FR2612930A1 - alpha Oligonucleotide probes - Google Patents

Abstract

The invention relates to chemical compounds consisting of an oligonucleotide or an oligodeoxynucleotide. According to the present invention, they contain a chain of nucleotides with unnatural anomeric alpha configuration. The invention also relates to an alpha nucleotide chain which is linked, via covalent bonding, to an effector, especially to an intercalating radical or to a reactive or photoactivable chemical radical. The invention also relates to an application of the compounds, an application according to which they concomitantly pair with a complementary beta oligonucleotide sequence.

Description

 The present invention relates to novel chemical compounds, as well as their application, especially as probes for detecting a defined sequence of DNA or RNA.

 The chemical compounds according to the present invention are constituted by an oligonucleotide or oligodeoxynucleotide having a sequence of nucleotides having the unnatural anomeric configuration as opposed to the natural anomeric configuration ss.

(a) et (b) représentent des nucléosides, il faut rajouter un phosphate en 5' pour obtenir des nucléotides.Thus, the formulas (a) and (b) respectively represent nucleosides a and ss

(a) and (b) represent nucleosides, a 5 'phosphate must be added to obtain nucleotides.

 The use of natural oligonucleotides as a probe, ie in all applications involving their pairing with a complementary oligonucleotide sequence for pharmacological purposes is known. However, the use of natural oligonucleotides has some difficulties.

 A first difficulty lies in the fact that it is necessary to provide sequences long enough for these hybrid sequences with their complementary target sequence, sufficiently stable.

 To reduce this length, it is necessary to add to the oligonucleotide, as proposed for the first time by C. HELENE et al., A chain carrying an intercalating agent, such as acridine.

These molecules bind more strongly to their complementary sequences, thanks to the excess energy produced by the intercalation of the covalent head in the hybrid formed.

 A second major difficulty of the use of natural oligonucleotides ss is the metabolic fragility of many oligonucleotide sequences vis-à-vis certain enzymes. However, we know that all living organisms contain many nucleases often very active.

 As is shown later, the compounds according to the present invention comprise oligonucleotide sequences which make it possible to overcome these disadvantages.

 Indeed, they offer the interesting property of pairing in parallel manner with a sequence of complementary natural ss oligonucleotides, whereas two complementary α sequences or two complementary ss sequences pair anti-parallel, and this parallel pairing is much more stable.

 In addition, these oligonucleotides have a very high resistance to enzymes, especially to nucleases.

 Under these conditions, multiple uses for biological and even pharmacological purposes already known for ss oligomers can be envisaged and this with more efficiency.

dans laquelle
les radicaux B peuvent être identiques ou différents et représentent chacun une base d'un acide nucléique éventuellement modifiée et rattachée au cycle glycosidique selon une configuration anomérique a non naturelle
les radicaux X peuvent être identiques ou différents et représentent chacun un oxoanion O un thioanion S # , un groupe alkyle, un groupe alcoxy, aryloxy, un groupe aminoalkyle, un groupe aminoalcoxy, un groupe thioalkyle;;
R et R', qui peuvent être identiques ou différents, représentent chacun un atome d'hydrogène ou un groupement -Y-z
Y représente un radical alcoylène droit ou ramifié -alk- ou un radical choisi parmi

avec U = O, N ou S. Among the compounds according to the present invention, it is therefore more particularly worth mentioning oligomeric compounds of the formula

in which
the radicals B may be identical or different and each represents a base of an optionally modified nucleic acid and attached to the glycoside ring in an unnatural anomeric configuration.
the radicals X may be the same or different and each represents an oxoanion O a thionanion S #, an alkyl group, an alkoxy group, an aryloxy group, an aminoalkyl group, an aminoalkoxy group, a thioalkyl group;
R and R ', which may be the same or different, each represent a hydrogen atom or a group -Yz
Y represents a straight or branched alkylene radical -alk- or a radical chosen from

with U = O, N or S.

or a radical -Y "-O-Y'- where Y" or Y 'can have the meanings given for Y
E can have the same meanings as
X;
J represents a hydrogen atom or a hydroxyl group
Z is a radical corresponding to an effector agent
n is an integer including O
L represents an oxygen atom, a sulfur atom or an -NH- group.

qui correspond à la formule développée

sur laquelle ont été mentionnées les extrémités (3') et (5').In this formula I, the condensed representation of the following nucleosides is used

which corresponds to the developed formula

on which the ends (3 ') and (5') have been mentioned.

 It should be noted that formula I represents a sequence of nucleotides that may be the same or different, n simply indicating the number of nucleotides included in the molecule; n is preferably a number between 1 and 50, and more preferably between 1 and 25.

 The effector radicals correspond to effector agents which are compounds known in nucleic acid techniques. These are, for example, compounds capable of "intercalating" in the structure of DNAs or RNAs.

 These intercalating agents are, in general, constituted by polycyclic compounds having a planar configuration such as acridine, furocoumarin, daunomycin, 1,10-phenanthroline, phenanthridinium, prophyrins, dipyrido derivatives ( 1,2-a: 3 ', 2'-d) imidazole, ellipticine or ellipticinium and their derivatives.

 These effector agents can also be reactive chemical radicals such as cleavage chemical radicals, that is to say that these radicals can directly or indirectly react to split a nucleotide chain. Preferably, these reactive chemical radicals will be activatable, for example by chemical or photochemical means.

Activatable cleavage reactive groups are, for example, compounds derivatives such as
ethylene-diamine-tetracetic acid,
Diethylene triamine pentaacetic acid,
porphyrins,
1,10-phenanthroline, psoralen and other aromatic groups absorbing near-UV and visible radiation.

 These chemically activatable groups in the presence of metal ions, oxygen and a reducing agent induce queues in nucleic acid sequences located in their vicinity.

 The radical B is constituted by a base of a nucleic acid linked to the sugar part of the nucleotide by an anomeric configuration a. It may be thymine, adenine, cytosine, guanine or uracil. But it is also possible to use modified nucleic acid bases, in particular halogenated or azidated, for example 5-bromo-uracil or 8-azidoadenine; or amino derivatives such as 2-aminoadenine and its substituted derivatives, for example on N6 with an aminoalkylene group or an azido phenylalkylene group; or guanine substituted on O, for example by a (U-alkylene) -9-acridine group or 8- (U-aminoalkyl) -amino-adenine and its derivatives substituted on the NH 2 in U by an acridine group.

 According to the present invention, therefore, are considered the usual bases, as well as the possibility of introduction of modified bases. "Effector" bases capable of covalently bonding to a complementary strand may be introduced.

Thus, the functionalization of C or T by an aziridine group at the 4-position leads to the formation of covalent CH 2 -CH 2 bridging between the two complementary strands on G and A, respectively.

 Thus, more particularly, the radical B is chosen from thymine, adenine, cytosine, guanine, 4-azido-cytosine or 4-azido-thymine and 8-azido-adenine, uracil, Bromo-uracil.

 The radical X, although it is preferably an oxoanion, may have other meanings when the radical X represents an alkyl group, it is preferably a lower alkyl group having 1 to 7 carbon atoms and, for example, ethyl, methyl or propyl groups; when the radical X represents an alkoxy group, it is preferably a lower alkoxy group having 1 to 7 carbon atoms, for example a methoxy or ethoxy group; when X represents an amino-alkyl or amino-alkoxy group, it may be a mono-substituted, disubstituted amino-amino group or an amino radical in the form of a quaternary ammonium salt.In these conditions, the substituents are preferably lower alkyl radicals as defined above; as for the alkyl or alkoxy chain connecting the amino group to the phosphorus, it is preferably a straight or branched chain having from 1 to 10 carbon atoms. When the alkyl or alkoxy radical is substituted by a nitrogenous heterocycle, it is in particular a 5-6 membered saturated ring having a nitrogen atom which may be quaternary.

Finally, when the radical X is a thioalkyl radical, it is preferably a lower thioalkyl radical, that is to say which has between 1 and 7 carbon atoms.

 The radical -alk- is preferably a straight or branched alkylene radical having from 1 to 10 carbon atoms.

 In particular, from the terminal alcohol functions 3 'or 5' of the oligomer a, the effector can therefore be introduced via a chain (CH 2) linked to a functionalization Z 'of various natures to the saccharide part of the oligomer .

From the formula
31
or -O-Z '- (CH2) n-effector (Z)
As will be seen from Z ', for example, a phosphate or methyl phosphonate of formula

or CH3
U = O, N or S an ether of general formula
3 'or - O @ CH2 # # CH2 # n-1 Z
51, an ester of general formula -O # CO # CH2 # n Z or a carbamate of general formula
-O #CO NH # CH2 # n Z
The present invention also relates to the above compounds in salt form with bases or acids, and compounds in racemic form, or in the form of purified or mixed R or S isomers.

 The present invention also relates to the above compounds in series D or L.

 In fact, all the advantage of the compounds according to the present invention, consisting of an α-oligonucleotide chain, is to be able to envisage their use independently of effector agents, in particular of intercalating agents, since these new oligonucleotide substances provide recognition. the complementary nucleic acid sequence with a very increased stability; the function of the intercalating agent being to increase the stability of the hybridization complex, its presence is no longer mandatory. However, an intercalating agent will further increase the stability of the thyldridation complex and a reactive group will lead by activation to hydrolysis of the complementary nucleic acid chain; this system thus making it possible to cut with high efficiency a nucleic acid chain at a previously chosen site.

 The compounds according to the invention, by their high specific affinity for complementary nucleic acid sequences, are therefore superior to current oligonucleotides whose affinity for complementary sequences is much lower.

 The compounds according to the present invention can therefore be used more effectively as hybridization probes, but also as purification components for specific sequences of DNA or RNA.

These compounds can also be used to detect mutations in a DNA or a
RNA more efficiently.

 The object of the present invention is also the application of these compounds to achieve the specific blocking of previously selected cell genes or pathogens (viruses, bacteria, parasites).

 The compounds of the present invention, by virtue of their property of binding strongly on the complementary nucleic sequence, and then of inducing cleavage of the nucleic acid chain at this site, can be used as sequence-specific artificial nucleases. They can be used as reagents in molecular biology or genetic engineering.

 The compounds according to the invention are therefore also usable as medicaments.

 The compounds according to the present invention can be prepared chemically by methods in which conventional phosphodiester, phosphotriester, phosphoramidite or hydrogenphosphonate syntheses are applied for B-anomers.

