Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K9/00—Peptides having up to 20 amino acids, containing saccharide radicals and having a fully defined sequence; Derivatives thereof
  • C07K9/006—Peptides having up to 20 amino acids, containing saccharide radicals and having a fully defined sequence; Derivatives thereof the peptide sequence being part of a ring structure
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
  • C07K17/02—Peptides being immobilised on, or in, an organic carrier
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
  • C07K17/02—Peptides being immobilised on, or in, an organic carrier
  • C07K17/06—Peptides being immobilised on, or in, an organic carrier attached to the carrier via a bridging agent
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
  • C07K7/50—Cyclic peptides containing at least one abnormal peptide link
  • C07K7/54—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring
  • C07K7/56—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring the cyclisation not occurring through 2,4-diamino-butanoic acid
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
  • C07K7/64—Cyclic peptides containing only normal peptide links

Definitions

• the present invention relates to the immobilization of recognition patterns on solid supports.
• the immobilization of molecules on solid supports can in particular enable the manufacture of biomolecule chips, in particular of sugar chips. These can especially be useful in methods of detection, analysis or screening.
• non-covalent immobilization methods which consist in adsorbing molecules on a surface via non-covalent interactions, in particular of the hydrophobic type, hydrogen bonds or ionic bonds.
• the surface used may in particular be glass covered with nitrocellulose or a polystyrene resin.
• covalently immobilized methods which consist in reacting a function present on the solid support with a function present on the molecule to form a covalent bond between the molecule and the support.
• Structures recognizing recognition patterns may be parts of molecules or compounds, molecules or compounds, or superstructures comprising such molecules, compounds, parts of molecules or compounds, including molecules or target compounds. These superstructures may be, for example, cells or microorganisms, such as bacteria or viruses.
• the inventors have discovered that a solid support on which the recognition motifs are fixed via a specific molecular chassis could make it possible to improve the detection threshold for certain structures, such as molecules or target compounds.
• the present invention relates to a solid support linked to at least one molecular chassis for multivalently binding at least one recognition pattern or having at least one pattern of recognition in a multivalent manner.
• the term “molecular chassis” is intended to mean a molecule that can be bound to a solid support and that it is bonded, in particular covalently, to at least one recognition unit.
• the term “multivalently bound” means a plurality of bonds, each connected to at least one recognition motif.
• the molecular chassis is “multivalently linked” is meant in the sense of the present invention that the molecular chassis is linked to several recognition patterns, in particular several times to the same pattern of recognition, in particular via several links.
• each recognition pattern is linked by a link to the molecular chassis.
• the term "recognition motif” is intended to mean any type of molecule or compound that can be recognized by, or form a complex with, at least one other molecule or compound, part of a molecule or compound.
• recognition motif is intended to mean any type of molecule or compound that can be recognized by, or form a complex with, at least one other molecule or compound, part of a molecule or compound.
• the compounds there may be mentioned especially the receptors, proteins, enzymes and molecules present on or in cells.
• this solid support or chip has an excellent detection threshold, in particular compounds or entities having an affinity with the recognition patterns, particularly with respect to the compounds or entities that can or must be detected by said support or chip.
• This detection threshold may be less than or equal to 1 mM, in particular less than or equal to 0.5 mM, in particular less than or equal to 0.2 mM, more particularly less than or equal to 0.1 mM, or even less than or equal to 0.08 mM, or even less than or equal to 0.05 mM, or even more particularly less than or equal to 0.01 mM.
• This detection threshold may also be less than or equal to 1000 microg / ml, in particular less than or equal to 500 microg / ml, in particular less than or equal to 200 microg / ml, more particularly less than or equal to 100 microg / ml, or even lower or equal to 50 microg / ml, or even less than or equal to 20 microg / ml, or even more particularly less than or equal to 10 microg / ml, by weight of compound or entity to be detected with respect to the volume of composition, such as a solution or a suspension in which it is present.
• Figure 1 shows schematically the plate obtained in Example 1.
• FIG. 2 is the image of a lactose-specific lectin-FITC direct labeling obtained in Example 2.
• FIG. 3 is the image of a direct lectin-FITC labeling specific for N-acetylgalactose.
• FIG. 4 schematically represents the plate obtained in example 4.
• FIG. 5 is the image of the plate obtained in Example 4 after specific Lectin-FITC labeling of N-acetylgalactose on the scanner.
