MXPA97001111A - Specific oligomeros of sequence union nucleic paraacidos and its use in antisent strategies - Google Patents

Abstract

This invention relates to oligomers consisting entirely or partially of 1,5-anhydrohexitol nucleoside analogs represented by the general formula (I), in which B is a heterocyclic ring, which is derived from a pyrimidic or purine base, such as cytosine, 5-methylcytosine, uracil and thymine, or derivatives thereof, or adenine, guanine, 2,6-diaminopurine, hypoxanthine and xanthine, or derivatives thereof;

Description

SPECIFIC OLIGOMERS OF SEQUENCE UNION FOR NUCLEIC ACIDS AND THEIR USE IN ANTICIPATED STRATEGIES The present invention relates to oligomers having nucleic acid binding properties, oligomers which consist wholly or partially of nucleoside analogs of 1,5-anhydrohexitol as monomeric units. This invention is further related to the use of the oligomers in antisense techniques and to a method of preparing the oligomers. Antisense techniques are based on the principle that the function of a sense strand encoding a DNA or RNA molecule can be blocked by a complementary antisense strand. Antisense techniques can be used for various applications, such as diagnosis, therapy, modification and isolation of DNA, etc. In these techniques, in addition to the stability of the antisense strand itself, the stability of the doublet or triplet formed by the sense and antisense strands, as well as the binding affinity of the antisense strand for the sense strand, are of importance. Like the sensitivity of the oligomer, the doublet or triplet to degrade enzymes, such as nucleases, is a relevant factor for effectiveness. Oligonucleotides are oligomers in which the monomers are nucleotides. The nucleotides are esters of REF: 24034 nucleoside phosphate, which are constituted by a pyrimidic or pyrimidic base and a sugar. The skeleton of each nucleotide consists of alternating sugar and phosphate groups. The stability and binding affinity of the nucleotides can, for example, be influenced by the modification of the base. The investigation in that direction (1-5) showed that such modifications only lead to a less stable doublet. Alterations in the skeleton or the incorporation of new structures in it led to greater stability of the nuclease but only had an adverse effect on its binding affinity for the complementary strands. The modification of the sugars led to a very limited increase in affinity for the target molecule (6-8). It is an object of the present invention to provide novel oligomers, which have improved stability and binding affinity compared to known oligomers. It has now been found that the oligomers, which consist entirely or partially of nucleoside analogues of 1,5-anhydro-2,3-dideoxy-D-arabino-hexitol, wherein hexitol is coupled via its 2-position to the heterocyclic ring of a Pyrimidic or purine base, they are able to bind to the oligonucleotides found in nature. The monomers of which the oligomers are at least partially compound are those represented by formula I: (D in which B is a heterocyclic ring, which was derived from a pyrimidic or purine base. The monomers are connected to each other via a phosphodiester bridge in formula II representing the structure of these oligomers, wherein B is a heterocyclic ring, which is derived from a pyrimidic or pyrrhic base and, where 1 is an integer from 0 to 15, and m are each integers from 1 to 15 but if k > 1, then it can be 0 and if m > 1, k can 5 be 0; and, where X represents oxygen or sulfur. All possible salts of the compound of formula II are included in 1-nvention. The monomers of formula I are the subject of European patent application No. 92201803.1. The oligomers of formula II are novel compounds. They present a Some resemblance to oligonucleotides consisting of the 2 '-deoxynucleosides found in nature, but the sugars of the monomers are elongated because a methyl group was incorporated between the oxide and a ring carbon, which was coupled to the base. According to the invention it has been found that the oligomers of formula II and their salts exhibit specific sequence binding to the natural oligomers represented by formula III where k is an integer and where B has the same designation as in formulas I and II. Therefore, a novel class of sequence-specific binding hybrids or polymers has been found. The fact that the oligomers according to the invention consist, at least partially, of pyranose nucleosides, which have a high binding affinity is very surprising. The study of the construction of oligonucleotides from monomeric pyranose nucleotides was undertaken years ago inter alia by the group of A. Eschenmoser et al. Eschenmoser investigated the natural selection of furanoses as building blocks of sugars for nucleic acids (9). However, he did not indicate the requirements that an adequate antisense molecule must satisfy to achieve a good binding to furanose-DNA that is found in nature. The present inventors however investigated which oligonucleotides similar to pyranose could be capable of forming two stable blocks with the natural DNA furanose (10, 11). Theoretically, a pyranose oligonucleotide has an advantageous free energy over a furanose oligomer due to the lower number of entropy changes during doublet formation. However, the pyranose-like oligonucleotides studied by the present inventors above were not capable or were not sufficiently capable of joining the complementary strands of the natural DNA furanose. Those pyranose-like oligonucleotides consisted of 2, 3-dideoxy-BD-erythro-hexopyranosyl nucleosides (formula V), 2,4-dideoxy-β-D-erythro-hexopyranosyl nucleosides (formula VI) and / or nucleosides of 3,4-dideoxy-β-D-erythro-hexopyranosyl (formula VII), respectively.

