Conjugates of antimicrobial agent and a linker moiety suitable for covalent attachment to materials and substrates

Abstract

The invention provides a conjugate of a compound of Formula (I) and a linker moiety of Formula (V), wherein Formulae (I) and (V) are as defined in the disclosure. The invention also provides a substrate having a derivative of the conjugate covalently attached thereto, methods of making the substrate and the conjugate, and substrates and conjugates obtainable by the methods of the invention.

Description

[0001] Conjugates of antimicrobial agent and a linker moiety suitable for covalent attachment to materials and substrates

[0002] The present invention relates to materials and substrates with antimicrobial agents, in particular small peptides, covalently attached thereto. Such materials find utility in a range of medical and industrial settings. The present invention further relates to conjugates of antimicrobial agents and a linker moiety suitable for covalent attachment to materials and substrates.

[0003] The rise of antibiotic resistance in common pathogens is a looming threat to patients worldwide. In medical settings, the issue is exacerbated by bacteria’s tendency to colonize and form resilient biofilm on abiotic surfaces such as implants, catheters and medical instruments. Biomaterial-associated infections with resistant bacteria already pose a heavy burden on the resources of medical clinics and result in bad health outcomes for the patients.

[0004] One approach could be to chemically attach substances that suppress bacterial growth, or binding, to the surface of biomaterials. Due to the covalent attachment, bacteria will experience a permanent, very high concentration of antibacterial substance in the close vicinity of the surface.

[0005] Antimicrobial peptides (AMPs) are a class of molecule with potential to replace classical antibiotics in these type of applications. These are amphiphilic peptides that can insert into the cell membrane(s) of bacteria, interact with the phospholipids and disturb membrane homeostasis. Membrane binding is facilitated by a high content of cationic amino acids lending AMPs selective binding to bacterial membrane, which have more anionic lipids than other cell membranes.

[0006] The use of natural AMPs for antibacterial products has been marginally successful; their length and dependence on native sequence makes their function sensitive to settings and enzymatic digestion, prone to cause off-target cytotoxicity, and lead to high production cost. Through studies of the structure-activity relationship for natural AMPs, novel peptides that are smaller, more potent and more stable have been designed. So-called small AMPs are made of a handful amino acids, mainly arginine (R) and tryptophan (W) providing the AMP with hydrophobic and cationic properties, respectively.

[0007] Several smart functionalized coatings that act as direct bacterial killers or as settlement repellents that prevent or limit the extent of bacterial colonization of the devices have been evaluated. For example, incorporating a slow-release antibiotic into the implant or coating via hydrolytically degradable multilayers or by bonding to graphene that sequentially deliver the antibiotic has been shown to increase tissue integration and lower infection occurrence. ZnO nanorods, superhydrophobic surfaces and noble metal nanoparticles have also all been evaluated for the purpose of preventing the formation of bacterial biofilm on medical devices and implants.

[0008] While many of the methods depend on release of an antibacterial component, some studies are also focused on developing coatings incorporating antimicrobial elements, including AMPs. One such study by Shriver-Lake et al. (Langmuir 2017, 33, 2878-2884) investigated the effect of linker length on cell capture by poly(ethylene glycol) (PEG) immobilized peptides of 14 to 27 amino acids in length. The PEG linkers varied from zero to 113 ethylene glycol units in length with mid-sized linkers seeming to perform best, although it was noted that the effects were peptide specific. These results contrasted with earlier reported studies where longer linkers were the best performers. Overall the teaching of the paper is that the best linker type and length for a given AMP cannot be predicted and there is no “one size fits all”.

[0009] The present inventors have developed a system which incorporates a highly effective and very small antimicrobial component and an optimized linker moiety which can be covalently attached to a substrate to generate a protective surface against colonizing microbes. It is an advantage of the present invention that the peptide-linker conjugate can be readily attached to a range of substrates (materials), through a controllable chemical reaction with reactive moieties on the substrate surface.

[0010] Summary of the invention

[0011] A first aspect of the invention provides a conjugate of a compound of Formula (I) as defined herein and a linker moiety of Formula (V),

[0012] N3-A-(CH2)m-NH-C(O)-Z-C*(O)- (V) wherein: the carbon marked with * forms an amide bond with the N-terminal amino group of the compound of Formula (I); A is absent or is an organic group comprising carbon, oxygen and hydrogen atoms and having a chain length of from 3 to 40 atoms; m is an integer of from 2 to 4, preferably 2 or 4;

[0013] Z is selected from the group consisting of -(CR4R5)p-(O)q-(CR4Rs)r-, -CR6=CR7-(CR4R5)S-, -(CR4R5)S-CR6=CR7-, -C(=CR8R9)-(CR4Rs)t- and -(CR4R5)t-C(=CR8R9)-; wherein q is 0 or 1 , preferably 1 ; p and r are each independently 0, 1 , 2 or 3, preferably 0, 1 or 2; and the sum of p, q and r is 2, 3 or 4, preferably 2 or 3, more preferably 3; s is 0 or 1 , preferably 0; t is 1, 2 or 3, preferably 1 or 2, more preferably 1 ; each R4 and Rs is independently selected from the group consisting of H, halogen, C1-C10 alkyl, C2-C10 alkenyl, aryl; -(CH2)i-eCOOH and -S-C(0)-Ci-Cio alkyl; or two R4 groups on adjacent -(CR4R5)- units (i.e. i.e. two R4 groups on adjacent alkylene units / an R4 group on one -(CR4R5)- unit and the R4 group on the next -(CR4R5)- unit) together form a carbocyclic ring or a heterocyclic ring; each Rs and R7is independently selected from the group consisting of H, halogen, C1-C10 alkyl, C2-C10 alkenyl, aryl; -(CH2)i-eCOOH and -S-C(0)-Ci-Cio alkyl; or Rs and R7together form a carbocyclic ring or a heterocyclic ring; each Rs and R9 is independently selected from the group consisting of H, halogen, C1-C10 alkyl, C2-C10 alkenyl, aryl; -(CH2)i-eCOOH and -S-C(0)-Ci-Cio alkyl.

[0014] A further aspect of the invention provides a conjugate of a compound of Formula (I) as defined herein and a linker moiety of Formula (VI)

[0015] N3-A-(CH2)m-NH-C(O)-Z-C*(O)- (VI) wherein: m, Z, p, q, r, s, t, and R4-R9 are as defined in the first aspect, and

[0016] A is absent or is an organic group comprising carbon, oxygen and hydrogen atoms; the -N**-A-(CH2)m-NH- unit has a number average molecular weight of 600 g / mol or less, wherein the nitrogen marked with ** corresponds to the nitrogen atom of the azide moiety which is bonded to the A group, or to the -(CH2)m- group if A is absent; and wherein the carbon marked with * in Formula (VI) forms an amide bond with the N-terminal amino group of the compound of Formula (I).

[0017] Further aspects of the invention are set out below.

[0018] Detailed Description

[0019] Compound of Formula (I)

[0020] The conjugates of the invention incorporate a compound of formula (I)

[0021] AA-AA-AA-X-Y (I) wherein, in any order, 2 of said AA (amino acid) moieties are cationic amino acids, preferably lysine or arginine but may be histidine or any non-genetically coded or modified amino acid carrying a positive charge at pH 7.0, and 1 of said AA is an amino acid with a large lipophilic R group, the R group having 14-27 non-hydrogen atoms and preferably containing 2 or more, e.g. 2 or 3, cyclic groups which may be fused or connected, these cyclic groups will typically comprise 5 or 6 non-hydrogen atoms, preferably 6 non-hydrogen atoms (in the case of fused rings of course the non-hydrogen atoms may be shared);

[0022] X is a N atom, which may be but preferably is not substituted by a branched or unbranched C1-C10 alkyl or aryl group, e.g. methyl, ethyl or phenyl, and this group may incorporate up to 2 heteroatoms selected from N, O and S; and

[0023] Y is selected from the group consisting of R1-R2-R3,

[0024] R1-R2-R2-R3, R2-R2-R1-R3, R1-R3 and R4 wherein:

[0025] R1 is C, O, S or N, preferably C;

[0026] R2 is C; each of R1 and R2 may be substituted by C1-C4 alkyl groups or unsubstituted, preferably Y is -R1-R2-R3 (in which R1 is preferably C) and preferably this group is not substituted, when Y is -R1-R2-R2-R3 or R2-R2-R1-R3 then preferably one or more of R1 and R2 is substituted;

[0027] R3 is a group comprising 1 to 3 cyclic groups each of 5 or 6 non-hydrogen atoms (preferably all C atoms but optionally also containing N, O or S), 2 or more of the cyclic groups may be fused; one or more of the rings may be substituted and these substitutions may, but will typically not, include polar groups, suitable substituting groups include halogens, preferably bromine or fluorine and C1-C4 alkyl groups; R3 incorporates a maximum of 15 non-hydrogen atoms, preferably 5-12, most preferably it is phenyl; and

[0028] R4 is an aliphatic moiety having 2-20 non-hydrogen atoms, preferably these are carbon atoms but oxygen, nitrogen or sulphur atoms may be incorporated, preferably R4 comprises 3-10, most preferably 3-6 non-hydrogen atoms and the moiety may be linear, branched or cyclic. If the R4 group comprises a cyclic group this is preferably attached directly to the nitrogen atom of X.

[0029] One AA in Formula (I) above, and AA2 in Formulae (ll)-(IV) below, comprises a large lipophilic R group. As used herein, the term “large” lipophilic R group denotes an R group as defined above, i.e. having 14-27 non-hydrogen atoms and preferably containing 2 or more, e.g. 2 or 3, cyclic groups which may be fused or connected, these cyclic groups will typically comprise 5 or 6 non-hydrogen atoms, preferably 6 non-hydrogen atoms (in the case of fused rings of course the non-hydrogen atoms may be shared).

[0030] Preferred compounds incorporate an R4 group which is linear or branched, in particular a linear or branched alkyl group including ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl and isomers thereof, hexyl and isomers thereof etc.; propyl, isopropyl, butyl and isobutyl are especially preferred.

[0031] In some embodiments, R4 is an aliphatic moiety (preferably an alkyl group) having 6-16 non-hydrogen atoms, preferably these are carbon atoms but oxygen, nitrogen or sulphur atoms may be incorporated, and the moiety may be linear, branched or cyclic.

[0032] In some preferred embodiments, R4 is an isopropyl group.

[0033] Of the R4 groups which comprise a cyclic group, preferred are molecules in which R4 is cyclohexyl or cyclopentyl.

[0034] Suitable non-genetically coded amino acids and modified amino acids which can provide a cationic amino acid include analogues of lysine, arginine and histidine such as homolysine, ornithine, diaminobutyric acid, diaminopimelic acid, diaminopropionic acid and homoarginine as well as trimethylysine and trimethylornithine, 4-aminopiperidine-4-carboxylic acid, 4-amino-1- carbamimidoylpiperidine-4-carboxylic acid and 4-guanidinophenylalanine.

[0035] The large lipophilic R group of the AA may contain hetero atoms such as O, N or S, typically there is no more than one heteroatom, preferably it is nitrogen. This R group will preferably have no more than 2 polar groups, more preferably none or one, most preferably none.

[0036] The compounds, which are preferably peptides, are preferably of formula (II)

[0037] AA1-AA2-AA1-X-Y (II) wherein:

[0038] AA1 is a cationic amino acid, preferably lysine or arginine but may be histidine or any non-genetically coded or modified amino acid carrying a positive charge at pH 7.0;

[0039] AA2 is an amino acid with a large lipophilic R group, the R group having 14-27 non-hydrogen atoms and preferably containing 2 or more, e.g. 2 or 3, cyclic groups which may be fused or connected, these cyclic groups will typically comprise 5 or 6 non-hydrogen atoms, preferably 6 non-hydrogen atoms; and

[0040] X and Y are as defined above.

[0041] Further preferred compounds include compounds of formulae (III) and (IV):

[0042] AA2-AA1-AA1-X-Y (III)

[0043] AA1-AA1-AA2-X-Y (IV) wherein AA1, AA2, X and Y are as defined above. Molecules of formula (II) are more preferred.

[0044] From amongst the above compounds certain are particularly preferred. In particular, compounds wherein the amino acid with a large lipophilic R group, conveniently referred to herein as AA2, is tributyl tryptophan (Tbt) or a biphenylalanine derivative such as Phe (4-(2-Naphthyl)) [also referred to herein as Bip (4-(2-Naphthyl)], Phe (4-(1 -Naphthyl)) [also referred to herein as Bip (4-(1-Naphthyl)], Bip (4-n-Bu), Bip (4-Ph) or Bip (4-T-Bu); Phe (4-(2-Naphthyl)) and Tbt being most preferred. In some preferred embodiments, the amino acid with a large lipophilic R group is tributyl tryptophan (Tbt).

[0045] Another preferred group of compounds are those wherein Y is -R1-R2-R3 as defined above, preferably wherein R1 and R2 are unsubstituted, most preferably wherein R1 and R2 are both carbon atoms.

[0046] A further preferred group of compounds are those in which -X-Y together is the group -NHCH2CH2Ph.

[0047] The compounds include all enantiomeric forms, both D and L amino acids and enantiomers resulting from chiral centers within the amino acid R groups and the C-terminal capping group “-X-Y”. p and y amino acids as well as a amino acids are included within the term 'amino acids', as are N-substituted glycines which may all be considered AA units. The compounds also include beta peptides and depsipeptides.

[0048] Most preferred compounds for incorporation into the conjugates of the invention are the following:

[0049] f-Bu represents a tertiary butyl group. This second compound incorporating the amino acid 2,5,7-Tris-tert-butyl-L-tryptophan is the more preferred. Analogues of this compound incorporating other cationic residues in place of Arg, in particular Lys, are also highly preferred. Analogues incorporating alternative C terminal capping groups as defined above are also highly preferred. The most preferred compound of Formula (I) has the structure

[0050] also referred to herein as AMC-109.

[0051] A further preferred group of compounds are those in which -X-Y together is selected from the group consisting of -NHCH(CH3)2, -NH(CH2)5CH3, -NH(CH2)3CH3, -NH(CH2)2CH3, -NHCH2CH(CH3)2, -NHcyclohexyl and -NHcyclopentyl, particularly preferred are compounds in which -X-Y is the group -NHCH(CH3)2or -NH(CH2)sCH3. A particularly preferred group of compounds are those in which -X-Y together is NHCH(CH3)2.

[0052] A preferred compound is a compound in which AAi is arginine, AA2is tributyl tryptophan and -X-Y together is NHCH(CH3)2.

