Method for functionalising a solid substrate with a peptide conjugate

Abstract

The present invention relates to a method for functionalising a solid support with a peptide conjugate comprising a benzophenone anchor head, a spacer arm and a peptide fragment. The invention also relates to a solid substrate that can be obtained by a functionalisation method according to the invention, as well as to the use of the solid substrate according to the invention.

Description

[0001] Method for functionalizing a solid support with a peptide conjugate

[0002] Technical field

[0003] The present invention relates to the field of functionalization of surfaces, such as textile or plastic surfaces, by peptides, in particular antimicrobial peptides.

[0004] Technological background

[0005] The medical industry faces many challenges. Key among them 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 strains that have developed antibiotic resistance. Therefore, it is now necessary to develop alternative methods to the use of disinfectants and antibiotics to eliminate biofilms and / or limit their occurrence.

[0006] To prevent the formation of biofilms on surfaces, one of the methods that has proven effective is the use of antimicrobial coatings on these surfaces. Bacteria do not easily develop resistance to these antimicrobial coatings, which have a mechanical and not only chemical action. Antimicrobial coatings can be obtained by functionalizing surfaces with molecules exhibiting antimicrobial properties. Patent application WO2014118779, for example, describes a method for functionalizing surfaces with bifunctional molecules comprising a catechol anchor and a peptide portion comprising a difluorophenylalanine dipeptide. The functionalization aims to confer antifouling properties to the surface, in particular 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.

[0007] It would be interesting to have new methods for functionalizing surfaces with peptide conjugates, said methods providing efficient anchoring of the peptide conjugates on the surfaces. The peptide conjugates should advantageously be adaptive, so that they can be used in a wide range of applications and with a wide range of peptides. Thus, it would be interesting to be able to implement functionalization methods 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.In this context, the inventors of the present invention have demonstrated that it is possible to effectively functionalize solid support surfaces with peptide conjugates comprising a benzophenone-type anchoring head, a spacer arm and a peptide fragment which can be attached to the spacer arm by different locations in its structure. This functionalization is particularly suitable for plastic and / or textile type supports.

[0008] Summary of the invention

[0009] Thus, the present invention relates to a 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) in which L is a spacer arm, n is 0 or 1, preferably 1, and

[0010] A is a peptide fragment, under conditions suitable for obtaining the anchoring of at least one peptide conjugate of formula (I) to the surface of the support.

[0011] 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.

[0012] 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 inhibitory peptide, preferably an antimicrobial peptide, or a fragment thereof.

[0013] In one embodiment, the peptide fragment is an antimicrobial peptide selected from the group consisting of peptides H-(RF)4-NH2 (SEQ ID NO:2), H-(RI)4-NH2 (SEQ ID NO:4) and H-(R)2-Palm.

[0014] 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 NRi group, in which Ri is chosen from a hydrogen atom, an aliphatic hydrocarbon radical comprising from 1 to 6 carbon atoms, a benzyl radical and a phenethyl radical, said chain possibly being unsubstituted or substituted. Preferably, L is an aliphatic carbon chain of formula (II) , in which m is an integer from 1 to 10, preferably from 4 to 6, in particular 5.

[0015] In one embodiment, the solid support is selected from the group consisting of a plastic support and a textile support.

[0016] In one embodiment, the functionalization method 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 contacting preferably being carried out by dipping, spraying and / or incubation; b. Irradiation, in particular UV irradiation, of the surface in contact with the solution or suspension of the 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 the at least one surface of the solid support with a solvent.

[0017] In one embodiment, the method further comprises at least one step chosen from the following steps: a step (i), before step (a) of contacting, of activating the at least one surface, preferably carried out by thermal activation, chemical activation and / or by irradiation;

[0018] - a step (i'), before step (a) of bringing into contact, of cleaning the at least one surface;

[0019] - a step (ii), after step (c) of rinsing, of centrifugation;

[0020] - a step (iii), after step (c) and, if present, after step (ii) of centrifugation, of aging, preferably carried out by vacuum aging and / or by heating. The present invention also relates to a solid support, at least one surface of which is coated with at least one peptide conjugate capable of being obtained, preferably obtained, by the functionalization method according to the invention.

