FR2686882A1 - Oligothionucleotides - Google Patents

Abstract

The subject of the present invention is chemical compounds consisting of an alpha or beta oligo-4'-thio-2'-deoxyribonucleotide or oligo-4'-thioribonucleotide, characterised in that they contain a chain of 4'-thio-2'-deoxyribonucleotides or 4'-thioribonucleotides respectively, it being possible for the chain to be optionally linked to an effector, especially a radical corresponding to an intercalating agent or a chemical or photoactivable radical, such as a radical carrying a functional group which reacts directly or indirectly with the nucleotide chains or a radical whose presence permits an easy and sensitive detection. It also relates to processes for preparing these compounds and their therapeutic or diagnostic applications or their applications as laboratory reagents.

Description

oligothionucleotides
The present invention relates to novel chemical compounds and their applications. The chemical compounds according to the present invention are constituted by an oligo 4'-thio-ribonucleotide or an oligo 4'-thio-2'desoxyribonucleotide comprising a sequence of 4 'thionucleotides.

 The compounds according to the invention may have the natural anomeric beta configuration or the unnatural anomeric alpha configuration.

(a) et (b) représentent des 4'-thionucléosides, il faut rajouter un phosphate en 5' poiir obtenir des nucléotides.Thus are represented by the formulas (a) and (b) respectively 4'-thionucleotides alpha (a) and beta (b)

(a) and (b) represent 4'-thionucleosides, it is necessary to add a phosphate in 5 'to obtain nucleotides.

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

 More specifically, the subject of the present invention is chemical compounds consisting of an oligo4'-thioribonucleotide or an oligo-4'-thio-2'-deoxyribonucleotide, characterized in that they comprise a sequence of 4'-thioribonucleotides or 4'-thioribonucleotides. thio-2 'deoxyribonucleotides respectively, which sequence may optionally be linked to an effector agent, in particular a radical corresponding to an intercalating agent or a chemical or photoactivatable radical, such as a radical carrying a function reacting directly or indirectly with nucleotide chains or a radical whose presence allows easy and sensitive detection.

In particular, the subject of the invention is the novel oligo-4'-thionucleotide derivatives, their preparation and their use especially as probes allowing the detection of a defined sequence of nucleic acids, as artificial nucleases specific for DNA sequences or RNA or as selective blocking agents for the expression of a gene whether it is endogenous (for example, oncogenic gene) or exogenous (for example DNA or
RNA from viruses, parasites or bacteria).

 In French patent applications FR 8301223 (2,540,122) and FR 8411795. (2,568,254) have been described chemical compounds consisting of an oligonucleotide or an oligodeoxynucleotide comprising a sequence of natural or modified nucleotides, that is to say to say beta-nucleotides, on which is fixed by a covalent bond at least one intercalating group, which have the property of selectively block the expression of a gene and which, therefore, are particularly useful in therapy as antiviral substances , antibiotics, antiparasitic or antitumor.

 In PCT International Application WO 83/01451 has been described a method for blocking the translation of messenger RNA (mRNA) into protein by hybridization of the mRNA with an oligonucleotide having the sequence complementary to the mRNA, the oligonucleotide being stabilized in phosphotriester form.

 In the international application WO 88/04301 have been described oligonucleotides of alpha anomeric configuration having parallel pairings with complementary sequences.

 It has also been described in the prior art derivatives of oligonucleotide compounds better resistant to enzymatic degradation whose phosphate part was modified thiophosphate or methylphosphonate in particular.

 However, these derivatives have a chirality at the phosphate level capable of generating insoluble diastereoisomers.

 It has now been found, and this is the subject of the present invention, that the substitution of oxygen by a sulfur on the sugar part of the nucleosides rather than on the phosphate group on the one hand, ensures the solubility of the oligomers and, on the other hand, could increase their penetration into the target cell, the substitution of oxygen by the sulfur element making the molecule more lipophilic.

 The 4'-thionucleotide derivatives form hybridization complexes with the RNA complementary sequences that are much more stable than those formed with DNA and that, with respect to the RNAs, the 4'-thionucleotide derivatives form Hybridization complexes more stable than natural beta-nucleotide derivatives.

dans laquelle
les radicaux B peuvent être identiques ou différents et représentent chacun une base d'un acide nucléique éventuellement modifiée, activable et/ou comportant un groupement intercalant
les radicaux X peuvant 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, un radical alkyle ou alcoxy substitué par un hétérocycle azoté ou un groupement -Y-Z
R et R', qui peuvent être identiques ou différents, représentent chacun un atome d'hydrogène ou un groupement -Y-Z ou Y'-Z'
Y et Y' identiques ou différents représentent chacun un radical alcoylène droit ou ramifié -alk- ou un radical choisi parmi

alk-O-alk

avec U = O, S ou N
En particulier, Y et Y' représentent un radical choisi parmi

avec U = O, S ou N
E peut avoir les mêmes significations que X excepté Y-Z ou
Y'-Z' ;
L représente O, S ou -NH-;;
n représente un nombre entier y compris 0
J représente un atome d'hydrogène ou un groupe hydroxy
Z et Z', identiques ou différents, représentent chacun un radical correspondant à un agent effecteur, notamment un radical correspondant à un agent d'intercalation ou un radical porteur d'une fonction qui réagit directement ou indirectement avec les chaînes de nucléotides ou un radical dont la présence permet une détection facile et sensible.Among the compounds according to the present invention, there may be mentioned more particularly the oligomeric compounds beta of formula. -

in which
the radicals B may be identical or different and each represent a base of an optionally modified, activatable and / or intercalating nucleic acid
the X radicals may be identical 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, an alkyl or alkoxy radical substituted by a nitrogen heterocycle or a group -YZ
R and R ', which may be identical or different, each represent a hydrogen atom or a group -YZ or Y'-Z'
Y and Y ', which are identical or different, each represent a straight or branched alkylene radical -alk- or a radical chosen from

alk-O-alk

with U = O, S or N
In particular, Y and Y 'represent a radical chosen from

with U = O, S or N
E can have the same meanings as X except YZ or
Y'-Z ';
L is O, S or -NH-;
n represents an integer including 0
J represents a hydrogen atom or a hydroxyl group
Z and Z ', which are identical or different, each represent a radical corresponding to an effector agent, in particular a radical corresponding to an intercalating agent or a radical carrying a function which reacts directly or indirectly with the nucleotide chains or a radical whose presence allows easy and sensitive detection.

qui correspond à la formule développée

sur laquelle ont été mentionnées les extrémités (3') et (5').In this formula I, the following condensed representation of nucleotides 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 4'-thionucleotides which may be identical or different, of D or L configuration, n simply indicating the number of nucleotides included in the molecule; n is preferably a number between I and 50, and more preferably between I and 25.

 In particular, mention is made of the products for which L represents oxygen; R and R 'represent hydrogen and B a natural nucleotide base, ie adenine, thymine, cytosine or guanine when J = H and adenine, uracil, cytosine and guanine when J = OH.

 Interleavers are known products in nucleic acid techniques; they are compounds capable of "intercalating" in the structure of DNAs or RNAs, that is to say capable of being inserted between the base plates of the nucleic acids.

 The intercalating agents may be chosen from polycyclic compounds having a planar configuration such as acridine and its derivatives, furocoumarin and its derivatives, daunomycin and the other anthracycline derivatives, 1,10-phenanthroline, phenanthridine and its derivatives, proflavine, porphyrins, derivatives of dipyrido [1,2-a 3 ', 2'-d] imidazole, ellipticine or ellipticinium and their derivatives and diazapyrene and its derivatives.

 The reactive chemical radicals can be radicals that can react directly or indirectly with a nucleotide chain to form a covalent bond or to modify it chemically, or to cut it. Preferably, these reactive chemical radicals are activatable, for example, chemically, biochemically or photochemically.

 The activatable reactive radicals may be chosen from ethylene-diamine tetraacetic acid, diethylene triaminepentaacetic acid, porphyrins, 1,10-phenanthroline, 4-azidoacetophenone, ethyleneimine and beta-chloroethylamine. psoralen and their derivatives, and aromatic compounds absorbing near ultraviolet or visible radiation or chemically reactive with the nucleic constituents.

 More particularly, the radicals that are chemically activatable in the presence of metal ions, oxygen and a reducing agent (ethylene-diamine-tetraacetic acid, diethylene-tri-amine-pentaacetic acid, porphyrin, phenanthroline) induce cleavage in sequences. nucleic acids located in their vicinity.

 By irradiation in the visible or near-ultraviolet region, it is possible to activate the derivatives which absorb these radiations and to carry out bridging reactions or photoinduced reactions (nucleic acid cleavage and modification) of the nucleic acids. on which the oligothionucleotide carrying the activatable group is attached.

- les radicaux dérivés de l'acide diéthylène-triamine-pentaacétique, - les radicaux dérivés de la méthylpyrroporphyrine de formule

- les radicaux dérivés de la phénanthroline de formule

- les radicaux dérivés de l'acridine ::

- les radicaux dérivés de la proflavine

Among the radicals Z and Z 'can be more particularly cited
radicals derived from ethylene diamine tetraacetic acid
formula

radicals derived from diethylenetriaminepentaacetic acid; radicals derived from methylpyrroporphyrin of formula

radicals derived from phenanthroline of formula

radicals derived from acridine ::

- radicals derived from proflavine

- les radicaux dérivés de l'azido-4 acétophénone de formule

R representing an amino (NH 2) or azido (N 3) group - radicals derived from biotin

the radicals derived from 4-azidoacetophenone of formula

 The radical B may preferably be chosen from natural nucleic bases (thymine, adenine, cytosine, guanine, uracil) but it is possible to use modified nucleic bases. Amino-2-adenine and its derivatives may be mentioned. substituted, for example, on the N6 azure atom by an aminoalkylene radical or by an azidophenylalkylene radical, the guanine substituted on the oxygen atom 06, for example, by a (w-alkylene) -9-acridine group (6-aminoalkyl) -amino-8-adenine and its derivatives substituted on the amino group in w by an acridine group, or the halogenated or azidated derivatives such as 5-bromo-uracil, 8-azadenadine, 7-deazaadenine, 7-deazaguanine. It is also possible to use derivatives of the nucleic bases comprising an intercalating group or a chemically or photochemically activatable group.

Thus, the functionalization of C or T by an aziridine group at the 4-position leads to the formation of covalent bridges
CH2-CH2 between the two complementary strands on respectively G and A.

Thus, more particularly, the radical B is then chosen from 4-azido-cytosine or 4-azido-thymine.

 Preferably, the radical X represents an oxoanion.

However, the radical X may represent an alkyl radical containing 1 to 7 carbon atoms (methyl, ethyl, propyl), an alkoxy radical in which the alkyl part contains 1 to 7 carbon atoms (methoxy, ethoxy, 2,2-dimethylpropyloxy). ), an aminoalkyl or aminoalkoxy radical of the general formula R1R2N-alk-A- in which A represents a bond or an oxygen atom, -alk- represents an alkylene radical containing 1 to 10 carbon atoms and R1 and R2, which are identical or different, represent a hydrogen atom or an alkyl radical containing 1 to 7 carbon atoms or form together with the nitrogen atom to which they are bound a saturated nitrogenous heterocycle with 5 or 6 members, it being understood that the group RR N can be quaternized, or an alkylthio radical whose alkyl part contains 1 to 7 carbon atoms.

 In the formula (I), 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 oligothionucleotide, the effector Z can therefore be introduced via a chain (CH 2) n linked to a functionalization Z 'of various natures to the saccharide part of the oligonucleotide. oligomer.

From the following formula
3 '
or -O-Z '- (CH2) n-effector (Z)
From Z1, for example, a phosphate or methyl phosphonate of formula

- un ester de formule générale :
-O-(CO)-(CH2)n-Z ou encore - un carbamate de formule générale :
-O-(CO)-(CH2)n-Z
La présente invention concerne également les composés précédents sous forme de sel avec des bases ou des acides, et les composés sous forme racémique, ou sous forme d'isomètres R ou S optiques purifiés ou en mélange, de formule (I).U = O, N or S - an ether of general formula

an ester of general formula
-O- (CO) - (CH2) nZ or else - a carbamate of general formula:
-O- (CO) - (CH2) nZ
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 optical isomers R or S, of formula (I).

 The present invention preferably relates to the above compounds of formula (I) in series D.

dans laquelle J et X sont définis comme dans la revendication 3 et B représente une base nucléique naturelle.In particular mention is made of D-oligothionucleotide compounds of general formula

wherein J and X are defined as in claim 3 and B is a natural nucleobase.

 The new products of general formula (I) can be prepared chemically by known methods and, in particular, those described in the known process applications and, in particular, those described in the French patent applications FR 8301223 (2540122) and FR 8411795 (2568254), WO 88/04301 and FR 88122648.