[0053] Compounds incorporated into the conjugates of the present invention are preferably peptides.

[0054] The compounds of formulae (I) to (IV) may be peptidomimetics and peptidomimetics of the peptides described and defined herein also represent compounds of use in accordance with of the present invention. A peptidomimetic is typically characterised by retaining the polarity, three dimensional size and functionality (bioactivity) of its peptide equivalent but wherein the peptide bonds have been replaced, often by more stable linkages. By 'stable' is meant more resistant to enzymatic degradation by hydrolytic enzymes. Generally, the bond which replaces the amide bond (amide bond surrogate) conserves many of the properties of the amide bond, e.g. conformation, steric bulk, electrostatic character, possibility for hydrogen bonding etc. Chapter 14 of "Drug Design and Development", Krogsgaard, Larsen, Liljefors and Madsen (Eds) 1996, Horwood Acad. Pub provides a general discussion of techniques for the design and synthesis of peptidomimetics. In the present case, where the molecule is reacting with a membrane rather than the specific active site of an enzyme, some of the problems described of exactly mimicking affinity and efficacy or substrate function are not relevant and a peptidomimetic can be readily prepared based on a given peptide structure or a motif of required functional groups. Suitable amide bond surrogates include the following groups: N-alkylation (Schmidt, R. et al., Int. J. Peptide Protein Res., 1995, 46,47), retro-inverse amide (Chorev, M and Goodman, M., Acc. Chem. Res, 1993, 26, 266), thioamide (Sherman D.B. and Spatola, A.F. J. Am. Chem. Soc., 1990, 112, 433), thioester, phosphonate, ketomethylene (Hoffman, R.V. and Kim, H.O. J. Org. Chem., 1995, 60, 5107), hydroxymethylene, fluorovinyl (Allmendinger, T. et al., Tetrahydron Lett., 1990, 31, 7297), vinyl, methyleneamino (Sasaki, Y and Abe, J. Chem. Pharm. Bull. 199745, 13), methylenethio (Spatola, A.F., Methods Neurosci, 1993, 13, 19), alkane (Lavielle, S. et. al., Int. J. Peptide Protein Res., 1993, 42, 270) and sulfonamido (Luisi, G. et al. Tetrahedron Lett. 1993, 34, 2391).

[0055] The peptidomimetic compounds will typically have 3 identifiable sub-units which are approximately equivalent in size and function to amino acids (AA units). The term 'amino acid' may thus conveniently be used herein to refer to the equivalent sub-unit of a peptidomimetic compound. Moreover, peptidomimetics may have groups equivalent to the R groups of amino acids and discussion herein of suitable R groups and of terminal modifying groups applies, mutatis mutandis, to peptidomimetic compounds.

[0056] As is discussed in the text book referenced above, as well as replacement of amide bonds, peptidomimetics may involve the replacement of larger structural moieties with di- or tripeptidomimetic structures and in this case, mimetic moieties involving the peptide bond, such as azole-derived mimetics may be used as dipeptide replacements. Peptidomimetics and thus peptidomimetic backbones wherein the amide bonds have been replaced as discussed above are, however, preferred.

[0057] Suitable peptidomimetics include reduced peptides where the amide bond has been reduced to a methylene amine by treatment with a reducing agent e.g. borane or a hydride reagent such as lithium aluminium-hydride. Such a reduction has the added advantage of increasing the overall cationicity of the molecule.

[0058] Other peptidomimetics include peptoids formed, for example, by the stepwise synthesis of amide-functionalised polyglycines. Some peptidomimetic backbones will be readily available from their peptide precursors, such as peptides which have been permethylated, suitable methods are described by Ostresh, J.M. et al. in Proc. Natl. Acad. Sci. USA(1994) 91 , 11138-11142. Strongly basic conditions will favour N-methylation over O-methylation and result in methylation of some or all of the nitrogen atoms in the peptide bonds and the N-terminal nitrogen.

[0059] Preferred peptidomimetic backbones include polyesters, polyamines and derivatives thereof as well as substituted alkanes and alkenes.

[0060] Linker moiety

[0061] As defined above, the conjugates of the invention may incorporate a linker moiety having the structure of Formula (V)

[0062] N3-A-(CH2)m-NH-C(O)-Z-C*(O)- (V)

[0063] The group “N3-” denotes an azide group, which may alternatively be depicted as The azide group may undergo an azide-alkyne cycloaddition, such as a Huisgen azide-alkyne cycloaddition, with a terminal or internal alkyne on the surface of a substrate resulting in covalent attachment of the linker to the surface of the substrate. Preferably, the reaction is a copper-catalysed azide-alkyne cycloaddition. These are examples of “click” reactions.

[0064] In the Z groups, -(CR4R5)p-(O)q-(CR4Rs)r-, -CR6=CR7-(CR4Rs)s-, -(CR4R5)S-CR6=CR7-, -C(=CR8R9)-(CR4Rs)t- and -(CR4Rs)t-C(=CR8R9)-, the bond projecting to the right denotes the point of attachment to carbonyl -C*(O)- whilst the bond projecting to the left denotes the point of attachment to the other carbonyl -C(O)-.

[0065] Preferably, Z is selected from the group consisting of -(CR4R5)p-(O)q-(CR4Rs)r-, -CR6=CR7-(CR4Rs)s- and -(CR4Rs)s-CR6=CR7-. More preferably Z is -(CR4Rs)p-(O)q-(CR4Rs)r-.

[0066] Preferably, each R4 and R5 is independently selected from the group consisting of H, halogen, C1-C10 alkyl, C2-C10 alkenyl, and aryl; or two R4 groups on adjacent -(CR4R5)- units together form a carbocyclic ring or a heterocyclic ring. More preferably each R4 and R5 is H or C1-C10 alkyl. Most preferably each R4 and R5is H.

[0067] Preferably, each Re and R7 is independently selected from the group consisting of H, halogen, C1-C10 alkyl, C2-C10 alkenyl, and aryl; or Re and R7 together form a carbocyclic ring or a heterocyclic ring. More preferably each Re and R7 is H or C1-C10 alkyl. Most preferably each Re and R7 is H.

[0068] Preferably, each Rs and R9 is independently selected from the group consisting of H, halogen, C1-C10 alkyl, C2-C10 alkenyl, and aryl. More preferably each Rs and R9 is H or C1-C10 alkyl. Most preferably each Rs and R9 is H.

[0069] Preferably, Z is selected from the group consisting of -CH2OCH2-, -(CH2)2-, -(CH2)3-, -(CH2)4-, -O(CH2)2-, -(CH2)2O-, -CH2-O-, and -O-CH2-. More preferably, Z is selected from the group consisting of -CH2OCH2-, -(CH2)2-, -(CH2)3- and -(CH2)4-. More preferably, Z is selected from the group consisting of -CH2OCH2-, -(CH2)2- and -(CH2)3-. Most preferably, Z is -CH2OCH2-.

[0070] Oxygen-containing Z groups may increase aqueous solubility of, and facilitate production of, the linker moiety and the conjugate of the invention. This may also provide higher surface wettability once the conjugate is bound to the surface of the substrate.

[0071] The term "alkyl", as used herein alone or as part of another group such as alkoxy, is intended to include both branched and straight-chain saturated aliphatic hydrocarbon groups having the specified number of carbon atoms. For example, "C1-C10 alkyl" is intended to include Ci, C2, C3, C4, C5, Ce, C7, Cs, C9, and C10 alkyl groups. Preferred alkyl group are Ci-Ce alkyl groups, more preferably C1-C4 alkyl groups. Examples of suitable alkyl groups include methyl (Me), ethyl (Et), propyl (e.g., n-propyl and isopropyl), butyl (e.g., n-butyl, isobutyl, f-butyl), and pentyl (e.g., n-pentyl, isopentyl, neopentyl). The term, "alkenyl" is intended to include hydrocarbon chains of either straight or branched configuration having the specified number of carbon atoms and one or more, preferably one to two, more preferably one, carbon-carbon double bonds that may occur in any stable point along the chain. For example, "C2-C10 alkenyl" is intended to include C2, C3, C4, C5, Ce, C7, Cs, C9, and C10 alkenyl groups. Examples of alkenyl include, but are not limited to, ethenyl, 1 -propenyl, 2-propenyl, 2-butenyl, 3-butenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 2-hexenyl, 3-hexenyl, 4- hexenyl, 5-hexenyl, 2-methyl-2-propenyl, and 4-methyl-3-pentenyl.

[0072] The term “halogen” (which may alternatively be referred to as “halo”) refers to chlorine, bromine, fluorine, and iodine, with chlorine or fluorine being preferred.

[0073] The term "aryl" (e.g. as in the definitions of R5-R9) refers to monocyclic or polycyclic (including bicyclic and tricyclic) aromatic hydrocarbons, including, for example, phenyl, naphthyl, anthracenyl, and phenanthranyl. Preferably, the term “aryl” denotes monocyclic and bicyclic aromatic groups containing 6 to 10 carbons in the ring portion (such as phenyl or naphthyl including 1-naphthyl and 2-naphthyl), more preferably the aryl group is phenyl. The aryl can be attached through any available carbon by replacement of a hydrogen on said carbon.

[0074] As used herein, the "carbocyclic ring" (which may alternatively be referred to as “carbocyclyl”) may be any stable 5-, 6-, 7-, or 8-membered monocyclic or bicyclic or 7-, 8-, 9-, 10-, 11-, 12-, or 13-membered bicyclic or tricyclic hydrocarbon ring, any of which may be saturated, unsaturated or aromatic. Examples of such carbocycles include, but are not limited to, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cycloheptenyl, adamantyl, cyclooctyl, cyclooctenyl, cyclooctadienyl, [3.3.0]bicyclooctane, [4.3.0]bicyclononane, [4.4.0]bicyclodecane (decalin), [2.2.2]bicyclooctane, fluorenyl, phenyl, naphthyl, indanyl, adamantyl, anthracenyl, and tetrahydronaphthyl (tetralin). As shown above, bridged rings are also included in the definition of carbocycle (e.g., [2.2.2]bicyclooctane). Carbocyclic rings include "aryl". A bridged ring occurs when one or more carbon atoms link two non-adjacent carbon atoms. Preferred bridges are one or two carbon atoms. It is noted that a bridge always converts a monocyclic ring into a tricyclic ring. Preferably, the carbocyclic ring is 5-10 membered, more preferably 5-7 membered. Preferred carbocycles that may be formed by two R4 groups together include cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl. Preferred carbocycles that may be formed by Re and R7 groups together include, cyclopentenyl, cyclohexenyl and phenyl. As used herein, the "heterocyclic ring" (which may alternatively be referred to as "heterocyclyl") may be any stable 5-, 6-, or 7-membered monocyclic or 5-, 6-, 7-, 8-, 9-, 10-, 11-, 12-, 13-, or 14-membered polycyclic (including bicyclic and tricyclic) heterocyclic ring that is saturated, unsaturated, or aromatic, and that contains carbon atoms and 1 , 2, 3 or 4 heteroatoms independently selected from N, O and S; and including any polycyclic group in which any of the above-defined heterocyclic rings is fused to a carbocyclic or an aryl (e.g., benzene) ring. The term "heterocyclic ring” includes non-aromatic ring systems, such as heterocycloalkyl and heterocycloalkenyl. The nitrogen and sulfur heteroatoms may optionally be oxidized ( / .e., N^O and S(O)p, wherein p is 0, 1 or 2). A nitrogen in the heterocycle may optionally be quaternized. It is preferred that when the total number of S and O atoms in the heterocycle exceeds 1 , then these heteroatoms are not adjacent to one another. It is preferred that the total number of S and O atoms in the heterocycle is not more than 1. Examples of heterocyclic rings include, piperazinyl, piperidinyl, piperidonyl, piperonyl, pyranyl, morpholinyl, tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl, and dihydrofuro[2,3-b]tetrahydrofuran. Further examples of heterocyclic groups include the heteroaryl groups: pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazinyl, furyl, quinolyl, isoquinolyl, thienyl, imidazolyl, thiazolyl, indolyl, pyrroyl, oxazolyl, benzofuryl, benzothienyl, benzthiazolyl, isoxazolyl, pyrazolyl, triazolyl, tetrazolyl, indazolyl, 1 ,2,4-thiadiazolyl, isothiazolyl, purinyl, carbazolyl, benzimidazolyl, indolinyl, benzodioxolanyl, and benzodioxane. Preferably, the heterocyclic ring is 5- 10 membered, more preferably 5-7 membered, and comprises 1-4 heteroatoms independently selected from N, O and S.

[0075] When two R4 groups on adjacent -(CR4R5)- units together form a carbocyclic ring or a heterocyclic ring, it is preferred that the R5 groups on said -(CR4R5)- units are H.

[0076] The chain length of the A group may alternatively be referred to as the backbone length. As used herein, the chain length refers to the shortest distance in terms of number of atoms between the atom of the A group which is bonded to the -(CFhjm- moiety and the atom of the A group which is bonded to the azide group. The atom of the A group bonded to the bonded to the -(CFhjm- moiety and the atom of the A group which is bonded to the azide group are included in the chain length. Hydrogen atoms are not considered to be atoms of the chain / backbone of the A group for the purpose of counting the chain / backbone length. The chain / backbone of the A group may be linear or branched, preferably the chain / backbone is linear.

[0077] When the A group is absent the azide is directly bonded to the alkylene group -(CH2)m- in Formula (V). However, preferably the A group is present.

[0078] Preferably, the chain length of the A group is from 3 to 25 atoms, more preferably from 3 to 21 atoms, more preferably from 5 to 20 atoms, more preferably from 6 to 18 atoms, more preferably from 6 to 15 atoms, more preferably from 6 to 12 atoms, most preferably from 9 to 12 atoms.

[0079] Preferably, the A group consists of carbon, oxygen and hydrogen atoms.

[0080] The A group is preferably polyoxygenated.

[0081] Preferably, the A group is hydrophilic. This may ensure that the linker, when covalently attached to the surface, raises the compound of Formula (I) off the surface (or away from the surface) of the substrate when exposed to an aqueous environment.

[0082] The A group is preferably a polyether or a single repeating unit or oligomer thereof. More preferably, the A group is a polyoxyalkylene or a single repeating unit or oligomer thereof.

