Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/66—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood sugars, e.g. galactose
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
  • C07H15/02—Acyclic radicals, not substituted by cyclic structures
  • C07H15/04—Acyclic radicals, not substituted by cyclic structures attached to an oxygen atom of the saccharide radical
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/54353—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand attached to the carrier via a chemical coupling agent
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates

Definitions

• the present invention relates to silanizing agents comprising a terminal saccharide group and to their use for the functionalization of solid supports.
• the present invention also relates to the solid supports functionalized by these silanisation agents (sugar chips), as well as to their use, in particular for the biological analysis and in particular for the screening of saccharide molecules or protein ligands of interest .
• silanisation agents sucrose chips
• the development of microarray technologies has led to a significant advance in programs related to functional genomics. Indeed, the miniaturization of DNA deposition or synthesis techniques has led to the realization of parallelized analyzes, thus multi-parameterizable, of DNA on chips. More recently, the emergence of proteomics has given rise to the concept of protein chips (Zhu and Sydner, Current Op, in Chem Biol., 2003, 7, 55-63). These allow parallel analysis of protein / ligand interactions.
• glycoconjugates that is to say any molecule having a glycan-type domain, such as glycoproteins, glycolipids, proteoglycans, and more generally any molecule containing carbohydrates
• these carbohydrates are molecules formed by the assembly of simple monomeric blocks. These assemblages can be of natural origin, and possibly fractionated, or of synthetic origin.
• the various functions of molecules belonging to the carbohydrate family rely on the ability of carbohydrate structures to interact with a very large number of molecules. The analysis of the mechanisms of recognition between carbohydrates and other molecules is a field of research in full emergence.
• sugar chips are either the result of a deposit on a given substrate of a natural or synthetic saccharide substance (ex situ synthesis), or the result of a supported multiparallel synthesis (combinatorial chemistry) of different oligosaccharide sequences (synthesis in vitro). situ), representative of the molecular diversity of some large families of endogenous glycoconjugates such as heparans for example.
• ex situ synthesis or the result of a supported multiparallel synthesis (combinatorial chemistry) of different oligosaccharide sequences (synthesis in vitro). situ), representative of the molecular diversity of some large families of endogenous glycoconjugates such as heparans for example.
• the invention which will be described below is part of this technology being particularly well suited to the manufacture of sugar chips, including allowing the attachment of saccharide molecules on solid supports in a preparation process easier to put than the known methods of the prior art.
• a spacer arm makes the link between the surface of the solid support and the terminal functional unit characterizing the biochip: oligopeptides, oligonucleotides (Osborn HMI et al, Tetrahedron, 1999, 55, 1807-1850; Stetsenko DA et al, Bioconjugate Chemistry, 2001, 12, 576-586), or oligosaccharides (U.S. Patent No. 6,579,725).
• This spacer can play several roles at once: it is a binding molecule, that is to say that it makes it possible to connect the surface of the solid support with a functional molecule (probe);
• the inventors have therefore set themselves the goal of providing new silanization agents which make it possible to overcome all the disadvantages set out above.
• they aimed to provide new silanization agents for functionalizing, in a single step, the surface of a solid support with molecules of saccharide nature and this in a simple, reliable, flexible method as to the nature and length of the spacer arm and finally less expensive than the methods of the prior art. It is on this occasion that the Inventors have developed what is the subject of the present invention.
• the present invention therefore firstly relates to a silanization agent with a saccharide terminal function, characterized in that it corresponds to the following formula (I): A-X-B (I) in which:
• A represents a probe molecule of saccharide nature
• X represents a spacer arm consisting of a carbon or heterocarbon chain comprising two ends, one of its two ends connecting, covalently, said spacer arm X to A and the other end covalently connecting said arm; spacer X to B, said chain having at least one ethylenic unsaturation between its two ends; it being understood that said chain can not contain several acetylenic unsaturations;
• the present invention thus provides a silanized saccharide molecule, which can act as a modular spacer arm, the different structures of which influence the reactivity of the arm, that is to say its chemical, electrochemical and / or steric behavior.