 The compounds of formula (I) can be prepared chemically by methods in which conventional phosphodiester, phosphotriester, phosphoramidite or hydrogen phosphonate syntheses are applied to oligonucleotides.

 In these processes, the 4'-thionucleotide chain is first prepared, the various non-used groups being protected, and finally the protecting groups are finally removed to obtain the desired products.

In particular, a process for synthesizing compounds according to the invention without an effector agent is described, characterized in that a synthesis supported by the phosphoroamidite method comprising a 3 'and 5' protection of the 4'-thionucleotides or oligo4 is carried out. '-thionucléo-
starting materials with for example dimethoxytrityl 5 'and
3 'methyl diisopropylaminophosphoramidite, functionalization of a solid support incorporating a 4'-thio derivative
nucleoside by a bond for example succinyl between the grouping
hydroxyl-3 'of the 4'-thionucleoside derivative and an amino group of
solid support, - the elongation of the oligothionucleotide chain in a reactor
synthesizer, - finally the stall, the deprotection and the purification of the oligothionu-
prolonged cleft.

 A process for synthesizing compounds incorporating an effector agent according to the invention is also mentioned, characterized in that one starts from an oligothionucleotide without a 5 'protected effector agent, the OH 3' terminus being reacted with a non-functional function. protected from 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.

 The 3 'OH terminus of the 5' protected oligomer can be esterified with an aminohexanoic acid, whose amine function is protected.

A solid phase method according to the phosphoramidite method, object of the present invention, comprises the following essential steps
A 4'-thionucleoside derivative protected by
via one of its hydroxyl functions at 3 'or, the
where appropriate, 2 'on a solid support.

- We assemble on this support in a synthesizer reactor
manual or automatic oligothionucleotide chain
protected by condensation of monomers consisting of
Protected 4'-thionucleosides substituted with groups
phosphoramidites in 3 ', where appropriate, the function
hydroxyl at 2 'riboses being protected by groups
Ctmp or TBDMS.

Oligothionucleotides are obtained after stalling
the oligomer obtained from the support and elimination of the groups
protectors.

 Suitably, the assembly of the oligothionucleotide is carried out by condensation, in the presence of an activating agent, of said monomers between their 3 'function and the 5' function of the immobilized 4'-thionucleoside compound for the first monomer or d a protected polythionucleotide intermediate compound attached to said immobilized thionucleoside compound for the following monomers.

 In particular, the activating agent for the condensation of said monomers may be chosen from tetrazole and its derivatives, such as para-nitrophenyl tetrazole.

avec R1 qui représente un alkyle en C1 à C7 éventuellement substitué identique ou différent, tel que CH3, (CH3)2CH. Advantageously, the 3 'phosphoramidite group of said monomers is an N, N dialkylaminophosphoramidite group of formula

with R1 which represents an optionally substituted C1 to C7 alkyl identical or different, such as CH3, (CH3) 2CH.

 R2 which represents optionally substituted C1-C7 alkyl, such as CH3, CH2CH2CN.

 In particular, the 3 'phosphoramidite group of said monomers is methyl diisopropylaminophosphoramidite (R 1 1 (CH 3) 2 CH and R 2 = CH 3) or 2-cyanoethyl diisopropylaminophosphite (R 1 = (CH 3) 2 CH and R 2 = CH 2 CH 2 CN).

 Suitably, the hydroxyl groups of said monomers and said immobilized 4'-thionucleoside compound can be protected at 5 'by the DmTr group.

The immobilized 4 'thionucleoside compound can be attached to the solid support via a divalent succinyl group between one of its groups
OH at 2 'or 3' which is thus esterified, and an amino group of the support.

 The solid support may consist of a long alkylamine chain attached to a polymer, a silica gel or porous glass beads.

As a functional group allowing the attachment or being bound on a solid support having an amino group, the group of formula

 with p = 1-5, wherein R4 represents H an activating group such as C6Cl5 or an NH radical bound to a solid support such as the NH radical of an alkylamine chain attached to a polymer, silica gel or silica gel. porous glass.

 An advantage of the compounds according to the present invention, constituted by an oligo-thio-nucleotide chain, is also to be able to envisage their use independently of effector agents, in particular intercalating agents, since these new oligonucleotide substances ensure the recognition of the complementary nucleic acid sequence, in particular RNA, 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.

 The compounds according to the invention, by their high specific affinity for complementary nucleic acid sequences, are indeed superior to the current oligonucleotides whose affinity for the complementary sequences is 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 at a DNA or RNA level more efficiently.

 The compounds of the present invention, by their property of binding strongly on the complementary nucleic acid sequence, and then of inducing the cleavage of the nucleic acid chain at this site, when they are coupled coupled to a cleavage agent, are usable. as artificial nucleases specific for sequences. They can be used as reagents in molecular biology or genetic engineering.

 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 according to the invention are therefore also usable as medicaments.

 The novel products of general formula (I) according to the invention and the products of general formula (I) in which L represents an oxygen atom, R and R 'each represent a hydrogen atom and B represents a natural nucleic base form hybridization complexes with RNA complementary sequences much more stable than those formed with DNA.

 In particular, the products of formula (I) in which J = OH (oligothioribonucleotides) more stably associate with complementary RNA sequences than the products of formula (I) in which J = H (oligothiodeoxyribonucleotides).

 Given that the targets of antisense molecules in therapies are the messenger RNA genes, this property of oligothioribonucleotides is quite interesting.

 This difference in stability of the hybridization complexes allows preferential inhibition of messenger RNAs and viral RNAs without any significant risk of causing undesirable effects on the genome DNA level.

 In addition, vis-à-vis the RNAs, the products according to the invention generally form more stable complexes than those obtained from natural oligonucleotides.

 The sequences of the oligothionucleotides, coupled or not with an intercalating agent or a chemically or photochemically activatable group, comprising a chain of pyrimidine nucleosides, are capable of binding to a DNA or RNA double helix comprising an adjacent purine base sequence. hydrogen bonded to the complementary sequence of the pyrimidine bases. This attachment involves the local formation of a triple helix in which the pyrimidine bases of the oligothionucleotide form hydrogen bonds (of the Hoogsteen or reverse Hoogsteen type) with the purine bases of the double helix. In this context, the oligothionucleotides form more stable complexes than the corresponding oligonucleotides and they make it possible to induce irreversible reactions (bridging, modification or cleavage) on an RNA or DNA double helix.

 The novel products of general formula (I) according to the invention and the products of general formula (I) in which L represents an oxygen atom, R and R 'each represent a hydrogen atom and B represents a natural nucleic base , which possess the property of binding strongly on the complementary nucleic sequence, can be used as probes to detect the presence of a complementary nucleotide chain. This detection is facilitated by the presence of a fluorescent group or a biotin group in these molecules.

The unnatural structure of the oligothionucleotides confers antigenicity properties thus making possible the preparation of specific antibodies directed against oligothionucleotides optionally coupled to an intercalating agent or against their complexes with a
DNA or natural RNA.

 For diagnostic or prognostic purpose, oligothionucleotides, optionally coupled to an intercalating agent, may be covalently attached to a solid support. Coupling to a luminescent marker or generating a colored, luminescent or fluorescent reaction makes it possible to use them as probes of DNA or RNA sequences for diagnostic or prognostic purposes.

 The oligothionucleotides according to the invention are much more resistant to nucleases than natural nucleoside derivatives.

This stability with respect to the enzymatic hydrolysis allows the use of the products of general formula (I) in in vivo or in vitro experiments in the presence of nucleases. As a result, the oligothionucleotides according to the invention have undeniable advantages over known beta-nucleoside derivatives.

 The oligothioribonucleotide compounds of formula (I) for which J = OH are more resistant to enzymatic degradation than the oligothiodeoxyribonucleotide compounds of formula I for which J = H.

 Therefore, as we have seen, another object of the present invention relates to the application of the novel products of general formula (I) according to the invention and products of general formula (I) in which L represents a hydrogen atom. oxygen, R and R 'each represent a hydrogen atom and B represents a natural nucleic base, in particular the products for which J = OH, to the specific blocking of previously selected cell genes or pathogens (viruses, bacteria, parasites), in particular the mRNAs.

 The novel products of general formula (I) according to the invention and the products of general formula (I) in which L represents an oxygen atom, R and R 'each represent a hydrogen atom and B represents a natural nucleic base , in particular the products for which J = OH, are particularly useful as medicaments making it possible to block the exogenous (viruses, bacteria, parasites) or endogenous (cellular genes, oncogenes) genes, in particular the mRNAs. The expression of these genes can be blocked by acting either directly on the DNA or the ART carrying the genetic information, or on the messenger ART, copy of the gene, while blocking any translation by hybridization or by hybridization then bypass or modification or cut of The messenger or viral ART chosen as target.

This blocking can be carried out using a product of general formula (I) according to the invention or a product of general formula (I) in which L represents an oxygen atom, R and R 'each represent a hydrogen atom and B represents a natural nucleotide base, in particular the products for which J = OH, the sequence of which is complementary to that of an uncomplexed region of an RNA or a
AND, and in particular, a messenger RNA. Hybridization, followed or not by bridging, modification or cleavage of messenger or viral ART, prevents the synthesis of ART or the corresponding protein or the expression of viral or parasitic functions.

 If this RNA or this protein is vital for the virus, the bacterium or the parasite, the products of general formula (I) according to the invention and the products of general formula (I) in which L represents an oxygen atom, R and R 'each represent a hydrogen atom and B represents a natural nucleic base, in particular the products for which J = OH, will constitute antiviral, antibacterial or antiparasitic drugs.

 If this RNA or protein is not vital to the body, it is possible to selectively remove the effects. In this case, the products of general formula (I) according to the invention and the products of general formula (I) in which L represents an oxygen atom, R and R 'each represent a hydrogen atom and B represents a natural nucleic acid, in particular the products for which J = OH, will constitute either anti-tumor drugs when the targeted gene or its messenger RNA encodes a protein involved in the cellular transformation, or drugs capable of suppressing the resistance character to antivirals, antibiotics or antiparasitics when the encoded protein is responsible for the inactivation of antibiotics, antivirals or antiparasitics.

 Specific cytotoxic effects may be obtained by the action of a product according to the invention or of a product of general formula (I) in which L represents an oxygen atom, R and R 'each represent a hydrogen atom and B represents a natural base, in particular the products for which J = OH, on a cell function essential to the target cell.

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

 Figure 1 shows the thermal stability curve of the betadT5 (SrT) duplex dT6 / betadC2A12C2 compared to that of the natural duplex betadT12 / betadC2A12C2.

 Figure 2 shows the enzymatic digestion curve of beta (SrT).

 In the following description, the abbreviation rS or Sr precedes a thioribunucleotide.

 I-SYNTEESE OF THIONUCLEOTIDES.

 I. The first syntheses of thiosugars were made between 1960 and 1970.

Two examples are given:
The synthesis of derivatives of 4-thio-D-ribofuranose is according to scheme 1:

SCHEME 1
This compound is obtained in 8 steps from = -Lyxose with an overall yield of 6%.

R. WHISTLER, W. E. DICK, T. INGLE, R. M. ROWELL and B. URBAS, J. Org.

Chem., 1964, 29, 3723-3725.

B. URBAS and R. L. WHISTLER, J. Org. Chem., 1966, 31, 813-816.

E.J. REIST, D. E. GUEFFROY and L. GOODMAN, J. Am. Chem. Soc., 1964, 86, 5658-5663.

R. L. WHISTLER and J. N. BeMILLER in "Methods in Carbohydrate Chemistry", R.

L. WHISTLER and M. WOLFROM, Eds., Academic Press, Inc., New York, 1962, Vol.

I, p.79.

The derivatives of 4-thio-2-deoxy-D-nbofuranose were obtained according to scheme 2

SCHEME 2
This 14-step synthesis leads to methyl 2-deoxy 4-thio-D-erythro-pentofuranoside with an overall yield of 8%.

Y. L. FU and M. BOBEK, J. Org. Chem., 1976, 41, 3831-3834.

- Y. L. FU and M. BOBEK, in "Nucleic Acid Chemistry", L. TOWSEND and R. S.

TIPSON, Eds., 1978, Part I pp. 183-194.

U. G. NAYAK and R. WHISTLER, Liebigs Ann. Chem., 1970, 741, 131-138.

Similarly, the 4-thio-D-arabinofuranose derivatives were obtained: R. L. WHISTLER, U. G. NAYAK and A. W. PERKIN, Jr., J. Org., Chem., 1970, 35, 519-521.

I. 2. The corresponding nucleosides were then synthesized:
a) In series 4'-thio-2'-deoxy-D-ribofuranose
YL FU and M. BOBEK in "Nucleic Acid Chemistry", L. TOWSEND, RL

TIPSON, Eds., John Wiley & Sons, New York, 1978, p. 317.

b) In series 4'-thio-D-ribofuramiasis
EJ REIST, DE GUEFFROY and L. GOODMAN, J. Am. Chem. Soc. , 1964, 86, 5658-5663.