[0083] Polyoxyalkylenes are polymers having the structure where Yi is an alkane-a,w-diyl (also known as an alkylene group) or substituted alkane-a,w-diyl group contributing at least two carbon atoms to the main chain (pages 1159-1160 of Glossary of class names of polymers, Pure Appl. Chem., Vol. 81 , No. 6, pp. 1131-1186, 2009. doi:10.1351 / PAC-REC-08-01-30). Variable ^” denotes to the number of repeating units and may be selected to provide a chain length as described above and / or a number average molecular weight as described below. As used herein, the term oligomer refers a moiety consisting of 2-4 repeating units. For example, an oligomer of a polyoxyalkylene would have the structure wherein n is 2-4. As used herein, the term polymer refers to a moiety having a greater number of repeating units than an oligomer, i.e.

[0084] 5 or more repeating units. Preferably, the polyoxyalkylene is selected from polyethylene glycol or polytetrahydrofuran (i.e. Yi is preferably -(CH2)2- or -(CH2)4-). More preferably, the polyoxyalkylene is polyethylene glycol. When the polyoxyalkylene group is polyethylene glycol, m is preferably 2. When the polyoxyalkylene group is polytetrahydrofuran, m is preferably 4.

[0085] The A group is preferably selected from polyethylene glycol, polytetrahydrofuran, or a single repeating unit or oligomer thereof, more preferably the A group is selected from polyethylene glycol or a single repeating unit or oligomer thereof.

[0086] Polyethylene glycols are preferred, for example for providing higher aqueous solubility to the conjugate, and higher surface wettability once the conjugate is bound to the surface of the substrate. However, polytetrahydrofurans may be used where lower aqueous solubility and / or surface wettability is desired.

[0087] -A-(CH2)m-NH- is preferably selected from the group consisting of: -(CH2CH2O)ni-(CH2)2-NH-, wherein n1 is from 1 to 13, or -(CH2CH2CH2CH2O)n2-(CH2)4-NH-, wherein n2 is from 1 to 8, more preferably -A-(CH2)m-NH- is -(CH2CH2O)ni-(CH2)2-NH-. n1 is preferably from 1 to 9, more preferably from 1 to 6, more preferably from 2 to 5, most preferably from 2 to 4. n2 is preferably from 1 to 5, more preferably from 2 to 4 or 2 to 3.

[0088] Preferably, at least 60 mol % (more preferably at least 70 mol %) of the -A-(CH2)m-NH- groups in the linker moiety have an n1 or n2 value that is at most 3 repeating units above or below the most abundant n1 or n2 value.

[0089] Preferably, at least 60 mol % (more preferably at least 70 mol %) of the -A-(CH2)m-NH- groups in the linker have an n1 or n2 value that is at most 2 repeating units above or below the most abundant n1 or n2 value.

[0090] The most abundant n1 or n2 values and the proportion of n1 or n2 values equal to or within 2 or 3 repeating units of the most abundant n1 or n2 values can be determined by conducting mass spectrometry, such as ESI-MS, optionally in combination with liquid chromatography or high-performance liquid chromatography (HPLC), on the conjugate of the compound of Formula (I) and the linker moiety.

[0091] Preferably, the -N**-A-(CH2)m-NH- unit in the compound of Formula (V) has a number average molecular weight (Mn) of 600 g / mol or less. The nitrogen marked with ** (i.e. N**) corresponds to the nitrogen atom of the azide moiety which is bonded to the A group (or the -(CH2)m- group if A is absent). More preferably, the -N**-A-(CH2)m-NH- unit has a number average molecular weight of 500 g / mol or less, more preferably 400 g / mol or less, more preferably 300 g / mol or less.

[0092] Preferably, the -N**-A-(CH2)m-NH- unit in the compound of Formula (V) has a Mnof at least 100 g / mol, more preferably at least 125 g / mol, more preferably at least 150 g / mol, more preferably at least 175 g / mol or at least 180 g / mol. These upper and lower limits for the Mnmay be combined in any manner. Thus, the -N**-A-(CH2)m-NH- unit preferably has a Mnof from 100 to 600 g / mol, more preferably from 100 to 500 g / mol, more preferably from 120 to 400 g / mol, more preferably from 150 to 300 g / mol, more preferably from 175 to 250 g / mol, most preferably from 180 to 225, such as around 200 g / mol.

[0093] The -N**-A-(CH2)m-NH- unit (and / or the linker of Formula (V), and / or the conjugate) may be monodisperse or polydisperse, but will typically be polydisperse.

[0094] The -N**-A-(CH2)m-NH- unit may have a dispersity (also known as a polydispersity index) value of from 1.000 to 1.15, preferably from 1.001 to 1.05 or 1.10, more preferably from 1.001 to 1.01 , more preferably from 1.001 to 1.007 or 1.001 to 1.005.

[0095] The conjugate of the compound of Formula (I) as defined herein and the linker may have a number average molecular weight (Mn) of from 1050 to 1550 g / mol, preferably from 1100 to 1550 g / mol, more preferably from 1100 to 1350 g / mol, most preferably from 1100-1200.

[0096] The conjugate of the compound of Formula (I) as defined herein and the linker may have a dispersity value of from 1 to 1.15, preferably from 1.001 to 1.10 or 1.05, more preferably from 1.001 to 1.01 , more preferably from 1.001 to 1.007 or 1.001 to 1.005.

[0097] Dispersity values are (also known as a polydispersity index) a measure of the breadth of the molecular weight distribution and is defined as Mw / Mnwhere Mwand Mnare respectively the weight average and number average molecular weight.

[0098] Mnand Mwvalues can be measured by size exclusion chromatography or by mass spectrometry, such as electrospray ionisation mass spectrometry (ESI-MS). The Mnand Mwvalues of the -N**-A-(CH2)m-NH- unit, and / or the conjugate, can be determined by conducting mass spectrometry, such as ESI-MS, optionally in combination with liquid chromatography or high-performance liquid chromatography (HPLC), on the conjugate of the compound of Formula (I) and the linker moiety of Formula (V). Furthermore, the Mnand Mwvalues of the -N**-A-(CH2)m-NH- unit typically correspond to those of the starting material (such as the polyoxyalkylene, e.g. polyethylene glycol or polytetrahydrofuran, or the oligomer thereof) that is used to make the linker according to the method of the invention.

[0099] Suitable mass spectrometry instruments and HPLC instruments are commercially available. For example, a Thermo scientific Orbitrap Exploris 120 Mass spectrometer with an electrospray ion source in positive ion mode may be used. A Thermo Scientific Vanquish LIHPLC system may also be used with Column: Accucore Vanquish C18+ 50x2.1 Particle size 1.5p. The eluent may be a mixture of acetonitrile and water with 0.1% trifluoroacetic acid added (such as a 98 / 2 v:v mixture of acetonitrile / water with 0.1% v:v trifluoroacetic acid).

[0100] A suitable size exclusion chromatography method for determining molecular weights may involve, dissolving the analyte (such as the conjugate described herein) in a suitable solvent (such as DMF) and introducing the solution into one or more size exclusion chromatography columns. The particle and pore size of the packing material of the chromatography column may be chosen to determine the molecular weight resolution. For example, a particle size of 5 pm and porosity of 100 Angstroms or 300 Angstroms may be used. Suitable chromatography columns are commercially available, for example from PSS Germany under the brand name PFG. A calibration curve may be used to determine the relationship between elution volume and molecular weight. The calibration curve may be prepared using molecular markers (such as PEGs) of known molecular weights.

[0101] For example, molecular weights may be determined by size exclusion chromatography (SEC) using a TOSOH EcoSEC HLC-8320GPC (Tokyo, Japan) equipped with an EcoSEC Rl detector (Tokyo, Japan). Polymer samples may be dissolved in DMF at a concentration of 2.5 mg / mL. DMF containing 0.01 M LiBr may be employed as mobile phase operating at a flow rate of 0.2 mL / min at 35 °C. Three different SEC columns, PSS PFG 5 pm; Microguard 100 A & 300 A from PSS (Mainz, Germany), providing a resolving ranging of 300-100 000 Da, may be used for molecular weight analysis. A conventional calibration method using narrow linear poly (ethylene glycol) standards (PSS, Mainz, Germany) with a molecular weight range between 106-44000 Da may be employed. Corrections for flow rate fluctuations may be made using toluene as an internal standard. PSS WinGPC Unity software version 7.2 (Mainz, Germany) may be used to process the collected data.

[0102] In Formula (V), group A is absent or is an organic group comprising carbon, oxygen and hydrogen atoms and having a chain length of from 3 to 40 atoms. Alternatively, group A may be absent or be an organic group comprising carbon, oxygen and hydrogen atoms (optionally having a chain length of from 3 to 40 atoms) and wherein the -N**-A-(CH2)m-NH- unit has a number average molecular weight of (Mn) of 600 g / mol or less.

[0103] Thus, the linker moiety may alternatively have the structure of Formula (VI)

[0104] N3-A-(CH2)m-NH-C(O)-Z-C*(O)- (VI) wherein: in the conjugate the carbon marked with * forms an amide bond with the N- terminal amino group of the compound of Formula (I);

[0105] A is absent or is an organic group comprising carbon, oxygen and hydrogen atoms; m and Z are as defined above for Formula (V); and the -N**-A-(CH2)m-NH- unit has a number average molecular weight of 600 g / mol or less, wherein the nitrogen marked with ** corresponds to the nitrogen atom of the azide moiety which is bonded to the A group, or to the -(CH2)m- group if A is absent.

[0106] For the avoidance of doubt, the preferred definitions above disclosed in relation to Formula (V) (including, but not limited to, the preferred definitions of Z, A, the number average molecular weight of the -N**-A-(CH2)m-NH- unit and polydispersity index values) may apply mutatis mutandis to Formula (VI).

[0107] The invention also provides a conjugate of a compound of Formula (I) as defined herein and a linker moiety of Formula (VI) in which the carbon marked with * in Formula (VI) forms an amide bond with the N-terminal amino group of the compound of Formula (I).

[0108] Coated substrate The present invention also provides a substrate with a derivative of the conjugate described above covalently attached thereto. A schematic representation of an example of this functionalised substrate is shown in Figure 1. The linker results in covalent attachment of the compound of Formula (I) to the substrate. In embodiments, the compound of Formula (I) remains attached to the substrate (at least the majority of the compound of Formula (I) remains attached to the substrate) when the substrate is in use, e.g. placed in or in contact with a human or animal body. As used herein the term “derivative of the conjugate” may refer to the part of the conjugate which remains after an azide-alkyne cycloaddition has taken place between the conjugate and a substrate comprising an alkyne moiety on the surface of said substrate. This part of the conjugate will not contain the azide moiety -N3, as this azide moiety reacts with the alkyne during the cycloaddition reaction to form a triazole ring. In this case, the derivative of the conjugate will covalently attached to the substrate via a triazole ring. Thus, the substrate may have a compound of Formula (I) as defined herein covalently attached thereto by a linking moiety as shown in Formula (Villa) or Formula (VI I lb) below.

[0109] Thus, in a further aspect, the present invention provides a substrate having a compound of Formula (I) as defined herein covalently attached thereto by a linking moiety as shown in Formula (Villa) or Formula (VI lib): in which in Formula (Villa) and the bond from Ring B intersected by the wiggly line in Formula (VI I lb) denotes the point of attachment to the substrate; the carbon marked with * forms an amide bond with the N-terminal amino group of the compound of Formula (I) and

[0110] A, Z and m are as defined elsewhere herein.

[0111] R10 is derived from the terminal or internal alkyne on the surface of a substrate, before the reaction with the conjugate of the invention, i.e. a compound having the structure

[0112] R10 may be hydrogen or an organic group comprising from 1 to 30 nonhydrogen atoms. The non-hydrogen atoms may preferably be selected from the group consisting of carbon, nitrogen and oxygen. Preferably, R10 may be selected from the group consisting of H, C1-C10 alkyl, C1-C10 alkenyl, C1-C10 alkoxy, -C(O)C C10 alkyl, -C(0)OCi-Cio alkyl, a carbocyclic group (preferably a 5-10 membered carbocyclic ring, more preferably phenyl) and a heterocyclic ring (preferably a 5-10 membered heterocyclic ring, such wherein said alkyl, alkenyl and alkoxy groups are optionally substituted with one or more substituents independently selected from halogen, hydroxy, amino, (C1-C10 alkyl)amino and di(Ci-Cio alkyl)amino.

[0113] Ring B in Formula (VI lib) is a cyclic group, optionally an 8-10 membered cyclic group, preferably a cycloalkenyl group, such as an 8-10 membered cycloalkenyl group. Ring B may be fused to one or more other carbocyclic or heterocyclic rings, preferably to one or more aryl rings (e.g. phenyl) or to one or more cycloalkyl rings (e.g. C3-C6 cycloalkyl, such as cyclopropyl). The one or more rings to which Ring B is fused may optionally be substituted, for example to modify solubility. For example, ring B may be a dibenzocyclooctene ring, such as: wherein the ring fusion to the triazole in

[0114] Formula (VI I lb) occurs at the dashed bonds and the bond intersected by the wiggly line in denotes the point of attachment to the substrate.

[0115] Ring B may optionally be substituted, preferably with one or more electron withdrawing substituents. Suitable substituents may independently be selected from halogen, hydroxyl, Ci-Ce alkoxy, or benzyl groups.

[0116] Ring B is typically formed when the substrate (before the reaction with the conjugate of the invention) comprises one or more cyclic alkyne moieties, such as cyclic alkyne moieties selected from cyclooctynyl, dibenzocyclooctyne, cyclononynyl or cyclodecynyl. These cyclic alkynes may react with the azide group in the conjugates of the invention via a strain-promoted azide alkyne cycloaddition (Chem. Rev. 2021 , 121 , 7122-7154).

[0117] The floating bond intersecting Ring B means that Ring B may be attached to the substrate via any available ring member, e.g. via any available carbon ring member.

[0118] Preferably Rio is H.

[0119] Preferably, the substrate has a compound of Formula (I) as defined herein covalently attached thereto by a linking moiety as shown in Formula (Villa).

[0120] Substrates include materials known for use in medical devices and industrial settings where is it desired to provide the material with antimicrobial resistance, in particular to reduce or prevent biofilm formation (biofouling) on the material.

[0121] Suitable substrates include polymers; metals; and inorganic substrates such as glass and ceramics.

[0122] As used herein the term metals includes alloys thereof. Suitable metals include titanium and alloys thereof such as titanium nickel alloys, chromium-cobalt alloys, aluminium, stainless steel and precious metals such as gold, silver and platinum. Preferred metals include titanium and alloys thereof such as titanium nickel alloys, chromium-cobalt alloys, aluminium and stainless steel. As set out above, metals such as aluminium with oxide layers are convenient as the surface hydroxyl moieties can be used for attachment of the alkyne moiety.

