Method for functionalizing a solid support with a peptide conjugate

Abstract

Method for functionalizing a solid support with a peptide conjugate. The present invention relates to a method for functionalizing a solid support with a peptide conjugate comprising a benzophenone anchoring head, a spacer arm, and a peptide fragment. It also relates to a solid support obtainable by a functionalization method according to the invention, as well as the use of the solid support according to the invention.

Description

Title of the invention: Method for functionalizing a solid support with a peptide conjugate technical field

[0001] The present invention relates to the field of functionalization of surfaces, such as textile or plastic surfaces, by peptides, in particular antimicrobial peptides. Technological background

[0002] The medical industry faces numerous challenges. Among the main ones are infection control and reducing the spread of microorganisms such as fungi, bacteria, and viruses. Some of these microorganisms can attach to surfaces and form biofilms. Beyond the medical field, biofilms also pose a threat in the food processing industry. Biofilms have traditionally been treated with antibiotics, but this widespread use of antibiotics has contributed to the emergence of antibiotic-resistant strains. Therefore, it is now necessary to develop alternative methods to the use of disinfectants and antibiotics to eliminate biofilms and / or limit their formation.

[0003] To prevent the formation of biofilms on surfaces, one method that has proven effective is the application of antimicrobial coatings to these surfaces. Bacteria do not easily develop resistance to these antimicrobial coatings, which have a mechanical action and not just a chemical one. Antimicrobial coatings can be obtained by functionalizing surfaces with molecules exhibiting antimicrobial properties.

[0004] Patent application WO2014118779 describes, for example, a method for functionalizing surfaces with bifunctional molecules comprising a catechol anchor and a peptide portion comprising a difluorophenylalanine dipeptide. The purpose of the functionalization is to impart antifouling properties to the surface, notably preventing the formation of biofilms on the surface. The catechol anchor allows grafting onto surfaces by adsorption. Such grafting is less robust than covalent grafting.

[0005] It would be advantageous to have new methods for functionalizing surfaces with peptide conjugates, said methods providing efficient anchoring of the peptide conjugates to the surfaces. The peptide conjugates should advantageously be adaptive, so as to be usable in a wide range of applications and with a wide range of peptides. Thus, it would be in It is interesting to be able to implement functionalization processes with peptide conjugates in which the nature of the peptide, its distance from the anchoring head, and its position of attachment to the anchoring head in particular can be controlled and / or adapted.

[0006] In this context, the inventors of the present invention have demonstrated that it is possible to efficiently functionalize solid substrate surfaces with peptide conjugates comprising a benzophenone-type anchoring head, a spacer arm, and a peptide fragment that can be attached to the spacer arm at various points in its structure. This functionalization is particularly well-suited to plastic and / or textile substrates. Summary of the invention

[0007] Thus, the present invention relates to a method of functionalizing at least one surface of a solid support, comprising bringing at least said surface of said solid support into contact with at least one peptide conjugate of formula (I)

[0008] [Chem.l] in which L is a spacer arm, n is 0 or 1, preferably 1, and A is a peptide fragment, under conditions adapted to obtain the anchoring of at least one peptide conjugate of formula (I) to the surface of the support.

[0009] In one embodiment A is a peptide fragment comprising from 2 to 80 amino acids, preferably from 3 to 30 amino acids, in particular from 7 to 20 amino acids.

[0010] In one embodiment, A is a peptide fragment selected from the group consisting of an antibiotic peptide, an antimicrobial peptide, an antifungal peptide, an anti-inflammatory peptide, a catalytic peptide, a biological receptor ligand peptide, an antibody and an enzyme inhibitor peptide, preferably an antimicrobial peptide, or a fragment thereof.

[0011] In one embodiment, the peptide fragment is an antimicrobial peptide selected from the group consisting of the peptides H-(RF)4-NH2, H-(RI)4-NH2 and H-(R)2-Palm.

[0012] In one embodiment, L is a saturated or unsaturated aliphatic hydrocarbon chain comprising from 1 to 10 carbon atoms, optionally interrupted or terminated by at least one of a heteroatom, in particular O or S, an aryl group, a C=O group, an SO2 group, an NR group, wherein R is selected from a hydrogen atom, an aliphatic hydrocarbon radical comprising from 1 to 6 carbon atoms, a benzyl radical, and a phenethyl radical, said chain being able to be unsubstituted or substituted. Preferably, L is a carbon aliphatic chain of formula (II) [Chem 2] O in which m is an integer from 1 to 10, preferably from 4 to 6, in particular 5.

[0013] In one embodiment, the solid support is chosen from the group consisting of a plastic support and a textile support.

[0014] In one embodiment, the functionalization process comprises the following steps: a. Contacting at least said surface of said solid support with a solution or suspension of at least one peptide conjugate of formula (I) as defined above in a solvent, the contact being carried out preferably by soaking, spraying and / or incubation; b. Irradiation, in particular UV irradiation, of the surface in contact with the solution or suspension of at least one peptide conjugate of formula (I), for a duration suitable for obtaining the grafting of all or part of the peptide conjugate of formula (I) onto the surface; and c. Rinsing all or part of at least one surface of the solid support with a solvent.

[0015] In one embodiment, the process further comprises at least one step selected from the following steps: • a step (i), before step (a) of contacting, of activation of at least one surface, preferably implemented by thermal activation, chemical activation and / or by irradiation; • a step (i'), before step (a) of contacting, of cleaning at least one surface; • a step (ii), after step (c) of rinsing, of centrifugation; • a step (iii), after step (c) and, if present, after step (ii) of centrifugation, of aging, preferably carried out by aging under vacuum and / or by heating.

[0016] The present invention also relates to a solid support, at least one surface of which is coated with at least one peptide conjugate that can be obtained, preferably obtained, by the functionalization process according to the invention.

[0017] The present invention also relates to the use of a solid support according to the invention for the manufacture of nanoparticles for diagnostics, for the functionalization of Elisa plates, for the manufacture of antifouling surfaces, for the manufacture of medical devices and / or for the manufacture of technical textiles. Brief description of the drawings

[0018] Figure 1 shows the percentages of inhibition of the two bacteria Escherichia coli and Staphylococcus aureus obtained by deposition of peptide conjugates according to the invention onto polycarbonate plates. Top left, conjugate 1a, top right, conjugate 1b, bottom left, conjugate 1a, and bottom right, conjugate 1b.

