METHOD FOR SYNTHESISING MACROMOLECULES IN SOLUTION FROM CARBOHYDRATE DERIVATIVE UNITS

Abstract

The invention relates to a method for synthesising macromolecules made up of units U that are mostly monosaccharides or monosaccharide derivatives, by successive elongation of a first unit U1 attached by a covalent bond to an anchor molecule soluble in organic solvents. The elongation takes place by coupling with a monomer or oligomer M having at least two functions. The method is characterised in that the anchor molecule comprises a polyolefin chain or a polyolefin oligomer or a polyalkene, with at least 5 monomer units, and preferably between 10 and 50 monomer units, the polyolefin chain preferably being a polyisobutene chain.

Claims

1. A method for synthesizing macromolecules made up of units U that are mostly monosaccharides or monosaccharide derivatives, which may be identical or different, said macromolecules having a first and a second end, said synthesis method proceeding by successive elongation of said second end by a monomer or oligomer M having at least two functions, and said method being characterized in that: in a first so-called anchoring step, the first unit U1 of said macromolecule, corresponding to a monomer M1 or a terminal unit of an oligomer M1, is attached by a covalent bond to an anchor molecule soluble in organic solvents, said covalent bond resulting from a reaction of a first of the functions of said monomer M1 or of said oligomer M1 with a function of said anchor molecule, the second termination possibly being another function of said monomer M1 or of said oligomer M1 that has been protected, prior to said reaction, by at least a first protecting group GP1, and possibly by a second protecting group GP2; in a second so-called deprotection step, one of said protecting groups GP1 or GP2 is removed, leaving a so-called free function on said monomer M1 or oligomer M1; in a third so-called coupling step, a second monomer M2 or oligomer M2, carrying at least one free function and at least one function protected by a protecting group GP3, is reacted so that its free function forms, by reaction with said free function of said first monomer M1 or oligomer M1, a covalent bond, thus creating a new molecule formed by said monomer M1 or oligomer M1, attached by its first end to said anchor molecule, and said monomer M2 or oligomer M2, attached to another of its terminations, and said method being characterized in that said anchor molecule comprises a polyolefin chain or a polyolefin oligomer or a polyalkene, with at least 5 monomer units, and preferably between 10 and 50 monomer units, said polyolefin chain being a branched chain, and preferably a polyisobutene chain.

2. The method according to claim 1, characterized in that an n-th monomer Mn or oligomer Mn is added by coupling.

3. The method according to claim 2, characterized in that after coupling the last monomer or oligomer, deprotection of the protecting groups of the macromolecule is carried out.

4. The method according to claim 3, comprising at least one step in which said macromolecule attached to said anchor molecule is separated from the reaction medium by extraction in an apolar organic solvent, and/or by extraction or washing with a polar solvent, and/or by filtration.

5. The method according to claim 4, comprising a step in which said macromolecule is fully deprotected.

6. The method according to claim 5, characterized in that said monosaccharide units or monosaccharide derivatives are pentose (and in particular nucleoside) or hexose derivatives.

7. The method according to claim 6, characterized in that the bond between two successive units is a bond of the osidic type (and preferably of the glycosidic type or of the phosphorylated type (in particular of the ose-1-phosphate type)) or of the carbohydrate type or of the N-heteroside type or of the S-heteroside type.

8. The method according to claim 7, characterized in that said anchor molecule has a mass average molecular mass between 300 and 20,000, and preferably between 500 and 15,000.

9. The method according to claim 8, characterized in that said polyolefin or polyolefin oligomer or polyalkene chain comprises a number of unsaturated carbon-carbon bonds not exceeding 5%, and preferably not exceeding 3%.

10. The method according to claim 9, characterized in that said polyolefin chain or polyolefin oligomer or polyalkene has been obtained by polymerization of a preferably biosourced monomer.

11. A use of a method according to claim 10, for synthesizing oligonucleotides or oligosaccharides.

Description

DETAILED DESCRIPTION

[0088] In the present invention, the terms below are used with the following meaning, which is in accordance with the terminology of the International Union of Pure and Applied Chemistry (IUPAC), and any other terms used should also be understood as defined by IUPAC.