• the probe molecule of saccharide nature constituting unit A of the compounds of formula (I) above may be of natural or synthetic origin, and may optionally be protected by one or more protective groups.
• This probe molecule may in particular be chosen from all the saccharide molecules to be fixed on a support, for example for reasons
• Elte ⁇ may in particular " be -synthetized in order to represent a molecule or biomolecule saccharide biological interest such as a heparan sulfate for example, or for the purpose of representing a saccharide chain acting itself as a spacer between a surface and a molecule or biomolecule of biological interest.
• the probe molecule of saccharide nature has a molecular weight of between 180 and 10 000 g / mol and even more preferably between 360 and 900 g / mol. It is preferably chosen from: i) monosaccharides and in particular from glucosamine, azidoglucosamine, D-ribose, D-xylose, L-arabinose, D-glucose, D-galactose, D-mannose , 2-deoxyribose, L-fucose, N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, N-acetylneuraminic acid, D-glucuronic acid, acid
• One, or more than one, of the hydroxyl and / or amine functions of the saccharide moieties of the probe molecule may be protected by one or more protective groups.
• These protecting groups are well known to those skilled in the art and are amply described in TW Greene et al, "Protective Groups in Organic Chemistry", Second Edition, A Wiley-Interscience Publication 1991.
• these protective groups are chosen from acetyl groups; benzyl; aryl and in particular aryl groups substituted with a radical R chosen from alkyl chains having from 1 to 40 carbon atoms; 2,2,2-trichloroethyloxycarbonyl (Troc); benzyloxycarbonyl (Z); trichloroacetamidate (TCA); tert-butyloxycarbonyl (BOC) and fluoranylmethoxycarbonyl (Fmoc).
• one or more of the hydroxyl and / or amine functions of the saccharide entities of the probe molecule may be substituted by one or more hydrophobic groups making it possible to make the spacer arm more specific and / or more selective towards the target molecule that will be fixed on the probe molecule and / or its role during the use of the spacer arm.
• the saccharide part can be rendered more or less hydrophobic.
• the anomeric part of the saccharide entities may be functionalized, like any glycosidic donor, by a group which will preferably be chosen according to the nature of the covalent bond ensuring the attachment of the Saccharide-type probe molecule at one of the two ends of the spacer arm X.
• the covalent bonds ensuring the attachment of each of the ends of the chain constituting the spacer arm X to the units A and B are derived from the reaction between a chemical function initially carried by the precursor of the spacer arm X and a complementary chemical function carried, on the one hand, by the probe-A molecule and on the other hand by the group silanized B.
• a chemical function initially carried by the precursor of the spacer arm X and a complementary chemical function carried, on the one hand, by the probe-A molecule and on the other hand by the group silanized B.
• the spacer arm X of the compounds of formula (I) according to the invention may be of variable length and structure. As has been seen above, it nevertheless still comprises at least one ethylenic unsaturation on the chain directly connecting A and B.
• the spacer arm X represents a linear or branched C 2 -C 40 alkyl or C 6 -C 40 aryl chain, said chain comprising at least one ethylenic unsaturation and which may optionally be interrupted by one or more heteroatoms selected from oxygen, nitrogen, sulfur and silicon, and / or one or more functions such as amide, oximes and tertiary amine functions and / or optionally substituted by one or more substituents ( preferably from 1 to 10 substituents) chosen from linear or branched C 2 -C 20 alkyl or C 6 -C 20 aryl chains, said chains optionally possibly being also interrupted by one or more heteroatoms chosen from oxygen, nitrogen, sulfur and silicon.
• the silanized group B is preferably chosen from the groups -Si (Ri) 3 , -SiRi (R 2 ) 2 and -SiRiR 2 R 3 in which the radicals R], R 2 and R 3 , independently of each other, represent a halogen atom such as fluorine or chlorine, Ci-C 4 alkoxy radical, a Ci-C 4 alkyl, amino or an ester radical.
• the methoxy and ethoxy radicals are particularly preferred and the alkyl radicals defined for the radicals R 1, R 2 and R 3 , the methyl and ethyl radicals are most particularly preferred.
