WO1997030064A1

WO1997030064A1 - Hexitol containing oligonucleotides and their use in antisense strategies

Info

Publication number: WO1997030064A1
Application number: PCT/EP1997/000762
Authority: WO
Inventors: Piet André Maurits Maria HERDEWIJN; Arthur Albert Edgard Van Aerschot
Original assignee: Stichting Rega Vzw
Priority date: 1996-02-16
Filing date: 1997-02-14
Publication date: 1997-08-21
Also published as: AU1874397A

Abstract

The present invention relates to novel oligomers comprising 1,5-anhydro-2,3-dideoxyhexitol nucleotide analogues and deoxyribose nucleotides. The invention further relates to complex consisting ofthe novel oligomer and a complementary single-stranded or double-stranded oligonucleotide. Pharmaceutical compositions, comprising the oligomer and/or the complex and the use of both in molecular biology and genetic engineering like hybridization, isolation of nucleic acids, site specific DNA modification and mapping.

Description

HEXITOL CONTAINING OLIGONUCLEOTIDES AND THEIR USE IN ANTISENSE STRATEGIES

Technical field

This invention relates to oligomers comprising or containing in part 1,5-anhydrohexitol nucleotide analogues which exhibit sequence-specific binding to complementary sequences of natural oligonucleotides. This invention further relates to the chemical synthesis of these oligomers and their use in antisense strategies which comprise diagnosis, hybridization, isolation of nucleic acids, site-specific DNA modification, and therapeutics

(This application makes use of nucleoside analogues protected by an European Patent Application n° 92.201803.1, dd. 18 juni 1992, and of monomer units for chain assembly as protected by an International Patent Application n° 94202342.5, dd. 17 august 1994).

Background

The last decade new strategies have been developed for sequence specific binding of nucleic acids. One of these strategies makes use of complementary constructs to the targeted sequence according to the Watson-Crick or the Hoogsteen rule of base pairing. Modifications in the oligonucleotide structure, with the purpose of obtaining new constructs which are able to hybridize strongly with complementary DNA or RNA, could be introduced either in the base moiety either in the sugar-phosphate backbone structure. The modifications which have been integrated into an oligonucleotide have been reviewed recently ( 1). With the exception of a few examples [i.e. 5-methylcytosine and 5-bromocytosine (2), 2-aminoadenine (3), 7-deazaadenine (4), 5-( 1-propynyl)-uracil and -cytosine (5)], incorporation of modified bases leads to less stable duplexes. The incorporation of new backbone structures into oligonucleotides generally enhances their nuclease stability and decreases their binding affinity for complementary sequences. Some sugar modifications [i.e. 2'-O-methylribonucleosides (6), carbocyclic nucleosides (7), α-nucieosides (8)] give modest enhanced target affinity. Although plenty of information is available about the duplex stability of oligonucleotides with alternative phosphate-containing linkages or phosphate-free backbones at a predetermined site (9), completely backbone modified oligonucleotides have seldom been synthesized. This is due to the unavailability of high yielding solid-support-based automated synthesis protocols for these functional groups. Two important exceptions are the morpholino-carbamate linked oligonucleotide (10) and the triple-helix forming polyamide linked acyclic nucleic acids (11). More recently oligodeoxyribonucleotide N3'→P5' phosphoramidates have been described as well, and are claimed to hybridize well to complementary oligonucleotides (12).

A European Patent Application n° 94202342.5 (dd. 17 august 1994) discloses the synthesis of phosphoramidite building blocks of hexitol nucleoside analogues, their assembly into oligonucleotides and the hybridization potential of this new class of oligonucleotide constructs, made up of or containing in part 1,5-anhydro-2,3-dideoxyhexitol nucleoside analogues, and phosphorylated at their 4'-end. The synthesis and antiviral activity of the afore mentioned nucleoside analogues themselves is subject of a European Patent Application n°92.201803.1, dd. 18 juni 1992. The present invention enlarges the diversity of possible constructs which can be envisaged for strong and selective hybridization with a complementary sequence as disclosed in the afore mentioned Patent Application n° 94202342.5, and further simplifies the synthesis of these constructs by elimination of the need for a 4'-terminal phosphate moiety.

Description of the invention

It is the object of the present invention to provide new oligomers, which have an improved stability and binding affinity as compared to the known oligomers.

