Oligoribonucleotide- and ribozyme-analoguer with terminal 3',3'- and 5',5'-bonds respectively.

The invention relates to oligoribonucleotide analogs with terminal 3'-3 'or 5'5' internucleotide linkages. This modification stabilizes the modified molecules, including ribozymes, without adversely changing their properties, including catalytic activities.

Antisense oligonucleotides are 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. Such oligonucleotides are increasingly being used to inhibit gene expression, mostly from a medical therapeutic point of view, 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. Whititesell, A. Rosolen, L. Neckers, Antisense Research and Development 1 (1991) 343).

Variations of the antisense principle are:<ul id="ul0001" list-style="none"><li>1. Triple helix-forming oligonucleotides: nucleic acid fragments which can form a triple helix on the DNA double strand and which modulate gene expression by inhibiting transcription (J.Chubb and M.Hogan, TIBTECH 10 (1992) 132-136).</li><li>II. Ribozymes: Ribonucleic acid fragments with enzymatic activity, which consists in that the target RNA, for example an m-RNA, is cleaved by the same after the specific binding of the ribozyme (TRCech, J.Am.Med. Assoc. 260 (1988) 3030).</li></ul>

In order for antisense oligonucleotides, triple helix-forming oligonucleotides and ribozymes to be used in biological systems, the following requirements must be met (E. Uhlmann, A. Peyman, Chem. Rev. 90 (1990, 543-584)):<ul id="ul0002" list-style="none"><li>1. on the one hand, they must be readily water-soluble, on the other hand they must easily pass through the lipophilic cell membrane,</li><li>2nd they must be sufficiently stable to breakdown within the cell, ie stable against nucleases,</li><li>3rd they must form stable hybrids with intracellular nucleic acids at physiological temperatures,</li><li>4th the hybridization must be selective; the difference between the dissociation temperature and an oligonucleotide that results in a mismatch must be sufficiently large that the latter can still be washed off specifically,</li><li>5. in the case of ribozymes, the catalytic activity must be retained.</li></ul>

Unmodified oligonucleotides, and especially unmodified oligoribonucleotides, are highly subject to nucleolytic degradation. Investigations were therefore carried out early on to structurally modify oligonucleotides in such a way that they better meet the above-mentioned requirements, in particular are better protected against nuclease degradation. For this purpose, a large number of oligonucleotide analogs were produced, sometimes 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 has recently been shown that 3'-3'- or 5'-5'-terminally linked oligodeoxynucleotides or their analogues have a significantly increased stability against nucleolytic degradation (1. H.Seeliger, A.Fröhlich, M.Montenarh: Nucleosides &amp; Nucleotides 10 (1991) 469-477; 2. H. Roesch, A. Froehlich, 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 synthetically easily accessible<ul id="ul0003" list-style="none"><li>a) is also able to stabilize the much more labile oligoribonucleotides against nucleases,</li><li>b) can stabilize ribozymes (oligoribonucleotides with special sequence requirements) against nucleases without impairing the catalytic activity.</li><li>c) Oligoribonucleotides or ribozymes, which were protected by chemical modification against nucleases, can additionally stabilize.</li></ul>

