Conjugates of antibacterial agents with linker moieties suitable for covalent attachment to materials and substrates

Abstract

The present invention provides conjugates of a compound of Formula (I) and a linker moiety of Formula (V), wherein Formula (I) and Formula (V) are as defined in the disclosure. The invention also provides substrates to which the derivatives of the conjugates are covalently attached, methods of making the substrates and the conjugates, and the substrates and conjugates obtainable by the methods of the invention.

Description

[0001] This invention relates to materials and substrates having antimicrobial agents (particularly small peptides) covalently linked thereto. Such materials have applications in a variety of medical and industrial settings. The invention also relates to conjugates of antimicrobial agents with connector portions suitable for covalently linking to materials and substrates.

[0002] The rise of antibiotic resistance among common pathogens poses an imminent threat to patients worldwide. This problem is exacerbated in healthcare settings, where bacteria tend to colonize and form resistant biofilms on non-biological surfaces such as implants, catheters, and medical devices. Biomaterial infections associated with drug-resistant bacteria have already placed a heavy burden on healthcare clinic resources and led to adverse patient health outcomes.

[0003] One approach might be to attach chemicals that inhibit bacterial growth or binding to the surface of a biomaterial. Due to covalent bonding, bacteria will experience a permanent, very high concentration of antimicrobial substances near the surface.

[0004] Antimicrobial peptides (AMPs) are a class of molecules that have the potential to replace traditional antibiotics in such applications. These are amphiphilic peptides that can insert into bacterial cell membranes, interact with phospholipids, and disrupt membrane homeostasis. Membrane binding is promoted by a high content of cationic amino acids, which makes AMPs selectively bind to bacterial membranes because bacterial membranes contain more anionic lipids than other cell membranes.

[0005] The use of natural AMPs in antimicrobial products has achieved some success; however, their length and dependence on the natural sequence make their function sensitive to environmental and enzymatic digestion, easily leading to off-target cytotoxicity and high production costs. By studying the structure-activity relationship of natural AMPs, researchers have designed new peptides that are smaller, more potent, and more stable. These so-called small AMPs consist of a small number of amino acids, primarily arginine (R) and tryptophan (W), which provide the hydrophobic and cationic properties of AMPs, respectively.

[0006] Several smart functionalized coatings have been evaluated as direct bactericides or colonization repellents to prevent or limit bacterial colonization on medical devices. For example, incorporating slowly released antibiotics into implants or coatings by hydrolyzing degradable multilayer structures or binding antibiotics to graphene for sequential antibiotic delivery has been shown to increase tissue integration and reduce infection rates. ZnO nanorods, superhydrophobic surfaces, and noble metal nanoparticles have also been evaluated for preventing bacterial biofilm formation on medical devices and implants.

[0007] While many approaches rely on the release of antimicrobial components, some studies have also focused on developing coatings containing antimicrobial elements, including AMPs. One such study by Shriver-Lake et al. (Langmuir 2017, 33, 2878-2884) investigated the effect of linker length on the capture of cells by polyethylene glycol (PEG)-fixed peptides ranging from 14 to 27 amino acids in length. The lengths of the PEG linkers ranged from zero to 113 ethylene glycol units, with medium-length linkers appearing to perform best, although it was noted that these effects were peptide-specific. These results contrast with earlier reported studies in which longer linkers performed best. Overall, the paper teaches that for a given AMP, the optimal linker type and length cannot be predicted, and there is no “universal solution.”

[0008] The inventors have developed a system comprising a highly efficient and very small antimicrobial component and an optimized linker portion that can be covalently attached to a substrate to create a protective surface against colonizing microorganisms. An advantage of this invention is that the peptide-linker conjugate can be easily attached to a variety of substrates (materials) through a controlled chemical reaction with a reactive portion on the substrate surface. Summary of the Invention

[0009] A first aspect of the invention provides a coupling of a compound of formula (I) as defined herein with a connector portion of formula (V).

[0010] in: Marked The carbon atom forms an amide bond with the N-terminal amino group of the compound of formula (I); A is an organic group that either does not exist or contains carbon, oxygen, and hydrogen atoms and has a chain length of 3 to 40 atoms; m is an integer from 2 to 4, preferably 2 or 4; Z is selected from the group consisting of -(CR4R5)p-(O)q-(CR4R5)r-, -CR6=CR7-(CR4R5)s-, -(CR4R5)s-CR6=CR7-, -C(=CR8R9)-(CR4R5)t- and -(CR4R5)tC(=CR8R9-); in q can be 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 R5 is independently chosen from H, halogen, C1-C 10 Alkyl, C2-C 10 Alkenyl, aryl, -(CH2) 1-6 COOH and -SC(O)-C1-C 10 Groups composed of alkyl groups; Alternatively, two R4 groups on adjacent -(CR4R5)- units (i.e., two R4 groups on adjacent alkylene units / one R4 group on a -(CR4R5)- unit and the next R4 group on a -(CR4R5)- unit) together form a carbocyclic or heterocyclic ring; Each R6 and R7 is independently selected from H, halogen, C1-C 10 Alkyl, C2-C 10 Alkenyl, aryl, -(CH2) 1-6 COOH and -SC(O)-C1-C 10 Groups composed of alkyl groups; Or R6 and R7 together form a carbon ring or heterocycle; Each R8 and R9 is independently selected from H, halogen, C1-C 10 Alkyl, C2-C 10 Alkenyl, aryl, -(CH2) 1-6 COOH and -SC(O)-C1-C 10 A group composed of alkyl groups.