The fact that specific sequence binding is found for the oligomers of formula II, which comprise pyranosides as building blocks of sugar is therefore even more surprising. By elongating the furan ring of the furanose compounds to a pyran ring, oligomers capable of binding to natural oligonucleotides are not produced. Thus, the effect of elongating the pentofuranosyl ring to a 1,5-anhydrohexitol ring could not be anticipated. The compounds according to the invention are therefore oligomers of nucleoside analogs, wherein a 1,5-anhydro-2,3-dideoxy-D-hexitol was coupled via its 2 position according to an arabino configuration to the heterocyclic ring of a pyrimidic or purine base. Oligomers consist of the above nucleoside analogs connected together as phosphate diesters or thiophosphate diesters. Oligomers can be represented by formula II in which k, 1, m, B and X have the designations stated above. The oligomers may be composed exclusively of hexitol nucleoside analogs of formula I (with 1 in formula II equal to zero) or may have natural 2'-deoxynucleosides interspersed or at the end of the molecule (with 1 in formula II being the same to one or more). Hexitol has the configuration (D) and the stoichiometry of the substituents is according to an arabino configuration. When group B is derived from a pyrimidic base, it can be either cytosine, 5-methyl cytosine, uracil or o-thi-na. When B is derived from a purine base this may be an adenine, guanine, 2,6-diaminopurine, hypoxanthine or xanthine ring, or a derivative of one of these. The nucleoside analogs, monomeric components of the present invention, can be prepared in different ways and one of the preparation methods is the subject of the European patent application No. 92.201803.1. These syntheses have also been described in Verheggen et al. (12) The assembly of the monomers in an oligomer follows the classical schemes and can be carried out either by the chemistry of the standard phosphoramidite (cf. reference 13) or by the chemistry of the H-phosphorate (compare reference 14). All procedures were conveniently carried out on an automated DNA synthesizer for standard oligonucleotide synthesis. For these standard conditions reference is made to the Methods in Molecular Biology (15). The preferred method is the phosphoramidite method, which makes use of hexitol nucleoside analog phosphoramidites as incoming building blocks for assembly in "6 'direction". Phosphoramidites are represented by formula VIII wherein B * is a protected basic portion suitable for oligonucleotide synthesis (eg, thymine, N4-benzoyl-cytosine, N6-benzoyladenine in N2-isobutyrylguanine, represented by formulas IX, X, XI and XII, respectively).

The products of formula VIII can be prepared according to standard procedures. The protection of the basic portions of cytosine, adenine or guanine is achieved following a transient protection strategy for the hydroxyl portions of the compounds of formula I (16). Preferably, however, the basic protection is carried out by acylation of the nucleoside analogs protected with 4,6-benzylidene la-d, which are intermediates in the synthesis of the monomers of the formula I set forth above. After acylation of the exocyclic amino functionality, the benzylidene moiety is removed with 80% acetic acid to obtain 3a-d. To obtain compound 3c, the p-nitro-phenylethyl group can be removed with DBU.

