EP4172168A1 - Method for synthesising macromolecules in solution from carbohydrate derivative units - Google Patents

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.

Description

PROCESS FOR SYNTHESIS IN SOLUTION OF MACROMOLECULES FROM UNITS OF CARBOHYDRATE DERIVATIVES

Technical Field of the Invention The present invention belongs to the field of the synthesis of organic macromolecules. More specifically, the present invention relates to a process for producing macromolecules of interest from units of monosaccharides or oligosaccharides or derivatives of such units. This process takes place in the liquid phase (solution) and uses soluble anchoring molecules in an organic medium and more particularly in non-polar solvents. These anchoring molecules (or liquid support) confer on the macromolecules (or intermediates) of interest, when they are linked to them: transient protection of at least one chemical function and excellent solubility in nonpolar organic solvents and consequently , purifications by simple extraction or by filtration on silica. The macromolecules targeted can in particular be macromolecules of biological interest, such as oligonucleotides and oligosaccharides, and they can contain units which are not mono- or oligosaccharides.

STATE OF THE ART The present invention relates to a new process allowing the synthesis of macromolecules comprising units of monosaccharides and / or oligosaccharides and / or their derivatives; these units can be or can comprise in particular pentoses or hexoses. Thus and more particularly, the present invention relates to the synthesis of oligonucleotides (or their derivatives) and the synthesis of oligosaccharides (or their derivatives).

Usually, the oligonucleotides are prepared, on a laboratory scale, by synthesis on a solid support, with automata. Many efforts have made it possible to improve this equipment at the industrial stage for more substantial productions (> 1 kilogram). However, since the end of the last century, therapeutic oligonucleotides have experienced a great boom triggering a real interest in these molecular species. To date, more than a hundred oligonucleotides are in clinical trials and eight drugs have been approved by the Food and Drug Administration (FDA) (see YS Sanghvi, "A Status Update for Modified Oligonucleotides for Chemotherapeutics Aviations" , Curr. Protoc. Nucleic Acid Chem., 2011, 46: 4.1.1-4.1.22.). The first oligonucleotide approved was fomivirsen or Vitravene ™ (see MD de Smet et al., “Fomovirsen - a phosphorothioate oligonucleotide for the treatment of CMV retinitis”, Ocul. Immunol. Intiamm., 1999, 7, 189-198 and ST Cooke. et al., “RNA-Targeted Therapeutics”, Cell Metabolism, 2018, 27, 714-739), an oligodeoxynucleotide consisting of 21 units linked by phosphorothioate bonds. It is used in the treatment of cytomegalovirus retinitis.

Later, Macugen ™ (pegaptanib sodium) and Kynamro ™ (mipomersen sodium) were approved successively in 2004 and 2013, respectively indicated for the treatment of the neovascular (wet) form of age-related macular degeneration (AMD). ) and in the treatment of homozygous familial hypercholesterolemia (see Sanghvi and Schulte, Curr. Opin. Drug Discovery Dev. 2004, 6, 765).

Until now, the synthesis of oligonucleotides in solid phase has made it possible to produce, in routine, oligonucleotides on the scale of the kilogram, with good qualities (see Sanghvi, YS (2019) "Large-scale automated synthesis oftherapeutic oligonucleotides : A status update ", published in S. Agrwal & MJ Gait (Eds.) Advances in nucleic acid therapeutic (pp. 453-473)). Although many optimization efforts have been made, the production methods of Oligonucleotides can still be improved The synthesis of oligonucleotides in the liquid phase is increasingly positioned as an efficient response to the production of oligonucleotides.

Whatever the phase (solid (heterogeneous) or liquid (solution)) where the reaction takes place, the synthesis of oligonucleotides is summed up in the formation of phosphoric ester bonds between nucleosides, in a defined order (see H. Lonneberg, "Synthesis of oligonucleotides on a soluble support", Beilstein J. Org. Chem., 2017, 13, 1368-1387 and A. Molina & YS Sanghvi, <r Liquid-Phase Oligonucleotide Synthesis: Past, Present, and Future Predictions ", Current Protocol in Nucleic Acid Chemlstry, 2019, 77 (1)). Often, the synthesis of oligonucleotides in the liquid phase (deoxyribonucleic acid (DNA) or ribonucleic acid (RNA)) begins with the attachment of the anchor molecule to the 3 'end of a nudeoside; elongation taking place from 3 'to 5'. At this stage, the first cycle of synthesis begins with the entry into play of a phosphorylated nucleoside derivative in the 3 'position (phosphoramidite or H-phosphinate or phosphotriester), which will react with the alcohol in the 5' position of the anchored nudeoside. on a synthetic support. With the exception of the alcohol entering into the reaction, all the other nucleophilic functions must be protected beforehand. Thus, the amines of the nucleic bases are preferentially protected by acylation (benzoyfe for cytosine and adenine, isobutyl for guanine) while the alcohol functions of deoxyribose and ribose are respectively masked by trityl ethers (dimethoxytrityl or monomethoxytrityl) in the 5 'position and / or by silyfe ethers (terf-butyldimethylsilyl or triisopropylsilyloxymethyl) in position 2 '.

Depending on the nature of the phosphorylated nudeoside chosen, the operating conditions of the coupling reaction are different. In the case of nucleoside phosphoramidites (see SL Beaucage & MH Caruthers, <r Dooxynuclaoside Phosphoramidites - A naw Class ofKay Intermadiatas for Deoxypdynudaotida Synthasis ”, Tetrahedron Letters, 1981, 22, 1859-1862) and of nucleosides H-phosphonates, the reactions of H-phosphonates coupling are followed by an oxidation reaction to lead to the corresponding phosphotriesters. In the case of nucleoside phosphates, the corresponding phosphotriesters are obtained by esterification with the preceding nudeoside (see C. B. Reese & Z. Pei-Zhuo, “Phosphotriestar Approach to tha Synthasis of Oligonucleotides: A Raappraisal”, J.

Chem. Soc., Perkin Trans. 1, 1993, 2291-2301, V. A. Efimov et al., “Application of naw catalytic phosphate protacting group for tha highly efficient phosphotriestar oligonucleotide synthasis”, Nudeic Acids Res. 1986, 14, 6525-6540, G. van der Marel et al., <R A Naw Approach to tha Synthasis of Phosphotriestar Intermadiatas of Nuclaosides and Nudeic Acids ”, Tetrahedron Lett., 1981, 22, 3887-3890 and E. de Vroom et al., <R Usa of a 1-hydroxybenzotriazole activatad phosphorylating raagant towards tha synthasis of short RNA fragments in solution ”, Nudeic Adds Res., 1986, 14, 5885-5900).

Various anchoring molecules have been described in the literature in relation to the synthesis of oligonucleotides in the liquid phase. Historically, the foundations for oligonucleotide synthesis have been laid by the work of Hayatsu and Khorana (see <r Daoxyribooligonudaotida Synythasis on a Potymar Support ", J. Am. Chem. Soc. 1966, 88, 13, 3182-3183) . This approach, inspired by the peptide synthesis methodology described by Merriefield, used a soluble polystyrene as the anchor molecule (5-0-monomethoxytrityl (rMMT)). However, this polystyrene becomes insoluble in organic solvents beyond three nucleotides, and as a result, the insulation of the product becomes poor.