[0011] Another aspect of the invention provides a coupling of a compound of formula (I) and a connector portion of formula (VI) as defined herein.

[0012] in: m, Z, p, q, r, s, t, and R4-R9 are as defined in the first aspect, and A is absent or is an organic group containing carbon, oxygen, and hydrogen atoms; The -N -A-(CH2) m The number-average molecular weight of the -NH- unit is 600 g / mol or less, wherein the -NH- unit is marked with The nitrogen atom corresponds to the nitrogen atom in the azide moiety, which is bonded to the A group, or, if A is absent, to -(CH2). m -group; and In formula (VI), the symbol is indicated as follows: The carbon atom forms an amide bond with the N-terminal amino group of the compound of formula (I).

[0013] Other aspects of the present invention are described below.

[0014] Detailed description

[0015] Compound of formula (I)

[0016] The couplings of the present invention include compounds of formula (I).

[0017] In this embodiment, in any order, two of the AA (amino acid) moieties are cationic amino acids, preferably lysine or arginine, but may also be histidine or any non-genetically encoded or modified amino acid carrying a positive charge at pH 7.0, and one of the AA is an amino acid having a highly lipophilic R group having 14-27 non-hydrogen atoms, preferably containing two or more (e.g., two or three) cyclic groups, which may be fused or linked, and these cyclic groups typically contain 5 or 6 non-hydrogen atoms, preferably 6 non-hydrogen atoms (in the case of fused rings, of course, non-hydrogen atoms can be shared); X is an N atom, which can be branched or unbranched C1-C. 10 Alkyl or aryl (e.g., methyl, ethyl, or phenyl) substitution, preferably unsubstituted, and the group may contain up to two heteroatoms selected from N, O, and S; and Y is selected from the group consisting of R1-R2-R3, R1-R2-R2-R3, R2-R2-R1-R3, R1-R3, and R4; in: R1 can be C, O, S or N, with C being preferred; R2 is C; Each R1 and R2 may be substituted with or not substituted with a C1-C4 alkyl group, preferably Y is -R1-R2-R3 (where R1 is preferably C), and preferably the group is not substituted. When Y is -R1-R2-R2-R3 or R2-R2-R1-R3, it is preferred that one or more of R1 and / or R2 are substituted. R3 is a group comprising 1 to 3 cyclic groups, each cyclic group having 5 or 6 non-hydrogen atoms (preferably all C atoms, but optionally also containing N, O, or S), and the two or more cyclic groups may be fused; one or more rings may be substituted, and these substituents may include, but do not typically include, polar groups, suitable substituents including halogens (preferably bromine or fluorine) and C1-C4 alkyl groups; R3 contains up to 15 non-hydrogen atoms, preferably 5-12, and most preferably phenyl; and R4 is an aliphatic moiety having 2-20 non-hydrogen atoms, preferably carbon atoms, but may also be doped with oxygen, nitrogen, or sulfur atoms. Preferably, R4 contains 3-10, and most preferably 3-6, non-hydrogen atoms, and this moiety can be straight-chain, branched, or cyclic. If the R4 group contains a cyclic group, the cyclic group is preferably directly attached to the nitrogen atom of X.

[0018] One of the AA groups in formula (I) above, and AA2 in formulas (II)-(IV) below, contains a large lipophilic R group. As used herein, the term "large" lipophilic R group means an R group as defined above, i.e., having 14-27 non-hydrogen atoms, preferably containing two or more (e.g., two or three) cyclic groups, which may be fused or linked, and which typically contain five or six non-hydrogen atoms, preferably six (in the case of fused rings, of course, non-hydrogen atoms may be shared).

[0019] Preferred compounds contain straight-chain or branched R4 groups, particularly straight-chain or branched alkyl groups, including ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl and its isomers, hexyl and its isomers, etc.; propyl, isopropyl, butyl and isobutyl are particularly preferred.

[0020] In some embodiments, R4 is an aliphatic moiety (preferably alkyl) having 6-16 non-hydrogen atoms, preferably carbon atoms, but may also be doped with oxygen, nitrogen or sulfur atoms, and the moiety may be straight-chain, branched or cyclic.

[0021] In some preferred embodiments, R4 is isopropyl.

[0022] In the R4 group containing cyclic groups, it is preferred that R4 is a molecule of cyclohexyl or cyclopentyl.