• FIG. 6 shows the functionalization of a glass plate by a molecular chassis, the oxidation, the deposition of ligands (respectively N-acetylgalactose and mannose), and then the revelation by labeling.
• the molecular chassis can have several links with recognition patterns, it can in particular be linked several times with several identical recognition patterns, or with several different recognition patterns.
• a molecular chassis that is multivalently linked to at least one recognition pattern can be represented as follows:
• CM represents a molecular chassis
• MR 1 , MR 2 , MR 3 , ..., MR n each represent a pattern of recognition identical or different, n representing an integer greater than 1, in particular greater than or equal to 2, in particular greater than or equal to 3, or even greater than or equal to
• a multivalent grafting by the ratio of number of links or links between the molecular chassis and recognition patterns / number of links or links between the molecular chassis and the solid support. In this case it is greater than 1, especially greater than or equal to 2, in particular greater than or equal to 3, or even greater than or equal to 4.
• the molecular chassis has at least two faces, in particular it has two faces.
• this molecular chassis may be a cyclopeptide, in particular defining two faces, an upper face and a lower face.
• the molecular chassis may have several recognition patterns grafted on its upper face, in particular several times the same pattern or different recognition patterns each grafted one or more times.
• the solid support is bonded to the molecular chassis, in particular by the lower face thereof, in particular by at least one covalent bond, more particularly by an oxime bond.
• the molecular chassis may be a cyclopeptide formed from 5, 10 or 14 amino acid residues, in particular 10 amino acids forming a cyclodecapeptide.
• This cyclopeptide may have at least one elbow, in particular two elbows, in particular to form the (L) PrO- (D) AA or (D) Pro-
• This cyclopeptide may also have a central symmetry.
• the cyclopeptide may have 10 or 14 amino acid residues and form two elbows, each elbow being formed by a combination (L) Pro- (D) AA or (D) Pro- (L) AA, wherein AA is an amino acid and preferably glycine, the two elbows being separated by three and / or five amino acid residues.
• amino acid residue of the elbow shown above by the acronym AA may be an amino acid residue other than proline and of opposite stereochemistry, it may be in particular the glycine residue.
• the elbows are separated by amino acid residues, in particular by an odd number of amino acid residues and in particular by three and / or five amino acids for a cyclodecapeptide and a cyclotetradecapeptide, respectively.
• the three and / or five amino acid residues may each have a chemical function protected orthogonally by a protecting group.
• the side chain protecting groups of these amino acids move alternately on either side of the median plane of said frame and define a so-called lower and upper face relative to this plane.
• the molecular chassis is a cyclodecapeptide of formula (I) below:
• Protected chemical entity means a chemical entity bearing a protective group. These groups are conventionally known to those skilled in the art and are described in reference works, especially T. Green's Protective Groups in Organic Synthesis, P.G. M. Wuts, Wiley-Interscience, New York, 1999.
• masked chemical entity means a chemical entity carrying a group or a residue that makes it possible to hide the said chemical entity.
• a residue may be an amino acid residue, for example serine.
• X 1 , X 2 , X 3 , X 4 and Y may represent entities carrying at least one functional group chosen from the group comprising the amine, hydroxyl, thiol and hydrazide functions, and in particular aldehyde and oxyamine.
• the solid support may in particular be in the form of a plate, in particular well plates, ball plates, in particular porous plates, in particular microbeads, capillaries or chambers, such as closed cavities constituting micro-components with micro-structured surfaces, nanostructures including carbon nanotubes.
• the solid support may in particular comprise, or consist of, at least one material selected from the group consisting of glass, silicon, semiconductor oxides, for example silicon oxide, plastic, gold, metal oxides , such as indium and tin oxide, soils-gels, rare earths, as well as organic assemblies (carbon-based), such as carbon nanotubes.
• the solid support can be linked directly or indirectly to the molecular chassis.
• directly linked is meant that a spacer is connected to each of the entities mentioned or that the connection is via at least one spacer.
• a spacer can be any type of molecule that can bind with the entities to which it must be attached.
• they may be molecules separating the two entities by 1 to 20 atoms, in particular by 2 to 15 atoms, in particular by 4 to 10 atoms.
• the spacer has a carbon skeleton, optionally comprising at least one heteroatom, for example oxygen, sulfur, nitrogen or phosphorus.
• the molecular chassis is bound to the solid support by at least one bond, in particular a covalent linkage, which may be chosen from the group comprising the ether, ester, amine, amide, thioether, oxime, phosphate, alkene, alkyne, hydrazide and disulfide linkages. .
• a covalent linkage which may be chosen from the group comprising the ether, ester, amine, amide, thioether, oxime, phosphate, alkene, alkyne, hydrazide and disulfide linkages.
• the solid support is linked to the molecular chassis via an oxime bond.
• the reasons for recognition may be different Among the recognition patterns that can be used according to the invention, mention may be made of the molecules of interest, in particular of biological interest.