For the phosphotriester method, the anucleotide derivatives of guanine are optionally protected at position o 6 by an additional group, preferably N, N-diphenylcarbamoyl.

 In the processes, the nucleotide chain is first prepared, the various non-used groups being protected, and finally the protecting groups are finally removed to obtain the desired products. This method has made it possible, for the first time, to obtain oligonucleotides comprising all the usual bases. This approach makes it possible to obtain oligonucleotides at desired lengths.

 The compounds of the invention may also be prepared by semi-synthetic methods, in particular using DNA or RNA fragments prepared by chemical engineering methods to which effector agents are then attached.

 Other features and advantages of the present invention will become apparent from the following examples.

 Example V is illustrated by FIGS. 1 and 2. Resistances to the S1 and phosphodiesterase nuclease enzymes respectively for the a and ss anomers are shown.

EXAMPLE I Synthesis of α [d (CATGCG)] and α-Ld (CGCATG); by
a phosphotriester method in solution
improved (see Diagrams I and II below)
The two unnatural hexadéoxyribonucleotides α- [d (CATGCG) 1 and α-d (CGCATG)] comprising only α-anomeric configuration nucleotide units; were obtained by a phosphotriester in improved solution. The starting materials were 4-soxyribonucleosides 3a-d (see Scheme I below).

The α-deoxynucleosides 3a and 3b (pyrimidine) were prepared by autoanomerization reactions followed by selective protections of the hydroxyl groups, while the two deoxynucleotides 3c and 3d (purine) were prepared by transglycosylation reactions. In the case of the α-nucleoside derivatives of guanine an additional group protecting the base was introduced in position O in order to avoid side reactions during assembly of the oligonucleotide. This group was N, N-diphenylcarbamoyl.

N-benzoyl-4-di-O-acetyl-3 ', 5' deoxy-2 '
cytidine was chosen as a sugar donor and was
reacted with the N-protected adenine and the
N-protected guanine in the presence of a Lewis acid
trimethylsilyl trifluoromethanesulfonate, in
acetonitrile at 700C. After reacting the donor
sugar with N-benzoyl-6 adenine the anomer at 1c, in
despite the fact that it was the majo nucleoside product
was not separated from the corresponding anomer
ss.

As a result, this anomeric mixture has been
treated with methanolic ammonia and deoxy
2 'adenosine (2c) was obtained in a yield of
27% after purification by column chromatography
Dowex 1. Then, the derivative (2c) has deoxy-2 'adenosine
has been converted into the 6-N-benzoyl derivative (3c) in a
70% yield.

In the same way, the reaction of trans
Glycosylation was attempted with N-acetyl-2 guanine.

However, very low yields of nucleo derivatives
2-N-acetyl guanine were obtained, true
similarly because of the low solubility of these drifts
in organic solvents. Better performance
in is obtained when one uses in the reaction
of transglycosylation the compound N-palmitoyl-2 guanine
who is more lipophilic.

After 50 minutes of reaction at 700C, an analysis
TLC of the reaction mixture indicated the formation of N-7 isomers as the majority products. Continuing heating, gentle transformation of N-7 isomers to N-9 isomers was observed after 6 hours of heating.

 The desired α-deoxy guanosine derivative was isolated in a yield of 26.6% after silica gel column chromatography and fractional crystallization.

 The enolizable lactam function of guanine residues disrupts the synthesis of oligodeoxynucleotides by the phosphotriester method because of secondary reactions of the guanine fragments which lead to a decrease in yield in the elongation of the guanine-containing chains. It is therefore necessary to protect the lactam function of guanine. N, N-diphenylcarbamoyl was chosen as the protecting group of the o6 position. It can be easily introduced in one step on the starting derivative dd and can be removed at the end of the phosphotriester synthesis, without additional deprotection step.

 The reaction between diphenylcarbamoyl chloride and diisopropylethylamine and product 1a in pyridine followed by addition of aqueous sodium hydroxide, provided after purification by Npalmitoyl-20-diphenylcarbamoyl-6a silica gel column chromatography. 2-deoxy-guanosine (3d) as a crystalline compound in a yield of 77%.

 Then the conversion of 3c and 3d products; of which only the base is protected, in the corresponding fully protected 4c and 4d nucleotides (Scheme II below) required a two-step protocol including selective tritylation of the 5 'hydroxyl by 4,4-dimethoxychloride trityl (1.5 molar equivalents) and phosphorylation of 3'-hydroxyl with triethylammonium (3-chloro-2-trityl-4-phenyl) (cyano-2-ethyl) phosphate (1.1 molar equivalents), chloride of mesitylenesulfonyl (2.2 molar equivalents) and 1-methylimidazole. This last step makes it possible to introduce 4-chloro-4-trityl phenyl as a phosphate-protecting group. Thanks to its lipophilic properties, this protecting group has been chosen to facilitate the purification and to increase the yields of the fully protected intermediates during the synthesis. oligonucleotides according to the phosphotriester method in solution. After purification by silica gel column chromatography, the two 4c and 4d nucleotide derivatives were isolated as diastereoisomeric mixtures in yields of 92 and 53%, respectively. Since the two target hexamines have a 3 'terminal guanine in their base sequence, the only 3' end unit for their syntheses is the O-benzoyl-3 'α-deoxy guanosine 5d derivative. Selective dimethoxytritylation of 3d followed by benzoylation and detritylation provides the 5'-OH 5d derivative in an overall yield of 81%.

 The synthesis of oligodeoxynucleotides fully protected α-Ed (CATGCG) λ and α-Ld (CGCATG) 3 was accomplished by repeating a synthetic cycle consisting essentially of three main steps.

Step (a) is a deprotection of the 5 'hydroxyl by treatment of a fully protected nucleotide or oligonucleotide intermediate with 2% benzenesulfonic acid in a mixture of methylene chloride and methanol (7 / 3) at OOC for 15 to 40 minutes. Step (b) is deprotection of the 3 'phosphodiester by removal of the cyanoethyl group from a fully protected nucleotide or oligonucleotide, with 10% tert-butylamine in anhydrous pyridine for 15-25 min . Step (c) is the condensation between the products of step (a) and of step (b) (-10 to 15% in a molar excess) in the presence of a coupling agent: (Mesitylene-2-sulfonyl) -3-nitro-1,2,4-triazole followed by purification of the products by silica gel column chromatography. The conditions and results of the coupling reactions are summarized in the following table.

A) ad (CATGCG) component component duration ren 3'-phosphodlaster 5'-hydroxy MSNT
(mmol) (mm) Product dem (mmol) (mmol) Dmtr-Ap # p HO-T # p-Cne 0.38 45 Dmtr-A # pT # p-Cne 90
(0.152) (0.1381
Dmtr-C # T # p HO-AE # pT ## p-Cne 0.35 40 Dmtr-C ## pA ## pT ## p-Cne 92 (0,138) (0,124) Dmtr-C ## p HO-G # - Bz 0.35 50 Dmtr-C ## pG-Bz 95
(0.137) (0.125) Dmtr-G ## p HO-C ## pG # -Bz 0.33 70 Dmtr-G ## pC ## pG # -Bz 89
(0.131) (0.119)
Dmtr-C ## pA ## pT ## p HO-G ## pC ## pG # -Bz 0.57 60 Dmtr-C ## pA ## p # T # pG ## pC ## pG # -Bz 97 (0.113 ) (0.096)
B) as (CGCATG) component component duration ren 3-phosphodlester 5'-hydroxyl MSNT
(mmol) (mm) Dementing product (mmol) (mmol) Dmtr-C ## p HO-C ## p-Cne 0.39 55 Dmtr-C ## pC ## p-Cne 80
(0.154) (0.1401 Dmtr-C ## p HO-G ## pC ## - Cne 0.31 60 Dmtr-C ## pG ## pC ## p-Cne 96
(0.124) (0.1121 Dmtr-T ## p HO-G # -Bz 0.34 45 Dmtr-T ## pC-Bz 95
(0.137) (0.125) Dmtr-A ## p HO-T ## pG # -Bz 0.28 60 Dmtr-A ## pT ## pG # -Bz 90 (0.112) (0.102)
Dmtr-C ## pG ## pC ## p HO-A ## pT ## pC # -Bz 0.54 45 Dmtr-C ## pG ## pC ## pA ## pT ## pG # -Bz 100 (0.108) (0.082 ) at
abbreviations: Dmtr: 4,4'-dimethoxy trityl, C *: N benzoyl-4 α deoxy-2 'cytidine, T *: α thymidine
A *: N-benzoyl-6α-deoxy-2'adenosine, G *: N
palmitoyl-20-diphenyl-6-carbamoyl deoxy-2 '
guanosine, p: 2-chloro-4-tritylphenylphosphate,
MSNT: 1- (mesitylene-2-sulfonyl) -3-nitro-1,2,4
triazole, Cne: 2-cyanoethyl, Bz: benzoyl.

 During the process of elongation of fully protected oligonucleotides, no significant difference (reaction time, formation of byproducts, etc.) was detected with respect to that of natural ss oligonucleotides.

 In other words, no significant anomeric effect appeared by the phosphotriester method.

 The total deprotection of the protected hexamines was carried out as follows: Treatment of 6-mer fully protected with 1M Nl, Nl, N3, N3-tetramethylguanidinium, 2-pyridine-carboxaldoximate in a dioxane-water mixture (7 / 1, v / v) at 700C for 24 hours, then with aqueous concentrated ammonia.

The result of this treatment was the simultaneous removal of diphenylcarbamoyl groups and 4-chloro-4-phenyl groups, and benzoyl and palmitoyl groups.

The resulting 5'-dimethoxytrityl oligonucleotides were then treated with. acetic acid for 15 minutes at room temperature. Then, purification was carried out by ion exchange column chromatography. The two α-Ed deprotected oligonucleotides (CATGCG) and c'-Ed (CGCATG) were obtained in yields of 59% and 52%, respectively.

 Thus, the phosphotriester method in solution has been successfully applied to the synthesis of oligonucleotides containing the usual four bases.

 Scheme I below reproduces the synthesis of the four 2-dioxynucleosides having their protected base (except T).

 The reaction conditions were as follows for the different steps of (a) to (h).

Synthesis of protected α-nucleosides: reaction conditions.