It has now been found that oligomers comprising or containing in part 1,5-anhydro-2,3- dideoxy-D -arabino-hexitol nucleotide analogues as the monomer units A, wherein the hexitol is coupled via its 2-position to the heterocyclic ring of a pyrimidine or purine base, provide such improved stability and binding affinity. The monomer units A are represented by the formula I:

wherein B is a heterocyclic ring which is derived from a pyrimidine or purine base and X represents oxygen or sulfur. Optionally these monomer units A are combined with 2'-deoxynucleotides (monomer units B), the latter represented by formula II:

wherein B is a heterocyclic ring which is derived from a pyrimidine or purine base and X represents oxygen or sulfur.

The monomers are connected to each other through a phosphodiester bridge with formula III representing the structure of these oligomers.

wherein m, n and p are integers ;

p≥2 ;

each m≥ 1, provided that mi may be 0

each n≥ 1 ; provided that np may be 0 except when p = 2,

each B independently is a heterocyclic ring which is derived from a pyrimidine or purine base ;

each X independently represents oxygen or sulfur ;

the 5'-end, respectively the 6'-end, of the oligonucleotide may optionally be dephosphorylated and the 3'-end, respectively the 4'-end, may optionally be phosphorylated;

All possible salts of the compound of formula III are included in the invention.

Optionally the oligomers may be comprised solely of monomer units A (formula I) as represented by formula IV.

wherein q is an integer with q≥ 2 ;

each X independently represents oxygen or sulfur ;

the 6'-end of the oligonucleotide may optionally be dephosphorylated;

and all possible salts thereof.

The oligomers of formula III and IV are new compounds. They display a certain similarity with oligonucleotides consisting of the natural 2'-deoxynucleosides represented by formula V

wherein k is an integer and wherein each B independently is a heterocyclic ring which is derived from a pyrimidine or purine base, and wherein the 5'-end of the oligonucleotide may optionally be dephosphorylated.

According to the invention it has been found that the oligomers of formula III and IV and their respective salts exhibit sequence specific binding to natural oligonucleotides represented by formula V or its 5'-dephosphorylated congener. A new class of hybridons or sequence- specific binding polymers has therefore been found.

Oligomers of formula III or IV may form a complex with another oligomer of formula III or IV, or they may form a complex with a complementary single-stranded or double-stranded natural oligonucleotide. Each strand may be of the same or different length.

The fact that sequence-specific binding is found for the oligomers of formula III as well as TV with the compounds of formula V, must be deemed surprising and has been discussed and explained in detail in the afore mentioned Patent Application n° 94202342.5. Enlarging the furan ring of furanose compounds to a pyran ring did not yield oligomers capable of binding natural oligonucleotides (compare ref. 13-14). Thus, the effect of enlarging the pentofuranosyl ring to a 1 ,5-anhydrohexitol ring could not be anticipated.

The compounds according to the invention are therefore oligomers comprising or containing partially nucleotide analogues wherein a 1,5-anhydro-2,3-dideoxy-D-hexitol is coupled via its 2-position according to an arabino-configuration to the heterocyclic ring of a pyrimidine or purine base. The oligomers consist of the above mentioned nucleotide analogues connected to each other as phosphate diesters or thiophosphate diesters The oligomers can be represented either by the formula lu or IV, wherein m, n, p, q, B and X have the above stated designations. The oligomers can be exclusively composed of the hexitol nucleotide analogues of the formula I (yielding oligomers of formula IV) or can have natural 2'-deoxynucleotides interspersed or at the end of the molecule (yielding oligomers of formula III). The hexitol has the (D)-configuration and the stereochemistry of both substituents is according to an (S) configuration.

When group B is derived from a pyrimidine base it can be either cytosine, 5-methylcytosine, uracil or thymine When B is derived from a purine base it can be either adenine, guanine, 2,6-diaminopurine, hypoxanthine or xanthine.

The invention also relates to the chemical synthesis of these oligomers and their salts and to their use in antisense strategies which comprise diagnosis, hybridization, isolation of nucleic acids, site-specific DNA modification and therapeutics. Chemical synthesis

The nucleoside analogues, monomer components of the present invention can be prepared in different ways and one of the preparation methods is subject of european patent application n° 92.201 803. 1. These syntheses have been described likewise ( 15). Assembly of the monomers into an oligomer follows the classical schemes and can be done either by standard phosphoramidite chemistry (compare ref. 16) or by H-phosphonate chemistry (compare ref. 17) All procedures are conveniently carried out on an automated DNA synthesizer as for standard oligonucleotide synthesis. For these standard conditions also compare e.g. ref. 18. The preferred method is the phosphoramidite method making use of the phosphoramidites of the hexitol nucleoside analogues as the incoming building blocks for assembly in the "6'-directien". The method of preparation for these phosphoramidite building units is subject of european patent application n° 94202342.5.