[0123] Inorganic substrates include glass and ceramics such as alumina and zirconia. Glass is a preferred inorganic substrate and may be prepared for attachment of the conjugate by silanization, i.e. the incorporation of silanol groups, which can then be modified with alkyne groups, e.g. using the silyl halides or silyl ethers described below.

[0124] Suitable polymers for use as the substrate include polyethylene (PE); polyurethane (Pll); polyamide; polyethylene terephthalate (PET); synthetic rubber (SR); polystyrene (PS); polyacrylates such as polyacrylic acid (PAA), polymethylmethacrylate (PMMA), poly(ethyl cyanoacrylate) and polyacrylamide; polyacrylonitrile (PAN); polyetheretherketone (PEEK), polylactic acid (PI_A), silicone, polysaccharide and polyglycolide (PGA). Preferred polymers include polyacrylates and Pll.

[0125] Substrates may be provided as coatings on other materials.

[0126] Suitable substrates include any material which can carry on its surface an alkyne moiety, the alkyne moiety is able to react with the azide part of the conjugate in a so-called Click reaction in order to covalently attach the compound of formula (I) and the linker to the substrate. The alkyne may be a terminal or internal alkyne, preferably it is a terminal alkyne. This reaction providing Formula (Villa) is illustrated in the Scheme below:

[0127]

[0128] As illustrated in the Scheme above, the carbon atoms of the alkyne group are incorporated into the triazole ring in Formula (Villa) and (Vlllb). For the avoidance of doubt, the substrate structure in Formula (Villa) and (Vlllb) will correspond to the non-alkyne portion of the substrate structure prior to the reaction with the conjugate of the invention.

[0129] The alkyne moiety can be incorporated into / onto the substrate through methods known in the art and described in the Examples herein for polyurethane (Pll) and glass. Thus, the substrate materials may need to adapted for incorporation of (or functionalised to include) the alkyne moiety.

[0130] For polymeric substrates, monomers comprising one or more side chains including the alkyne moiety can be incorporated into the polymer structure and / or side chains can be reacted with alkyne-containing compounds to introduce the alkyne moiety.

[0131] For example, alkyne-containing compounds can be joined to side chains via ester or amide linkages. This approach can be used to introduce alkyne moieties to substrates formed from polyacrylates, e.g. polyacrylic acid.

[0132] When the substrate is formed from a metal or an alloy which comprise a surface oxide layer (such as titanium, aluminium or alloys thereof) surface hydroxyl groups on the oxide layer can be reacted with alkyne-containing compounds to introduce the alkyne moiety. Surface oxide layers typically comprise surface hydroxide groups when hydrated.

[0133] Glass substrates typically comprise surface silanol groups which can also be reacted with alkyne-containing compounds to introduce the alkyne moiety.

[0134] Metal substrates (such as noble metals, for example gold, silver and platinum) can be functionalised to include amino groups. These amino groups can then be reacted with alkyne-containing compounds to introduce the alkyne moiety.

[0135] Silyl halides (such as silyl chlorides) comprising one or more alkyne- containing groups and / or silyl ether compounds comprising one or more alkoxy groups and one or more alkyne-containing groups are examples of alkyne- containing compounds suitable for reacting with polymer side chains or substrates comprising silanol, hydroxyl and / or amino groups on the surface of the substrate.

[0136] An example of a suitable silyl ether compound no. 870987-68-1).

[0137] Alkyne moieties can also be introduced onto polymer side chains or substrates comprising silanol, hydroxyl and / or amino groups on the surface stepwise by first reacting with a silyl halide or silyl ether compound comprising a functional group (such as an amino group) which can subsequently be reacted with an alkyne-containing compound. For example, (aminopropyl)trimethoxysilane may be reacted with suitable polymer side chains or with substrates comprising surface hydroxyl, silanol or amino groups to provide an immobilised amino groups. The immobilised amino group can then react with an alkyne containing moiety (such as 5-hexynoic acid) via an amidation reaction to immobilise the alkyne groups.

[0138] Alkyne-containing compounds, such as alkyne-containing thiols, which form self-assembled monolayers (SAMs) on substrates (such as noble metals, for example gold, silver and platinum) can also be used to incorporate the alkyne moiety.

[0139] Pll is a preferred substrate and alkyne functionalised diols may be used as chain extenders and to provide a modified Pll surface with alkyne groups available to take part in a Click reaction with the conjugate of the invention.

[0140] The Pll may comprise aromatic or aliphatic diol monomers comprising one or more alkyne groups (such as acetylene). Suitable diols include 2,2-di(prop-2- ynyl)-propane-1 ,3-diol (DPPD) and 3,5-bis(hydroxymethyl)-1-propargylbenzene (PBM), with PBM being preferred

[0141] Preferably, the length of the chain between the carbon marked with # in Formula (Villa) or (Vlllb) and the surface of the substrate, or between the carbon marked with # and the backbone of the polymer (such as when the substrate is a polymer comprising a side chain which contains one or more alkyne moieties and / or a side chain which has been modified to include one or more alkyne moieties) is 25 atoms or fewer, more preferably 20 atoms or fewer, more preferably 10 atoms or fewer.

[0142] This chain length refers to the shortest distance in terms of number of atoms between the carbon marked with # and the surface of the substrate, or between the carbon marked with # and the backbone of the polymer, such as when the substrate is a polymer comprising a side chain which contains one or more alkyne moieties and / or a side chain which has been modified to include one or more alkyne moieties. This chain will typically comprise atoms from the compound used to introduce the alkyne group onto the surface of the substrate prior to reaction with the conjugate of the invention, or atoms from the side chain comprising one or more alkyne groups or from the side chain as modified to include one or more alkyne groups (when the substrate is a polymer comprising a side chain which contains one or more alkyne moieties and / or a side chain which has been modified to include one or more alkyne moieties). Hydrogen atoms are not considered to be atoms of this chain. The carbon marked with # is not considered part of this chain for the purpose of calculating this chain length. Where the alkyne moiety is attached to the substrate via surface exposed hydroxyl or silanol groups, or via amino groups bonded to the surface of the substrate (e.g. using a silyl chloride or silyl ether compound as described above) the oxygen of the hydroxyl or silanol group or the nitrogen of the amino group, is included for the purpose of calculating the chain length. If a SAM is used to attach the alkyne moiety to the surface of the substrate the atom of the alkyne-containing compound forming the SAM which is bonded to the surface of the substrate is included for the purpose of calculating the chain length.

[0143] Preferably, the distance between the carbon marked with # in Formula (Villa) or (Vlllb) and the surface of the substrate is 30 Angstroms or less, preferably 25 Angstroms or less, preferably 20 Angstroms or less, more preferably 10 Angstroms or less. This distance may be between the carbon marked with # in Formula (Villa) or (Vlllb) and the surface of the substrate when the substrate is exposed to aqueous conditions. Typically, the linking moiety shown in Formula (Villa) or (Vlllb) will lift the compound of Formula (I) off (or away from) the surface when exposed to aqueous conditions.

[0144] Preferably, the derivative of the conjugate (i.e. the conjugate once covalently attached to the surface) is homogeneously distributed over the surface (such as on the micron scale).

[0145] Thus, preferably the alkyne groups are homogeneously distributed over the surface of the substrate (such as on the micron scale) prior to the click reaction to attach the conjugate of the invention.

[0146] The distribution of conjugate derivatives on the surface of the substrate after the click reaction and / or the distribution of alkyne groups on the surface prior to the click reaction can be assessed by Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS).

[0147] In a further aspect, the present invention provides a method of making a substrate with a compound of Formula (I) covalently attached thereto, which method comprises reacting a conjugate as defined herein with a substrate having alkyne groups on the surface thereof, under conditions which allow a cycloaddition reaction to take place between the alkyne and an azide group on the conjugate. The invention also provides a substrate with a derivative of the conjugate covalently attached thereto which is obtainable by (or obtained by) this method.

[0148] The substrates described herein may be or be formed into a medical device or coating on a medical device.

[0149] Preferably, the contact angle of the substrate with the derivative of the conjugate described above covalently attached thereto, is less than 90°, more preferably 80° or less, more preferably 70° or less. The contact angle may be determined by the sessile drop method. The drop applied may be distilled water, a suitable drop volume may be 3-10 pL, such as around 5 pL, and the drop shape may be analysed 10-30 seconds, such as around 10 seconds, after surface deposition. The drop shape may be analysed under ambient conditions (such as at 18-25 °C and a relative humidity of 50-90%). Commercially available instruments and software may be used for deposition and evaluation of drop shape (such as a DSA100 instrument and DSA3 software from Kruss GmbH (Hamburg, Germany)).

[0150] An exemplary method for determining contact angle may involve extraction of the substrate comprising the covalently attached derivative of the conjugate in acetone / EtOH over-night followed by careful washing with EtOH. A DSA100 instrument from Kruss GmbH (Hamburg, Germany) with DSA3 software (Kruss GmbH, Hamburg, Germany) may then be used for the deposition and image evaluation of drop shape. A drop volume of 5 pL may be deposited using ultrapure and deionized water (resistivity > 18.2 MQ cm) and the drop shape may be measured 10 seconds after surface deposition. The analysis may be performed under ambient temperature and humidity. Contact angles may be reported as the average of at least six measurements.

[0151] A further aspect provides a method for attaching the conjugate of the invention to a substrate comprising one or more alkyne moieties on the surface of the substrate, the method comprising reacting the conjugate with the substrate comprising one or more alkyne moieties. Suitable substrates comprising one or more alkyne moieties are described above. In this aspect, the substrate starting material will comprise the one or more alkyne groups, which subsequently react with the azide to form the triazole ring shown in the structure of Formula (Villa) or (VI I lb) above. As set out above, this reaction is an azide-alkyne cycloaddition, such as a Huisgen azide-alkyne cycloaddition. Preferably, the reaction is a copper-catalysed azide-alkyne cycloaddition (CuAAC) or a strain-promoted azide- alkyne cycloaddition (SPAAC), more preferably CuAAC. These are examples of “click” reactions and suitable reaction conditions are known. For example, suitable reaction conditions are described in the Examples. The invention also provides a substrate with a derivative of the conjugate covalently attached thereto which is obtainable by (or obtained by) this method.

[0152] The substrates of the invention described herein may be or be formed into a medical device or coating on a medical device. Suitable medical devices include implantable devices (including orthopedic implants such as hip and knee implants, as well as dental implants), surgical and other medical instruments and single use devices such as syringes. Of particular interest are stents, catheters, lines, fasteners, pins and staples, dressings, patches, prosthetic liners and sutures, Pll catheters are especially preferred devices according to the present invention. Methods for making conjugates

[0153] Another aspect of the invention provides a method (Method A) of making a conjugate of a compound of Formula (I) and a linker moiety as described herein, the method comprising coupling a compound of Formula (IX):

[0154] N3-A-(CH2)m-NH-C(O)-Z-C*(O)OH (IX) to a compound of Formula (I) to form an amide bond between the carbon marked with * and the N-terminal amino group of the compound of Formula (I), wherein A, Z and m may be as defined elsewhere herein.

[0155] The step of coupling may involve activating the carboxylic acid group in the compound of Formula (IX) with a suitable peptide coupling reagents. Suitable peptide coupling reagents are known in the art, e.g. HBTLI.

[0156] The compound of Formula (IX) may be produced by a method comprising, converting a compound of Formula (X)

[0157] HO-A-(CH2)m-OH (X) to a compound of Formula (XI)

[0158] N3-A-(CH2)m-N3(XI) converting the compound of Formula (XI) to a compound of Formula (XII) N3-A-(CH2)m-NH2(XII) and reacting the compound of Formula (XII) with a 5-7 membered cyclic anhydride to provide the compound of Formula (IX).

[0159] The compound of Formula (X) may be converted to the compound of formula (XI) by activating the OH groups towards nucleophilic substation (e.g. by converting the OH groups into better leaving groups) and then reacting the activated intermediate with an azide nucleophile source, such as sodium azide. Examples of leaving groups to which into which the OH groups may be converted include tosylate and mesylate. Preferably, the compound of formula (X) is bis- tosylated and then converted to a compound of Formula (XI)

[0160] The compound of Formula (XI) may preferably be converted to a compound of Formula (XII) using a reducing agent, such as triphenylphosphine (e.g. via the Staudinger Reduction). This step conveniently generates an asymmetric intermediate because once one of the azide moieties has been reduced to an amine, the molecule moves from an organic to an aqueous phase where no further reduction can take place. The inventors have found that this ability to move from one phase to the other is dependent on the length of the compound of Formula (XI) and so restricts “m” and the length of moiety “A” and thus the length of the linker / conjugate as defined herein.

[0161] The compound of Formula (X) may preferably have a Mnof 600 g / mol or less. More preferably, the compound of Formula (X) has a number average molecular weight of 500 g / mol or less, more preferably 400 g / mol or less, more preferably 300 g / mol or less. Preferably, the compound of Formula (X) has a Mnof at least 100 g / mol, more preferably at least 125 g / mol, more preferably at least 150 g / mol, more preferably at least 175 g / mol or at least 180 g / mol. These upper and lower limits for the Mnmay be combined in any manner. Thus, the compound of Formula (X) preferably has a Mnof from 100 to 600 g / mol, more preferably from 100 to 500 g / mol, more preferably from 120 to 400 g / mol, more preferably from 150 to 300 g / mol, more preferably from 175 to 250 g / mol, most preferably from 180 to 225, such as around 200 g / mol.

[0162] The compound of Formula (X) may have a dispersity (also known as a polydispersity index) value of from 1.000 to 1.15, preferably from 1.001 to 1.05 or 1.10, more preferably from 1.001 to 1.01 , more preferably from 1.001 to 1.007 or 1.001 to 1.005.

[0163] Preferably, the compound of Formula (X) is polyethylene glycol, polytetrahydrofuran, or an oligomer thereof, more preferably PEG or an oligomer thereof.