The primary hydroxyl function of the 1,5-anhydrohexitol analogues 3a-d can be protected with a dimethoxy-trityl group to produce 4a-d. The conversion to the phosphoramidite building blocks 5a-d, suitable for incorporation into an oligonucleotide chain can be effected with 2-cyanoethyl N, N-diisopropylchlorophosphoro-amidite. Supports containing a 1,5-anhydrohexitol analog can be prepared by acylating compounds 4a-d producing 6a-d, which can be coupled to the amino function of any long chain alkylamino controlled pore glass (CCAA). CPG) or a polystyrene with suitable amino functionalities (for example Tentagel®-RAPP Poly ere) using a carbodiimide, and producing 7a-d (for the functionalization of supports v. Ref 17). After assembly, the obtained oligonucleotides are separated from the support and deprotected by treatment with ammonia for 16 hours at 55 ° C. The purification of the oligomers obtained from the formula II set forth above can be carried out in several ways (18). The preferred method is the purification by FPLC of anion exchange at a basic pH of 12 to break all possible secondary structures (10). Desalting can be carried out by simple gel filtration techniques followed by lyophilization. All acceptable salts can be prepared in a conventional manner. la- 2a-d a: B = timin-l-yl a: B * = timin-l- b: B = adenin-9-yl b: B * = N6-benzoyladenin-9-yl 7a-b: R2 - J- CH2CH2CONH - CPG O c: B = N 2 -sobutyryl-06- (2- (p-nitro-c: B * = N 2 -isobutyrylguanin-9-yl phenyl) ethyl) guanin-9-yl d: B * = N 4 -benzoycycline-1 -ilo d: B = cytosin-l-yl CPG = controlled pore glass (solid support) (i) 80% HOAc; (ii) dimethoxytrityl chloride, pyridine; (iii) N, N-diisopropylethylamine, 2-cyano-N, N- "diisopropylchlorophosphoramidite, CH2C12; (iv) DMAP, succinyl anhydride, pyridine; (v) preactivated LCAA-CPG, DMAP, Et3N, 1- (3- < ± Letilap nopropyl) -3-ethylcarboaliimi'da. HCl, pyridine, As stated above, the oligomers exhibit sequence specific binding by natural oligonucleotides. They show a stronger binding to a complementary natural oligodeoxynucleotide than the unmodified sequences and are rotated with much greater biochemical stability. Thus, they can be used advantageously for antisense strategies comprising diagnosis, hybridization, isolation of nucleic acids, modification of site-specific DNA and the therapeutic and antisense strategies that currently continue with natural oligodeoxynucleotides.

E.JEMPLOS The compounds according to the invention as well as their chemical synthesis and the preparation of the starting materials are further illustrated in the following examples, which however are not intended to limit the invention. The following abbreviations were used: FABMS = fast atomic bombardment mass spectrometry Thgly = thioglycerol NBA = nitrobenzyl alcohol The synthesis of the nucleoside analogs of 1,5-anhydro-2,3-dideoxy-2-substituted-D-arabino-hexitol and its derivatives protected with 4,6-O-benzylidene has been described by Verheggfjn et al. (12) E. EXAMPLE 1 Nucleoside analogs protected by bases 1. 1. 1, 5-anhydro-2- (N6-benzoyladenin-9-yl) -2, 3-dideoxy-D-arabinohexitol (3b) To a solution of 2.3 g (6.51 mmol) of 1,5-anhydro-4,6-benzylidene-2- (adenin-9-yl) -2,3-dideoxy-D-arabino-hexitol in 20 ml of dry pyridine, 0.9 ml (7.8 mmol) of benzoyl chloride were added at 0 ° C. After stirring for 4 hours at room temperature, the mixture was cooled on an ice bath and 2 ml of H20 was added thereto. After the addition of 1.5 ml of a concentrated NH3 solution (33% g / v) and stirring for an additional 45 minutes at room temperature, the mixture was evaporated. The residue was purified by column chromatography (CH2-Cl2-MeOH, 98: 2) yielding 1.92 g (4.19 mmol, 64% yield) of 1,5-anhydro-4,6-benzylidene-2 (N6) -benzoyladenin-9-yl) -2, 3-dideoxy-D-arabinohexitol. This was further treated with 100 ml of 80% acetic acid at 60 ° C for 5 hours to remove the benzylidene portion. Evaporation, coevaporation with toluene and purification by column chromatography (CH2-Cl2-MeOH, 95: 5 tot 90:10) yielded 1.10 g (2.98 mmol, yield 71%) of the compound mentioned in the title of this example. UV (MeOH) ^ "298nm (e = 20200) FABMS (Thgly, NaOAc) m / e: 392 (M + Na) + .240 (B + 2H) + 1 E NMR (DMSO-dβ 1.94 (m, 1H, H -3'ax), 2.32 (m, 1H.H-3'eq), 3.21 (m, 1H, H-5 '), 3.42-3.76 (m, 3H, H-4', H-6 ', H -6"), 3.90 (dd, 2J = 131iz, 1H, H-l'ax), 4.27 (dd, 2J = 12.2Hz, 1H, H-l'eq), 4.67 (t, J = 5.7Hz, 1H , 6 '= OH), 4.88-5.00 (m, 2H, H-2', 4 '-OH), 7.47-7.68 (, 3H, aromatic H), 8.00-8.07 (m, 2H, aromatic H) 8.60 ( s, 1 H), 8.73 (s, 1 H) (H-2, H-8) ppm 13 C NMR (DMSO-dβ) d 635.8 (C-3 '), 50.7 (C-2'), 60.5 60.7 (C -4 ', C-.6'), 67.9 (C-1 '), 83.1 (C-5'), 125.1 (C-5), 128.5 (Co, Cm), 132.5 (Cp), 133.6 (Cx) , 143.5 (C-8), 150.3 (C-4), 151.4 (C-2), 152.4 (C-6) ppm. 1. 2. l, 5-Anhydro-2,3-dideoxy-2- (N2-isobutyrylguanin-9-yl-arabinohexitol (3c) The alkylation of N2-isobutyryl-06- [2- (p-nitrophenyl) ethyl] guanine (1.85 g, 7.5 mmol) with 1,5-anhydro-4,6-benzylidene-3-dideoxy-D-glucitol (1.18 g, 5 mmol) yielded 1.35 g of crude 1,5-anhydro-4,6-benzylidene-2,3-dideoxy-2- (N 2 isobutyl): il-guanin-9-yl) -D-arabinohexitol after the removal of the p-nitrophenylethyl group with 1.5 ml (10 mmol) of D3U in anhydrous pyridine for 16 hours and purification by flash column chromatography (CH2C12-MeOH, 99: 1 to 97: 3). Hydrolysis of the benzylidene portion with 100 ml of 80% HOAc (5 hours at 60 ° C) gave the desired compound 3c (610 mg, 1.74 mmol, total yield 34%) after column chromatography (CH2Cl2-MeOH , 90:10).