[0023] Suitable non-genetically encoded amino acids and modified amino acids that can provide cationic amino acids include analogs of lysine, arginine, and histidine, such as homolysine, ornithine, diaminobutyric acid, diaminopimelic acid, diaminopropionic acid, and homoarginine, as well as trimethyllysine and trimethylornithine, 4-aminopiperidine-4-carboxylic acid, 4-amino-1-formamidinylpiperidine-4-carboxylic acid, and 4-guanidinophenylalanine. The highly lipophilic R group of the AA may contain heteroatoms such as O, N, or S, typically no more than one heteroatom, preferably nitrogen. The R group preferably has no more than two polar groups, more preferably none or one, and most preferably none.

[0024] The compound (preferably a peptide) is preferably of formula (II).

[0025] in: AA1 is a cationic amino acid, preferably lysine or arginine, but it can also be histidine or any non-genetically encoded or modified amino acid that carries a positive charge at pH 7.0; AA2 is an amino acid with a highly lipophilic R group having 14-27 non-hydrogen atoms, preferably containing two or more (e.g., two or three) cyclic groups, which may be fused or linked, and these cyclic groups typically contain five or six non-hydrogen atoms, preferably six; and X and Y are as defined above.

[0026] Further preferred compounds include compounds of formulas (III) and (IV):

[0027] AA1, AA2, X, and Y are as defined above. The molecule of formula (II) is preferred.

[0028] Among the above compounds, some are particularly preferred. In particular, the amino acid having a large lipophilic R group (conveniently referred to herein as AA2) is tributyltryptophan (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 are most preferred. In some preferred embodiments, the amino acid having a large lipophilic R group is tributyltryptophan (Tbt).

[0029] Another preferred group of compounds are those in which Y is -R1-R2-R3 as defined above, preferably in which R1 and R2 are not substituted, and most preferably in which R1 and R2 are both carbon atoms.

[0030] Another preferred group of compounds are those in which -XY together form the group -NHCH2CH2Ph.

[0031] The compounds include all enantiomers, including D- and L-amino acids, as well as enantiomers formed by a chiral center within the R group of an amino acid and a C-terminal capping group "-XY". The term "amino acid" includes β- and γ-amino acids, as well as α-amino acids, and also includes N-substituted glycine, which can all be considered AA units. The compounds also include β-peptides and condensates.

[0032] The most preferred compounds for incorporation into the couplings of the present invention are as follows:

[0033] t-Bu represents tert-butyl. The second compound incorporating the amino acid 2,5,7-tri-tert-butyl-L-tryptophan is more preferred. Analogs of this compound incorporating other cationic residues (especially Lys) in place of Arg are also highly preferred. Analogs incorporating alternative C-terminal capping groups as defined above are also highly preferred. The most preferred compound of formula (I) has the following structure. It is also referred to as AMC-109 in this article.

[0034] Another preferred group of compounds are those in which -XY are selected together from -NHCH(CH3)2, -NH(CH2)5CH3, -NH(CH2)3CH3, -NH(CH2)2CH3, -NHCH2CH(CH3)2, -NHcyclohexyl, and -NHcyclopentyl, with particular preference for compounds in which -XY is the group -NHCH(CH3)2 or -NH(CH2)5CH3. A particularly preferred group of compounds are those in which -XY together is NHCH(CH3)2.

[0035] A preferred compound is one in which AA1 is arginine, AA2 is tributyltryptophan, and -XY together form NHCH(CH3)2.

[0036] The compound incorporated into the coupling agent of the present invention is preferably a peptide.

[0037] Compounds of formulas (I) through (IV) can be peptide mimics, and peptide mimics of peptides described and defined herein also represent compounds usable according to the invention. Peptidomimics are typically characterized by retaining the polarity, three-dimensional size, and functionality (bioactivity) of their peptide equivalents, but in which the peptide bonds have been replaced, usually with more stable linkages. “Stable” means more resistant to enzymatic degradation by hydrolases. Typically, the bonds replacing the amide bonds retain many properties of the amide bonds, such as conformation, spatial volume, electrostatic properties, hydrogen bonding potential, etc. Chapter 14 of “Drug Design and Development” (Krogsgaard, Larsen, Liljefors and Madsen (Eds) 1996, Horwood Acad. Pub) provides a general discussion of peptide mimic design and synthesis techniques. In this case, since the molecule reacts with the membrane rather than with the specific active site of the enzyme, some issues of precisely mimicking affinity and potency or substrate function are irrelevant, and peptide mimics can be readily prepared based on a given peptide structure or the motif of the desired functional group. Suitable amide bond substitutes include the following groups: N-alkylation (Schmidt, R. et al., Int. J. Peptide Protein Res., 1995, 46, 47), trans-reverse amide (Chorev, M and Goodman, M., Acc. Chem. Res., 1993, 26, 266), thioamide (Sherman D.B. and Spatola, AFJ Am. Chem. Soc., 1990, 112, 433), thioesters, phosphonates, ketomylidene groups (Hoffman, RV and Kim, HOJ Org. Chem., 1995, 60, 5107), hydroxymethylene, fluorovinyl groups (Allmendinger, T. et al., Tetrahydron Lett., 1990, 31, 7297), vinyl groups, methylene amino groups (Sasaki, Y and Abe, J. Chem. Pharm). Bull. 1997 45, 13), methylene thio (Spatola, AF, Methods Neurosci, 1993, 13, 19), alkanes (Lavielle, S. et. al., Int. J. Peptide Protein Res., 1993, 42, 270) and sulfonamides (Luisi, G. et al. Tetrahedron Lett. 1993, 34, 2391).

