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

(74) Proxy

Hoechst AG, D-65926 Frankfurt am Main, DE Heinz Hartmut Seliger, Elchingen, DE

Flavio Ramalho Ortigao, Ulm, DE Hannelore Rosch, Eroach, DE

Rudi Rosch, Erbach, DE

Bemd Krist, Ulm, DE

Bryns Patentkontor AS, 0106 Oslo

<td>(54) Designation</td><td>Oligoribomicleotides with terminate 3'-3 'or 5'-5 'junctions, their use in the manufacture of a pharmaceutical composition and medicament containing these nucleotides</td>

<td>(56) Cited publications</td><td>EP 464638 Seliger et al. (1991), CA 117: 184152 Seliger et al. (1991), CA 115: 275574 Ramalho et al. (1992), CA 119: 174846 Pieken et al. (1991), Science, 253: 314-317</td>

<td>(57) Summary</td><td>Oligoribonucleotide analogs are described down to terminal 3'-3 'and 5'5'-internukleotldsammenføyninger. This modification stabilizes the thereby altered molecules, including ribozymes, without altering the properties, including any catalytic activities, in a negative direction.</td>

in

The present invention relates to oligoribonucleotide analogs having terminal 3'-3 'and 5'-5 'internucleotide assemblies, their use in the manufacture of a pharmaceutical and medicament containing these nucleotides. This modification stabilizes the molecules thus altered, including ribozymes, without adversely altering their properties, including any catalytic activities.

Antisense oligonucleotides are nucleic acid fragments whose sequence is complementary to the coding or sense sequence of a messenger RNA, respectively. to codogen strand of DNA. Such oligonucleotides are increasingly used t o 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. 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 during formation can form a triple helix on the DNAdouble strand and which by inhibition modulate the transcription of gene expression (J. Chubb and M. Hogan, TIBTECH 10 (1992) 132-136).

II. Ribozymes: Ribonucleic acid fragments with enzymatic activity which consist of target RNA, e.g. an m-RNA, after the specific binding of the ribozyme is cleaved by the same (TR Cech, J. Am. Med. Assoc. 260 (1988) 3030).

In order for antisense oligonucleotides, triple helix-forming oligonucleotides and ribozymes to be used in biological systems, the following preconditions must be met (E. Uhlmann, A. Peyman, Chem. Rev. 90 (1990) 543584):

1. They must on the one hand be well water-soluble, but on the other hand easily cross the lipophilic cell membrane,

2. they must be sufficiently degradable inside the cell, ie stable against nucleases,

3. they must form stable hybrids with intracellular nucleic acids at physiological temperatures,

4. the hybridization must be selective; the difference in dissociation temperature of an oligonucleotide which gives a mismatch must be large enough for the latter to be specifically washed off,

5. in the case of ribozymes, the catalytic activity must be maintained.

Unmodified oligonucleotides and especially unmodified oligoribonucleotides are highly subject to nucleolytic degradation. Therefore, studies were carried out at an early stage to modify the oligonucleotides structurally so that they better meet the above requirements, in particular are better protected against nuclease degradation. For this purpose, a large number of oligonucleotide analogs were prepared, in part by enormously complicated synthetic methods (1. E. Uhlmann, A. Payman, Chem. Fox. 90 (1990) 543-584; 2. J. Goodchild, Bioconjugate Chem. 1 (1990) 165-187).

Recently it was shown that 3'-3'- resp. 5 '-5' -terminally joined oligodeoxynucleotides resp. their analogues show a significantly increased stability against nucleolytic degradation (1. H. Seliger, A. FrShlich, M. Montenarh: Nucleosides &amp; Nucleotides 10 (1991) 469-477; 2. H. Rbsch, A. Frohlich, J. Ramalho- Ortigao, J. Flavio, M. Montenarh, H. Seliger: EP 0464638A2). Surprisingly, it has now been found that the same, synthetically readily available, terminal joining type

a) is also able to stabilize the much more labile oligoribonucleotides against nucleases,

b) is able to stabilize ribozymes (oligoribonal cleotides with special sequence requirements) against nucleases without affecting the catalytic activity,

c) is also able to stabilize oligoribonucleotides resp. ribozymes which, by chemical modification, are protected against nucleases.