[0019] Fig. 2 is the photo showing the fluorescence of the 3 wells of the plate functionalized in Example 3. Detailed description

[0020] Definitions

[0021] According to the present invention, a “spacer arm” is a chemical group comprising at least one atom, preferably 1 to 10 atoms.Preferably, a spacer arm is an aliphatic hydrocarbon chain, saturated or unsaturated, comprising from 1 to 10 carbon atoms, optionally interrupted and / or terminated by at least one of a heteroatom, in particular O or S, an aryl group, a C=O group, an SO2 group, an NR group; in which R; may in particular be selected from a hydrogen atom, an aliphatic hydrocarbon radical comprising from 1 to 6 carbon atoms, a benzyl radical and a phenethyl radical, said chain being able to be unsubstituted or substituted by one or more radicals, said radicals being able in particular to be selected from halogen atoms (F, Cl, Br or I), the hydroxyl group, saturated aliphatic hydrocarbon chains comprising from 1 to 4 carbon atoms, benzyl radicals, phenethyl radicals, polyethylene oxide (PEG) radicals and polyalanine radicals.

[0022] A "peptide fragment" is a chain of at least two amino acids linked together by peptide and / or pseudopeptide bonds. Preferably, the peptide fragment comprises from 2 to 80 amino acids, preferably from 2 to 40 amino acids, preferably from 3 to 40 amino acids, preferably from 3 to 30 amino acids, preferably from 4 to 30 amino acids, preferably from 4 to 20 amino acids, in particular 7 to 20 amino acids.

[0023] An "amino acid" is a molecule comprising at least one carboxylic acid (COOH) group and one amine group, and at least one carbon atom linking this carboxylic acid group and this amine group. The amino acids used in the present invention may include, in particular, natural and / or synthetic amino acids.

[0024] Natural amino acids include in particular the following amino acids: glycine (Gly, G), alanine (Ala, A), valine (Val, V), leucine (Leu, L), isoleucine (Ile, I), serine (Ser, S), threonine (Thr, T), phenylalanine (Phe, F), tyrosine (Tyr, Y), tryptophan (Trp, W), cysteine ​​(Cys, C), methionine (Met, M), proline (Pro, P), aspartic acid (Asp, D), asparagine (Asn, N), glutamine (Gin, Q), glutamic acid (Glu, E), histidine (His, H), arginine (Arg, R) and lysine (Lys, K). The preferred natural amino acids according to the present invention are the L-series amino acids.

[0025] Synthetic amino acids are all non-natural amino acids. These include the following amino acids: beta-alanine, allylglycine, tert-leucine, norleucine, 3-aminoadipic acid, 2-aminobenzoic acid, 3-aminobenzoic acid, 4-aminobenzoic acid, 2-aminobutanoic acid, 4-amino-l-carboxymethyl piperidine, 1-amino-l-cyclobutanecarboxylic acid, 4-aminocyclohexaneacetic acid, 1-amino-l-cyclohexanecarboxylic acid, (1R,2R)-2-aminocyclohexanecarboxylic acid, (1R,2S)-2-aminocyclohexanecarboxylic acid, (1S,2R)-2-aminocyclohexanecarboxylic acid, (1S,2S)-2-aminocyclohexanecarboxylic acid, and (1S,2S)-2-aminocyclohexanecarboxylic acid. 3-aminocyclohexanecarboxylic acid, 4-aminocyclohexanecarboxylic acid, (1R,2R)-2-aminocyclopentanecarboxylic acid, (1R,2S)-2-aminocyclopentanecarboxylic acid, 1-amino-1-cyclopentanecarboxylic acid, 1-amino-1-cyclopropanecarboxylic acid, 3-aminomethylbenzoic acid,4-Aminomethylbenzoic acid, 2-Aminobutanoic acid, 4-Aminobutanoic acid, 6-Aminohexanoic acid, 1-Aminoindane-l-carboxylic acid, 2-Aminoisobutyric acid, 4-Aminomethylphenylacetic acid, 4-Aminophenylacetic acid, 3-Amino-2-Naphthoic acid, 4-Aminophenylbutanoic acid, 4-Amino-5-(3-Indolyl)-Pentanoic acid, (4R,5S)-4-Amino-5-Methylheptanoic acid, (R)-4-Amino-5-Methylhexanoic acid, (R)-4-Amino-6-Methylthiohexanoic acid, (S)-4-Amino-Pentanoic acid, (R)-4-Amino-5-Phenylpentanoic acid 4-aminophenylpropionic acid, (R)-4-aminopimeric acid, (4R,5R)-4-amino-5-hydroxyhexanoic acid, acid, (R)-4-amino-5-hydroxypentanoic acid, (R)-4-amino-5-(p-hydroxyphenyl)-pentanoic acid, 8-aminooctanoic acid, (2S,4R)-4-aminopyrrolidine-2-carboxylic acid, acid (2S,4S)-4-aminopyrrolidine-2-carboxylic acid, azetidine-2-carboxylic acid, (2S,4R)-4-benzylpyrrolidine-2-carboxylic acid, (S)-4,8-diaminooctanoic acid, tert-butylglycine, gamma-carboxyglutamate, beta-cyclohexylalanine, citrulline, 2,3-diaminopropionic acid, hippuric acid, homocyclohexylalanine, moleucine, homophenylalanine, 4-hydroxyproline, indoline-2-carboxylic acid, isonipecotic acid, alpha-methylalanine, naphthylanine, nicopetic acid, norvaline, octahydroindole-2-carboxylic acid, ornithine, penicillamine, phenylglycine, acid 4-Phenylpyrrolidine-2-carboxylic acid, pro-pargylglycine, 3-pyridinylalanine, 4-pyridinylalanine, 1-pyr-rolidine-3-carboxylic acid, sarcosine, statins, tetrahydroisoquinoline-1-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, tranexamic acid, 4,4-difluoroproline, 4-fluoroproline, 1'-alpha-(3,4-difluorobenzyl)-proline, gamma-(3,4-difluorobenzyl)-proline, alpha-(trifluoromethyl)phenylalanine, hexafluoroleucine, 5,5,5-trifluoroleucine, 6,6,6-trifluoronorleucine, 2-(trifluoromethyl)leucine, 2-(trifluoromethyl)norleucine, 4,4,4-trifluorovaline, 4,4,4,4',4',4'-hexafluorovaline, pentafluorophenylalanine, 2,3-Difluorophenylalanine, 2,4-Difluorophenylalanine, 2,5-Difluorophenylalanine, 2,6-Difluorophenylalanine, 3,4-Difluorophenylalanine, 3,5-Difluorophenylalanine, 3,3-Difluoro-3-(4-fluorophenyl)alanine, 2,3-Difluorophenylglycine, 2,4-Difluorophenylglycine, 2,5-Difluorophenylglycine, 3,4-Difluorophenylglycine, 4,4-Difluoroethylglycine, 4,4,4-Trifluoroethylglycine, 4-Fluorotryptophan, 5-Fluorotryptophan, 6-Fluorotryptophan, 5-Methyltryptophan, S-Tritylcysteine, Selenocysteine, Selenomethionine, Ethinine, P-(2-thienyl)alanine, beta-chloroalanine, thiazolylalanine, triazo-lalanine, p-fluorophenylalanine, o-fluorophenylalanine, m-fluorophenylalanine, dihydroxyphenylalanine, 2,5-dihydrophenylalanine, thioproline, pi-pecolic acid, canavanine, indospicin, 3,4-dehydroproline, histidinol and hexafluoronorleucine, and their like or derivatives.