Synthesis of the afore mentioned oligonucleotide constructs of formula III makes use of standard solid supports (e.g. controlled pore glass) functionalized with an appropriately protected 2'-deoxynucleoside. Following standard procedures and making use of well-known phosphoramidite monomers of 2'-deoxynucleosides on one hand and phosphoramidite building units of 1,5-anhydro-2,3-dideoxyhexitol analogues as subject of european patent application n° 94202342.5 on the other hand, assembly of any composition of formula III can be obtained. Following removal of the dimethoxytrityl protecting group of the last coupled 1,5-anhydro-2,3-dideoxyhexitol analogue or 2'-deoxynucleotide. the primary hydroxyl group optionally can be phosphorylated by well-known commercially available reagents (e.g. phosphorylation reagent of Glen Research) or can be left intact.

Synthesis of the afore mentioned oligonucleotide constructs of formula IV can be done starting from solid supports functionalized with an appropriately protected 1,5-anhydro-2,3-dideoxyhexitol analogue. The synthesis of such supports is outlined below. Following standard solid phase procedures and using the afore mentioned phosphoramidite building units of 1,5-anhydro-2,3-dideoxyhexitol analogues, constructs of any composition of formula IV can be obtained. Following removal of the dimethoxytrityl protecting group of the last coupled 1 ,5-anhydro-2,3-dideoxyhexitol analogue, the primary hydroxyl group optionally can be phosphorylated by well-known commercially available reagents (e.g phosphorylation reagent of Glen Research) or can be left intact.

The assembly of the constructs of formula III and IV most conveniently is performed on an automated DNA synthesizer using phosphoramidite chemistry. Alternatively different chemistry can be used like the phosphonate strategy, and assembly can be done in solution with either of both strategies This is exemplified by synthesis of a dimer in solution by the phosphonate strategy as described further.

Solid supports containing a 1,5-anhydrohexitol analogue can be prepared by succinylation of the compounds la-d yielding 2a-d, which can be coupled to the amino function of either long chain alkylamino controlled pore glass (CCAA-CPG) or a suitable amino functionalized polystyrene (e g. Tentagel^R - RAPP Polymere) making use of a carbodiimide and yielding 3a-d (for functionalization of supports compare ref. 19).

After assembly, the obtained oligonucleotides are cleaved from the support and deprotected by ammonia treatment for 16 h at 55°C. Purification of the obtained oligomers of the above stated formula's III or IV can be accomplished in several ways (compare e.g. ref. 20). The preferred method is purification by anion-exchange FPLC at a basic pH of 12 to disrupt all possible secondary structures (compare e.g. ref 13). Desalting is done by simple gel filtration techniques followed by lyophilization. All acceptable salts can be prepared in a conventional manner.

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 are not however intended to limit the invention.

Abbreviations : LSIMS liquid secondary ion mass spectrometry, Thgly thioglycerol, TEA triethylamine, TEAB triethylammonium carbonate, TEAA triethylammonium acetate.

Synthesis of the 1,5-anhydro-2,3-dideoxy-2-substituted-D-arabino-hexitol nucleoside analogues and of their 4,6-O-benzylidene protected derivatives has been described by Verheggen et al. ( 15) Base protection, dimethoxytritylation and succinylation of these hexitol nucleoside analogues likewise has been described in patent application nº EU 94202342.5. 1,5-anhydro-6-O-monomethoxytrityl-2-(thymin- 1-yl)-2,3-dideoxy-D-arabinohexitol-4-yl-hydrogenphosphonate, triethylammonium salt (6a)