• recognition motifs mention may be made of the molecules chosen from the group comprising sugars, and in particular mono- or oligosaccharides, nucleic acids, peptides, proteins, as well as “mixed” molecules, such as glycopeptides, glycoproteins or phospholipids, or organic molecules, particularly those of therapeutic or diagnostic value, and a mixture thereof.
• Glucose Fructose, Galactose, Mannose, Rhamnose, Fucose, Glucosamine, Galactosamine, Mannosamine, N-acetylglucosamine, N-acetylgalactosamine, N-acetylmannosamine, and glucuronic acid.
• Galacturonic acid Mannuronic acid, N-acetylneuraminic acid, 3-deoxy-D-ma.m2 ⁇ -2-octulosonic acid.
• Recognition patterns may be related to the molecular chassis directly or indirectly.
• the recognition units may be linked to the molecular framework by at least one covalent bond, which may be chosen from ether, ester, amine, amide, thioether, oxime, phosphate, alkene, alkyne, hydrazide and disulfide linkages.
• the recognition motifs are linked to the molecular chassis via an oxime bond.
• the subject of the invention is also a method of manufacturing a solid support comprising at least one molecular chassis enabling presenting or having at least one pattern of recognition in a multivalent manner, comprising at least the step of grafting on the solid support at least one molecular chassis for presenting or having patterns of recognition multivalently on a support.
• grafted is intended to mean that a bond, in particular of covalent type, is formed between two chemical entities.
• the molecular chassis-recognition pattern complexes are grafted onto the solid support.
• This strategy consists of individually synthesizing and purifying the molecular chassis-recognition pattern complexes, in particular the molecular-sugar chassis, and then grafting them onto the solid support.
• the recognition patterns can be grafted onto the molecular chassis by a chemical bond resulting from the condensation of a function carried by the molecular chassis and a function carried by the recognition pattern.
• bonds making it possible to graft recognition motifs on the molecular frameworks mention may be made of the amide, ester, ether, amine, oxime, phosphate, alkene, alkyne, hydrazide and disulphide linkages.
• the molecular chassis comprises at least one oxyamine or aldehyde bond capable of reacting with at least one function present on the solid support, in particular to form an oxime bond.
• Recognition patterns can be grafted onto the molecular chassis, especially when the latter includes amino acid residues, using oxyamine chemistry, especially in the case where the recognition motifs are sugars.
• the molecular chassis may carry a carbonyl derivative group (aldehyde or ketone) and the sugar may be modified in the anomeric position by an oxyamine function (-ONH 2 ), or vice versa the sugar may carry a carbonyl function, in particular on its reducing end, and the molecular chassis can carry an oxyamine function (-ONH 2 ).
• At least one reactive function carried by the molecular chassis is protected or masked, in particular by a serine residue.
• the upper face of the molecular chassis carries one or more serines
• these can be oxidized, in particular by sodium periodate, so as to obtain glyoxylic aldehyde functions (-CO-CHO).
• the recognition motifs may in particular be sugars carrying an oxyamine function capable of reacting with the aldehyde functions of the molecular frameworks to form oximes bonds.
• the recognition motifs can be grafted onto the molecular chassis via a spacer.
• a spacer Among the types of possible grafting, mention may be made of the reaction of an aldehyde function present on the solid support with an oxyamine function present on the lower face of the molecular chassis. This reaction is, in general, effective and selective, it leads to the formation of an oxime bond.
• the solid support is bonded to the molecular chassis via an oxime bond.
• the grafting step of the molecular chassis carrying the recognition patterns on the solid support may be carried out by the deposition of solution drops comprising the molecules molecular chassis-recognition patterns, either manually, which gives a diameter of pads of about 1 mm, or by an automaton, which reduces the size of the pad, for example to 180 microns.
• the molecular chassis is grafted onto the solid support, and then the recognition motifs are then grafted onto the molecular chassis.
• This method of manufacturing a solid support allowing a multivalent presentation of recognition patterns may comprise at least the following steps: grafting the molecular frame onto the solid support, graft the recognition patterns to the molecular chassis.
• This method may further comprise at least one of the following steps:
• This embodiment is particularly interesting insofar as it can make it possible to produce a support having a large variety of recognition patterns from a single molecular chassis.
• the functions intended to react with the recognition patterns do not react with the functions present on the solid support, either by their very nature or because they are protected or masked.
• the immobilization of the molecular chassis on the solid support can be done by the reaction of a function carried by the solid support with at least one function carried by the molecular chassis, in particular located on the underside of the molecular chassis.
• the function carried by the solid support is an aldehyde function