[0026] The term “side chain” refers to the fragment attached to the alpha carbon of an amino acid. For example, the side chains of natural amino acids such as glycine, valine, alanine, and aspartic acid correspond to the hydrogen atom, the isopropyl group, the methyl group, and the CH2COOH group, respectively. The side chains of other amino acids may be included in the definition of an amino acid side chain, such as those of the following amino acid: 4-amino tetrahy- dropyran-4-carboxylic acid, allylglycine, diaminobutyric acid, diaminopropionic acid, aminoserine, aminobutyric acid, aminobutylglycine, phenylglycine, 4-chlorophenylalanine, 4-fluorophenylalanine, 4-nitrophenylalanine, citrulline, cyclohexylalanine, thienylalanine, and their like.

[0027] The side chains of amino acids can be protected by protecting groups (P), and more particularly by N-protectors, O-protectors, or S-protectors when these chains contain the corresponding heteroatoms. The protection of certain reactive functions of peptides is obligatory during the synthesis of said peptides.

[0028] “Protective groups (P)” are groups known to those skilled in the art. These protective groups and their use are described in works such as, for example, Greene, “Protective Groups in Organic Synthesis”, Wiley, New York, 2007, 4th edition; Harrison et al., “Compendium of Synthetic Organic Methods”, Vol. 1 to 8 (J. Wiley & Sons, 1971 to 1996). In addition, peptide synthesis techniques are described in Paul Lloyd-Williams, Fernando Albericio, Ernest Giralt, "Chemical Approaches to the Synthesis of Peptides and Proteins", CRC Press, 1997 or Houben-Weyl, "Methods of Organic Chemistry, Synthesis of Peptides and Peptidomimetics", Vol E 22a, Vol E 22b, Vol E 22c, Vol E 22d., M. Goodmann Ed., Georg Thieme Verlag, 2002.Protecting groups attached to a nitrogen atom will be designated as N-protecting groups; protecting groups attached to a sulfur atom will be designated as S-protecting groups; and protecting groups attached to an oxygen atom will be designated as O-protecting groups. For example, a hydroxyl group may be protected by a trityl group, or a carboxylic acid may be protected as a tert-butyl ester. In the case of solid-state synthesis, the resin serves as the protecting group for the C-terminal carboxylic acid function. The protection of the amino group (i.e., the "alpha amine") of the amino acid may be achieved, for example, by a tert-butyloxycarbonyl (Boc-) group or a 9-fluorenylmethyloxycarbonyl (Fmoc-) group. The protection is carried out according to the methods known in Article 1.In the case of protecting the functional groups of natural amino acids, the resulting amino acids are synthetic until the protective group(s) are removed, thus releasing the so-called natural amino acid. The removal of the protective group(s) is also carried out according to the processes known in art.

[0029] A “solid support” or “substrate” is an object of which at least one surface is solid, and of which at least this surface can be functionalized by the invention.

[0030] The term “plastic” refers to a substrate or surface comprising a mixture containing a base material that is a polymer, or a mixture of polymers. The plastic substrate or surface may have been obtained by molding, extrusion, or shaping, preferably hot and under pressure, of said mixture comprising a polymer.

[0031] The term “textile” refers to an assembly of yarns or fibers, advantageously bonded together, forming a solid and insoluble entity. Thus, and appropriately, a textile can be a fabric, advantageously obtained by weaving or knitting yarns, or a nonwoven fabric obtained by assembling fibers. The textile material can be, in particular, a fabric, or a synthetic material such as a plastic. The term textile includes, in particular, medical textiles, civilian textiles, technical textiles, and filtration membranes.

[0032] By "functionalization" or "anchoring" is meant the covalent or non-covalent attachment of at least one peptide conjugate to the surface of the solid support, preferably by means of the attachment, in particular the covalent attachment, of the benzene motif of the conjugate to said surface.

[0033] By "degree of functionalization" is meant the quantity or density of peptide conjugate of formula (I) which is anchored to the surface of the solid support functionalized according to the invention.

[0034] Peptide conjugate of formula (I)

[0035] The peptide conjugate used in the functionalization process according to the invention is a conjugate of formula (I)

[0036] [Chem.3] in which L is a spacer arm, n is 0 or 1, and A is a peptide fragment.