The invention therefore relates to oligoribonucleotides of the formula I.<chemistry id="chem0001" num="0001"><img file="EP0593901A2_D0001.tif" /></chemistry> wherein<dl id="dl0001"><dt>R¹</dt><dd>Is hydrogen or a radical of the formula II<chemistry id="chem0002" num="0002"><img file="EP0593901A2_D0002.tif" /></chemistry></dd><dt>R²</dt><dd>Is hydrogen or a radical of the formula III,<chemistry id="chem0003" num="0003"><img file="EP0593901A2_D0003.tif" /></chemistry> however, at least one of the radicals R¹ or R² represents a radical of the formula II or III;</dd></dl> B stands for a base, for example natural bases such as adenine, thymine, cytosine, guanine or unnatural bases, for example purine, 2,6-diaminopurine, 7-deazaadenine, 7-deazaguanine, N⁴, N⁴-ethanocytosine or their prodrug forms; R³ independently of one another OH, hydrogen, O (C₁-C₁₈) alkyl, O (C₂-C₁₈) alkenyl, F, NH₂ or whose prodrug forms and N₃, but at least one R³ radical is different from H and R³ is preferably OH, hydrogen, O (C₁-C₆) alkyl, O (C₂-C₆) alkenyl, F, NH₂.<dl id="dl0002"><dt>W and W '</dt><dd>independently represent oxygen or sulfur;</dd><dt>Z and Z '</dt><dd>independently of one another O⁻; S⁻; C₁-C₁₈ alkoxy, preferably C₁-C₈ alkoxy, particularly preferably C₁-C₃ alkoxy, especially methoxy; C₁-C₁₈ alkyl, preferably C₁-C₈ alkyl, particularly preferably C₁-C₃ alkyl, especially methyl; NHR⁴, with R⁴ = preferably C₁-C₁₈ alkyl, particularly preferably C₁-C₈ alkyl, in particular C₁-C₄ alkyl, or C₁-C₄ alkoxy-C₁-C₆ alkyl, preferably methoxyethyl; NR⁴R⁵, wherein R⁴ is as defined above and R⁵ is preferably C₁-C₁₈-alkyl, particularly preferably C₁-C₈-alkyl, in particular C₁-C₄-alkyl, or wherein R⁴ and R⁵ together with the nitrogen atom carrying them are a 5-6-membered heterocyclic Ring means which can additionally contain a further heteroatom from the series O, S, N, such as morpholine;</dd></dl> in which<dl id="dl0003"><dt>X</dt><dd>stands for OH, H, F, Cl, Br, NH₂, N₃, OC (O) - (C₁-C₁₈) alkyl, OC (O) - (C₂-C₁₈) alkenyl, OC (O) - (C₂-C₁₈) Alkynyl, OC (O) - (C₆-C₁₈) aryl, O- (C₁-C₁₈) alkyl, O- (C₂-C₁₈) alkenyl, O- (C₂-C₁₈) alkynyl, O- (C₆-C₁₈) aryl 'P (O) YY', where Y and Y 'are defined as Z and Z'. In Formula II, R3 and X together can form a cyclic phosphoric diester.</dd></dl>

X is preferably OH, H, F, particularly preferably OH.    and<dl id="dl0004"><dt>n</dt><dd>is an integer from 5-60, preferably 10-40 and particularly preferably 15-25,</dd></dl> and their physiologically tolerable salts.

Aryl in this context should be understood to mean, for example, phenyl, phenyl substituted (1-3 times) with C₁-C₆-alkyl, C₁-C₆-alkoxy and / or halogen.

The oligoribonucleotides of the formula I are preferred. Also preferred are oligoribonucleotides of the formula I in which R² is a radical of the formula III and R¹ is hydrogen; R¹ or R² represents a radical of the formulas II or III; or R² is hydrogen and R¹ is a radical of the formula II, where either W or Z in the latter case do not mean oxygen.

Oligoribonucleotides of the formula I in which W is oxygen or Z and W are both oxygen may also be mentioned in particular.

In particular, oligoribonucleotides of the formula I may also be mentioned, the base sequence B¹, B², ..... Bⁿ meets the sequence requirements for ribozymes.

Here, "hammerhead ribozymes" (eg Uhlenbeck, Nature 328 (1987) 596; Haseloff, Gerlach, Nature 334 (1988) 585), the "hairpin ribozymes" (eg Hampel et al., Nucl. Acids. Res. 18 (1990) 299) the "human hepatitis α-virus ribozyme" (e.g. Branch, Robertson, Proc. Natl.Acad.Sci. USA 88 (1991) 10163) and the "external guide sequence for RNase P" (e.g. Forster, Altman, Science 249 (1990) 783), but especially the "hammerhead ribozymes".

Oligoribonucleotides of the formula I are very particularly preferred, in which R² is a radical of the formula III and R¹ is hydrogen. Oligoribonucleotides of the formula I may also be mentioned which are additionally substituted by groups which promote intracellular uptake, which serve as reporter groups in vitro or in vivo, and / or groups which, when the oligoribonucleotide hybridizes to biological DNA or RNA, or attack RNA molecules with binding or cleavage.