• ester functions defined for the radicals R 1 , R 2 and R 3 mention may in particular be made of the acetoxy radical.
• silanized terminal unit B there may be mentioned trimethoxysilyl, triethoxysilyl, trimethylsilyl and triethylsilyl groups.
• A is chosen from monosaccharides, oligosaccharides and polysaccharides, and even more particularly from oligomers such as Glc-Glc-Glc; Lac-Lac or Gal-Gal-Gal-Gal-Gal in which the abbreviations GIc, Lac and GaI respectively denote glucose, lactose and galactose;
• X represents a carbon chain having from 2 to 40 carbon atoms, comprising at least one ethylenic unsaturation, said chain being linear or branched, and optionally interrupted by one or more rings and / or one or more functions such as the amide functions, oximes and tertiary amines;
• B represents a trimethoxysilyl or triethoxysilyl group.
• formula (I) there may be mentioned the compounds corresponding to formulas (1-1) and (1-2) below:
• silanizing agents of formula (I) above can be easily prepared according to the principles of organic synthesis well known to those skilled in the art depending on the nature of the units A, X and B.
• these silanizing agents can generally be prepared by a simple assembly of the units A, X and B, said units being either previously prepared or commercially available, it being understood that said units contain the chemical functions suitable for the formation of a covalent bond on the one hand between the unit A and one end of the spacer arm X and, on the other hand, between the unit B and the other end of the spacer arm X.
• the reactions used are generally conventional glycosylation reactions of A on X followed by hydrosilylation reactions (of Karstedt for example) to hang B.
• the silanizing agents of formula (I) according to the invention can be used to the functionalization of solid supports.
• the present invention therefore relates to the use of at least one silanizing agent of formula (I) as defined above, for the functionalization of solid supports, and in particular for the manufacture of sugar chips.
• silanization agents of formula (I) advantageously makes it possible to rapidly modify the surface of solid supports by a stable layer carrying easily sacrificial saccharide-type probe molecules of the support, given the presence of at least one ethylenic unsaturation on the spacer arm "X of the compounds of formula (I) according to the invention.
• the compounds of formula (I) according to the invention have the following advantages:
• the unit X of the compounds of formula (I) according to the invention allows in particular the connection with a very wide range of saccharides, oligosaccharides or polysaccharides.
• the compounds of formula (I) can be used either as spacer arm of a first saccharide unit, for example an oligosaccharide unit, which will be attached to unit X and then it is possible to grow (combinatorial chemistry on solid support) or for the attachment of presynthesized saccharide probe molecules (attachment to the X unit, in the anomeric position of their reducing part).
• the spacer arm is cleavable: thanks to the presence of at least one ethylenic unsaturation on the unit X compounds of formula (I) that can be easily opened and targeted to isolate the sugar from the solid phase, and this under conditions that do not alter the integrity of the saccharide probe molecule.
• ozonolysis, Grubbs metathesis or dihydroxylation followed by oxidative cleavage of diol-osmylation (OsO 4 , NaIO 4 ) as well as other known mild chemical cleavage reactions can be used as the cleavage method. the skilled person.
• the present invention also relates to a method for preparing a solid support functionalized by probe molecules of saccharide nature, characterized in that it comprises at least one step of silanization of at least one surface of a -solid support -with a solution of at least one silanizing agent of formula (I) in an organic solvent.
• the organic solvent is preferably chosen from trichlorethylene, toluene, and lower alcohols such as ethanol or methanol, these solvents optionally being supplemented with a basic compound such as triethylamine or N 5 N-diisopropylethylamine (DIEA). ).
• the contacting of the solid support with the solution of the silanizing agent of formula (I) is preferably carried out at a temperature of between 4 and 80 ° C., for about 1 to 48 hours.
• the substrate is then rinsed with the reaction solvent or with chloroform and dried, preferably with nitrogen.
• This method has the advantage of being simple to implement and to combine the silanization step with the step of functionalization of the solid support, whereas the methods known from the prior art required at least three successive steps; that is to say a first step of silanization of the surface of the solid support with a molecule having a functional group allowing the attachment of a spacer arm in a second step and finally the attachment of a saccharide probe molecule in a third step, for example according to a glycosylation reaction.
