EP1718657A1

EP1718657A1 - Molecular spacer, production method thereof and uses of same on an analysis chip with molecules or biomolecules

Info

Publication number: EP1718657A1
Application number: EP05728104A
Authority: EP
Inventors: Véronique PERETTI; Françoise Vinet; David Bonnaffe
Original assignee: Commissariat a lEnergie Atomique CEA
Current assignee: Commissariat a lEnergie Atomique et aux Energies Alternatives CEA
Priority date: 2004-02-25
Filing date: 2005-02-22
Publication date: 2006-11-08
Also published as: WO2005085263A1; US20070190570A1; FR2866649A1; US20080044838A2; CN1922195A; JP2007523945A

Abstract

The invention relates to a molecular spacer, to a method of fixing a molecular unit to a solid support and to the use of said spacer on analysis chips with molecules or biomolecules. According to the invention, the spacer has formula (I) wherein: X0, X4 = C, O, S, Se, N, P, As; X1-3 = C, O, N, S, Se, P, As, or aryl, heteroaryl at C1-6; Z1-2 = C-R, Si-R, N, P and As, where R = alkyl at C1-6; R1-3 = H, or alkyl, aryl, heteroaryl at C1-6; [Gp] = protective group of >N; n, m and n = integers = 1; [Sup] = H or a silanised solid support; and [mo] = H or a molecular unit which is intended to be fixed covalently by means of the spacer to the silanised solid support.

Description

MOLECULAR SPACER ARM, MANUFACTURING METHOD, AND USES ON AN ANALYSIS CHIP WITH MOLECULES OR BIOMOLECULES

DESCRIPTION

Technical Field The present invention relates to a molecular spacer arm, to a process for preparing the spacer arm connecting a molecular unit to a solid support, as well as to the use of this spacer arm on molecular analysis chips or biomolecules. In the following description, the references in square brackets [] refer to the list of references at the end of the description. The analysis chips targeted by the present invention are more particularly, but not exclusively, biochips and microsystems dedicated to biological analysis. They fall into three categories: DNA chips, lab-on-chip ("Lab-On-Chip") and cell chips ("Cell-On-Chip"). Currently, a new type of biochip is emerging: the sugar chip ("Glycochip"). This biochip is either the result of a deposit of a natural or synthetic substance, or the result of a supported multi-parallel synthesis (combinatorial chemistry) of different oligosaccharide sequences, representative of the molecular diversity of certain large families of endogenous glycoconjugates, for example heparan sulfates. The present invention is particularly well suited to this new type of biochip, in particular allowing the attachment of these molecules to biochip supports by an efficient and simplified chemistry process compared to the prior art. The molecules or biomolecules, called in the present “molecular units”, which can be fixed on a solid support by means of the spacer arm of the present invention can be for example nucleic acids (DNA or RNA), sugars, glycoproteins, glycolipids, etc. Still other examples are given below.

PRIOR ART In the majority of biochips, a spacer makes the link between the solid support [2] and, for example, the probes of oligopeptides, of oligonucleotides [3] or of oligosaccharides [4]. This spacer can play several roles at the same time: binding molecule, spatial distance arm, place of cleavage of the probe, etc. The proximity of the support to the target recognition sites by the probes can indeed hinder or even prevent the probe / target recognition, and therefore adversely affect the accuracy and the quality of analysis of the biochips. This is especially true when the probes are small, for example in the case of sugar fleas. The equation of principle below indicates the general diagram of the formation then of the cleavage of

1 'spacer, in which X' represents a solid support, and X '' a molecular unit. Precursor of the spacer arm Su pport activated ^ ^ ~ -N m olecule to be fixed hooking

Fixing of the tool to be fixed on the solid support by means of the formed space arm

Rupture of the spacer arm X '1 I N-

; ^ι Many spacer arms have been made to date, but these have a number of unresolved drawbacks. Indeed, their structure implies a severe limitation of the chemical processes usable for their fixation on the solid support and / or they do not allow to fix easily any type of biological molecule and / or they are so chemically stable that once fixed on the support solid their cleavage to recover the biological molecule cannot be done easily, and can lead to the deterioration of the latter or of the support. Document [4] (US-A-6, 579, 725) describes a spacer arm fixing oligosaccharides. This spacer arm, although more effective than those of the even more prior art, does not make it possible to solve all the aforementioned problems at the same time. It can also be noted that its length, its functionality, its reactivity and its size cannot always be generated at will.