[0037] In formula (I), the spacer arm L is present (n=1) or absent (n=0), preferably it is present (n=1). The presence of this spacer arm can, in particular, help to move steric hindrance away from the surface when the peptide fragment is bulky, for example when it includes side chains.

[0038] The nature of the spacer arm can vary to a wide extent. In some embodiments, L is a saturated or unsaturated aliphatic hydrocarbon chain comprising from 1 to 10 carbon atoms, optionally interrupted or terminated by at least one of a heteroatom, in particular O or S, an aryl group, a C=O group, an SO2 group, an NR group, wherein R is selected from a hydrogen atom, an aliphatic hydrocarbon radical comprising from 1 to 6 atoms of carbon, a benzyl radical or a phenethyl radical, said chain being either unsubstituted or substituted.

[0039] In some embodiments, L is a carbon aliphatic chain of formula (II)

[0040] [Chem.4] O O in which m is an integer from 1 to 10, preferably from 4 to 6, in particular 5.

[0041] The ends of the spacer arm L which are attached respectively to the benzophenone and the peptide fragment A are typically made up of functional groups allowing the attachment of the different entities by chemical reactions well known in the art, such as peptide bond formation reactions.

[0042] In one embodiment, the bond between the spacer arm L and the peptide fragment A is at the N-terminal end of the peptide fragment A. In another embodiment, the bond between the spacer arm L and the peptide fragment A is at the C-terminal end of the peptide fragment A. In yet another embodiment, the bond between the spacer arm L and the peptide fragment A is at a side chain of the peptide fragment A.

[0043] In one embodiment, the peptide conjugate comprises a single peptide fragment and a single benzophenone head. In other embodiments, the peptide conjugate may comprise a single peptide fragment and several benzophenone heads, for example 2, 3, 4 or 5 benzophenone heads preferably linked at different positions on the peptide fragment.

[0044] The peptide fragment may comprise from 2 to 80 amino acids, preferably from 2 to 40 amino acids, preferably from 3 to 40 amino acids, preferably from 3 to 30 amino acids, preferably from 4 to 30 amino acids, preferably from 4 to 20 amino acids, and in particular from 7 to 20 amino acids. In one embodiment, the peptide fragment A is not the GRGDSP fragment. In one embodiment, the peptide fragment A is not the RGD fragment. In one embodiment, the peptide fragment A is neither the GRGDSP fragment nor the RGD fragment.

[0045] In one embodiment, the peptide fragment A is an unmodified peptide. In another embodiment, the peptide fragment is a modified peptide, for example, a peptide whose end(s) not bound to the spacer arm is / are substituted and / or protected by a protecting group. For example, the end The COOH of the peptide can be substituted by a fatty acid, such as palmitic acid.

[0046] In one embodiment, the peptide fragment A is chosen from the group consisting of a linear natural peptide strand, a linear synthetic peptide strand, a linear protected natural peptide strand, a linear protected synthetic peptide strand, a linear natural pseudopeptide strand, a linear synthetic pseudopeptide strand, a linear protected natural pseudopeptide strand, and a linear protected synthetic pseudopeptide strand.In another embodiment, the peptide fragment A is chosen from the group consisting of a natural cyclic peptide fragment, a synthetic cyclic peptide fragment, a natural protected cyclic peptide fragment, a synthetic protected cyclic peptide fragment, a natural cyclic pseudopeptide fragment, a synthetic cyclic pseudopeptide fragment, a natural protected cyclic pseudopeptide fragment and a synthetic protected cyclic pseudopeptide fragment.

[0047] The peptide fragment A of the peptide conjugate of formula (I) may comprise one or more peptides, peptide strands and / or peptide fragments as described in this description.

[0048] In one embodiment, the peptide fragment A is chosen from the group consisting of the peptides H-(RF)4-NH2, H-(RI)4-NH2 and H-(R)2-Palm, Palm denoting a modification of the terminal arginine by a palmitic acid.

[0049] Advantageously, the peptide fragment A has a particular property, in particular a particular chemical or biological activity. Thus, the peptide fragment A can be selected from the group consisting of an antibiotic peptide, an antimicrobial peptide, an antifungal peptide, an anti-inflammatory peptide, a catalytic peptide, a biological receptor ligand peptide, an antibody, and an enzyme inhibitor peptide. Preferably, the peptide fragment A is selected from the group consisting of an antibiotic peptide, an antimicrobial peptide, an antifungal peptide, an anti-inflammatory peptide, and a catalytic peptide. In particular, the peptide fragment A is selected from the group consisting of an antibiotic peptide, an antimicrobial peptide, and an antifungal peptide.

[0050] In one embodiment, the peptide fragment A is an antimicrobial peptide, preferably a cationic antimicrobial peptide, with an overall positive charge and comprising at least one hydrophobic residue.

[0051] The particular property of the peptide fragment A is advantageously unaffected by its grafting to the spacer arm and the benzophenone head; this particular property is therefore also a particular property of the corresponding peptide conjugate. Similarly, the particular property of the peptide conjugate is advantageously unaffected by its anchoring to the surface of the solid support; this particular property is therefore also a particular property of the functionalized surface. of the corresponding solid support.

[0052] In one embodiment, the peptide fragment A is an antimicrobial peptide selected from the group consisting of the peptides H-(RF)4-NH2, H-(RI)4-NH2 and H-(R)2-Palm, Palm denoting a modification of the terminal arginine by a palmitic acid.

[0053] The peptide conjugates according to the invention can be synthesized by any suitable technique known in the art, in particular by successive couplings of the spacer arm with benzophenone and with the peptide fragment A.

[0054] The peptide conjugate and the peptide fragment A can notably be synthesized by classical peptide synthesis techniques. Peptide synthesis is classically carried out by activating the carboxylic acid function of an amino acid, or a chain of amino acids, using a coupling agent. This activated acid is brought into contact with an amino acid, or a chain of amino acids, whose terminal amine is not protected, thus resulting in the formation of an amide bond, also called a peptide bond. The coupling conditions and the coupling agents used are very well known to those skilled in the art and are described, for example, in works such as Greene, "Protective Groups in Organic Synthesis", Wiley, New York, 2007, 4th edition; Harrison et al., "Compendium of Synthetic Organic Methods", Vol. 1 to 8 (J. Wiley & Sons, 1971 to 1996).In addition, peptide synthesis techniques are described in Paul Lloyd-Williams, Fernando Albericio, Ernest Giralt, "Chemical Approaches to the Synthesis of Peptides and Proteins", CRC Press, 1997 or Houben-Weyl, "Methods of Organic Chemistry, Synthesis of Peptides and Peptidomimetics", Vol E 22a, Vol E 22b, Vol E 22c, Vol E 22d., M. Goodmann Ed., Georg Thieme Verlag, 2002.