[0038] Peptide-like compounds typically possess three identifiable subunits that are roughly equivalent in size and function to amino acids (AA units). Therefore, the term "amino acid" can be conveniently used herein to refer to the equivalent subunits of peptide-like compounds. Furthermore, peptide-like compounds may possess groups equivalent to the R groups of amino acids; the discussion of suitable R groups and terminal modification groups in this paper is further elaborated below. After making necessary modifications ( mutatis mutandis This is applicable to peptide-like compounds.

[0039] As discussed in the cited literature above, in addition to replacing the amide bond, peptide mimics may involve replacing larger structural portions with dipeptide or tripeptide-mimicking structures. In this case, the mimicking portion involving the peptide bond, such as azole-derived mimics, can be used as the dipeptide replacement. However, preferred are peptide mimics in which the amide bond has been replaced as described above, and the resulting peptide mimicking backbone.

[0040] Suitable peptide-mimicking compounds include reduced peptides, in which the amide bond has been reduced to methyleneamine by treatment with a reducing agent (e.g., borane or hydride reagents such as lithium aluminum hydride). This reduction also has the added advantage of increasing the overall cationicity of the molecule.

[0041] Other peptide mimics include peptide analogs formed, for example, through stepwise synthesis of amide-functionalized polyglycine. Some peptide mimic backbones can be readily prepared from their peptide precursors, such as fully methylated peptides; a suitable method is described by Ostresh, JM, et al. in Proc. Natl. Acad. Sci. USA (1994) 91, 11138-11142. Strongly basic conditions favor N-methylation rather than O-methylation and result in the methylation of some or all nitrogen atoms in the peptide bond, as well as the N-terminal nitrogen.

[0042] Preferred peptide backbones include polyesters, polyamines and their derivatives, as well as substituted alkanes and olefins.

[0043] Connector section

[0044] As defined above, the coupling of the present invention may include a connector portion having a (V) structure.

[0045] The group "N3-" represents an azide group, which can also be described as... .

[0046] The azide group can undergo an azide-alkyne cycloaddition reaction with terminal or internal alkynes on the substrate surface, such as the Huisgen azide-alkyne cycloaddition reaction, resulting in a linker covalently attached to the substrate surface. Preferably, the reaction is a copper-catalyzed azide-alkyne cycloaddition reaction. These are examples of "click" reactions.

[0047] In the Z group, –(CR4R5)p-(O)q-(CR4R5)r-, -CR6=CR7-(CR4R5)s-, -(CR4R5)s-CR6=CR7-, -C(=CR8R9)-(CR4R5)t-, and -(CR4R5)tC(=CR8R9)-, the right-protruding bond indicates a bond with the carbonyl group -C. The (O)- bond indicates a connection point with another carbonyl group -C(O)-, while the bond protruding to the left indicates a connection point with another carbonyl group -C(O)-.

[0048] Preferably, Z is selected from the group consisting of –(CR4R5)p-(O)q-(CR4R5)r-, -CR6=CR7-(CR4R5)s- and -(CR4R5)s-CR6=CR7-. More preferably, Z is -(CR4R5)p-(O)q-(CR4R5)r-.

[0049] Preferably, each R4 and R5 is independently selected from H, halogen, C1-C. 10 Alkyl, C2-C 10 The group consisting of alkenyl and aryl groups; or two R4 groups on adjacent -(CR4R5)- units together forming a carbocyclic or heterocyclic ring. More preferably, each R4 and R5 is H or C1-C. 10 Alkyl group. Most preferably, each of R4 and R5 is H.

[0050] Preferably, each R6 and R7 is independently selected from H, halogen, C1-C. 10 Alkyl, C2-C 10 The group consisting of alkenyl and aryl groups; or R6 and R7 together forming a carbocyclic or heterocyclic ring. More preferably, each R6 and R7 is H or C1-C. 10 Alkyl group. Most preferably, each of R6 and R7 is H.

[0051] Preferably, each R8 and R9 is independently selected from H, halogen, C1-C. 10 Alkyl, C2-C 10 The group consisting of alkenyl and aryl groups. More preferably, each R8 and R9 is H or C1-C. 10 Alkyl group. Most preferably, each of R8 and R9 is H.

[0052] 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-. Even more preferably, Z is selected from the group consisting of -CH2OCH2-, –(CH2)2-, and -(CH2)3-. Most preferably, Z is -CH2OCH2-.

[0053] The oxygen-containing Z-group can increase the water solubility of the joint portion and coupling of the present invention and promote its production. Once the coupling binds to the substrate surface, it can also provide higher surface wettability.