To 270 μL (3 mmol) of phosphorus trichloride in 30 mL of anhydrous CH₂Cl₂ cooled on an icebath, was added 3 3 mL (30 mmol) of N-methylmorpholine and 690 mg (10 mmol) of 1,2,4-triazole. The mixture was stirred for 30 min at room temperature, subsequently cooled again, and 320 mg (0 6 mmol) of 1,5-anhydro-6-O-monomethoxytrityl-2-(thymin-1-yl)-2,3-dideoxy-D-arabinohexitol (4a) dissolved in 10 mL of CH₂Cl₂.was added dropwise over 15 min. The mixture was stirred for 10 min. more at room temperature and poured into 25 mL of 1 M TEAB buffer. After separation of both layers, the aqueous phase was extracted once more with CH₂Cl₂ and the organics were dried and purified by flash chromatography on 20 g of silica gel (gradient of CH₂Cl₂-TEA 99: 1 to CH₂Cl₂-TEA-MeOH 89: 1 : 10). Product containing fractions were washed once with 25 mL of 1 M TEAB buffer and dried, affording 344 mg (0 495 mmol, 82%) of the title product 6a as a white foam.

LSIMS (ThGly doped with NaOAc) m/z 659 (M+3Na⁺, 1), 637 (M+2Na⁺, 2), 273 (MMTr⁺, 100)

¹H NMR (CDCl₃): δ 1.27 (t, 9H, J=7Hz, CH₂CH₃), 1.74 (s, 3H, CH₃), 1.94-2.11 (m, 1H, H-3'_ax), 2.61 (dm, 1H, H-3"_eq), 3.00 (q, 6H, CH₂CH₃), 3.34 (dd, 1H, J= 4.5 and 10.4Hz, H-5'), 3.45-3.60 (m, 2H, H-6', H-6"), 3.79 (s, 3H, OCH₃), 3.89 (dd, 1H, J= 3.4 and 13.4Hz, H-1'_ax), 4.16 (d, 1H, J= 13.6Hz, H-1'_eq), 4.57 (m, 1H, H-4'), 4.73 (m, 1H, H-2'), 6.66 (d, 1H, J=625 Hz, P-H), 6.83 (d, J - 8.7, 2H, aromatic H), 7.15-7.55 (m, 12H, aromatic H), 8.05 (s, 1H, H-6), 9.50 (br s, 1H, NH) ppm.

¹³C NMR (CDCI₃) δ 8.5 (CH₂CH₃), 12.7 (CH₃), 35.2 (C-3'), 45.3 (CH₂CH₃), 50.4 (C-2'), 55.1 (OCH₃), 62 7 (C-6'), 64.2 (C-4', d, J=3.8Hz), 68.4 (C-1'), 80.4 (C-5', d, J=8Hz), 86.2 (Ph₃C), 1 10.5 (C-5), 138.5 (C-6), 151.2 (C-2), 163.9 (C-4), 1 13 0, 126.8, 127.4, 127.7, 130 4, 135.5, 144 3, 158 4 (aromatic C) ppm.

1,5-anhydro-6-O-monomethoxytrityl-2-(N⁶-benzoyladenin-9-yl)-2,3-dideoxy-D-arabinohexitoI-4-yl-hydrogenphosphonate, triethylammonium salt (6b)

To 3 15 μ L (3.5 mmol) of phosphorus trichloride in 30 mL of anhydrous CH₂Cl₂ cooled on an icebath, was added 3 85 mL (35 mmol) of N-methylmorpholine and 830 mg ( 12 mmol) of 1 ,2,4-triazole The mixture was stirred for 30 min at room temperature, subsequently cooled again, and 450 mg (0.7 mmol) of 1,5-anhydro-6-O-monomethoxytrityl-2-(N°-benzoyladenin- 9-yl)-2,3-dideoxy-D-arabinohexitol (4b) dissolved in 10 mL of CH₂CI₂. was added dropwise over 15 min. The mixture was stirred for 10 min. more at room temperature and poured into 25 mL of 1 M TEAB buffer. After separation of both layers, the aqueous phase was extracted once more with CH₂Cl₂ and the organics were dried and purified by flash chromatography on 20 g of silica gel (gradient of CH₂Cl₂-TEA 99 1 to CH₂Cl₂-TEA-MeOH 89.1 :10). Product containing fractions were washed once with 25 mL of 1 M TEAB buffer and dried, affording 5 16 mg (0 64 mmol, 92%) of the title product 6b as a white foam

LSIMS (ThGly doped with NaOAc) m/z 722 (M+3Na⁺, 2), 750 (M+2Na⁺, 8), 273 (MMTr⁺,

100)

¹H NMR (CDCI₃): δ 1.22 (t, 9H, J=7Hz, CH₂CH₃), 2. 10-2.29 (ddd, 1H, H-3'_ax), 2.82 (dm,