UV (MeOH)? Mc? 273nm FABMS (Thgly, NaOAc) m / e: 352 (M + H) + * H NMR d 1.11 (d, J = 6.7 Hz, 6H, CH3), 1.93 (m, 1H, H -3'ax), 2.11-2.38 (m, 1H, H-3'eq), 2.80 (q, 1H, CHMe-2), 3.25 (m, 1H, H-5 '), 3.42-3.78 (m, 3H, H-4 ', H-6', H-6"), 3.89 (dd, 2J = 13Hz, 1H, H-l '), 4.21 (dd, 2J = 13Hz, 1H, Hl"), 4.69 13C NMR d 19.4 (CH3), 34.5 (CHMe2), 35.8, (C-3 '), 50.5 (C-2'), 60.5, 60.7 (C-4 ', C-6'), 67.9 (C-1 ' ), 83.1 (C-5 '), 116.7 (C-5), 141 7 (C-8), 152.0 (C-4), 153.0 (C-2), 159.8 (C-6), 175.2 (C = 0) ppm.

EASEM 2 Dimetoxytrylation of nucleoside analogues 2. 1. l, 5-Anhydro-6-0-dimethoxytrityl-2- (timin-1-yl) -2,3-dideoxy-D-arabinohexitol (4a) The 1,5-anhydro-2- (timin-2-yl) -2,3-dideoxy-D-arabinohexit: ol (3a) (330 mg, 1.29 mmol) was dissolved in 20 ml of anhydrous pyridine, and added 480 mg (1.42 mmol) of dimethoxytrityl chloride. The mixture was stirred overnight at room temperature, diluted with 100 ml of CH2C12 and washed twice with 100 ml of saturated NaHCO3 solution. The organic layer was dried, evaporated and coevaporated with toluene. The resulting residue was purified by column chromatography (with a gradient of 0 to 3% MeOH in CHC13 containing 1% triethylamine) to yield 373 mg (0.67 mmol, 52%) of the title compound as a foam. FABMS (Thgly, NaOAc) m / e: 581 (M + Na)? 127 (B + 2H) + 1K NMR (CDC13): d 1.60-2.50 (m, 2H, H-3 ', H-3"), 1.91 (s, 3H, CH3), 3.12-3.62 (m, 2H, H-5', H-4 '), 3.77 (s, 6H, 2x OCH3 ), 3.65-4.17 (m, 4H, H-6 ', H-6", H-l', Hl"), 4.53 (s, 1H, H-2 '), 4.88 (d, 1H, J = 5.1 , Hz 4'-OH), 6.81 (d, J = 8.7, 4H, aromatic H), 7.09-7.53 (m, 9H, aromatic H), 8.09 (s, 1H, H-6), 9.10 (s broad 1H , NH) ppm 13 C NMR (CDCl 3) d 12.5 (CH 3), 35.5 (C-3 '), 50.7 (C-2'), 54.9 (OCH 3), 62.4, 63.1 (C-4 ', C-6') , 68.2 (C-1 '), 81.1 (C-5'), 86.0 (Ph3C), 110.0 (C-5), 138.4 (C-6), 151.0 (C-2), 163.8 (C-4), 112.9, 126.6, 127.5, 127.8, 129.7, 135.6, 144.6, 158.3 (C 5 aromatic) ppm. 2. 2. l, 5-Anhydro-6-0-dimethoxytrityl-2- (N6-benzoylansin-9-yl) -2, 3-dideoxy-D-arabinohexitol (4b) I0 A solution of 370 mg (1 mmol) of the nucleoside 3b and 400 mg (1.2 mmol) of dimethoxytrityl chloride in 25 ml of dry pyridine was stirred at room temperature for 16 hours. The mixture was diluted with 100 ml of CH2C12 and washed two i5 times with saturated NaHCO3 solution. The organic layer was dried, evaporated and coevaporated with toluene. The residue was purified by column chromatography (0 to 3% MeOH in CH 2 C 12 with 0.2% pyridine) to obtain 400 mg (0.6 mmol, 63% yield) of compound 4b as a foam.