[0054] The term "alkyl," used alone or as part of another group such as an alkoxy group, is intended to include branched and straight-chain saturated aliphatic hydrocarbon groups having a specified number of carbon atoms. For example, "C1-C..." 10 "Alkyl" is intended to include C1, C2, C3, C4, C5, C6, C7, C8, C9 and C 10 Alkyl groups. Preferred alkyl groups are C1-C6 alkyl groups, more preferably C1-C4 alkyl groups. Suitable examples of alkyl groups include methyl (Me), ethyl (Et), propyl (e.g., n-propyl and isopropyl), butyl (e.g., n-butyl, isobutyl, tert-butyl), and pentyl (e.g., n-pentyl, isopentyl, neopentyl).

[0055] The term "alkenyl" is intended to include straight-chain or branched hydrocarbon chains having a specified number of carbon atoms and one or more (preferably one to two, more preferably one) carbon-carbon double bonds that can appear at any stable point in the chain. For example, "C2-C 10 "Alkenyl" refers to compounds containing C2, C3, C4, C5, C6, C7, C8, C9, and C6. 10 Alkenyl. Examples of alkenyl groups include, but are not limited to, vinyl, 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.

[0056] The term "halogen" (also known as "halogenated") refers to chlorine, bromine, fluorine, and iodine, with chlorine or fluorine being preferred.

[0057] The term "aryl" (as defined in R5-R9, for example) refers to monocyclic or polycyclic (including bicyclic and tricyclic) aromatic hydrocarbons, including, for example, phenyl, naphthyl, anthracene, and phenanthrene. Preferably, the term "aryl" indicates a monocyclic or bicyclic aryl (e.g., phenyl or naphthyl, including 1-naphthyl and 2-naphthyl) containing 6 to 10 carbon atoms in the ring moiety, more preferably phenyl. Aryl groups can be linked by any available carbon atom (by replacing a hydrogen atom on that carbon).

[0058] As used herein, a “carbocyclic ring” (also referred to as a “carbocyclic group”) can be any stable 5, 6, 7, or 8-membered monocyclic or bicyclic ring, or a 7, 8, 9, 10, 11, 12, or 13-membered bicyclic or tricyclic hydrocarbon ring, wherein any ring can be saturated, unsaturated, or aromatic. Examples of such carbocyclic rings 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 (decahydronaphthalene), [2.2.2]bicyclooctane, fluorenyl, phenyl, naphthyl, indanyl, adamantyl, anthracene, and tetrahydronaphthalene (tetrahydronaphthalene). As shown above, bridged rings are also included in the definition of a carbocyclic ring (e.g., [2.2.2]bicyclooctane). Carbocyclic rings include “aryl”. A bridged ring is formed when one or more carbon atoms connect to two non-adjacent carbon atoms. Preferably, the bridge consists of one or two carbon atoms. It is important to note that the bridge always transforms a monocyclic ring into a tricyclic ring. Preferably, the carbon ring is 5-10 quintiles, more preferably 5-7 quintiles. Preferred carbon rings formed by two R4 groups together include cyclopentyl, cyclopentenyl, cyclohexyl, and cyclohexenyl. Preferred carbon rings formed by R6 and R7 groups together include cyclopentenyl, cyclohexenyl, and phenyl.

[0059] As used herein, a “heterocycle” (also referred to as a “heterocyclic group”) can be any stable 5, 6, or 7-membered monocyclic ring, or a 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14-membered polycyclic (including bicyclic and tricyclic) heterocycle, which is saturated, unsaturated, or aromatic, and contains a carbon atom and 1, 2, 3, or 4 heteroatoms independently selected from N, O, and S; and includes any polycyclic group in which any heterocycle defined above is fused to a carbon ring or an aryl (e.g., benzene) ring. The term “heterocycle” includes non-aromatic ring systems, such as heterocyclic alkyl and heterocyclic alkenyl groups. Nitrogen and sulfur heteroatoms may optionally be oxidized (i.e., N→O and S(O)p, where p is 0, 1, or 2). Nitrogen in the heterocycle may optionally be quaternized. Preferably, when the total number of S and O atoms in the heterocycle exceeds 1, these heteroatoms are not adjacent to each other. Preferably, the total number of S and O atoms in the heterocycle does not exceed 1. Examples of heterocycles include: piperazinyl, piperidinyl, piperidinoneyl, piperinyl, pyranyl, morpholinyl, tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl, and dihydrofurano[2,3-b]tetrahydrofuranyl. Further examples of heterocycles include heteroaryl groups: pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazinyl, furanyl, quinolinyl, isoquinolinyl, thiopheneyl, imidazolyl, thiazolyl, indolyl, pyrroleyl, oxazolyl, benzofuranyl, benzothiopheneyl, benzothiazolyl, isoxazolyl, pyrazolyl, triazolyl, tetrazolyl, indolyl, 1,2,4-thiadiazolyl, isothiazolyl, purinyl, carbazoleyl, benzimidazolyl, indololinyl, benzodioxanepentyl, and benzodioxane. Preferably, the heterocycle is 5-10 quinary, more preferably 5-7 quinary, and contains 1-4 heteroatoms independently selected from N, O and S.

