WO1995016696A2 - Modified oligonucleotides, method for producing them and use as active substances - Google Patents

Abstract

The invention pertains to 10 to 50-mer oligonucleotides which, with the attachment of appropriate radicals that carry basic groups, are capable of forming amphoteric ions via intramolecular ammonium salts. Oligonucleotides modified in this way do not lose their ability to pair with their complementary strand. Such oligonucleotides can be expected to show a greater nuclease resistance and better membrane penetrability than natural oligonucleotides. This yields important benefits for antisense therapy.

Description

 Modified oligonucleotides, process for their preparation and use as active substances

Oligonucleotides offer promising new approaches, especially for the treatment of viral and cancer diseases. A limiting factor in their use is that such compounds as polyanions penetrate membranes poorly and can only reach the site of action with difficulty. There are a number of approaches to solving this problem, especially by producing appropriate constructs - for example by incorporating them into liposomes, coupling to polylysine or incorporating them into virus envelopes - a simple solution has not yet been achieved. The present invention relates to oligonucleotides which, by attaching suitable residues which carry basic groups, are able to form zwitterions via intramolecular ammonium salts. Oligonucleotides modified in this way do not lose the ability to pair with the appropriate counter strand and, in terms of their stability, which can be determined by CD melting point, are superior to the structurally related modified 2-O-ethyl type oligonucleotides. For such self-neutralizing oligonucleotides, both a higher nuclease resistance and a better membrane passage can be expected compared to natural oligonucleotides.

This provides important advantages in antisense therapy.

The claimed oligonucleotides are 10-50 mer compounds with the sequence of nucleotides determined by their intended use, in which the individual nucleotides are linked to one another via a phosphate or phosphothioate group and in which one, several or all nucleotides, preferably in the case of the nucleotides at the 3'-end, the 2'-oxygen atom via a spacer group, preferably an alkylene radical having a length of 4-7 carbon atoms, with a basic amino group, preferably a free amino group, or one substituted with lower alkyl primary amino group and which has the general formula

in welcher n = 10 bis 50 ist, in welcher B für eine der natürlichen Nukleotidbasen mit der durch die jeweilige Anwendung bestimmten Abfolge steht, in welcher X = O oder S bedeutet, in welcher R = H oder eine basische Aminoalkoxygruppe, vorzugsweise mit einem Alkylenrest A in der Länge von 4 - 7 Kohlenstoffatomen und vorzugsweise mit einer freien Aminogruppe, oder mit einer mit Niederalkyl R' substituierten primären Aminogruppe in beliebiger Kombination bedeu¬ tet, wobei zumindest in einem der Oligonukleotidbausteine R ungleich H sein muß und in wel¬ chen die basischen Aminoalkylgruppen in beliebigen Positionen, vorzugsweise am 3'-Ende ste¬ hen.

in which n = 10 to 50, in which B for one of the natural nucleotide bases with the is the sequence determined by the respective application, in which X = O or S, in which R = H or a basic aminoalkoxy group, preferably with an alkylene radical A in the length of 4-7 carbon atoms and preferably with a free amino group, or with a primary amino group substituted with lower alkyl R 'means in any combination, where at least in one of the oligonucleotide building blocks R must not be H and in which the basic aminoalkyl groups are in any positions, preferably at the 3' end.

The subject compounds can be prepared by known oligonucleotide synthesis methods, for example in an automated solid-phase synthesis, the nucleotide building blocks provided with the basic residue being provided with an unstable protective group on the amino group of the residue, which can be split off after oligonucleotide synthesis has taken place.

Examples:

Example 1: General preparation instructions for the oligonucleotides: The oligonucleotide synthesis is carried out in an automatic DNA synthesizer with commercially available amidite reagents for the standard nucleotides according to the usual preparation instructions. The coupling yields are determined by measuring the absorption of the detritylation solutions. A 0.1 M solution in acetonitrile is prepared from the modified nucleotide building blocks, 100-200 mg of molecular sieve 4A are added and the mixture is left to stand for 1 hour at room temperature. The modified oligonucleotides are synthesized on a 0.2 μ-molar scale according to a protocol which is set to 2.4 sec with regard to the delivery times of the amidite solutions and to 300 - 600 sec for the coupling times. After completion of the synthesis and the cleavage from the carrier material, the samples are held at 55 ° for 8 to 12 hours, then the ammoniacal solution is transferred to Eppendorf vessels and evaporated to near dryness on the Speed-Vac at room temperature. The residue is taken up twice more with 1 ml of water, evaporated and finally subjected to microfiltration in approximately 500 μl of solution. The filtrate is evaporated to dryness, taken up in 300 μl of water and the crude yield is determined by measuring the UV absorption at 260 nm. The oligonucleotide solution obtained in this way can finally be purified by HPLC, chromatography being carried out either on a precolumn (for example New-guard Rp-8) and a subsequent HPLC column (for example Aquapore Rp 300) or on a cartridge (for example Aquapore Octyl Prep 10) .