FABMS (Thgly, NaOAc) m / e: 694 (m + Na) \ 240 (B + 2H) \ 2. 3. 1, 5-Anhydro-6-0-dimethoxytrityl-2- (N2-isobutyrylguanin-9-yl) -2,3-dideoxy-D-arabinohexitol (4c) To a solution of 580 mg (1.65 mmol) of nucleoside 3c and 670 mg (2.0 mmol) of dimethoxytrityl chloride in 25 ml of dry pyridine was stirred at room temperature for 16 hours. The mixture was diluted with 100 ml of CH2C12 and washed twice with 100 ml of saturated NaHCO3 solution. The organic layer was dried, evaporated and coevaporated with toluene. The residue was purified by column chromatography with a gradient of 0 to 3% MeOH in CH 2 C 12 with 0.2% pyridine content to obtain 770 mg (1.18 mmol, 71% yield) of compound 4c as a foam. FABMS (NBA) m / e: 654 (M + H) \ 2. 4. Preparation of amidite building blocks (5a-c) A mixture of the protected 6'-O nucleoside (0.5 mmol), 3 equivalents of dry N, N-diisopropylethylamine and 1.5 equivalents of 2-cyanoethyl-N, N-diisopropylchlorophosphoramide-dita in 2.5 ml of dry CH2C12 was stirred at room temperature during 3 hours. After the addition of 0.5 ml of EtOH and an additional stirring for 25 minutes, the mixture was washed with 5% NaHCO3 solution (15 ml) and saturated NaCl solution, dried and evaporated. Flash column chromatography with Et3N gave the amidite as a white foam, which was dissolved in a small amount of dry CH2C12 and added dropwise to 100 ml of cold n-hexane (-50 ° C). The precipitate was isolated, washed with n-hexane, dried and used as such for DNA synthesis. The following table gives the elution solvent and the yield after precipitation for the different amidites: solvent compound yield ratio FABMS (NBA) of solvent m / e 5a n-hexane / acetate 23: 75: 2 62% 759 (M + H) * ethyl / triethylamine 5b n-hexane / acetate 50: 48: 2 65% 872 (M + H) * ethyl / triethylamine 5c n-hexane / acetone / 55: 43: 2 56% 854 (M + H) * triethylamine E «TEMPLE 3 Succinylation of 6-0 protected nucleoside analogs 3. 1. l, 5-Anhydro-6-0-dimethoxytrityl-4-0-succinyl-2- (timin-1-yl) -2,3-did «_ -soxy-D-arabinohexitol (ßa) A mixture of 80 mg (0.14 mmol) 4a, 9 mg (0.07 mmol) of DMAP and 43 mg (0.14 mmol) of succinic anhydride in 5 ml of anhydrous pyrid was stirred at room temperature for 24 hours. Since the reaction was not completed, an additional amount of 43 mg (0.43 mmol) was added and the mixture was stirred for another 24 hours. The solution was evaporated and coevaporated with toluene. The residue was dissolved in CH2C12, the organic layer was washed with saturated NaCl solution and water, dried and evaporated to give 78 mg (0.12 mmol, 86% yield) of 6a as a white foam. 3. 2 l, 5-Anhydride-6-0-dimethoxytrityl-4-0-succinyl-2- (N-benzoyladenin-9-yl) -2,3-dideoxy-D-arabinohexitol (6b) The same procedure as described for 6a was used for the synthesis of 6b. An amount of 260 mg (0.39 mmol) of 4b produced 256 mg (0.33 mmol, 85% yield) of the title compound as a foam.