(a) trimethylsilyl trifluoromethanesulfonate
N, O-bis (trimethylsilyl) acetamide / acetonitrile (b) sodium methoxide / methanol or aqueous sodium hydroxide / water / tetrahydrofuran / methanol (c) trimethylsilyl trifluoromethanesulfonate,
N, O-bis (trimethylsilyl) acetamide, 6-N-benzoyl
adenine / acetonitrile (d) methanolic ammonia (e) trimethylsilyl chloride / pyridine
benzoyl, concentrated solution of ammonia (f) trimethylsilyl trifluoromethanesulfonate, N, O
bis (trimethylsilyl) acetamide, N-palmitoyl-2 guanine /
acetonitrile (g) diphenylcarbamoyl chloride / pyridine (h) aqueous sodium hydroxide / ethanol.

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<tb><SEP> -o
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 T: Thymine.

Cbz: 4-N-benzoylcytosine.

Abz: 6-N-benzoyladenine.

Gpal, dpc: 2-N-Palmitoyl-6-O-diphenylcarbamoylguanine.

Scheme I
Scheme II below reproduces the synthesis of the four deoxy-2 'nucleosides a, having their base protected (except T).

The reaction conditions were as follows in steps (a) to (d) (a) dimethoxy-4,4'-trityl chloride / pyridine (b) (4-trityl-2-chlorophenyl) (2-cyanoethyl) phosphate
of triethylammonium / mesitylene chloride-2
sulfonyl (c) benzoyl chloride / pyridine (d) benzenesulfonic acid 2% / methylene chloride
methanol (7: 3, v / v).

Abz: 6-N-Benzoyladenine
Gpal, dpc: 2-n-Palmitoyl-6-O-diphenylcarbamoyl-5d B = Gpal, guanine dpc
Tr: Triphenylmethyl
Scheme II
1) Preparation of the 2'-deoxy-adenosine (2c) (Scheme I)
This compound was prepared as described by
T. Yamaguchi and Cal. in Chem. Pharm. Bull. 32, 1441
(1984). This compound was obtained in a yield of
27.3% for a melting point of 212.5-2130C.

2) Preparation of N-benzoyl-6α-deoxy-2'adenosine (3c)
(diagram II)
Using a similar three-step process
to that described for the corresponding s anomers
for example in Journal Amer. Chem. Soc., 104, 1316
(1982), this product was prepared from (2c) (2.01 g, 8 mmol). At the end of the third step, that is, after treatment with aqueous ammonia for 30 minutes followed by evaporation, the residue was dissolved in water (200 ml). The solution was washed with diethyl ether (2 x 70 ml) and evaporated to dryness. The remaining residue was mixed with methanol (50 ml) and filtered, and the filtrate evaporated to dryness. The residue was purified by silica gel column chromatography (100g). The appropriate fractions were combined and evaporated. The residue was crystallized from ethanol-acetone. Filtration gave 1.99 g (70%) of 2. The melting point is 163-1640C.

 The analytical results were as follows: Anal. calc. for 17H17O4N5: c.

57.46; n, 4.82; N, 19.71, obtained: C, 57.30: H, 4.56; N, 19.75. UV
# maxH2O (nm (#)): 280 (21,500); # maxpH2.1 (nm (#)): 253 (10,900), 284
(24,000), [&alpha; [D20 + 59] (c = 1, DMF). 1H-NMR (DMSO-d6) @ 2.44
(m, 1H, H2-); 2.8 (m, 1H, H2 '); 3.49 (m, 2H, H5, and H5 #); 4.21
(M, 1H, H4); 4.37 (m, 1H, H3,); 4.86 (t, 1H, OH5,); 5.54 (d, 1H, OH3,); 6.50 (dd, 1H, H1., J-2.58 and 7.57 Hz); 7.52-8.06 (m, 5H, Ar-N); 8.69
det 8.74 (2s, 2H, H2 and Hg); 11.14 (brs, 1H, NH).

 The proton NMR spectra were obtained on a Bruker WBUM 360 apparatus.

3) Preparation of Di-O-acetyl-3 ', 5' N-palmitoyl-2
a 2'-deoxy guanosine (id) (scheme I)
Bis-trimethylsilylacetamide (7.4 ml, 300 mmol) was added to a solution of di-O-acetyl3 ', 5'-N-benzoyl-4-deoxy-cytidine (15 g, 36 mmol) and N-palmitoyl. -2 guanine (37.4 g, 96 mmol) in anhydrous acetonitrile (220 mL). The mixture was heated at 700 ° C. for one hour and trimethylsilyl triflate (8 ml, 47 mmol) was added. The mixture was stirred at 700C for six hours. The solvent was removed under reduced pressure and the residue was dissolved with methylene chloride (750 ml). Cold aqueous sodium hydrogencarbonate (70 ml) was added and after vigorous stirring, the mixture was filtered by suction.

The organic layer was separated and dried with anhydrous sodium sulfate and the solvent was removed under reduced pressure. The residue (Rf 0.44 and 0.57, silica gel TLC, CH 2 Cl 2 -CH 3 OH 9: 1, v / v) was chromatographed on a silica gel column (250 g) using increasing proportions (0-5%) of methanol in the chloroform as eluent. Fractions containing material with a Rf of 0.44 (N-9a and ss isomers) were combined and evaporated to dryness.

The residue was dissolved in hot ethanol and cooled, the ald anomer was crystallized to give 5.655 g (26.6%) of colorless crystals; melting point 129-1300C.

Anal. Calc. for C30H47N5O7: c, 61.10; H, 8.03; N, 11, B, C, 60.96; H, 8.19; N, 12.06. UV #max @@@ (mn (#)); 258 (18,300); 279 (13,900). [&alpha;] D20 + 19 (c = 1, DMF) 1H-MHR (CDCl3) # 0.86 (t, 3H, (CH2) 14-CH3); 1.20-1.40 (m, 24H, - (CH 2) 12-CH 3); 1.71 (m, 2H,
C (= o) -CH 2 -CH 2 -; 2.03 and 2.1 (2s, 6H, 2CH3-C (= o) -); 2.48 (t, 2H,
C (=) - CH2- (CH2) 13-); 2.56 (m, 1H, H2 @); 2.84 (m, 1H, H2,); 4.18-4.28 (m, 2H, H5, and H5 +); 4.52 (m, 1H, H4,); 5.28 (m, 1H, H3,); 6.23 (dd, 1H, H1, J = 1.89 and 7.47 Hz); 7.92 (s, 1H, H8); 8.77 and 12 (2br s, 2H, 2 NH).

4) Preparation of N-palmitoyl-2 O-diphenylcarbamoyl
6α-deoxy-2'guanosine (3d) (Scheme I)
Diphenylcarbamoyl chloride (2.192 g, 9.46 mmol) and diisopropylethylamine (1.23 mL, 7.1 mmol) were added to a solution of ID (2.536 g, 4.3 mmol) in anhydrous pyridine. (11 ml). The resulting mixture was stirred for 2.75 hours at room temperature and (41 ml) pyridine and (21.5 ml) ethanol were added. The reaction mixture was stirred for 10 min at 0OC. A solution of 2 N aqueous sodium hydroxide (21.5 ml) was added and after further stirring for 10 min at 0 ° C the reaction mixture was neutralized with acetic acid (2.65 ml). Then, the reaction mixture was poured into water (150 ml) and the products were extracted with methylene chloride (4 x 50 ml). The combined organic layers were evaporated to dryness under reduced pressure and the residue was fractionated by silica gel column chromatography (50g). Fractions containing pure 3d were combined and evaporated to dryness. The residue was crystallized from ethylacetate (2.323 g, 77%), mp 134-1350 ° C.

Anal. Calc. for
C39H52N6O6: C, 66.83; H, 7.48; N, 11.99; obtained C, 66.53; H, 7.66; N, 11.70. [&alpha;] D20 + 33- (c = 1, DMF). UV #maxETOH (nm (#)) 225 (37,900), 277 (13,900), 257 (shoulder). 1H-NMR (DMSO-d6) δ 0.8S (t, 3H, (CH 2) 14-CH 3); 1.18-1.35 (m, 24H, (CH 2) 12-CH 3); 1.58 (m, 2H, C (= o) -CH 2 -CH 2 -); 2.41-2.48 (m, 3H, H2 and (CH2) 13); 2.76 (m, 1H, H2,); 3.48 (m, 2H, H5, and H5 +); 4.21 (m, 2H, H4,); 4.35 (m, 1H, H3,); 4.86 (t, 1H, OH5,); 5.43 (d, 1H, OH3,); 6.33 (dd, 1H, H1, J = 2.36 and 7.67 Hz); 7.30-7.51 (m, 10H, ArH); 8.66 (s, 1H, H8); 10.65 (s, 1H, C (= o) -NH-).

5) Preparation of N-palmitoyl-2 O-diphenylcarbamoyl-6
0-benzoyl-3 '&alpha;2'-deoxyquanosine (5d) (Scheme II)
Dimethoxy-4,4'trityl chloride (0.39 g) (1.15 mmol) was added to a solution of 3d (0.7 g,
1 mmol) in dry pyridine (3.5 ml). The mixture
resulting was stirred for two hours at room temperature
and benzoyl chloride (0.152 ml, 1.3 mmol) was added. The mixture was stirred for an additional 4.4 hours and 1 ml of water.
been added. After five minutes of stirring, the mixture
of reaction was poured into hydrogen carbonate
saturated aqueous sodium (40 ml) and the products were
extracted with methylene chloride (4 x 30 ml).

The combined organic layers were evaporated
to dryness under reduced pressure. The residue was
dissolved in methylene chloride-methanol (7/3,
v / v, 20 ml). To the resulting cooled solution (0 C),
has been added a cooled solution of benzene
sulfonic acid 10% (w / v) in the term melanqe (5ml). After 14 minutes
stirring at OOC, the reaction mixture was neutral
read with aqueous sodium hydrogencarbonate
(20 ml) and shared between sodium hydrogencarbonate
Saturated aqueous solution (40 ml) and methylene chloride
(4 x 30 ml) The combined organic sunsets were
evaporated to dryness and the residue was fractionated
born by chromatography on silica gel column (30g).

Fractions containing pure 5d product were combined
and evaporated. The residue was crystallized from
methanol to give colorless crystals (0.65 g, 81%), bp 144-146 C.Anal.Calc.for C46H56N6O7: C, 68.64; H.