[0021] The present invention also relates to the use of a solid support according to the invention for the manufacture of nanoparticles for diagnosis, 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.

[0022] Brief description of the drawings

[0023] Figure 1 shows the percentages of inhibition of the two bacteria Escherichia coli and Staphylococcus aureus obtained by depositing peptide conjugates according to the invention on polycarbonate plates. Top left, conjugate 1a, top right, conjugate 1b, bottom left conjugate 1a and bottom right conjugate 1b.

[0024] Figure 2 is the photo showing the fluorescence of the 3 wells of the plate functionalized in example 3.

[0025] Detailed description

[0026] Definitions

[0027] 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 a saturated or unsaturated aliphatic hydrocarbon chain 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 NRi group, in which Ri may in particular be chosen from a hydrogen atom, an aliphatic hydrocarbon radical comprising from 1 to 6 carbon atoms, a benzyl radical and a phenethyl radical, said chain possibly being unsubstituted or substituted by one or more radicals, said radicals possibly in particular being chosen 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.

[0028] 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 from 7 to 20 amino acids.

[0029] An “amino acid” is a molecule comprising at least one carboxylic acid function (COOH) and one amine function, and at least one carbon atom linking this carboxylic acid function and this amine function. The amino acids used in the present invention may in particular be natural and / or synthetic amino acids.

[0030] Naturally occurring amino acids include, but are not limited to, the following: 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.

[0031] Synthetic amino acids are all unnatural amino acids. They 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-1-carboxymethyl piperidine, 1-amino-1-cyclobutanecarboxylic acid, 4-aminocyclohexaneacetic acid, 1-amino-1-cyclohexanecarboxylic acid, (1R,2R)-2-aminocyclohexanecarboxylic acid, (1R,2S)-2-aminocyclohexanecarboxylic acid, (1S,2R)-2-aminocyclohexanecarboxylic acid, (1S,2S)-2-aminocyclohexanecarboxylic acid, 3-aminocyclohexanecarboxylic acid, 4-aminocyclohexanecarboxylic acid, (lR,2R)-2-aminocyclopentanecarboxylic acid, (IR,2S)-2-aminocyclopentanecarboxylic acid, 1-amino-l-cyclopentanecarboxylic acid, 1-amino-l-cyclopropanecarboxylic acid, 3-aminomethylbenzoic acid,4-aminomethylbenzoic acid, 2-aminobutanoic acid, 4-aminobutanoic acid, 6-aminohexanoic acid, 1-aminoindan-1-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, (R)-4-amino-5-hydroxypentanoic acid, (R)-4-amino-5-(p-hydroxyphenyl)-pentanoic acid, 8-aminooctanoic acid, (2S,4R)-4-amino-pyrrolidine-2-carboxylic acid, (2S,4S)-4-amino-pyrrolidine-2-carboxylic acid, azetidine-2-carboxylic acid, (2S,4R)-4-benzyl-pyrrolidine-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, naphthyl-alanine, nicopetic acid, norvaline, octahydroindole-2-carboxylic acid, ornithine, penicillamine, phenylglycine, 4-phenylpyrrolidine-2-carboxylic acid, propargylglycine, 3-pyridinylalanine, 4-pyridinylalanine, acid l-pyrrolidine-3-carboxylic acid, sarcosine, statins, tetrahydroisoquinoline-1-carboxylic acid, l,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, tranexamic acid, 4,4-difluoroproline, 4-fluoroproline, alpha-(3,4-difluo- robenzyl)-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-difluorophen- nylglycine, 4,4-difluoroethylglycine, 4,4,4-trifluoroethylglycine, 4-fluorotryptophan, 5-fluorotryptophan, 6-fluorotryptophan, 5-methyltryptophan, S-tritylcysteine, selenocysteine, selenomethionine, ethioneine, P-(2-thienyl)alanine, beta-chloroalanine, thiazolylalanine, triazolalanine, p-fluorophenylalanine, o-fluorophenylalanine, m-fluorophenylalanine, dihydroxyphenylalanine, 2,5-dihydrophenylalanine, thioproline, pipecolic acid, canavanine, indospicine, 3,4-dehydroproline, histidinol and hexafluoronorleucine, and their analogues or derivatives.