Examples of groups which promote intracellular uptake are lipophilic residues such as alkyl residues, for example with up to 18 carbon atoms or cholesteryl, or thiocholesteryl (E. Uhlmann, A. Peyman, Chem. Rev. 90 (1990) 543-584; J Goodchild, Bioconjugate Chem. 1 (1990) 165-187; B. Oberhauser, E. Wagner, Nucl.Acids Res. 20 (1992) 533; C.MacKellar et al. Nucl.Acids Res. 20 (1992) 3411) or conjugates that use natural carrier systems, such as Bile acid or peptides for the corresponding receptor (eg receptor-mediated endocytosis). Examples of reporter groups are fluorescent groups (eg acridinyl, dansyl, fluoresceinyl) or chemiluminescent groups such as acridinium ester groups.

Examples of oligonucleotide conjugates that bind and / or cleave to nucleic acids can be found in the citations below. (E. Uhlmann, A. Peyman, Chem. Rev. 90 (1990) 543-584; J. Goodchild, Bioconjugate Chem. 1 (1990) 165-187; Helene, Toulme, Biochim. Biophys, Acta 1049 (1990) 99 ). Conjugate partners include Acridine, psoralen, chloroethylaminoaryl, phenanthridine, azidophenazyl, azidoproflavin, phenazine, phenanthroline / Cu, porphyrin / Fe, benzo [e] pyridoindole, EDTA / Fe (Mergny et al, Science 256 (1992) 1681).

The characteristic structural modification of the oligoribonucleotides according to the invention is that the internucleotide linkages at the two chain ends are changed, ie instead of biological 3'-5 'linkages they are 3'-3' or 5'-5 'linkages. Surprisingly, it was found that this minimal structural modification is sufficient to stabilize such compounds against nuclease degradation without adversely affecting other properties, for example enzymatic activities.

As will be described below, the minor structural modification results in hybridization behavior that is almost the same as that of the biological oligoribonucleotides. This also results in the general applicability of these compounds as inhibitors of gene expression.

The compounds of the formula I are prepared in the same way as the synthesis of biological oligonucleotides in solution or preferably on a solid phase, if appropriate with the aid of an automatic synthesizer. The invention therefore also relates to a process for the preparation of the oligoribonucleotides of the formula I, characterized in that<ul id="ul0004" list-style="none"><li>a) a nucleotide unit with 3 'or. 5'-terminal phosphorus (III) or phosphorus (V) groups or their activated derivative with a further nucleotide unit with 3'- or 5'-terminal free hydroxy group or</li><li>b) the oligonucleotide is built up in the same way by fragments, in the oligonucleotides obtained according to (a) or (b), if appropriate, one or more protective groups temporarily introduced to protect other functions are split off and the oligonucleotides of the formula I thus obtained are optionally converted into their physiologically acceptable salt .</li></ul>

A carrier resin to which the first nucleoside monomer is attached via the 5'-OH group is used as the starting component for the production of oligoribonucleotides with a terminally inverted 3'3 'bond for the solid phase synthesis. To produce this component, one uses methods known from the literature (T. Atkinson, M Smith in Oligonucleotide Synthesis, MJ Gait (ed), 35-49 (1984)) carrier resin, preferably silica gel or "controlled pore glass", which is functionalized with amino groups. It is reacted with a nucleoside derivative which is protected on the nucleobase and on the 3'-OH group and had previously been converted into 5'-p-nitro-phenylsuccinate. Acyl groups, for example benzoyl, isobutyryl or phenoxyacetyl, are preferably used as base protecting groups. The 3'-position is preferably protected by the dimethoxytrityl protective group, which can be introduced according to MDMatteucci, MHCaruthers, Tetrahedron Letters 21 (1980) S.3243-3246.