 - E.J. REIST, D. E. GUEFFROY and L. GOODMAN, Chem. Ind., 1964, 13641365.

 B. URBAS and R. L. WHISTLER, J. Org. Chem., 1966, 31, 813-816.

- Mr BOBEK, RL WHISTLER and A. BLOCH, J. Med. Chem., 1970, 23, 411-413
- M. BOBEK, A. BLOCH, R. PARTHASARATHY and RL WHISTLER, J. Med.

Chem., 1975, 18, 784
- Mr BOBEK, RL WHISTLER and A. BLOCH, J. Med. Chem., 1972, 15, 168171.

N. OTOTANI and RL WHISTLER, J. Med. Chem., 1974, 17,535
MW PICKERING, JT WITKOWSKI and RK ROBBINS, J. Med. Chem., 1976, 19, 841-842.

- AKM ANISUZZAMAN and MD AMIN, J. Bangladesh Acad. Sci., 1978, 2 59-64
c) In other series:
RGS RITCHIE and WA SZAREK, J. Chem. Soc. Chem. Commun., 1973, 686.

- EJ REIST, LV FISCHER and L. GOODMAN, J. Org. Chem., 1968, 33,189
RL WHISTLER, LW DONER and UG NAYAK, J. Org. Chem., 1971, 36, 108
These nucleosides are obtained by the traditional routes of nucleoside synthesis in oxygenated series.

Either by the heavy metal salt method:
Treatment of the chloromercuric heterocycle with the corresponding chlorosugar.

- Mr BOBEK, R. L. WHISTLER and A. BLOCH, J. Med. Chem., 1972, 168-171.

- D. E. GUEFFROY and L. GOODMAN, Chem. & Ind., 1964, 1364-1365.

- E.J. REIST, M. BOBEK, R. L. WHISTLER and A. BLOCH, J. Med. Chem., 1970, 411-413.

E.J. REIST, D. E. GUEFFROY and L. GOODMAN, J. Am. Chem. Sac., 1964, 86 5658-5663.

- E.J. REIST, L. V. FISCHER and L. GOODMAN, J. Org. Chem., 1968, 33, 189-192.

 Either by the method of HILBERT and JOHNSON, - by condensation of the pyrimidine base with the corresponding thiochlorosugar.

B. URBAS and R. L. WHISTLER, J. Org. Chem., 1966, 31813-816.

by condensation of the silylated derivatives of the bases and the corresponding thiochlorosucres.

M. BOBEK, A. BLOCH, R. PARTHASARATHY and R. L. WHISTLER, J.

Med., Chem., 1975, 784-787.

R. WHISTLER, L. W. DONER and U. G. NAYAK, J. Org. Chem., 1971, 36, 108110.

N. OTOTANI and R. L. WHISTLER, J. Med Chem., 1974, 17 535-537.

 by the fusion reaction of 3-cyano-1,2,4-triazole and 1,2,3,5-tetra-O-acetyl-4-thio-D-ribofuranose.

M. V. PICKERING, J. T. WITKOWSKI and R. K. ROBINS, J. Med. Chem., 1976, 841-842.

by the method of VORBRUGGEN and NIEDBAILLA by condensation of adenine and 1,2,3,5 tetra-O-acetyl thioribofuranose in the presence of catalyst
FRIEDEL and CRAFTS.

- A. K. M. ANISUZZAMAN and M. D. AMEN, J. Bangladesh Acad. Sci., 1978, 59-64.

Recently, the synthesis of thionucleosides has again been of interest.

Thus, 2 ', 3', 5 'tn-O-benzyl 4'-thio-ss-D-xylofuranosyl uracil was obtained by an original route according to scheme 3: - MW BREDENKAMP, CW HOLZAPFEL and AD SWANEPOEL, Tetrahedron
Lett., 1990, li, 2759-2762, MW BREDENKAMP, CW HOLZAPFEL and AD SWANEPOEL, S. Afr. J.

Chem., 1991, 44, 31-33.

(i = dibutyl tin oxide, ii = MsCI, iii = BnCl, iv = tetra-butylairnonium iodide, barium carbonate, v = Hg (OAc) 2 / AcOH, vi = modified HILBERT JOHNSON method.)
SCHEME 3
The synthesis of pyrimidine analogues of 4'-tbio 2'-deoxy nucleosides has been published.

- R. R. DYSON, P. L. COE and R. T. WALKER, J. Med. Chem., 1991 34, 2782-2786.

by the method of HORTON and MARKOVS.

D. HORTON and R. A. MARKOVS, Carbohydrate Res., 1980, 80,356.

It is illustrated in Figure 4:

SCHEMA 4
In a similar manner, 2'-deoxy-4'-thiocytidine, 2'2'-deoxy-4'-thiouridine and 4'thiothymidine were obtained by JA SECRIST III et al. (JA SECRIST, KN

TIWARI, JM RIORDAN and JA MONTGOMERY, J. Med. Chem., 1991, 34, 2361
2366); (JA MONTGOMERY and JA SECRIST III, PCT Int Appl. WO 9104,033) to
from 2-deoxy-4-thio-ss-erythro-pentafuranose synthesized according to BOBEK et al. (Y.

L. FU, M. BOBEK, J. Org. Chem., 1976, 41, 3831-3834.) Using the TMSTf as
coupling catalyst.

 The synthesis of 4'-thio 2'-deoxy-D-ribofuranose has been the subject of a patent and publications: - European Patent Application, 0421 777 Al R. T. WALKER.

- R. R. DYSON, P. L. COE and R. T. WALKER, J. Chem. Soc. Chem. Comm., 1991,741-742.

- R. DYSON, P. L. COE and R. T. WALKER, Carbohydrate Res., 1991, 216, 237-248.

(i = HCl/MeOH, ii = BuBr/THF, iii = BnSH/HCl, iv = Ph3P/DEAD/BzOH/THF, v = MeONa/
MeOH / CH2Cl2, vi = MsCl I Pyr., vii = Nal I CO3Ba / Acetone, viii = BCl3 / CH2C12, ix = p
toluoylCl, x = Ac2O/H2SO4)
SCHEMA 5
Des ribothionucléosides derivés des pyrimidines ont fait l'objet du même brevet. Mais la synthèse du thioribofurannose de départ a été réalisée selon la méthode de E. J. REIST.It is described in Figure 5:

(i = HCl / MeOH, ii = BuBr / THF, iii = BnSH / HCl, iv = Ph3P / DEAD / BzOH / THF, v = MeONa /
MeOH / CH2Cl2, vi = MsCl I Pyr, vii = Nal I CO3Ba / Acetone, viii = BCl3 / CH2Cl2, ix = p
toluoylCl, x = Ac2O / H2SO4)
SCHEME 5
Ribothionucleosides derived from pyrimidines were the subject of the same patent. But the synthesis of the starting thioribofuranose was carried out according to the method of EJ REIST.

E.J. REIST, D. E. GUEFFROY and L. GOODMAN, J. Am. Chem. Soc., 1964, 84-5658.

I. 3. SYNTHESIS OF SYNTHESIS OF 4'-THIO-BETA-D-RIBOFURANNOSE
NECESSARY FOR THE PRODUCTION OF THIOOLIGOMERS.

Synthesis of L-thioLyxofuranose and D-thioRibofuranose in 7 steps from D-ribose and L-lyxose was performed respectively with 29 and 21% yield respectively. This synthesis is illustrated in Figure 6.

(i = MeOH, HCl, Li = BnBr, KOH, iii = HCl, H2O, dioxane, iv = BnSH, HCl, v = MesCL, pyr, vi =
NBu4I, BaCO3; vii = Hg (OAc) 2)
SCHEME 6
Our synthesis for thio-D-ribofuranose uses L-lyxose as a starting material as in the WHISTLER synthesis (page 19). This strategy is called strategy C1 ---- C4. In fact, the sulfur atom is first introduced into the anomeric position and a nucleophilic substitution SN2 between the sulfur atom carried by the anomeric carbon (C1) and the C4 carbon carrying an activated OH by a Mesyl group. (Ms) is then carried out.This strategy applied to the synthesis of 4-thio-D-ribofuranose and 4-thio-L-lyxofuranose resembles that which WALKER used to achieve the 2-desoxy-D-ribofuranose series ( Patent
Eur. Pat. 0421 777 A 1 page 6). For example, WALKER product 5 (Scheme 5) is similar to our product (Schema 6) in L-Lyxofuranose series.

13 B = T 14a 14 B = T 18 <t 18ss
B = U 15 &alpha;, 15ss B = U 19 &alpha;, 19ss
B = C 18 &alpha;, 18ss B = C 20 &alpha;
B = A 17a 17, 'B = A 21 &alpha;, 21ss
(i = BSA, TMSTf, CH3CN, ii = BBr3, CH2Cl2)
SCHEME 7
Synthesis of thionucleosides of uracil, thymine and cytosine was performed using bis-trimethylsilyl acetamide (BSA) as silylating agent and trimethylsily trifluoromethanesulfonate (TMSTf) as coupling agent. The nucleosides a and B were obtained with 74, 77 and 70% of yield respectively. The synthesis of the adenine thionucleosides was carried out by SnCl4 in acetonitrile. Nucleosides a and B are obtained with 58% yield (Scheme 7).

II. SYNTHESIS OF OLIGO-THIONUCLEOTIDES ON SOLID SUPPORT.

The supported synthesis of B oligothioribonucleotides was carried out using a GENE model APPLIED BIOSYSTEM 318A synthesizer according to the phosphoramidite method.

It is similar to that described for series B oligodeoxyribonucleotide by
CARUTHERS et al. (SL BEAUCAGE and MH CARUTHERS, Tetrahedron Lett., 1981, 22, 1859-1862, LJ Mc BRIDE and MH CARUTHERS, Tetrahedron Lett., 1983, 24, 245-248.) And to that described by USMAN et al. oligoribonucleotides (N. USMAN, KK

OGILVIE, M. Y. JIANG and R. J. CEDERGREN, J. Am. Chem. Soc., 1987, 109, 78457854.).

This synthesis required:
I) The preparation of synthons B thioribonucleoside phosphoramidites corresponding to the bases T, U, C, A and G.

 II) Functionalization of the solid support incorporating a thioribonucleoside B derivative.

 III) The elongation of the oligothioribonucleotide chain using an automatic synthesizer.

 IV) Finally, at the end of the required number of synthesis cycles, the oligothionucleotide was removed from its support, deprotected and purified.

 II.1 - Synthesis of phosphoramidite of beta-thioribonucleoside 26 a-d.

He. 1. Synthesis of 5'-DmTr-2'-O-TBDMS derivatives (Scheme 8)
The major problem in the chemical synthesis of oligoribonucleosides is the presence of the 2 'hydroxyl in the ribose or thioribose ring which requires selective protection.

The selection of a protecting group for the hydroxyl in 2 'must correspond to several criteria, it must be stable:
under the coupling conditions during the deprotection of the 5 'protecting groups to allow the extension of the chain.

 during oxidation in the case of the phosphite triester method.

 during the masking following the coupling.

 during the deprotection of the protective groups of the exocyclic amines.

 during the deprotection of phosphates.

 and in the case of syntheses on a solid support during the cleavage of the oligomer of the support.

Finally this protecting group must be removed under very mild conditions to avoid an attack of the 2 'hydroxyl function released on the adjacent phosphodiester linkage.

A large number of strategies for protecting the hydroxyls in 2 'have been developed and illustrate the difficulties in the choice of the protection scheme for hydroxyl groups.

(C. B. REESE, Nucleotide Nucleotides, 1985, 4, 117-127, R. KIERZECK, M. H.

CARUTHERS, C. E. LONGFELLOW, D. SWINTON, D. H. TURNIER and S. M.

FREIER, Biochemistry, 1986, 25, 7840-7846; S. IWAI and E. OHTSUKA, Nucleic Acids
Res., 1988, 16, 9443-9456;
C. LEHMANN, YZ XU, C. CHRISTODOULOU, ZK TAN and MJ GAIT, Nucleic
Acids Res., 1989, 17, 2379-2390; T. TANAKA, S. TAMATSUKURI, and M. IKEHARA, Nucleic Acids R., 1986, 14, 6265-6279.).

The use of alkylsilyl protecting groups and in particular tert-butyldimethylsilyl (TBDMS) for the 2 'hydroxyl facilitated the synthesis of oligoribonucleotides. The TBDMS group is sufficiently stable under acidic or basic conditions and is easily removed by tetra-butyl ammonium fluoride in THF for use in solid phase strategy (N. USMAN, K. K. OGILVIE, M. Y. JIANG and R. L.