[0032] The term "side chain" represents the moiety carried by the alpha carbon of an amino acid. For example, the side chains of naturally occurring amino acids such as glycine, valine, alanine, and aspartic acid correspond to the hydrogen atom, isopropyl, methyl, and CH2COOH groups, 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 acids: 4-amino tetrahydropyran-4-carboxylic acid, allylglycine, diamino butyric acid, diamino propionic acid, aminoserine, aminobutyric acid, amino butylglycine, phenylglycine, 4-chlorophenylalanine, 4-fluorophenylalanine, 4-nitrophenylalanine, citrulline, cyclohexylalanine, thienylalanine, and their like.

[0033] The side chains of amino acids can be protected by protective groups (P) and more particularly N-protective, O-protective or S-protective when these chains contain the corresponding heteroatoms. The protection of some of the reactive functions of peptides is mandatory during the synthesis of said peptides.

[0034] "Protective groups (P)" are groups known to those skilled in the art. These protecting 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 carried by a nitrogen atom will be referred to as N-protecting groups; protecting groups carried by a sulfur atom will be referred to as S-protecting groups, and protecting groups carried by an oxygen atom will be referred to as O-protecting groups. For example, a hydroxyl may be protected by a trityl group, or a carboxylic acid may be protected as a tert-butyl ester. In the case of solid-support synthesis, the resin serves as the protecting group for the C-terminal carboxylic function. Protection of the amino group (i.e., the "alpha amine") of the amino acid may be effected, for example, by a tert-butyloxycarbonyl (Boc-) group or a 9-fluorenylmethyloxycarbonyl (Fmoc-) group. The protection is effected according to methods known in the art.In the case of the protection of functional groups of natural amino acids, the resulting amino acids are synthetically processed until the protective group(s) are removed, thereby releasing the so-called natural amino acid. The removal of the protective group(s) is also carried out according to methods known in the art.

[0035] 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.

[0036] The term "plastic" means a support or surface comprising a mixture containing a base material which is a polymer, or a mixture of polymers. The plastic support or surface may have been obtained by molding, extrusion or shaping, preferably hot and under pressure, said mixture comprising a polymer.

[0037] The term "textile" refers to an assembly of threads or fibers, advantageously joined together or to each other, forming a solid and insoluble entity. Thus and suitably, the textile may be a fabric, advantageously obtained by weaving or knitting threads, or a non-woven obtained by assembling fibers. The textile material may in particular be a fabric, or a synthetic material such as a plastic material. The term textile includes in particular medical textiles, civilian textiles, technical textiles and filtration membranes.

[0038] 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 benzophenone unit of the conjugate to said surface.

[0039] 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. Peptide conjugate of formula (I)

[0040] The peptide conjugate used in the functionalization process according to the invention is a conjugate of formula (I) in which L is a spacer arm, n is 0 or 1, and A is a peptide fragment.

[0041] 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 make it possible to move the steric hindrance away from the surface when the peptide fragment is bulky, for example when it comprises side chains.

[0042] The nature of the spacer arm can vary widely. In certain 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 NRi group, in which Ri is chosen from a hydrogen atom, an aliphatic hydrocarbon radical comprising from 1 to 6 carbon atoms, a benzyl radical or a phenethyl radical, said chain possibly being unsubstituted or substituted.

[0043] In some embodiments, L is an aliphatic carbon chain of formula (II) in which m is an integer from 1 to 10, preferably from 4 to 6, in particular 5.

[0044] The ends of the spacer arm L which are attached respectively to the benzophenone and to 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.