by formula I

Accordingly, the invention relates to oligoribonucleotides characterized in which

R¹

R²

<img file="NO307656B1_D0001.tif" />

<img file="NO307656B1_D0002.tif" />

<img file="NO307656B1_D0003.tif" />

means hydrogen (I) or a residue of formula II

<img file="NO307656B1_D0004.tif" />

Z '-pw

<img file="NO307656B1_D0005.tif" />

(II) means hydrogen

<img file="NO307656B1_D0006.tif" />

a residue of formula III (III)

B stands for a base, such as natural bases such as adenine, thymine, cytosine, guanine or unnatural bases, such as purine, 2,6-diaminopurine, 7-deazaadenine, 7deazaguanine, N⁴, N⁴-ethanocytosine resp. their prodrug forms;

R³ independently of one another means OH, hydrogen or F, and not more than one R³-rest is equal to H;

W and W 'independently of one another denote oxygen or sulfur;

Z and Z 'independently of one another mean O^-; S ~; C 1 -C 6 alkoxy, preferably C 1 -C 6 alkoxy, preferably^Cl^-C18 "alkyl, preferably C 1 -C 6 alkyl, NHR⁴, with R⁴ = C 1 -C 18 alkyl, or C 1 -C 4 alkoxy-C 1 -C 6₎-alkyl, preferably methoxyethyl; REF⁴R⁵, where R⁴ is as defined above and R 2 preferably represents C 1 -C 6 alkyl, particularly preferably C 1 -C 6 alkyl, or wherein R⁴ and R⁵together with the nitrogen atom bearing them means a 5-6 membered heterocyclic ring, which may additionally contain an additional heteroatom from the group O, S, N, such as e.g. morpholine;

where X stands for

OH, H, F, Cl, Br, NH₂, N₃ , O-C (O) - (C 1 -C 10) -alkyl, O-C (O) - (C₂-C₁₈) -alkenyl, OC (O) - (C₂-C₁₈) alkynyl, O-C (O) - (C₆-C₁₈) aryl, O- (C 1 -C 6)₁₈) -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 form a cyclic phosphoric acid diester, preferably X represents OH, H, F, n represents an integer of 5-60, and especially preferred

15-25, as well as their physiologically acceptable salts.

By aryl is meant in this context e.g. phenyl, phenyl substituted (1-3 times) with C 1 -C 4 alkyl, C₁-C₆-alkoxy and / or halogen.

Preferred are the oligorlbonucleotides of formula I.

Furthermore, oligoribonucleotides of formula I are preferred, wherein R² stands for a residue of formula III and R¹ means hydrogen; R¹ respec tively. R² stands for a residue of formula II resp. III; or R² represents hydrogen and R 2 represents a residue of formula II, whereby either W or Z in the latter case does not mean oxygen and X represents OH or H.

In particular, oligoribonucleotides of formula I should be mentioned, in which W stands for oxygen or Z and W both represent oxygen.

In particular, oligoribonucleotides of formula I, the base order B of which is mentioned¹, B²...... Bⁿ corresponds to the sequence requirements for ribozymes. Particular emphasis should be placed on Hammerhead ribozymes (e.g. Uhlenbeck, Nature 328 (1987) 596; Haseloff, Gerlach, Nature 334 (1988) 585), Hairpin ribozymes (e.g. Hampel et al., Nucl. Acids. Res. 18 (1990) 299), '' Human Hepatitis a-Virus Ribozyme (e.g. Branch, Robertson, Proc. Natl. Acad. Sci. USA 88 (1991) 10163) and external guide sequence for RNase P '(f e.g., Forster, Altman , Science 249 (1990) 783), especially the Hammerhead rlbozymes.