E «EMPLO 4 Production of oligonucleotides 4. 1 Preparation of solid support A mixture of 80 μmol of succinates (6a, b), 400 mg of preactivated LCAA-CPG (17), 5 mg (40 mmol) of DMAP, 35 μl of Et3N and 153 mg of (800 μmol) 1- (3- dimethylaminopropyl) -3-ethylcarbodii ida.HCl in 4 ml of anhydrous pyridine was sonicated first for 5 minutes and then stirred at room temperature for 16 hours. After shaking, the solid support of CPG was filtered and washed successively are pyridine, methanol and CH2C12 followed by drying under vacuum. The unreacted sites on the surface of the support were crowned using 1.5 ml of 1-methylimidazole in THF (Applied Biosystems) and 1.5 ml of acetic anhydride-lutidine-THF 1: 1: 8 (Applied Biosystems). After stirring for 4 hours at room temperature, the solid support was filtered, washed with CH2C12 and dried under vacuum. The colorimetric analysis with dimethoxytrityl indicated a charge of 18.5 μmol / g for 7a and 21.5 μmol / g for 7b. -_ 4.2 DNA synthesis The oligonucleotide synthesis was performed on an ABI 381A DNA synthesizer (Applied Biosystems) using the phosphoramidite (final dimethoxytrityl) method. The obtained sequences were deprotected and excised from the solid support by treatment with concentrated ammonia (55 ° C, 16 hours). After purification on a NAP-10® column (Sephadex G25-DNA grade, O Pharmacia), eluted with buffer A (see below), the purification was carried out on a mono-Q® HR 10 / anion exchange column. 10 (Pharmacia) with the following gradient system [A = 10 mM NaOH, pH 12.0, 0.1 M NaCl; B = 1.0 mM NaOH, pH 12.0, 0.9 M NaCl; the gradient used depends on the oligonucleotide, flow rate 2 ml / min]. The low pressure liquid chromatography system consisted of a Merck-Hitachi L6200 A Intelligent Pump, a Mono Q® HR 10/10 column (Pharmacia), a Uvicord SJI 2138 UV detector (Pharmacia-LKB) and a recording device. The fraction that 0 contained the product was desalted on a NAP-10® column and lyophilized.

E TTEMPLO 5 Melting temperatures The oligomers were dissolved in the following buffer: 0.1 M NaCl, 0.02 M potassium phosphate pH = 7.5, 0.1 mM EDTA. The concentration was determined by measuring the absorbance at 260 nm at 80 ° C and assuming that the nucleoside analogs of 1,5-anhydrohexitol have the same extinction coefficients in the denatured state as the natural nucleosides. For the adenine monomers e = 15000 For the thymine monomers e = 8500 For the guanine monomers e = 12500 For the cytosine monomers e = 7500 The concentration in all the experiments was approximately 4 μM of each strand. The melting curves were determined with a Uvikon 940 Spectrophotometer. The cuvettes were thermostabilized with circulating water through the cuvette holder and the temperature of the solution was measured with a resistance thermometer directly immersed in the cuvette. Temperature control and data collection were performed automatically with an IBM / Pc AT compatible computer. The samples were heated and cooled at a speed of 0.2 ° C / min and no differences were observed between the hot and cold fusion curves, the melting curves were evaluated taking into account the first derivative of the absobancia curve against the temperature curve. Examples of the oligonucleotides synthesized together with their melting points are given in Tables 1 to 4.