SUMMARY OF THE INVENTION The present invention makes it possible precisely to solve the abovementioned problems of the prior art at a single time by providing a molecular spacer arm of formula (I) below:

(D - in which the substituents X °; X ¹ ; X ² ; X ³ X "Z ¹ ; Z ² ; R ¹ ; R ² ; and R ³ are such that: X ° and X ⁴ are each chosen independently of the others substituents from C, O, N, S, Se, P, As, Si; X ¹ ; X ² ; and X ³ are each independently chosen from the other substituents from C, O, N, S, Se, P, As, Si and from an aryl and a heteroaryl each comprising, for example, from 2 to 20 carbon atoms; • Z ¹ and Z ² are each chosen independently of the other substituents from CR, Si-R, C, N, P and As, where R is an alkyl comprising for example from 1 to 40 carbon atoms; • R ¹ ; R ² ; and R ³ are each chosen independently of the other substituents from H, an alkyl, an aryl and a heteroaryl each comprising from 2 to 20 carbon atoms;

• [Gp] represents a protective group for the secondary amine -N- or a molecule participating in the functionality of the spacer arm;

- in which n, m and p are whole numbers, each greater than or equal to 1 and chosen independently of one another, preferably so that 1 ≤ n, m and p ≤ 40;

- in which [Sup] represents H or a solid silanized support on which said spacer arm can be covalently attached; and

- in which [mo] represents H or a molecular unit intended to be covalently attached via said spacer arm to said solid silanized support. Of course, X ° to X ⁴ are atoms forming the skeleton of the spacer arm of the present invention, radicals chosen for example from H, O, alkyl, aryl, and a heteroaryl each comprising from 2 to 20 carbon atoms which can be attached to these atoms. This spacer arm (I) can be used, in general, to fix a molecular unit [mo] on a solid support [Sup], for example to make a biochip, or more advantageously a sugar chip, where [mo] is generally a molecule functionalizing said biochip. According to the invention, preferably, in the spacer arm [1] as defined above:

• X ° and X ⁴ can each be chosen independently of the other substituents from C, 0, N, S, Si; and or

• X ¹ ; X ² ; and X ³ may each be independently chosen from the other substituents from C, O, N, S, Si and from aryl and heteroaryl each comprising, for example, from 2 to 10 carbon atoms; and or

• Z ¹ and Z ² may each be chosen independently of the other substituents from C, N, CR, Si-R, where R is an alkyl containing from 1 to 30 carbon atoms, preferably from 1 to 20 carbon atoms, preferably from 1 to 10 carbon atoms; and / or • R ¹ ; R ² ; and R ³ may each be independently chosen from the other substituents from H, an alkyl, an aryl and a heteroaryl each comprising from 2 to 10 carbon atoms. According to the invention, n, m and p can also be chosen independently of each other so that 1 ≤ n, m and p ≤ 30, preferably so that

1 ≤ n, m and p ≤ 20, and more preferably so that 1 ≤ n, m and p ≤ 10. By way of example, according to the invention, in the spacer arm [1] as defined above above, X ° and X ⁴ are C; X ¹ ; X ² ; and X ³ are C; Z ¹ and Z ² are C;

R ¹ ; R ² ; and R ³ are H. According to the invention, the protective group

[Gp] can be any of the protective groups of secondary amines known to those skilled in the art. It is preferably chosen so that it resists the synthetic chemistry of the spacer arm, of its attachment to the support and of its attachment with the molecular unit [mo]. It can be chosen for example from Ac, Bn (benzyl), an aryl group (R) in Ci _{at 40Λ} Troc, z, ICA, BOC, Fmoc, etc., to form with the secondary amine of the spacer arm (I) one of the following chemical groups (> N- indicates the protected secondary amine):> N-Ac: acetamide (> N-CO- e);

> N-Bn: benzylamide;

> N-R: C1 to 40 arylamide;

> N-Troc: 2, 2, 2-trichloroethyl carbamate (> N-

C (0) 0CH ₂ CC1 ₃ ); > Nz: benzyl carbamate (> NC (O) OCH ₂ Ph);

> N-TCA: trichloroacetamidate (> N-C0-CC1 ₃ );

> N-BOC: t-butyl carbamate (> NC (O) OCMe ₃ );

> N-Fmoc: 9-fluorenylmethyl carbamate:

(Ph = phenyl and Me = methyl) Preferably, according to the invention, the protective group is chosen from Ac, BOC or a C1 to 40 aryl group. According to the invention, the molecule [Gp] participating in the functionality of the spacer arm can be, for example, alkyl or a Ci to 40 aryl, for example in Ci to 30 for example in Ci to 20 or in Ci to 10 • It can be any substituent, not necessarily protective, which can participate in the functionality of the spacer arm when is used. It may for example be a hydrophobic group, making it possible to make the spacer arm more specific and / or more selective with respect to the molecule [mo] to be fixed, and / or its role during the use of the spacer arm, for example on a sugar or protein chip. According to the invention, the solid support can for example be any support that can be silanized. They can be, for example, plates, beads or capillaries. It can for example be based on silica, glass, or other materials known to a person skilled in the art, for example for manufacturing the supports or surfaces of biochips. The silanization of the support can be carried out by any process known to those skilled in the art. According to the invention, the molecular unit [mo] can be a natural or synthetic molecule. It can be any molecule which must be fixed on a support, for example for analytical reasons. It can be a small molecule, for example having a molecular weight ranging from approximately 180 to 400,000 g.mol ^-1 . In the case of a sugar, [mo] may for example have a molecular weight ranging from 180 to 10,000 g.mol ^-1 . When it is a protein or a peptide, [mo] can for example have a molecular weight ranging from 5500 to 400000 g.mol ^-1 , generally ranging from 5500 to 220 000 g.mol ^-1 (weight of most proteins). This molecular unit [mo] can for example be chosen from monosaccharides, oligosaccharides, poly-oligosaccharides, glyco-conjugates, peptides, proteins, enzymes, glycoproteins, lipids, fatty acids, glycolipids, glycolipoproteins, etc. Among the monosaccharides, mention may be made of glucose, glucosamine, azidoglucosamine, D-ribose, D— xylose, L-arabinose, D-glucose, D-galactose, D-mannose, 2-deoxy-D-ribose, L-fucose , N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, N-acetylneuraminic acid, D-glucuronic acid, L-iduronic acid, D-sorbitol, D-mannitol, etc. Among the oligosaccharides, mention may be made of sucrose, lactose, fragments of heparan sulfates, saccharide fragments of heparin, of chondroitin, of dermatan sulfates, Lewis antigens, etc. Among the poly-oligosaccharides, there may be mentioned saccharide parts of heparan sulfates, heparin, chondroitin, dermatan sulfates, etc. Among the glyco-conjugates, there may be mentioned heparanes sulfates, heparin, chrondroitin, dermatans sulfates, etc. Among the peptides and proteins, mention may be made of chemokines, cytokines, insulin, fibrinogen, myosin, hemoglobin, etc. Among the enzymes, there may be mentioned oxidoreductases, transferases, hydrolases, lyases, isomerases, ligases. Among the glycoproteins, mention may be made of glycoproteins, mention may be made of immunoglobulin G, hyaluronic acid, etc. Among the lipids, mention may be made of hydrolysable lipids: fats (glycerol + 3 fatty acids), waxes (fatty acid + fatty alcohols), sterol esters (sterol + fatty acids), phospholipids (phosphatidic acids (glycerol, 2 acids fatty + phosphate)), phosphalides (glycerol + 2 fatty acids

+ phosphate), sphingolipids (sphingosine + fatty acid + phosphate + amino alcohol); non hydrolyzable lipids: alkanes, carotenoids, sterols

(cholesterol), steroids (estradiol, testosterone), acids (fatty acids), eicosanoids, etc. Among the fatty acids, there may be mentioned arachidonic acid, linoleic acid, linolenic acid, lauric acid, nervonic acid, palmitic acid, oleic acid, etc. Among the glycolipids, there may be mentioned galactosyl-ceramic, glucosyl-ceramide, gangliosides, cerebrosides (fatty acid + sphingosine + 1 sugar), gangliosides (fatty acid + sphingosine + many sugars, neuraminic acid), etc. Among the glycolipoproteins, there may be mentioned MPB83 (trade mark), GLP19 (trade mark) and IRBP (trade mark). The present invention also relates to a method of covalent attachment of a molecular unit [mo] to a solid support via a spacer arm, advantageously that of the present invention. The process can comprise the following stages: (i) reduction of the nitrile function of a compound of formula:

(ii) formation of an aldehyde function from an allyl function of a biological molecule of formula:

(iii) reductive amination between said reduced nitrile function followed by protection of the secondary amine formed and said aldehyde function to obtain an activated biological molecule for its fixation on the support, said activated biological molecule being of formula:

(iv) silanization of a solid support, and functionalization of the solid support silanized with a molecule of formula:

(v) metathesis reaction between the molecule functionalizing the support and the activated biological molecule to form a spacer arm according to the invention connecting the biological molecule and the support. In this process, the substituents X °; X ¹ ; X ² ;

X ³ ; X ⁴ ; Z ¹ ; Z ² ; R ¹ ; R ² ; R ³ ; and [mo] are as defined above. According to the invention, the compound of formula

may for example be an allylated sugar, [mo] being said sugar. This allylated sugar can be obtained by any process known to a person skilled in the art which does not alter the sugar. It may, for example, be the process described in document [5]. According to the invention, the secondary amine can also be protected by a protective group. Thus, the method of the invention can also comprise a step of fixing a protective group [Gp] on the secondary in innate function. The protective group can be as defined above. Its attachment to the secondary amine can be done by any chemical process known to those skilled in the art, for example according to one of the processes described in document [7]. To implement the various steps of this process of the invention, the conventional organic chemistry processes known to those skilled in the art can be used. Thus, by way of example, for the nitrile reduction step, the method described in document [6] can be used. For the step of forming an aldehyde function from an allylated function of a biological molecule, the ozonolysis method described in document [5] can be used. For the reductive amination step followed by protection of the nitrogen between the reduced nitrile and said aldehyde function to obtain an activated biological molecule, the process described in the document