Most particularly preferred are oligoribonucleotides of formula I, wherein R² stands for a residue of formula III and R 2 means hydrogen. Furthermore, mention should be made of oligoribonucleotides of formula I, which are additionally substituted with groups which promote intracellular uptake, which in vitro or in vivo serve as reporter groups, and / or groups which, by hybridizing the oligoribonucleotide to biological DNA or RNA, attack these DNA cells. or the RNA molecules during binding or cleavage.

Examples of groups that favor intracellular uptake are lipophilic residues such as alkyl residues, e.g. 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 con jugates which utilize natural carrier systems, such as e.g. bile acid or peptides for the corresponding receptor (eg receptor-mediated endocytosis). Examples of reporter groups are fluorescent groups (e.g. acridinyl, dansyl, fluoresceinyl) or chemiluminescent groups such as e.g. akridlniumestergrupper.

Examples of oligonucleotide conjugates that bind to nucleic acids and / or cleave these are found in the following references. (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, azldofenazyl, azldoproflavin, phenazlin, phenanthroline / Cu, porphyrin / Fe, benzopyridoindole, EDTA / Fe (Mergny et al., Science 256 (1992) 1681).

The characteristic structural modification of the oligoribonucleotides according to the invention consists in that the internucleotide junctions of the two chain ends are altered, i.e. instead of biological 3'-5 'junctions are 3'-3' and 5'-5'-joints. Surprisingly, it was found that the minimal structural modification is sufficient to stabilize such compounds against nuclease degradation without adversely altering other properties, e.g. enzymatic activities.

As described below, only the minimal structural modification causes a hybridization behavior that is approximately similar to that of biological oligoribonucleotides. This also results in the general utility of these compounds as inhibitors of gene expression.

The compounds of formula I are prepared in the same way as the synthesis of biological oligonucleotides in solution or preferably in solid phase, optionally with the aid of an automatic synthesizer. A process for the preparation of oligoribonucleotides of formula I is carried out by

a) converts a nucleotide unit with 3 'or 5 'terminal phosphorus (III) or phosphorus (V) moieties or their activated derivatives with an additional nucleotide moiety of 3' and 3 ', respectively. 5 'terminal free hydroxy group or

b) builds up the oligonucleotide of fragments in the same way, in the oligonucleotides obtained after (a) or (b) one or more protecting groups temporarily introduced for protection of other functions are cleaved and the oligonucleotides of formula I thus obtained are optionally transferred to their physiologically acceptable salt. .

As starting components for the preparation of oligoribonucleotides with terminal inverted 3'3 'binding, a carrier resin to which the first nucleoside monomer is attached via the 5'-OH group is used for the solid phase synthesis. For the preparation of this component a carrier resin is used which is prepared by a method known from the literature (T. Atkinson, M. Smith in Oligonucleotide Synthesis, MJ Gait (ed.), 35-49 (1984)), preferably silica gel or controlled pore glass, which is functionalized with am ino groups. It is reacted with a nucleoside derivative protected on the nucleobase or on the 3'-OH group, which has been previously converted to 5'-p-nitro-phenylsuccinate. As base protecting groups, acyl groups are preferably used, e.g. benzoyl, isobutyryl or phenoxyacetyl. The 3 'position is preferably protected by the dimethoxytryltyl protecting group, which can be introduced according to MD Matteucci, MH Caruthers, Tetrahedron Letters 21 (1980) pp.3243-3246.

The further construction of the oligoribonucleotide chain up to the penultimate chain link takes place by methods known from the literature (Beaucage, Iyer, Tetrahedron 48 (1992)