[0055] Functionalization method

[0056] The functionalization process according to the invention is applicable to at least one surface of a solid support. In one embodiment, the surface functionalized by the process according to the invention is the total surface of the solid support. In another preferred embodiment, the surface functionalized by the process according to the invention is only a portion of the total surface of the solid support.

[0057] The solid support, a surface of which is functionalized according to the invention, can be of any material benefiting from functionalization by the peptide conjugate of formula (I). It can in particular be a plastic support or a textile support, preferably a plastic support.

[0058] In the case where the peptide fragment A of the peptide conjugate of formula (I) has a particular property, in particular a particular chemical or biological activity, the functionalization of at least one surface of the solid support makes it possible to confer the same property to at least one surface of the support.

[0059] In one embodiment, the process is carried out with a single peptide conjugate of formula (I). In other embodiments, the process is carried out with at least two different peptide conjugates, either one after the other or in a mixture. For example, the process can be carried out with at least two peptide conjugates comprising two different peptide fragments, or with at least two peptide conjugates comprising the same peptide fragment but different spacer arms, in particular spacer arms of different lengths.

[0060] Contacting at least one surface with at least one peptide conjugate of formula (I) can be achieved by any suitable technique. In particular, contact can be achieved by dipping, spraying, and / or incubating at least one surface with a solution or suspension of the peptide conjugate of formula (I) in a solvent. The solvent can be an organic solvent, an inorganic solvent, or a mixture of such solvents. The solvent can, in particular, be an alcohol, such as ethanol. In one embodiment, the peptide conjugate of formula (I) is soluble in the solvent used. Examples include spin-coating, dip-coating, casting, laminar flow deposition, and aerospray deposition.

[0061] A person skilled in the art can determine the parameters such as the duration and / or temperature of the contact, taking into account, in particular, the nature of the surface of the solid support, the structure of the peptide conjugate of formula (I), and / or the desired degree of functionalization. For example, the contact time may correspond to the time required for the complete evaporation of the solvent in which the conjugate is dissolved and / or suspended.

[0062] The functionalization process can allow anchoring on the surface of the solid support at least one layer, preferably a monolayer, of the peptide fragment(s) of the peptide conjugate(s) of formula (I).

[0063] In one embodiment, the functionalization process according to the invention comprises the following steps, preferably in this order: a. Contacting at least said surface of said solid support with a solution or suspension of at least one peptide conjugate of formula (I) in a solvent, the contacting being carried out preferably by soaking, spraying and / or incubation; b. Irradiation of the surface in contact with the solution or suspension of at least one peptide conjugate of formula (I), for a duration suitable for achieving the anchoring of all or part of the peptide conjugate of formula (I) to the surface; and c. Rinsing all or part of at least one surface of the solid support with a solvent.

[0064] The contacting step a. can be implemented as described above for the contacting step.

[0065] Step b. of irradiation allows, in particular, the activation of the benzophenone head of the peptide conjugate of formula (I) and its anchoring to the surface of the solid support. Preferably, this involves irradiation with ultraviolet (UV) light. Those skilled in the art are able to adjust the irradiation parameters, such as the irradiation time, intensity, and / or wavelength, according to, in particular, the nature of the solid support surface, the structure of the peptide conjugate of formula (I), and / or the desired degree of functionalization. Step b. of irradiation can be carried out either in the presence of the solvent of the solution or suspension brought into contact with the support in step a., or after evaporation or drying of all or part of the solvent, in particular after evaporation or drying of all the solvent.

[0066] Rinsing step c allows, in particular, the removal of excess peptide conjugate of formula (I) that has not bonded to the surface of the solid support after irradiation step b. Rinsing can be carried out with one or more solvents, either simultaneously or sequentially. The solvent for rinsing step c can be an organic solvent, an inorganic solvent, or a mixture of such solvents. The rinsing step can be performed once or several times, in particular 2, 3, 4, 5, or 10 times, with the same solvent or with different solvents. Preferably, rinsing step c removes all of the peptide conjugate of formula (I) that is not bonded to the surface after step b.

[0067] In one embodiment, the functionalization process further comprises, prior to the contacting step (a), a step (i) of activating at least one surface. This activation step notably improves the subsequent anchoring of the peptide conjugate of formula (I) to the surface, for example by increasing the anchoring rate and / or increasing the degree of functionalization. The nature of the activation to be implemented depends on the nature of the solid support surface. This activation may, in particular, be thermal activation, by exposure to a temperature above ambient temperature (15 to 25°C), chemical activation, by contact with a chemical agent enabling activation, and / or activation by irradiation.

[0068] In one embodiment, the functionalization process further comprises, prior to the contacting step a, a cleaning step (i') of at least one surface. The purpose of this step is to remove any substance present on the surface that could prevent or weaken subsequent functionalization by the peptide conjugate(s). The cleaning step (i') can be carried out, for example, using of a solvent such as an alcohol, in particular ethanol or isopropanol.

[0069] In one embodiment, the functionalization process further includes, after step c. of rinsing, a step (ii) of centrifugation, preferably also including the separation of the pellet and the centrifugation supernatant, the solid support being in the centrifugation pellet.

[0070] In one embodiment, the functionalization process further comprises, after step (c) of rinsing, and after step (ii) of centrifugation if present, an aging step (iii). Step (iii) may, in particular, be an aging step at reduced pressure (under vacuum) and / or an aging step by heating to a temperature above ambient temperature. In one embodiment, step (iii) comprises aging at reduced pressure followed by aging by heating.