[0089] The term “carbohydrate” comprises monosaccharides, oligosaccharides and polysaccharides as well as compounds derived from monosaccharides by reduction of the carbonyl function (especially the aldehyde or ketone function), by oxidation of at least one functional group at the end of the chain to a carboxylic acid, or by replacement of one or more hydroxyl groups with a hydrogen atom, an amino group, a thiol group or with any similar atom. This term also includes derivatives of such compounds.

[0090] The term “monosaccharide” refers to the monomer of carbohydrates.

[0091] The term “glycosylamine” means a compound in which a carbohydrate is linked to an amine group in an anomeric position.

[0092] The term “nucleoside” refers to a ribosyl or deoxyribosyl derivative of some pyrimidine or purine bases, more precisely glycosylamines consisting of a nucleobase linked to the anomeric carbon atom of a pentose residue, generally ribose (ribonucleoside) or deoxyribose (deoxyribonucleoside), by a glycosidic linkage from the N.sup.1 atom of a pyrimidine or the N.sup.9 atom of a purine.

[0093] The term “nucleotide” refers to an organic molecule that is the building block of a nucleic acid such as DNA or RNA; this molecule is made up of a nucleic base, a five-carbon ose, and finally one to three phosphate groups.

[0094] The term “protecting group” refers to a molecule used for reversible protection of a functional group during a chemical reaction to make said functional group non-reactive in said chemical reaction process which would have transformed said unprotected functional group.

[0095] The term “functional group” means an atom or group of atoms that can react with other functional groups. Examples of functional groups are the following: aldehyde, carboxylic acid, phosphoric acid, phosphonic acid, sulfonic acid, primary or secondary amine, ketone, alkyl halide, hydrazine, hydroxylamine, hydroxyl, isocyanate, isothiocyanate, thiol.

[0096] The term “macromolecule” refers to a high molecular mass molecule designed from a succession of low molecular mass units. These units can be linked individually and successively to form a chain; an oligomer comprising several of these units can also be coupled. Generally speaking, these units can be identical or different. The macromolecules can be of synthetic or biological origin or a combination of both. They may comprise one or more functional groups. The term “macromolecule” as used herein includes polymers; in the case of a polymer, said unit is called a monomer.

[0097] The term “polymer” refers to a macromolecule comprising repeating structural units, namely monomers, connected by chemical bonds in a linear, circular, branched, cross-linked, dendrimeric manner or a combination thereof. It is understood that a polymer may, for example, also comprise at least one functional group. A polymer is called a “homopolymer” if the polymer is made up of the same monomers and is called a “copolymer” if the polymer is made up of different monomers.

[0098] According to one characteristic of the invention, which will be described in greater detail below, the anchor molecules or solubilizing protecting groups or anchor matrix are polyolefins, or more precisely polyolefin oligomers (polyolefins being also called polyalkenes) and derivatives thereof, that is they carry at least one functional group.

[0099] In one preferred embodiment, the method according to the invention uses polyolefins, or more precisely oligomers of polyolefins (polyolefins being also called polyalkenes), and their derivatives as anchor molecule or protecting group or anchor matrix, of several functional groups of various monomers, at least monofunctional, linked by a covalent bond (ester, amide, ether, thioether or any other suitable chemical functions), making the new monomeric derivative soluble in apolar liquid phase. Polyolefin molecules comprise a chain of carbon atoms linked by single bonds. They may include branches consisting of identical or different, but preferably identical, alkyl groups. Preferably, the polymers consist of a number of monomer units of at least 10 and preferably between 15 and 50. Homopolymers are preferred, but copolymers (saturated or unsaturated) can be used. In the case of unsaturated polymers or copolymers, the number of unsaturated bonds in the chain of carbon atoms advantageously does not exceed 5%, and preferably does not exceed 3%.

[0100] In one preferred embodiment, these are derivatives of polyisobutenes (PIBs), a class of polymers known since the 1930s, but derivatives of polypropylenes can also be used.