[0045] In one embodiment, the linkage between the spacer arm L and the peptide fragment A is at the N-terminus of the peptide fragment A. In another embodiment, the linkage between the spacer arm L and the peptide fragment A is at the C-terminus of the peptide fragment A. In another embodiment, the linkage between the spacer arm L and the peptide fragment A is at a side chain of the peptide fragment A.

[0046] 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 multiple benzophenone heads, for example, 2, 3, 4, or 5 benzophenone heads preferably linked to different positions on the peptide fragment.

[0047] 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, in particular from 7 to 20 amino acids. In one embodiment, the peptide fragment A is not the GRGDSP fragment (SEQ ID NO: 1). In one embodiment, the peptide fragment A is not the RGD fragment. In one embodiment, the peptide fragment A is neither the GRGDSP fragment (SEQ ID NO: 1) nor the RGD fragment.

[0048] In one embodiment, peptide fragment A does not comprise an amino acid capable of reacting with benzophenone, such as methionine and / or tryptophan.

[0049] In one embodiment, 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 linked to the spacer arm are substituted and / or protected by a protecting group. For example, the COOH terminus of the peptide may be substituted by a fatty acid, such as palmitic acid.

[0050] In one embodiment, peptide fragment A is selected 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, peptide fragment A is selected from the group consisting of a cyclic natural peptide fragment, a cyclic synthetic peptide fragment, a cyclic protected natural peptide fragment, a cyclic protected synthetic peptide fragment, a cyclic natural pseudopeptide fragment, a cyclic synthetic pseudopeptide fragment, a cyclic protected natural pseudopeptide fragment, and a cyclic protected synthetic pseudopeptide fragment.

[0051] 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 the present description.

[0052] In one embodiment, the peptide fragment A is selected from the group consisting of the peptides H-(RF)4-NH2(SEQ ID NO:2), H-(RI)4-NH2(SEQ ID NO:4) and H-(R)2-Palm, Palm designating a modification of the terminal arginine by a palmitic acid.

[0053] Advantageously, the peptide fragment A has a particular property, in particular a particular chemical or biological activity. Thus, the peptide fragment A may 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.

[0054] In one embodiment, peptide fragment A is an antimicrobial peptide, preferably a cationic antimicrobial peptide, with an overall positive charge and comprising at least one hydrophobic residue.

[0055] The particular property of the peptide fragment A is advantageously not affected by its grafting to the spacer arm and to 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 not affected by its anchoring on the surface of the solid support, this particular property is therefore also a particular property of the corresponding functionalized surface of the solid support.

[0056] In one embodiment, peptide fragment A is an antimicrobial peptide selected from the group consisting of peptides H-(RF)4-NH2(SEQ ID NO:2), H-(RI)4-NH2(SEQ ID NO:4) and H-(R)2-Palm, Palm designating a modification of the terminal arginine by a palmitic acid.

[0057] In one embodiment, the peptide conjugate is selected from the group consisting of the conjugate defined by SEQ ID No. 3 and the conjugate defined by SEQ ID No. 5.

[0058] 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 the benzophenone and with the peptide fragment A.

[0059] The peptide conjugate and the peptide fragment A can in particular be synthesized by conventional peptide synthesis techniques. Peptide synthesis is conventionally carried out by activating the carboxylic acid function of an amino acid, or a chain of amino acids, by using a coupling agent. This activated acid is placed in the presence of 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 as well as the coupling agents used are very well known to those skilled in the art and 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 Alberi cio, 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.

[0060] Functionalization process

[0061] The functionalization method according to the invention applies to at least one surface of a solid support. In one embodiment, the surface functionalized by the method according to the invention is the total surface of the solid support. In another preferred embodiment, the surface functionalized by the method according to the invention is only a part of the total surface of the solid support.

[0062] The solid support, one surface of which is functionalized according to the invention, may be of any material benefiting from functionalization by the peptide conjugate of formula (I). It may in particular be a plastic support or a textile support, preferably a plastic support.

[0063] 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 the at least one surface of the solid support makes it possible to confer the same property on the at least one surface of the support.

[0064] In one embodiment, the method is carried out with a single peptide conjugate of formula (I). In other embodiments, the method is carried out with at least two different peptide conjugates, either one after the other or as a mixture. For example, the method 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.