7.01; N, 10.44; obtained c, 65.37: H, 7.04; N. 10.35. @ H-NHR
(CDCl3) @ 0.87 (t, 3H, (CH2) 14-CH3); 1.18-1.36 (m, 24H, (CH 2) 12-CH 3); 1.66-1.74 (m, 2H, C (= o) CH2 = CH2-); 2.67 (pseudo t, 2H, C (= o) -CH 2 (CH 2) 13); 2.96-3.03 (m, 3H, H2., H2- and OH5.); 3.87-3.90 (m, 2H, H5.

 and H5 @); 4.56 (m, 1H, H4,); 5.61 (m, 1H, H3,); 6.53 (pseudo t, 1H, H1., Japp = 4.3 Hz); 7.21-7.68 (m, 15H, ArH); 8.22 (s, 1H, NH); 8.27 (s, 1H, H8).

6) General method of preparation of 0-dimethoxytrityl-5 '
has deoxyribonucleoside 3'-O- (2-chloro-4-trityl)
phenyl) (2-cyanoethyl) phosphates
4,4'-Trityl-dimethoxy chloride (1.091 g, 3.22 mmol) was added to a solution of protected base-deoxyribonucleoside (2.8 mmol) in dry pyridine (7 ml) and the The mixture was stirred at room temperature until complete disappearance of the nucleoside starting material (2 to 2.5 h).

Then triethylammonium (2-chloro-4-trityl-phenyl) (2-cyanoethyl) phosphate (2.06 g, 3.5 mmol), mesitylene-2-sulphonyl chloride (1.531 g, 7 mmol) and N Methylimidazole (0.56 ml, 7 mmol) was added successively, and the reaction mixture was stirred at
room temperature for one hour and a mixture
water (2 ml) and pyridine (5 ml) was added.

After an additional stirring of 15 minutes, the mixture
of reaction was poured into dihydrogenphosphate
0.2 M aqueous sodium (150 ml) and extracted with
methylene chloride (3 x 60 ml). Orga layers
were washed successively with
0.2 M aqueous sodium dihydrogenphosphate (75 ml)
and aqueous ammonium hydrogencarbonate 0.6 M
(1% relative to sodium chloride, 75 ml). The
organic layer was evaporated to dryness under
reduced pressure and the residue was fractionated by
silica gel column chromatography (60g).

The appropriate fractions were combined and evaporated
to dryness and freeze-dried from benzene.

For O-dimethoxytrityl-5'-N-benzovl-6
2'-deoxy-adenosine 3'- (2-chloro-4-trityl phenyl)
(2-cyanoethyl) phosphate (4c).

The yield of diastereoisomeric mixture
was 92%, Anal. talc. for C66H56ClN6O9P, 0.5 H2O: C. 68.77: H. 4.98; N, 7.29; got
C, 68.85; H. 4.99: N, 7.35. @ lp-NMR (CDCl3) d -7.88 and -7.96. 1H
NMR (CDCl3) δ 2.58-2.65 (m, 2H, CH2-CN); 2.95 (m, 1H, H2-); 3.08 (m, 1H, H2,); 3.22-3.47 (m, 2H, H5, and-H5 +); 3.76 and-- 3.77 (2s, 6H, 2
O-CH3); 4.05-4.16 (m, 2H, PO-CH 2 -); 4.71 and 4.83 (2 pseudo t, 1H, H4.); 5.33 (m, 1H, H3,); 6.71 (pseudo t, 1H, H1, Japp "4.9 Hz), 5.8-8 (m, 36H, ArH), 8.28 and 8.30 (2s, 1H, H2 or H8), 8.74 and 8.79 (2s, 1H). , H8) or H-): 8.97 and s (2s, 1H, NH).

For O-dimethoxytrityl-5 'N-palmitoyl-2
O-diphenylcarbamoyl-6α-deoxy-2 'guanosine 3' - [(chloro
2-trityl-4-phenyl) (2-cyanoethyl) 3-phosphate (4d).

The yield of diastereoisomeric mixture was 53%, filai. Calc. for C88H91ClN7O11P:
C, 70.98; H, 6.16 N, 6.58; obtained C, 71.26;
H, 6.18; N, 6.47. 31p-NMR (CDCl3) @ -8.15 and -8.52. 1H-NMR
(CDCl3) @ 0.87 (t, 3H, (CH2) 14-CH3); 1.15-1.33 (m, 24H, (CH 2) 12-CH 3); 1.68-1.73 (m, 2H, C (= o) -CH 2 -CH 2 -); 2.41-2.48 (m, 2H, CH 2-CN); 2.652.74 (m, 2H, C (= o) -CH 2 -CH 2 -); 3.03 (m, 2H, H2, and H2-); 3.19-3.46
(m, 2H, H5, and H5 +); 3.75 and 3.76 (25, 6H, 20-CH3); 3.83-3.95
(m, 2H, PO-CH2-): 4.65 and 4.82 (2 pseude, 1H, H4,); 5.31
(M, 1H, H3,); 6.56 (pseudo t, 1H, H1., Japp "3.3 Hz); 6.80-7.43
(M, 41H, Ar); 7.91 and 7.97 (2s, 1H, NH); 8.17 and 8.23 (2s, 1H, H8).

7) General method of assembly of oligodeoxyribonucleo
tides a
The fully protected oligonucleotides have been assembled by repeating a cycle of selective reactions including detritylation, decyanoethylation and condensation, these steps being conventional.

8) General method of deprotection of oligodeoxyribonu
cleotides a containing protected G residues.

 An oligonucleotide solution was completely protected (0.058 mmol) of 2-pyridinealdoxime (1.117 g, 9.15 mmol) and N1, N1, N31N3-tetramethylguanidine (1.15 mL, 9.15 mmol) in a dioxane-dioxane mixture. water (7: 1, v / v, final volume adjusted to 9.15 ml) was heated at 700 ° C for 24 hours. The solution was evaporated and the residue was dissolved in aqueous ammonia at 500C for 5 hours. Then, the solution was evaporated and the residue was dissolved in 80% acetic acid (16 ml). After 15 minutes of acid treatment, the solution was washed with methylene chloride (5 x 20 ml).

The aqueous solution was evaporated to dryness and the products were chromatographed on a column (2.25 cm) of DEAE-Sephadex A25 (HCO3-) using a triethylammonium hydrogencarbonate buffer (pH 7.5 linear gradient). 0.001 M to 1M per 1000 ml) as eluent. 9 ml fractions were collected and the appropriate fractions were combined and evaporated. The remaining residue was treated with a 50 W Dowex (Na +) yielding the desired oligonucleotide as the sodium salt. Both α-Fd (CATGCG) and α-Ed (CGCATG) oligonucleotides were obtained in yields of 59 to 52%, respectively. analysis
HPLC on C18 column indicated purities greater than 98%.

EXAMPLE II Synthesis of hexaribonucleotide a-CpUpCp
CpCpU by the phosphotriester method
(see diagrams III and IV below)
REFERENCES 1. SS JONES, B. RAYNER, CB REESE, A. UBASAWA and
M. UBASAWA, Tetrahedron, 36, 3075 (1980).

2. RA SANCHEZ and THE ORGEL,
J. Mol. Biol., 47, 531 (1970).

3. WT MARKIEWICZ,
J. Chem. Res., Synop. 24 (1979) and J. Chem. Res.

 Miniprint, 181 (1979).

4. JB CHATTOPADHYAYA and CB REESE,
Tet. Letters, 5059 (1979).

The synthesis of hexaribonucleotide
CpUpCpCpCpU described in the following was carried out according to the phosphotriester method in solution. It is similar to that described by CB REESE et al. (1) for the natural series B. It required the preparation of the nucleoside synthon a constituting the 3 'end of the oligonucleotide, the synths a and Sb for the extension of the oligonucleotide chain and the synthon 7b constituting the end 3 'of the oligonucleotide.

The two partially protected triribonucleotides 12 and 16 were prepared according to scheme IV, and then assembled to produce the fully protected hexaribonucleotide 17. After deprotection, the α-CpUpCpCpCpU hexaribonucleotide was purified and characterized.

a B: CYTOSINE a B ': N-BENZOYL-4 CYTOSINE (CBz) b B: URACILE b B': URACILE (U)
THP: TETRARYDROPYRANYL-2 SCHEMAIII
Dmtr: DIMETHOXY-4,4 'TRITYL
Ar: CHLORO-2 PHENYL
Bz: BENZOYL

THP: TETRANYDROPYRANMYL-2 Dmtr: DINETMOXY-4,4 'TRITYL
Bz: BENZOYL
Ar: CNLO @ O-2-PNEMYL
And: ETHYL
i) O-CMLOROPNEMYL PMOSPNORO @ L- (TRIAZOL-1,2,4 IDE) / ACETONIYRILE / PYRI @ INE THEN WATER / TRLETHYLAMIME / PYRIDINE ii) MESITYLENESULFONYL-1 NITRO-3 TRIAZOLE-1,2,4 (MSNT) / PYRIDINE.

iii) TRIFLUDNOACETIC ACID 0.5% / DICHLORONETHANE AT 0 C OR ACID BENZENESULFONIOUE 2% / DICHLONOMETRANE
METNANOL AT 0 C
Scheme IV 1) Preparation of the nucleoside synthons 5a, 5b, 6a
and 7b
They were prepared from 1a and 1b ribonucleosides obtained according to methods described in the literature (2). The selective introduction of the acid-labile tetrahydropyranyl-2 (THP) protecting group was carried out according to the following method (3) (see scheme III). After treating the compound 1b with tetraisopropyl-1,1,3,3-dichloro-1,3-disiloxane in a dimethylformamide-pyridine mixture, the derivative obtained 2b is treated with tetrahydropyran in the presence of trifluoroacetic acid to give 3b. the latter is subjected to a treatment with a solution 1t1 of tetrabutylammonium fluoride in tetrahydrofuran. The tetraisopropyl-1,1,3,3-disilyl protection is thus selectively removed and the compound 5b is obtained.

 Cytidine &alpha; is treated analogously to that previously described with however the following modification which makes it possible to introduce a benzoyl protection in position 4 of the cytosine base. After introduction of the tetrahydropyranyl group, the compound 3a is treated with benzoyl chloride in a solution of pyridine and the resulting compound 4a is treated with a 1M solution of tetrabutylammonium fluoride in tetrahydrofuran to give, after purification, the desired compound 5a. The nucleoside derivative 6a was obtained after tritylation of α by dimethoxy-4,4'trityl chloride in a pyridine medium.