[0101] These anchor molecules employed in the method according to the invention are preferably in the form of functionalized derivatives. The preceding Schemes 4 and 5 show a number of PIB derivatives with their functionalizations that are suitable for carrying out the present invention.

[0102] According to one characteristic of the invention, these anchor molecules are linked to a monomer (or oligomer) unit, by a covalent bond such as amide, ether, thioether, thioester ester, sulfonylhydrazide or acylhydrazide (non-exhaustive list). This assumes that the PIB derivatives involved are suitably functionalized. This functionalization of the anchor molecules is as a general rule in the terminal position, namely preferably at one of the ends of the carbon atom chain.

[0103] According to the invention, the multifunctional monomers (or oligomers) can be functionalized with PIB derivatives via a covalent bond, in the form of ester, ether, thioether, thioester or any other chemical functions compatible with the present method. This acts as a solubilizing protecting group for the monomers (or oligomers).

[0104] Polyolefin oligomers used as anchor molecules are typically characterized by a mass average molecular mass, but “pure” oligomers that have identical molecules of a given chain length can also be used.

[0105] The reaction between the anchor molecule and the monomer (or oligomer) leads to a new molecule with a low water solubility (< 30 mg/mL).

[0106] According to another characteristic of the invention, PIB functionalization, by chemical reactions, leads to various molecular structures, capable of acting as solubilizing protecting groups, of an at least bifunctional molecule (or intermediate) of interest, during a multi-step synthesis. It is implied that the protecting group is also at least monofunctional. Otherwise, the chemical function not involved in the linkage between the PIB derivative and the molecule (or intermediate) of interest have to be inert or suitably protected, to avoid any spurious products or side reactions.

[0107] According to another characteristic of the invention, chemical functions of the monomer (or oligomer) not involved in the covalent bond with the PIB derivative, directly or not, have to be passive or suitably protected, to avoid formation of undesirable products.

[0108] In a further aspect of the invention, the molecule resulting from the reaction between the PIB derivative and a monomer (or oligomer), via the formation of a covalent bond, directly or not, is characterized in that it has a low solubility in water (< 30 mg/mL). Stated differently, the PIB derivative acts as a solubilizing molecule.

[0109] According to another characteristic of the invention, the molecule resulting from the reaction between the PIB derivative and a monomer (or oligomer), via the formation of a covalent bond, directly or not, is characterized in that the PIB derivative significantly increases the solubility of the monomer (or oligomer) in apolar solvents (cyclohexane, heptane(s), hexane(s) or aromatic solvents) or any other suitable solvent. Thus, the new monomeric (or oligomeric) derivative has a selective solubility (a high partition coefficient) for an apolar solvent during a liquid-liquid extraction (in the presence of water or a water/ethanol or water/acetonitrile mixture), making the purification method simple, fast and cheap.

[0110] The protection reaction between the PIB derivative and a monomer (or oligomer), via the formation of a covalent bond, directly or indirectly (spacer), is carried out in any solvent or inert liquid that can dissolve the reactants, at an appropriate temperature. Applicable solvents, pure or as mixtures, include, but are not limited to, halogenated or non-halogenated hydrocarbons. Preferred solvents are dichloromethane and toluene (alone or in the presence of N-N-dimethylformamide).

[0111] Depending on the free chemical functions of the monomer (or oligomer) and the anchor molecule, various chemical reactions are possible. The formation of the new monomer or oligomer derivative can therefore be carried out according to all methods known to the skilled person. By way of non-exhaustive examples, applicable reactions include esterification reactions, amidation reactions or etherification reactions. Consequently, the reaction conditions (solvent(s), temperature(s), concentration(s), duration(s)) have to be adapted for each protection reaction.

[0112] Depending on the chemical nature of the bond between the monomer (or oligomer) and the anchor molecule, the deprotection steps can be carried out using reaction conditions known to the person skilled in the art. Without being exhaustive, saponification, hydrolysis and hydrogenolysis can be mentioned. More precisely, the method for solubilizing a suitably protected and anchored monomer (or oligomer) via a covalent bond according to the invention is characterized in that it is solubilized in an organic solvent. Stated differently, the anchor molecule acts as a solubilizing molecule and a protecting group of a chemical function of the monomer (or oligomer).

