Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H3/00—Compounds containing only hydrogen atoms and saccharide radicals having only carbon, hydrogen, and oxygen atoms
  • C07H3/02—Monosaccharides
• • 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
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
  • Y02P20/00—Technologies relating to chemical industry
  • Y02P20/50—Improvements relating to the production of bulk chemicals
  • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

• MOLECULAR SPACER ARM MANUFACTURING METHOD, AND USES ON AN ANALYSIS CHIP WITH MOLECULES OR BIOMOLECULES
• 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.
• 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”).
• Lab-On-Chip lab-on-chip
• Cell-On-Chip cell chips
• 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.
• 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
• 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:
• [Gp] represents a protective group for the secondary amine -N- or a molecule participating in the functionality of the spacer arm;
• 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;
• 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.
• 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.
• n, m and p can also be chosen independently of each other so that 1 ⁇ n, m and p ⁇ 30, preferably so that
• 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 40 ⁇ 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-R C1 to 40 arylamide
• N-TCA trichloroacetamidate (> N-C0-CC1 3 );
• N-BOC t-butyl carbamate (> NC (O) OCMe 3 );
• the protective group is chosen from Ac, BOC or a C1 to 40 aryl group.
• 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.
• 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.
• the molecular unit [mo] can be a natural or synthetic molecule.
• [mo] 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.
• 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.
• oligosaccharides mention may be made of sucrose, lactose, fragments of heparan sulfates, saccharide fragments of heparin, of chondroitin, of dermatan sulfates, Lewis antigens, etc.
• poly-oligosaccharides there may be mentioned saccharide parts of heparan sulfates, heparin, chondroitin, dermatan sulfates, etc.
• glyco-conjugates there may be mentioned heparanes sulfates, heparin, chrondroitin, dermatans sulfates, etc.
• chemokines cytokines
• insulin fibrinogen
• fibrinogen myosin
• myosin myosin
• hemoglobin etc.
• enzymes there may be mentioned oxidoreductases, transferases, hydrolases, lyases, isomerases, ligases.
• glycoproteins mention may be made of glycoproteins, mention may be made of immunoglobulin G, hyaluronic acid, etc.
• 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
• sphingolipids sphingosine + fatty acid + phosphate + amino alcohol
• non hydrolyzable lipids alkanes, carotenoids, sterols
• cholesterol cholesterol
• steroids estradiol, testosterone
• acids fatty acids
• eicosanoids etc.
• fatty acids there may be mentioned arachidonic acid, linoleic acid, linolenic acid, lauric acid, nervonic acid, palmitic acid, oleic acid, etc.
• 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.
• 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:
• the secondary amine can also be protected by a protective group.
• 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].
• the conventional organic chemistry processes known to those skilled in the art can be used.
• the method described in document [6] can be used for the nitrile reduction step.
• the ozonolysis method described in document [5] can be used for the step of forming an aldehyde function from an allylated function of a biological molecule.
• 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
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• ozonolysis a metathesis of Grubbs (Grubbs catalyst), or a dihydroxylation followed by an oxidative cut of diolosmylation (Os0, NaI0)
• Os0, NaI0 oxidative cut of diolosmylation
• 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] .
• 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.
• 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].
• 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.
• 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).
• 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).
• 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,
• 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).
• 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,
• 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 4 ⁇ 3 Si, M 232.39, 100 ⁇ l ): 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).
• Example F Metathesis reaction (reaction F) The chemical process used is described in document [9].
• the silanized CPG beads (9) (2 g, 30 ⁇ mol / g) are stirred in dichloromethane (20 ml), under a nitrogen atmosphere.
• the sugar-spacer system (5) (300 ⁇ mol,> 5 eq) is then added, in the middle, with a Grubbs catalyst (6 ⁇ mol, 5 mg,
• Example G Cleavage of the probe (reaction G)
• the chemical process used is described in document [10].
• system (10) solid support-spacer of the present invention-oligosaccharide 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).
• ozone O3 is bubbled into the reaction medium until a blue color appears.
• 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.
• 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,
• [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 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)
• 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:
• Example M In this example, [mo] is a sialic acid.

Landscapes

• Health & Medical Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Immunology (AREA)
• Engineering & Computer Science (AREA)
• Molecular Biology (AREA)
• Organic Chemistry (AREA)
• Hematology (AREA)
• General Health & Medical Sciences (AREA)
• Biochemistry (AREA)
• Urology & Nephrology (AREA)
• Biotechnology (AREA)
• Biomedical Technology (AREA)
• Food Science & Technology (AREA)
• Physics & Mathematics (AREA)
• Analytical Chemistry (AREA)
• Medicinal Chemistry (AREA)
• Microbiology (AREA)
• General Physics & Mathematics (AREA)
• Pathology (AREA)
• Genetics & Genomics (AREA)
• Cell Biology (AREA)
• Saccharide Compounds (AREA)
• Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)

Applications Claiming Priority (2)

Publications (1)

Family

ID=34834238

Family Applications (1)

Country Status (6)

Family Cites Families (2)

• 2004
  • 2004-02-25 FR FR0450346A patent/FR2866649A1/fr not_active Withdrawn
• 2005
  • 2005-02-22 CN CNA2005800055588A patent/CN1922195A/zh active Pending
  • 2005-02-22 EP EP05728104A patent/EP1718657A1/de not_active Withdrawn
  • 2005-02-22 JP JP2007500273A patent/JP2007523945A/ja active Pending
  • 2005-02-22 WO PCT/FR2005/050117 patent/WO2005085263A1/fr not_active Application Discontinuation
  • 2005-02-22 US US10/590,137 patent/US20080044838A2/en not_active Abandoned

Non-Patent Citations (1)

Also Published As

Similar Documents

Legal Events