The further construction of the oligoribonucleotide chain up to the penultimate chain link takes place according to methods known from the literature (Beaucage, Iyer, Tetrahedron 48 (1992) 2223), preferably using nucleoside-3'-phosphorous acid ester amides or nucleoside-3 protected on the 5'-OH group by dimethoxytrityl groups '-H-phosphonates. The 2'-hydroxy group is preferably protected by the tert-butyldimethylsilyl group. (M. Lytttle et al., J. Organ. Chem. 56 (1991) 4608; Scaringe et al., Nucl. Acids Res. 18 (1990) 5433). The 2'-amino group (synthesis of compounds with R³ = NH₂) is preferably protected with the trifluoroacetyl group (Benseler et al., Nucleosides &amp; Nucleotides 11 (1992) 1333. As the last chain link, another one on the 3'-OH group, preferably used with dimethoxytrityl protected nucleoside 5'-phosphorous ester amide or nucleoside H-phosphonate. The illustration of such an oligoribonucleotide chain with terminally twisted internucleotide bonds is shown schematically below. (Phosphoramidite cycle for the preparation of oligonucleotides with 3'-3 'and 5'-5' linkages at the ends). The representation of oligoribonucleotides with 3'-3 'or 5'-5' linkages is carried out accordingly.<chemistry id="chem0004" num="0004"><img file="EP0593901A2_D0004.tif" /></chemistry> The incorporation of 2'-modified ribonucleotide units such as, for example, 2'-O-alkyl 2'-deoxyribonucleotides (Iribarren et al., Proc. Natl. Acad. Sci. USA 87 (1990) 7747; Sproat, Lamond in oligonucleotides) also takes place according to methods known from the literature and Analogues: F. Eckstein, Ed., IRL Press, Ocford 1991); 2'-F- and 2'-NH₂-2'deoxyribonucleotides (Benseler et al., Nucleosides &amp; Nucleotides 11 (1992) 1333; Pieken et al., Science 253 (1991) 314; Olsen et al., Biochemistry 30 (1991 ) 9735).

For structure and sequence analysis, the oligoribonucleotides are labeled as described in Example 4 below. This is done by radioactive labeling, preferably with the help of 5'-γ³²P-ATP / polynucleotide kinase. This radioactive labeling takes place on the free 5'-OH group, ie opposite an oligonucleotide with only biological 3'-5 'linkages at the opposite end of the nucleotide chain.

The sequences with a 3'-3 'inversion have a 5'-OH group on both sides and are therefore partially phosphorylated on both sides.

The oligonucleotides of the formula I are used for hybridization-chemical methods of regulating or suppressing the biological function of nucleic acids based on the addition to double- or single-stranded nucleic acids or their cleavage, and for selectively suppressing the expression of viral genome functions and for the prophylaxis and therapy of virus functions, to suppress oncogene function and to treat cancer.

The behavior of an oligoribonucleotide of the formula I constructed according to the invention and dissolved in blood serum can be regarded as a measure of the stability in vivo. The general test is described in Example 4. In contrast to the 3'-5'-oligoribonucleotides, the oligoribonucleotides according to the invention are degraded much more slowly.

Example 5 demonstrates that the oligoribonucleotides according to the invention which meet the sequence requirements for hammerhead ribozymes do not differ in their enzymatic activity from the unmodified ribozymes.

Description of the figures:

<dl id="dl0005"><dt>Fig. 1:</dt><dd>Stability of p53-INV in serum</dd><dt>Fig. 2:</dt><dd>Kinetics of substrate cleavage using modified ribozymes</dd><dt>Fig. 3:</dt><dd>Nucleolytic degradation of p53-1 and p53-INV</dd><dt>Fig. 4:</dt><dd>Nucleolytic degradation of Fp 53-INV and p53-F (U, C), INV</dd></dl>

Example 1:

Synthesis of 2'-fluoro-2'desoxynucleoside units (Appendix 1) 5'-O- (dimethoxytrityl) -2'-fluoro-2'-deoxyuridine

In a 50 ml Schlenk flask 0.5 g (about 2 mmol) of 2'-fluoro-2'-deoxyuridine with two 10 ml abs. Pyridine coevaporated. The dried nucleoside is treated with 25 ml abs. Pyridine was added and 0.66 g (about 2.2 mmol) of dimethoxytrityl chloride and 10 mg of 4-dimethylaminopyridine were added at room temperature. After three hours, 1 ml of methanol was added to the mixture and then concentrated to dryness in vacuo. The remaining oil is taken up in 50 ml of methylene chloride (deacidified with aluminum oxide) and extracted three times with 50 ml of water. The organic phase is dried over sodium sulfate. After the methylene chloride had been stripped off, the crude product remained as a solid foam. To remove higher tritylated constituents and the tritanol, the crude product was at 40-50 ° C with 30 ml abs. Digested benzene. 0.75 g corresponding to 67% of the theory of a white solid is obtained.

