribonucleotides of the formula I are those in which R
2
is a radical of the formula III and R
1
is hydrogen.
Furthermore, mention may be made of oligoribonucleotides of the formula I which are additionally substituted by groups which favor intracellular uptake, which act in vitro or in vivo as reporter groups, and/or groups which, on hybridization of the oligoribonucleotide onto biological DNA or RNA, interact with these DNA or RNA molecules with binding or cleavage.
Examples of groups which favor intracellular uptake are lipophilic radicals such as alkyl radicals, for example with up to 18 carbon atoms, or cholesteryl, or thiocholesteryl (E. Uhlmann,

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