The aminoalkyl-modified building block required as a starting product for the synthesis of the oligonucleotides can be produced as follows. 50 g of adenosine are dried for 20 hours at 80 ° C. in a HV over phosphorus pentoxide and partially dissolved by heating in 200 ml of anhydrous DMF. The suspension is cooled to 0 ° C. and deprotonated with 9 g of washed sodium hydride for 30 minutes. 60.5 g of N- (6-iodohexyl) trifluoroacetic acid amide are added and the mixture is heated to 40 ° C. within one hour. After 5 hours, the reaction is stopped and the reaction mixture is evaporated to dryness in vacuo. The residue is koevapo- several times with toluene riert, digested in diethyl ether and unreacted starting material separated by vacuum flash chromatography (5-20% methanol in dichloromethane). In the subsequent chromatographic separation, 25.9 g of 2'-0- [6- (trifluoroacetylamino) hexyl)] - adenosine are obtained, which is protected according to known processes on the adenine amino group with a phenoxyacetyl group and on 5'-0 with a dimethoxytrityl group and is converted to the building block for oligonucleotide synthesis by reaction with (2-cyanoethoxy) -N, N-diisopropylmonochlophophoramidite. Example 2: Melting points of base-modified oligonucleotides

The circular dichroism spectra were recorded over a wavelength range of 320-210 nm at 10 ° C and with a double accumulation. The temperature-dependent determination of the melting curves was carried out over a temperature range of 0 ° -80 ° C with a temperature increase of 50 ° C / hour. 0.15M NaCl, 0.01 M Tris, HCl pH 7.0 was used as the buffer solution. The melting points of the oligonucleotides were determined mathematically by forming the first derivative from that curve which was obtained when the maximum ellipticity change was plotted as a function of the temperature change. A * = base-modified adenosine with aminohexyl residue

A * A ^* A ^M A * A "A ^* AAAAAA 29.5 ^I C

AAAA * A * A "A ^* A * A * AAA 3 1.7 ° C

A * A * A "A ^* A * AAAAAAA 30.7 = C

A * A * A * A * A A A A A A A A 31.3 ° C

A * A ^* A ^* AAAAAAAAA 32.6'C

A ^* A * AAAAAAAAAA 33.5'C

A * A A A A A A A A A A A 35.7 ° C

for comparison: dA6 = 18.5 °, dA7 = 19.0 °, dA8 = 22.2 °, dA9 = 29.2 °, dA10 = 31.3 °, dA1 1 =

33.2 ° C for comparison: A (2'-0-ethyl) A (2'-0-ethyl) A (2'-0-ethyl) A (2'-0-ethyl) A (2'-0 -Ethyl)

A (2'-0-ethyl) A A A A A A = 28.4 ° C.

Claims

Patent claims: 1. Oligonucleotides of the general formula

in which n = 10 to 50, in which B represents one of the natural nucleotide bases with the sequence determined by the respective application, in which X = O or S, in which R = H or a basic aminoalkoxy group, preferably with an alkylene radical A in the length of 4-7 carbon atoms and preferably with a free amino group, or with a primary amino group substituted with lower alkyl R 'in any combination, whereby at least in one of the oligonucleotide building blocks R must be not equal to H and in which one basic aminoalkyl groups in any positions, preferably at the 3 'end. 2. Process for the preparation of the compounds of the general formula I, characterized in that nucleotide units provided with a removable protective group on the aminoalkyl group are used in an oligonucleotide synthesis. 3. The method according to claim 2, characterized in that the oligonucleotide synthesis is a solid phase synthesis according to the Phophitamidverfahren, in which, depending on the choice of the oxidation conditions, phosphates or Phophothioate can be obtained. 4. The method according to claim 2, characterized in that a removable protecting group for the aminoalkoxy group is selected, which can be split off selectively and optionally in one step with the amino protecting groups on the nucleic acid residues. 5. The method according to claim 4, characterized in that the removable protective group for the aminoalkyl group is a trifluoroacetyl group. 6. Use of the oligonucleotides according to claim 1 as active substances against viral and cancer diseases.