[7] can be used. For the stage of silanization of the solid support and of its functionalization, the method described in document [8] can be used. For the metathesis reaction, the method described in document [10] can be used. The spacer arm of the present invention can therefore be created from three parts which are linked on the one hand by reductive amination followed by nitrogen protection on the side of the molecular unit, and on the other hand during a Grubbs metathesis reaction. Grubbs methatesis is for example described in document [11]. The nitrogen atom which is inserted into the carbon chain has several advantages: obtained during the attachment of two links, it is in the form of a secondary amine which can be protected in different ways to give a reactivity particular to the spacer. This advantageously modular function on a case-by-case basis by different protective groups or by a molecule participating in the functionality of the spacer arm, makes it possible to vary and control the hydrophilicity or the hydrophobicity of the spacer arm and to control its steric bulk. It is also advantageously possible to modulate the electrophilic / nucleophilic or acid / basic character of this part of the spacer arm: the nature of the protective groups of the nitrogen atom is therefore preferably chosen with the aim of optimizing reaction conditions , interactions, characterization operations or analyzes, whether before or after cleavage of the spacer to release the molecular unit. For example with an acetyl group, due to its small size, a small steric bulk is obtained, which allows optimization of molecular recognition when using the spacer arm, for example on a molecule chip. For example too, with a group butyl, a hydrophobic carbon substituent is obtained which makes this part of the spacer arm hydrophobic, which allows for example a more specific, more selective recognition of hydrophilic proteins with respect to the hydrophilic parts of the spacer arm ([mo]).

The present invention therefore provides a modular spacer arm (or “spacer”), the various structures of which influence the reactivity of the arm, that is to say its chemical and / or electrochemical and / or steric behavior. The present invention can be carried out in a simple and effective manner and the spacer advantageously has the following three properties, in particular when it is used for the manufacture of sugar chips: - first of all, the spacer does indeed have the function of arms making it possible to move the sweet chain away from the solid surface which supports this chain. - then the spacer is a cleavable arm: it is possible to easily and targetedly open the spacer to isolate the sugar from the supported phase. - Finally, the spacer, due to its low level of chemical functionality, remains inert under many reaction conditions carried out during organic syntheses on sweet units for example and during the use of sugar chips. In addition to the aforementioned advantages, the inventors noted the following during the various experiments of implementation of the present invention: - The spacer arm makes it possible to overcome the steric problems due to the presence of the solid support. It makes it possible to study, under good steric conditions, the protein / sugar interactions on the sugar chips obtained. It solves the problems of steric hindrance which have arisen in the prior art when approaching the protein towards the sweet ligands and which hinder future potential interactions. - The length of the arm is flexible: a judicious choice of functional counterparts of different sizes, in particular by the choice of starting reagents, makes it possible to prepare spacers of different sizes. - It is not only possible to choose the distance between the sugar chain and the solid support but also to control the hydrophilic or hydrophobic character of this part of the space by means of the protective grouping. - The simplicity of the chemical structure of 1 "spacer gives it properties of chemical non-reactivity during the numerous organic reactions during its manufacture and during the use of the sugar chip. - The spacer, due to its absence of interactive chemical functions has no influence on potential interactions with other molecules, when the system is used as part of a sugar chip or more generally a small molecule chip. - The spacer can be cleaved in a precise and selective manner at the level of its C = C double bond, under reaction conditions which do not alter the biological molecule, for example oligosaccharide. Indeed, one can conveniently use for cleavage for example an ozonolysis (0 ₃ ), a metathesis of Grubbs (Grubbs catalyst), or a dihydroxylation followed by an oxidative cut of diolosmylation (Os0, NaI0), and others mild chemical reactions known to those skilled in the art. - The fact that the spacer is easily cleavable, and that this cut does not modify the structure of the sugar, makes it possible to carry out structural and conformational analytical checks of the isolated oligosaccharide chain. It is also easy to calculate the quantity of sugar probes attached (“loading”) to the solid support during the sugar synthesis. One of the advantages of this spacer, in comparison with that described in document [1] (1 octenediol for example) lies in the adaptability of its length, its functionality, its reactivity and the steric hindrance which can be generated at will. The inventors also note that the spacer of the present invention allows the connection with a very wide range of saccharides, oligosaccharides or polysaccharides which are very often presynthesized and protected in the anomeric position of their reducing part by an allyl group. In one step, these sweet units can indeed be transformed to bind directly to the spacer. Thus, this spacer is advantageously compatible with many sweet molecules already synthesized and described in the literature, for example in documents [7], [12] and [13]. The present invention can for example be used for the manufacture of a sugar chip, for example a chip capable of identifying by screening oligosaccharide sequences recognizing a particular protein, for example according to the technique described in document [1] . In this application, the present invention makes it possible to optimize the screening methods and therefore to have more efficiently and quickly molecules for therapeutic or biotechnological purposes. It can be expected that this capability will also exist in the other applications of the present invention. The present invention can also be used on biochips where a spacer arm must make the link between the solid support and probes of oligopeptides, oligonucleotides and / or oligosaccharides. In particular, the spacer arm of the present invention can be used on an oligopeptide chip such as that described in the document [3], on an oligosaccharide chip such as that described in document [4]. Other characteristics and advantages will appear to those skilled in the art on reading the examples which follow, given by way of illustration.