[0113] The monomer (or oligomer), suitably protected and covalently bound to an anchor molecule (of various kinds), is characterized in that its solubility in water is low (< 30 mg/mL). Stated differently, the anchor matrix acts as a solubilizing entity. This derivatization significantly increases the solubility of the new molecule to the point that it becomes soluble in apolar organic solvents. Consequently, monomers (or oligomers) anchored to a PIB derivative have a high partition coefficient (selective solubility (or selective distribution)) for the apolar organic phase during a liquid-liquid extraction in the presence of cyclohexane or heptane(s) or hexane(s) and water or a water/ethanol or water/acetonitrile mixture, allowing a simple and fast purification.

[0114] The present invention opens the possibility of convergent synthesis of long oligomers, which can be achieved by using at least two suitably protected oligomer fragments, at least one of which is bound to an anchor molecule.

[0115] Reaction scheme 6 below shows a complete sequence for producing an oligonucleotide. More precisely, in a first so-called anchoring step, a first monomer unit (in this case a deoxyribose derivative) protected by a protecting group (in this case DMTr) is attached to a liquid support molecule according to the invention; the product obtained is purified by liquid-liquid extraction. In a second so-called deprotection step, the protecting group (DMTr) is removed and scavenged in an acidic medium in the presence of a scavenger and the product obtained is purified by liquid-liquid extraction. It is preferable to perform anchoring and deprotection (with scavenging) without isolation of the intermediate protected alcohol.

##STR00006##

Scheme 6: Synthesis of Oligonucleotides

[0116] After the deprotection step, the protecting groups can be derivatized, preferably in situ, to form compounds soluble in a polar solvent (or mixture thereof). Thus, the monomer (or oligomer) anchored to a PIB derivative whose primary alcohol is protected by a trityl group is cleaved in an acidic medium in the presence of a scavenger of the corresponding carbocation (trityl) for making it soluble in the aqueous (or polar) phase. Scavengers for the trityl carbocation can advantageously be selected from the group consisting of: thioglycolic acid, 3-mercaptoproprionic acid, 3-mercapto-1-propanesulfonic acid, cysteine, thiomalic acid, mercaptosuccinic acid. An example of this deprotection is represented in Scheme 7 below.

##STR00007##

Scheme 7: Deprotection of a DMTr Protecting Group

[0117] In a third so-called coupling /oxidation (sulfurization) step, a second monomer unit (in this case a phosphorylated ribose derivative) protected by a protecting group (in this case DMTr) is introduced. In the case of phosphoramidite chemistry, the coupling reaction is carried out in the presence of tetrazole or any other appropriate reagents (benzylthio-1H-tetrazole (BTT), 4,5-dicyanoimidazole), followed by an oxidation reaction (metachloroperbenzoic acid (mCPBA), iodine, 2-butanone peroxide). A dinucleotide is thus obtained; the reagents and side products are separated by extraction.

[0118] In a fourth step called general deprotection, this dinucleotide, which is still bound to the liquid support molecule according to the invention, can be deprotected and then detached from this support. Alternatively, it can enter a new cycle for the addition of a third unit, and so on.

[0119] Reaction scheme 8 below shows a complete sequence for producing an oligosaccharide. More precisely, in a first so-called anchoring step, a first monomer unit (in this case a hexose derivative) protected or not by a first, stronger protecting group (GP) and by a second, more labile protective or not protecting group, called temporary (GPt) is attached to a liquid support molecule on the anomeric position according to the invention; this compound is purified by extraction. In a second so-called selective deprotection step, said second protecting group (GPt) is removed, and the compound is purified by extraction. In a third so-called glycosylation step, a second monomer unit (in this case a protected or unprotected donor glycosyl derivative) is added and coupled to said first unit to form an anchored disaccharide; the reactants and by-products are removed by liquid-liquid extraction. In a fourth so-called overall deprotection step, this disaccharide is deprotected and then separated from the liquid support. Alternatively, it can enter a new cycle, after selective deprotection of a functional group, if necessary, to initiate a new cycle, and so on.