[0060] When two R4 groups on adjacent -(CR4R5)- units together form a carbon ring or heterocycle, the R5 group on the -(CR4R5)- unit is preferably H.

[0061] The chain length of group A can also be referred to as the skeletal length. As used in this article, chain length refers to the length of group A connected to -(CH2). m - The shortest distance (in atoms) between a partially bonded atom and an atom in group A bonded to the azide group. (The atom in group A is bonded to -(CH2)) m Partially bonded atoms and atoms bonded to azide groups are included in the chain length. Hydrogen atoms are not considered as chain / skeletal atoms of group A when calculating chain / skeletal length. The chain / skeletal structure of group A can be straight or branched; preferably, it is straight.

[0062] When group A is absent, the azide group is directly bonded to the alkylene group (CH2) in formula (V). m However, the presence of an A group is preferred.

[0063] Preferably, the chain length of group A is 3 to 25 atoms, more preferably 3 to 21 atoms, more preferably 5 to 20 atoms, more preferably 6 to 18 atoms, more preferably 6 to 15 atoms, more preferably 6 to 12 atoms, and most preferably 9 to 12 atoms.

[0064] Preferably, the A group is composed of carbon, oxygen, and hydrogen atoms. The A group is preferably polyoxide-based.

[0065] Preferably, the A group is hydrophilic. This ensures that when the linker is covalently attached to the surface, it elevates the compound of formula (I) above (or away from) the substrate surface when exposed to an aqueous environment.

[0066] Group A is preferably a polyether or a single repeating unit or oligomer thereof. More preferably, Group A is a polyoxyethylene or a single repeating unit or oligomer thereof.

[0067] Polyoxyethylene is a polymer having the following structure: Y1 is an alkane-α,ω-diyl (also known as an alkylene) or a substituted alkane-α,ω-diyl that contributes at least two carbon atoms to the main chain (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). The variable “n” represents the number of repeating units and can be selected to provide the chain length described above and / or the number-average molecular weight described below. In this document, the term “oligomer” refers to a portion consisting of 2–4 repeating units. For example, oligomers of polyoxyethylene have the structure: Where n is 2-4. In this paper, the term "polymer" refers to the portion of an oligomer with more repeating units than an oligomer, i.e., 5 or more repeating units.

[0068] Preferably, the polyoxyethylene is selected from polyethylene glycol or polytetrahydrofuran (i.e., Y1 is preferably –(CH2)2– or –(CH2)4–). More preferably, the polyoxyethylene is polyethylene glycol. When the polyoxyethylene group is polyethylene glycol, m is preferably 2. When the polyoxyethylene group is polytetrahydrofuran, m is preferably 4.

[0069] Group A is preferably selected from polyethylene glycol, polytetrahydrofuran, or a single repeating unit or an oligomer thereof; more preferably, Group A is selected from polyethylene glycol or a single repeating unit or an oligomer thereof.

[0070] Polyethylene glycol is preferred, for example, to provide higher water solubility to the conjugate and higher surface wettability once the conjugate binds to the substrate surface. However, in cases where lower water solubility and / or surface wettability are required, polytetrahydrofuran can be used.

[0071] -A-(CH2) m -NH- is preferably selected from the group consisting of: -(CH2CH2O) n1 -(CH2)2-NH-, where n1 is from 1 to 13, or -(CH2CH2CH2CH2O) n2 -(CH2)4-NH-, where n2 is from 1 to 8, more preferably -A-(CH2) m -NH- is -(CH2CH2O) n1 -(CH2)2-NH-.

[0072] n1 is preferably from 1 to 9, more preferably from 1 to 6, still more preferably from 2 to 5, and most preferably from 2 to 4.

[0073] n2 is preferably from 1 to 5, more preferably from 2 to 4 or 2 to 3.

[0074] Preferably, at least 60 mol% (more preferably at least 70 mol%) of the -A-(CH2) m -NH- groups in the linker moiety have n1 or n2 values within up to 3 repeating units above or below the most common n1 or n2 value.

[0075] Preferably, at least 60 mol% (more preferably at least 70 mol%) of the -A-(CH2) m -NH- groups in the linker moiety have n1 or n2 values within up to 2 repeating units above or below the most common n1 or n2 value.

[0076] The most common n1 or n2 value, and the proportion of n1 or n2 values equal to or within 2 or 3 repeating units above or below the most common n1 or n2 value, can be determined by mass spectrometry (such as ESI-MS) of the conjugate of the compound of formula (I) with the linker moiety, optionally in combination with liquid chromatography or high performance liquid chromatography.