1H, H-3"_eq), 2.88 (q, 6H, CH₂CH₃), 3.35 (dd, 1 H, J= 4.7 and 10.3Hz, H-5'), 3 .56 (dd- 1H, J=1 .8 and 10.3Hz, H-6'), 3.62-3.74 (m, 1H, H-6"), 3.79 (s, 3H, OCH3), 4.03 (dd, 1H, J= 2.4 and 13 .0Hz, H-1'_ax), 4.37 (d, 1H, J= 13.0Hz, H- 1'_eq), 4.46 (m, 1H, H-4'), 5.04 (m, 1H, H-2'), 6.56 (d, 1H, J=625.3Hz, P-H), 6.84 (d, J = 8.7, 2H, aromatic H), 7.15-7.65 (m, 15H, aromatic H), 8.05 (d, 2H, aromatic H), 8.73 (s, 1H) and 8.79 (s, 1H) (H-2, H-8), 9.37 (br s, 1H, NH) ppm.

¹³C NMR (CDCI₃) δ 8.4 (CH₂CH₃), 35.8 (C-3'), 45.2 (CH₂CH₃), 50.6 (C-2'), 55.1 (OCH₃), 62.7 (C-6'), 64.3 (C-4', d, J=5.5Hz), 69.0 (C-1'), 80.7 (C-5', d, J=9.3Hz), 86.3 (Ph₃C), 122.4 (C-5), 142.7 (C-8), 149.5 (C-4), 151.7, 152.3 (C-2, C-6), 166.8 (CO) ppm + aromatic signals.

1,5-anhydro-4-O-benzoyl-2-(thymin-1-yl)-2,3-dideoxy-D-arabinohexitoI (5a)

An amount of 225 mg (0 42 mmol) 1,5-anhydro-6-O-monomethoxytrityl-2-(thymin-1-yl)-2,3-dideoxy-D-arabinohexitol (4a) was dissolved in 10 mL of anhydrous pyridine and treated with 100 μL (0.84 mmol) of benzoyl chloride overnight at room temperature. The mixture was concentrated, poured into 25 mL CH₂Cl₂ and washed twice with a 5% NaHCO₃ solution. The organic phase was dried, evaporated and coevaporated twice with toluene. The obtained foam was treated with 40 mL of a 80% aqueous acetic acid solution for 1 h at 60°C Evaporation and coevaporation with toluene left an oil which was purified by flash chromatography on 20 g of silica gel (gradient of CH₂Cl₂ to CH₂Cl₂-MeOH 94:6). Besides some 4'-O, N³-diben-zoylated product, 108 mg (0.3 mmol, 71%) of the title product 5a was isolated as a white foam.

LSIMS (ThGly) m/z 383 (M + Na⁺, 10), 361 (MH⁺, 20), 239 (MH⁺ - BzOH, 50), 127 (BH₂ ⁺), 105 (C₇H₅O, 100).

1,5-anhydro-4-O-benzoyl-2-(N⁶-benzoyladenin-9-yl)-2,3-dideoxy-D-arabinohexitol (5b)

An amount of 257 mg (0.4 mmol) of 4b was coevaporated with pyridine and subsequently dissolved in 20 mL of anhydrous pyridine after which 1 10 μL (0.8 mmol) of benzoyl chloride was added. The mixture was stirred overnight at room temperature and quenched with 2 mL of methanol. After addition of some NaHCO₃ , the mixture was concentrated and partitioned between CH₂Cl₂ and 5% of aq NaHCO₃. The organic phase was washed once more with 75 mL of aq. NaHCO₃ and dried on Na₂SO₄. Evaporation left an oil which was coevaporated with toluene. The foam was treated with 40 mL of 80% aq HOAc and left at RT for 1 h. Evaporation gave 400 mg of a light brown foam which was purified on silica gel with a methanol gradient in CH₂Cl₂ (100 to 96:4). The product containing fractions were pooled affording 86 mg (0.18 mmol, 45%) of the title product 5b as a white powder.

LSIMS (ThGly) m/z 474 (MH⁺, 25), 240 (BH₂ ⁺, 7).