[0071] Solid support

[0072] Another object of the present invention is a solid support of which at least one surface is coated with at least one peptide conjugate, which can be obtained, preferably obtained, by the functionalization process according to the invention.

[0073] In some embodiments, only one surface of the solid support is functionalized by a functionalization process according to the invention. In other embodiments, at least two surfaces of the solid support, preferably all surfaces of the solid support, are functionalized by a functionalization process according to the invention.

[0074] In other words, in certain embodiments, only a portion of the surface of the solid support is functionalized by a functionalization process according to the invention. Thus, for example, 100% of the surface of the solid support is functionalized by a functionalization process according to the invention. In other embodiments, less than 100%, less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, or less than 5% of the surface of the solid support is functionalized by a functionalization process according to the invention.

[0075] Use of solid support

[0076] The solid support according to the invention and / or the solid support that can be obtained, preferably obtained, by a functionalization process according to the invention, can be used in a wide variety of applications.

[0077] Among the possible uses, we can mention the manufacture of nanoparticles for diagnostics, the functionalization of Elisa plates, the manufacture of antifouling surfaces, the manufacture of medical devices and / or the manufacture of technical textiles.

[0078] Thus, a final object of the invention is the use of a solid support according to the invention, of a solid support obtained or capable of being obtained by a functionalization process according to the invention, and / or of a functionalization process according to the invention, for the manufacture of nanoparticles for diagnostics, for the functionalization of Elisa plates, for the manufacture of antifouling surfaces, for the manufacture of medical devices and / or for the manufacture of technical textiles.

[0079] The following examples illustrate the invention more precisely, and should be considered as illustrative and not limiting examples of the invention. Examples

[0080] Preparation of monomers substituted by a peptide

[0081] The peptides are synthesized on solid support using a Symphony X type peptide synthesizer (Protein Technologies, Inc., USA) in Fmoc / tert-butyl strategy using nitrogen bubbling as the stirring method for the coupling and deprotection cycles of the Fmoc groups in the N-terminal position or in solution in Boc / Bzl strategy.

[0082] The syntheses are carried out on a 0.25 mmol scale using Fmoc-Rink-Amide polystyrene resin (481 mg / 0.52 mmol / g) or 2-chlorotrityl chloride resin (892 mg, 0.28 mmol / g). The standard deprotection-coupling cycle for each residue comprises six steps: Washing the resin with 5 mL of dimethylformamide (DMF) (3 x 30 sec). Deprotection of the Fmoc group with 5 mL of 20% piperidine in DMF (3 x 3 min). Washing the resin with 5 mL of DMF (3 x 30 sec). The Fmoc-AA residue was coupled for 60 min using HATU (l-[Bis(dimethylamino)methylene]-lH-l,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate) and N,N-diisopropylethylamine DIEA as the coupling agent. At the end of the coupling cycle, a capping step was performed with 5 mL of 10% acetic anhydride (Ac2O) in DMF for 7 min, followed by washing the resin with 5 mL of DMF (3 x 30 sec).

[0083] Cleavage of the resin (Fmoc-Rink-Amide) is carried out for 2 x 60 minutes in a cleavage cocktail (trifluoroacetic acid TFA / triisopropylsilane TIS / water H2O 95 / 2.5 / 2.5) for the unprotected sequences and for 2 x 60 minutes in a cleavage cocktail (acetic acid AcOH / 2,2,2-Trifluoroethanol TFE / dichloromethane DCM 10 / 20 / 70) for the protected sequences (2-chlorotrityl chloride resin). Once the resin has been filtered, the solution is evaporated under vacuum and the peptide is precipitated in diethyl ether. The precipitated peptide is centrifuged (3500 RPM) and the supernatant is discarded. (X3). The peptide in the form of TFA salts is then solubilized in a water H2O / acetonitrile ACN mixture before being frozen and lyophilized.

[0084] The peptides are analyzed by UPLC chromatography and ESLMS mass spectrometry, equipped with a BEH C18 column (WATERS), 150*2.1 mm (150 x 2.1 mm) (flow rate: 0.6 ml / min). Solvents A and B are at 0.1% TFA in water and at 0.1% TFA in acetonitrile.

[0085] The peptides were purified on a WATERS HPLC 4000 instrument equipped with a UV 486 detector and a Vydac Denali 10 pm C18 120 Å (310 x 25 mm) column at a flow rate of 50 mL / min. The solvents used were 0.1% TFA in water (buffer A) and 0.1% TFA in acetonitrile (buffer B).

[0086] Example 1: Synthesis of peptide conjugates according to the invention

[0087] Example a. Peptide conjugate comprising the H-(RFL-NH2) peptide

[0088] [Chem.5]

[0089] The antibacterial peptide H-(RF)4-NH2 was synthesized on a Rink Amide resin (load 0.52 mmol / g, synthesis scale: 0.25 mmol) using an Fmoc / tBu strategy. Each coupling was followed by deprotection of the N-terminal Fmoc group. The peptide was then functionalized on a support after the introduction of a spacer (or "linker"). The Fmoc-Ahx-OH spacer (6-aminohexanoic acid protected by an Fmoc group) was introduced into DMF in the presence of 3 equivalents of HATU and 6 equivalents of DIEA. The resin was washed (3*DMF, 1*methanol MeOH, and 1*dichloromethane DCM). 4-Benzoylbenzoic acid was coupled in DMF in the presence of 3 equivalents of HATU and 6 equivalents of DIEA. The resin was washed (3*DMF, 1*MeOH, and 1*DCM). The resin was then cleaved in a 50 / 47.5 / 2.5 DCM / TFA / H2O mixture for 2 x 1 h.The "cleavage" solution was concentrated under reduced pressure, then the antibacterial anchor peptide was precipitated in diethyl ether and finally purified by preparative HPLC (Vydac Denali 10 pm C18 120 Å column (310 x 25 mm) using a 20 to 60% gradient of buffer B in 40 min.).