[0164] Any 5-7 membered cyclic anhydride may be used to convert the compound of Formula (XII) to the compound of Formula (IX). In this context “5-7 membered” denotes the number of atoms in the ring containing the anhydride functional group -C(O)-O-C(O)-. The other ring members may be selected from oxygen and carbon atoms and the ring containing the anhydride functional group may be saturated or unsaturated. The cyclic anhydride may comprise one or more substituents, for example as defined above for group Z. Preferably, the cyclic anhydride may have the structure wherein Z is as defined above for Formula

[0165] (V). Examples of suitable cyclic anhydrides include glycolic anhydride, succinic anhydride, glutaric anhydride, adipic anhydride, methyl succinic anhydride, phthalic anhydride, maleic anhydride, 1,2-cis-cyclohexanedicarboxylic anhydride, homophthalic anhydride, octenylsuccinic anhydride, S-acetylmercaptosuccinic anhydride, itaconic anhydride, citraconic anhydride, dimethylmaleic anhydride, cisaconitic anhydride, 2-(2’-carboxyethyl) maleic anhydride, and 1-methyl-2-(2’- carboxyethyl) maleic anhydride. Preferably the cyclic anhydride is glycolic anhydride.

[0166] Another aspect provides a method (Method B) of preparing conjugate of a compound of Formula (I) as defined herein and a linker moiety, the method comprising converting a compound of Formula (X)

[0167] HO-A-(CH2)m-OH (X) wherein A is absent or is an organic group comprising carbon, oxygen and hydrogen atoms (preferably a polyoxyalkylene [more preferably PEG or PTHF] or a single repeating unit, or oligomer thereof); m is an integer of from 2 to 4, preferably 2 or 4; and wherein the compound of Formula (X) has a Mnof 600 g / mol or less; to a compound of Formula (XI)

[0168] N3-A-(CH2)m-N3(XI) converting the compound of Formula (XI) to a compound of Formula (XII)

[0169] N3-A-(CH2)m-NH2(XII) and reacting compound of Formula (XII) with a 5-7 membered cyclic to provide a compound of Formula (IX)

[0170] N3-A-(CH2)m-NH-C(O)-Z-C*(O)OH (IX) and coupling the compound of Formula (IX) to a compound of Formula (I) to form an amide bond between the carbon marked with * in Formula (IX) and the N-terminal amino group of the compound of Formula (I).

[0171] For the avoidance of doubt, the Mnand dispersity values for the compound of Formula (X) disclosed above in relation to Method A apply equally to this aspect of the invention. The details on the coupling step between Formula (IX) and Formula (I), and the steps of: converting the compound of Formula (X) to the compound of Formula (XI), converting the compound of Formula (XI) to the compound of Formula (XII) and the nature of the cyclic anhydride, disclosed above in relation to method A also apply equally to this aspect of the invention.

[0172] For the avoidance of doubt, the definitions above disclosed in relation to Formula (V) (including, but not limited to, the preferred definitions of Z, A, the number average molecular weight and polydispersity index values) may apply mutatis mutandis to the methods of the invention and the conjugates obtained by or obtainable by the methods of the invention (including Method A and Method B).

[0173] Another aspect provides the conjugate of a compound of Formula (I) and a linker moiety, obtainable by (or obtained by) the methods of the invention (including Method A and Method B).

[0174] The conjugate obtainable by (or obtained by) the methods of the invention (including Method A and Method B) may be attached to a substrate comprising one or more alkyne moieties on the surface of the substrate, by reacting the conjugate with the substrate comprising one or more alkyne moieties. As set out above, this reaction is an azide-alkyne cycloaddition. The invention also provides a substrate with a derivative of the conjugate covalently attached thereto which is obtainable by (or obtained by) this method.

[0175] Peptide synthesis

[0176] The compounds of Formula (I) for use in the conjugates of the invention may be synthesised in any convenient way. Generally, the reactive groups present (for example amino, thiol and / or carboxyl) will be protected during overall synthesis.

[0177] The final step in the synthesis will thus be the deprotection of a protected derivative of the invention.

[0178] In building up a peptide, one can in principle start either at the C-terminal or the N-terminal although the C-terminal starting procedure is preferred.

[0179] Methods of peptide synthesis are well known in the art but for the present invention it may be particularly convenient to carry out the synthesis on a solid phase support, such supports being well known in the art.

[0180] A wide choice of protecting groups for amino acids are known and suitable amine protecting groups may include carbobenzoxy (also designated Z), t- butoxycarbonyl (also designated Boc), 4-methoxy-2,3,6-trimethylbenzene sulphonyl (Mtr) and 9-fluorenylmethoxy-carbonyl (also designated Fmoc). It will be appreciated that when the peptide is built up from the C-terminal end, an amine- protecting group will be present on the a-amino group of each new residue added and will need to be removed selectively prior to the next coupling step.

[0181] Carboxyl protecting groups which may, for example be employed include readily cleaved ester groups such as benzyl (Bzl), p-nitrobenzyl (ONb), pentachlorophenyl (OPCIP), pentafluorophenyl (OPfp) or t-butyl (OtBu) groups as well as the coupling groups on solid supports, for example methyl groups linked to polystyrene.

[0182] Thiol protecting groups include p-methoxybenzyl (Mob), trityl (Trt) and acetamidomethyl (Acm). A wide range of procedures exists for removing amine- and carboxyl- protecting groups. These must, however, be consistent with the synthetic strategy employed. The side chain protecting groups must be stable to the conditions used to remove the temporary a-amino protecting group prior to the next coupling step.

[0183] Amine protecting groups such as Boc and carboxyl protecting groups such as tBu may be removed simultaneously by acid treatment, for example with trifluoroacetic acid. Thiol protecting groups such as Trt may be removed selectively using an oxidation agent such as iodine.

[0184] The present invention will now be further described by way of the following non-limiting Examples and with reference to the Figures in which:

[0185] Figure 1 Top: Alkyne functionalized diols fur use in the Pll materials of Example 1. Bottom: Schematic representation of Click mediated AMP functionalization of a PU- film incorporating alkyne-containing monomers.

[0186] Figure 2 Structures of the modified AMP prepared in Example 1 and AMC-109.

[0187] Figure 3 CLSM images of coated plastic films after incubation with S. epidermidis as described in Example 1. Left image: Control PU6 film. Right image: PU6 film coated with AMC-25-04 with a much lower bacterial load.

[0188] Figure 4 Fluorescence micrographs of coated PU-catheters as described in Example 1. Upper row illustrates catheter section coated with PU6. The lower row shows catheters coated with PU6 with AMC-25-04 covalently attached after incubation with S. epidermidis and staining.

[0189] Figure 5 Green color (first and third bars from left) denotes control experiments in which azide conjugated D-mannose was bound to the glass surface, blue color (second and fourth bars from left) denotes experiments with WT E. coli in which AMC-25-04 was bound to the glass surface. Statistical significance was tested by Student’s T-test where n.s. denotes not significant, * p<0.05, ** p<0.01, *** p<0.005 and **** p<0.001. The bars show the average and SE of median GRs measured before (“Initial GR”) and after (“Final GR”) the transition time was reached in the individual experiment.

[0190] Figures 6 and 7 are mass spectra of the conjugates produced in Example 3. The number average molecular weight of the PEG starting materials were 200 g / mol (Figure 6) and 400 g / mol (Figure 7).

[0191] Figure 8 Antibacterial efficacy of the coated substrate produced in Example 4 (right) vs control (left) evaluated using the standard shake flask test ASTM E2149- 13a and the Certika data evaluation approach. Example 1 - Biocompatible polyurethane coatings incorporating a surface tethered antimicrobial peptide

[0192] As outlined in Figure 1, a polyurethane surface with alkyne moieties is functionalized by a peptide-linker-azide conjugate through a CuAAC (Copper- Catalyzed Azide-Alkyne Cycloaddition) reaction , often described as a Click reaction. The linker is a short polyethylene glycol (PEG) moiety that is monodisperse (n=3) or polydisperse, and the PEG-linker has an azide moiety at one end, whereas the other end is modified as a carboxylic acid for connection to the N-terminus of the antimicrobial peptide.

[0193] MATERIALS AND METHODS

[0194] Peptide synthesis

[0195] Peptides were prepared by Amicoat A / S and used as received at a purity in excess of 95 %. Briefly, AMC-109 was prepared according to literature procedures (Svenson J, Stensen W, Brandsdal BO, Haug BE, Monrad J, Svendsen JS. Biochemistry. 2008, 47, 3777-88 DOI: 10.1021 / bi7019904).

[0196] Peptides were prepared using standard Fmoc-protocols coupled to the PEG-linker either on resin or in solution.

[0197] The amino-azido derivative of PEG200, prepared from PEG200 as described by Jiang et al. (ACS chemical neuroscience. 2018, 9, 100-6 DOI: 10.1021 / acschemneuro.7b00111), was reacted with diglycolic anhydride to provide a bifunctional polydisperse PEG-linker with an azido terminus and a carboxylic acid terminus according to (Pat.US2009 / 0203584A1 p15).

[0198] This linker (1.0 eq.) was subsequently coupled to AMC-109 (1.0 eq.) using HBTU (1.2 eq.) and TEA (4.8 eq.) providing the final product (AMC-25-04). The crude peptide was purified by preparative HPLC and lyophilized to yield a TFA-salt. Purity (HPLC): > 95%.

[0199] A small amount of monodisperse AMC-25-04 was also prepared and used for optimizing the CuAAC reaction in solution.

[0200] The peptides were purified using reversed-phase HPLC on a Supelco Ascentis C18 column (10 pm, 21.2 x 100 mm) with a mixture of water and acetonitrile (both containing 0.1 % TFA) as eluent. The purity of the peptides was analyzed by RP- HPLC using a Supelco Ascentis Express C18 column (2.7 pm, 3.0 x 100 mm) and positive ion electrospray mass spectrometry on a Thermo Orbitrap mass spectrometer.

[0201] Synthesis of AMC-08-89 (comparative).

[0202] The peptide was synthesized on 2-chlorotrityl resin by standard solid phase Fmoc-protocols using HBTLI and DIPEA as activating agent, except for the last coupling where Boc-Trp(Boc)-OH were used to ensure that the N-terminus remained protected upon cleavage of the peptide from the resin. The fully protected peptide, Boc-Trp(Boc)-Arg(Pbf)-Trp(Boc)-Arg(Pbf)-Phe-Gly-OH was liberated from the resin using acetic acid:trifluoroethanol:DCM (1 :1:8). The PEG-linker, 2-[2-(2-azidoethoxy)ethoxy]ethanaminium tosylate, IRIS Biotech PEG4980) was coupled to the C-terminus of the protected peptide using HBTLI and DIPEA, before the final and exhaustive deprotection using TFA:H2O:triisopropylsilane (95:2.5:2.5). The crude peptide was purified by preparative HPLC and lyophilized to yield TFA- salt. Purity (HPLC): 87%. ESI-MS: (MH+ , calculated for C51 H70N 1808: 1063.57, observed 1063.5700). The structure of AMC-08-89 is WRWRFG-NHCH2CH2(OCH2CH2)2-N3.

[0203] Synthesis AMC-25-01 (comparative).

[0204] The peptide was synthesized on Rink Amide AM resin using standard solid phase Fmoc-protocols (Coin, I., Beyermann, M. & Bienert, M. Solid-phase peptide synthesis: from standard procedures to the synthesis of difficult sequences. Nat Protoc 2, 3247-3256 (2007)). The linker was introduced to the peptide by coupling 11-azido-3,6,9-trioxaundecanoic acid (TCI, A2293) to the N-terminus of the Rink amide bound peptide using HCTU and DI PEA as activating agent. The crude peptide was purified by preparative HPLC and lyophilized to yield the TFA-salt. Purity (HPLC): 92%. ESI-MS: (MH+, calculated for C51H69N17O9: 1064.55, observed 1064.5549). The structure of AMC-25-01 is N3-(CH2CH2O)3-CH2C(O)-FRWRW-NH2.

[0205] Antibacterial evaluation in vitro

[0206] The minimal inhibitory concentrations (MIC) of the peptides were determined against Staphylococcus aureus (ATCC 29213), Escherichia coli (ATCC 25922) and Staphylococcus epidermidis (RP62a), via serial dilution. Briefly, overnight colonies were used to prepare 2 x 108cfu / mL stock solutions. The bacterial solution was additionally diluted 1 :100 in MHB to generate final inoculums (2 x 106cfu / mL). Peptides were dissolved in DMSO to a concentration of 20 mg / mL serially diluted to suitable concentration (128 to 2 pg / mL) in microtiter plates (10 pL in each well). The bacterial inoculum (90 pL) was added to each well, before the plates were incubated at 37°C for 20 hours to obtain satisfactory growth. Bacterial growth was analyzed by visual examination and OD measurement at 600 nm. The MIC was defined as the lowest antimicrobial concentration inhibiting the visible growth of the bacterium investigate (a 50 % reduction in OD was set as the limit). Each peptide concentration was analyzed in duplicate. DMSO in growth medium (0.6 %) was used as negative control and medium without DMS was employed as blank. Gentamicin sulfate and AMC-109 were used as positive controls.

[0207] Polyurethane synthesis

[0208] Polytetrahydrofuran (PTHF) (CAS 25190-06-1 , Mw= 950 - 1050 g / mol and Mw= 2825 - 2976 g / mol) and diisocyanates isophorone diisocyanate (I PDI , CAS 4098- 71-9), toluene diisocyanate (TDI, CAS 584-84-9), 4,4'-methylenebis(phenyl isocyanate) (MPI, CAS 101-68-8) and hexamethylene diisocyanate (HDI, CAS 822- 06-0) were obtained from Sigma-Aldrich, Sweden. Glycerol eth oxy late- co- propoxylate triol (CAS, 51258-15-2), 1 ,3-propanediol (CAS, 504-63-2), 2-ethylhexyl diphenyl phosphate (CAS, 1241-94-7) and anhydrous (<50 ppm) dimethylformamide (DMF) was also provided by Sigma-Aldrich, Sweden. The dimethyltin neodecanoate catalyst was provided by Avison GmbH, Germany. The PTHF were dried for 16 h at 60 °C under vacuum prior to use, while glycerol ethoxylate-co-propoxylate triol and the diisocyanates were used as received. The 3,5-bis(hydroxymethyl)-1-propargylbenzene (PBM) and 2,2-di(prop-2-ynyl)propane- 1 ,3-diol (DPPD) chain extenders were received from Amicoat A / S and stored under dry conditions in an exicator before used.