[0077] Preferably, the number average molecular weight (Mn) of the -N -A-(CH2) m -NH- units in the compound of formula (V) is 600 g / mol or less. The nitrogen marked (i.e., N ) corresponds to the nitrogen atom of the azide moiety bonded to the A group (if A is absent, bonded to the -(CH2) m - group). More preferably, -N -A-(CH2) m The number-average molecular weight of the -NH- unit is 500 g / mol or less, more preferably 400 g / mol or less, and even more preferably 300 g / mol or less. Preferably, the -N- unit in the compound of formula (V) has a molecular weight of 500 g / mol or less, more preferably 400 g / mol or less, and even more preferably 300 g / mol or less. -A-(CH2) m The Mn content of the -NH- unit is 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 Mn can be combined arbitrarily. Therefore, -N -A-(CH2) m The -NH- unit preferably has 100 to 600 g / mol of Mn, more preferably 100 to 500 g / mol, more preferably 120 to 400 g / mol, more preferably 150 to 300 g / mol, more preferably 175 to 250 g / mol, and most preferably 180 to 225 g / mol, for example about 200 g / mol.

[0078] -N -A-(CH2) m -NH- units (and / or connectors of formula (V), and / or couplings) can be monodisperse or polydisperse, but are usually polydisperse.

[0079] -N -A-(CH2) m The dispersion (also known as polydispersity index) value of the -NH- unit can be from 1.000 to 1.15, preferably from 1.001 to 1.05 or 1.10, more preferably from 1.001 to 1.01, and even more preferably from 1.001 to 1.007 or 1.001 to 1.005.

[0080] The number-average molecular weight (Mn) of the coupling of the compound of formula (I) and the linker as defined herein can be from 1050 to 1550 g / mol, preferably from 1100 to 1550 g / mol, more preferably from 1100 to 1350 g / mol, and most preferably from 1100 to 1200 g / mol.

[0081] The dispersion value of the coupling of the compound of formula (I) and the linker as defined herein can be from 1 to 1.15, preferably from 1.001 to 1.10 or 1.05, more preferably from 1.001 to 1.01, and even more preferably from 1.001 to 1.007 or 1.001 to 1.005.

[0082] The dispersion value (also known as the polydispersity index) is a measure of the width of the molecular weight distribution, defined as M. w / M n M w and Mn These are weight-average molecular weight and number-average molecular weight, respectively.

[0083] M n and M w The value can be measured by size exclusion chromatography or mass spectrometry (e.g., electrospray ionization mass spectrometry, ESI-MS). -N -A-(CH2) m M of -NH- units and / or couplings n and M w The value can be determined by mass spectrometry (e.g., ESI-MS) of the conjugate of compound (I) and the linker moiety of formula (V), optionally combined with liquid chromatography or high-performance liquid chromatography. Furthermore, -N -A-(CH2) m The Mn and Mw values ​​of the -NH- unit typically correspond to those values ​​of the starting material (e.g., polyoxyethylene, such as polyethylene glycol or polytetrahydrofuran, or oligomers thereof) used to prepare the connector according to the method of the present invention.

[0084] Suitable mass spectrometers and HPLC instruments are commercially available. For example, a Thermo Scientific Orbitrap Exploris 120 mass spectrometer with a positive ion mode electrospray ionization source can be used. Alternatively, a Thermo Scientific Vanquish UHPLC system can be used, with an Accucore Vanquish C18+ 50x2.1 column and a particle size of 1.5 μm. The eluent can be a mixture of acetonitrile and water, with the addition of 0.1% trifluoroacetic acid (e.g., acetonitrile / water mixed at a 98 / 2 (v:v) ratio and containing 0.1% (v:v) trifluoroacetic acid).

[0085] A suitable size exclusion chromatography method for determining molecular weight may involve dissolving the analyte (such as the conjugates described herein) in a suitable solvent (such as DMF) and introducing that solution into one or more size exclusion columns. The particle size and pore size of the column packing material can be selected to determine the molecular weight resolution. For example, a particle size of 5 μm and a porosity of 100 Å or 300 Å can be used. Suitable columns are commercially available, such as the PFG brand from PSS, Germany. A calibration curve can be used to determine the relationship between elution volume and molecular weight. The calibration curve can be prepared using molecular markers of known molecular weight (such as PEGs).

[0086] For example, molecular weight can be determined by size exclusion chromatography (SEC) using a TOSOH EcoSEC HLC-8320GPC (Tokyo, Japan) equipped with an EcoSEC RI detector (Tokyo, Japan). Polymer samples can be dissolved in DMF at a concentration of 2.5 mg / mL. DMF containing 0.01 M LiBr can be used as the mobile phase at a flow rate of 0.2 mL / min at 35 °C. Three different SEC columns from PSS (Mainz, Germany) can be used: PSS PFG 5 μm; Microguard 100 Å and 300 Å, providing a resolution range of 300–100,000 Da for molecular weight analysis. Conventional calibration methods can be used with narrowly distributed linear polyethylene glycol standards (PSS, Mainz, Germany) with a molecular weight range of 106–44,000 Da. Toluene can be used as an internal standard to correct for flow rate fluctuations. Collected data can be processed using PSS WinGPC Unity software version 7.2 (Mainz, Germany).

[0087] In formula (V), group A is absent or is an organic group containing carbon, oxygen, and hydrogen atoms with a chain length of 3 to 40 atoms. Alternatively, group A may be absent or is an organic group containing carbon, oxygen, and hydrogen atoms (optionally having a chain length of 3 to 40 atoms), and wherein -N -A-(CH2) m The number-average molecular weight (Mn) of the -NH- unit is 600 g / mol or less.