LEDERGEN, J. Am. Chem. Soc., 1987, 109, 7845-7854; N. USMAN, K.

NICOGHOSIAN, RL CEDERGREN and KK OGILVIE, Proc. Natl. Sci. USA, 1988, 85, 5764-5768; SA SCARINGE, CF FRANCKLYN and N. USMAN, Nucleic Acids
Res., 1990, 18, 5433-5441, KK O'GILVIE and MJ NEMER, Tetrahedron Lett., 1980, 21, 4159; RT PON and KK OGILVIE, Nucleotide Nucleotides, 1984, 3, 485; RT

PON and K. K. OGILVIE, Tetrahedron Lett., 1984, 25, 713.).

Thus, the protection scheme used for the oxygenated nucleosides was applied in series B oligothioribonucleotide (Scheme 8).

Namely the protections
Benzoyl for adenine and cytosine, isoButyl for guanine as protection for exocyclic amino groups.

 Dimethoxytrityl (DmTr) to protect the 5 'primary hydroxyls.

 tert-butyldimethylsilyl (TBDMS) to protect the secondary hydroxyl in 2 '.

 - Methoxy for the protection of phosphates.

The first step makes it possible to protect the 5 'free hydroxyl with a labile acidic group DmTr (Scheme 8).

Thus, the nucleoside derivative 22 is treated with dimethoxytrityl chloride (1.27 molar equivalents) in anhydrous pyridine under an inert atmosphere. After customary treatment of the reaction mixture, the dimethoxytrityl derivative 23 is purified by chromatography on silica gel and isolated with yields ranging from 70 to 80%.
The physicochemical characteristics of these derivatives 23 (NMR, Mass) are in agreement with the proposed structures.

(i = DmTrCI, pyr, ii = TBDMSC1, pyr.)
The abbreviations have the following meanings: aB = T = Thymine b- B = U = Uracil.

c- B = CBz = N-Benzoyl-4-Cytosine.

d = B = ABz = N-Benzoyl-6-Adenine.

e-B = GPal = N-Palmitoyl-2-guanine.

Dmtr = Dimethoxy-4,4'-trityl. iPr = Isopropyl. Bz = Benzoyl. TBDMS = tert butyldimethylsilyl. (iPr) 2 P (Cl) OMe = Chloro, (N, N diisopropylamino, methoxyphosphine). .

SCHEME 8
The second step (Scheme 8) consists of the protection of the secondary hydroxyl in 2 '.

During silylation, a mixture of 2'-O-silyl and its 3'-O-silyl regioisomer is obtained (KK OGILVIE and DW ENTWISTLE, Carbohyd Res., 1981, 89, 203-210;
KK OGILVIE, AL SCHIFMAN and CL PENNEY, Can. J. Chem., 1979, 57, 2230;
GH HAKIMELAHI, ZA PROBA and KK OGILVIE, Can. J. Chem., 1982, 60, 1106.). These isomers must be carefully separated on a silica gel column in order to obtain the 2'-O-TBDMS 24 derivative with a purity greater than 99.95% measured by HPLC.

Indeed, if this is not the case, the subsequent steps will lead us to the expected 5'-3 'oligothionucleotide mixtures with the undesirable 5'-2' isomer.

Accordingly, the purity of 5'-O-DmTr-2'-O-silyl 24 is paramount for good synthesis.

Thus, obtaining compounds 5'-DmTr-2'-O-TBDMS 24 was first performed according to the method of OGILVIE et al. (KK OGILVIE, AL SCHIFMAN and CL PENNEY,
Can. J. Chem., 1979, 57, 2230.) which consists in condensing 23 with tert-butyldimethylsilyl chloride (TBDMSC1) (1.2 molar equivalent) in pyridine in the presence of imidazole (2.6 molar equivalents). At the end of the reaction, a mixture of derivative 2'-O-TBDMS 24 and its isomer 3'-O-TBDMS 25 is obtained. The expected compound 24 is separated from the mixture by chromatography on a column of silica gel.

It is then possible, by an equilibration reaction, to obtain 24 from its 3'-O-TBDMS isomer (SS JONES and CB REESE, J. Chem Soc., Perkin Trans I, 1979, 2762.). This migration of the TBDMS group is intramolecular and is carried out via an intermediate having a pentavalent silicon atom. Isomerization of the 3'-O derivative
TBDMS obtained previously after a first chromatographic separation is carried out in ethanol at room temperature overnight. The two position isomers thus obtained are again separated by silica gel column chromatography. This isomerization operation followed by purification is repeated three times.

However the yields of 5'-DmTr-2'-O-TBDMS 24 obtained by this method (40 to 50% depending on the bases) did not seem satisfactory to us. We used another, more recent, developed by OGILVIE (G. H. HAKIMELAHI, Z. A. PROBA and K. K.

OGILVIE, Can. J. Chem., 1982, 60, 1106) which allows the preferential introduction of the
TBDMS at the 2 'position of ribonucleosides B. This technique recommends the use of silver salts.

Thus, 5'-dimethoxytrityl 23 is condensed with TBDMS chloride (1.3 molar equivalents) in the presence of silver nitrate (1.2 molar equivalents) and 3.7 equivalents of pyridine in anhydrous THF. After purification by chromatography on a silica gel column, 56% yield is obtained at 24 and 27% at 5'-DmTr-3'-O-TBDMS 25.

As a result, 5'-DmTr-2'-O-TBDMS 24 is obtained under these new conditions in a single step with a better yield, but the selectivity of introduction of the 2 'TBDMS group is not improved.

II.1.2 Synthesis of the Phosphoramidite Derivatives 26 ad (Scheme 9)
The N-protected four N-protected phosphoribonucleosides 26a-d are then obtained from the corresponding N-protected 5'-DmTr-2'-O-TBDMS B thioribonucleosides 24 (Scheme 9).

The nucleoside derivative 24 was treated with chloro N, N diisopropylamino methoxy phosphine (2.5 molar equivalents) in the presence of N, N, N diisopropyl ethylamine (DIEA) in dichloromethane under an inert atmosphere. The phosphoramidite derivative 26 was purified by chromatography on silica gel and lyophilized in benzene, the phosphoramidites 26 ad are obtained with yields ranging from 78 to 80% depending on the nature of the base.

i = (iPr) 2P (Cl) OMe, DIEA, DMAP, cH2Cl2)
SCHEME 9
Analyzes of the 31p and 1H NMR spectra of 26 clearly show that we obtain a single pair of diastereoisomers which establishes the regioisomeric purity of the compounds obtained.

Indeed, some authors (R. KIERZEK, MH CARUTHERS, CE LONGFELLOW,
D. SWINION, DH TURNER and SM FREIER, Biochemistry, 1986, 25, 7840-7846.) Had hypothesized that silyl groups at the 2 'position were not stable under the conditions of phosphorylation. However, it has been shown (WK KOHLER,
W. SCHLOSSERM, G. CHARUBALA and W. PFLEIDLERER, Chemistry and Biology of
Nucleosides and Nucleotides, Academic Press, New York, 1978, pp 347-358; KK

OGILVIE and DW ENTWISTLE, Carbohydrate Res., 1981, 89, 203-210; N. USMAN,
KK OGILVIE, MY JIANG and RJ CEDERGREN, J. Am. Chem. Soc., 1987, 109, 7845-7854.) That alkylsilyl groups migrate in protic solvents such as methanol or in aqueous solutions such as pyridine / water mixture, but these silyl groups are stable in anhydrous solvents and especially in non-protic bases such as anhydrous pyridine (KK OGILVIE, DW ENTWISTLE,
Carbohydrate Res., 1981, 89, 203-210; W. KOHLER, W. SCHLOSSER, G.

 CHARUBALA and W. PFLEIDERER, Chemistry and Biology of Nucleosides and Nucleotides, Academic Press, New York, 1978, pp 347-358.). On the other hand, it has been clearly demonstrated in ribonucleotide series that the use of 2'-O-silyl ribonucleosides in the synthesis of oligoribonucleotides leads exclusively to 5'-3 'bonds (K.K.

OGILVIE, M.J. NEMER, Tetrahedron Lett., 1980, 21, 4159; R. T. PON and K. K.

OGILVIE, Tetrahedron Lett., 1984, 25, 713; R. T. PON, and K.

 K. OGILVIE, Nucleoside Nucleotides, 1984, 3, 485; J. GAREGG, I. LINDH, I.

STAWINSKI, and R. STROMBERG, Tetrahedron Lett., 1986, 27, 4055-4058). The synthesis of phosphoramidites is not stereospecific and the formation in equal amounts of the two diastereoisomers is due to the chirality of the phosphorus atom of R or S configuration. The formation of these two products is not annoying insofar as subsequent oxidation reactions suppress this chirality.

II.2. EXPERIMENTAL PART
General Method for the Preparation of 5'-O-Dimethoxytrityl N-acyl 4'-thio-B-Dribonucleosides 23a-d.

To a solution of N-acyl B-D-thioribonucleoside 23 a-d (immolate) in anhydrous pyridine (5.6 ml) is added dimethoxytrityl chloride (1.27 mmol, 430 mg). The reaction mixture is stirred at ambient temperature under an inert atmosphere for 90 to 120 min and then methanol (1 ml) is added. After stirring for a further 10 minutes, the reaction mixture is poured into a saturated aqueous solution of sodium bicarbonate (25 ml) and the products are extracted with dichloromethane (2 × 20 ml). The organic phases are washed with water (2 × 100 ml) and then dried over sodium sulphate and filtered. The filtrate is evaporated off under reduced pressure and the residue obtained is chromatographed on a column of silica gel. The elution is carried out with a CH 2 Cl 2 / MeOH: 98/2 mixture in the presence of 1% triethylamine; the fractions containing the product are evaporated to dryness and the nucleosides 23 a-d are obtained in the form of an oil.

1- [4'-thio-5'-O-dimethoxytrityl-ss-D-ribofuranosyl] Thymine 23a
Yield: 68%.

FAB mass spectrometry> 0 NOBA m / z = 577 [M + H] +, 303 [DmTr] +
1H NMR (DMSOd6, 360MHz) δ 11.24, (s, 1H, NH); 7.44, (s, 1H, H6); 6.87-7.30, (m, 13H, H aromatics); 5.83, (d, 1H, H1, J1.2 '= 7.0); 5.53, (d, 1 H, OH 2,, JOH, 2 '= 5.8); 5.30, (d, 1H, OH3 ', JOH, 3' = 4.7); 4.14, (dd, 1H, H2, J2 ', 1 = 6.9, J2', 3 '= 3.0); 4.00, (t, 1H, H3, J3 ', 2' = 3.0, J3 ', 4' = 3.0); 3.69, (s, 6H, OMe); 3.30, (dd, 1H, H4 '); 3.13, (m, 2H, H 5, H 5 -); 1.60, (s, 3H, CH 3).

1- [4'-thio-5'-O-dimethoxytrityl-ss-D-ribofuranosyl] Uracil 23 b.

Yield 70%.

1H NMR (DMSOd6, 360 MHz) δ 11.31, (s, 1H, NH); 7.70, (d, 1H, H6, J6.5 = 8.0), 7.40-6.89, (m, 13H, aromatic H of the dmTr group); 5.84, (d, 1H, H1, J1 ', 2' = 5.7), 5.53, (d, 1H, OH2 ', JOH2', 2 '= 5.6), 5.48, (d, 1H, Hs, J5.6 = 8.0); 5.26, (d, 1H,
OH3 ', JOH3', 3 '= 4.45); 4.02, (m, 2H, H2, H3 '); 3.74, (s, 6H, OMe); 3.31, (m, 3H,
H4 ', H5', H5 ").

Mass spectrometry FAB> 0 NOBA m / z = 563 [M + H] +, 303 [DmTr] + 1- [4'-thio-5'-O-dimethoxytrityl-ss-D-ribofuranosyl] N-4-benzoylCytosine 23 c
Yield 69%.

1H NMR (DMSOd6, 250 MHz) S 11.32, (s, 1H, NH); 8.40, (d, 1H, H6, J6.5 = 7.5); 8.01, (d, 2H, Ho of the benzoyl group); 7.63, (d, 1H, Hs, J5.6 = 7.3); 7.44, (m, 17H,
Aromatic H of the group DmTr and Hm, p of the benzoyl group); 5.88, (d, 1H, H1 ',
J1 ', 2' = 4.1); 5.73, (d, 1H, OH2 ', JOH, 2' = 5.2); 5.27, (d, 1H, OH3s, OJH, H3 '= 5.7); 4.06, (m, H 2 ', J 2', 1 '= 4.0, J 2', 3 '= 3.7, J 2', OH = 5.2); 4.03, (m, H3 ', J3', 2 '= 3.7, J = 5.3, J3', OH = 5.7); 3.76, (s, 6H, OCH 3 from the DmTr group); 3.43, (m, 3H,
H4 ', H5' and H5 ").