[0065] The contacting of the at least one surface with the at least one peptide conjugate of formula (I) may be carried out by any suitable technique. In particular, the contacting may be carried out by dipping, spraying and / or incubation of the at least one surface with a solution or suspension of the peptide conjugate of formula (I) in a solvent. The solvent may be an organic solvent, an inorganic solvent, or a mixture of such solvents. The solvent may in particular be an alcohol, such as ethanol. In one embodiment, the peptide conjugate of formula (I) is soluble in the solvent used. In particular, spin-coating, dip-coating, casting, laminar flow deposition and aerospray deposition techniques may be mentioned.

[0066] A person skilled in the art is able to determine the parameters such as the duration and / or the temperature of the contact depending in particular on 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 duration of contact may correspond to the time required for the total evaporation of the solvent in which the conjugate is dissolved and / or suspended.

[0067] The functionalization process can make it possible to anchor 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).

[0068] In one embodiment, the functionalization method 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 preferably being carried out by dipping, spraying and / or incubation; b. Irradiating the surface in contact with the solution or suspension of the at least one peptide conjugate of formula (I), for a duration suitable for obtaining the anchoring of all or part of the peptide conjugate of formula (I) on the surface; and c. Rinsing all or part of the at least one surface of the solid support with a solvent.

[0069] Step a. of contacting can be implemented as described above for the contacting step.

[0070] The irradiation step b. allows in particular the activation of the benzophenone head of the peptide conjugate of formula (I) and its anchoring on the surface of the solid support. Preferably, this involves irradiation with ultraviolet (UV) rays. A person skilled in the art is able to adjust the irradiation parameters such as the duration of irradiation, the intensity and / or the wavelength of the irradiation depending in particular on 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. The irradiation step b. can be carried out either in the presence of the solvent of the solution or the 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.

[0071] The rinsing step c. makes it possible in particular to remove the excess peptide conjugate of formula (I) which has not anchored to the surface of the solid support at the end of the irradiation step b. The rinsing can be carried out with one solvent or with several solvents, either simultaneously or one after the other. The solvent of the rinsing step c. can be an organic solvent, an inorganic solvent or a mixture of such solvents. The rinsing step can be carried out once or several times, in particular 2, 3, 4, 5 or 10 times, with the same solvent or with different solvents. Preferably, the rinsing step c. makes it possible to remove all of the peptide conjugate of formula (I) which is not anchored to the surface at the end of step b.

[0072] In one embodiment, the functionalization method further comprises, before step a. of contacting, a step (i) of activating the at least one surface. This activation step makes it possible in particular to improve the subsequent anchoring of the peptide conjugate of formula (I) to the surface, for example by increasing the anchoring speed and / or by increasing the degree of functionalization. The nature of the activation to be implemented depends on the nature of the surface of the solid support. This activation may in particular be a thermal activation, by exposure to a temperature above room temperature (15 to 25°C), a chemical activation, by contacting with a chemical agent allowing activation, and / or an activation by irradiation. In one embodiment, the functionalization method further comprises, before step a. of contacting, a step (i') of cleaning the at least one surface.This step aims to remove any substance present on the surface which could prevent or weaken subsequent functionalization by the peptide conjugate(s). The cleaning step (i') can be carried out for example using a solvent such as an alcohol, in particular ethanol or isopropanol.

[0073] In one embodiment, the functionalization method further comprises, after the rinsing step c., a centrifugation step (ii), preferably also comprising the separation of the pellet and the centrifugation supernatant, the solid support being in the centrifugation pellet.

[0074] In one embodiment, the functionalization process further comprises, after rinsing step c., and after centrifugation step (ii) if present, an aging step (iii). Step (iii) may in particular be a reduced pressure aging step (under vacuum) and / or a heating aging step at a temperature above room temperature. In one embodiment, step (iii) comprises reduced pressure aging followed by heating aging.

[0075] Solid support

[0076] Another subject of the present invention is a solid support of which at least one surface is coated with at least one peptide conjugate, capable of being obtained, preferably obtained, by the functionalization method according to the invention.