Other polymers have been studied, such as cellulose (Biochemistry 1968, 7, 8, 2809-2813) or polyvinyl alcohol (H. Schott et al., <R Uquid-Phasa-Synthasa von

Oligothymidylphosphatan ”, Die Makromolekulare Chemie, 1973, 173, 247-251). In in these two cases, during the incorporation of the first nucleoside on the support, a so-called capping reaction, of the free (non-functionalized) hydroxyl groups, is necessary in order to avoid the presence of truncated oligonucleotides at the end of the synthesis. In the 1990s, another category of polymer was widely studied as an anchor molecule: polyethylene glycol (or PEG). With this type of soluble supports, the Bonora group has successfully synthesized oligonucleotides on a gram scale while considerably reducing the handling time (see "HELP (High Efficlency Liquid Phase) new oligonucleotide synthesis on soluble potymeric support" , Nucleic Acids Research, 1990,18, 3155-3159 and “Large scale, liquid phase synthesis of oligonucleotides by the phosphoramidite approach”, Nucleic Acids Research, 1993, 21, 1213-1217).

More recently, Livingston's group has developed and used membranes for molecular separations by nanofiltration (see PRJ Gaffhey et al., “Uquid-Phase Synthesis of 2'-Methyl-RNA on a Homostar Support through Organi - Solvent Nanofiltration”, Chem. Eur. J., 2015, 21, 9535-9543 and JF Kim et al., “Organic Solvent Nanofiltration (OSN): A New Technology Platform for Liquid-Phase Oligonucleotide Synthesis (LPOS)”, Org. Proc. Res. Dev., 2016, 20, 1439-1452). They make it possible to facilitate and accelerate the stages of purification of the growing oligonucleotides.

In general, the use of polyethylene glycols (PEG) as an anchoring matrix has several advantages such as: the use of acetonitrile as a solvent; obtaining oligonucleotides of good purity; simplified purifications (at each stage); the possibility of achieving reasonable amounts of oligonucleotides; versatility at the level of coupling reactions (phosphoramidites, H-phosphonate (see KJ Padiya et al., "Large Scale, Liquid Phase Oligonucleotide Synthesis byAlkyl H-phosphonate Approach", Bioorg. Med. Chem., 2000, 8, 337 -342) and phosphates) and the reduction in the quantity of monomers involved. The main obstacles to the use of PEGs on an industrial scale are: the large number of precipitation stages; excessive consumption of solvents and the cost of nanofiltration membranes.

In 2017, the use of an adamantane derivative was described as an anchoring molecule for the synthesis of oligonucleotides in liquid phase (A. Schwenger et al., “R Solution- Phase Synthesis of Branched Oligonucleotides with up to 32 Nucleotides and the Reversible Formation of Materials ”, Eur. J. Org. Chem., 2017, 5852-5864). An advantage of this support lies in the purification by simple extraction. The main drawback of this anchoring molecule is its low factor E (which expresses the waste / product mass ratio desired) because a large volume of solvent is necessary for the various stages of purification by extraction. The same research group described a tetrasubstituted adamantane derivative as an anchor molecule for the synthesis of oligonucleotides in the liquid phase. Here too, the large volume of solvent required for the purifications and the excess of phosphoramidite monomers are hindering its development at industrial level.

In 2006, the first ionic anchoring molecule, in this case of the imidazolium type, was described (RA Donga et al., “A Novel Approach to Oligonucleotide Synthesis Using an Imidazolium Ion Tag as a Soluble Support”, J. Org Chem., 2006, 71, 7907-7910). Purification of the elongating oligonucleotide is accomplished by precipitation and extraction. In an Industrial vision, this liquid support is weighted by the large volume of solvent necessary for the purifications of the growing oligonucleotide.

Soluble β-cyclodextrin anchoring molecules, based on D-glucopyranose, have demonstrated their efficiency for the synthesis of oligonucleotides in the liquid phase (A. Gimenez Molina et al., “Acetylated and Methylated β-Cydodextnns as Viable Soluble Supports for the Synthesis of Short 2-Oligodeoxynbo-nudeotides in Solution ”, Molécules, 2012, 17, 12102-12120). The advantages associated with these soluble supports are their cost and a reduction in the quantity of monomers required for the coupling steps. The weak points of these matrices lie in the level of purification. In fact, for each cycle, purification by chromatography column is compulsory.

Another category of anchor molecules, consisting of derivatives of functionalized pentaerythritol, has been described. They have been used for DNA synthesis (V. Kungurtsev et al., “Solution-Phase Synthesis of Short Oligo-2'-deoxyribonucleotides by Using Clustered Nucleosides as a Soluble Support”, (Eur. J. Org. Chem. ., 2013, 6687-6693) and RNA (A. Gimenez Molina et al., “Solution phase synthesis of short oligoribonucleotides on a precipitative tetrapodal support”, Beilstein J. Org. Chem., 2014, 10, 2279- 2285 and Current Organic Synthesis, 2014, 12, 202-207). The advantages of this liquid support are: the possibility of carrying out the elongation reactions according to the chemistry phosphoramidites or phosphotriesters; the stability of the matrix; the presence of four anchoring sites; precipitation purifications.

The main obstacles to the industrialization of this liquid support are the use of two hydrophobic protecting groups (in 2 'and 5'), during the synthesis of RNA, and the use of non-commercial monomers and the numerous purifications by precipitation. .

Gallic acid derivatives, as a soluble carrier, have also been used for the production of oligonucleotides in solution (see JP 2010-275254 and WO 2013/179412 as well as the publications by S. Kim et al., <R Liquid -Phase RNA Synthesis by Using Alkyl-Chain- Soluble Support ”, Chem. Eur. J., 2013, 19, 8615-8620, and by T. Shoji et al.,“ Synthesis of Conjugated Oligonucleotide in Solution Phase Using Alkyl-chain -soluble Support ”, Chem. - Lett., 2014, 43, 1251-1253). Using these, in combination with phosphoramidite chemistry, the synthesis of RNA (21-mer) was achieved in excellent yield (26%) and good purity (78%). However, the numerous purification steps (> 50) make these anchoring molecules unsuitable for industrial use.

In the same vein, the anchoring molecules called Ajiphase ™ have been described for the production of oligonucleotides (see S. Katayama & K. Mirai, “r Uquid-phase synthesis of oligonucleotides”, published in S. Obika & M. Sekine (Eds.), “Synthesis of therapeutic oligonucleotides” (pp. 83-95), Springer Singapore, 2018). The use of Ajiphase ™ as an anchor molecule for the production of oligonucleotides has certain advantages: the anchored oligomers can be purified by simple precipitation in acetonitrile or methanol; the number of phosphoramidite equivalents is low (<2 equivalents); the overall yield is improved and the consumption of solvents is reduced. However, the main limitation of this process remains the purification (one precipitation per cycle).