Mass spectrometry FAB> 0 NOBA m / z = 666 [M + H + +, 517 [M + H-CONH]] +, 303 [DmTrj + 1- [4'-thio-5'-O-dimethoxytrityl-ss-D- ribofuranosyl] N6-benzoylAdenine 23 d
Yield 70%
1H NMR (DMSOd6, 250 MHz) δ 11.25, (s, 1H, NH); 8.63, (s, 1H, H8); 8.57, (s, 1H,
H2); 8.04, (d, 2H, Ho of the benzoyl group); 7.51, (m, 13H, aromatic H of the DmTr group); 6.92, (d, 3H, Hm, p of the benzoyl group); 5.98, (d, 1H, H1R, J1, 2, = 58); 5.75, (d, 1H, OH 2 ', JOH, 2' = 3.5); 5.45, (d, 1H, OH3 ', JOH, 3' = 2.5); 4.70, (m, 1H, H2 '); 4.26, (m, 1H, H3 '); 3.74, (s, 6H, OCH3 from the DmTr group); 3.53, (m, 3H, H4, H5 and H5 -).

FAB mass spectrometry> 0 NOBA m / z = 690 [M + H] +, 303 [DmTrj +
General method for the preparation of 2'-O-tert-butyldimethylsilyl-4'-thio-5'-O-dimethoxytrityl-N-acyl-ss-D-ribonucleosides 24 ae and their isomer 3 '-TBDMS 25 ae.

To a solution of 5'-O-dimethoxytrityl 4'-thio N-acyl B-D-ribonucleoside 23a-d (immolate) in anhydrous HF (10 ml) is added silver nitrate (1.2 mmol, 203 mg). The reaction mixture is then stirred for 5 minutes at room temperature before the addition of tert-butyldimethylsilyl chloride (TBDMSCI, 1.3 mmol, 195 mg) and anhydrous pyridine (3.7 mmol, 0.193 ml). The stirring at room temperature is then prolonged for 24 h, then the heterogeneous solution is filtered before being poured into a 5% aqueous solution of sodium bicarbonate (30 ml). The products are extracted with dichloromethane (3 × 20 ml) and the organic phases are washed with water then dried over sodium sulphate and filtered. The filtrate is evaporated to dryness and the residue is chromatographed on a column of silica gel using as eluent a mixture of CH2Cl2 / AcOEt: 95/5 then of CH2Cl2 / AcOEt: 80/20. The fractions containing the desired product (2'-O-TBDMS, 24 m) are combined and evaporated to dryness. The 3'-O-TBDMS isomer α-d is then obtained.

1- [2'-O-tert-butyldimethylsilyl-4'-thio-5'-O-dimethoxytrityl-ss-D-ribofuranosyl]
Thymine 24a.

Yield: 33%.

FAB mass spectrometry> O NOBA m / z = 691 [M + H + +, 713 1M + Na] +.

1H NMR (DMSOd6, 300MHz): # 11.36, (s, 1H, NH); 7.58, (s, 1H, H6); 7.33-6.90, (m, 13H, H aromatics); 5.89, (s, 1H, H1, J1 ', 2' = 6.1); 5.27, (m, 1H, OH3 '); 4.20, (dd, 1H, H2 ', J2 #, 1 # = = 6.10, J2', 3 '= 3.5); 3.97, (m, 1H, H3 '); 3.73, (s, 6H, OMe); 3.36, (m, 1H, H4 '); 3.25, (m, 2H, H5 ', H5-); 1.58, (s, 3H, CH3); 0.81, (s, 9H, tert-butyl); d, 6H, Me 2 Si).

1- [2'-O-tert-butyldimethylsilyl-4'-thio-5'-O-dimethoxytrityl-ss-D-ribofuranosyl]
Uracil 24 b.

Yield 56%.

Tf = 114 C.

1H NMR (DMSOd6, 360 MHz): δ 11.20 (s, 1H, NH); 7.81, (d, 1H, H6, J6.5 = 8.1); 7.30-6.89, (m, 13 H, aromatic H of the group dmTr) 5.84, (d, 1H, H1, J1 ', 2 # = 5.5); 5.50, (d, 1H, Hs, J5.6 = 8.1); 5.23, (d, 1H, OH3 ', JOH3', 3 '= 4.7); 4.11, (q, 1H,
H2 ', J2 = 5.5, J2,, 3' = 3.5); 3.95, (m, 1H, H3 '); 3.74, (s, 6H, OMe); 3.38, (m, 2H, H4, H5, J4, 5, = 3.28, (m, 1H, H5-); 0.82, (s, 9H, tert-butyl); 0.05, (m, 6H, 2 Si).

Mass spectrometry FAB> NOBA m / z = 677 [M + H] +, 619 [M + H-tert-butane] +, 303 [dmTr] + 1- [2'-O-tert-butyldimethylsilyl-4 'thio-5'-O-dimethoxytrityl-ss-D-ribofuranosyl]
N4-BenzoylCytosine 24c.

Yield 32%.

1H NMR (DMSOd6, 250 MHz): # 11.31, (s, 1H, NH); 8.51, (d, 1H, H6, J6.5 = 7.3); 8.01, (d, 2H, Ho of the benzoyl group); 7.51, (m, 14H, aromatic H of the group
DmTr and H5); 6.93, (d, 3H, Hm, p of the benzoyl group); 5.93, (d, 1H, H1, J1 ', 2' = 3.0); 5.20, (d, 1H, OH3 ', OJH, H3' = 5.0); 4.14, (m, 1H, H2 '); 4.00, (m, 1H, H3 '); 3.76, (s, 6H, OCH 3 from the DmTr group); 3.38, (m, 3H, H4 ', H5' and H5 "), 0.86, (s, 9H, tert-butyl), 0.60, (d, 6H, Me2Si).

Mass spectrometry FAB> 0 NOBA m / z 780 [M + H + +, 303 [DmTr] + 1- [2'-O-tert-butyldimethylsilyl-4'-thio-5'-O-dimethoxytrityl-ss-D-ribofuranosyl] ]
N6-benzoylAdenine 24 d.

Yield 35%
1H NMR (DMSOd6, 250 MHz): δ 11.25, (s, 1H, NH); 8.64, (s, 1H, H8); 8.61, (s, 1H,
H2); 8.04, (d, 2H, Ho of the benzoyl group); 7.51, (m, 13H, aromatic H of the DmTr group); 6.95, (d, 3H, Hm, p of the benzoyl group); 6.01, (d, 1H, H1X,
J1 ', 2' = 5.8); 5.48, (d, 1H, OH3 ', JOH, 3' = 2.5); 4.70, (m, 1H, H2 '); 4.26, (m, 1H, H3 '); 3.75, (s, 6H, OCH 3 from the DmTr group); 3.53, (m, 3H, H4 ', H5' and H5-), 0.82, (s, 9H, tert-butyl), 0.50, (d, 6H, Me2Si).

Mass spectrometry FAB> 0 NOBA m / z 804 [M + H] +, 303 [M + H] + 1- [2'-O-tert-butyldimethylsilyl-4'-thio-5'-O-dimethoxytrityl-ss-D] -ribofurannosyl]
Thymine a.

Yield 13%.

1H NMR (DMSOd6, 360 MHz): S, 11.34, (s, 1H, NH); 7.46, (s, 1H, H6); 7.89-7.30, (m, 13H, aromatic H); 5.87, (d, 1H, Hf, J1 ', 2' = 7.8); 5.46, (d, 1H, OH2 ', JOH, 2' = 5.1); 4.13, (m, 1H, H3 '); 4.11, (m, 1H, H2 ', J2', 1 '= 7.8, 8, OJH, 2 # = 5.1); 3.73, (s, 6H, OMe); 1.66, (s, 3H, CH3); 0.84, (s, 9H, ten-butyl); 0.50, (d, 6H, Me 2 Si).

1- [3'-O-tert-butyldimethylsilyl-4'-thio-5'-O-dimethoxytrityl-ss-D-ribofuranosyl]
Uracil 25 b.

Yield 27%
Tf = 109C.

1 H NMR (DMSOd6, 360 MHz): δ 11.30, (s, 1H, NH); 7.70, (d, 1H, H6, J6.5 = 8.0); 7.30-6.89, (13 H, aromatic H group-ment dmTr); 5.84, (d, 1H, H1 ', J1', 2 '= 7.3); 5.57, (d, 1H, H5, J5.6 = 8.0); 5.46, (d, I H, OH 2s, JOH 2 ', 2' = 5.1); 4.12, (t, 1H, H3, J3 ', 2, = 3.1, J3', 4 '= 3.1); 4.03, (m, 1H, H2 '); 3.74, (s, 6H, OMe); 3.41, (m, 1H, H4 '); 3.21, (m, 2H, H5 ', H5-); 0.08, (s, 9H, tert-butyl), 0.02, (m, 6H, Me2Si).

FAB mass spectrometry> O NOBA m / z 677 [M + H +, 619 [M + H-tert-butane] +, 303 [dmTr] +.

This compound was brought into contact with a 0.25% ethanol / triethylamine (v / v) solution for 12 hours at room temperature to give an equal proportion of a mixture 24b and 25b.

1- [3'-O-tert-butyldimethylsilyl-4'-thio-5'-O-dimethoxytrityl-ss-D-ribofuranosyl]
N4-benzoyl Cytosine 25 c.

Yield 31%.

1H NMR (DMSOd6, 250 MHz): δ 11.33, (s, 1H, NH); 8.40, (d, 1H, H6, J6 s = 7.4); 8.01, (d, 2H, Ho of the benzoyl group); 7.60, (m, 14H, aromatic H of the group
DmTr and Hs); 6.92, (d, 3H, Hm, p of the benzoyl group); 5.92, (d, 1H, H 21, J ', 2' = 5.3); 5.60, (d, 1H, OH2 ', JOH, 2' = 5.0); 4.15, (m, 2H, H2 ', H3'); 3.75, (s, 6H, OCH 3 from the DmTr group); 3.38, (m, 3H, H4, HsX and H5-); 0.79, (s, 9H, tert-butyl); 0.00, (d, 6H,
2 Si).

Mass spectrometry FAB> NOBA m / z 780 [M + H] +, 303 [DmTrj + 1-C3'-O-terr-butyldimethylsilyl-4'-thio-5'-O-dimethoxytrityl BD-ribofuranosyl]
N6-benzoyl adenine d.

Yield 29%.

1H NMR (DMSOd6, 250 MHz): δ 11.25, (s, 1H, NH); 8.64, (s, 1H, H8); 8.61, (s, 1H,
H2); 8.04, (d, 2H, Ho of the benzoyl group); 7.51, (m, 13H, aromatic H of the DmTr group); 6.95, (d, 3H, Hm, p of the benzoyl group); 6.01, (d, 1H, H1,
J1 ', 2' = 5.8); 5.79, (d, 1H, OH2, JOH, 2 '= 3.5); 4.70, (m, 1H, H2,); 4.26, (m, 1H, H3 '); 3.75, (s, 6H, OCH 3 from the DmTr group); 3.53, (m, 3H, H4 ', H5' and H5 "), 0.82, (s, 9H, tert-butyl), 0.01, (d, 6H, Me2Si).

FAB mass spectrometry> O NOBA m / z 804 [M + H] +, 303 [M + H] +
General method of preparation of BD-ribonucleoside phosphoramidites ad.

To a solution of the compounds 24 ad (1 mmol) in anhydrous dichloromethane (3.8 ml) are added under argon inert atmosphere N, N, N-diisopropylethylamine (4 mmol, 0.7 ml), chloro N, N-diisopropylaminomethoxyphosphine (2.5mmole), 0.48ml) and the 4
N, N-dimethylaminopyridine (0.2mmol, 24.4mg). The reaction mixture is stirred for 40 to 90 minutes at room temperature and is then diluted with ethyl acetate (35 ml). The resulting solution is washed with brine (4xS0ml) and then with water (2x50ml). The organic phase is dried over sodium sulfate, filtered and evaporated under reduced pressure.

The residue is chromatographed on a column of silica gel and the elution is carried out using a mixture of cyclohexane, dichloromethane and triethylamine (100/0 / 0.1 to 50/50 / 0.1). The products 26 a-d are obtained in the form of a white powder after lyophilization in benzene.

1- [2'-O-tert-butyldimethylsily-3'-N, N-diisopropylmethoxyphosphoramidite-4'thio5'-O-dimethoxytrityl-ss-D-ribofuranosyl] Thymine 26a (diastereoisomeric mixture)
Yield: 89%.

31p NMR (CD3CN): s, 151.32 and 150.03.