4-N-acetyl-2'-fluoro-2'-deoxycytidine

In a 100 ml Schlenk flask, 1 g (approx. 4 mmol) of 2'-fluoro-2'-deoxycytidine hydrochloride is washed twice with 20 ml of abs. Pyridine and twice with 10 ml of abs. Acetonitrile coevaporated. The dried nucleoside material is suspended in 40 ml of abs. DMF and adds 0.6 ml (ca.4.4 mmol) acetic anhydride. 0.5 ml (4.4 mmol) of abs are added dropwise over the course of a day. Triethylamine to the approach. The solvent is then removed in an oil pump vacuum. The crude product is washed with 50 ml of dieethyl ether and then dried. The purification was carried out by column chromatography (silica gel 60H, column 4 × 10 cm, eluent methylene chloride with 0.1% pyridine, gradient methanol); the product is eluted at 8% methanol. When the solvent is stripped off, 0.83 g (71% of the theoretical yield) of product remains.

5'-O- (Dimethoxytrityl) -4-N-acetyl-2'-fluoro-2'-deoxycytidine

In a 100 ml Schlenk flask, 4 mmol of 4-N-acetyl-2'-fluoro-2'-deoxycytidine with 25 ml abs. Pyridine was added and 1.3 g (about 4.4 mmol) of dimethoxytrityl chloride and 20 mg of 4-dimethylaminopyridine were added at room temperature. After three hours, 1 ml of methanol was added to the mixture and then concentrated to dryness in vacuo. The remaining oil is taken up in 50 ml of methylene chloride (deacidified with aluminum oxide) and extracted three times with 50 ml of water. The organic phase is dried over sodium sulfate. After the methylene chloride had been stripped off, the crude product remained as a solid foam. To purify the crude product, it is chromatographed on silica gel 60H (column 2 × 20 cm, eluent methylene chloride with 0.1% pyridine, gradient methanol). The product was eluted with 3% methanol in methylene chloride. After the solvent has been stripped off, 1.39 g (59% of theory) of a white foamy solid remain.

Phosphorous acid ester amides of the 2'-fluoro-2'-deoxynucleosides

1 mmol of the protected monomer is abs in 5 ml. Methylene chloride and 1 ml abs. Dissolved diisopropylamine. 1.2 mmol of chloro-N, N-diisopropylamino-β-cyanoethoxyphosphine are added dropwise under argon with a disposable syringe. After an hour, the conversion is almost quantitative and the reaction can be stopped with 0.1 ml of methanol. The mixture is taken up in 20 ml of ethyl acetate and extracted three times with 20 ml of saturated NaCl solution. The organic phase is dried over sodium sulfate and the solvent is stripped off. The crude product is taken up in 5 ml of methylene chloride and 400 ml of abs. Petroleum ether precipitated at room temperature. After the precipitation has been collected, it is dried on an oil pump and stored at -20 ° C.

5'-O- (Dimethoxytrityl) -4-N-acetyl-2'-fluoro-2'-deoxycytidine-diisopropylamino-β-cyanoethoxyphosphine:

Batch: 0.59 g (1 mmol) 5'-O- (dimethoxytrityl) -4-N-acetyl-2'-fluoro-2'-deoxycytidine Yield: 0.60 g (0.78 mmol, 78% of theory)

5'-O- (Dimethoxytrityl) -2'-fluoro-2'-deoxyuridine-diisopropylamino-β-cyanoethoxyphosphine:

Batch: 0.55 g (1 mmol) of 5'-O- (dimethoxytrityl) -2'-fluoro-2'-deoxyuridine Yield: 0.61 g (0.83 mmol, 83% of theory)

Example 2:

Loading of CPG 10-1400 carrier material with 3'-O-dimethoxytrityl deoxyribonucleoside units (Appendix 2)

3'-O-DMTr-deoxyribonucleoside 5'-O-succinate

<dl id="dl0006"><dt>Approach:</dt><dd>1.0 mmol 3'-O-DMTr-dN 0.8 mmol succinic anhydride (80 mg) 0.5 mmol dimethylaminopyridine (61 mg)</dd></dl> The reaction of succinic anhydride with the 5'-OH group of the deoxyribonucleosides was carried out in 5 ml of absolute pyridine with DMAP as catalyst overnight at room temperature. After the reaction was complete, the solution was concentrated and the pyridine was removed by azeotropic distillation with toluene three times. The residue was taken up in dichloromethane, washed with 10% ice-cold citric acid solution and H₂O and the organic phase was evaporated in vacuo. The crude product was dissolved in about 3 ml of toluene and precipitated in 200 ml of n-hexane.