[0077] In some embodiments, only one surface of the solid support is functionalized by a functionalization method 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 method according to the invention.

[0078] In other words, in certain embodiments, only a portion of the surface of the solid support is functionalized by a functionalization method according to the invention. Thus, for example, 100% of the surface of the solid support is functionalized by a functionalization method 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 method according to the invention.

[0079] Using solid support

[0080] The solid support according to the invention and / or the solid support capable of being obtained, preferably obtained, by a functionalization process according to the invention, can be used in a wide variety of applications.

[0081] Possible uses include the manufacture of nanoparticles for diagnosis, the functionalization of Elisa plates, the manufacture of antifouling surfaces, the manufacture of medical devices and / or the manufacture of technical textiles.

[0082] 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 diagnosis, 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.

[0083] The following examples illustrate the invention more specifically, and should be considered as illustrative and not limiting of the invention.

[0084] Examples

[0085] Preparation of peptide-substituted monomers

[0086] Peptides are synthesized on solid support using a Symphony X peptide synthesizer (Protein Technologies, Inc., USA) in Fmoc / tert-butyl strategy using nitrogen bubbling as a stirring method for coupling cycles and deprotection of Fmoc groups in the N-terminal position or in solution in Boc / Bzl strategy.

[0087] The syntheses were carried out on a 0.25 mmol scale on Fmoc-Rink-Amide polystyrene resin (481 mg / 0.52 mmol / g) or 2-chlorotrityl chloride (892 mg, 0.28 mmol / g). The standard deprotection-coupling cycle for each residue consisted of six steps: Wash 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). Wash the resin with 5 mL of DMF (3 x 30 sec). Coupling of the Fmoc- AA residue for 60 min using HATU (1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate) and N,N-diisopropylethylamine DIEA as coupling agent. At the end of the coupling cycle, a capping step is performed with 5 mL of 10% acetic anhydride AC2O in DMF for 7 min, then the resin is washed with 5 mL of DMF (3 x 30 sec).

[0088] Cleavage of the resin (Fmoc-Rink-Amide) is carried out 2 x 60 minutes in a cleavage cocktail (trifluoroacetic acid TFA / triisopropylsilane TIS / water H2O 95 / 2.5 / 2.5) for the deprotected sequences and 2 x 60 minutes in a cleavage cocktail (acetic acid AcOH / 2,2,2-Trifluoroethanol TFE / dichl oromethane DCM 10 / 20 / 70) for the protected sequences (2-chlorotrityl chloride resin). Once the resin is filtered, the solution is evaporated under vacuum and the peptide is precipitated in diethyl ether. The precipitated peptide is centrifuged (3500 RCF) and the supernatant is discarded. (X3). The peptide in the form of TFA salts is then solubilized in a water ftO / acetonitrile ACN mixture before being frozen and lyophilized.

[0089] Peptides were analyzed by UPLC chromatography and ESI-MS 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 were 0.1% TFA in water and 0.1% TFA in acetonitrile.

[0090] Peptide purification was performed on a WATERS HPLC 4000 equipped with a 486 UV detector and a Vydac Denali 10 pm C18 120 Å column (310 x 25 mm) 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).

[0091] Example 1: Synthesis of peptide conjugates according to the invention

[0092] Example 1a. Peptide conjugate comprising the peptide H-4-NH2 (SEQ ID NO. 2)

[0093] The antibacterial peptide H-(RF)4-NH2 (SEQ ID No. 2) was synthesized on a Rink Amide resin (loading 0.52 mmol / g, synthesis scale: 0.25 mmol) using the Fmoc / tBu strategy. Each coupling was followed by deprotection of the N-terminal Fmoc group. The peptide was then functionalized on a support after introduction of a spacer (or "linker"). The Fmoc-Ahx-OH spacer (6-hexanoic amino 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 mixture of DCM / TFA / H2O 50 / 47.5 / 2.5 for 2X1 h.The “cleavage” solution was concentrated under reduced pressure and 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 gradient of 20 to 60% buffer B in 40 min.).