1 H NMR: δ 7.99, (s, 1H, NH), 7.65, (d, 1H, H 6); 7.35-6.80, (m, 13H, aromatic H); 5.97, (m, 1H, H1 '); 4.25, (m, 1H, H2 '); 4.13, (m, 1H, H3 '); 3.78, (s, 6H, OMe); 3.68, (m, 1H, H4 '); 3.54, (m, 2H, H5, H5-); 3.35 (m, 3H, MeO phosphoramidite group); 3.25, (m, 2H, CH isopropyl groups); 1.60, (m, 3H, CH 3); 1.15, (m, 12H, CH 3 isopropyl groups), 0.86, (s, 9H, ten-butyl), 0.05, (m, 6H, CH 3);
H, Me 2 Si) FAB mass spectrometry 0 NOBA m / z = 852 [M + H] + 1- [2'-O-tert-butyldimethylsilyl-3'-N, N-diisopropylmethoxyphosphoramidite4thio-5'-O -dimethoxytrityl-BD-ribofuranosyU Uracil 26b (diastereoisomeric mixture)
Yield 78%.

31 P NMR (CDCl 3): s 150.65 and 150.50.

1H NMR (CD3CN, 250 MHz): δ 7.99, (s, 1H, NH), 7.22, (m, 1H, H6); 7.30-6.89, (m, 13 H, aromatic H of the DmTr group); 5.90, (m, 1H, H1 '); 5.50, (m, 1H, Hs); 4.13, (m, 2H, H2 ', H3'); 3.80, (s, 6H, OMe from the DmTr group); 3.58, (m, 3H, H4 ', H5',
H5 "); 3.42, (m, 2H, 2 CH isopropyl groups); 3.30 (m, 3H, MeO of the DmTr group); 1.19, (m, 12H, CH3 isopropyl groups); 0.84, (s 9H, tert-butyl) 0.05 (m, 6H, Me 2 Si).

FAB mass spectrometry> NOBA m / z = 534 [M + H-DmTrH] + 1- [2'-O-tert-butyldimethylsilyl-3'-N, N-diisopropylmethoxyphosphoramidite4thio-5'-O-dimethoxytrityl] ss-D-ribofuranosyl] N4benzoyl Cytosine 26c.

Yield 75%.

31 P NMR (CD3CN): δ 151.30 and 150.05
Mass spectrometry FAB> O PEG m / z 941 [M + H] +, 637 [M + H-DmHrH] 303 [D, Tr] +.

1- [2'-O-tert-butyldimethylsilyl-3'-N, N-diisopropylmethoxyphosphoramidite4'thio-5'-O-dimethoxytrityl-ss-D-ribofuranosyl] N6benzoyl Adenine d.

Yield 71%.

31 P NMR (CDCl 3): s, 151.26 and 150.01
Mass spectrometry FAB> O PEG m / z 964 [M + H] +, 600 [M + H-Dm Hr] 303 [DmTrJ +.

 He. 3 Functionalization of the solid support (Diagram 10).

The solid support or LCA-CPG (Long Chain Alkylamine Controlled Pore Glass) P-1 was first activated by a solution of trichloroacetic acid at 3% in dichloromethane at room temperature for 2 to 3 hours in order to release the greater number of amino groups, which leads to the maximum of reactive sites on the surface of the glass beads. The support thus activated P-2 is then functionalized by coupling with succinic anhydride in pyridine in the presence of 4-DMAP, which leads to P-3. In the next step, the 3'-silyl nucleoside is used since it was obtained during the silylation reaction of the thioribonucleosides and is not necessary for the synthesis of oligothionucleotides containing the 3'-5 'phosphodiester linkage. Thus, P-3 activated with 1 (3-dimethylaminopropyl) 3-ethylcarbodiimide (DEC) in the presence of 4-DMAP is reacted in anhydrous pyridine with 5'-O-DmTr-3'-OTBDMS ss-D. -thioribonucleoside 25. The unused sites of the support are masked with piperidine. A last step of masking with acetic anhydride in the presence of collidine in THF leads to P-6. The amount of nucleoside bound to the support is determined by UV spectrophotometry by measuring the amount of dimethoxytrityl cation released after treatment with a solution of
10% trichloroacetic acid in dichloromethane. The degree of functionalization varies from 21 to 38 pmol per gram of support depending on the nature of the nucleoside base.

(i = TCA3%, CH2Cl2, ii = succinic anhydride, DMAP, Pyr, iii = 51o-DrnTr4'-thio-3'o-TBDMS-I3-
D-ribonucleoside, DMAP, NEt3, DEC Pyr .; iv = pentachlorophenol;
v = piperidine; vi = Ac1O 0.5M in THF)
SCHEME 10
II. 4. Automated Synthesis of Thiooligonucleotides (Scheme 11).

Thiooligonucleotides were synthesized on a DNA synthesizer (Applied Biosystems
model 381 A). The elongation cycle has four important steps:
Detritylation of the nucleoside or oligonucleotide attached to the solid support.

 Condensation of tetrazole-activated phosphoramidite on the free 5 'hydroxyl of the nucleoside or oligonucleotide.

 Masking of 5'-hydroxyl functions that have not reacted.

Oxidation of phosphitetriester functions in phosphotriester.

SCHEME 11
Each synthesis was carried out on a column containing 40 mg of solid support. The yield of each incorporation is 98% evaluated by measuring the concentration of the dimethoxytrityl cations recovered after each coupling by treatment of the support with trichloroacetic acid.

Washings are carried out between each step. and the duration of an elongation cycle is 21.1 minutes for the synthesis of the 4'-thio-BD-ribonucleotide homododecamer (BrSU12) (Table 1).

<Tb>

Number <SEP> Step <SEP> Time <SEP> (sec.) <SEP> Solvent <SEP> and <SEP> Reagents
<tb><SEP> 1 <SEP> Dry <SEP> and <SEP> 24 <SEP> MeCN-Argon
<tb><SEP> Wash
<tb><SEP> 2 <SEP> Detritylation <SEP> 80 <SEP> TCA <SEP> to <SEP> 3% <SEP> in <SEP> CH2Cl2 <SEP>
<tb><SEP> 3 <SEP> Dry <SEP> and <SEP> 74 <SEP> MeCN-Argon
<tb><SEP> Wash
<tb><SEP> 4 <SEP> Coupling <SEP> 915 <SEP> amidite <SEP> 0.1M (24eq.) + MeCN <SEP>
<tb><SEP> + tetrazole <SEP> 0.5M (10eq.) + MeCN
<tb><SEP> 5 <SEP> Drying <SEP> 45 <SEP> argon
<tb><SEP> 6 <SEP> Masking <SEP> 23 <SEP> Methyimidazole THF
<tb><SEP> Ac2O / lutidine / THF :: 1/1/8
<tb><SEP> 7 <SEP> Drying <SEP> 11 <SEP> argon
<tb><SEP> 8 <SEP> Oxidation <SEP> 43 <SEP> 12 <SEP><SEP> 0.1M <SEP> in <SEP> THF / Pyr. / H2O <SEP>
<tb><SEP> 40/10/1 <SEP>
<tb><SEP> 9 <SEP> Washing <SEP> 53 <SEP> MeCN
<Tb>
TABLE 1: Elongation cycle used for synthesis of BrSU12.

 II. 5. Stall, deprotection and purification of the thiooligomer.

After having carried out the deprotection steps of methoxyphosphate diesters with thiophenol, the stalling of the solid support dodecamer with ammonia, the deprotection of the hydroxyls with TBAF, the thiooligomer was desalted on a G-25 exclusion column.
DEAE Sephadex where on an A-25 Sephadex DEAE ion column and purified by reverse phase HPLC.

The synthesis of the 4'-thio-oligonucleotides uses the solid support synthesis method in methoxyphosphoroamidite strategy, well known in the oxygenated series.

However, the modified oligomers obtained are quite new to date.

 III. EXPERIMENTAL PART.

Example I: Synthesis of the homododecamer BrSU12.

1-1 Synthesis of the solid support:
The solid support P-1 (2.415 g) is suspended with a solution (50 ml) of 3% trichloroacetic acid in anhydrous CH 2 Cl 2. After 4 h stirring, 50 ml of a solution of triethylamine and diisopropylamine (9: 1) is added and after 5 minutes of stirring the solution is filtered. The solid support P-2 thus obtained is washed with dichloromethane (2 × 50 ml) and then with ether (50 ml) and dried.

The solid support P-2 is then suspended in anhydrous pyridine (14.5 ml) in the presence of succinic anhydride (0.48 g) and DMAP (0.0805 g). The suspension is stirred for 20 hours and then filtered. The solid support P-3 is then washed with anhydrous pyridine (50 ml), dichloromethane (2 x 50 ml) and ether (2 x 50 ml) and then dried under vacuum.

Condensation of the solid support P-3 with 1- [3'-O-tert-butyldimethylsilyl-2'-hydroxy-4'thio-5'-O-dimethoxytrityl-BD-ribofuranosyU Uracil 25b
A mixture of P-3 (2,415 g) 1- [3'-O-tert-butyldimethylsilyl-2'-hydroxy-4'thio-5'-O-dimethoxytrityl-ss-D-ribofuranosyl] Uracil 25b (333 mg, 1 eq) of DMAP (29.5 mm, 0.5 eq) of triethylamine (0.146 mg, 202 l, 0.003 eq.) and DEC (920 mg, 10 eq.) in 29 ml of Anhydrous pyridine is stirred for 3 days at room temperature.

Pentachlorophenol (321 mg, 2.5 eq.) Is then added and the mixture is left stirring for another 24 hours. The solid support is then filtered, rinsed with anhydrous pyridine (2 x 50 ml) then with dichloromethane (2 x 50 ml) and ether (2 x 50 ml).

Immediately afterwards, the solid support P-5 is treated with 25 ml of piperidine. After stirring at room temperature for 24 hours, the solid support is filtered, washed with dichloromethane (2 × 50 ml) and then with ether (2 × 50 ml) and dried under vacuum.

The dry solid support (2.415 g) is placed in the presence of a solution of acetic anhydride (0.5 M) in TE (15 ml) and a solution of collidine (0.5 M) in THF ( 15 ml).

After 4 hours of reaction, the solid support is washed with anhydrous pyridine (2 × 50 ml), dichloromethane (2 × 50 ml), TNF (2 × 50 ml) and ether (2 × 50 ml). ).

P-6 thus obtained is dried under vacuum.

I-2) Determination of the functionalization rate of the solid support P-6.

This determination is carried out by assaying the DmTr groups released in acidic medium.

Exactly 38.0 mg of P-6 are weighed and suspended in 3.4 ml of a solution of para-toluenesulphonic acid (0.1 M) in acetonitrile. The mixture is stirred for 15 minutes and then sonicated for 2 minutes.

The volume is then adjusted to 10 ml with the 0.1 M solution of para-toluenesulfonic acid in acetonitrile. 0.3 ml of this solution is taken to which 5 ml of the 0.1 M solution of para-toluenesulphonic acid is added. Reading the OD (0.317 OD unit) at 500 nm allows us to calculate the degree of functionalization of the support P-6 which in this case is 21 μmol / g of solid support.

 I-3) Automated synthesis of the homododecamer BrSU12.

The synthesis is carried out on a column containing 39.8 mg of solid support functionalized at 21 mol / g of resin, ie 0.835 μmol (1 eq) of 4'-thiouridine.

Each elongation cycle requires an excess of phosphoramidite synthon (20 pmol, 24%), ie 240 μmol in total for the synthesis of the dodecamer. The synthesizer taking 200 μl of a 0.1 M solution of synthon in acetonitrile by incorporation.

The duration of an elongation cycle is 21.1 minutes (Table 1).

The coupling step was considerably increased (900 s against 45 s in the case of a deoxynucleoside) because of the lower reactivity of the ribonucleotide synthon.

The determination of the dimethoxytrityl cations released at each detritylation stage made it possible to evaluate the average efficiency of incorporation of the thioribonucleotide unit at 98.7%.

 I-4) Stall, deprotection and purification of thio homododecamer BrSU12.

The solid support carrying the thio-homododecamer BrSU12 is treated with 5 ml of a solution of thiophenol, triethylamine, dioxane 1, 2 (1/2/2) for half an hour at room temperature.

The solution of thiophenol is then filtered and the support washed with a solution of 32% ammonia in ethanol 95 (3/1: v / v) (3 x 500 l) to unhook the thio-homododecamer from the solid support. The solution thus obtained is evaporated, taken up in 500 ml of water and then freeze-dried.

The oligomer is then dissolved in 300 l of a solution of TBAF (1.1 M) in THF. The reaction mixture is stirred for 24 hours. The reaction is then stopped with 300 μl of an aqueous solution of ammonium acetate (0.05 M).

The solution is evaporated to dryness, coevaporated 3 times with water and the residue chromatographed on Sephadex G-25 exclusion gel column. The fractions containing thiooligomer are pooled, evaporated to dryness and analyzed by HPLC.

Analysis and purification of the thio-oligomer BrSU12 obtained.