Examples

1) By way of example, the synthesis of a length spacer corresponding to a chain of fourteen carbons is set out below, using operating protocols chosen from those accessible to those skilled in the art. References in bold refer to the reaction scheme below. The compounds chosen are: a monosaccharide (1) of the glucose type (1) (N-acethylglucosamine (GlcNac): allyl sugar in position 1) constituting the molecular unit [mo]; 4-pentenenitrile (3) carrying the nitrile function to be reduced; and 7-octenyltrimethoxysilane (8) for the functionalization of the support. The solid support consists of beads (6) Controlled Pore Glass (CPG) (trademark) based on silica. The reaction scheme below summarizes all of the chemical reactions undertaken in these examples for fixing an oligosaccharide (1) on a support (6) by means of a spacer arm in accordance with the present invention. These chemical reactions are indicated by the letters A to F. An example of a cleavage reaction of the spacer arm is presented in Example G below. On this reaction scheme, the groups

“R” indicated on the sugar have not been deliberately differentiated to simplify the representation. These

“R” represent substituents, identical or different from one another at different positions on the sugar-forming cycle, and possibly the substituents generally encountered on sugars. In the particular example presented here, the sugar used being N-acethylglucosamine, the skilled person has no difficulty in identifying the substituents "R" at the different positions of the compounds (1), (2), (5) and (10).

Example A: Activation of the oligosaccharide (reaction A) An ozonolysis reaction is used in this example. The process used is described in document [5] The allylated sugar in the anomeric position (1) (0.93 mmol) is dissolved in 5 ml of a mixture of dichloromethane and methanol (1/1): the medium is immersed in a cold bath at a temperature of -78 ° C (acetone + dry ice). Ozone O3 must then dabble in the solution: as soon as the blue color appears (characteristic of an excess of ozone), the ozone is replaced by argon (or nitrogen). Once the reaction is complete, the medium is made reducing by adding diethylsulphide Me ₂ S (4.65 mmol, 5 eq): dimethyl sulphoxide DMSO is then formed. The medium slowly rises to room temperature overnight, then is evaporated under vacuum: the organic residue is taken up in diethyleter Et ₂ 0, and washed with water. The organic phases are evaporated under vacuum, then co-evaporated with toluene. The crude product is purified by chromatography on a column of silica gel (eluent: petroleum ether / ethyl acetate: 8/2). Thus, the aldehyde (2) is obtained with a yield of 75%.

Example B: Reduction of a nitrile (reaction B) The chemical process used is described in document [6]. The lithium aluminum hydride LiAlH ₄ (381 mg, 10.03 mmol, 1 eq) is introduced into the freshly distilled diethylether (20 ml). 4-pentenenitrile (3) (814 mg, 1 ml,

10.03 mmol) is added slowly to the stirred reaction medium under a nitrogen atmosphere, at a temperature of 0 ° C

(ice bath). Agitation should continue for about 20 minutes at room temperature. Then water (0.4 ml), then a 20% sodium hydroxide solution in water (0.3 ml), and finally another amount of water (1.4 ml) are added: these additions must be carried out with great care because the neutralization can be violent. When the diethyl ether solution is decanted from the white inorganic residue, the supernatant is extracted. The white solid (residue) is washed twice with diethyl ether, and the organic phases are combined. A 3M hydrochloric acid solution is added to this organic phase to obtain an acidic pH (pH <7): the unreacted 4-pentenenitrile remains in the ethereal phase, while the amine passes into the aqueous phase. After extraction, the aqueous phase is therefore preserved and is added a 3 M NaOH sodium hydroxide solution to pass to a basic pH (pH> 7): the amino product will then pass into the ethereal phase during this new extraction. The ethereal phase thus extracted is dried over magnesium sulphate (MgS0 ₄ ), then evaporated under vacuum (rotary evaporator). The crude amine (4) is then purified by fractional distillation (ball oven, T≈96 ° C ± 9 ° C). ^X E NMR analysis (Brùcker AM 250): 5.82 (ddt, ³ J _tr a _ns = 18 Hz, ³ J _cis = 13 Hz,

³ J (H ^I ) = 6.5 Hz, 1H, CH =), 5.00 (m, 2H, CH ₂ =), 2.70 (t,

³ Jn) = 6.5 Hz, 2H, CHin), 2.10 (ttd, ³ J (Hn) = 6.5 Hz,

³ J (HC) = 6.5 Hz, ³ J (H ₂ C -) = 1.5 Hz, 2H, CHi), 1.70 (s, 2H, NH ₂ ), 1.56 (quint., ³ J (H _π ) = ³ J (H _m ) = 6.5 Hz, 2H, CH _IT .).