• a step of inactivation (“capping" non-glycosylated sites) was then necessary, which is not the case according to the method of the present invention.
• the solid supports that can be functionalized with the silanizing agents of formula (I) according to the invention are preferably chosen from glass, silica or other materials known to those skilled in the art as being capable of being used. silanized.
• These solid supports have at least one flat surface or not, smooth or structured, and may be for example in the form of blade, flat or well plate, capillary or porous ball or not.
• the present invention therefore also relates to solid supports characterized in that they comprise at least one functionalized surface with one or more silanizing agents of formula (I) as defined above.
• Such carriers constitute sugar chips which are for example capable of being used for the identification, by screening, of saccharide molecules and in particular oligosaccharide sequences recognizing a particular protein of interest, for example using the method described in international application WO-A-03/008927.
• sugar chips according to the present invention can also be used for the identification, by screening, of ligands, for example protein ligands recognizing a saccharide of interest.
• the subject of the present invention is finally a method for screening saccharide molecules, and in particular oligosaccharide sequences, or, respectively, protein ligands, characterized in that it comprises at least one contacting step of a solid support comprising at least one surface functionalized with at least one silanizing agent of formula (I) as defined previously with a solution containing one or more potential oligosaccharide molecules or, respectively, one or more potential protein ligands.
• the functionalized solid supports in accordance with the present invention make it possible to optimize the screening methods and thus to dispose more efficiently and more rapidly of molecules for therapeutic or biotechnological purposes.
• the invention also comprises other provisions which will emerge from the description which follows, which refers to an example of preparation of a compound of formula (I) according to the invention as well as an example of functionalization of a solid support with a compound of formula (I) according to the invention.
• the spacer arm (3) is obtained in two successive steps: 2-vinyl-3-chloro-tetrahydrofuran (2) is accessible from 2,3-dichlorotetrahydrofuran (1) by treatment with a Grignard reagent according to the method described by L. Crombie, and RD
• the compound (3) is obtained in a yield of 93%.
• the preparation of the glucose derivative (5) is carried out according to the following reaction scheme B:
• the glucose derivative (5) (thioglycoside) is obtained from D-Glucose in two successive stages which are conventional reactions of the chemistry of sugars:
• This glycosylated spacer arm is prepared according to the following Reaction Scheme C:
• the coupling of these two molecules is a glycosylation reaction of a protected glucose on an unsaturated chain.
• the thioglycoside (5) 500 mg, 1.13 mmol, 1 eq.
• the unsaturated spacer arm (3) 127 mg, 1.13 mmol, 1 eq.
• Molecular 630 mg. This mixture is stirred for 30 minutes at room temperature, then the reaction medium is brought to a temperature of -30 ° C.
• N-iodosuccinimide (NIS) 510 mg, 2.26 mmol, 2 eq.
• NIS trifluoromethanesulfonic acid
• the substrate then . It was washed with deionized water and then soaked for 1 hour in 0.2 N hydrochloric acid. was again washed with deionized water and then dried in an oven at a temperature of 80 ° C. for 30 minutes
• the rehydrated substrate was soaked in 10 ml of a solution of trichlorethylene (TCE) containing 10 mM (21 mg) of compound of formula (I-1) as prepared in Example 1 above. After 1 night at ambient temperature, the substrate was silanized with the compound of formula (I-1) according to the invention.
• TCE trichlorethylene
• the thus functionalized substrate was then washed with TCE, with ethanol and finally with chloroform. It was then dried in an oven for 30 minutes at a temperature of 50 ° C.
• the functionalized substrate was stored under an inert atmosphere (argon or nitrogen).
• This substrate can then be used for the preparation of a sugar chip (growth of oligosaccharides on the silanized substrate thus prepared) or of any other molecule chip or biomolecule (in this case, the sweet silane which functions the substrate becomes a spacer for the attachment of any new molecule or biomolecule, natural or synthetic).

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