HPLC analysis of the thiooligomer was performed on a Beckman C-18 RP 3 IL x column
LODS Ultrasphere under the following gradient conditions:
A: 10% acetonitrile in a 0.05 M aqueous solution of triethylammonium acetate.

B: 15% acetonitrile in a 0.05 M aqueous solution of triethylammonium acetate.

Gradient: A # B # B # A
20 '10' 5 '
Analysis time: 30 '.

Under these conditions, it is found that the thiooligomer is pure at 92.73%.

A purification of BrSU12 on a Nucleosyl semi-preparative column is then carried out with a gradient: A # B # B # A
15 '10' 5 '
Under these conditions, BrSU12 is obtained with a purity of 94.42% sufficient for subsequent studies.

Example II Synthesis of dodecamper B (SrT) 1 dT11.

 11-1) Synthesis of the solid support.

The solid support functionalized at 1 Immole for 35 mg of 2'-deoxythymidine resin as well as the synthons 1- [5'-O-DmTr-3'-ON, N-diisopropylamino cyanoethyl phosphine, 2'-deoxy-BD-ribofuranosyl thymine are marketed by Applied Biosystems.

II-2) Automated synthesis of B (SrT) 1 dT11.

The synthesis is carried out on a column containing 35 mg of solid support, ie 1 immole of 2'-deoxythymidine. Each elongation cycle requires an excess of phosphoramidite synthons (20 mol, 20 eq), ie 220 mol of 2'-deoxythymidine synthons and 20 mol of 4'-thio-thymidine synthon 5. The synthesizer taking up 200 Immoles by incorporation of a 0.1 M solution of each synthon in acetonitrile. The duration of a cycle of incorporation of a deoxythymidine unit is 5.5 minutes. While it is 20.1 minutes for a synthesis cycle of the synthon 4'-thiothymidine.

The increase in the duration of the incorporation cycle of the chimeric synthon is due to the coupling step of 909 'against 36' for the synthon 2'-deoxythymidine.

The determination of the dimethoxytrityl cations released at each detritylation stage made it possible to evaluate the average incorporation efficiency of the 2'-deoxythymidine unit at 98.5% and 93.5% for the synthon 4'-thiothymidine.

II-3) Stall, deprotection and purification of B (SrT) 1 dT11
The solid support carrying oligomer B (SrT) 1 dT11 is treated with a solution of ammonia (32%) in ethanol 95 (3/1, v / v) (3 × 500 μl) for 3 cycles of 20 minutes. each to remove the oligomer from its support, then the dodecamer is incubated for 2 hr in a dry oven in the same mixture to deprotect the cyanoethyl and methoxy functions.

The oligomer is then evaporated under vacuum, taken up in 500 ml of water and freeze-dried.

The deprotection of the TBDMS groups is carried out according to the protocol developed in Example I.

The residue obtained is chromatographed on a gel column of DEAE Sephadex A-25. Fractions containing the oligomer are pooled, evaporated to dryness and analyzed by HPLC.

Analysis and purification of B (SrT) 1 dT11 by HPLC.

HPLC analysis of the oligomer was performed on a Beckman C 18 RP 3 CL column.
Ultrasphere under the following gradient conditions:
A: 6% acetonitrile in a 0.05 M triethylammonium acetate buffer

B: 20% acetonitrile in a 0.05 M triethylammonium acetate buffer

A # B # B
15 '10'
Analysis time: 25 mm.

Under these conditions, the oligomer is 49.3% pure.

Purification of B (SrT) 1 dT11 is then performed by semi-preparative HPLC with a Nucleosyl column under the following gradient conditions: A # B # B
15 '5'
The 12-mer then has a retention time of 12.99 min and a purity higher than 99
Example Lit: Synthesis of dodecamer 8 dT5 (SrT) dT6 III-1) Synthesis of the solid support.

The solid support defined in Example II was used.

III-2) Automated synthesis of B dT5 (SrT) dT6.

The same synthesis conditions developed for the synthesis of B (SrT) 1 dTI1 were used.

The determination of the dimethoxytrityl cations released at each detritylation stage made it possible to evaluate the average incorporation efficiency of the 2'-deoxythymidine unit at 99.3% and at 96.1% for the synthon 4'-thiothymidine.

III-3) Stall and purification of B dT5 (SrT) dT6
The treatment of this oligomer was carried out according to the protocol developed in Example II. HPLC analysis and purification performed under the same gradient conditions revealed a retention time of 13.18 minutes and a spectrophotometric purity at 260 nm of 100.
IV. HYBRIDIZATION PROPERTY of B-4'-thio-oligoribonucleotides
UV spectrophotometry makes it possible to study nucleic acids and to
evidence of duplex formation, with melting temperature being one of the characteristics
a duplex. Stacking and base pairing of nucleic acids are
accompanied by a decrease in UV absorption (Hypochromicity).
UV spectrophotometry, it is possible to control the formation or dissociation of a
duplex. (W.SANGER, Principles of Nucleic Acid Structure, Springer-Verlag, .New
York, 1984, pp. 141-149). When the temperature of a solution containing a double
helix (DNA or RNA) is slowly increased, UV absorption
along the dissociation.The inflection point of the sygmoldal
Absorbance variation as a function of temperature is called melting temperature or
Tm and corresponds to the co-existence of separate strands and paired strands in a 50/50 ratio
Analysis of the melting curves of the duplexes B-4'-thiooligoribonucleotide B-
oligodeoxyrib onucleotide and B-4'-thiooligoribonucleotide / B-oligoribonucleotide
will define the thermal stability of such duplexes by measuring the temperature
fusion. These hybridization experiments are carried out respecting a stoichiometry 1 /
1 between the two complementary strands, in the presence of a 1 M Nazi buffer, 10 mM
Sodium cacodylate.A high concentration of NaCl favors, in fact, the pairings
by solvating the negative charges around the phosphorus atoms of both
oligonucleotide. So we did these hybridization experiments in another
buffer: 0.1 M NaCl, 10 mM sodium cacodylate.

Iv. 1. RESULTS AND DISCUSSION
Examples I and II: BSrU12 / polyrA and BrU12 / polyrA duplex fusion experiments.

These experiments make it possible to highlight the influence of the sulfur atom in
4 'position of the modified dodecamer on the thermal stability of the duplex by comparing the
Tm obtained with those of the natural duplex.

Table 2 summarizes the melting temperatures obtained in the two experiments.

<Tb>

Concentration <SEP> ssSrU12 / polyrA <SEP>'C<SEP> ssrU12 / polrA <SEP>' C
<tb><SEP> in <SEP> NaCl <SEP> Hypochromicity <SEP>% <SEP> Hypochromicity <SEP>%
<tb><SEP> 1M <SEP> 46 C <SEP> 25% <SEP> in <SEP> courses
<tb><SEP> 0.1M <SEP> 33 C <SEP> 25% <SEP> in <SEP> courses
<Tb>
Table 2
Melting temperatures of dodecamer duplexes
Concentration 3 μM in each oligomer.

The analysis of these results shows that the BS rU12 / polyrA duplex exhibits good
thermal stability represented by the Tm of 46 C obtained at a concentration of 1 M
NaCl.

Examples m and IV: BSrU12 / ssdC2A12C2 duplex fusion experiments and
ssrU12 / ssdC2A12C2.

The melting temperatures obtained in the two experiments are summarized in
table 3.

<Tb>

Concentration <SEP> ssSrU12 / ssdC2A12C2 <SEP>'C<SEP> ssrU12 / ssdC2A12C2 <SEP>' C
<tb><SEP> in <SEP> NaCl <SEP> Hypochromicity <SEP>% <SEP> Hypochromicity <SEP>%
<tb><SEP> 1M <SEP> 27 C <SEP> 9.3% <SEP> in <SEP> courses
<tb><SEP> 0.1M <SEP> 5 C <SEP> 5.6% <SEP> in <SEP> courses
<Tb>
Table 3
Melting temperatures of dodecamer duplexes
Concentration 3, μM in each oligomer
Example IV: Autohybridization experiment of the ssSrU12 oligomer

In order to verify the previous results, it is necessary to ensure that the dodecamer
ssSrU12 does not lead to any autohybridization. For this, we carried out an experiment
BSrU12 dodecamer auto-hybridization at two NaCl concentrations (1 M, and 0.1
M). Hybridization curves do not have the typical sigmoidal appearance but
obey the equation A = k + T where A is the absorbance at 260 nm, T the temperature and k
a constant. No inflection point can be visualized on such a line. We
We can therefore conclude that the dodecamer BSrU12 does not self-match.

Comparison of BSrU12 / ssdC2A12C2 duplex melting temperatures (Table
2) and ssSrU12 / polyrA (Table 3) shows a thermal stability of the homoduplew
RNA / RNA (Tm = 46 C) significantly higher than that of heteroduplex
RNA / DNA (Tm = 27c), on the other hand, no self-hybridization of BSrU12 was
noted.

Example VI and VII: Fusion Experiment of BdT5 (SrT) duplexes dT6 / BdC2A12C2 and ssdT12 / ssdC2A12C2
This experiment makes it possible to evaluate the thermal stability of the duplex ssdT5 (SrT) dT6 /
BdC2A12C2 compared to that of the natural duplex BdT12 / BdC2A12C2 (Figure 1).

The melting temperatures of these duplexes are indicated in
Table 4.

<Tb>

Concentration <SEP> ssdTs (SrT) dT6 <SEP> / <SEP> BdC2Al2C2 <SEP> C <SEP> ssdT12 / <SEP> BdC2A12C2 <SEP> C <SEP>
<tb><SEP> in <SEP> NaCI <SEP> Hyperchromicity <SEP>% <SEP> Hyperchromicity <SEP>% <SEP>
<tb><SEP> 1M <SEP> 40 <SEP> C <SEP> 18 <SEP>% <SEP> 45C <SEP> 20.5 <SEP>% <SEP>
<Tb>
Table 4
BdT5 duplex temperature (SrT) dT6 / BdC2Al2C2
and ssdT12 / ssdC2A12C2
Concentration in each oligomer 10, tM
In view of these results, it appears that the thermal stability of the modified duplex is lower than that of the natural duplex. However, the 5 (C) difference between the two melting temperatures is too small to conclude that there is a mismatch, because some authors (Y.KAWASE, SIWAI, H.INOUE, K.MIURA and E.OKTSUKA, Nucleic Acid
Research, 1986, 14, 7727) have shown that the existence of a single mismatch in a duplex of 13 nucleotide units results in a decrease in Tm greater than
C.

It can therefore be concluded that the 4'-thio-nucleotide unit pairs well with the 2 '
deoxyadenosine but with a lower affinity than 2'-deoxythymidine
as confirmed by the difference in Tm of 5 ° C obtained (Table 4).

IV. 2. EXPERIMENTAL PART
General Method for Hybridization Experiments
A - Determination of complementary oligomers.

In order to perform the hybridization experiment, it is necessary to know the concentration in each oligomer to respect the 1: 1 stoichiometry between the two complementary strands. According to the BEER-LAMBERT law A = elc, the absorbances of the oligomers were measured at 80 ° C in order to eliminate the stacking phenomenon of the bases creating a disturbance of the hyperchromicity due to the existence of tertiary structures. It is therefore possible to know the molecular extinction coefficients of the dodecamers at 80 C according to the relation e265 (SrU12) = 12 and 265 (rU).

B - Hybridization conditions.

Hybridization experiments are performed with a UVIKON 810 spectrophotometer (KONTRON), coupled to a compatible IBM microcomputer. The temperature is controlled by a HUBER PD 415 temperature programmer connected to a thermostated bath (HUMER MINISTAT). The UV cuvettes are 1 cm long quartz and a continuous circulation of nitrogen is used for temperatures below 20 e Ce Before the experiment, 3 tLM (Examples I to V) or 10 M (Examples VI and VII) Additional oligomers are combined in a sufficient amount per 1000 μl 1M NaCl buffer and 10 mM sodium cacodylate. adjusted to pH 7 (Examples I-VII). This mixture is heated at 80 ° C for 1 hour, then cooled to -20 ° C at a temperature gradient of 0.5 ° C per minute. Absorbance and temperature values are collected every 30 seconds.