Carrier loading

<dl id="dl0007"><dt>Approach:</dt><dd>0.8mmol 3'-O-DMTr-dN-5'-O-succinate 0.8 mmol p-nitrophenol (112 mg) 2.0 mmoles of dicyclohexylcarbodiimide 3 g of aminopropylated CPG 10-1400</dd></dl> The protected succinylated deoxytribonucleoside was added to a solution of p-nitrophenol in 5 ml of absolute dioxane and 0.2 ml of pyridine and then DCCI was added as a condensing agent. The reaction was complete after 3 hours. The precipitated dicyclohexylurea was suctioned off under argon and the filtrate was added directly to a suspension of the functionalized carrier material in 15 ml of absolute DMF. 0.8 ml of triethylamine was added and the mixture was shaken overnight. The loaded carrier was then suctioned off, washed with methanol and ether and dried in a desiccator. To block unreacted amino groups, the loaded support was shaken for 1 hour at room temperature with a solution of 1 ml of acetic anhydride and 50 mg of dimethylaminopyridine in 15 ml of absolute pyridine, then suction filtered, washed with methanol and ether and dried.

Example 3:

Synthesis of oligoribonucleotides with 2'-fluoro-2'-deoxyuridine units and a 3'-3'-phosphodiester bond at the 3'-terminus

The synthesis of the modified "hammerhead" ribozyme (Tab. 1) was carried out on a 0.2 μmol scale using the DNA synthesizer Gene Assember Puls from Pharmacia. The support material for the synthesis was functionalized with the deoxyadenosine component suspended via the 5'-hydroxyl group; In synthesis, this results in an inverted structure at the 3'-terminus of the oligonucleotide. The presentation was carried out according to the standard protocol for oligoribonucleotide synthesis in the phosphorous acid ester amide method.<img file="EP0593901A2_D0005.tif" /> fN: 2'-fluoro-2-deoxynucleosides rN: ribonucleosides The phosphorous ester amides of the 2'-fluoro-2'-deoxynucleosides were used 0.12 M in acetonitrile. In the chain extension step, 0.1 ml of the amidophosphite is reacted with 0.37 ml of the tetrazole solution (0.5 M) with the carrier-bound 5'-hydroxyl groups of the oligonucleotide. After a coupling time of 12 minutes, capping is carried out as standard, oxidized and detritylated in preparation for the next coupling step. The coupling yields averaged 99%.

After the synthesis, the support is transferred to a screwable Eppendorf reaction vessel to split off the base-labile protective groups and to split the suspension. The mixture was incubated at 55 ° C. for 12 hours with 2 ml of a 3: 1 mixture of 32% ammonia and ethanol. The supernatant solution was removed, cooled to -20 ° C and carefully lyophilized. The dry residue was suspended in 0.4 ml of a 1.1 M TBAF solution in THF and incubated for a further 16 hours at room temperature. The reaction was stopped by adding the same volume of a triethylammonium acetate buffer (TEAA buffer). The solution was cooled to -70 ° C and carefully concentrated to 0.4 ml. After adding 40 µl sodium acetate, 1.4 ml ethanol and 5 µl acetic acid, the product was precipitated overnight at -20 ° C. The sample was centrifuged and the supernatant discarded. The dry oligonucleotide was extracted with a 1: 1 mixture of formamide blue marker and water to apply it to an acrylamide gel (20%, 7M urea). In order to identify and cut out the product band, the gel was covered with cellophane films. Elution was carried out at 40 ° C. with ammonium acetate solution. After 5 hours the solution was precipitated as described above. The oligonucleotide was washed with 70% ethanol, resuspended in 70% ethanol and stored at -70 ° C.

Example 4:

Investigation of the stability of the modified ribozymes in the blood serum test

The ribozyme p53 and the modified oligoribonucleotides p53-INV and Fp53-INV were radioactively labeled enzymatically by T4 polynucleotide kinase in the presence of (-³²p) -dATP (specific activity: 4500 Ci (mmol). The sequences with a 3'-3 ' -Inversion have a 5'-hydroxy group on both sides and are therefore partially phosphorylated at both ends.