2223), preferably using nucleoside-3'-phosphoric acid ester amides or nucleoside-3'-H-phosphonates protected on the 5'-OH group with dimethoxytrityl groups. The 2 'hydroxy group is preferably protected by the tert-butyldimethylsilyl group (M. Lyttle et al., J. Org. Chem. 56 (1991) 4608; Scaringe et al., Nucl. Acids Res. 18 (1 990) 5433). The 2'-amino group (synthesis of compounds with R 2 = ΝΗβ) is preferably protected with the trifluoroacetyl group (Benseler et al., Nucleosides &amp; Nucleotides 11 (1992) 1333). As the last chain link, a nucleoside-5'-phosphoric acid esteramide or 3 '-phosphoric acid ester protected with dimethoxytrityl is again used. nucleoside H-phosphonate. The preparation of such an oligoribonucleotide chain with terminally twisted internucleotide bonds is shown schematically in the following. (Phosphoramidite cycle for the preparation of oligonucleotides with 3'-3 'and 5'-5' junctions at the ends.) The preparation of oligoribonucleotides with 3'-3 'or 5'-5' junctions takes place correspondingly.

<img file="NO307656B1_D0007.tif" />

1. Detritylation uMTfO

DUTrO

DUTrO

<img file="NO307656B1_D0008.tif" />

<img file="NO307656B1_D0009.tif" />

DMTiO

2. Activation addition

<img file="NO307656B1_D0010.tif" />

end seal

Oxidation on '

<img file="NO307656B1_D0011.tif" />

itylation after n condensations

OR * 1 I

RO-P = O

<img file="NO307656B1_D0012.tif" />

end seal

oxidation

2. Activation and addition o R^{J 1}

OMTtO R

According to literature-known methods, the incorporation of 2 '-modified ribonucleotide units such as e.g. 2'-O-alkyl 2 '-deoxyrylbonucleotides (Iribarren et al., Proc. Natl. Acad Sci. USA 87 (1990) 7747; Sproat, Lamond in Oligonucleotides and Analogues: F. Eckstein, ed., IRL Press, Ocford 1991 ); 2'-F and 2'-NHg-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 analyzes, the oligorlbonucleotides are labeled terminally, as described in the following Example 4. This is done by radiolabeling, preferably by means of »- r32p_ATP / p_Olynukl_COkinase. This radiolabeling takes place on the free 5'-OH group, i.e. opposite an oligonucleotide with only biologi cal 3'-5 'junctions 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 formula II are used for hybridization chemical processes based on the addition of double or single chain nucleic acids, respectively. their cleavage for regulation or suppression of biological function of nucleic acids as well as for selective suppression of the expression of viral genome functions and for prophylaxis and therapy of viral functions, for suppression of oncogenic function and for the treatment of cancers.

As a measure of the stability in vivo, the behavior of an oligoribonucleotide of formula I built up in blood serum according to the invention can be considered. The general test is described in Example 4. The oligorlbonucleotides of the invention degrade much more slowly in contrast to the 3'-5 'oligorlbonucleotides.

Example 5 demonstrates that the oligoribonucleotides of the invention, which meet the sequence requirements for Hammerhead ribozymes, in terms of enzymatic activity do not differ from the unmodified ribozymes.

Description of the figures:

Fig. 1: Stability of serum p53-INV.

Fig. 2: Kinetics of substrate cleavage in modified

Fig.

Fig.

3:

4:

ribozymes.

Nucleolytic degradation of p53-1 and p53-INV.

Nucleolytic degradation of Fp 53-INV and p53F (U, C), INV.

Example 1:

Synthesis of 2'-fluoro-2 'deoxynucleoside units (Appendix 1)

5'-0- (dimethoxytrityl) -2'-fluoro-2'-desoksyuridin

In a 50 ml swing flask, co-evaporate 0.5 g (approx. 2 mmol)

2'-fluoro-2'-deoxyuridine with 2 times 10 ml abs. pyridine. The dried nucleoside is taken up with 25 ml of abs. pyridine and mixed at room temperature with 0.66 g (about 2.2 mmol) of dimethoxytrityl chloride and 10 mg of 4-dimethylaminopyridine. After three hours, the mixture was added with 1 ml of methanol and then evaporated in vacuo to dryness. The residual oil is taken up in 50 ml of methylene chloride (acidified over alumina) and extracted three times with 50 ml of water. The organic phase is dried over sodium sulfate. After removal of the methylene chloride, the crude product remained as a solid foam. To remove higher tritylated constituents and the tritanol, the crude product was digested at 40-50 ° C with 30 ml of abs. benzene. 0.75 g was obtained, corresponding to 67% of the theoretical amount of a white solid.