The same experiment is carried out using a sufficient amount for 1000 μl of 0.1 M NaCl hybridization buffer, 10 mM sodium cacodylate adjusted to pH 7 (Examples
I and V) in order to evaluate the thermal stability of the duplex in this buffer, which favors less the pairings
EXAMPLE I Thermal Stability of BSrU12 / PolyrA Duplex II Oligomer Assay
Concentration of BSrU12 = 0.351mM
Concentration of polyrA = 4.61 mM 1-2 Hybridization conditions 3 μM (8.55 l) of ssSrU12 and 3 M (7.79 l) of polyrA are combined with 983.6 l of hybridization buffers (1 M NaCl, 10 mM sodium cacodylate or 0.1 M NaCl, 10 mM sodium cacodylate)
Example II: Thermal stability of the duplex ssrU12 / polyrA in progress
Example m: Thermal stability of BSrU12 / BdC2A12C2 duplex
III-1 Determination of oligomers
Concentration of BSrU12 = 0.351 mM
Concentration of ssdC2A12C2 = 0.369 mM
III-2 Hybridization Conditions 3 M (8.55 l) of ssSrU12 and 3 M (8.11 l) of ssdC2A12C2 are brought into contact with 983.3% of the hybridization buffers (1 M NaCl, 10 mM cacodylate sodium or 0.1M
NaCl, 10 mM sodium cacodylate)
Example IV: Thermal stability of duplex BrU12 / BdC2A12C2 (in progress)
IV-1 Determination of oligomers
BrU12 concentration =
Concentration of BdC2A12C2 = 0.369 mM 111-2 Hybridization Conditions 3 μM (4) of ssrU12 and 3 M (8.11 μl) of BdC2A12C2 are brought into contact with 4 of the hybridization buffers (1 M NaCl, 10 mM cacodylate sodium or 0.1 M NaCl, 10 mM sodium cacodylate)
Example V: Autohybridization experiment of ssSrU12 oligomer
V-1 Determination of oligomers
Concentration of ssSrU12 = 0.351 mM
V-2 Hybridization conditions
6 μM (17.10 μl) of ssSrU12 are exposed to 986.9 l of hybridization buffers
(1 M NaCl, 10 mM sodium cacodylate or 0.1 M NaCl, 10 mM sodium cacodylate)
Example VI: Thermal Stability of the BdT5 (SrT) duplex dT6 / ssdC2A12C2
VI-1 Determination of oligomers
SsdT concentration (SrT) dT6 = 1.33 mM
Concentration of ssdC2A12C2 = 0.369 mM
VI-2 Hybridization Conditions 10 μM (7.49 l) of BdTs (SrT) dT6 and 10 m (22.5 l) of ssdC2A12C2 are placed in the presence of 970 μl of 1M NaCl hybridization buffer, 10 mM cacodylate. sodium.

Example VII: Thermal stability of the duplex BdT12 / BdC2A12C2
VII-1 Determination of oligomers
Concentration of ssdT12 = 1.196 mM
Concentration of ssdC2A12C2 = 0.369 mM
VII-2 Hybridization Conditions 10 g (8.36 l) of BdT12 and 10 μM (22.5 l) of BdC2A12C2 are placed in the presence of 969.1 l of 1 M NaCl hybridization buffer, 10 mM cacodylate. sodium
V. ENZYMATIC STUDIES
The recognition of nucleic acids by enzymes is largely structural. The replacement of the natural heteroatom of the sugar of an oligonucleotide confers to it a strong resistance to degradation by nucleases.

The study of the hydrolysis of 4'-thio-oligoribonucleotides B by these nucleases confirms that the replacement of intracyclic oxygen with a sulfur atom confers a better enzymatic stability.

Example I: Study of the enzymatic degradation of dodecamer B (SrT) dT11 by phosphodiesterase of calf's spleen.

The phosphodiesterase of calf's spleen. is an exonuclease that degrades oligomers from their 5 'free ends thus releasing nucleotide 3' phosphate.

This study will allow us to evaluate the resistance to enzymatic degradation induced by the presence in the 5 'position of a 4'-thionucleotide unit in oligomer B (SrT) dT11 compared with the enzymatic degradation of the natural oxygenated homologue BdT12
The comparative stability of the two dodecamers BdT12 and B (SrT) dT11 towards phosphodiesterase hydrolysis of calf spleen is shown in Table 5.

The difference in behavior between the two dodecamers is important, indeed only 23% of the oligomer B (SrT) dT11 was hydrolysed in 25 minutes whereas in the same period of time the 12-mer BdT12 was degraded to 93%

<tb> .Dodecamer <SEP> Degradation <SEP> at <SEP> 0 <SEP> min <SEP> Degradation <SEP> at <SEP> 25 <SEP> min
<tb><SEP> BdT12 <SEP> 0% <SEP> 93 <SEP>% <SEP>
<tb> B (SrT) dT11 <SEP> 0% <SEP> 23% <SEP>
<Tb>
Table 5
Kinetics of degradation of BdT12 and B (SrT) dT11 by phosphodiesterase of calf spleen
The evolution over time of the enzymatic degradation of the two dodecamers was followed by HPLC. We have been able to trace the enzymatic digestion curve of B (SrT) dT11 as a function of time: A = Ao -kt (Figure 2, A) where: -Ao is the initial area of 12-mer -A is the area from 12-sea to instant t
-t is the time
-k is the first order rate constant of the degradation
This curve (Figure 2) allows to calculate, using the regression software
curvilinear EUREKA, k = 0, .016min ~ 1 and the half-life time of the dodecamer tl / 2 = In
2 / k = 43 min
In the same way, the enzymatic digestion curve of BdT12 as a function of time: A = Ao could be plotted. (Figure 13). It is noted that the kinetics of degradation of
BdT12 is much faster than B (SrT) dT11; indeed the half-life time is 1 minute for the natural dodecamer.

The kinetics of enzymatic degradation of the modified oligomer B (SrT) dT11 by the 5 'end has a half-life time much greater than that of BSrU12 degradation. These results show that the introduction of a thioribonucleotide unit at the 5 'end of an oligomer stabilizes the latter with respect to the phosphodiesterase of calf spleen.

EXAMPLE 1 Study of Enzymatic Degradation of Dodecamer B (SrT) dT11 by Calf Spleen Phosphodiesterase

An oligonucleotide solution (4.2.3 absorbance units at 260 nm) is diluted in 0.125 M ammonium acetate buffer (pH 7.0), 2.5 mM EDTA buffer and 0.0625% Tween 80 buffer. (80 μL). A commercial solution of phosphodiesterase from calf's spleen (2 4, final concentration 0.008 units / ml) is added and the mixture is incubated at 37 ° C. At specified times, aliquots are taken (5 4), heated to 100 ° C. C for one minute and analyzed by HPLC.

HPLC analysis conditions.

The analyzes were carried out on a PVDI column (Polyvinylimidazole SPCC-Shandon) protected by a precolumn and a prefilter. The elution is carried out according to a linear gradient in 20 minutes from 0.1 M to 0.4 M KCl in 20 mM KH 2 PO 4 at pH 6 containing 20% acetonitrile. The flow rate was set at 1 ml / min and U.V. detection at 260 nm.

Claims (21)

 1. Chemical compounds consisting of an oligo-4'-thio-2'desoxyribonucleotide or an oligo 4'-thioribonucleotide, characterized in that they comprise a sequence of 4'-thio-2'deoxyribonucleotides or 4'-thioribonucleotides respectively, linkage optionally being linked to an effector agent, in particular a radical corresponding to an intercalation agent or a chemical or photoactivatable radical, such as a radical carrying a function reacting directly or indirectly with the nucleotide chains or a radical whose the presence allows easy and sensitive detection.  2. Chemical compounds according to claim 1 consisting of an oligo 4'-thioribonucleotide or an oligo 4'-thio-2'-deoxyribonucleotide, characterized in that they comprise a sequence of 4-thioribonucleotides or 4-thio-2'- deoxyribonucleotides respectively not bound to an effector radical.  3. Compounds according to claim 1 or 2 characterized in that they have the formula

 in which  the radicals B may be identical or different and each represent a base of an optionally modified, activatable nucleic acid and / or having a group intercalating and attached to the glycoside ring in an unnatural alpha anomeric configuration  the radicals X may be identical or different and each represents an oxoanion O, a thionanion S, an alkyl group, an alkoxy group, a thioalkyl group, an alkyl or alkoxy radical substituted by a nitrogen heterocycle or a group -Y-Z  R and R ', which may be identical or different, each represent a hydrogen atom or a group -Y-Z or Y'-Z'  Y and Y ', which are identical or different, each represent a straight or branched alkylene radical -alk- or a radical chosen from

 alk-O-alk

 with U = O, S or N  E can have the same meaning as X except Y-Z or Y'-Z ';  J represents a hydrogen atom or a hydroxy group;  Z and Z ', which are identical or different, each represent a radical corresponding to an effector agent, in particular a radical corresponding to an intercalating agent or a radical carrying a function which reacts directly or indirectly with the nucleotide chains or a radical whose presence allows easy and sensitive detection.  L represents an oxygen atom, a sulfur atom or an -NH- group.  n is an integer including 0  4. Compounds according to claim 3, characterized in that Y and Y 'represent a radical chosen from

 with U = O, S or N  5. Compounds according to claim 3, characterized in that L represents oxygen, R and R 'represent a hydrogen atom and B represents a natural nucleic base.  6. New derivative according to one of claims 3 to 5, characterized in that the intercalation agent Z or Z 'is selected from polycyclic compounds having a planar configuration.  7. New derivative according to claim 6, characterized in that the intercalation agent Z or Z 'is chosen from acridine and its derivatives, furocoumarin and its derivatives, daunomycin and other derivatives of anthracycline, l, 10-phenanthroline, phenanthridine and its derivatives, proflavine, porphyrins, dipyrido [1,2-a: 3'-d] imidazole derivatives, ellipticine or ellipticinium and their derivatives and diazapyrene and derivatives.  8. New derivative according to one of claims 3 to 5, characterized in that the reactive chemical groups Z or Z 'are chosen from ethylene-diamine tetraacetic acid, diethylenetriaminepentaacetic acid, porphyrins, l, 10 -phenanthroline, 4-azidoacetophenone, ethyleneimine, beta-chloroethylamine, psoralen and their derivatives.  9. New derivative according to one of claims 3 to 8, characterized in that the radical B is a radical corresponding to the thymine, adenine; 2-aminoadenine and its substituted derivatives, cytosine, guanine and its substituted derivatives, uracil, 5-bromo-uracil, 8-azadenaden, 7-deazaadenine, 7-deazaguanine, 4-azido -cytosine, 4-azido-thymine and derivatives of these bases having an intercalating group and / or a chemically or photochemically activatable group.  10. Compounds according to one of claims 1 to 9, characterized in that they are oligothioribonucleotide compounds of formula I for which J = OH.  11. Preparation process or oligothionucleotide, according to one of claims 3 to 10, characterized in that syntheses are applied to phosphodiester, phosphotriester, phosphoramidite or hydrogenophosphonate starting from 4thionucleosides and in that they are prepared completely protected derivatives and that the protective groups are eliminated.  12. Process for synthesizing compounds without an effector agent according to one of Claims 2 to 10, characterized in that a synthesis supported by the phospohoroamidite method comprising a 3 'and 5' protection of the 4 'thionucleotides or trace 4'thionucléotides  starting material with for example dimethoxytrityl 5 'and diisopropyla  3 'methyl minophosphoramidite, functionalization of a solid support incorporating a 4'thio derivative  nucleoside by a bond for example succinyl between the grouping  hydroxyl-3 'of the 4'-thionucleoside derivative and an amino group of  solid support, - the elongation of the oligothionucleotide chain in a reactor  synthesizer, - finally the stall, the deprotection and the purification of the oligothio  extended nucleotide.  13. A method for synthesizing compounds incorporating an effector agent according to one of claims 3 to 10, characterized in that one starts from an oligothionucleotide without 5 'protected effector agent, which is reacted with the 3' OH-terminus. with an 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. The method of claim 13, characterized in that the 3 'OH terminus of the 5' protected oligomer is esterified with an aminohexanoic acid, the amine function is protected.  15. Application of the compounds according to one of claims 3 to 10, characterized in that said compounds are used as hybridization probes or as purification components for specific sequences of DNA or RNA in single strand or double helix for diagnostic or prognostic purpose.  16. Application of the compounds according to one of claims 3 to 10, characterized in that the compounds are used to detect mutations at a DNA or an RNA.  17. Application of the compounds according to one of claims 3 to 10, characterized in that these compounds are used to carry out specific blocking of previously selected cell genes or pathogens, such as viruses, bacteria or parasites.  18. Application of a derivative according to one of claims 3 to 10 for selectively blocking the expression of a gene either by hybridization, by selective cleavage, or by chemical modification of the gene or its Messenger RNA of cellular, viral, bacterial or parasitic origin.  19. Application of a derivative according to one of claims 3 to 10 for selectively blocking the expression of a viral RNA either by hybridization, by cleavage or by chemical modification of the viral ART or messenger RNA.  20. Application of the compounds according to one of claims 3 to 10, characterized in that they can be used as artificial nucleases specific sequences.  21. As medicaments, compounds according to one of claims 3 to 10, used as antiviral, antitumor, antibiotic or antiparasitic or in any pathology where a gene is abnormally expressed either by mutation or by deregulation.