[0090] ESI-MS (m / z): [M+H]+ theoretical C75Hio3N22Oi4: 1552.87, experimental: 1552.98

[0091] HPLC (retention time, min): 3.73

[0092] Example 1b: Peptide conjugate comprising the peptide H-(RI)2-NH2

[0093] [Chem.6]

[0094] The antibacterial peptide H-(RI)4-NH2 was synthesized on a Rink Amide resin (charge 0.52 mmol / g, synthesis scale: 0.25 mmol) using an Fmoc / tBu strategy. Each coupling was followed by deprotection of the N-terminal Fmoc group. The peptide was then functionalized on a support after the introduction of a spacer (or "linker"). The Fmoc-Ahx-OH spacer was introduced into DMF in the presence of 3 equivalents of HATU and 6 equivalents of DIEA, and the resin was washed (3*DMF, 1*MeOH, and 1*DCM). 4-Benzoylbenzoic acid was coupled into DMF in the presence of 3 equivalents of HATU and 6 equivalents of DIEA, and the resin was washed (3*DMF, 1*MeOH, and 1*DCM). The resin was then cleaved in a DCM / TFA / H2O mixture of 50 / 47.5 / 2.5 for 2 x 1 h.The "cleavage" solution was concentrated under reduced pressure, then the antibacterial anchor peptide was precipitated in diethyl ether and finally purified by preparative HPLC (Vydac Denali 10 qm C18 120 Â column (310 x 25 mm) using a 20 to 60% gradient of buffer B in 40 min.).

[0095] ESI-MS (m / z): [M+H]+ theoretical C75Hio3N22Oi4: 1416.80, experimental: 1496.98

[0096] HPLC (retention time, min): 3.63

[0097] Example: Peptide conjugate comprising the H-(R)2-Palm peptide

[0098] [Chem.7]

[0099] The antibacterial peptide H-(R)2-Palm was synthesized on a Rink resin (charge 0.52 mmol / g, synthesis scale: 0.25 mmol) previously functionalized with a benzotriazole using an Fmoc / tBu strategy. Each coupling was followed by deprotection of the N-terminal Fmoc group.

[0100] Rink-amide resin was pre-treated with piperidine (20% in DMF) and washed (3*DMF, 1*MeOH and 1*DCM). 4-Amino-3-nitrobenzoic acid was The reaction was coupled in DMF in the presence of 3 equivalents of HATU and 6 equivalents of DIEA. The reaction was stirred for 2 hours, then the resin was washed (3 DMF, 1 MeOH, and 1 DCM), capped with acetic anhydride (15 mL of a 10% v / v solution in DCM), and then washed again (2 DCM, 3 DMF, 1 MeOH, and 1 DCM). The nitro group reduction was carried out in a solution of 5 g SnCl2 (2H2O) and 900 µL of 1,8-diazabicyclo[5.4.0]undec-7-ene DBU per 10 mL of DMF, which was added to the resin under nitrogen bubbling for 10 minutes. The reaction was stirred for 15 hours with the syringe open and washed (3 DMF, 3 DCM).

[0101] The introduction of the peptide sequence -(R)2- was carried out using an Fmoc / tBu strategy. Each coupling was followed by deprotection of the N-terminal Fmoc group. The peptide was then functionalized on a support after the introduction of a spacer (or "linker"). The Fmoc-Ahx-OH spacer was introduced into DMF in the presence of 3 equivalents of HATU and 6 equivalents of DIEA, and the resin was washed (3*DMF, 1*MeOH, and 1*DCM). 4-Benzoylbenzoic acid was coupled into DMF in the presence of 3 equivalents of HATU and 6 equivalents of DIEA, and the resin was washed (3*DMF, 1*MeOH, and 1*DCM). The resin was then treated with isoamyl nitrite (10 equivalents) for 90 minutes (DCM wash x5). The nucleophile (4 equivalents / in this case the hexadec y lamin) in solution in DCM in the presence of DIEA (8 equivalents) was bubbled with nitrogen 20 minutes before the end of cyclization, then was introduced immediately onto the resin.The reaction was agitated, inducing the cleavage of the peptide from the resin.

[0102] The peptide was then deprotected in a DCM / TFA / H2O 50 / 47.5 / 2.5 mixture for 2x1 h. The "deprotection" solution was concentrated under reduced pressure and then the antibacterial peptide was precipitated in diethyl ether and finally purified by preparative HPLC (Vydac Denali 10 pm Cl8 120 Â column (310 x 25 mm) using a gradient of 20 to 60% of buffer B in 40 min).

[0103] ESI-MS (m / z): [M+H]+ theoretical C43H75NiO8: 876.22 experimental: 876.19

[0104] HPLC (retention time, min): 5.63

[0105] Example 2: Deposition of peptide conjugates according to the invention on a substrate

[0106] The deposition of the three peptides obtained in Example 1 was carried out by dip-coating, i.e. by dipping / withdrawing at a constant speed of the substrate in a solution of the anchor peptide in an ethanolic solution.

[0107] Each peptide was solubilized in an ethanolic solution (95%).

[0108] After a few minutes of stirring, a clear and stable solution was obtained. This The solution was deposited onto a clean polycarbonate substrate by immersion. The immersed plastic plates were then dried and irradiated under UV light for 15 minutes (360 nm, 250 W). After irradiation, the plates were washed under sonication in water and ethanol baths (4 x 10 minutes).

[0109] Example 3: Grafting benzophenone onto a support

[0110] The fluorescent dansyl-benzophenone block of formula Chem 8 below was synthesized by classical techniques. [YES] [Chem. 8]

[0112] A 96-well polystyrene plate was first rinsed with distilled water and ethyl alcohol, then dried. A 0.1M solution of dansylbenzophenone compound was loaded into the first three wells. The plate was irradiated for 180 seconds under UV light at a power of 120 mW / cm². The plate was then rinsed with distilled water. Figure 2 shows a photograph of the plate, which exhibits fluorescence in the three functionalized wells.

[0113] This result proves that the benzophenone head grafts onto the surface of the wells of the 96-well plate.

[0114] Example 4: Determination of the antibacterial activity of substrates treated with antimicrobial peptides according to the invention

[0115] Antibacterial activity according to ISO 22196:2011 is intended to evaluate the antimicrobial activity of plastic products or non-porous surfaces treated with antimicrobial agents. The method used in this study focuses solely on evaluating antibacterial activity.