4-N-acetyl-2'-fluoro-2'-desoksycytidin

In a 100 ml volumetric flask, co-evaporate 1 g (ca. 4 mmol) of 2'fluoro-2'-deoxycytidine hydrochloride twice with 20 ml of abs. pyridine and twice, each time with 10 ml, abs. acetonitrile. The dried nucleoside material is suspended in 40 ml of abs. DMF and 0.6 ml (about 4.4 mmol) of acetic anhydride are added. During one day, 0.5 ml (4.4 mmol) of abs are added dropwise. triethylamine to the mixture. The solvent is then removed in an oil pump vacuum. The crude product is washed with 50 ml of diethyl ether and then dried. The purification is carried out by column chromatography (silica gel 60H, column 4 x 10 cm, eluent methylene chloride with 0.1 pyridine, gradient methanol); the product is eluted at $ 8 methanol. After removal of the solvent, 0.83 g of product remained (71% theoretical yield).

5'-O- (dimethoxytrityl) -4-N-acetyl-2'-fluoro-2'-deoxyxytidine in a 100 ml volumetric flask 4 mmol of 4-N-acetyl-2'-fluoro-'-deoxycytidine with 25 ml abs . pyridine and mixed at room temperature with 1.3 g (about 4.4 mmol) of dimethoxytrityl chloride and 20 mg of 4-dimethylaminopyridine. After 3 hours, the mixture was added with 1 ml of methanol and then evaporated in vacuo to dryness. The residual oil is taken up in 50 ml of methylene chloride (acid removed over alumina) and extracted three times with 50 ml of water. The organic phase is dried over sodium sulfate. After stripping off the methylene chloride, the crude product remained as a solid foam. To purify the crude product, it is chromatographed on silica gel 60H (column 2 x 20 cm, eluent methylene chloride with 0.1% pyridine, gradient methanol). The product was eluted with 3% methanol in methylene chloride. After removal of the solvent, 1.39 g (59% of theory) of a white foamy solid remained.

Phosphoric acid ester amides of 2'-fluoro-2'-deoxynucleosides mmol of the protected monomers are dissolved in 5 ml of abs. methylene chloride and 1 ml abs. dlisopropylamin. Under argon, 1.2 mmol of chloroN, N-diisopropylamino-p-cyanoethoxyphosphine are added dropwise with a one-way syringe. After one hour, the reaction is almost quantitative and the reaction can be quenched with 0.1 ml of methanol. The mixture is taken up in 20 ml of ethyl acetate and extracted three times, each time with 20 ml of saturated NaCl solution. The organic phase is dried over sodium sulfate and the solvent is removed. The crude product is taken up in 5 ml of methylene chloride and precipitated from 400 ml of abs. petroleum seats at room temperature. After removal of the precipitate, it is dried under an oil pump vacuum and stored at -20 ° C.

5'-O- (dimethoxytrityl) -4-N-acetyl-2'-fluoro-2'-deoxycytidine diisopropylamino-p-cyanoethoxyphosphine in:

Mixture: 0.59 g (1 mmol) of 5'-O- (dimethoxytrityl) -4-N-acetyl2'-fluoro-2'-deoxycytidine

Yield: 0.60 g (0.78 mmol, 78 av> of theoretical)

5'-O- (dimethoxytrityl) -2'-fluoro-2'-deoxyuridine diisopropylamino-p-cyanoethoxyphosphine:

Mixture: 0.55 g (1 mmol) of 5'-O- (dimethoxytrityl) -2'-fluoro-2-deoxyuridine

Yield: 0.61 g (0.83 mmol, 83 avο of theoretical)

Example 2:

Coating of CPG 10-1400 Carrier Material with 3'-O-Dimethoxytrityl-Deoxyribonucleoside Units (Appendix 2)

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

Mixture: 1.0 mmol 3'-O-DMTr-dN

0.8 mmol succinic anhydride (80 mg)

0.5 mmol dimethylaminopyridine (61 mg)

The reaction of succinic anhydride with the 5'-OH group of deoxyribonucleoside was carried out in 5 ml of absolute pyridine with DMAP as catalyst overnight at room temperature. After complete reaction, the solution was evaporated and the pyridine was removed by three azeotropic distillation with toluene. The residue was taken up in dichloromethane, washed with ice-cold citric acid solution and H 2 O and the organic phase was rotated in vacuo. The crude product was dissolved in approx. 3 ml of toluene and precipitated in 200 ml of n-hexane.

Carrier coating in

Mixture: 0.8 mmol 3'-O-DMTr-dN-5'-O-succinate

0.8 mmol p-nitrophenol (112 mg)

2.0 mmol dicyclohexylcarbodlimide g aminopropylated CPG 10-1400

The protected succinylated deoxytriribonucleoside was added to a solution of p-nitrophenol in 5 ml of absolute dioxane and 0.2 ml of pyridine and then DCCl was added as a condensing agent. After 3 hours the turnover was complete. The precipitated dicyclohexylurea was filtered off with suction 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 coated support was then filtered off with suction, washed with methanol and ether and dried in a desiccator. To block unreacted amino groups, the coated support was shaken with a solution of 1 ml of acetic anhydride and 50 mg of dimethylaminopyridine in 15 ml of absolute pyridine for 1 hour at room temperature, then filtered off with suction, 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 3 'terminal

The synthesis of the modified Hammerhead ribozyme (Table 1) was performed on a 0.2 pmol scale with the DNA synthesizer Gene Assembler Puls from Pharmacia. The support material for the synthesis was functionalized with the deoxyadenosine building block attached above the 5 'hydroxyl group; in the synthesis, this resulted in an inverted structure at the 3 'terminal of the oligonucleotide. The preparation was performed according to the standard protocol for olgoribonucleotide syntheses in the phosphoric acid ester amide method.

p53-INV: 5'-r (AAAGA UCUGA UGAGG CCGUU AGGCC GAAAC AGGG) -3'-3'-dA-5 '

Fp53-INV: 5'-rArArArGrA fUrCfUrGrA fUrGrArGrG rCrCrGfUfU rArGrGrCrC rGrArArArC rArGrGrG-3'-3'-dA-5 '

Table 1:

fN: 2'-fluoro-2-deoxynucleosides rN: ribonucleosides

The phosphoric acid ester amides of 2'-fluoro-2'-deoxynucleosides were used 0.12 M in acetonitrile. In the chain extension step, 0.1 ml of amidophosphite is reacted with 0.37 ml of the tetrazole solution (0.5 M) with the carrier-bonded 5'-hydroxyl groups of the oligonucleotide. After a coupling time of 12 minutes, it is cut in the standard manner, oxidized and detrylated to prepare for the next coupling step. The coupling yields averaged 99 1o.

For cleavage of the base-labile protecting groups and for cleavage of the attachment, the support is transferred after synthesis into an Eppendorf reaction beaker which can be screwed on. At 55 ° C, incubate for 12 hours with 2 ml of a 3: 1 mixture of 32% ammonia and ethanol. The above solution was raised, cooled to -20 ° C and gently lyophilized. The dry residue was suspended in 0.4 ml of a 1.1 M TBAF solution in

THF and incubated for an additional 16 hours at room temperature. The reaction was quenched by the addition of the same volume of a trethylethylammonium acetate buffer (TEAA buffer). The solution was cooled to -70 ° C and carefully evaporated to 0.4 ml. After adding 40 μl of sodium acetate, 1.4 ml of ethanol and 5 μl of acetic acid, the product precipitated overnight at -20 ° C. The sample was centrifuged and the supernatant discarded. The dry oligonucleotide was taken up in a 1: 1 mixture of formamide blue marker and water to add it to an acrylamide gel in? (20 ,ο, 7M urea). To identify the product bands and to cut out, the gel was covered with cellophane foils. Elution took place at 40 ° C with ammonium acetate solution. After 5 hours, the solution precipitated as described above. The oligonucleotide was washed with 70% ethanol, resuspended in 70% ethanol and stored at -70 ° C.