[0209] Flexible segmented thermoplastic polyurethane

[0210] For synthesis of the prepolymer, dried PTHF (-0.005 mol) dissolved in DMF (40 mL) was added to a three necked round-bottom flask (100 mL) fitted with a burette, condenser and a gas tight mechanical stirrer. The flask was placed in an oil-bath at 80 °C, purged with nitrogen and stirred with a teflon blade. Diisocyanate (MDI, TDI or HDI) was then charged to the flask at an NCO:OH ratio of 2:1. Dimethyltin decanoate (DMTND) catalyst (0.4 w % based on total monomer mass) dissolved in DMF (10 mL) was added and the reaction was carried out until the theoretical isocyanate content was reached as determined by following the disappearance of the isocyanate absorbance at 2270 cm-1using ATR FT-IR. For the final polymer synthesis 0.005 mol (molar ratio NCO:OH 1:1) of PBM was dissolved in DMF (10 mL) and added to the prepolymer. Polymerization was carried out at 80 °C under nitrogen purge until a stable molecular mass of the final polymer was reached as determined by size exclusion chromatography (SEC). The reaction mixture was precipitated in ice cold Milli-q water, washed three times in water and once in ethanol. The finished polymer was dried in vacuum at 70°C until stable mass was reached (typically 20-24 h). Thin films (=25 pm) for characterizations and functionalization were prepared on PET substratum (Kodak ESTAR, Rochester, USA). The PU polymer was dissolved in BHT stabilized (0.025%) tetrahydrofuran (THF, VWR chemicals, France) at 1 w / v%, and films were prepared using a 200 pm film applicator followed by solvent evaporation at ambient RT overnight. Thicker films (=250 pm) were prepared on polytetrafluoroethylene (PTFE) foil (ESSKA, Sweden) using a 10% w / v% PU solution in THF and a 500 pm film applicator. After preparation, solvent was allowed to evaporate for at least 3 days. Before analysis, the PU-film was removed from the PTFE foil and cut into suitable pieces.

[0211] Quartz crystal microbalance with dissipation monitoring (QCM-D)

[0212] The time resolved peptide coupling during the Click reaction were evaluated by QCM-D. An E1 QCM-D instrument from Q-sense (Gothenburg, Sweden) was used for the immobilization measurements. The coupled peptide mass (ng cm-2) was calculated from the frequency shift during coupling using the Sauerbrey relation (Eq. 1) in which is a sensor specific constant (17,7), nr =3, which is the odd overtone number used for calculations and Z is the shift in resonance frequency:

[0213] Gold (Au) coated sensor surfaces (Q-Sense, Gothenburg, Sweden) were washed for 10 min at 80°C in a basic piranha solution containing 5:1 :1 Milli-q water, NH3 (24 %, Sigma-Aldrich, Sweden) and H2O2 (30 %, Sigma-Aldrich, Sweden) followed by a wash in excess of Milli-q water before drying under N2 (g). To create coated sensor surfaces, polymer was dissolved (1 % w / v) in THF and applied to the clean sensor surfaces by spin-coating (50 pL, 2000 rpm, 1 min). After THF solvent evaporation, the sensor surface was mounted in the instrument and Milli-q water was pumped over the surface (0.1 mL / min). After 20 min, the AMC-peptides (1 pM), CuSO4 (10 pM) and ascorbate (20 pM) was injected and the shift in mass during surface coupling was followed. The coupling reagents were removed by washing with Milli-q after 16 h. The AMC-109 peptide is missing the azide functionality and was therefore used as control.

[0214] CuAAC- reaction In solution

[0215] Acetonitrile, trifluoroacetic acid (TFA), sodium ascorbate salt and copper sulfate pentahydrate (CUSO4 5H2O) was purchased from Sigma Aldrich, Sweden. PBM and AMC-25-04 were provided by Amicoat AS, Norway. Probing of optimal coupling conditions were performed with ranging concentrations of ascorbic acid and copper sulfate. For a typical solution reaction, 250 pL of aqueous PBM (3.2 mM) was mixed with 250 pL of peptide (3.2 mM) also dissolved in deionized water. Ranging amounts of ascorbic acid (0.16-20 mM) and copper sulfate (0.032-3.2 mM) were added as aqueous solutions at 250 pL each to reach a final volume of 1 mL. The reactions were monitored for 40 h at ambient temperatures employing a Perkin Elmar Flexar HPLC fitted with a Flexar binary pump. The compounds were separated using gradient elution (from 25 to 40 % acetonitrile) over 60 min using mixtures of acetonitrile and deionized water (0.1 % TFA in each solvent). 10 pL of the reaction mixture was injected onto the column (Quasar C18 column 250 mm x 4.6 mm inner diameter, 5 pm particle size) heated at 30°C in a Flexar Peltier LC Column Oven that housed it. The signal was detected and processed with a Flexar PDA Plus Detector with a 10 mm flow cell. Data was collected at both 254 nm and 280 nm with the data processed primarily being from the 254 nm readings.

[0216] Verification and quantification of the formation of the solution Click products was also performed using MS. The samples were separated by LC (Acquity LIPLC I Class, Waters) using a Acquity LIPLC CSHT C18 1.7 pm column at 70°C. The gradient employed was from 95:5 of 0.1 % formic acid 0.05 % TFA in water:0.1 % formic acid 0.05 % TFA in acetonitrile to 5:95 in 2 min at a flow rate of 0.8 mL / min. The analytes were detected by ESI in positive ion mode by MS (G2-S QTOF, Waters) and data were evaluated using the MassLynx 2.1 (Waters) software.

[0217] On alkyne functionalized PU

[0218] Pieces of PU-films were prepared and placed in an Eppendorf tube. The PU-films were washed first in EtOH for 5 min followed by Milli-Q water (3 x 5 minutes). The Cu (II) solution, the ascorbic acid and the peptide solution were then added to the tube. For the initial studies, the Cu concentration was varied between 2-1000 nM, the ascorbate between 4 - 2000 nM and the peptide concentration was varied between 0.05 - 10 pM. After controlled time at ambient room temperature (RT) the reactants were removed and the PU-films were washed in Milli-Q water (3 x 5 minutes). Films were then allowed to dry in RT for 1 h before placed in refrigerator until either subjected for chemical, physical or biological tests. For most subsequent coating experiments, a molar ratio of peptide:CuSO4:ascorbic acid of 1:2:10 was employed with overnight incubation to ensure good coupling. The presence of peptide on the surfaces prepared was established using XPS, Tof-SIMS, static contact angle measurements and QCM-D.

[0219] In vitro antibacterial evaluation of peptide coated materials

[0220] Microscopy images of peptide coated surfaces

[0221] Overnight colonies of Staphylococcus epidermidis (RP62a) and S. aureus (ATCC 29213), were used to prepare a 0.5 McFarland (1 x 108CFU / mL) solution in 0.5 % NaCI and further diluted in TSB (Sigma Aldrich, Missouri, USA), with 1 % glucose to 105CFU / mL. 50 pL of bacterial solution was applied to the test material which was placed in an incubation chamber and incubated at 37°C for 24 h. After incubation, the test material was rinsed once with PBS and placed in a well plate and for 5 min on an orbital shaker (100 rpm). The PBS was exchanged and the plate was further shaken for 5 min. The film films were left in fresh PBS until stained and imaged., The viability of bacteria was evaluated by staining with LIVE / DEAD® BacLight™ Bacterial Viability Kit (Thermo Scientific, Massachusetts, USA). Attached bacteria and formed biofilms was observed using a Leica confocal microscope LSM800 and a Zeiss Axio Observer fluorescent microscope. Two replicates of each treatment were analysed at two different positions (150 x 150 pm). The full experiment was repeated once starting with a new McFarland bacterial solution. Data presented are the grand mean from the two experiments (± 95 % confidence interval). Significant differences between control and peptide functionalized surface was evaluated by Mann-Whitney non-parametric unpaired u- test. Images were evaluated using Imaged software (ver. 1.52a, NIH, USA) by first splitting the red and green channels followed by transform to binary and a particle analysis setting cut-off of 5 pixels2and a circularity of 0.02 - 1.0. The threshold was set so orange coloured bacteria was found in viable (green) channel.

[0222] Microscopy images of peptide coated catheters

[0223] Overnight cultures of S. epidermidis RP62a were diluted 1:100 in TSB with 1 % glucose. Tubes were cut in pieces of 1 cm, and further horizontally divided, and submerged in the bacterial solution. The coated catheters were incubated overnight, rinsed in PBS and stained with LIVE / DEAD® BacLight™ Bacterial Viability Kit (Thermo Scientific, Massachusetts, USA). Images were acquired using the ZEISS Axio Zoom.V16 Fluorescence microscope.

[0224] RESULTS AND DISCUSSION

[0225] Table 1 shows the intrinsic antimicrobial efficacy of the peptides determined as minimum inhibitory concentration (MIC) values. AMC-109 was highly active against all tested bacterial strains.

[0226] AMC-109 was coupled to the corresponding linker molecules resulting in AMC-25- 04 (Figure 2). It is notable that the MIC-values for AMC-25-04, whether monodisperse or the polydisperse version derived from PEG 200, provided identical antimicrobial efficacy. Table 1. Minimal inhibitory concentration1(MIC) of the peptides evaluated.

[0227] 1(|jg / mL), literature values from (Bagheri M, Beyermann M, Dathe M. Immobilization reduces the activity of surface-bound cationic antimicrobial peptides with no influence upon the activity spectrum. Antimicrobial Agents Chemotherapy. 2009, 53, 1132-41).

[0228] Adding the linker to the AMC-109 significantly lowered the antimicrobial activity. Nevertheless, given the high initial antimicrobial activity of AMC-109, AMC-24-04 remained highly active with MIC-values ranging from 16-64 pg / mL against the included bacterial strains.

[0229] As shown in Table 1, the comparative conjugates AMC-08-89 and AMC-25-01 were inactive following the addition of the linker.

[0230] Click chemistry in solution

[0231] Solution formation of the PBM-peptide conjugate peptides from AMC-25-04 was rapid (<10 minutes) at optimized reaction conditions and these were also employed in the 2D polymer format to attach the peptide to the polymerized PBM. Verification of covalent peptide attachment to the polymers was established with XPS, Tof- SIMS, static contact angle measurements and reaction conditions and quantification was studied with QCM-D.

[0232] PU material

[0233] A number of different PU materials were considered and evaluated and the material referred to herein as PU6 was chosen for coupling and testing. PU6 is a flexible thermoplastic polymer presented in Table 2.

[0234] Table 2. Composition of PU6

[0235] 11 ,3-propanediol,2Glycerol ethoxylate-co-propoxylate triol

[0236] Polymerization and characterization of PU6

[0237] Optimization of the polymerization conditions was supported by IR-spectroscopy following the disappearance of the isocyanate group during synthesis of the prepolymer and final polymer as well as the characterization of the final PU-films. The conversion in the polymerization reaction was further studied by SEC, by monitoring the increased molecular mass as a function of polymerization time. Both FT-IR and SEC analysis showed that polymers with acceptable molecular mass was generated by 1 h of polymerization. The molecular mass (number average and weight average) of the polymer is presented in Table 3.

[0238] Table 3. Molecular mass aAII values presented are means of two measurementsbAII values presented are means of four measurements with error being 95 % conf. int.

[0239] Coatings of PU6 were rapidly prepared by dissolving the polymer, in for example THF, followed by an applicator or a spray coating procedure. This process of making coatings did not change the mechanical properties of the polymer.

[0240] Coupling of peptides to PU6

[0241] To accurately quantify both the kinetics of attachment and final peptide surface density, QCM-D was employed. In a typical QCM-D experiment a rapid increase in associated mass upon injection of peptide and coupling reagents was observed. The rapid increase in mass was then followed by a slow steady increase. The gain in mass could either be physically adsorbed components or covalently bound peptide or a mixture of both. After different time intervals the sample chamber was washed with 2 mL of buffer. The washing procedure removed about 30 % of the attached peptide, but after wash a stable level of attached peptide was observed. In addition to normal single wash procedure an intensive wash procedure was performed by flushing 3 x 5 mL of buffer followed by 2 mL wash in Milli-q:DMSO 1:1. It was shown that this extensive procedure did not remove more peptide and single wash step was sufficient (data not shown).

[0242] The AMC-109 peptide lacks the azide functionality and should thus not be possible to anchor to the PU surface employing Click chemistry. The peptide was removed upon one single washing step, demonstrating the non-covalent adsorption to PU with the use of this peptide.

[0243] The QCM-D result indicate that the AMC-25-04 becomes covalently bonded under Click conditions. It is also an important observation with regards to the bioactivity studies of the prepared materials as the immobilization experiments clearly show that AMC-109 adsorbed to the PU6 surface but was easily removed after a single wash and it is therefore unlikely that a potential release of adsorbed peptides will interfere with the biological studies. in vitro Antibacterial evaluation

[0244] To evaluate the antimicrobial potency of the developed PU6, coated and control polymer, were inoculated with biofilm producing bacterium Staphylococcus epidermidis RP62A. The PU films were prepared on plastic films using an applicator for controlled film thickness as described in Materials&Methods section. After incubation, the PU films were rinsed once in PBS and stained with LIVE / DEAD™ BacLight™ and examined by confocal microscopy as shown in Figure 3.

[0245] The control surface displayed a well-established live bacterial biofilm after incubation. This finding contrasted with the PU6 coated with AMC 25-04, which was very sparsely colonized, and the bacteria observed were mainly dead. The difference in colonization between the materials is substantial and indicates that the optimized AMC 25-04 peptide retains activity when covalently linked and suggest that the anchoring point allows for the peptide to retain a bioactive conformation. Failure to ensure sufficient rotational and structural freedom have been shown to generate surfaces that instead attract bacteria. AMC-25-04 has a PEG-spacer that also may interfere with bacterial attachment and adhesion and assist the removal of biofilm upon washing.

[0246] To probe the versatility of this functional antimicrobial layer a format was also assessed employing commercially available lumbar drainage catheters which were coated on the outside and inoculated with bacteria. The catheters were coated with PU6-AMC 25-04 and controls with an identical PU6 film, without peptide attached was also included as negative control. Coated catheters were cut in 1 cm pieces or cut longitudinally in halves and incubated in S. epidermidis overnight cultures that were diluted 1:100 in fresh TSB with glucose. Tubes were incubated for 24 h at 37 °C. After incubation, the tube samples were rinsed with PBS once and stained with LIVE / DEAD™ BacLight™ and examined by fluorescence microscopy as shown in Figure 4.

[0247] As can be seen in the Figure 4 the catheters appear green in the pictures due to autofluorescence, but the bacterial biofilm is clearly discernible on the control catheter in the top three panels of Figure 4. As shown in the lower three pictures, no colonization or biofilm formation on the exterior of the peptide coated catheters was observed. A slight bacterial growth occurs at the inside of the AMC 25-04 coated catheter, where there is no peptide coating, (in particular at the ends). The control catheter was heavily colonized with bacteria and a thick biofilm was established. The biofilm was to a large degree composed of live (green colored) bacteria, but dead bacteria were also observed in the images of the control catheters. No quantification could be performed due to the 3D-format of the coated catheter. The initial antimicrobial evaluation illustrates how the peptide coating can be efficiently used to increase the resilience of the flexible Pll towards bacterial biofilm establishment.