[0094] The conjugate obtained has the formula as presented above, and corresponds to SEQ ID No. 3. E SI-MS (m / zy. [M+H] + theoretical C75H103N22O14: 1552.87, experimental: 1552.98 HPLC (retention time, min): 3.73

[0095] Example lb: Peptide conjugate comprising the peptide H-(RI)4-NH2 (SEQ IP No. 4)

[0096] The antibacterial peptide H-(RI)4-NH2 (SEQ ID No. 4) was synthesized on a Rink Amide resin (loading 0.52 mmol / g, synthesis scale: 0.25 mmol) using the Fmoc / tBu strategy. Each coupling was followed by deprotection of the N-terminal Fmoc group. The peptide was then functionalized on a support after 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, the resin was washed (3*DMF, 1*MeOH and 1*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 DCM / TFA / H2O 50 / 47.5 / 2.5 mixture for 2X1 h.The “cleavage” solution was concentrated under reduced pressure and 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 gradient of 20 to 60% buffer B in 40 min.).

[0097] The conjugate obtained has the formula as presented above, and corresponds to SEQ ID No. 5. E SI-MS (m / zy. [M+H] + theoretical C75H103N22O14: 1416.80, experimental: 1496.98 HPLC (retention time, min): 3.63

[0098] Example: Peptide conjugate comprising the peptide H-(R)2-Palm

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

[0100] The Rink-amide resin was pretreated with piperidine (20% in DMF) and washed (3*DMF, 1*MeOH and 1*DCM). 4-Amino 3-nitrobenzoic acid was coupled in DMF in the presence of 3 equivalents of HATU and 6 equivalents of DIEA. The reaction was left stirring for 2 hours and 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 (2*DCM, 3*DMF, 1*MeOH and 1*DCM). The reduction of the nitro group was carried out in a solution of 5g of SnCh(2H2O) and 900 μL of l,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 15h, syringe opened and washed (3*DMF, 3*DCM).

[0101] The introduction of the peptide sequence -(R)2- was carried out using the Fmoc / tBu strategy. Each coupling was followed by deprotection of the N-terminal Fmoc group. The peptide was then functionalized on a support after 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, 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, 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 hexadecylamine) in solution in DCM in the presence of DIEA (8 equivalents) was bubbled with nitrogen 20 minutes before the end of the cyclization, then was immediately introduced onto the resin.The reaction was stirred inducing 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 C18 120 Å column (310 x 25 mm) using a gradient of 20 to 60% buffer B in 40 min).

[0103] ESI-MS (m / z): [M+H] + theoretical C43H75N10O8: 876.22 experimental: 876.19

[0104] HPLC (retention time, min): 5.63 Example 2: Deposition of peptide conjugates according to the invention on a substrate

[0105] The deposition of the three peptides obtained in Example 1 was carried out by dip-coating, i.e. by dipping / removing at a constant speed the substrate in a solution of the anchor peptide in an ethanolic solution.

[0106] Each peptide was solubilized in an ethanolic solution (95%).

[0107] After a few minutes of stirring, a clear and stable solution was obtained. This solution was deposited on a clean polycarbonate substrate by dipping. The dipped plastic plates were then dried and irradiated under UV for 15 minutes (360 nm, 250W). After irradiation, the plates were washed under sonication in water and ethanol baths (4X10 minutes).

[0108] Example 3: Grafting of benzophenone onto a support

[0109] The fluorescent dansyl-benzophenone block of formula below was synthesized by conventional techniques.

[0110] A 96-well polystyrene plate was first rinsed with distilled water and ethyl alcohol, then dried. A 0.1M solution of dansyl-benzophenone compound was placed in the first three wells. The plate was irradiated for 180 seconds under UV at a power of 120 mW / cm 2 The plate was then rinsed with distilled water. Figure 2 shows a photo of the plate, which shows fluorescence from the 3 functionalized wells.

[0111] This result proves that the benzophenone head grafts onto the surface of the wells of the 96-well plate.