The present invention also relates to oligosaccharides, formerly called "carbohydrates". They are involved in several biological processes and play an essential role in the living world In fact, sugars are involved in the structure of essential molecules such as nucleic acids (ribose and deoxyribose). Oligosaccharides and polysaccharides are made up of monosaccharides linked together by a glycosidic bond. With a view to understanding the roles of sugars in the living world, chemists have made conceptual and practical advances allowing access to increasingly complex oligosaccharides (M. Panza et al., <R Automated Chemical Oligosaccharide Synthesis: Novel Approach to Traditional Challenges ”, Chem. Rev. 2018, 118, 17, 8105-8150 and M. Guberman & PH Seeberger, “Automated Glycan Assembly: A Perspective”, J. Am. Chem. Soc. 2019, 141, 14, 5581-5592). Since Memfield's work in the 1960s on peptide synthesis on a solid support, many chemists have developed similar methods, suitable for the synthesis of oligosaccharides, in order to benefit from the advantages of this methodology, in this case : the use of excess reagents and purifications by simple washing of the solid support. However, this oligosaccharide synthesis approach involves some problems such as: the choice of the solid support, the design of the spacer, the selection of the protecting groups, the follow-up of the reactions, the choice of the donor or acceptor monomers and the final release. of the target oligosaccharide of the matrix (see PH Seeberger & SJ Danishefsky, “Solid-Phase Synthesis of Oligosaccharides and Glycoconjugates by the Glycai Assembly Method: A Five Year Retrospective”, Acc. Chem. Res., 1998, 31, 685- 695; PH Seeberger & W.-C. Haase, “Solid-Phase Oligosaccharide Synthesis and Combinatorial Carbohydrate Libraries”, Chem. Rev., 2000, 100, 4349-4393 and PH Seeberger, “Automated oligosaccharide synthesis”, Chem. Soc. Rev. 2008, 37, 19-28). In 1973, the first synthesis of a trisaccharide on a solid support was described (see JM Frechet & C. Schuerch, "Solid-Phase Synthesis of Oligosaccharides. I. Prapapration of the Solid Support. Poly [p- (1-propen- 3-ol-1-yl) styrene] ”, J. Am. Chem. Soc., 1971, 93, 492-496; Frechet, JM in“ Polymer-Supported Reactions in Organic Synthesis ”, Hodge, P., Sherrington, DC, Eds .; John Wiley & Sons Ltd: Chichester, UK, 1980, pp 407-434 and Carbohydr. Res., 1972, 399-412).

Inspired by this work, Seeberger et al., Designed a machine allowing the automated synthesis of oligosaccharides (PH Seeberger, “Soiid Phase Oligosaccharide Synthesis”, J. Carbohydr. Chem., 2002, 21, 613-643 and M. Weishaupt et al., “Solid Phase Synthesis of Oligosaccharides”, published in “Methods in Enzymology”, 2010, 478, 463-484). Thus, they described the synthesis of several complex oligosaccharides such as derivatives of β-glucan dodecamers.

The use of glycai for glycosidic coupling has been explored on a solid support (JT Randolph et al., “Major Simplifications in Oligosaccharide Synthesis Arising from a Solid-Phase Based Method: An Application to the Synthesis of the Lewis b Antigen ”, J. Am. Chem. Soc., 1995, 117, 5712-5719; C. Zheng et al., "Solid Support Oligosaccharide Synthesis: Construction of β-Unked Oligosaccharides by Coupling of Glycal Derived Thioethyl Glycosyi Donors", J. Org. Chem., 1998, 63, 1126-1130 and KA Savin et al. , “A New Polymer Support Silylene Linking Method for Hindered Hydroxyl · Bearing Systems”, J. Org. Chem., 1999, 64, 4183-4186). This concept of glycosidic coupling is based on the activation of glycal with an electrophile ( dimethyldioxirane for example).

It follows from what has just been exposed that the main challenges for the synthesis of oligosaccharides in the solid phase are: the use of excess monomer units, the impossibility of controlling the anomerism of each monomer introduced, the extrapolation to all types of glycosidic bonds, the low kinetics of the cleavage of the spacers, the complex synthesis of the spacers, the need for special equipment (likely to drop to around - 20 ° C) and production capacities weak.

In short, the production of macromolecules, oligonucleotides and oligosaccharides, on an industrial scale at a lower cost and with a fair environmental footprint, is illusory with current methodologies. This is precisely the object of the present invention. Indeed, the inventors have developed a range of protective groups (or anchoring molecules or solubilizing molecules or anchoring matrices) capable of allowing the synthesis of oligonucleotides and oligosaccharides at low cost, low environmental impact and low complexity. In other words, the use of these anchoring molecules makes it possible to combine the advantages of synthesis in liquid phase and synthesis in solid phase (the homogeneity of the reaction medium, due to the fact that the supports are soluble, this which allows linear kinetics; the possibility of reducing the quantity of expensive reagents as well as the solvents; the possibility of implementing large-scale reactions (batch or batch); simplified purifications of the macromolecules during elongation, of the reagents in excess and by-products. These purifications are based specifically on the physicochemical properties and the nature of the anchor molecule, they are most often carried out by the technique of liquid-liquid extraction which is based on the distribution selective (or partition coefficient or distribution coefficient) of solutes in two almost immiscible liquids.

OBJECT OF THE INVENTION The present invention proposes to resolve the difficulties left in the prior art, in particular as regards the purification technique by, on the one hand, the derivation protective groups which could be soluble in nonpolar solvents and on the other hand, by the use of polyolefins or oligomers of polyolefins or polyalkenes, capable of causing selective solubility during the production of biological macromolecules (oligonucleotides and oligosaccharides) in liquid phase (or solution). Indeed, the inventors have found that the use of polyolefins, and in particular of polyisobutenes (PIB) derivatives, as protecting groups or anchoring molecules or liquid supports or anchoring matrices, allows the synthesis of these macromolecules in solution. (halogenated and / or non-halogenated solvents) while facilitating their purification by liquid-liquid extraction.

The present invention therefore relates to the synthesis of macromolecules by successive elongation of various units, which are predominantly carbohydrate derivatives which may be identical or different. Said macromolecules can in particular be oligonucleotides or oligosaccharides.