¹³ C NMR analysis (Brùcker AM 250): 138.6 (CH =), 114.6 (CH ₂ =), 42.0 (CH ₂ -N), 33.1 (CH ₂ , 31.4 (CH ₂ ).

Example C: Reductive amination (reaction C) The chemical process used is described in document [7]. The aldehyde (2) (20.87 mmol) is dissolved in dimethylformamide (1.2 ml) freshly distilled on calcium hydride (CaH ₂ ): the medium is stirred and the amine (4) (31.30 mmol, 2 eq) is added. After about twenty minutes, sodium cyanoborohydride NaBH ₃ CN (83.47 mmol, 4 eq) is added to the mixture, which is left to stir at ambient temperature overnight. If the reaction is not completed, it is possible to add NaBH ₃ CN (1 eq). Then, when the reaction is complete, pyridine (2.4 ml) and acetic anhydride Ac ₂ 0 (83.47 mmol, 2 eq / amine) are added to the mixture. When the reaction is complete (about 1 hour after the addition), the crude compound is extracted with diethyl ether and with water. The combined organic phases are dried over magnesium sulfate (MgSO ₄ ), then filtered, evaporated in vacuo then co-evaporated with toluene. Compound (5) is then purified by chromatography on silica gel (gradient of cyclohexane / ethyl acetate eluent from 7/3 to 5/5).

Example D: Functionalization of the solid support (reactions D and E) The chemical process used is described in document [7]. Controlled Pore Glass beads (6) (CPG,

500 A, 2 g) are shaken very gently for

2 hours at room temperature in a NaOH sodium hydroxide solution (700 mg) in EDI deionized water (6 ml) and 99% EtOH ethanol (8 ml). Then the balls are centrifuged, the supernatant is extracted, the beads are washed thoroughly with EDI to reach a neutral pH. The beads are then dried under vacuum (rotary evaporator), and remain for 1 hour at room temperature in a hydrochloric acid solution (HCl 0.2 N) before being washed with water, centrifuged, dried and then placed in the water. oven for 15 minutes at 80 ° C. They are then washed with ethanol, then with toluene (centrifuge). They are then dried before participating in the next silanization step, the reaction mixture of which has been prepared just before use. The CPG beads (7) are introduced into a mixture of toluene (45 ml), triethylamine Et ₃ N (1.35 ml) and 7-octenyltrimethoxysilane (8) (CnH ₂ 4θ ₃ Si, M 232.39, 100 μl ): the reaction medium is set at 80 ° C for 16 hours (oven). The beads are extracted from the mixture by centrifugation, and are rinsed with ethanol several times and then dried (rotary evaporator). They are then subjected to a temperature of 110 ° C for 3 hours to carry out the crosslinking step (oven). The silanization and crosslinking steps being thus carried out, it is necessary to neutralize the residual acidity and hydrophilicity of the surface silanols which have not reacted during the silanization step (“end-capping”). A solution of trimethylsilyl chloride IMSC1 (109 mg, 130 μl) and triethylamine Et ₃ N (506 mg, 700 μl) in the dichloromethane DCM (10 ml) is added to the silanized CPG beads, and the mixture is left under gentle stirring for 2 hours at 25 ° C. The beads are then rinsed thoroughly with dichloromethane (centrifuge), then with acetonitrile (centrifuge). They are then dried under vacuum

(rotary evaporator), and put in the oven (80 ° C) to complete the drying. The silanized beads (9) are thus obtained.

Example F: Metathesis reaction (reaction F) The chemical process used is described in document [9]. The silanized CPG beads (9) (2 g, 30 μmol / g) are stirred in dichloromethane (20 ml), under a nitrogen atmosphere. The sugar-spacer system (5) (300 μmol,> 5 eq) is then added, in the middle, with a Grubbs catalyst (6 μmol, 5 mg,

0.1 eq). The reaction medium is then brought to reflux, ie a temperature of 44 ° C. After 6 hours, another portion of Grubbs catalyst (6 μmol, 5 mg, 0.1 eq) is added. The mixture is kept at 44 ° C. for another 6 hours, then is brought back to room temperature. The beads are filtered, and washed thoroughly with dichloromethane and ethanol (centrifuge). The beads are then evaporated in vacuo to be dried. The sweet beads (10) are thus obtained. Reaction scheme of examples A to F