 Finally, the nucleoside construct zb, constituting the 3 'end of the oligonucleotide, was obtained in three steps from cytosine a. These three steps are: a) selective tritylation of the 5'-hydroxyl by dimethoxy-4,4'-trityl chloride, then b) benzoylation of 2 'and 3' sections by benzoyl chloride and finally c) selective deprotection 5'-hydroxyl by treatment with 2% benzenesulfonic acid in dichloromethane-methanol (7/3, v / v). The overall performance of these three steps is 67%.

2) Fully Hexaribonucleotide Assembly
teged a-CpUpCpCpCpU
Triribonucleoside diphosphate 16 and triribonucleoside triphosphate 12 were prepared according to Scheme III. Each assembly cycle comprises a phosphorylation step for introducing a phosphodiester group at the 3 'position of a partially protected nucleoside or nucleotide derivative and a coupling step between the phosphodiester-3' compound obtained in the preceding step and a nucleoside or nucleotide derivative whose OH-5 'function is free. In some cases (see Scheme II), an additional detritylation step is necessary to deprotect this OH-5 'function.

 The 3 'phosphorylation steps were carried out by treatment with O-chlorophenylphosphorodi- (triazol-1,2,4-ide) (4) of an OH-3' derivative dissolved in an acetonitrile-pyridine mixture. anhydrous, followed by hydrolysis in the presence of triethylamine. Thus, O-aryl phosphate-3 '8a, 10 and 12 derivatives were obtained with yields greater than 90%.

 The coupling steps between an O-arylphosphate-3 'derivative and a nucleoside or nucleotide derivative 5'-OH were carried out in solution in anhydrous pyridine in the presence of coupling agent: mesitylenesulfonyl-1-nitro-3-triazole 1,2,4 (MSNT) (1).

 Thus, for example, to a solution of triribonucleoside triphosphate 12 (0.334 mmol) and triribonucleoside diphosphate 16 (6.278 mmol) in anhydrous pyridine (2 ml), MSNT (1.67 mmol) is added.

The mixture is stirred for 55 minutes at room temperature and then poured onto a solution of sodium hydrogencarbonate (50 ml). The resulting mixture is extracted with dichloromethane (3 x 30 ml). The organic phases are combined and evaporated under reduced pressure. The residue is purified by column chromatography on silica gel (eluent: dichloromethanemethanol). The fractions containing the desired product are combined, evaporated under reduced pressure and the residue is precipitated with petroleum ether (58% yield).

3) Deprotection, purification and characterization of
lthexaribonucleotide aCpUpCpCpCpU
The fully protected hexaribonucleotide 17 was subjected to the following treatments. 1M tetramethylguanidinium 1-nitro benzyl benzoximate
in a dioxane-water mixture (1/1, v / v) for 18 hours
hours.

 . ammonia concentrated for 5 hours at 50 ° C.

. hydrochloric acid (pH 2) for 4 days at room temperature
ambient temperature.

 At the end of these treatments, the α-CpUpCpCpCpU deprotected oligonucleotide was purified by DEAE-Sephadex anion exchange column chromatography (HCO3-> form using a solution of triethylammonium hydrogen carbonate (0.01M linear gradient ## 1M over 1000 ml).

 The deprotection efficiency was 56%.

 A fraction (about 5 units of OD 100) of α-CpUpCpCpCpU oligoribonucleotide was enzymatically degraded by snake venom phosphodiesterase and alkaline phosphate. High performance liquid chromatography analysis of the products of digestion indicated complete degradation to auridine and α-cytidine in a 1: 2.1 ratio.

EXAMPLE III
Supported synthesis of oligodeoxyribonucleotides a
The supported synthesis of α-oligodeoxyribonucleotides, which is described below, proceeds according to the phosphoroamidite method. It is similar to that described for the natural series ss by Caruthers et al. (SL Beaucage and MH Caruthers, Tetrahedron
Lett., 22, 1859-1862 (1981) and LJ McBride and MH

Caruthers, Tetrahedron Lett., 24, 245-248 (1983)). It required the preparation (1) of 2a-d-deoxynucleoside-phosphoramidite synthons corresponding to the four bases T, C, A and G according to the following scheme

The abbreviations have the following meanings: B = T = Thymine b B = CBZ = N-benzoyl-4 cytosine c B = ABZ = N-benzoyl-6 adenine
d B = Pal d B = N-palmitoyl2 guanine.

Dmtr: Dimethoxy-4,4'-trityl iPr. isopropyl Bz: benzoyl
This synthesis also required the functionalization of a solid support (2) incorporating an α-deoxyribonucleoside derivative.

In a second step, the elongation of the oligonucleotide chain (3) was carried out with the aid of a
manual or automatic synthesizer. Finally, at the end of
number of rings of synthesis cycles, the oliaonucleotide
was unhooked, deprotected and purified.

Preparation of deoxyribonucleoside phosphoramidites
2a-d.

The four α-deoxyribonucleoside phosphoramic
N-protected 2a-d were obtained from the
N-protected O-dimethoxytrityl-5'-deoxyribonucleosides
corresponding 1a-d. The nucleoside derivative 1 has been
treated with chloro N, N-diisopropylamino phosphoramidite
methyl (2.5 molar equivalents) in the presence of
N, N, N-diisopropylethylamine (DIEA) in dichloro
methane under an inert atmosphere. The phosphoramidite derivative
obtained 2 was purified by gel chromatography of
silica and isolated with yields ranging from 79% to
95%. Analysis of the four phosphoramidite derivatives
2a-d by 31P-NMR indicates purity greater than 97%.

The general method of preparation of derivatives
N, N-diisopropylamino-3'-aminopro-2'-amiodoronephosphoramidite
nucleosides 2a-da was the next.

To a solution of O-dimethoxytrityl-5'N
protected α-deoxynucleoside 1 (1 mmol) and N, N, N-diiso
propylethylamine (4 mmol) in dichloromethane
anhydrous (3 ml) is added, under argon and through
syringe, methyl chloro N, N-diisopropylamino phosphoramidite (2.5 mmol). The reaction mixture was stirred at room temperature for 10 minutes and then transferred to a separatory funnel containing ethyl acetate (35 ml). The mixture is extracted with brine (4 x 80 ml) and the organic phase is dried over anhydrous sodium sulphate and evaporated under reduced pressure. The evaporation residue is purified by chromatography on silica gel (10 g) using as eluent a hexane-dichloromethane mixture containing 1% triethylamine. The fractions containing the pure product are combined and evaporated under reduced pressure.

The residue is precipitated in n-hexane (50 ml) cold (-780) from a solution in toluene (5 ml) or freeze-dried from a solution in benzene (15 ml). Compounds 2a-d are obtained in the form of white powders and stored under argon at -200.

The results of analyzes of the four phosphoramidite derivatives 2a-d by 31 P-NMR are as follows: 2a: yield 88% 31 P-NMR # 150.9
1H-NMR 6.89 (broad s, 1H, H-N3); 7.60 and 7.57 (2d, 1H, H6);
7.46-6.79 (m, 13H, ArH); 6.34 (pseudo d, 1H, H1,, Japp = 7.5
Hz); 4.63-4.45 (m, 2H, H3, and H4,); 3.80 (s, 6H, CH 3 -OAr);
3.68-3.42 (m, 2H, H5, and H5-); 3.32 and 3.31 (2d, 3H, CH3-0-P,
J = 13.3 Hz); 3.25 (m, 2H, N (CH- (CH 3) 2 2 2), 2.86-2.65 (m, 1H,
H2,); 2.30 - 2.14 (m, 1H, H2 -); 1.92 (s, 3H, (H3C) 5); 1.17-1.01
1m, 12H, (C H 3) 2-cH) 2N).

2b: 79% yield. 31P-NMR # 150.42 and 151.7
1H-NMR δ 8.62 (broad s, 1H, H-N4); 8.12 and 8.11 (2d, 1H, H6
J5 6 = 7.2 Hz); 7.90 (d, 1H, H5); 7.66-6.82 (m, 18H, ArH);
6.35 (pseudo t, 1H, H1, t api = 5.5 Hz); 4.72 and 4.63 (2 pseu
do, 1H, H3,); 4.54-4.47 (m, 1H, H4,); 3.80 (s, 6H, CH 3 -O Ar);
3.52-3.35 (m, 2H, H5, and H5 ,,); 3.29 and 3.23 2d, 3H, CH 3 -O-P,
J = 13.3 Hz); 3.29-3.10 (m, 2H, N (CH- (CH 3) 2) 2); 2.93-2.71 (m, 1H,
H2,); 2.53-2.36 (m, 1H, H2 ,.); 1.12-0.98 Cm, 12H, t (CH3) 2-CH32N).

2c: 95% yield. 31P-NMR # 150.81 and 150.91
1 H NMR 6 8.91 (s, 1H, NH); 8.84 and 8.59 (2d, 2H, H and H8)
8.04-6.81 (m, 18H, ArH); 6.74-6.68 (m, 1H, H1,); 4.67 -4.56
(m, 2H, H3, and H4,); 3.80 (s, 6H, CH 3 -OAr); 3.61-3.43 (m, 2H,
H5, and H5.,); 3.28 and 3.26 (2d, 3H, CH3-OP, J = 13 Hz);
3.40-3.16 (m, 2H, N (CH- (CH 3) 2) 2); 3.0-2.9 (m, 1H, H2,);
2.72-2.53 (m, 1H, H 2 O); 1.16-1.01 (m, 12H, t (CH3) 2-CH) 2N).

:: 87% yield 31P-NMR # 150.76 and 150.97
1H-NMR # 11.88 (broad s, 1H, NH); 8.28 and 8.24 (2 s, 1H, H8);
7.46-6.82 (m, 14H, NH and ArH); 6.28 (pseudo t, 1H, H1,, Japp =
7.8 Hz); 4.70-4.44 (m, 2H, H3, and H4,); 3.80 (s, 6H, CH 3 -O Ar);
3.76-3.41 (m, 2H, H5, and H5,); 3.31 and 3.25 (2d, 3H, CH 3 -O-P, J =
13.5H); 3.36-3.12 (m, 2H, N (CH- (CH 3) 2) 2); 2.94-2.80 Xm, 1H, H2,);
2.61-2.36 (m, 3H, H2, and (CH2) 13-CH2-C (= o)); 1.70 (m, 2H, CH2-
CH2-C (= O)); 1.25 (m, 24H, CH3- (CH2) 12); 1.17-1.08 (m, 12H,
((CH3) 2-CH) 2N); 0.88 (m, 3H, CH3- (CH2) 14).