A first object of the present invention is a process for the synthesis of macromolecules composed of U units which are predominantly monosaccharides or monosaccharide derivatives which may be identical or different, said macromolecules having a first and a second end, said synthesis process proceeding by successive elongation of said second end by an M monomer or oligomer having at least two functions, and said process being characterized in that:

- in a first step called anchoring, the first unit U1 of said macromolecule, corresponding to an M1 monomer or a terminal unit of an M1 oligomer, is fixed by a covalent bond (ether, ester or any other functions compatible with the present process ) to an anchoring molecule soluble in organic solvents, said covalent bond resulting from a reaction of a first of the functions of said M1 monomer or of said M1 oligomer with a function of said anchor molecule, the second termination is possibly a another function of said M1 monomer or of said M1 oligomer having been protected, prior to said reaction, by at least one first protective group GP1, and possibly by a second protective group GP2 and an umpteenth protective group GPn;

- in a second step called deprotection, one of said protective groups GP1 or GP2 or GPn is removed, leaving on said monomer M1 or said oligomer M1 at least one free chemical function; - in a third step called coupling, reacting a second M2 monomer or M2 oligomer, carrying at least one free chemical function and at least one function chemical protected by a protective group GP3, so that its free chemical 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 termination to said anchor molecule, and said M2 monomer or M2 oligomer, attached to another of its termini, and said method being characterized in that said anchor molecule comprises a polyolefin chain or an oligomer of polyolefin 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.

Thus, an nth Mn monomer or Mn oligomer can be added by coupling; this process makes it possible to synthesize macromolecules.

The method comprises at least one step in which said macromolecule fixed to said anchoring molecule is separated from the reaction medium by the liquid-liquid extraction technique in an apolar organic solvent with a polar solvent (or a mixture of polar solvent) immiscible . The real challenge is therefore to maintain the selective solubility of the anchored M1 monomer or oligomer after the deprotection and coupling reactions with respect to the by-products of said reactions.

It is the branched nature of the polyolefin chain of the anchor molecule which gives the latter its excellent solubility in nonpolar solvents and its very low solubility in polar solvents, which are necessary to allow separation of the molecule from anchoring of the reaction medium by liquid-liquid extraction.

The method may include a step in which said macromolecule is completely deprotected. After the coupling of the last monomer or oligomer, the protective groups of the macromolecule are removed. Said monosaccharide units or monosaccharide derivatives are in particular derivatives of pentoses (and in particular of nucleosides) or of hexoses. Among the pentoses, mention may be made, for example, of nucleosides, such as C-nucleosides, acyclic nucleosides, carboxylic nucleosides, imino-C-nucleosides, nucleosides having modified bases; all of these molecules must be used in a suitably protected form. Other usable monosaccharide derivatives are osamines (amino sugars), such as glucosamine, galactosamine, mannosamine, meglumine.

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.

In one embodiment of the method, some of the U units may not be monosaccharides or monosaccharide derivatives. In particular, these U units can be selected from amino acids and lipids (isoprene derivatives).

Said anchor molecule advantageously has a mass average molecular mass of between 300 and 20,000, and preferably between 500 and 15,000. Its polyolefin chain is an oligomer or a polymer constructed from monomers which are preferably identical.

In an advantageous embodiment, said polyolefin or polyolefin or polyalkene oligomer chain has been obtained by polymerization of a monomer. This monomer is advantageously biobased.

In a particular embodiment, said polyolefin or polyolefin or polyalkene oligomer chain comprises a number of unsaturated carbon-carbon bonds not exceeding 5%, and preferably not exceeding 3%. A second object of the invention is the use of a process according to any one of the embodiments of the invention for the synthesis of oligonucleotides or oligosaccharides.

In the case of oligonucleotides, the monomer unit typically consists of a suitably protected phosphorylated nucleoside (or oligonucleotide chain) (scheme 1). This unit will react with the alcohol in the 5 'position of a nucleoside (or of an oligonucleotide chain) suitably protected on the nucleic base and / or in the 2' position of the sugar and anchored in the 3 'position on a support. liquid. [Chem 1]

Diagram 1

In the case of oligosaccharides, the monomeric unit typically consists of a donor glycosyl (or of an oligosaccharide chain) suitably protected or not and carrying a Z group, in the anomeric position, which may be without s Limit therein to a cyclic hemiacetal, an acetate, a thioglycoside, a phosphate or an imidate. The alcohol function of an acceptor glycoside (or of an oligosaccharide chain) suitably protected or not and anchored on a liquid support will react with the said monomer unit (diagram 2).

[Chem 2] Scheme 2 In general, the method according to the invention can be implemented with identical or different units. By way of example, it is thus possible to synthesize an oligosaccharide which is a homopolymer, that is to say which consists of identical monosaccharide units. It is also possible to synthesize an oligosaccharide from different monosaccharide units. It is also possible to use units which are disaccharides, trisaccharides or longer oligosaccharides.

Likewise, oligonucleotides can be synthesized from identical or different phosphorylated nucleoside (or oligonucleotide) units.

By way of example, to prepare oligonucleotides, 3 'nucleosides (2-cyanoethyl · Ν, Ν-dialkylphosphoramidite) or 3' nucleosides - (H-phosphonates) or nucleoside phosphates (di or triesters) can be used. . In the synthesis of oligosaccharides we can use donor glycosyls suitably protected or not. In both cases, the coupling reactions known to those skilled in the art are applied.

The method according to the invention also makes it possible to synthesize mixed polysaccharides comprising monosaccharide units and nucleotide units.

The method according to the invention also makes it possible to introduce into the macromolecule units which are or do not include either oligosaccharides or oligonucleotides.

An essential characteristic of the process according to the invention is the use of anchoring molecules or solubilizing molecules or anchoring matrices. They must be soluble in an apolar solvent. They consist of a polyolefin chain having at least 5 monomer units, and preferably between 10 and 50 units; they are polyolefins or oligomers of polyolefins or polyalkenes. Advantageously, the anchoring molecules are functionalized at at least one of their ends to allow protection or attachment of the initial monomer (or oligomer) unit.

Said anchor molecule may include in each of its units identical or different alkyl groups, which are preferably selected from the group formed by methyl and ethyl. Said polyolefin chain advantageously has an average molecular weight of between 300 and 20,000, and preferably between 500 and 15,000. Said polyolefin chain may comprise a number of unsaturated carbon-carbon bonds not exceeding 5%, and preferably not exceeding 3%. Preferably it is a polyisobutene chain (abbreviated as PIB). Diagram 3 shows certain PIBs which can be used within the framework of the invention; in these formulas, X represents a spacer which carries the function intended to react with the first unit U1 of the macromolecule to achieve anchoring on the liquid support.

[Chem 3]

Diagram 3

The method according to the invention provides access to high purity macromolecules. This process induces an advantageous E factor (environmental factor) owing to the fact that the purifications are carried out by the liquid-liquid extraction technique, with quantities of reasonable solvent, which makes it possible to minimize the stages of purification by chromatography columns; resulting in financial savings. Thus, reactions making it possible to derive by-products soluble in nonpolar solvents can be carried out before the liquid-liquid extraction.

In a particular embodiment, said anchor molecule comprises a polyolefin chain (or is a polyolefin chain) terminated by at least one group selected from the group (spacer) formed by an aliphatic chain (branched or not and unsaturated or not) , a (hetero) cyde and / or a (hetero) aryl (substituted or not).

In an advantageous embodiment, the mass average molecular mass of the anchoring molecules, apart from their terminal functionalization, is between 300 and 20,000, and preferably between 500 and 15,000. Above a mass average molecular mass around 20,000, these molecules may have a viscosity that is too high, which could limit their solubility in organic solvents.