##STR00008##

Scheme 8: Synthesis of Oligosaccharides

[0120] Among anchor molecules, those that are able to be manufactured by a polymerization technique from simple monomers are preferred. This is the case of polyisobutenes (PIBs), which represent one particularly preferred type of anchor molecules. The monomer of polyisobutenes, namely isobutene, can be industrially manufactured from biosourced feedstocks, and PIBs can be prepared from the biosourced isobutene by simple polymerization. Thus, the present invention can be implemented with biosourced anchor molecules, and in particular with biosourced PIB.

[0121] The concept of biosourced content is defined in ISO 16620-1:2015 “Plastics - Biosourced Content - Part 1: General Principles”, including a definition of the terms “biosourced synthetic polymer”, “biosourced synthetic polymer content”, “biosourced carbon content” and “biosourced mass content”, as well as in ISO 16620-2:2015 “Plastics - Biobased Content - Part 2: Determination of Biobased Carbon Content” and ISO 16620-3:2015 “Plastics - Biosourced Content - Part 3: Determination of Biobased Synthetic Polymer Content”, for methods of determining and quantifying biosourced nature.

[0122] Advantageously, anchor molecules used in the present invention have a biosourced carbon content greater than 90%, preferably greater than 93%, and even more preferably greater than 95%.

[0123] The method according to the invention has many advantages.

[0124] A first advantage is that it allows to obtain matrix-bound oligomers with good purity by simple liquid-liquid extraction in an apolar organic solvent and water or a water/ethanol or water/acetonitrile mixture or by filtration on silica, causing the removal of by-products (salts, excess reagents or any other molecular species) that are not bound to the polyolefin or polyalkene oligomer derivatives. Apolar organic solvents such as cyclohexane, heptane(s), hexane(s) which have flash points < 15° C., are appropriate for solubilizing the polyolefin or polyolefin oligomer or polyalkene derivatives during extraction or washing. The method according to the invention thus facilitates purification steps and produces less waste (effluents and stationary phase).

[0125] A second, particularly interesting, advantage is the possibility of automating the method according to the invention.

[0126] A third advantage is the possibility to recycle extraction solvents as well as anchor molecules (polyolefins or oligomers of polyolefins or polyalkenes), especially on an industrial scale. Indeed, these protecting groups can be easily removed at the end of the synthesis by reactions usually used in organic synthesis (such as hydrolysis, saponification, hydrogenolysis or any other reaction compatible with the present method) and recycled. This proves that the method according to the invention is in line with green or sustainable chemistry, contrary to current production methods.

[0127] A fourth advantage of the invention lies in the possibility of accessing large oligomers, either by modulating the size of the anchor molecule, or by moving towards convergent synthesis, or by introducing one or more anchor molecules onto monomeric units.

[0128] A fifth advantage is the possibility to control the purity of the oligomer during the synthesis, at each step by different analytical techniques such as mass spectrometry, high performance liquid chromatography, proton or carbon-13 nuclear magnetic resonance.

[0129] A sixth advantage is the possibility to implement, on an industrial scale, without expensive equipment.

[0130] Thanks to their high purity, macromolecules produced by this method can be used as pharmaceuticals, cosmetics, phytosanitary products or agri-food products, or to access any of these products.

[0131] Yet another advantage is that the preferred anchor molecules, namely polyisobutene derivatives, can be prepared from biosourced isobutene, as explained above.

EXAMPLES

[0132] The following examples illustrate the synthesis of some functionalized anchor molecules that can be used to implement the method according to the invention.

[0133] Unless otherwise indicated, known PIB derivatives have been prepared from precursors and methods described (Tetrahedron, 2005, 61, 12081) and commercial reagents.