¹ H NMR (CDCI₃) δ 2.21-2.39 (ddd, 1 H, H-3'_ax), 2.84-2.98 (dm, J= 13.5Hz, 1H, H-3"_eq),

3.64-3.91 (m, 3H, H-5', H-6', H-6"), 4. 13 (dd, 1H. J= 2.6 and 13. 1Hz, H- 1'_ax), 4.52 (dm, 1H,

J= 12.5Hz, H-1'_eq), 5.07-5. 14 (m, 1 H, H-2'). 5. 18-5 .33 (m, 1 H, H-4'), 7.35-7.65 (m, 6H, aromatic H), 7.91 -8.08 (m, 4H, aromatic H), 8.70 (s, 1 H) and 8.80 (s, 1H) (H-2, H-8), 9.63

(br s, 1H, NH) ppm.

¹³C NMR (CDCI₃) δ 33.1 (C-3'), 50.7 (C-2'), 61.4 (C-6'), 64.4 (C-4'), 69.1 (C-1'), 80.5 (C- 5'), 123.2 (C-5), 142.0 (C-8), 149.6 (C-4), 15 1 .7 (C-6), 152.5 (C-2), 164.8 and 165.4 (2 ×

CO) ppm + aromatic signals.

[1,5-anhydro-2-(thymin-1-yl)-2,3-dideoxy-D-arabinohexitoI]-(4-6)-[1,5-anhydro-2-(adenin-9-yl)-2,3-dideoxy-D-arabinohexitol]-phosphate, sodium salt (8)

A portion of 86 mg (0.18 mmol) of 5b and 145 mg (0.21 mmol) of the thymine phosphonate 6a were coevaporated twice with anhydrous pyridine, and dissolved in a 6 mL mixture of anhydrous pyridine and acetonitrile. Then 1 mL (0.5 mmol) of a dilution of pivaloyl chloride in pyridine was added and the reaction mixture was stirred for 10 min at room temperature under a nitrogen atmosphere. Subsequently 2 mL of a 4% I₂ (w/v) solution in pyridine-water (96:4) was added and stirring continued for another 10 min. at room temperature. The mixture was diluted with 100 mL of CH₂Cl₂ and washed with 50 mL of 10% aqueous Na₂S₂O₃ and with 50 mL of 1 M TEAB . The organic phase was dried, evaporated and purified by column chromatography on 20 g of silica gel (gradient of CH₂Cl₂-TEA 99: 1 to CH₂Cl₂-TEA-MeOH 93: 1 :6) affording 200 mg (0.17 mmol, 95%) of the fully protected dimer.

LSIMS (ThGly doped with NaOAc) m/z 1 108 (M+2Na⁺, 2), 273 (MMTr⁺, 100)

The white foam obtained was dissolved in 32 mL of a 3 : 1 mixture of cone ammonia and ethanol and heated overnight at 40°C. The mixture was evaporated and coevaporated with 1,4-dioxane. The residue was dissolved in 20 mL of 80% aqueous acetic acid and heated at 60 °C for 1 h. After evaporation, the residue was partitioned between 10 mL of 10 mM TEAA solution and 10 mL of ether. The aqueous phase was washed 3 times more with 10 mL of diethylether and concentrated and purified by reverse phase HPLC on a polystyrene-divinyl-benzene support (PLRP-S, 250 × 9 mm) with an acetonitrile gradient in 0.1 M TEAA. Product containing fractions were pooled and the TEAA salt was exchanged for the sodium salt by chromatography on a Dowex 50X8-200 cation exchange resin under its sodium form. Lyophylization afforded 52 mg (S5 μmol, 47% overall) as a white voluminous powder.

LSIMS (ThGly) m/z 62S (M sodium salt + Na⁺, 30), 606 (M sodium salt + H⁺, 4).

REFERENCES

1 . Beaucage, S.L.; Iyer, R.P. Tetrahedron 1993, 49, 6123-6194.