Certain PIB derivatives used in the context of the present invention are commercially available as ligands for homogeneous catalysis. By way of example, one can use 2-methyl-3- [polyisobutyl (12)] propanol (mass average molecular weight 757, including terminal functionalization) or 4- [polyisobutyl (18)] phenol (mass weight average molecular weight 1104, including terminal functionalization) which are distributed, respectively, under the references 06-1037 and 06-1048 by the company Strem Chemicals. These two molecules are polyisobutenes derivatives whose chain is terminated, respectively, by a -CH2-C (CH3) (H) -CH2-OH group (ie isopropanol) and by a -CH2-C (CH3) 2- group. C6H4OH (ie phenol).

According to one characteristic of the invention, the anchoring molecules act as protective groups, alcohol functions and / or any other functions which need to be inert under the conditions of the process which is the subject of the present invention.

In the case of the synthesis of oligonucleotides, the alcohol function in position 3 ′ of ribose / deoxyribose and / or an amine of the nucleic base can be masked, simultaneously or not, with at least one solubilizing protective group.

In the case of the synthesis of oligosaccharides, the initial anomeric function is anchored (protected) with a solubilizing protecting group. These derivations allow the solubilization of the monomers and oligomers in nonpolar solvents such as cyclohexane, heptane (s) or hexane (s).

According to another characteristic of the invention, the use of anchoring molecules soluble in organic solvents as described above (and more particularly the use of polyolefins), and insoluble in certain polar solvents (such as water and / or ethanol and / or acetonitrile), facilitates the purification of the anchored monomers and oligomers by simple liquid-liquid extraction or simple filtration on silica. According to yet another characteristic of the invention, commercially available anchoring molecules are used, or else anchoring molecules which can be synthesized simply and directly from commercially available precursors, in particular certain polyisobutenes derivatives. In other words, conventional protecting groups can be linked to PIB, such as benzyl, silyl or carboxylic acid derivatives.

Diagram 4 below shows some structures, which are not exhaustive, capable of playing the role of a protective group in the synthesis of oligonucleotides.

[Chem 4]

Diagram 4

Scheme 5 below shows some structures, not exhaustive, capable of playing the role of a protective group in the synthesis of oligosaccharides. [Chem 5]

Diagram 5

According to yet another characteristic of the invention, the initial monomer (or oligomer) is fixed to the anchoring molecule according to reactions known as a function of the chemical functions in an appropriate solvent, such as mixtures of solvents (halogenated or not). , and at a temperature between - 50 ° C and 150 ° C.

According to yet another characteristic of the invention, the chemical functions of the monomer (or oligomer), incompatible with this process, can be temporarily masked by appropriate protective groups such as ttertbutyldimethylsilyl (abbreviated TBDMS or TBS), dimethoxytrityl (abbreviated DMTr ) or any other protective group compatible with the present process. According to yet another characteristic of the invention, after the deprotection step, the protective groups can be derived, preferably in situ, to form compounds soluble in a polar solvent (or a mixture of polar solvent). Thus, the monomer (or oligomer) anchored on a GDP derivative whose primary alcohol is protected by a trityl group is cleaved in an acidic medium in the presence of a trapping agent (scavengers) of the corresponding carbocation (trityl) making it possible to make it soluble in aqueous (or polar) phase. Trityl carbocation trapping agents include, but are not limited to thioglycolic acid, 3-mercaptoproprionic acid, 3-mercapto-1-propanesulfonic acid, cysteine or thiomalic acid (or mercaptosuccinic acid). According to yet another characteristic of the invention, said anchoring molecule reacts with a first suitably protected monomer (or oligomer), leading to a covalent bond, such as an ester or O-glycoside bond, between these two molecular species.

According to yet another characteristic of the invention, the monomers having chemical functions incompatible with the reaction conditions of the elongation cycle (or iteration) can be temporarily masked by a solubilizing protective group. In this case, depending on the length of the target macromolecule, the chemical functions may be masked by one or more solubilizing protective groups and by one or more conventional protective groups such as: benzoyl, acetyl, tert-butyledimethylsilyl.

According to yet another characteristic of the invention, the process for synthesizing macromolecules is carried out using units of monomers (or oligomers) linked to an anchor molecule as a starting point.

In the case of the synthesis of oligonucleotides, the first cycle of elongation begins with a selective deprotection in an acidic medium and in the presence of a trapping agent, if it is a trityl derivative, of the protecting group in position 5 'of the anchored nucleoside.

In the case of the synthesis of oligosaccharides, the first cycle of elongation begins with a reaction between a donor glycosyl suitably protected or not and a glycoside anchored and deprotected on the alcohol function which will be involved in the reaction. Again, if the protective group is a trityl derivative, it is deprotected in an acidic medium in the presence of a trapping agent

According to yet another characteristic of the invention, the integration of a unit (or iteration) of monomer requires several steps.

In the case of the synthesis of oligonucleotides, two steps are necessary if they are nucleoside phosphate (coupling and deprotection of the alcohol in the 5 'position), and three steps if they are nucleoside phosphoramidites or H-phosphonates (coupling, oxidation and deprotection of alcohol in the 5 'position).

In the case of the synthesis of oligosaccharides, two steps are necessary (coupling and deprotection). According to yet another characteristic of the invention, said macromolecule is formed of n monomer units, a chemical function of which is linked to an anchoring molecule. During the course of the process, the oligomer chain grows by successive elongation (or cycle) or iteration, and during each of these steps a unit of monomer or oligomer is added to the end of the free alcohol.

According to yet another characteristic of the invention, it is possible to use in said chain of oligomers natural and / or non-natural and / or synthetic monomers (or oligomers).

According to yet another characteristic of the invention, it is possible to use in said chain of oligomers one or more units of suitably protected monomer analogs (or oligomers) such as morpholino-phosphonated monomers or N-Acetyl monosaccharide monomers. .

According to yet another characteristic of the invention, at least one step in which said oligomer chain is purified from the reaction medium by extraction in an apolar organic solvent immiscible with water (or immiscible with a water / ethanol mixture or a water / acetonitrile mixture) or by filtration on silica. As the non-polar organic solvent, preference is given to hydrocarbons having only carbon and hydrogen atoms, such as linear alkanes, branched alkanes and cycloalkanes, and particularly preferred are cyclohexane, heptanes and hexanes.

According to yet another characteristic of the invention, the process makes it possible to obtain oligomers of high purity, after total deprotection and detachment of the anchoring molecules, to be used according to their destination, for example as an active principle for tests. preclinical, clinical care or any other application.

According to yet another feature of the invention, the anchor molecules can be reused (recycled) in the process according to the invention.

According to yet another characteristic of the invention, the extraction solvents can be reused (recycled) in the process according to the invention.

The method according to the invention can be automated and / or carried out in flow chemistry. detailed description

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

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

The term "monosaccharide" denotes the monomer of carbohydrates.

The term "glycosylamine" means a compound in which a carbohydrate is linked to an amine group in the anomeric position.