Example 1: General O-Arylation Procedure

[0134] ##STR00009##

To a mixture of PIB—CH.sub.2—CH(CH.sub.3)—CH.sub.2—OMs derivative (1 equivalent) and phenol (3 equivalents) in a toluene/N-N-dimethylformamide (1/1) (0.1 M) mixture, potassium carbonate (5 equivalents) has been added, and then the reaction medium has been heated at 120° C. for 16 h and then cooled to room temperature. The reaction medium has been extracted three times with cyclohexane and acetonitrile/water or ethanol/water (90/10) mixture, washed with brine, dried over sodium sulfate, filtered, concentrated under reduced pressure and the residue purified by silica filtration, if necessary, to yield the corresponding O-aryl derivative.

Example 2

[0135] ##STR00010##

The anchored aldehyde (1 equivalent), has been dissolved in a (0.1 M) tetrahydrofuran/ethanol (1/1) mixture, and then cooled to 0° C. for 5 minutes. Sodium borohydride (3 equivalents) has been added to the reaction medium (in small portions), and then the reaction medium has been stirred at room temperature for 30 minutes. The reaction medium has been concentrated under reduced pressure, and then 1N sodium hydroxide solution and cyclohexane have been successively added to the residue. The organic phase has then been washed with water, brine, dried over sodium sulfate, filtered and concentrated under reduced pressure to yield the corresponding benzyl alcohol.

Example 3

[0136] ##STR00011##

The anchored methyl ester (1 equivalent) has been dissolved in a (0.1 M) tetrahydrofuran/DMSO/water (8/1/1) mixture. Lithium hydroxide (3 equivalents) has been added to the reaction medium, and then the reaction medium has been stirred at room temperature for 12 h. The reaction medium has been extracted three times with cyclohexane and washed successively with hydrochloric acid solution (1N), ethanol/water mixture (90/10), brine, dried over sodium sulfate, filtered, concentrated under reduced pressure and the residue has been purified by silica filtration, if necessary, to yield the corresponding carboxylic acid.

Example 4

[0137] ##STR00012##

To a mixture of PIB-phenol derivative (1 equivalent) and methyl 4-(bromomethyl)benzoate (3 equivalents) in a (0.1 M) toluene/N-N-dimethylformamide (1/1) mixture has been added potassium carbonate (5 equivalents) then the reaction medium has been heated at 120° C. for 16 h then cooled to room temperature. The reaction medium has been extracted three times with cyclohexane and washed successively with an acetonitrile/water or ethanol/water (90/10) mixture, brine, dried over sodium sulfate, filtered, concentrated under reduced pressure, and the residue is purified by filtration on silica, if necessary, to yield the corresponding O-aryl derivative.

Example 5

[0138] ##STR00013##

To a solution of the PIB-phenol derivative (1 equivalent) in toluene (0.1 M), under stirring and at room temperature, succinic anhydride (2 equivalents), and then triethylamine (3 equivalents) have been added. The reaction medium has been heated to 60° C. for 16 h and then cooled to room temperature. After addition of a (1N) hydrochloric acid solution, the reaction medium has been extracted three times with cyclohexane and the organic phase has been washed successively with an ethanol/water (90/10) mixture, brine, dried on sodium sulfate, filtered, concentrated under reduced pressure and the residue has been purified by filtration on silica, if necessary, to yield the corresponding ester.

Example 6

[0139] ##STR00014##

To a solution of the PIB-phenol derivative (1 equivalent) in toluene/DMF (1/1) (0.1 M), under stirring and at room temperature, 5-fluoro-2-nitrobenzaldehyde (3 equivalents), and then potassium carbonate (3 equivalents) have been added. The reaction medium has been heated to 80° C. for 48 h and then cooled to room temperature. The reaction medium has been extracted three times with cyclohexane and washed with an ethanol/water (90/10) mixture, brine, dried over sodium sulfate, filtered, concentrated under reduced pressure and the residue is purified by silica filtration, if necessary, to yield the corresponding aryl ether.