The labeled ribozymes were treated with fresh human serum.<dl id="dl0008"><dt>Approach:</dt><dd>1 pmol phosphorylated ribozyme 20 µl serum</dd></dl> The samples were incubated at 37 ° C. After the following times, 2 µl were removed and phenolized: p53: 0, 1, 2, 5, 10, 15, 30, 60 min. p53-INV and Fp53-INV: 0, 1, 2, 5, 10, 15, 30, 60, 120, 240 min.

The phenolized samples were lyophilized, taken up in 95% formamide loading buffer and electrophoresed on a 20% polyacrylamide gel with 8 M urea at 55 ° C.

The intensity of the bands on the X-ray film was determined using a laser densitometer (see Fig. 1). The half-lives of the investigated ribozymes (t1 / 2 = 30 min for Fp53-INV, 1 min for P53-INV and &lt;&lt; 1 min for the biological oligoribonucleotide p-53 (see Example 5) clearly show the protective effect of terminal inversion .

Example 5

A 20-mer substrate oligoribonucleotide, SB-1 5'-r (GC CCC UGU CAU CUU UUG UCC) -3 'was enzymatically treated with T4 polynucleotide kinase in the presence of 32 P-ATP (specific activity: 4500 Ci / mmol on the 5th '- radioactively marked end. The cleavage reaction of SB-1 by various ribozymes was carried out as follows: The reaction conditions were 50mM Tris HCl, pH 7.5, 20mM MgC₂ at 50 ° C. The substrate concentration SB-1 was 0.025 µM (Varies to 0.05, 0.1 and 0.25 µM), the ribozyme concentration was 0.02 µM. A sample was taken at 1 min, 5 min, 10 min, 15 min and 30 min over 30 min and loading buffer was added. The samples were electrophoresed on a 20% polyacrylamide gel (8M urea) at 55 ° C. The decrease in the intensity of the bands of SB-1 was determined on the X-ray film using a laser densitometer. The following were used as ribozymes: p53-INV; Fp53-INV (see above); p53 and Fp53<img file="EP0593901A2_D0006.tif" /> The speed of the splitting of SB-1 does not differ when using p53 and p53-INV. Fp53 and Fp53-INV also have the same activity, but this is about a factor 5 less than that of p53.

Example 6

Substrate cleavage and kinetic measurements

The preliminary initial rates of the reaction were measured with 40 nM substrate and 4 nM enzyme in 50 nM Tris-Cl (pH 7.5). The reaction was started by adding 10 nM MgCl₂. The amount of the cleavage product at 55 ° C was measured after 1, 2, 5, 10 and 15 minutes.

From this experiment the K_m- Approximately determined value. More precise measurements of the initial reaction rates were carried out according to Suelter, CH (1985) in "A practical Guide Enzymology", J. Wiley New York, 231. Here, 40 nM enzyme were carried out in six separate reactions, each with 25 nM, 50 nM, 100 nM, 200 nM, 500 nM and 1000 nM substrate. After certain time intervals, an aliquot of 2 μl was removed and the reaction was stopped by adding phenol. The samples were then separated on a denaturing gel (20% PAGE, 7 M urea) and analyzed.<dl id="dl0009"><dt>Substrate:</dt><dd>SB-1 (example 5)</dd><dt>Ribozymes:</dt><dd>p53-INV (example 3)<img file="EP0593901A2_D0007.tif" /></dd></dl>