Example G: Cleavage of the probe (reaction G) The chemical process used is described in document [10]. When the system (10) (solid support-spacer of the present invention-oligosaccharide chain) has been obtained, it is possible to cleave the spacer, without denaturing the sugar chain. The experimental protocol is described in document [5]. The chemical equation is as follows:

The sweet CPG beads (10) are stirred slowly in a 1/1 dichloromethane / methanol mixture. The medium is brought to a temperature of -78 ° C (acetone + liquid nitrogen). Next, ozone O3 is bubbled into the reaction medium until a blue color appears. Then, argon bubble for a few minutes in the mixture, before neutralizing the medium with dimethylsulfide and then the reaction medium is left to return to room temperature overnight. The beads are taken up in diethylether, filtered, rinsed several times with diethylether and with water. The beads (12) are then put aside, and the supernatant is extracted (diethyl ether / water), the organic phase is dried over magnesium sulfate MgSO ₄ , evaporated in vacuo and co-evaporated with toluene. The product (11) is then obtained.

2) Other molecules according to the invention were manufactured using the operating protocols exposed above (examples A to F). These molecules are detailed in the following examples.

Example H: In this example, [mo] is an RGD peptide (Arg-

Gly-Asp) attached to the spacer arm of the invention by its C-terminal end. The support is the same as in the operating protocol described above. Thus, in this example, the molecule (1) of the reaction scheme of Examples A to F is replaced by RGD. Beads on which the RGD peptide is attached are thus obtained. They have the formula:

[mo]: RGD peptide hooked by C-terminal (Arg-Gly-Asp)

Example I: This example uses the same [mo] as in Example H, but attached to the spacer arm of the invention by its N-terminal end. Moreover,

in of the spacer arm of the invention, X ¹ is C and n = 20. By C is understood, of course, hydrogenated carbon. The beads obtained have the formula:

[mo] is an RDG peptide hooked by the N-terminus Example J: In this example, [mo] is a “sialyl- is is”. The support is the same as in the operating protocol described above. The protective group is Boc. Its fixing chemistry is known to those skilled in the art. Beads on which the sialyl-lewis has been attached are thus obtained. They have the formula:

[mo] is a sialyl-lewis a, with "1" = H or CH ₃

Example K:

In this example, [mo] is a sulfated compound. The support is the same as in the operating protocol described above. Beads on which the sulfated compound is attached are thus obtained. They have the formula:

[mo] is a sulphated compound, Z being a protective group (for example Gp defined in the part "description of the invention)

In another protocol, the carbon X ⁴ was replaced by a sulfur atom. The corresponding beads were obtained.

Example L: In this example, [mo] is a protected sugar. The support is the same as in the operating protocol described above. Beads on which the protected sugar is hung are thus obtained. They have the formula:

protected sugar

Example M: In this example, [mo] is a sialic acid.

The support is the same as in the operating protocol described above. Beads on which sialic acid is attached are thus obtained. They have the formula:

Sialic acx

Bibliographic references

[I] WO-A-03/008927: Dukler, N. Dotan, A. Shtavi, A. Gargir. [2] H.M.I. Osborn, T.H. Khan, Tetrahedron, 1999, 55, 1807-1850. [3] D.A. Stetsenko, M.J. Gai, Bioconjugate Chemistry, 2001, 12, 576-586. [4] US-A-6,579,725: P. H. Seeberger, R.B. Andrade. [5] R. Roy, C.A. Laferrière, Canadian Journal of Chemistry, 1990, 68, 2045-2054. [6] L.H. Amundsen, L.S. Nelson, Journal of the American Chemical Society, 1951, 13, 242-244. [7] J.F. Tolborg, K.J. Jensen, Chemical Communication, 2000, 147-148.

[8] F. Vinet, A. Hoang, EN 00 16940.

[9] K. Biswas, D.M., Coltart, S.J. Danishefsky, Tetrahedron Letters, 2002, 43, 6107-6110. [10] C. Sylvain, A. Wagner, C. Miokowski, Tetrahedron Letters, 1997, 38, 1043-1044.

[II] Q.J. Plante, E.R. Palmacci, P. H. Seeberger, Science, 2001, 291, 1523-1527.

[12] P.H. Seeberger, Chem. Corn., 2003, 1115-1121. [13] D.M. Ratner, E.R. Swanson, P.H. Seeberger, Org. Lett. 2003, 4717-4720.