The term “nucleoside” denotes a ribosyl or deoxyribosyl type derivative of certain pyrimidine or purine type bases, and more specifically glycosylamines consisting of a nucleic base linked to the anomeric carbon atom of a pentose residue, generally of ribose (ribonucleoside) or deoxyribose (deoxyribonucleoside), through a glycosidic bond from the N ¹ nitrogen atom of a pyrimidine or the N ⁹ atom of a purine.

The term "nucleotide" denotes an organic molecule which is the basic building block of a nucleic acid such as DNA or P RNA; this molecule is composed of a nucleic base, of an ose with five carbon atoms, and finally of one to three phosphate groups. The term "protecting group" denotes a molecule used for the reversible protection of a functional group during a chemical reaction to render said functional group unreactive in said chemical reaction process which would have transformed said unprotected functional group.

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

The term "macromolecule" denotes 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; can also coupling an oligomer comprising several of these units. In general, these units can be identical or different. Macromolecules can be of synthetic, biological, or a combination of both. They can include 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.

The term "polymer" denotes a macromolecule comprising repeating structural units, i.e. monomers, linked by chemical bonds in a linear, circular, branched, crosslinked, dendrimeric manner or a combination thereof. It is understood that a polymer can for example also comprise at least one functional group. A polymer is called "homopolymer" if the polymer is composed of the same monomers and is called "copolymer" if the polymer is composed of different monomers.

According to one characteristic of the invention, which will be described in greater detail below, the anchoring molecules or solubilizing protecting groups or anchoring matrix are polyolefins, or more precisely polyolefin oligomers (polyolefins also being called polyalkenes) and their derivatives, i.e. they carry at least one functional group. In a preferred embodiment, the process according to the invention uses polyolefins, or more precisely polyolefin oligomers (polyolefins also being called polyalkenes), and their derivatives as anchoring molecule or protective group or anchoring matrix, 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 non-polar liquid phase. Polyolefin molecules consist of a chain of carbon atoms connected by single bonds. They can comprise branches made up of identical or different alkyl groups, but preferably identical. Preferably, the polymers consist of a number of monomer units of at least 10 and preferably between 15 and 50. Homopolymers are preferred, but co-polymers (saturated or not) 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%. In a preferred embodiment, these are polyisobutenes (PIB) derivatives, a class of polymers known since the 1930s of the last century, but polypropylene derivatives can also be used.

These anchoring molecules used in the process according to the invention are preferably in the form of functionalized derivatives. Schemes 4 and 5 above show a number of GDP derivatives with their functionalizations which are suitable for carrying out the present invention.

According to one characteristic of the invention, these anchoring 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 engaged GDP derivatives are suitably functionalized. This functionalization of the anchor molecules is generally in the terminal position, that is to say preferably at one of the ends of the chain of carbon atoms.

According to the invention, the multifunctional monomers (or oligomers) can be functionalized with derivatives of PIB via a covalent bond, in the form of ester, ether, thioether, thioester or any other chemical functions compatible with the present invention. process. This amounts to playing a role of protective group solubilizing monomers (or oligomers).

Polyolefin oligomers used as anchor molecules are typically characterized by a weight average molecular weight, but "pure" oligomers which have identical molecules of a given chain length can also be used.

The reaction between the anchor molecule and the monomer (or oligomer) results in a new molecule with low solubility in water (<30 mg / ml).

According to another characteristic of the invention, the functionalization of the PIB, by chemical reactions, leads to various molecular structures, capable of playing the role of solubilizing protective groups, of an at least bifunctional molecule (or intermediate) of interest, during a multi-step synthesis. It is understood that the protecting group is also at least monofunctional. Otherwise, the chemical function not involved in the bond between the GDP derivative and the molecule (or Intermediate) of interest must be inert or suitably protected, to avoid any parasitic products or side reactions.

According to another characteristic of the invention, the chemical functions of the monomer (or of the oligomer) not involved in the covalent bond with the GDP derivative, directly or indirectly, must be passive or suitably protected, in order to avoid the formation of products. Unwanted.

In a further aspect of the invention, the molecule resulting from the reaction between the derivative of PIB 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). It is in this sense that the derivative of PIB acts as a solubilizing molecule.

According to another characteristic of the invention, the molecule resulting from the reaction between the derivative of PIB and a monomer (or oligomer), via the formation of a covalent bond, directly or indirectly, is characterized in that the derivative of PIB significantly increases the solubility of the monomer (or oligomer) in nonpolar 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 of a water / ethanol mixture or even water. / acetonitrile), making the purification process simple, fast and inexpensive.

The protective 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 which can dissolve the reactants, at a appropriate temperature. Applicable solvents, pure or in mixtures, include, but are not limited to, halogenated and non-halogenated hydrocarbons. The preferred solvents are dichloromethane and toluene (alone or in the presence of N-N-dimethylformamide).

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 the methods known to those skilled in the art. By way of non-exhaustive examples, the reactions applicable include esterification reactions, amidation reactions or etherification reactions. Consequently, the reaction conditions (solvent (s), temperatures), concentration (s), time (s)) must be adapted for each protection reaction.

Depending on the chemical nature of the bond between the monomer (or the oligomer) and the anchor molecule, the deprotection steps can be carried out using reaction conditions known to those skilled in the art. Without being exhaustive, we can cite saponification, hydrolysis and hydrogenolysis. More specifically, the method of 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. It is in that the anchor molecule acts as a solubilizing molecule and a protecting group for a chemical function of the monomer (or an oligomer).

The monomer (or oligomer), suitably protected and bound to a covalently anchoring molecule (of various kinds), is characterized in that its solubility in water is low (<30 mg / ml). It is in this sense that the anchor matrix acts as a solubilizing entity. This derivation significantly increases the solubility of the new molecule to such an extent that it becomes soluble in nonpolar organic solvents. Consequently, the monomers (or oligomers) anchored on a derivative of PIB have a high partition coefficient (selective solubility (or selective distribution)) for the nonpolar organic phase during a liquid-liquid extraction in the presence of cyclohexane or heptane ( s) or hexane (s) and water or a water / ethanol mixture or else water / acetonitrile, thus allowing simple and rapid purification. The present invention opens up the possibility of convergent synthesis of long oligomers, which can be achieved using at least two suitably protected oligomer fragments, at least one of which is linked to an anchor molecule.

Reaction Scheme 6 below shows a complete sequence to produce an oligonucleotide. More precisely, in a first step called anchoring, a first unit of monomer (in this case a derivative of deoxyribose) protected by a protective 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 step called deprotection, the protective group (DMTr) is removed and trapped in an acidic medium in the presence of a scavenger and the product obtained is purified by extraction. liquid-liquid. It is preferable to carry out anchoring and deprotection (with entrapment) without isolation of the intermediate protected alcohol. Scheme 6: Synthesis of oligonucleotides.

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

Figure 7: Deprotection of a DMTr protective group

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

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, he can enter a new cycle for the addition of a third unit, and so on.