Determination of the kinetic parameters

The initial reaction rate was determined at 5 different substrate concentrations for the early phase of the progression curve at the time when the product formation rate was linear (after 4 minutes). Figure 2 shows typical results from these kinetic measurements. In our experiments, since the reactions were started by adding divalent cations to the reaction mixture and the formation of the enzyme conformation was therefore not permitted, a lag phase was usually observed 5 to 10 minutes after the start of the reaction. The following enzymatic parameters were determined from an Eadie-Hofstee order. <tables id="tabl0001" num="0001"><table frame="all"><title>Table 2</title><tgroup cols="5" colsep="1" rowsep="0"><colspec colnum="1" colname="col1" colwidth="31.50mm" /><colspec colnum="2" colname="col2" colwidth="31.50mm" /><colspec colnum="3" colname="col3" colwidth="31.50mm" /><colspec colnum="4" colname="col4" colwidth="31.50mm" /><colspec colnum="5" colname="col5" colwidth="31.50mm" /><thead valign="top"><row><entry namest="col1" nameend="col1" align="left">Ribozyme</entry><entry namest="col2" nameend="col2" align="center">K_m (nM)</entry><entry namest="col3" nameend="col3" align="center">V_Max (min⁻¹)</entry><entry namest="col4" nameend="col4" align="center">K_cat (min⁻¹)</entry><entry namest="col5" nameend="col5" align="center">K_cat/ K_m (µM⁻¹ min⁻¹)</entry></row></thead><tbody valign="top"><row><entry namest="col1" nameend="col1" align="left">p53-1</entry><entry namest="col2" nameend="col2" align="right">24</entry><entry namest="col3" nameend="col3" align="char" char=".">8.6</entry><entry namest="col4" nameend="col4" align="char" char=".">0.21</entry><entry namest="col5" nameend="col5" align="char" char=".">8.75</entry></row><row><entry namest="col1" nameend="col1" align="left">p53-INV</entry><entry namest="col2" nameend="col2" align="right">230</entry><entry namest="col3" nameend="col3" align="char" char=".">52.3</entry><entry namest="col4" nameend="col4" align="char" char=".">1.30</entry><entry namest="col5" nameend="col5" align="char" char=".">5.65</entry></row><row rowsep="1"><entry namest="col1" nameend="col1" align="left">p53-F (U, C), INV</entry><entry namest="col2" nameend="col2" align="right">180</entry><entry namest="col3" nameend="col3" align="char" char=".">40.9</entry><entry namest="col4" nameend="col4" align="char" char=".">1.02</entry><entry namest="col5" nameend="col5" align="char" char=".">5.56</entry></row></tbody></tgroup></table></tables>

Example 7

mark

Substrate and enzymes were ratio-labeled with [γ-32 P] ATP and polynucleotide kinase. Unincorporated nucleotides were removed by phenol extraction followed by ethanol precipitation.

Test to determine degradation

The kinetics of degradation with modified ribozymes was determined by dissolving the radioactively labeled oligoribonucleotides in combined, fresh and undiluted human serum with a final concentration of 20,000 cpm / µl. After an initial sample was taken, the reaction mixture was incubated at 37 ° C. After certain periods of time, 1 μl aliquots were removed and the reaction with phenol was stopped. After phenol extraction and ethanol precipitation, the samples were suspended in 80% formamide containing 20nM EDTA, 0.01% bromophenol blue and 0.01% ethylene cyanot. The cleavage products were separated on a 20% polyacrylamide gel (PAGE) with 7 M urea.

The results of the incubation of p53-1 and p53-INV are shown in FIG. 3. It can be seen from the figure that an inverted structure at the 3 'terminus alone brings about an improvement in the stability of the ribozymes in the presence of serum from less than 10 seconds to several minutes.

The experiments were carried out with 1 pmol of unmodified (p53-1) and modified (p53-INV) ribozymes in 10 µl undiluted human serum at 37 ° C. The numbers on the edge of the gel correspond to the positions of corresponding length standards.

The results of the incubation of Fp53 and p53-F (U, C), INV are shown in FIG. 4. It can be seen from this that p53-F (U, C), INV no degradation was observed after an incubation of 4 hours in undiluted serum. Less than 10% degradation was observed after 48 hours. The experiments were carried out with 1 pmol modified ribozymes, where Fp53 has an inverted structure at the 3'-terminus and is fluorinated at the positions U₆, U₈, U₁₁, U₁₉ and U₂₀. p53-F (U, C), INV is additionally fluorinated on the cytosine residues C₇ and C₃₀.

Annex 1:

Synthesis of 2'-fluoro-2'desoxycytidine (A) and 2'-fluoro-2'-deoxyuridinephosphoric acid ester amide (B)

<chemistry id="chem0005" num="0005"><img file="EP0593901A2_D0008.tif" /></chemistry>

Appendix 2:

Synthesis of 3'-O-DMTr-deoxyribonucleoside-5'-O-succinyl-p-nitrophenyl ester and loading of the controlled - pore - glass support material

<chemistry id="chem0006" num="0006"><img file="EP0593901A2_D0009.tif" /></chemistry><img file="EP0593901A2_D0010.tif" /><img file="EP0593901A2_D0011.tif" /><img file="EP0593901A2_D0012.tif" />