[0248] CONCLUSIONS

[0249] We have developed novel functionalized polyurethane polymers allowing the covalent attachment of short antimicrobial peptides via Click chemistry to combat the establishment of bacterial biofilms. Azide-functionalized antimicrobial peptides were successfully tethered to the coatings via a cycloaddition reaction in a rapid and homogenous fashion to generate uniform peptide surfaces. The repelling performance of the peptide coated materials was illustrated against biofilms of S. epidermidis on coated plastic films and was further shown on coated commercial catheters.

[0250] Example 2 - Analysis of bacterial growth with antimicrobial peptides covalently attached to glass

[0251] Methods

[0252] Synthesis of AMC-25-04

[0253] Amino-azido derivate of PEG200 was prepared as described by Jiang et al. (supra) and reacted with diglycolic anhydride to provide azido-PEG-COOH linker. One equivalent (EQLIIV) azido-PEG-COOH was coupled to 1 EQIIIV AMC-109 (Amicoat A / S, Norway) using 1.2 EQUIV HBTU and 4.8 EQUIV TEA to gain AMC-25-04. The crude peptide was purified by preparative HPLC and lyophilized to yield TFA-salt. The purity was >95% as determined by HPLC. The resultant product was monodisperse.

[0254] Microfluidic channel assembly and silanization

[0255] Microfluidic channels were prepared by mounting a square glass capillary with ID 0.8x0.8 mm2(VitroCom, USA) on a microcopy slide using UV-curable adhesive (NOA 68, Norland Products inc., USA). The capillary was connected to tubing at its both ends using Gel-loading Pipette Round Tips (VWR). The channels (and coverslip glass surfaces used for fluorescence microscopy and TOF-SIMS analysis) were cleaned by immersion overnight with Hellmanex III cleaning solution (2%, Hellma GmbH, Germany) followed by immersion in Sulfuric acid (2M, Sigma-Aldrich 99.9%) for one hour, followed by extensive rinsing with water (Milli-Q, Merck Life Science). The cleaned glass was rinsed with ethanol (99.5%, Solveco, Sweden) and then silanized by immersion in 10% solution of O-(Propargyloxy)-N- (triethoxysilylpropyl)urethane (90%, ABCR, Germany) in ethanol (99.5%, Solveco, Sweden) for one hour.

[0256] Click-chemistry modifications of glass surfaces

[0257] The silanized capillaries and coverslips were modified with AMC-25-04 or a- mannose-PEG3-azide(>95%, Sigma-Aldrich) or Azide-fluor 488 (>90%, Sigma- Aldrich) using copper-catalyzed alkyne-azide cycloaddition (click-chemistry) (Kolb, H. C., Finn, M. G. & Sharpless, K. B. Angewandte Chemie International Edition 40, 2004-2021 (2001). The glass modified with the alkynated silane was conjugated to the different azidated molecules by immersion for 10 minutes in click reaction solution containing 33 pM azidated reactant, 17 mM Guanidine hydrochloride (Sigma-Aldrich), 75 pM CuSO4 (Sigma-Aldrich), 250 pM Tris(3- hydroxypropyltriazolylmethyl)amine (THPTA, Tokyo Chemical Industry Co., Ltd.) and 500 pM Ascorbic acid (Merk) diluted in PBS buffer (pH 7.4).

[0258] Bacteria and growth media

[0259] Versions of the E. coli wild-type (WT) strain MG 1655 was used throughout all experiments. WT E. coli were provided with green fluorescence (GFP) and resistance to Kanamycin by transformation with plasmid pBE1-mGPFmut2. The bacteria were kept in deep-frozen glycerol stocks and on weekly basis plated and grown on LB-Agar. For live-cell microscopy experiments, a single colony was selected from a plate, inoculated into LB media supplemented with 50 pg / ml Kanamycin and grown overnight at 37°C. The overnight cultures were gently centrifuged to remove aggregated bacteria. The supernatant, which typically had ODeoo^O.I, was diluted 1:1 in fresh LB media supplemented with Kanamycin and grown at 37°C until 0.35< OD6oo<0.6.

[0260] Live-cell microscopy

[0261] The microfluidic channel was mounted under a microscope (Axioskop 20, Carl Zeiss Microscopy) equipped with a stage heated to 37°C. The channel was connected to a syringe pump (NE-300, New Era Pump Systems) via polypropylene tubing. A pipette tip attached to the tubing by UV-curable adhesive (NOA 68, Norland Products Inc., USA) worked as adapter between the tubing and the pipette tip attached to the channel. The lower surface of the channel was imaged using a water-immersion objective (W plan-apochromat 63x / 1.0, Carl Zeiss Microscopy) and footage was acquired using a microscope camera (Axiocam 305 Colour, Carl Zeiss Microscopy) at 2 fps. Bacteria in LB media (see above) were taken directly from the incubator, transferred to a syringe and injected into the channel, first 10 minutes at a flow rate of 100 pl / min to equilibrate the system, and then at the lower flow rate of 20 pl / min to promote bacterial binding. The syringe was then replaced with a new syringe containing LB media, or LB media supplemented with 100 pM AMPs, which was injected at flow rate 100 pl / min for approximately 3 hours.

[0262] Image analysis and segmentation

[0263] Image analysis of footage was done with an automated workflow written in Matlab (MATLAB Version: 9.13.0.2049777 [R2022b], The MathWorks Inc., USA) featuring functions of the Image Processing Toolbox. For analysis of GR, non-bound bacteria were excluded from analysis by averaging every 20 consecutive frames, reducing the effective frame rate from 2 fps to 0.1 fps. For analysis of bacteria’s small motions around their major axis, footage was analyzed at the original frame rate during 2 minutes.

[0264] Images were corrected for uneven illumination and background was subtracted. The resulting image was used to construct a mask by subtraction of a user-set constant value, two rounds of median filtering, marker-controlled watershed segmentation, and applying shape / size criteria. Using this mask, properties of the individual bacteria were extracted, the length was measured as the major axis of the best-fitting centroid. Bacteria in subsequent frames were stitched together to trajectories if their footprints overlapped and their length changed <25%. To handle issues with bacteria temporarily “disappearing” from the segmented mask, e.g. due to focus drift, a bacterium remained a member of the same trajectory although it was not visible for a short time if it reappeared with unaltered appearance.

[0265] Otherwise, new trajectories start if bacteria divided or appeared on new positions.

[0266] Analysis of growth rate and mean binding time

[0267] The evolution of population growth rate, GR(t), and cell length, L(t), and the overall mean binding time, T%, were extracted from trajectories of growing cells using Matlab (MATLAB Version: 9.13.0.2049777 [R2022b], The MathWorks Inc., USA) featuring functions of the Curve Fitting Toolbox and the Statistics and Machine Learning Toolbox. The momentaneous GR of a bacterium was determined by linear regression to size data extending + / - 5 min around each time point of a trajectory. Trajectories shorter than 10 min, and the first and last 5 min of each trajectory (which are prone to noise), were thus not included. The experimental time axis was divided into 20 min intervals and the mean GR for each bacterium within each time interval was calculated. Results

[0268] Coating of microfluidic channels with AMPs through a Click reaction

[0269] AMC-109 is a synthetic tripeptide AMP that has proven to be efficient against common bacterial and fungal pathogens. To make the AMP surface coating of this pharmacophore, we created the peptide AMC-25-04 by attaching an azide- terminated short poly(ethylene) glycol PEG linker to the N-terminal of AMC-109 featuring cyclic anhydride.

[0270] Bacterial binding and growth experiments were done using microfluidic channels of glass. The inner walls of the capillary were modified with a silane presenting an alkyne functional group that allows orthogonal binding of azide-functional molecules through copper-catalyzed click reaction. Fluorescence microscopy showed that azide-conjugated fluorophore readily bound to silane-modified surfaces in the presence of all reagents needed for click reaction, but very sparsely if copper ions were omitted confirming the reactivity of the silane modification (data not shown).

[0271] Two types of molecules were attached to the glass surfaces using click chemistry. Azide-conjugated D-mannose was used as a control. Wild type E. coli can readily bind to D-mannose coated surfaces via their type-1 fimbriae. AMP-coated surfaces were made by attachment of AMC-25-04.

[0272] Bacterial response to AMP-coated surfaces

[0273] Wild type E. coli was found to bind readily to surfaces coated with AMC-25-04. Right after binding, the initial GR was attenuated by 26% relative to WT E. coli growing on mannose-coated surfaces (Figure 5). GRs however increase over time towards control levels. Growth rates (GR) were calculated in each time point from the gradient of the length versus time plots, and the average values obtained for each bacterium were binned.

[0274] Example 3 - Synthesis of AMC-109 conjugates with linkers comprising PEG 200 and PEG 400

[0275] 0,0 -Bis(tosyloxy)polyethylene glycols (56-1, 57-1)

[0276] Polyethylene glycol Mn 200 (3.0 g, 0.015 mol, 1.0 eq.) was dissolved in 60 mL DCM, and cooled down on an ice-bath. KOH (6.73 g, 0.12 mol, 8.0 eq.) and 4- toluenesulfonyl chloride (8.6 g, 0.045 mol, 3.0 eq.) were added and stirred overnight at room temperature. To the solution, 40 mL of ice-cold water was added to stop the reaction and extracted with DCM (30 mL x 3). The organic phase was washed with brine, then dried with MgSO4, filtered, and concentrated under vacuum. The crude product was purified to remove excess of 4-toluenesulfonyl chloride using flash chromatography (DCM / MeOH). This resulted in 56-1 (7.249 g, 95 %) as a colorless oil.1H NMR (400 MHz, CDCI3) 6 7.79 (dd, J = 8.2, 1.5 Hz, 4H), 7.34 (d, J = 8.0 Hz, 4H), 4.18 - 4.11 (m, 4H), 3.69 - 3.51 (m, 14H), 2.44 (s, 6H).

[0277] Synthesis of 57-1 followed the same protocol for 56-1 using polyethylene glycol Mn 400 (3.0 g, 0.0075 mol, 1.0 eq - . Sigma Aldrich, product no. 202398). The crude product was purified using flash chromatography (DCM / MeOH) which resulted in 57-1 (4.516 g, 85 %) as a light-yellow oil.1H NMR (400 MHz, CDCI3) 6 7.84 - 7.80 (m, 4H), 7.36 (d, J = 8.1 Hz, 4H), 4.18 (dd, J = 5.5, 4.2 Hz, 4H), 3.74 - 3.57 (m, 30H), 2.47 (s, 6H).

[0278] 0,0 -Bis(2-azidoethyl)polyethylene glycols (56-2, 57-2)

[0279] 56-1 (12.68 g, 0.0249 mol, 1.0 eq.) was dissolved in 50 mL DMF, and NaNs (4.867 g, 0.0748 mol, 3.0 eq.) was added slowly while stirring. The solution was heated up to 80-90 °C with reflux and was let stirring overnight. The reaction was stopped by adding 50 mL of ice-cold water and was extracted with Et20 (50 mL x 3). The organic phase was dried with MgSCL, filtrated, and concentrated under vacuum. Residues of DMF was removed with lyophilization. This resulted in 56-2 (5.560 g, 89 %) as a colorless oil and was not purified for the next synthesis step.1H NMR (400 MHz, CDCI3) 6 3.74 - 3.63 (m, 14H), 3.39 (td, J = 5.1 , 2.4 Hz, 4H).

[0280] Synthesis of 57-2 followed the same protocol for 56-2 using 57-1 (4.516 g, 0.0064 mol, 1.0 eq.) with a few modifications during workup. The crude product was extracted with EtOAc:MeOH (10:1 , 30 mL x 3) and lyophilization was not required as all DMF was removed under vacuum. This resulted in 57-2 (2.187 g, 76 %) as a colorless oil and was used without further purification.1H NMR (400 MHz, CDCI3) 6 3.70 - 3.61 (m, 30H), 3.39 (t, J = 5.1 Hz, 4H).

[0281] O-(2-Aminoethyl)-O '-(2-azidoethyl)polyethylene glycols (56-3, 57-3)

[0282] N34^ / O^X^'NH2

[0283] 56-2 (2.161 g, 1.0 eq.) was dissolved in 20 mL Et20. To the solution, 20 mL 1 M HCI and triphenylphosphine (2.263 g, 0.0086 mol, 1.0 eq.) were added. The reaction was stirred at high-speed overnight at room temperature. White solids were formed of triphenylphosphine oxide (TPPO) and were removed with filtration. The water phase was washed with EtOAc to remove traces TPPO and starting material. The water phase was cooled down on ice-bath and KOH was added to the water phase until pH > 12 was obtained. The product was extracted from water phase using DOM (20 ml x 5), dried over MgSO4, filtered, and concentrated under vacuum. This resulted in 56-3 (1.590 g, 82 %) as a colorless oil and was not purified for the next synthesis step.1H NMR (400 MHz, CDCI3) 6 3.71 - 3.61 (m, 12H), 3.51 (td, J = 5.2, 3.7 Hz, 2H), 3.40 - 3.36 (m, 2H), 2.86 (td, J = 5.2, 2.1 Hz, 2H).

[0284] Synthesis of 57-3 followed the same protocol for 56-3 using 57-2 (2.126 g, 0.0047 mol, 1.0 eq.) which resulted in 57-3 (1.382 g, 69 %) as a colorless oil and was used without further purification.1H NMR (400 MHz, CDCI3) 6 3.69 - 3.63 (m, 28H), 3.51 (tq, J = 4.7, 2.4 Hz, 2H), 3.38 (t, J = 5.0 Hz, 2H), 2.87 (dd, J = 6.4, 4.0 Hz, 2H).