Reaction Scheme 8 below shows a complete sequence to produce an oligosaccharide. More precisely, in a first step called anchoring, a first unit of monomer (in this case a hexose derivative) protected or not by a first protective group, more resistant (GP) and by a second protective group or not, more labile, called temporary (GPt) is attached to a liquid support molecule in the anomeric position according to the invention; this compound is purified by extraction. In a second step called selective deprotection, said second protective group (GR) is removed and then the compound is purified by extraction. In a third step called glycosylation, a second unit of monomer (in this case a derivative of glycosyl donor protected or not) is added and coupled to said first unit to form a anchored disaccharide; reagents and side products are removed by liquid-liquid extraction. In a fourth step called global deprotection, 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.

Figure 8: Synthesis of oligosaccharides.

Among the anchoring molecules, preference is given to those which are capable of being produced by a polymerization technique from simple monomers. This is the case with polyisobutenes (PIB), which represent a particularly preferred type of anchoring molecule. The monomer of polyisobutenes, namely isobutene, can be manufactured industrially from bio-based raw materials, and PIBs can be prepared from bio-based isobutene by simple polymerization. Thus, the present invention can be implemented with biobased anchoring molecules, and in particular with biobased PIB. The concept of biobased content is defined in standard ISO 16620-1: 2015 <r Plastics - Biobased content - part 1: General principles ”, in particular by a definition of the terms“ biobased synthetic polymer ”,“ biobased synthetic polymer content ” , “Biobased carbon content” and “biobased mass content”, as well as in standards ISO 16620-2: 2015 “Plastics - Biobased content - part 2: Determination of biobased carbon content” and ISO 16620-3: 2015 “Plastics - Bio-based content - part 3: Determination of the content of bio-based synthetic polymer”, for the methods for determining and quantifying the bio-based character.

Advantageously, the anchoring molecules used in the context of the present invention have a biobased carbon content greater than 90%, preferably greater than 93%, and even more preferably greater than 95%.

The method according to the invention has many advantages.

A first advantage is that it makes it possible to obtain oligomers bound to a matrix with good purity by simple liquid-liquid extraction in an apolar organic solvent and water or a water / ethanol mixture or else water / acetonitrile or by filtration on silica, causing the elimination of by-products (salts, excess reagents or any other molecular species) which are not bound to the derivatives of polyolefins or oligomers of polyolefins or polyalkenes. Nonpolar organic solvents such as cyclohexane, heptane (s), hexane (s) which have flash points <15 ° C, are suitable for solubilizing the derivatives of polyolefins or of oligomers of polyolefins or polyalkenes during l extraction or washing. The process according to the invention therefore makes it possible to facilitate the purification steps and to produce less waste (effluents and stationary phase).

A second advantage, which is particularly interesting, is the possibility of automating the process according to the invention.

A third advantage is the possibility of recycling the extraction solvents as well as your anchoring molecules (polyolefins or oligomers of polyolefins or polyalkenes), in particular on an industrial scale. Indeed, these protecting groups can be easily removed at the end of synthesis by reactions usually used in organic synthesis (such as hydrolysis, saponification, hydrogenolysis or any other reaction compatible with the present process) and recycled. This proves that the process according to the invention is in line with green or sustainable chemistry, unlike current production methods. 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 molecules. (s) anchoring on monomeric units. A fifth advantage is the possibility of controlling the purity of the oligomer during synthesis, at each step by the different analysis techniques such as mass spectrometry, high performance liquid chromatography, nuclear magnetic resonance of the proton or carbon-13. A sixth advantage is the possibility of implementing, on an industrial scale, without expensive equipment.

Thanks to their high purity, the macromolecules produced by this process can be used as pharmaceuticals, cosmetics, phytosanitary products or agrifood products, or to access any of these products.

Yet another advantage is that the preferred anchor molecules, namely the polyisobutene derivatives, can be prepared from bio-based isobutene, as explained above.

Examples:

The following examples illustrate the synthesis of certain functionalized anchor molecules which can be used to implement the method according to the invention. 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 [Chem 9]

Has a mixture of the derivative of PIB-CH2-CH (CH3) -CH2-OMs (1 equivalent) and phenol (3 equivalents) in a toluene / N -N -dimethylformamide (1/1) (0.1 M) mixture, a Potassium carbonate (5 equivalents) was added then the reaction medium was heated at 120 ° C for 16 h then cooled to room temperature. The reaction medium was extracted three times with cyclohexane and with an acetonitrile / water or ethanol / water mixture (90/10), washed with brine, dried over sodium sulfate, filtered, concentrated under reduced pressure and the residue is purified by filtration. on silica, if necessary, to lead to the corresponding O-aryie derivative.

Example 2: [Chem 10]

The anchored aldehyde (1 equivalent) was dissolved in a tetrahydrofuran / ethanol (1/1) (0.1 M) mixture then cooled to 0 ° C for 5 minutes. Sodium borohydride (3 equivalents) was added to the reaction medium (in small portions) then the reaction medium was stirred at room temperature for 30 minutes. The reaction medium was concentrated under reduced pressure and then a 1N sodium hydroxide solution and cyclohexane were successively added to the residue. The organic phase was then washed with water, with brine, dried over sodium sulfate, filtered and concentrated under reduced pressure to yield the corresponding benzyl alcohol. Example 3: [Chem 11]

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

Example 4. [Chem 12]

To a mixture of the derivative of RIB-phenol (1 equivalent) and of methyl 4- (bromomethyl) benzoate (3 equivalents) in a mixture of toluene / NN-dimethylformamide (1/1) (0.1 M) was added carbonate of potassium (5 equivalents) then the reaction medium was heated at 120 ° C. for 16 h then cooled to room temperature. The reaction medium was extracted three times with cyclohexane and washed successively with an acetonitrile / water or ethanol / water mixture (90/10), 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: [Chem 13] To a solution of the RIB-phenol derivative (1 equivalent) in toluene (0.1 M), with stirring and at room temperature, was added succinic anhydride (2 equivalents) followed by triethylamine (3 equivalents). The reaction medium was heated at 60 ° C. for 16 h then cooled to room temperature. After adding a hydrochloric acid solution (1N), the reaction medium was extracted three times with cyclohexane and the organic phase was washed successively with an ethanol / water mixture (90/10), brine, dried. over sodium sulfate, filtered, concentrated under reduced pressure and the residue is purified by filtration over silica, if necessary, to yield the corresponding ester.

Example 6:

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

Example 7:

The anchored aldehyde (1 equivalent) was dissolved in a tetrahydrofuran / ethanol (1/1) (0.1 M) mixture then cooled to 0 ° C for 5 minutes. Sodium borohydride (3 equivalents) was added to the reaction medium (in small portions) then the reaction medium was stirred at room temperature for 30 minutes. The reaction medium was concentrated under reduced pressure and then a 1N sodium hydroxide solution and cyclohexane were successively added to the residue. The organic phase was then washed with water, with brine, dried over sodium sulfate, filtered and concentrated under reduced pressure to yield the corresponding benzyl alcohol. Example 8; [Chem 16]

To a solution of the PIB-alcohol derivative (1 equivalent) in dichloromethane (0.1 M) with stirring, under an inert atmosphere and at room temperature, were added succinic anhydride (1.1 equivalent) then, triethylamine (1, 2 equivalent). The reaction medium was heated at 40 ° C. for 18 h then cooled to room temperature.