Example 7

[0140] ##STR00015##

The anchored aldehyde (1 equivalent) has been dissolved in a (0.1 M) tetrahydrofuran/ethanol (1/1) mixture, and then cooled to 0° C. for 5 minutes. Sodium borohydride (3 equivalents) has been added to the reaction medium (in small portions) and then the reaction medium has been stirred at room temperature for 30 minutes. The reaction medium has been concentrated under reduced pressure, and then 1N sodium hydroxide solution and cyclohexane have been successively added to the residue. The organic phase has then been washed with water, brine, dried over sodium sulfate, filtered and concentrated under reduced pressure to yield the corresponding benzyl alcohol.

Example 8

[0141] ##STR00016##

To a solution of the PIB-alcohol derivative (1 equivalent) in dichloromethane (0.1 M) under stirring, under an inert atmosphere and at room temperature, succinic anhydride (1.1 equivalent), and then triethylamine (1.2 equivalent) have been added. The reaction medium has been heated to 40° C. for 18 h and then cooled to room temperature.

[0142] At this stage, 5′-O-(4,4′-dimethoxytrityl)thymidine (1.1 equivalent), ethyl-(N,N-dimethylamino)-propylcarbodiimide hydrochloride (EDCI) (1.1 equivalent) and 4-(N,N-dimethylamino)-pyridine (DMAP) (0.5 equivalent) have been successively added to the reaction medium; then the reaction medium has been heated at 40° C. for 18 h. The reaction medium has been evaporated and then extracted with cyclohexane, washed 3 times with an ethanol/water (90/10) mixture, brine, dried over sodium sulfate, filtered, concentrated under reduced pressure and the residue has been purified by silica filtration, if necessary, to yield the corresponding anchored thymidine.

Example 9

[0143] ##STR00017##

To a solution of the PIB-carboxylic acid derivative (1 equivalent) in dichloromethane under stirring, inert atmosphere and at room temperature 5′-O-(4,4′-dimethoxytrityl)thymidine (1,1 equivalent), ethyl-(N,N-dimethylamino)-propylcarbo-diimide hydrochloride (EDCI) (1.1 equivalent) and 4-(N,N-dimethylamino)-pyridine (DMAP) (0.5 equivalent) have been added, and then the reaction medium has been heated to 40° C. for 18 h. The reaction medium has been evaporated and then extracted with cyclohexane, washed 3 times with an ethanol/water (90/10) mixture, brine, dried over sodium sulfate, filtered, concentrated under reduced pressure and the residue has been purified by silica filtration, if necessary, to yield the corresponding anchored thymidine.

Example 10

[0144] ##STR00018##

To a solution of the anchored 5′-O-(4,4′-dimethoxytrityl)thymidine derivative (1 equivalent) in a (0.1 M) THF/H.sub.2O mixture, at room temperature, mercaptosuccinic acid (5 equivalents) then dichloroacetic acid (5% V) have been added, and then the reaction medium has been stirred for 1 h. The reaction medium has been evaporated, and then extracted with cyclohexane, washed 3 times with ethanol/water mixture (90/10), brine, dried on sodium sulfate, filtered, concentrated under reduced pressure to yield the deprotected derivative of corresponding anchored thymidine.

Example 11

[0145] ##STR00019##

To a solution of the PIB-carboxylic acid derivative (1 equivalent) in dichloromethane (0.1 M) under stirring in an inert atmosphere and at room temperature, 5′-O-(4,4′-dimethoxytrityl)thymidine (1.1 equivalent), ethyl-(N,N-dimethylamino)-propylcarbodiimide hydrochloride (EDCI) (1.1 equivalent) and 4-(N,N- dimethylamino)-pyridine (DMAP) (0.5 equivalent) have been added. Then the reaction medium has been heated at 45° C. for 18 h and then evaporated.

[0146] The residue has been solubilized in a (0.1 M) THF/H.sub.2O (9/1) mixture, at room temperature, and then mercaptosuccinic acid (5 equivalents), dichloroacetic acid (5% V) have been successively added then the reaction medium has been stirred at room temperature for 1 h. The reaction medium has been evaporated, and then extracted with cyclohexane, washed 3 times with an ethanol/water (90/10) mixture, a saturated sodium bicarbonate solution, brine, dried over sodium sulfate, filtered, concentrated under reduced pressure to yield the deprotected derivative of the corresponding anchored thymidine.