Table 1 Melting points of the oligonucleotides with a single anhydrohexitol nucleoside (A *, T *) incorporated (measured at a NaCl concentration of 0.1 M) to half an A13 / T3 doublet.

It is clear from Table 1 that the incorporation of 1,5-anhydro-2- (adenine-9-yl) -2,3-dideoxy-D-arabinohexitol into an oligodeoxy diadelate gives almost identical helical transitions to the insertion of a 2'- b natural deoxyadenosine. It should be mentioned, however, that a mismatch in a doublet of oligodeoxydacylate / oligotimidine has a great effect on the stability of the doublet. Conversely, replacing thymidine with 1,5-anhydro-2,3-dideoxy-2-: o (timin-1-yl) -D-arabinohexitol in an oligotimidylate gives a substantial decrease in the melting temperature . In contrast to the previous observations of our laboratory with 2, 4-dideoxy-β-D-erythro-hexopyranosyl nucleoside where a bad coupling A * .G [A *: 9-2,4-15 dideoxy-ß-D -eritrohexopyranosyl) adenine] gives a more stable hybridization than a base pairing A * .T [A *: 9-2,4-dideoxy-β-D-erythrohexopyranosyl) adenine] (11) where there is no alteration based on the specificity of pairing with the 1, 5-anhydrohexitol nucleosides when the oligodeoxydacylate / oligotimidine doublet is used as a model.

Table 2 Melting temperature of completely modified oligonucleotides and modified oligonucleotides at both ends, determined at 0.1 M NaCl. (1) measured at 284 nm The oligoA * and the oligoT * of a single strand both show an ordered structure but, in contrast to the results at high salt concentration (the results are not shown) the polyT * does not show the same tendency for the formation of homodoblets. This was demonstrated by the more or less linear increase in UV absorption with temperature, both for oligoA * and oligoT *. An equimolar mixture of oligoT * and oligodeoxy diadenylate shows a melting temperature of 45 ° C with a hypochromicity of 49% when measured at 284 nm. It is known that, by changing the salt concentration, structural transitions occur in the DNA and this is clearly the case. The oligoT *: oligodeoxydadenylate association was favored at lower salt concentrations while the formation of oligoT * homodoblets was favored at high salt concentrations. The thermal behavior of the complex at 260 nm, however, indicates that the oligoT *: oligodeoxydacylate association is not a classical helical transition. At 260 nm, the hypochromicity decreases first, showing a minimum at 46 ° C (the melting point observed at 484 nm) and then increases. In the same way, the completely modified mixed sequences (two hexamers and a dodecamer) containing nucleoside analogs of adenine (A *) and guanine (G *) were evaluated.

Table 3 Fully modified hexamer melting temperatures Sequence (equimolar mixture with complement) Tm (° C) (16) 6'-A * G * G * A * G * A * 31.2 (17) 5'-AGGAGA 10.0 (18) 6'-G * A * G * A * G * A * 14.7 (19) 5'-GAGAGA 9.5 determined at 1 M NaCl, 20 mM KH2P04 pH 7.5, 0.1 mM EDTA The doublets were formed with the complementary sequences 5'-TCTCCT (20) for 16 and 17, and 5'-TCTCTC (21) for 18 and 19 respectively. Although some of those melting points of the sequence could be determined for the hexamers, the thermal denaturation of those oligonucleotides was studied in 1 M NaCl (with a K content: 20 mM HP04 pH 7.5 and 0.1 mM EDTA). The most important phenomenon is the clear formation of a doublet between the oligonucleotides similar to pyranose and their natural counterparts. In addition, those modified doublets are more stable than the control doublets that consist of Watson-Crick base pairs exclusively.

. Surprisingly, however, the difference is large in the melting temperature for sequences 16 (Tm = 31.2 ° C) and 17 (Tm = 14.7 ° C) with their antiparallel complementary oligonucleotides. Where both modified oligonucleotides contain 3 G * 'and 3A * differing only in sequence order, the melting temperature for 16 doubles that of 18. This sequence-dependent effect is only marginally reflected by control oligonucleotides 17 and 19 .