At this stage, were successively added to the reaction medium: 5'-0- (4,4'-dimethoxytrityl) thymidine (1.1 equivalent), ethyl- (N-dNim-ethylamino) propylcarbodiimide hydrochloride (EDCI) (1.1 equivalent) and A- (N, N-dimethylamino) -pyridine (DMAP) (0.5 equivalent); then the reaction medium was heated at 40 ° C for 18 h. The reaction medium was evaporated then extracted with cyclohexane, washed 3 times with an ethanol / water mixture (90/10), brine, dried over sodium sulfate, filtered, concentrated under reduced pressure and the residue is purified by filtration through silica, if necessary, to lead to the corresponding anchored thymidine.

Example 9: [Chem 17]

To a solution of the PIB-carboxylic acid derivative (1 equivalent) in dichloromethane with stirring, under an inert atmosphere and at room temperature, was added 5 -0- (4,4'-dimethoxytrityl) thymidine (1.1 equivalent) , ethyl) NN-dimethylamino) -propylcarbodiimide hydrochloride (EDCI) (1.1 equivalent) and 4 - (N, N- dimethylamino) -pyridine (DMAP) (0.5 equivalent) then the reaction medium was heated at 40 ° C. for 18 h. The reaction medium was evaporated then extracted with cyclohexane, washed 3 times with an ethanol / water mixture (90/10), brine, dried over sodium sulfate, filtered, concentrated under reduced pressure and the residue is purified by filtration through silica, if necessary, to lead to the corresponding anchored thymidine.

Example 10: [Chem 18] To a solution of the derivative of 5'-0- (4,4'-dimethoxytrityl) thymidine anchored (1 equivalent) in a THF / H2O mixture (0.1 M), at room temperature, were added mercaptosuccinic acid (5 equivalents) then dichloroacetic acid (5% V) then the reaction medium was stirred for 1 h. The reaction medium was evaporated then extracted with cydohexane, washed 3 times with an ethanol / water mixture (90/10), brine, dried over sodium sulfate, filtered, concentrated under reduced pressure to yield the deprotected derivative of thymidine corresponding anchor.

Example 11: [Chem 19]

To a solution of the PIB-carboxylic acid derivative (1 equivalent) in dichloromethane (0.1 M) with stirring under an inert atmosphere and at room temperature, were added 5'-0- (4,4'-dimethoxytrityl) thymidine (1 , 1 equivalent), ethyl - (/ V, / V-dimethylamino) - propylcarbodiimide hydrochloride (EDCI) (1.1 equivalent) and 4 - {N, N- dimethylamino) - pyrldine (DMAP) (0.5 equivalent). Then the reaction medium was heated at 45 ° C. for 18 h and then evaporated.

The residue was dissolved in a THF / H2O (9/1) (0.1 M) mixture at room temperature, then were successively added mercaptosucdnic acid (5 equivalents), dichloroacetic acid (5% V) then reaction medium was stirred at room temperature for 1 h. The reaction medium was evaporated then extracted with cyclohexane, washed 3 times with an ethanol / water mixture (90/10), a saturated solution of sodium bicarbonate, brine, dried over sodium sulfate, filtered, concentrated under reduced pressure. to lead to the deprotected derivative of the corresponding anchored thymidine.

Example 12: [Chem 20]

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

At this stage, the residue was dissolved in a THF / H2O (9/1) (0.1 M) mixture at room temperature, then were successively added mercaptosuccinic acid (5 equivalents), dichloroacetic acid (5%). V) then the reaction medium was stirred at room temperature for 1 h. The reaction medium was evaporated then extracted with cyclohexane, washed 3 times with an ethanol / water mixture (90/10), an aqueous solution of sodium bicarbonate (10%), brine, dried over sulfate. sodium, filtered, concentrated under reduced pressure to yield the deprotected dimeric derivative of the corresponding anchored thymidine.

Example 13; [Chem 21]

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

Example 14: [Chem 22]

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

Example 15: [Chem 23]

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

Example 16: [Chem 24]

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

Claims

1. Process for the synthesis of macromolecules composed of U units which are predominantly monosaccharides or monosaccharide derivatives which may be identical or different, said macromolecules having a first and a second end, said synthesis process proceeding by successive elongation of said second end by a monomer or oligomer M having at least two functions, and said process being characterized in that:

- in a first step called anchoring, the first unit U1 of said macromolecule, corresponding to an M1 monomer or a terminal unit of an M1 oligomer, is attached by a covalent bond to an anchoring molecule soluble in organic solvents, said covalent bond resulting from a reaction of a first of the functions of said M1 monomer or of said M1 oligomer with a function of said anchoring molecule, the second termination is possibly another function of said M1 monomer or of said M1 oligomer having been protected beforehand to said reaction, by at least a first protection group GP1, and possibly by a second protection group GP2;

- in a second step called deprotection, one of said GP1 or GP2 protection groups is removed, leaving on said M1 monomer or said M1 oligomer a so-called free function;

- in a third step called coupling, a second M2 monomer or M2 oligomer is reacted, carrying at least one free function and at least one function protected by a GP3 protecting group, so that its free function forms, by reaction with said free function of said first M1 monomer or M1 oligomer, a covalent bond, thus creating a new molecule formed by said M1 monomer or M1 oligomer, attached by its first termination to said anchor molecule, and said M2 monomer or M2 oligomer, attached on another of its terminations, and said method being characterized in that said anchoring 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. Method according to claim 1, characterized in that an n-th Mn monomer or Mn oligomer is added by coupling.

3. Method according to one of claims 1 or 2, characterized in that after the coupling of the last monomer or oligomer, the deprotection of the protective groups of the macromolecule is carried out.

4. Method according to any one of claims 1 to 3, comprising at least one step in which said macromolecule attached to said anchoring 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. Method according to any one of claims 1 to 4, comprising a step in which said macromolecule is completely deprotected.

6. Method according to any one of claims 1 to 5, characterized in that said units of monosaccharides or monosaccharide derivatives are derivatives of pentoses (and in particular of nucleosides) or of hexoses.

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

8. Method according to any one of claims 1 to 7, characterized in that said anchor molecule has a mass average molecular weight of between 300 and 20,000, and preferably between 500 and 15,000.

9. Method according to any one of claims 1 to 8, characterized in that said polyolefin or polyolefin or polyalkene oligomer chain comprises a number of unsaturated carbon-carbon bonds not exceeding 5%, and preferably not exceeding 3%. .

10. A method according to any one of claims 1 to 9, characterized in that said polyolefin or polyolefin or polyalkene oligomer chain has been obtained by polymerization of a monomer, preferably biobased.

11. Use of a process according to any one of claims 1 to 10 for the synthesis of oligonucleotides or oligosaccharides.