(2) Functionalization of the solid support
It was carried out by the establishment of a succinyl linkage between the hydroxyl-3 'group of the N-acyl-O-dimethoxytrityl-5' a-deoxynucleoside derivative 1 and the amino group of the solid support according to the following scheme

DCC: dicyclohexylcarbodiimide
DMAP: dimethylaminopyridine
(catalyst)

 Compound 1 was previously converted to the pentachlorophenyl succinate-3 '4 derivative and then reacted with the amino group of the long chain alkylamine attached to porous glass beads (LCA-CPG) (SP Adams, KS Kavka, EJ Wykes, SB

Holder and G. R. Galluppi, J. Am. Soc., Ic, 66 (1.83,).

 The amount of nucleoside bound to the solid support was determined by spectrophotometric assay of the dimethoxytrityl cation released by treatment with a 10% solution of trichloroacetic acid in dichloromethane. The degree of functionalization varies from 25 to 38 moles per gram depending on the nature of the nucleoside base.

(3) Synthesis of &alpha; -d (TACGGCCAGT)
The decanucleotide ad (TACGGCCAGT) was synthesized manually. The 0-dimethoxytrityl-5'-alpha-thymidine covalently bound to the LCA-CPG support (about 1 ssmole) is introduced into a small reactor. The elongation of this oligonucleotide was carried out by a succession of nine synthesis cycles (see Table III) below).

 In addition to washes, each cycle comprises detritylation (step 4), condensation (step 12), acetylation (step 13) and oxidation (step 15). After the required number of cycles, the support was treated with thiophenol / triethylamine / dioxane (1/2/3, v / v / v) mixture for one hour to remove the methyl group from the phosphates. The support was then treated with concentrated ammonia at 600 for 12 hours to remove the protective groups of the heterocyclic amines and detach the nucleotide products from the solid support. The desired oligonucleotide, still carrying the dimethoxytrityl group at its 5 'end, was purified by high performance liquid chromatography on graft phase ClaI column with a yield of 20%. Treatment with 80% acetic acid made it possible to deprotect its 5 'end. Finally, the purified decanucleotide was subjected to enzymatic degradation by snake venom phosphodiesterase and alkaline phosphatase. High performance liquid chromatography analysis of the products of digestion indicated complete degradation to α-thymidine, α-deoxy-2'-cytidine, α-deoxy-2'-adenosine and α-deoxy-2'-guanosine in a ratio of 2.1 / 3.2 / 1.8 / 3.3 (Theoretical ratio: 2/3/2/3).

 These results show that oligodeoxyribonucleotides comprising only nucleotide units of α-anomeric configuration can be rapidly synthesized by the phosphoramidite method on a solid support.

 The protocol for the manual synthesis of α-oligonucleotides on porous glass beads (CPG) was as follows.

 The reactor was loaded with 30 mg of functionalized support (25-38 ssmol / g).

 The volume corresponding to each step is 1 ml, unless otherwise indicated.

TABLE III

<tb> Step <SEP> Reagent <SEP> or <SEP> Solvents <SEP> Time
<tb> 1, <SEP> 2, <SEP> 3 <SEP> CH2CLz <SEP> 10 <SEP> s <SEP> by <SEP> step
<tb> 4 <SEP> DCA / CH2CL2 <SEP> (1/50, <SE> v / v) <SEP> 3 <SEP> mn
<tb> 5, <SEP> 6 <SEP> CH2 <SEP> CL2 <SEP> 10 <SEP> s <SEP> by <SEP> step
<tb> 7, <SEP> 8, <SEP> 9, <SEP> 10,
<tb> 11 <SEP> CH3CN <SEP> 10 <SEP> s <SEP> by <SEP> step <SEP>
<tb> 12 <SEP> 0.1M <SEP> phosphoramidite <SEP> * <SEP> 5 <SEP> mn
<tb><SEP> (0.2 <SEP> ml) <SEP> and <SEP> 0.4M <SEP> tetra
<tb><SEP> zole <SEP> (0.2 <SEP> ml) <SEP> in <SEP> ac
<tb><SEP> tonitrile
<tb> 13 <SEP> DMAP / THF / lutidine <SEP> 2 <SEP> mn
<tb><SEP> (6/90/10, <SEP> p / v / v), <SEP> then
<tb><SEP> 0.1 <SEP> ml <SEP> of anhydride <SEP> acne
<tb><SEP> tick
<tb> 14 <SEP> THF / lutidine / H2O <SEP> 2/2/1, <SEP> 1 <SEP> mn
<tb><SEP> v / v / v)
<tb> 15 <SEP> 0.1M <SEP> l2 <SEP> in <SEP> THF / lutidi <SEP> 30 <SEP> s
<tb><SEP> ne / H2O <SEP> (2/2/12, <SEP> v / v / v)
<tb> 16, <SEP> 17, <SEP> 18,
<tb> 19 <SEP> CH3 <SEP> CN <SEP> 10 <SEP> s <SEP> by <SEP> step
<tb> * In the case of G and for reasons of solubility,
a 0.05 M solution of phosphoramide is used.

In this case, step 12 is performed a second
times before going to step 13.

EXAMPLE IV: Automatic synthesis of oligodeoxyribo
nucleotides a
A gene synthesizer, model 380 from Ap? Lied
Biosystems, was used. Due to the low solubility of the phosphoramidite derivative of G in acetonitrile, a 0.05M solution of this phosphoramidite, permanently maintained at 370C, was used. The standard synthesis program has been modified so as to repeat the steps corresponding to the incorporation of G and to ensure a high coupling efficiency (greater than 98%).

 The oligonucleotide α-d (TAAAAGGGTGGGAATC) was thus obtained (62 units of OD260). Its purity and size were verified by gel electrophoresis under denaturing conditions.

EXAMPLE V Enzymatic Stability of Oligonucleotides
a- and ss- [d (CATGCG)] vis-à-vis some
nucleases.

 Three nucleases were used: an endonuclease, the S1 nuclease which is specific to single-stranded DNAs; a 5'-exonuclease, the phosphodiesterase of calf's spleen; and a 3'-exonuclease, the phosphodiesterase of snake venom. Hyperchromicity at 260 mm and HPLC were used to monitor enzymatic hydrolysis. No significant changes in absorbance occurred within 20 minutes when α-oligomer was incubated with S1 nuclease, while ss oligomer gave an increase in absorbance with duration.

 The absorption W does not allow a quantitative comparison between the oligomers ss and a with respect to their degradation. We therefore confirmed these results by HPLC analysis. The disappearance of the peak corresponding to hexanucleotide was recorded during incubations with nuclease S1 at 370C. There were no more hexamers after 10 minutes of incubation, whereas only 7% of hexamer a was degraded (Table 11).

 The comparative stability of the two hexamers a and ss with respect to the hydrolysis by the phosphodiesterase of calf is illustrated in Table II below.

The difference between the two anomeric oligomers is striking: no change in oligomer a appeared in 10 minutes, while 89% of the oligomer ss was hydrolysed at the same time.

TABLE II
Hydrolysis of [d (CATGCT) α- or S-166 mmol each) versus time S1 nuclease (93 units / ml) and calf spleen phosphodiesterase (0.013 units / ml) at 370 ° C. Fraction of hexamer remaining after 1 or 10 minutes.

Nuclease S1 Phosphodiesterase
duration (min.) of calf's rate
duration (min.)
0 1 10 0 10 ss- [d (CATGCG)] 100% 1.3% 0 100% 11% &alpha; - [d (CATGCG)] 100% 98% 93% 100% 104%
The stability of the two anomeric hexamers vis-a-vis the hydrolysis by phosphodiesterase of snake venom was studied by following the UV absorbance at 260 mm.

At low enzyme concentrations, oligonucleotide a was slowly hydrolysed, while degradation of the corresponding ss oligonucleotide was very clear.

1) Comparison of the substrates Ed (CATGCG) a- and f3- by
compared to different nucleases.

HPLC Analysis: The HPLC was a Waters
Associates and was equipped with a C 18 Bondapak column.

Isocratic elution was performed with the following system: 27% acetonitrile in 20 mM ammonium acetate <pH 6.5) and the detection procedure at 260 nm.

a) S1 nuclease.

 This nuclease was branded GIBCO BRL.

 A unit solubilized with 1 g of DNA denatured in 1 min at 370C.

A solution of oligonucleotide α- or oligonucleotide -ss- (50 nmol) in water (30 μl) was mixed with the following buffer (30 μl): 0.5 M sodium acetate (pH 4 , 7), 3M sodium chloride,
0.1M zinc acetate. This volume was completed with 300 l of water. S1 nuclease (28 units, 3 μl) was then added. The mixture was incubated at 370C and the aliquots (20L) were collected in a timely manner and mixed with the blocking buffer: 20mM potassium phosphate (pH 6.5, 180L), before being heated at 1000C for 5 minutes. Similar treatment was done for control experiments. Volumes of 50 wl were injected.

b) Phosphodiesterase of calf's spleen.

 A procedure similar to that described above was followed, except that said phosphodiesterase (4.10-3 units, 1 ssl) was added in place of S1 nuclease in 0.1 M ammonium acetate (pH 6.5). The phosphodiesterase of calf spleen was supplied by BOEHRINGER (MANNHEIN). One hydrolysis unit 1 mol. thymidine-3'-nitrophenylphosphate in one minute at 250C. The reaction was stopped in water instead of potassium phosphate solution.

UV spectrophotometry.

 A Uvikon spectrophotometer at 810 equipped with a thermostated system operating at 370C was used. The total reaction volume was 1 ml. Oligonucleotides (60 nmol) were added and the absorbance at 260 nm was adjusted to zero. S1 nuclease (1 ssl, 9.3 units) was added and the absorbance at 260 nm was recorded as a function of time. The analogous procedure was used with the snake venom phosphodiesterase also provided by BOEHRINGER -0 (1 μL, 3.10 units).