Table 4 Fusion temperatures for fully modified dodecamers containing A * and G * Sequence (equimolar mixture with complement) Tm with 24 (° C) (22) 6'-A * G * G * G * A * G * A * G * G * A * G * A * 64.8 (23) 5 '-AGG GAG AGG AGA 49.0 determined at NaCl ^ O.l M (24) 5 '-TCT CCT CTC CCT Observing the dodecameters, an increase in the stability of the completely modified oligonucleotides compared with their control sequence 23 can be noticed again with an increase in the melting temperature of 16 ° C, when both sequences are evaluated with its antiparallel complementary sequence 24.

REFERENCES I. Beaucage, S.L. & Iyer, R.P., Tetrahedron 49, 6123-6194 (1993) 2. Sanghvi et al., Nucleosides and Nucleotides 10, 345-346 (1991) 3. Chollet et al., Chemica Scripta 26, 37-40 (1986) 4. Seela, F. & Kehne, A., Biochemistry 24, 7556-7561 (1985) . Wagner et al., Science 260, 1510-1513 (1993) 6. Inoue et al., Nucleic Acids Res. 15, 6131-6148 (1987) 7. Perbost et al., Biochem. Biophys. Res. Commun. 165, 742-747 (1989) 8. Gagnor et al., Nucleic Acids Res. 15, 10419-10436 (1987) 9. Esche iaoser, A., Mash & Appl. Chem. 65, 1179-1188 (1993) 10. Augustyns et al., Nucleic Acid Res. 20, 4711-4716, (1992) II. Augustyns et al., Nucleic acids Res. 21, 4670-4676, (1993) 12. Verheggen et al., J. Med. Chem. 36, 2033-2040 (1993) 13. Matteucci in Caruthers, J. Am. Chem. Soc. 103, 3185-3191 (1981) 14. Froehler et al., Nucí. Acids Res. 14, 5399-5407 (1986) . Methods in Molecular Biology, vol. 20, Protocols for Oligonucleotides and Analogs, S. Agrawal ed., Humana Press, Toto a, New Jersey, U.S.A. 16. Ti et al., J. Am. Chem. Soc. 104, 1316-1319 (1982) 17. Pon et al., Biotechniques 6, 768-775 (1988) 18. Methods in Molecular Biology vol. 26, hoofdstuk 9"Analysis and Purification of synthetic oligonucleotides by CLAP"; S. Agrawel ed., Humana Press, Totowa, New Jersey, USA It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention is that which is clear from the present description of the invention. Having described the invention as above, property is claimed as contained in the following:

Claims (9)

1. The oligomers, characterized in that they fully or partially comprise 1, 5-anhydrohexitol nucleoside analogs represented by the general formula I wherein B is a heterocyclic ring, which was derived from a pyrimidic or purine base.

2. The oligomers according to claim 1, characterized by the general formula II - .. in which B is a heterocyclic ring, which is derived from a pyrimidic or purine base, and in which k, 1, and m are each integers from 0 to 15, with the proviso that k and m are at minus one; but if k > 5 1, then m can be 0; and if m > 1, k can be 0; and, wherein X represents oxygen or sulfur, and the salts thereof.

3. The oligomers according to claim 1 or 2, characterized in that the heterocyclic ring is selected from the group consisting of cytosine, 5-methylcytosine, uracil and thymine, or derivatives thereof.

4. The oligomers according to claim 1 or 2, characterized in that the heterocyclic ring is selected from the group consisting of adenine, guanine, 2,6-diaminopurine, hypoxanthine and xanthine, or derivatives thereof.

5. The oligomers according to any of the preceding claims, characterized in that the compound of formula I has the configuration (D) and the substituents are localized in the arabino configuration. 5 ß.

The oligomers according to any of claims 1-5, characterized in that they are used in antisense techniques.

7. The oligomers for use according to claim 6, characterized in that the antisense techniques comprise diagnosis, hybridization, isolation of nucleic acids, modification of DNA directed to a site and therapy.

8. A method for preparing the oligomers of formula II, characterized in that it comprises coupling a suitable amount of monomers of formula I.

9. The phosphoramidites of the general formula VIII characterized in that B * is a protected base, for use in the preparation of the oligomers of claim 1.