[0112] Example 4: Determination of the antibacterial activity of substrates treated with antimicrobial peptides according to the invention

[0113] 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 only on the evaluation of antibacterial activity.

[0114] 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.

[0115] Polycarbonate material (6 cm x 5 cm rectangle)

[0116] Such a study ideally uses 5 cm x 5 cm samples on 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 medium enumeration technique. Comparison of the bacterial concentrations obtained between the treated and untreated material makes it possible to define the antibacterial activity of the tested formulation.

[0117] The study conducted here was carried out on samples in the form of 6 cm x 5 cm rectangles 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.

[0118] Each conjugate 1a, 1b and 1c as synthesized in Example 1 was deposited on the surface of the polycarbonate plates by depositing a solution of 0.02 to 4 mg / mL.

[0119] The results were able to show a significant inhibition with a reduction of Staphylococcus aureus growth up to 99.87% (Tables 1 and 2 and Figure 1). Table 1 corresponds to Staphylococcus aureus, Table 2 corresponds to Escherichia coli.

[0120] In Tables 1 and 2, PC denotes polycarbonate. Peptide conjugates are designated by reference to the example in which their synthesis is described (1a, 1b and 1c).

[0121] Table 1

[0122] Table 2

[0123] Figure 1 illustrates these results. Significant inhibition of both types of bacteria was achieved (up to 99.87% growth reduction for Staphylococcus aureus, and up to 97.0% growth reduction for Escherichia coll) for the polycarbonate samples functionalized according to the invention compared to the non-functionalized polycarbonate samples.

[0124] The antibacterial activity of the peptide conjugates 1a, 1b and 1c is therefore efficiently transferred to the polycarbonate support onto which they are grafted.

Claims

Claims 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) in which L is a spacer arm, n is 0 or 1, preferably 1, and A is a peptide fragment, under conditions suitable for obtaining the anchoring of at least one peptide conjugate of formula (I) to the surface of the support.

2. Method for functionalizing at least one surface of a solid support according to claim 1, in which 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.

3. A method of 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 catalyst peptide, a biological receptor ligand peptide, an antibody and an enzyme inhibitor peptide, preferably an antimicrobial peptide, or a fragment thereof.

4. Method for functionalizing at least one surface of a solid support according to any one of claims 1 to 3, in which the peptide fragment is an antimicrobial peptide chosen from the group consisting of the peptides H-(RF)4-NH2(SEQ ID No. 2), H-(RI)4-NH2(SEQ ID No. 4) and H-(R)2-Palm.

5. Method for functionalizing at least one surface of a solid support according to any one of claims 1 to 4, in which 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 NRi group, in which Ri is chosen from a hydrogen atom, an aliphatic hydrocarbon radical comprising from 1 to 6 carbon atoms, a benzyl radical or a phenethyl radical, said chain possibly being unsubstituted or substituted, preferably L is an aliphatic carbon chain of formula (II) in which m is an integer from 1 to 10, preferably from 4 to 6, in particular 5.

6. Method for functionalizing at least one surface of a solid support according to any one of claims 1 to 5, in which the solid support is chosen from the group consisting of a plastic support and a textile support.

7. A method for functionalizing at least one surface of a solid support according to any one of claims 1 to 6, 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 one of claims 1 to 5 in a solvent, the contacting preferably being carried out by dipping, spraying and / or incubation; b. Irradiation, in particular UV irradiation, of the surface in contact with the solution or suspension of the 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 the at least one surface of the solid support with a solvent.

8. Method according to claim 7, further comprising at least one step chosen from the following steps: - a step (i), before step (a) of contacting, of activating the at least one surface, preferably implemented by thermal activation, chemical activation and / or by irradiation; - a step (i'), before step (a) of bringing into contact, of cleaning the 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.

9. Solid support of which at least one surface is coated with at least one peptide capable of being obtained, preferably obtained, by the functionalization process according to any one of claims 1 to 8.

10. Use of a solid support according to claim 9, for the manufacture of nanoparticles for diagnosis, 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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