• the function carried by the molecular chassis is an oxyamine, which leads to the formation of an oxime bond.
• This grafting step can be done by depositing a solution comprising the molecular frame on the solid support.
• the deposit can take place on the entire surface of this support or only in certain places.
• This step of grafting the molecular chassis may be followed by a step which makes it possible to mask the reactive functions of the unreacted solid support with the molecular chassis, for example by bringing the solid support into contact with a solution of hydroxylamine for hide the unreacted aldehyde functions.
• At least one recognition pattern is grafted onto the molecular chassis by reaction with at least one reactive function of said molecular chassis
• the method according to the invention may furthermore comprise a saturation step which may consist of absorbing a protein which does not specifically recognize the recognition motif, such as, for example, bovine serum albumin (BSA).
• BSA bovine serum albumin
• This step may in particular make it possible to avoid the non-specific absorption of proteins or targets on the surface during the step of recognizing the recognition pattern, for example by the protein to be detected.
• This saturation step can make it possible to reduce the background noise.
• the invention also relates to a chip comprising at least one solid support as defined above or obtained by a method as defined above.
• residues likely to act as a recognition motif mention may be made of the osidic residues involved in numerous pathologies, such as cancer (presence of tumor markers based on sugars), AIDS, or from pathogenic aggressions. and bacterial, pathogenic or bacterial agents may have on their surfaces recognition patterns, such as receptors, saccharide pattern.
• recognition patterns such as receptors, saccharide pattern.
• the invention can also be used in the context of the detection of pathogens in water or in air.
• the invention may also be useful in drug discovery, by recognizing antagonists or cell receptor agonists based on the recognition of sugars in the context of high throughput screening.
• the invention can also be used for studying the specificity and affinity of natural but also synthetic sugars.
• the typing of the cells and / or proteins involved in the recognition within the organism and a correlation with the structure of the sugar can also be envisaged.
• the present invention further relates to the use of molecular chassis for multivalently binding at least one recognition pattern or having at least one recognition pattern in a multivalent manner to functionalize a surface, particularly with sugars.
• Example 1 Preparation of a chip for detecting lectin
• an aqueous solution (A) comprising 30 ⁇ M of compound (A) of the following formula (II) z
• line A represents the spots obtained with solution (A)
• line B represents the spots obtained with solution (B)
• the line C represents the spots obtained with the solution (C)
• the line D represents the spots obtained with the solution (D).
• Direct labeling of a chip of Example 1 is then carried out with lectin-FITC specific for lactose. Specific detection of said lectin is then observed by the part of the chip presenting in a multivalent manner, in this case tetravalent, the lactose recognition pattern. The result is shown in FIG. 2. It can be observed in FIG. 2 that only the spots obtained with the molecular frameworks bearing four lactose units detect lactose-specific FITC lectins at 30 ⁇ M.
• Example 3 detection of lectin-FITC specific for N-acetylgalactose by a chip of Example 1
• Example 4 Preparation of a solid support on which is grafted a molecular chassis and then patterns of recognition
• An aqueous solution (E) comprising 50 ⁇ M of a molecular chassis (E) having the formula (II) in which X 1 , X 2 , X 3 and X 4 each represents a serine residue, and Z represents -NHCOCH 2 ONH 2 .
• a glass plate carrying aldehyde groups is functionalized by dipping it into the molecular chassis solution (E).
• a so-called saturation step is then performed by reacting the aldehyde functions of the unreacted glass plate with hydroxylamine, by dipping said plate in a 10 mM hydroxylamine solution.
• the serines are then oxidized to aldehydes by soaking the plate in a solution of 10 mM sodium periodate for 60 minutes.
• the resulting plate is shown schematically in FIG.
• Example 5 Preparation of a solid support on which is grafted a molecular chassis and recognition patterns
• An aqueous solution (E) comprising 50 ⁇ M of a molecular chassis (E) having the formula (II) in which X 1 , X 2 , X 3 and X 4 each represents a serine residue, and Z represents -NHCOCH 2 ONH 2 .
• a glass plate carrying aldehyde groups is functionalized by dipping it into the molecular chassis solution (E) for 30 minutes.
• a so-called saturation step is then performed by reacting the aldehyde functions of the unreacted glass plate with hydroxylamine by dipping said plate in a 10 mM hydroxylamine solution.
• the serines are then oxidized to aldehydes by soaking the plate in a solution of 10 mM sodium periodate for 60 minutes.
• FIG. 6 represents:
• Step 1 Functionalization of the glass plate by the molecular chassis (E) and saturation by NH 2 OH - Step 2: Oxidation of the serine residues into aldehyde
• Step 3 Deposit of drops of a 50 ⁇ M solution of N-acetylgalactose (line 1) or of mannose (line 2) carrying a -O-NH 2 function
• Step 4 Indirect labeling, Line 1 biotinylated lectin specific for N-acetylgalactose followed by CY3 streptavidin and line 2 biotinylated lectin specific for mannose and streptavidin CY3 (concentration 10 ⁇ g / ml).