Claims

1. Molecular spacer arm of the following formula (I)

- in which X ° and X ⁴ are modular substituents so as to allow a bond of [mo] and

[Sup] via said spacer arm, X ° and X ⁴ being different from H and each chosen independently of the other substituents of the spacer arm from C, O, N, S, Se, P, As, Si; and - in which the substituents X ¹ ; X ² ; X ³ ; Z ¹ ; Z ² ; R ¹ ; R ² ; and R ³ are such that:

• X ¹ ; X ² ; and X ³ are each independently chosen from the other substituents from C, O, N, S, Se, P, As, Si and from aryl and heteroaryl each comprising from 2 to 20 carbon atoms;

• Z ¹ and Z ² are each chosen independently of the other substituents from CR, Si-R, C, N, P and As, where R is an alkyl comprising from 1 to 40 carbon atoms;

• R ¹ ; R ² ; and R ³ are each chosen independently of the other substituents from H, an alkyl, a aryl and a heteroaryl each having 2 to 20 carbon atoms;

- in which n, m and p are whole numbers, each greater than or equal to 1 and chosen independently of one another, preferably so that 1 ≤ n, m and p ≤ 40; - in which [Sup] represents H or a solid silanized support on which said spacer arm can be covalently attached; and

- in which [mo] represents H or a molecular unit intended to be covalently attached via said spacer arm to said solid silanized support.

2. Spacer arm according to claim 1, in which • X ° and X ⁴ are chosen independently of the other substituents from C, O, N, S, Si; and or

• X ¹ ; X ² ; and X ³ are chosen independently of the other substituents from C, O, N, S, Si and from an aryl and a heteroaryl each comprising from 2 to 10 carbon atoms; and / or Z ¹ and Z ² are chosen independently of the other substituents from C, N, CR, Si-R, where R is an alkyl comprising from 1 to 30 carbon atoms; and or • R ¹ ; R ² ; and R ³ are chosen independently of the other substituents from H, an alkyl, an aryl and a heteroaryl each comprising from 2 to 10 carbon atoms.

3 spacer arm according to claim 1, wherein the protective group [Gp] is. chosen from Ac, Benzyle, an aryl group in Ci to C ₄₀ r Troc, z, TCA, BOC, F oc.

4. Spacer arm according to claim 1, in which the solid support [Sup], when present, is chosen from a plate, a ball or a capillary.

5. A spacer arm according to claim 1 or 4, in which [Sup] is based on silica or glass.

6. A spacer arm according to claim 1, in which [mo], when it is present, is a molecule having a molecular weight ranging from 180 to 400,000 g.mol ^-1 .

7. The spacer arm according to claim 1, in which [mo], when it is present, is chosen from monosaccharides, oligosaccharides, poly-oligosaccharides, glyco-conjugates, peptides, proteins, enzymes, glycoproteins, lipids, fatty acids, glycolipids, glycolipoproteins.

8. A spacer arm according to claim 1, wherein [mo], when present, is a sugar.

9. Use of a spacer arm according to any one of claims 1 to 8 for fixing on a solid silanized support [Sup] a molecular unit [mo].

10. Use according to claim 9, in which [mo] is a molecule having a molecular weight ranging from 180 to 400,000 g.mol ^-1 .

11. Use according to claim 9, in which [mo] is chosen from monosaccharides, oligosaccharides, poly-oligosaccharides, glyco-conjugates, small natural or synthetic molecules; and [Sup] represents a solid silanized support to which the spacer arm is capable of being fixed.

12. Use according to claim 9, in which [Sup] is chosen from a plate, beads or a capillary.

13. Use according to claim 12, in which [Sup] is based on silica or glass.

14. Use according to any one of claims 9 to 13 for the manufacture of a biochip.

15. Use according to any one of claims 9 to 13 for the manufacture of a sugar chip.

16. A method of covalent attachment of a molecular unit [mo] to a support by means of a spacer arm, said method comprising the following steps: (i) reduction of the nitrile function of a compound of formula:

(v) metathesis reaction between the molecule functionalizing the support and the activated biological molecule to form a spacer arm according to the invention connecting the biological molecule and the support; process in which the substituents X °; X ¹ ; X ² ; X ³ ; X ⁴ ; Z ¹ ; Z ² ; R ¹ ; R ² ; R ³ ; and [mo] are as defined in claim 1.

17. The method of claim 16, wherein the compound of formula

is an allylated sugar, [mo] being said sugar.

18. The method of claim 16, wherein [Sup] is selected from a plate, a ball or a capillary.

19. The method of claim 16 or 18, wherein [Sup] is based on silica or glass.

20. The method of claim 16, wherein [mo] is a molecule having a molecular weight ranging from 180 to 400,000 g. ol ^-1 .

21. The method of claim 16, in which [mo] is chosen from monosaccharides, oligosaccharides, poly-oligosaccharides, glyco-conjugates, peptides, proteins, enzymes, glycoproteins, lipids, fatty acids , glycolipids, glycolipoproteins.

22. The method of claim 16, wherein [mo] is a sugar.

23. The method of claim 16, further comprising a step of fixing a protective group [Gp] on the secondary amine function.

24. The method of claim 23, wherein [Gp] is selected from Ac, benzyl, aryl _Ci-4 o, Troc, z, TCA, BOC, Fmoc.

25. Use of a method according to any one of claims 16 to 24 for the manufacture of a biochip.

26. Use of a method according to any one of claims 16 to 24 for the manufacture of a sugar chip.