FIG. 1 represents the UV absorbance curves at 260 nm as a function of time for the oligonucleotides Ud (CATGCG)] R- and a- respectively. A buffer solution consisted of 0.05 M sodium acetate (pH 4.7), 0.3 M sodium chloride and 0.01 M zinc acetate.
S1 nuclease (0.3 units / ml) was added to 60 μM hexa
mother & alpha - or 6--
Figure 2 shows the absorbance
UV at 260 nm according to time. The buffer solution
consisted of 0.1 M tris-HCl (pH 9) and chloride
0.01 M magnesium. At time t = 0 min of the phosphodiesterase of
snake venom (3.10- units / ml) has been added to
66 M hexamer a- or S- at 370C.

b) la séquence ss-[d(GTACGC)] présente deux pics plus larges
à 13,65 et 12,58 ppm.Là encore, l'intensité relative
et des considérations de symétrie suggèrent que ceci
est dû à l'appa:iement anti-parallèle

c) le mélange des séquences des ard(CATGCG) et
ss-[d(GTACGC)] (1:1) présente six pics imino à
14,22 , 13,83 , 13,23 , 13,02 , 12,73 and 12,57 ppm
et un large pic à 13,66 ppm. Puisque les déplacements
chimiques du premier ensemble de six signaux sont
tous différents de ceux observés in (a) et (b)
(excepté pour 6 = 12,57 lequel est présent à la fois
pour les séquences a- et ss-), il semble raisonnable
d'associer ces six pics à ceux des protons imino prove
nant de l'appariement de &alpha;-[d(CATGCG)]-ss-[d(GTACGC)]. EXEMPT VI: Parallel pairing of two strands a and ss
From the synthesized sequences, including the four bases, the results below prove that the sequences a and ss are paralleled pairwise, while two complementary sequences a pair are anti-parallel. On the other hand, the ss match is much more stable than the match ss
Indeed, the NMR spectrum of the imino protons at 40C of these solutions in D2O-H2O reveals that a) the sequence &alpha; - [d (CATGCG)] has two well-defined peaks
finished at 12.57 and 14.17 ppm intensity: 1) pro
coming from the anti-parallel matching

b) the sequence ss- [d (GTACGC)] has two larger peaks
at 13.65 and 12.58 ppm. Again, the relative intensity
and symmetry considerations suggest that this
is due to the anti-parallel

c) the mixing of the ard sequences (CATGCG) and
ss- [d (GTACGC)] (1: 1) has six imino peaks at
14.22, 13.83, 13.23, 13.02, 12.73 and 12.57 ppm
and a broad peak at 13.66 ppm. Since the displacements
chemicals of the first set of six signals are
all different from those observed in (a) and (b)
(except for 6 = 12.57 which is present at a time
for the sequences a- and ss-), it seems reasonable
to associate these six peaks with those of protons imino prove
the pairing of &alpha; - [d (CATGCG)] - ss- [d (GTACGC)].

The number of imino proton signals observed is
more in accordance with parallel matching (A,
only with an antiparallel pairing (B)

EXAMPLE VII Association of a thymidine tetramer a
with an intercalation agent
A thymidine tetramer covalently linked through a pentamethylene chain to a 2-N-methyl-9-hydroxyellipticinium acetate derivative was synthesized in three steps according to the following scheme.

<tb><SEP> Dmtr-a <SEP> (TTT- <SEP> t <SEP> Dmtr-NH (CH, <SEP>) <SEP> COOH
<tb><SEP> step <SEP> 1 <SEP> DCC, <SEP> DMAP
<tb><SEP> Dmtr-a (TTTT) çC, (CH2) <SEP> NH-Dmtr
<tb><SEP> ri <SEP> s
<tb><SEP> step <SEP> 2 <SEP><SEP> AcOH <SEP> 80 <SEP>%
<tb><SEP> fl <SEP> P
<tb><SEP> a <SEP> TTTT-OC (CH,) <SEP> NH,
<tb> step <SEP> 3 <SEP> CH3 <SEP> + <SEP> / <SEP> CH3 <SEP> H202, <SEP> peroxidase
<tb> HO
<tb><SEP> CH3 <SEP> a <SEP> T4C5OPC
<tb><SEP> H <SEP> CH3 <SEP> a <SEP> T4CsoPC
<Tb>
Step 1: Binding of the Pentamethylene Arm on the Thymidine Tetramer a.

 The 3'-OH-terminus of the 5'-protected tetramer with a dimethoxytrityl group (Dnttr) is esterified with an aminocaproic acid, the terminal residue of which is blocked with Dmtr. The reaction is carried out at room temperature in pyridine with dicyclohexylcarbodiimide (DCC) as coupling agent and dimethylaminopyridine (DMAP) as catalyst.

The reaction is complete after three hours.

 - blape 2: Deprotection.

ainsi obtenu, est purifié à travers une colonne en phase inverse. A ce point, le rendement des réactions (1+2) est égal à 65 %.Dmtr groups are removed with 80% acetic acid. The resulting product

thus obtained, is purified through a reverse phase column. At this point, the reaction yield (1 + 2) is 65%.

 Step 3: Binding of the Ellipticine Derivative with the Modified Tetramer

 In this step, we used an enzyme system: horseradish peroxidase -HzO ,.

de formule suivante

a T4CnOPC
La réaction est effectuée dans un tampon Phosphate pH 7 et le produit final est purifié sur une colonne C 18 en phase inverse et par HPLC.The ellipticin derivative N-methyl-2-hydroxy-9-elligticinium (NMHE) is oxidized by this enzymatic reaction, resulting in an O-quinone derivative of NMHE. This electrophilic intermediate then undergoes the attack of the amino function carried by the modified tetramer. An oxazole nucleus is thus created, leading to the fluorescent compound

of following formula

at T4CnOPC
The reaction is carried out in Phosphate pH 7 buffer and the final product is purified on a reverse phase C 18 column and by HPLC.

1) Preparation of O-benzovyl-3'-thymidine
389.7 mg (1.15 mmol) of 4-4'-dimethoxytritylchloride was added to a solution of 2'-a-thymidine
(1 mmol and 242.2 mg) in anhydrous pyridine
(2.5 ml). The resulting mixture was stirred for three hours at room temperature and methanol (1 ml) was added. After additional stirring for 15 minutes, the melanae was poured into 40 ml of saturated aqueous sodium hydrogen carbonate and extracted with methylene chloride (3 x 20 ml). The combined organic layers were evaporated to dryness and the residue fractionated by chromatography using acetone-methylene chloride (0-40%) as eluent, fractions containing pure O-dimethoxvtrityl-5'-thymidine. were collected and evaporated to dryness. The residue was precipitated from ether (less than 400C) (514.6 mg 94.5%).

 42.2 mg of (0.30 mmol) benzoyl chloride was added to a solution of 5'-O-dimethoxytrityla-thymidine (0.25 mmol 0.136 g) in anhydrous pyridine (1 ml). The solution was stirred for one hour at room temperature and 0.5 ml of water was added.

After stirring for 15 minutes, the reaction mixture was poured into 20 ml of saturated aqueous sodium hydrogencarbonate and extracted with methylene chloride (3 x 10 ml). The organic layers were evaporated to dryness. The residue was dissolved in methylene chloride-methanol (7-3 v / v; 5 ml) and 10% (v / v) benzenesulfonic acid in methylene chloride was also added. stirring at room temperature, the reaction mixture was neutralized with 2 ml of saturated aqueous sodium hydrogen carbonate and partitioned between 20 ml of saturated sodium-aqueous hydrogen carbonate and (3 x 15 ml) of methylene chloride. The combined organic phases were evaporated to dryness and the residue was fractionated by chromatography (10 g). Fractions containing 3'-O-benzoyl-α-thymidine derivative were pooled and evaporated. The residue was precipitated from ether (less than 400C) to give a colorless amorphous powder (0.075 g, 87%).

2) Preparation of 5'-O-dimethoxytrityl- &alpha; - F (Tp) T
To 0.393 g (0.15 mmol) of a solution of Dmtr-α- (TP) 3T-Bz protected in water-dioxane (1/7, v / v, 5 ml) was added 0.6106 g. (5mmol) pyridine2-aldoxime and 0.627ml (5mmol) N1, N1, N3, N3-tetramethylguanidine. The resulting solution was heated at 70 ° C. for 9.5 h, then cooled and evaporated to dryness. The residue was dissolved in tetraklammonium hydrogen carbonate (TEAB, 50 ml, 50 mmol) and was washed with diethyl ether (2 x 25 ml). The aqueous solution was evaporated to dryness and the residue was chromatographed on a reverse phase silica gel Cla column (1.2 x 10 cm) using a gradient of methanol (0 to 50%) in 1 ml. water (0.1 M relative to TEAB). The appropriate fractions were combined and evaporated to dryness. Lyophilization of the residue gave the desired compound as a colorless powder (0.223 g) in 84% yield.

3) Preparation of compound &alpha;-( Tp) 3T 3'-O - # - aminohexanoate
To a solution of 58-O-dimethoxytryl-α- (TP) 3T (0.223 mg, 0.13 mmol) and N-dimethoxytrityl # -aminohexanoic acid (0.281 g, 0.65 mole) in anhydrous pyridine (3 ml) was added 0.2678 g (1.3 mmol) of dicyclohexylcarbodiimide and 0.0793 mg (0.65 mmol) of N, N-dimethylamino-4-pyridine. The resulting solution was stirred for three and a half hours at room temperature. 1 ml of water was then added and the mixture was filtered. The filtrate was evaporated to dryness and the residue was precipitated from diethyl ether (this latter operation was repeated three times). The precipitate was dissolved in 80% acetic acid (5 ml) and the residue was dissolved. solution was kept at room temperature for 20 minutes. The mixture was extracted with diethyl ether, evaporated to dryness. To the residue was added toluene and the resulting mixture was evaporated to dryness again. The residue was purified by reversed phase silica gel column chromatography (1.2 x 10 cm) using a step gradient of methanol (0 to 40%) in water. Fractions containing the product were evaporated to dryness and the residue freeze-dried. The desired compound was obtained as a colorless powder (0.130 g) for a yield of 65%.

4) Preparation of

 The amounts of reagent used were as follows.

For 130 mg (0.082 mmol of a- (Tp) 3T-3'OE-aminohexanoate and 11 mg (0.032 mmol) of 2-methyl, 9-hydroxyellipticinium acetate were dissolved in 50 ml of 0.02 M phosphate buffer pH 7. The solution was maintained with continuous stirring at room temperature 0.8 mg of peroxidase (horseradish) was added to the mixture After the dissolution of the enzyme, 1.5 ml of a 20 mM solution of H 2 O 2 was added. The solution was filtered and evaporated to dryness and the residue was prepurified by reverse phase (0 to 60%) Lighrosorbe RP 18 column chromatography in water. Combined and evaporated to dryness The product was purified by HPLC The product was passed through a column
Dowex 50 W (NH4 + form). After lyophilization, the final compound obtained was a yellow powder of 2.7 mg in 5% yield.