Example 4:

Stability study of modified ribozymes in 25 blood serum tests

The ribozyme p53 and the modified oligorlbonucleotides p53INV and Fp53-INV were radiolabeled enzymatically by T4 polynucleotide kinase in the presence of (-32β) _dATP (specific 5 ° activity: 4500 Cl (mmol). The sequences with a 3'-3 ' inversion has a 5 'hydroxy group on both sides and is consequently phosphorylated in part at both ends.

The labeled ribozymes were treated with healthy human serum.

Mixture: 1 pmol phosphorylated ribozyme pl serum

The samples were incubated at 37 ° C. After the following times, 2 μl was taken out in each case 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 separated electrophoretically on a polyacrylamide gel with 8 M urea at 55 ° C.

The intensity of the bands on the X-ray film was determined by means of a laser densitometer (see Fig. 1). The half-lives of the tested ribozymes (tl / 2 = 30 minutes for Fp53INV, 1 minute for P53-INV and &lt;&lt; than 1 minute for the biological oligoribonucleotide p-53 (see Example 5) clearly show the protective effect of the terminate inversion.

Example 5:

A 20-mer substrate oligoribonucleotide, SB-1 5'-r (GC CCC UGU CAU CUU UUG UCC) -3 'was radiolabeled enzymatically with T4 polynucleotide kinase in the presence of θ ^ Ρ-ΑΤΡ (specific activity: 4500 Ci / mmol) at the 5 'end. The cleavage reaction of SB-1 was performed using various ribozymes as follows: The reaction conditions were 50 mM Tris HCl, pH 7.5, 20 mM MgCa at 50 ° C. The substrate concentration SB-1 was 0.025 pM (varied between 0.05, 0.1 and 0.25 pM), the ribozyme concentration was 0.02 pM. Within 30 minutes, a sample was taken at 1 minute, 5 minutes, 10 minutes, 15 minutes and 30 minutes and mixed with loading buffer. The sample was separated electrophoretically on a 20% polyacrylamide gel (8M urea) at 55 ° C. The reduction in the intensity of the bands of SB-1 was determined on the X-ray film by means of a laser densitometer. As ribozymes it was used:

p53 INV; Fp53-INV (p. In any case above); p53 and Fp53.

p53: 5'-r (AAGA UCUGA UGAGG CCGUU AGGCC GAAC AGGGA) -3 '

Fp53: 5'-r (AAAGA fUCfUGA fUGAGG CCGfUfU AGGCC GAAAC AGGGA) 3 '

The rate of cleavage of SB-1 does not differ when using p53 and p53-INV. Fp53 and Fp53-INV also have the same activity, which, however, is approx. a factor of 5 lower than that of p53.

Example 6;

Substrate cleavage and kinetic measurements

The measurement of the preliminary start rate of the reaction was performed with 40 nM substrate and 4 nM enzyme in 50 nM TrlS-Cl (pH 7.5). The reaction was started by adding 10 nM MgCl 3₂. The amount of the decomposition product was measured at 55 ° C after 1, 2, 5, 10 and 15 minutes.

From this experiment, K_m-value determined approximately. More accurate measurements of the initial velocities were performed according to Suelter, CH (1985) in A Practical Guide Enzymology * ', J. Wiley, New York, 231. For this purpose, 40 nM enzyme was performed in six separate reactions with in each case 25 nM, 50 nM, 100 nM, 200 nM, 500 nM and 1000 nM substrate. After certain time intervals, a 2 μl aliquot was withdrawn and the reaction was stopped by the addition of phenol. The samples were then separated on a denaturing gel (20 PAGE, 7 M urea) and analyzed.