Example 12

[0147] ##STR00020##

The deprotected derivative of the anchored thymidine (1 equivalent) and DMT-dT phosphoramidite (2 equivalents) have been co-evaporated 3 times with anhydrous toluene and then dried under vacuum. To the residue under inert atmosphere dichloromethane (0.1 M) then a benzylthio-1H-tetrazole (BTT) solution (4.5 equivalents) in (0.01 M) acetonitrile have been added and the reaction medium has been stirred for 16 h at room temperature. mCPBA (3 equivalents) has been added to the reaction medium then stirred for 1 h and then evaporated.

[0148] At this stage, the residue has been solubilized in a (0.1 M) THF/H.sub.2O (9/1) mixture, at room temperature, then mercaptosuccinic acid (5 equivalents), dichloroacetic acid (5% V) have been successively added, and then the reaction medium has been stirred at room temperature for 1 h. The reaction medium has been evaporated, and then extracted with cyclohexane, washed 3 times with an ethanol/water (90/10) mixture, an aqueous sodium bicarbonate solution (10%), brine, dried over sodium sulfate, filtered, concentrated under reduced pressure to yield the dimeric deprotected derivative of the corresponding anchored thymidine.

Example 13

[0149] ##STR00021##

To a solution of the PIB-benzoic acid derivative (1 equivalent) in dichloromethane (0.1 M) under stirring, under an inert atmosphere and at room temperature, N-hydroxysuccinimide (1.1 equivalent), ethyl-(N,N-dimethylamino)-propylcarbodiimide hydrochloride (EDCI) (1.1 equivalent) and 4-(N,N-dimethylamino)-pyridine (DMAP) (0.1 equivalent) have been added, and then the reaction medium has been heated to 40° C. for 30 min and then cooled to room temperature. Hydrazine hydrate has been added to the reaction medium and then heated to 40° C. for 30 min. The reaction medium has been evaporated, and then extracted with cyclohexane, washed 3 times with an ethanol/water (90/10) mixture, brine, dried over sodium sulfate, filtered, concentrated under reduced pressure to yield the corresponding PIB-acyl hydrazide derivative.

Example 14

[0150] ##STR00022##

To a solution of the PIB-aryl derivative (1 equivalent) in dichloromethane (0.1 M) under stirring, under an inert atmosphere and at room temperature, chlorosulfuric acid (1.1 equivalent) has been added dropwise, then the reaction medium has been stirred at room temperature for 30 min. Hydrazine hydrate (3 equivalents) has been added to the reaction medium and then stirred for 30 min. The reaction medium has been evaporated, and then extracted with cyclohexane, washed 3 times with an ethanol/water (90/10) mixture, brine, dried over sodium sulfate, filtered, concentrated under reduced pressure to yield the corresponding PIB-sulfonyl hydrazide derivative.

Example 15

[0151] ##STR00023##

To the PIB-acyl hydrazide (1 equivalent) and 2,3,4,6-tetra-O-benzyl-D-glucopyranose (1.5 equivalent) mixture in a (0.1 M) DMF/THF (9/1) mixture, acetic acid (0.01 equivalent) has been added and then the reaction mixture has been heated to 90° C. for 18 h. The reaction medium has been evaporated and then extracted with cyclohexane, washed 3 times with ethanol/water mixture (90/10), brine, dried over sodium sulfate, filtered, concentrated under reduced pressure to yield the corresponding PIB-glycosylhydrazide derivative.

Example 16

[0152] ##STR00024##

To the PIB-acyl hydrazide (1 equivalent) and 2,3,4,-tri-O-benzyl-D-glucopyranose (1.5 equivalent) mixture in a (0.1 M) DMF/THF (9/1) mixture, acetic acid (0.01 equivalent) has been added, and then the reaction mixture has been heated at 90° C. for 18 h. The reaction medium has been evaporated, and then extracted with cyclohexane, washed 3 times with an ethanol/water (90/10) mixture, brine, dried over sodium sulfate, filtered, concentrated under reduced pressure to yield the corresponding PIB-glycosylhydrazide derivative.