Landscapes

• Chemical & Material Sciences (AREA)
• Organic Chemistry (AREA)
• Genetics & Genomics (AREA)
• Biochemistry (AREA)
• Biophysics (AREA)
• General Health & Medical Sciences (AREA)
• Health & Medical Sciences (AREA)
• Medicinal Chemistry (AREA)
• Molecular Biology (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Life Sciences & Earth Sciences (AREA)
• Peptides Or Proteins (AREA)
• Apparatus Associated With Microorganisms And Enzymes (AREA)
• Investigating Or Analysing Biological Materials (AREA)
• Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Applications Claiming Priority (2)

Publications (1)

Family

ID=37433950

Family Applications (1)

Country Status (6)

Families Citing this family (4)

Family Cites Families (2)

• 2006
  • 2006-02-20 FR FR0601472A patent/FR2897616B1/fr not_active Expired - Fee Related
• 2007
  • 2007-02-20 JP JP2008555833A patent/JP2009527755A/ja active Pending
  • 2007-02-20 US US12/224,191 patent/US20090221449A1/en not_active Abandoned
  • 2007-02-20 WO PCT/FR2007/000307 patent/WO2007096517A1/fr active Application Filing
  • 2007-02-20 CA CA002643042A patent/CA2643042A1/fr not_active Abandoned
  • 2007-02-20 EP EP07731015A patent/EP1994046A1/de not_active Withdrawn

Non-Patent Citations (1)

Also Published As

Similar Documents

Legal Events