Substrate: SB-1 (Example 5)

Ribozyme: p53-INV (Example 3) p53-F (U, C), INV: 5 ' AUG AGG CCG UUA GGC CGA AAC AGG G) -3 '

Determination of the kinetic parameter

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). Typical results for these kinetic measurements are shown in Fig. 2. Since the reactions in the present experiment were started by adding divalent cations to the reaction mixture and the formation of the enzyme conformation was thus not allowed, a Lag phase was usually observed 5 to 10 minutes after the start of the reaction.

The following enzymatic parameters were determined from an EadieHof stee record.

Table 2

<td>ribozyme</td><td>^Km (nM)</td><td>V max (my*¹)</td><td>^Kcat (min *¹)</td><td>^Kcat /^Km (pM '¹ my*¹)</td>

<td>p53-1</td><td> 24</td><td> 8.6</td><td> 0.21</td><td> 8.75</td>

<td>P53-INV</td><td> 230</td><td> 52.3</td><td> 1.30</td><td> 5.65</td>

<td>p53 F (U, C), INV</td><td> 180</td><td> 40.9</td><td> 1.02</td><td> 5.56</td>

Example 7:

Marking

Substrate and enzymes were radiolabeled with [ύ-³²Ρ] ΑΤΡ and polynucleotide kinase. Unincorporated nucleotides were removed by phenol extraction with subsequent ethanol precipitation.

Test for determination of nephrectomy

The kinetics of degradation with modified ribozymes were determined by dissolving the radiolabeled oligoribonucleotides in a combined, fresh and undiluted human serum with a final concentration of 20,000 cpm / μl. After a starting sample was taken, the reaction mixture was incubated at 37 ° C. After certain sections of time, 1 μl aliquots were taken and the reaction was quenched with phenol. After phenol extraction and ethanol precipitation, the samples were suspended in 80% formamide containing 20 nM EDTA, 0.01% bromophenol blue and 0.01 hylencyano. 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. The figure shows that an inverted structure at the 3 'terminal alone already causes a stability improvement for ribozymes in the presence of serum in less than 10 seconds to several minutes. .

The experiments were performed with 1 pmol of unmodified (p53-1) and modified (p53-INV) ribozymes in 10 μm undiluted human serum at 37 ° C. The numbers on the gel edge 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 appears that for p53-F (U, C), INV after an incubation of 4 hours in undiluted serum was not observed degradation. After 48 hours, less than 10% degradation was observed. The experiments were performed with 1 pmol modified ribozymes, whereby Fp53 has an inverted structure at the 3 'terminal and is fluorinated at the positions, Ug, Ull. Ui9 and U₂₀· P53-F (U, C), INV is additionally fluorinated on cytosine residues C7 and C3Q.

Attachment 1:

Synthesis of 2'-fluoro-2'-deoxyethylidene- (A) and 2'-fluoro-2'-deoxyuridine phosphoric acid ester amide (B)

<img file="NO307656B1_D0013.tif" />

<img file="NO307656B1_D0014.tif" />

6.

BMTrO

Phosphylethylation (1) / HN'Pr,

<img file="NO307656B1_D0015.tif" />

I α

IN

NcccHtir-o * ^ ζ

Appendix 2:

Synthesis of 3 * -O-DMTr-deoxyuronbonucleosyl-5 * -O-succinyl-pintrophenyl ester and loading of the Controlled-Pore-Glass support material

<img file="NO307656B1_D0016.tif" />

DMTrO

0 »C '' C» O

V- /

II

C-0 $ c

I OOMTr

OH

<img file="NO307656B1_D0017.tif" />

HO

-Qw »

<img file="NO307656B1_D0018.tif" />

benzoyladenine

benzoylcytosine - isobutyrylguanine

DMTr = 4,4 '- dimethoxytrityl

<img file="NO307656B1_D0019.tif" />

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IN

NO,

<img file="NO307656B1_D0020.tif" />

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