[0116] This study is intended to evaluate the antibacterial activity of peptide-grafted polycarbonate materials against the untreated reference according to the recommendations of ISO 22196:20111 for a contact time of 24 hours.

[0117] Polycarbonate material (6 cm x 5 cm rectangle)

[0118] Such a study ideally uses 5 cm x 5 cm samples onto which a known concentration of the microorganism to be tested has been deposited. After incubation for 24 hours at 35°C, the quantity of viable microorganisms is assessed by the agar plate enumeration technique. Comparing the bacterial concentrations obtained between the treated and untreated material allows the antibacterial activity of the tested formulation to be determined.

[0119] The study carried out here was performed on samples in the form of 6 rectangles cm x 5 cm in accordance with the standard. The tests were carried out against the bacterial strains prescribed by the standard and very frequently involved in infections, Escherichia coli ATCC 8739 and Staphylococcus aureus ATCC 6538P.

[0120] Each conjugate la, 1b and le as synthesized in Example 1 was deposited on the surface of the polycarbonate plates by deposition of a solution of 0.02 to 4 mg / mL.

[0121] The results showed significant inhibition with a reduction in Staphylococcus aureus growth of up to 99.87% (Tables 1 and 2 and [Fig. 1]). Table 1 corresponds to Staphylococcus aureus, Table 2 corresponds to Escherichia coli.

[0122] In Tables 1 and 2, PC denotes polycarbonate. Peptide conjugates are designated by reference to the example in which their synthesis is described (la, 1b and le).

[0123] [Tables 1] Reference Time Geometric Mean (CFU / cm²) LoglO Mean (CFU / cm²) R (number of logs) % reduction Oh Negative Control 1.62E+O₄ 4.21 White PC 1.42E+O₄ 4.15 24 h White PC 1.19E+O₄ 4.07 PC / Peptide 1.54E+O₁ 1.19 2.9 99.87 PC / Peptide 1b 7.08E+O₂ 2.85 1.2 94.0 PC / Peptide 1b 6.34E+O₃ 3.8 0.3 46.7

[0124] [Tables2] Reference Time Geometric Mean (CFU / cm²) Log1O Mean (CFU / cm²) R (number of logs) % reduction Oh Negative Control 1.35E+O4 4.13 White PC 1.42E+O4 4.15 24 h White PC 2.12E+O6 6.33 PC / Peptide 1.22E+O6 6.09 0.2 42.6 PC / Peptide 1b 6.31E+O4 4.80 1.5 97.0 PC / Peptide 1.24E+O6 6.09 0.2 41.7

[0125] Fig. 1 illustrates these results. Significant inhibition of both types of bacteria was obtained (up to 99.87% reduction in growth for Staphylococcus aureus, and up to 97.0% reduction in growth for Escherichia coli) for polycarbonate samples functionalized according to the invention compared to non-functionalized polycarbonate samples.

[0126] The antibacterial activity of the peptide conjugates la, 1b and le is therefore efficiently transferred to the polycarbonate support on which they are grafted.

Claims

Demands

1. Method for functionalizing at least one surface of a solid support, comprising bringing at least said surface of said solid support into contact with at least one peptide conjugate of formula (I) [Chem.9] in which L is a spacer arm, n is 0 or 1, preferably 1, and A is a peptide fragment, and in which L has the formula (II) [Chem. 10] O O

2.

3.

4. in which m is an integer from 1 to 10, preferably from 4 to 6, in particular 5, under conditions adapted to obtain the anchoring of at least one peptide conjugate of formula (I) to the surface of the support. A method for functionalizing at least one surface of a solid support according to claim 1, wherein A is a peptide fragment comprising 2 to 80 amino acids, preferably 3 to 30 amino acids, in particular 7 to 20 amino acids. A method for functionalizing at least one surface of a solid support according to claim 1 or claim 2, wherein A is a peptide fragment selected from the group consisting of an antibiotic peptide, an antimicrobial peptide, an antifungal peptide, an anti-inflammatory peptide, a catalytic peptide, a biological receptor ligand peptide, an antibody and an enzyme inhibitor peptide, preferably an antimicrobial peptide, or a fragment thereof. A method for functionalizing at least one surface of a substrate solid according to any one of claims 1 to 3, wherein the peptide fragment is an antimicrobial peptide selected from the group consisting of the peptides H-(RF)4-NH2, H-(RI)4-NH2 and H-(R)2-Pahn.

5. A method for functionalizing at least one surface of a solid support according to any one of claims 1 to 4, wherein the solid support is chosen from the group consisting of a plastic support and a textile support.

6. A method for functionalizing at least one surface of a solid support according to any one of claims 1 to 5, comprising the following steps: a. Contacting at least said surface of said solid support with a solution or suspension of at least one peptide conjugate of formula (I) as defined in any one of claims 1 to 4 in a solvent, the contact being carried out preferably by soaking, spraying and / or incubation; b. Irradiating, in particular UV irradiation, the surface in contact with the solution or suspension of at least one peptide conjugate of formula (I), for a time suitable for obtaining the grafting of all or part of the peptide conjugate of formula (I) onto the surface; and c. Rinsing all or part of the at least one surface of the solid support with a solvent.

7. A method according to claim 6, further comprising at least one step selected from the following steps: • a step (i), before step (a) of contacting, of activating at least one surface, preferably carried out by thermal activation, chemical activation and / or by irradiation; • a step (i'), before step (a) of contacting, of cleaning at least one surface; • a step (ii), after step (c) of rinsing, of centrifugation; • a step (iii), after step (c) and, if present, after step (ii) of centrifugation, of aging, preferably carried out by aging under vacuum and / or by heating.

8. Solid support having at least one surface coated with at least one peptide that can be obtained, preferably obtained, by the functionalization process according to any one of claims 1 to 7.

9. Use of a solid support according to claim 8, for the manufacture of nanoparticles for diagnostics, for the functionalization of ELISA plates, for the manufacture of antifouling surfaces, for the manufacture of medical devices and / or for the manufacture of technical textiles.

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