EXAMPLE VIII Matching α-T4-C5-OPC with a Polv (rA)
The interaction between α-T4-Cs-OPC and poly (rA) was studied using W absorption and fluorescence. The W spectrum of a-T4-C5-OPC has two maximums at 271 nm and 317 nm. In addition, this compound is fluorescent with excitation and maximum emission respectively at 318 nm and 525 nm.

 In the presence of increasing amounts of poly (rA), the α-T4-Cs-OPC spectrum (0.05 mM in 10 mM sodium cacodylate, 100 mM NaCl, pH 7) is modified. Hypochromicity occurs between 260 nm and 320 nm and corresponds to the combination of α-T4-C5-OPC with the poly (rA) sequence. At 270 nm, duplex formation between tetra-a-thymidilate and poly (rA) can be followed, while at 310 nm the interaction of the oxazolopyridocarbazole moiety is monitored. a-T4-Cs-free OPC on the initial absorbance of a-T4-C5-OPC, as a function of the ratio of the concentration of poly (rA) expressed in nucleotides, on the concentration of oxazolopyridocarbazole derivative, it can be deduced the stoichiometry of the interaction, which corresponds to a bound α-T4-C5-OPC molecule for 4 adenine residues.

Under the same conditions, no association between a-T4 and poly (rA) was detected.

 It is also possible to study the association of &alpha; -T4-C5-OPC with poly <rA) as a function of temperature. Once the complex was formed at 80C, the UV spectrum of free α-T4-C5-OPC was gradually recovered by raising the temperature. Two isobestic points appear at 347 nm and 324 nm. For temperatures greater than 260C, the absorbance at 317 nm is greater than that corresponding to free α-T4-Cs-OPC at 80C. This indicates that a detachment of the α-T4-Cs-OPC molecule occurs when the temperature is high.

The temperature corresponding to 50% dissociation of the complex is equal to 24.50C at 270 nm and 260C at 310 nm.

This result could be compared to that observed for complexes formed between 6-T4-Cs-OPC and poly (rA).

In the latter case, the dissociation temperature of the complex, under the same conditions, was 160C.

 The fluorescence of the OPC fragment of the ss-T4-C5-OPC molecule is not changed after formation of the complex with poly (rA). This suggests that this fragment is probably not intercalated between the newly formed base pairs. This is another difference between a- and S-T4-Cs-OPC, since in the latter case the interaction between the substance and the poly (rA) causes an increase in fluorescence.

Moreover, by varying the concentration of α-T4-Cs-OPC, and by measuring the corresponding temperature Tm (Tm represents the dissociation temperature at 50% of the complex), the thermodynamic parameters of its interaction with poly (rA) were determined. These parameters are deduced from the following equation: 1 / Tm = S / H + 2.3R / H logCm equation in which Tm represents the half-dissociation temperature of the complex,
Cm represents the concentration of α-T4-C5-free OPC t = Tm and R is equal to 2 calories / mol / K.

Tm was measured at 270 nm and 310 nm, and the following values were obtained at 270 nm: AH = - 38.7 kcal / mole, AS = 105.3 cal / mole and
AG at 2980K = - 7.2 kcal / mole.

at 310 nm: AH = - 36.8 kcal / mole, AS = 98.8 cal / mole and
AG at 2980K = - 7.4 kcal / mole.

In conclusion, it has been possible to synthesize a new molecule: α-T4-C5-OPC, in which a potential intercalating agent having an oxazolopyridocarbazole structure is covalently linked via a pentamethylene arm to a tetrathymidilate non-natural consisting exclusively of α-anomeric nucleotide units. UV absorption and fluorescence indicate that the oligonucleotide fragment provides the recognition elements necessary to bind selectively to the complementary sequence by base sequence. In comparison with the S-anomer there are two significant differences: a) the anomeric configuration has an effect
additional stabilization in the formation of the
complex with poly (rA) b) the oxazolopyridocarbazole residue does not seem to interfere
stall in the duplex formed by basic matching
between the oligonucleotide a- and poly (rA).

EXAMPLE IX: Resistance of the α-T4-C5-OPC Substrates with respect to
of an S1 nuclease.

S1 endonuclease degradation has been studied in comparison with a- and ss-T4-C5-OPC by
HPLC. Peak intensity was monitored as a function of nuclease reaction time S1 at 370C. After 20 minutes of reaction, the compound completely disappeared when the anomeric configuration of the oligonucleotide chain is ss, while it is still intact after 30 minutes when the anomeric configuration is a.

HPLC analysis was undertaken on a C18 -Bondapak column. Isocratic elution was performed with the following system: 31% acetonitrile in 20 mM ammonium acetate (pH 6.5, 5 mM relative to tetrabutylammonium chloride). Detection
UV was performed at 270 nm.

 Analysis conditions: a solution of α- or ε-T4-Cs-OPC (33 nmol) in water (8 μl) was mixed with the following buffer: 0.5 M sodium acetate (pH 4,7), 3M sodium chloride and 0.1M zinc acetate.

The volume of the reaction mixture was supplemented to 300 ml with water and then S1 nuclease (Gibco
BRL, 28 units, 3 Al) has been added. The mixture was incubated at 37 ° C and aliquots (20 μl) were removed at different times, but immediately mixed with blocking buffer: 20 mM sodium phosphate (pH 6.5, 180 l) and heated at 1000C for 5 minutes. . Similar treatments were performed for each control experiment. For each HPLC analysis, a volume of 50 was injected.

Claims (20)

 1. Chemical compounds consisting of an oligonucleotide or an oligodeoxynucleotide, characterized in that they comprise a sequence of nucleotides with unnatural anomeric configuration a.  2. Compounds according to claim 1, characterized in that the sequence of nucleotides a is covalently linked to an effector agent, in particular to an intercalating radical or to a reactive or photoactivatable chemical radical.  3. Compounds according to one of the preceding claims, characterized in that they have the formula

 in which  the radicals B may be identical or different and each represents a base of an optionally modified nucleic acid and attached to the glycoside ring in an unnatural anomeric configuration.  the radicals X may be the same or different and each represents an oxoanion a thionanion S (3, an alkyl group, an alkoxy group, aryloxy, an aminoalkyl group, an aminoalkoxy group, a thioalkyl group;  R and R ', which may be identical or different, each represent a hydrogen atom or a group -Y-Z  Y represents a straight or branched alkylene radical -alk- or a radical chosen from

 with U = O, S or N or a radical -Y "-O-Y'- where Y" or Y 'can have the meanings given for Y  E can have the same meanings as X;  J represents a hydrogen atom or a hydroxyl group  Z is a radical corresponding to an effector agent  n is an integer including O  L represents an oxygen atom, a sulfur atom or an -NH- group.  4. Compounds according to claim 3, characterized in that fYE represents a radical chosen from

 with U = O, S or N  5. Compounds according to one of the claims  preceding, characterized in that Z is a radical  intercalant derived from polycyclic compounds having a  flat configuration.  6. Compounds according to one of the claims  preceding, characterized in that the radical inter  Calming is derived from acridine, furocoumarin,  daunomycin, 1,10-phenanthroline, phenanthridin  nium, porphyrins, derivatives of dipyrido  (1,2-a: 3 ', 2'-d) imidazole, ellipticine or ellipticinium and derivatives of these intercalating agents.  7. Compounds according to one of the preceding claims, characterized in that the reactive chemical groups are chosen from the derivatives of ethylenediamine-tetraacetic acid, diethylenetriamine-pentaacetic acid, porphyrins, 1,10-phenanthroline, psoralen.  8. Compounds according to one of the preceding claims, characterized in that the radical B is chosen from thymine, adenine, cytosine, guanine, 4-azido-cytosine or 4-azido-thymine and 8 -azido-adenine, uracil, 5-bromo-uracil.  9. Compounds according to one of the preceding claims, characterized in that they consist of an α-oligonucleotide, covalently linked, in particular via a pentamethylene chain to a 2-N-methyl-9-hydroxy derivative. ellipticinium acetate.  10. Preparation process or oligonucleotide a according to one of the preceding claims, characterized in that phos syntheses are applied.  ohodiester, phosphotriester, phosphoramide or hydrogeno  conventional phosphonate for ss anomers, but  starting from nucleosides a.  11. Process for the preparation of oligonucleotide  according to one of the preceding claims, characterized  in that the synthesis is applied to phosphotriester  classical for SS anomers, but in which the  a-nucleotide derivatives of guanine are protected in  position 06 by an additional group, preferably  N, N-diphenylcarbamoyl.  12. Process for synthesizing compounds without  effector agent according to one of the preceding claims  dentes, characterized in that a synthesis is made  supported by the phosphoroamidite method  so  a protection in 3 'and 5' nucleotides or oligo  starting nucleotides with for example dimethoxy  5 'trityl and diisopropylaminophosphoramidite  methyl in 3 ',  the functionalization of a solid support incorporating  a nucleoside derivative by a link for example  succinyl between the hydroxyl-3 'group of the derivative  a nucleoside and an amino group of the support  solid, - the elongation of the oligonucleotide chain in  a synthesizer reactor, - finally the stall, the deprotection and the purifica  of the prolonated oligonucleotide.  13. A method of synthesizing compounds incorporating an effector agent according to one of the preceding claims, characterized in that one starts from an oligonucleotide without 5 'protected effector agent, which is reacted with the OH 3' terminus with a unprotected function of a difunctional arm whose second function is protected and after deprotection of the resulting product, said product is linked to the effector agent by the second free function of the difunctional arm.  14. Process according to Claim 13, characterized in that the 3 'OH terminus of the 5' protected oligomer is esterified with an aminohexanolic acid, the amine function of which is protected.  15. Application of the compounds according to one of the preceding claims, characterized in that they parallel pair with a sequence of oligonucleotide ss complementary.  16. Application of the compounds according to claim 15, characterized in that said compounds are used as hybridization probes or as purification components for specific sequences of DNA or RNA.  17. Application of the compounds according to one of claims 15 and 16, characterized in that the compounds are used to detect mutations at a DNA or an RNA.  18. Application of the compounds according to one of claims 15 to 17, characterized in that these compounds are used to achieve specific blocking of previously selected cell genes or pathogens, such as viruses, bacteria or parasites.  19. Application of the compounds according to one of claims 15 to 18, characterized in that they can be used as artificial nucleases specific sequences.  20. As medicaments, compounds according to one of the preceding claims.