2. Sanghvi et al. Nucleoside and Nucleotides 1991, 10, 345-346.

3. Chollet et al. Chemica Scripta 1986, 26, 37-40.

4. Seela, F.; Kehne, A. Biochemistry 1985, 24, 7556-7561 .

5. Wagner et al. Science 1993, 260, 15 10- 15 13.

6. Inoue et al. Nucleic Acids Res. 1987, 15. 613 1-6148.

7. Perbost et al. Biochem. Biophys. Res. Commun. 1989, 165, 742-747.

8. Gagnor et al. Nucleic Acids Res. 1987, 15, 10419-10436.

9. Varma, R.S. Synlett 1993, 621.

10. Wang et al. Tetrahedron Lett. 1991, 32, 7385-7388.

11. Egholm et al. J. Am. Chem. Soc. 1992, 114, 1895-1897.

12. Gryaznov and Chen, J. Am. Chem. Soc. 1994, 116, 3143-3144.

13. Augustyns et al. Nucleic Acids Res. 1992, 20, 471 1-4716.

14. Augustyns et al. Nucleic Acids Res. 1993. 21, 4670-4676.

15. Verheggen et al., J. Med. Chem. 1993, 36, 2033-2040.

16. Matteucci and Caruthers, J. Am. Chem. Soc. 1981, 103, 3 185-3191.

17. Εroehler et al., Nucleic Acids Res. 1986, 14, 5399-5407.

18. Methods in Molecular Biology, vol. 20, Protocols for oligonucleotides and analogs, S.

Agrawal ed., Humana Press, Totowa, New Jersey, U.S.A.

19. Pon et al., Biotechmques 1988, 6, 768-775.

20. Methods in Molecular Biology vol 26, chapter 9 "Analysis and purification of synthetic oligonucleotides by HPLC", S Agrawal ed., Humana Press, Totowa, New Jersey, USA.

Claims

1. An oligomer comprising 1 ,5-anhydro-2,3-dideoxyhexitol nucleotide analogues and deoxyribose nucleotides having the general formula :

wherein m, n and p are integers ;

p≥2 ;

each m≥ 1, provided that mi may be 0

each n≥ 1 ; provided that np may be 0 except when p = 2;

each X independently represents oxygen or sulfur ;

the 5'-end. respectively the 6'-end, of the oligonucleotide may optionally be dephosphorylated and the 3 '-end, respectively the 4'-end, may optionally be phosphorylated,

and all possible salt forms thereof.

2. An oligomer according to claim 1 wherein p > 2.

3. An oligomer according to claim 1 wherein m 1≥ 1 and n_p = 0 such that there is at least one 1,5-anhydro-2,3-dideoxyhexitol moiety at each end of the oligomer

4. An oligomer comprising 1,5-anhydro-2,3-deoxyhexitol nucleotide analogues having the general formula

wherein q is an integer with q > 2 ;

each X independently represents oxygen or sulfur ;

the 6'-end of the oligonucleotide may optionally be dephosphorylated;

and all possible salt forms thereof.

5. An oligomer as claimed in any one of claims 1 to 4, wherein the heterocyclic ring B, when derived from a pyrimidine base, is selected from the group consisting of cytosine, 5- methylcytosine, uracil or thymine.

6. An oligomer as claimed in any one of claims 1 to 4, wherein the heterocyclic ring B, when derived from a purine base, is selected from the group consisting of adenine, guanine, 2,6- diaminopurine, hypoxanthine or xanthine.

7. An oligomer as claimed in any one of claims 1 to 4, wherein the heterocyclic ring B is cytosine, thymine, adenine or guanine.

8. An oligomer as claimed in any one of claims 1 to 7 wherein the 1,5-anhydro-2,3- dideoxyhexitol nucleotide analogues have the (D) configuration and the stereochemistry of both substituents is according to the (S) configuration.

9. A complex comprising a first oligomer as claimed in any one of claims 1 to 8 and either a complementary single-stranded or double-stranded natural oligonucleotide or a self- complementary second oligomer as claimed in any one of claims I to 9, wherein each of the strands in the complex may be of the same or of a different length.

10. An oligomer as claimed in any one of claims 1 to 8 or a complex according to claim 9 for use as a medicine.

1 1. A pharmaceutical composition comprising as an active ingredient a therapeutically effective amount of an oligomer as claimed in any one of claims 1 to S or a complex as claimed in claim 9, and a pharmaceutically acceptable carier.

12. A process of preparing a pharmaceutical composition as claimed in claim 1 1 , characterized in that a therapeutically effective amount of an oligomer as claimed in any one of claims 1 to 8 or a complex as claimed in claim 9, is intimately mixed with a pharmaceutically acceptable carrier.

13. The use of an oligomer as claimed in any one of claims 1 to 8 or of a complex as claimed in claim 9, in molecular biology and genetic engineering.

1 4 . Uses according to claim 13 comprising hybridization, isolation of nucleic acids (isolation of DNA and RNA fragments), site-specific DNA modification, mapping.