[0285] O-(2-Azidoethyl)-O-[2-(diglycolyl-amino)ethyl]polyethylene glycols (56-4, 57-4)

[0286] To a solution of 56-3 (5.322 g, 0.0238 mol, 1.0 eq.) in 50 mL DCM, diglycolic anhydride (5.515 g, 0.047 mol, 2.0 eq.) and DMAP (0.533 g, 0.0047 mol ,0.2 eq.) was added and the reaction was stirred overnight. Upon completion confirmed by MS, ethylenediamine (2.856 g, 0.047 mol, 2.0 eq.) was added and stirred overnight. Another 30 mL of DCM was added along with 20 mL of 1M HCI, and the organic phase was washed with 1M HCI (20 mL x 3). The collected water phase was extracted DCM (20 mL x 5). The organic phase was dried with MgSC>4, filtered, and concentrated under vacuum. This resulted in 56-4 (5.703 g, 70 %) as a light-pink oil and was used without further purification.1H NMR (400 MHz, CDCI3) 6 8.80 (s, 1 H), 4.12 (s, 2H), 4.09 (s, 2H), 3.61 (tt, J = 9.0, 2.3 Hz, 12H), 3.56 - 3.51 (m, 2H), 3.46 (q, J = 5.3 Hz, 2H), 3.34 (td, J = 5.1, 1.6 Hz, 2H).

[0287] Synthesis of 57-4 followed the same protocol for 56-4 using 57-3 (1.382 g, 0.0033 mol, 1.0 eq.) which resulted in 57-4 (1.589 g, 90 %) as light-pink oil and was used without further purification.1H NMR (400 MHz, CDCI3) 6 7.65 (s, 1 H), 4.17 (s, 2H), 4.12 (s, 2H), 3.73 - 3.60 (m, 28H), 3.59 - 3.54 (m, 2H), 3.51 (q, J = 5.0 Hz, 2H), 3.39 (t, J = 5.1 Hz, 2H).

[0288] O-(2-Azidoethyl)-O-[2-(diglycolyl-amino)ethyl]polyethylene glycols with AMC-

[0289] 109 (56-5, 57-5) To a solution of 56-4 (505 mg, 1.485 mmol, 1.3 eq.) in 25 mL DMF, HBTLI (519, 1.370 mmol, 1.2 eq.) and triethylamine (554 mg, 5.482 mmol, 4.8 eq.) were added. The reaction was stirred for 15 minutes at room temperature followed by adding AMC-109 (900 mg, 1.1421 mmol, 1.0 eq.). The reaction was left stirring at room temperature and monitored by MS. Unreacted AMC-109 was observed and another 505 mg of 56-4 were added along with equivalent HBTU and triethylamine, then left stirring for 2 days upon completion. 100 mL of H2O was added and was extracted with EtOAc (3 x 150 mL). The combined organic layers were then washed with H2O (5 x 50 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude yield was 1.5 g. The crude product was then purified on C18 RP-HPLC which resulted in 56-5 varying dispersity as a white powder. The high-resolution ESI (+ve mode) mass spectrum fitted exactly the molecular formula and was used to determine dispersity of each purified fraction.

[0290] Following the same protocol for 56-5 at a 0.635 mmol scale of AMC-109 (500 mg, 1.0 eq.) with 57-4 (446 mg, 0.825 mmol, 1.3 eq.) applying a few modifications. It was not needed to add an additional amount of 57-4 as the reaction was complete after 2 days, and traces of DMF were removed with lyophilization. The crude product was purified on C18 RP-HPLC which resulted in 57-5 (385 mg, 46 %) as a white powder. The high-resolution ESI (+ve mode) mass spectrum fitted exactly the molecular formula.

[0291] Mass spectra for 56-5 and 57-5 are shown in Figures 6 and 7 respectively.

[0292] ESI-MS

[0293] The following instruments were used to provide the mass spectra in Figures 6 and 7:

[0294] Thermo scientific Orbitrap Exploris 120 Mass spectrometer with an electrospray ion source in positive ion mode

[0295] Thermo Scientific Vanquish UHPLC system

[0296] Column: Accucore Vanquish C18+ 50x2.1 Particle size 1.5p. The eluent is a mixture of acetonitrile water with 0.1% trifluoroacetic acid added. Example 4 - Preparation and testing of polyacrylate coated cellulose materials with AMC-25-04 covalently attached thereto.

[0297] AMC-25-04 was prepared as described in the earlier Examples.

[0298] Non-woven cellulose materials were prepared either by wet spinning commercial grade viscose (80 grams per square meter) with carboxymethyl cellulose, or by taking a cotton / flax / hemp / wood pulp and using a blowing technique as described in J. Appl. Polym. Sci. 2020, DOI: 10.1002 / APP.48339.

[0299] The cellulose substrates were coated with polyacrylic acid through plasma polymerization of acrylic acid monomers (parameters: Flow:3 seem, Pressure: 20- 25 mtorr, Frequency: 135 kHz, Power: 10 W, Time: 2 min). The resultant crosslinked coating had a thickness of abut 20nm and was homogeneously distributed. About 12% of the -COOH functionality was preserved for modification in order to attach the peptide.

[0300] The carboxylate groups on the surface were converted into an acetylene ester using standard coupling reactions, before using click chemistry as described in the earlier Examples to attach the AMC-25-04 peptide.

[0301] Antibacterial efficacy of the coated substrate was evaluated using the standard shake flask test ASTM E2149- 13a and the Certika data evaluation approach. According to this technique, bacterial growth was optically determined every 30 min over a period of 48 h for the AMC-25-04 coated substrate as compared to a control which had been coated with the polyacrylic acid but did not include the AMC-25-04. The antimicrobial activity was calculated from the time needed to reach an optical density (OD) of 0.2 for the coated substrate relative to the control. A time difference (net onset OD) of 6 h represents a reduction of >99.9% (3 log) of bacteria on the substrate surface, and a net onset of 8 h represents a reduction of 99.99% (4 log). The AMC-25-04 sample exhibited a very impressive delay of over 9 hours relative to control (see Figure 8).

Claims

CLAIMS1. A conjugate of a compound of Formula (I) as defined herein and a linker moiety of Formula (V), wherein compound of Formula (I) has the structure:AA-AA-AA-X-Y (I) wherein, in any order, 2 of said AA (amino acid) moieties are cationic amino acids and 1 of said AA is an amino acid with a lipophilic R group, the R group having 14-27 non-hydrogen atoms;X is a N atom, which may be substituted by a branched or unbranched C1-C10 alkyl or aryl group, which group may incorporate up to 2 heteroatoms selected from N, O and S; andY is selected from the group consisting of R1-R2-R3,R1-R2-R2-R3, R2-R2-R1-R3, R1-R3 and R4 wherein:R1 is C, O, S or N;R2 is C; each of R1 and R2 may be substituted by C1-C4 alkyl groups or unsubstituted;R3 is a group comprising 1 to 3 cyclic groups each of 5 or 6 non-hydrogen atoms, 2 or more of the cyclic groups may be fused; one or more of the rings may be substituted; R3 incorporates a maximum of 15 non-hydrogen atoms; andR4 is an aliphatic moiety having 2-20 non-hydrogen atoms, said moiety being linear, branched or cyclic; and wherein the linker moiety of Formula (V) has the structure:N3-A-(CH2)m-NH-C(O)-Z-C*(O)- (V) wherein: the carbon marked with * forms an amide bond with the N-terminal amino group of the compound of Formula (I);A is absent or is an organic group comprising carbon, oxygen and hydrogen atoms and having a chain length of from 3 to 40 atoms;m is an integer of from 2 to 4;Z is selected from the group consisting of -(CR4R5)p-(O)q-(CR4Rs)r-, -CR6=CR7-(CR4R5)S-, -(CR4R5)S-CR6=CR7-, -C(=CR8R9)-(CR4Rs)t- and -(CR4R5)t-C(=CR8R9)-; wherein q is 0 or 1 ; p and r are each independently 0, 1, 2 or 3; and the sum of p, q and r is 2, 3 or 4; s is 0 or 1 ; t is 1, 2 or 3; each R4 and Rs is independently selected from the group consisting of H, halogen, C1-C10 alkyl, C2-C10 alkenyl, aryl; -(CH2)i-eCOOH and -S-C(0)-Ci-Cio alkyl; or two R4 groups on adjacent -(CR4R5)- units together form a carbocyclic ring or a heterocyclic ring; each Rs and R7is independently selected from the group consisting of H, halogen, C1-C10 alkyl, C2-C10 alkenyl, aryl; -(CH2)i-eCOOH and -S-C(0)-Ci-Cio alkyl; or Rs and R7together form a carbocyclic ring or a heterocyclic ring; each Rs and R9 is independently selected from the group consisting of H, halogen, C1-C10 alkyl, C2-C10 alkenyl, aryl; -(CH2)i-eCOOH and -S-C(0)-Ci-Cio alkyl.

2. The conjugate of claim 1 , wherein the chain length of the A group is from 3 to 25 atoms.

3. The conjugate of claim 2, wherein the chain length of the A group is from 6 to 18 atoms.

4. The conjugate of any preceding claim, wherein the A group is a polyether or a single repeating unit or oligomer thereof.

5. The conjugate of any preceding claim, wherein the A group is a polyoxyalkylene or a single repeating unit or oligomer thereof, wherein the polyoxyalkylene is selected from polyethylene glycol or polytetrahydrofuran.

6. The conjugate of any preceding claim, wherein the -N**-A-(CH2)m-NH- unit in the compound of Formula (V) has a number average molecular weight (Mn) of 600 g / mol or less, wherein the nitrogen (N**) marked with ** corresponds to the nitrogen atom of the azide moiety which is bonded to the A group or the -(CH2)m- group if A is absent.

7. The conjugate of claim 6, wherein the -N**-A-(CH2)m-NH- unit has a dispersity value of from 1.000 to 1.15.

8. The conjugate of any preceding claim, wherein Z is -(CR4R5)p-(O)q-(CR4R5)r-.

9. The conjugate of any preceding claim, wherein each R4and Rs is independently selected from the group consisting of H, halogen, C1-C10 alkyl, C2-C10 alkenyl, and aryl; or two R4groups on adjacent -(CR4Rs)- units together form a carbocyclic ring or a heterocyclic ring.

10. The conjugate of claim 9, wherein each R4and Rs is H.11 . The conjugate of any preceding claim, wherein Z is selected from the group consisting of -CH2OCH2-, -(CH2)2-, -(CH2)3-, -(CH2)4-, -O(CH2)2-, -(CH2)2O-, -CH2-O-, and -O-CH2-.

12. The conjugate of any preceding claim, wherein the compound of Formula (I) is a compound of Formula (II)AA1-AA2-AA1-X-Y (II) wherein:AA1 is a cationic amino acid;AA2 is an amino acid with the lipophilic R group, the R group having 14-27 non-hydrogen atoms; andX and Y are as defined in claim 1.

13. The conjugate of any preceding claim, wherein the compound of Formula (I) has the structural formula14. A substrate having a derivative of the conjugate of any preceding claim covalently attached thereto.

15. The substrate of claim 14 wherein the compound of Formula (I) is covalently attached to the substrate by a linking moiety as shown in Formula (Villa) orFormula (VII lb):in whichin Formula (Villa) and the bond from Ring B intersected by the wiggly line in Formula (VI I lb) denotes the point of attachment to the substrate; the carbon marked with * forms an amide bond with the N-terminal amino group of the compound of Formula (I);A, Z and m are as defined in any of claims 1-11 ; ring B in Formula (VII lb) is a cyclic group, such as an 8-10 membered cyclic group, optionally wherein ring B is fused to one or more other carbocyclic or heterocyclic rings and wherein ring B is optionally substituted, preferably with one or more substituents independently selected from halogen, hydroxyl, Ci-Ce alkoxy, or benzyl groups; and wherein Rio is hydrogen or an organic group comprising from 1 to 30 non-hydrogen atoms, preferably wherein Rio is selected from the group consisting of H, C1-C10 alkyl, C1-C10 alkenyl, C1-C10 alkoxy, -C(0)Ci-Cio alkyl, -C(O)OCi-C alkyl, a carbocyclic group and a heterocyclic ring, wherein said alkyl, alkenyl and alkoxy groups are optionally substituted with one or more substituents independently selected from halogen, hydroxy, amino, (C1-C10 alkyl)amino and di(Ci-Cio alkyl)amino, more preferably wherein Rwis H.

16. The substrate of claim 14 or claim 15, wherein the substrate is selected from polymers; metals; and inorganic substrates such as glass and ceramics.

17. The substrate of claim 16, wherein the metal is selected from titanium and alloys thereof, chromium-cobalt alloys, aluminium, stainless steel and precious metals such as gold, silver and platinum, and / or wherein the polymer is selected from polyethylene (PE); polyurethane (Pll); polyamide; polyethylene terephthalate (PET); synthetic rubber (SR); polystyrene (PS); polyacrylates such as polyacrylic acid (PAA), polymethylmethacrylate (PMMA), poly(ethyl cyanoacrylate) and polyacrylamide; polyacrylonitrile (PAN);polyetheretherketone (PEEK), polylactic acid (PLA), silicone, polysaccharide and polyglycolide (PGA).

18. The substrate of any of claims 14-17, wherein the substrate is provided as a coating on another material.

19. The substrate of any of claims 15-18, wherein the length of the chain between the carbon marked with # in Formula (Villa) or (VI I lb) and the surface of the substrate, or between the carbon marked with # and the backbone of the polymer when the substrate is a polymer, is 25 atoms or fewer.

20. The substrate of any of claims 15-19, wherein the distance between the carbon marked with # in Formula (Villa) or (VI 11 b) and the surface of the substrate is 30 Angstroms or less.21 . A method of making a substrate with a compound of Formula (I) covalently attached thereto, the method comprising reacting a conjugate as defined in any of claims 1-13 with a substrate having alkyne groups on the surface thereof under conditions which allow a cycloaddition reaction to take place between the alkyne and the azide group of the conjugate.

22. A substrate with a derivative of the conjugate as defined in any of claims 1-13 covalently attached thereto which is obtainable by the method of claim 21.

23. A method of making the conjugate of any of claims 1-13, the method comprising coupling a compound of Formula (IX):N3-A-(CH2)m-NH-C(O)-Z-C*(O)OH (IX) to a compound of Formula (I) to form an amide bond between the carbon marked with * and the N-terminal amino group of the compound of Formula (I), wherein A, Z and m are as defined in any of claims 1-11.

24. The method of claim 23, wherein the compound of Formula (IX) is produced by a method comprising converting a compound of Formula (X)HO-A-(CH2)m-OH (X) to a compound of Formula (XI)N3-A-(CH2)m-N3(XI) converting the compound of Formula (XI) to a compound of Formula (XII)N3-A-(CH2)m-NH2(XII) and reacting the compound of Formula (XII) with a 5-7 membered cyclic anhydride to provide the compound of Formula (IX).

25. A conjugate comprising the compound of Formula (I) which is obtainable by the method of claim 23 or claim 24.

Images