[0088] Therefore, the joint portion can have the structure of formula (VI).

[0089] in: In the coupling, marked with The carbon atom forms an amide bond with the N-terminal amino group of the compound of formula (I); A is absent or is an organic group containing carbon, oxygen, and hydrogen atoms; m and Z are defined as above for equation (V); and -N -A-(CH2) m The number-average molecular weight of the -NH- unit is 600 g / mol or less, wherein the -NH- unit is marked with The nitrogen atom corresponds to the nitrogen atom of the azide portion bonded to group A, or, if A is absent, to the nitrogen atom bonded to -(CH2). m - The nitrogen atom of the azide moiety bonded by the group.

[0090] To avoid any doubt, the preferred definitions disclosed above for formula (V) (including but not limited to the preferred definitions of Z and A, -N) - Number-average molecular weight and polydispersity index of A-(CH2)m-NH- units) in the process of After necessary modifications (mutatis mutandis) Applicable to formula (VI).

[0091] This invention also provides couplings of compounds of formula (I) as defined herein with a connector portion of formula (VI), wherein formula (VI) is indicated by... The carbon atom forms an amide bond with the N-terminal amino group of the compound of formula (I).

[0092] Coating substrate

[0093] The present invention also provides a substrate having a derivative of the above-mentioned coupling compound covalently linked thereto. A schematic diagram of an example of such a functionalized substrate is shown below. Figure 1 The connector allows the compound of formula (I) to be covalently attached to the substrate. In embodiments, when the substrate is in use (e.g., placed in or in contact with a human or animal), the compound of formula (I) remains attached to the substrate (at least a majority of the compound of formula (I) remains attached to the substrate). As used herein, the term "derivative of the coupling" can refer to a portion of the coupling remaining after an azide-alkyne cycloaddition reaction occurs between the coupling and the substrate containing an alkyne moiety located on the surface of the substrate. This portion of the coupling will not contain the azide moiety -N3, because the azide moiety reacts with the alkyne in the cycloaddition reaction to form a triazole ring. In this case, the derivative of the coupling will be covalently attached to the substrate via the triazole ring. Thus, the substrate can have the compound of formula (I) as defined herein covalently attached thereto via a connecting portion of formula (VIIIa) or formula (VIIIb) as shown below.

[0094] Therefore, in another aspect, the present invention provides a substrate having a compound of formula (I) as defined herein covalently connected thereto by a linking portion of formula (VIIIa) or formula (VIIIb) as shown below: (VIIIa) (VIIIb) Among them, in equation (VIIIa) And in equation (VIIIb), the bond from ring B that is crossed by the wavy line represents the connection point with the substrate; Marked The carbon atom forms an amide bond with the N-terminal amino group of compound (I); and A, Z, and m are defined elsewhere in this document.

[0095] R 10 Compounds derived from terminal or internal alkynes on the substrate surface, having the following structure prior to reaction with the coupling compound of the present invention: .

[0096] R 10 It can be hydrogen or an organic group containing 1 to 30 non-hydrogen atoms. The non-hydrogen atoms are preferably selected from the group consisting of carbon, nitrogen, and oxygen. Preferably, R 10 Optional free H, C1-C 10 Alkyl, C1-C 10 alkenyl, C1-C 10 Alkoxy, C(O)Cl-C 10 Alkyl group, -C(O)OC1-C 10 Alkyl groups, carbocyclic groups (preferably 5-10 membered carbon rings, more preferably phenyl) and heterocyclic groups (preferably 5-10 membered heterocycles, for example...) The group consisting of alkyl, alkenyl, and alkoxy groups, wherein the alkyl, alkenyl, and alkoxy groups are optionally selected independently from halogen, hydroxyl, amino, (C1-C2) groups. 10 alkyl)amino and di(C1-C) 10 Substitution of alkyl)amino groups.

[0097] In formula (VIIIb), ring B 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 with one or more other carbocyclic or heterocyclic rings, preferably with one or more aryl rings (e.g., phenyl) or with one or more cycloalkyl rings (e.g., C3-C6 cycloalkyl, such as cyclopropyl). The one or more rings fused to ring B may optionally be substituted, for example, to change solubility. For example, ring B may be a dibenzocyclooctene ring, for example: In formula (VIIIb), fusion with the triazole ring occurs at the dashed bond, and the bond intersecting the wavy line indicates the connection point with the substrate.

[0098] Ring B may be optionally substituted, preferably by one or more electron-withdrawing substituents. Suitable substituents may be independently selected from halogens, hydroxyl groups, C1-C6 alkoxy groups, or benzyl groups.

[0099] When the substrate (before reacting with the conjugate of the present invention) contains one or more cyclic alkyne moieties (e.g., cyclic alkyne moieties selected from cyclooctynyl, dibenzocyclooctynyl, cyclononynyl, or cyclodecynyl), a ring B is typically formed. These cyclic alkynes can react with the azido group in the conjugate of the present invention via a strain-promoted azido-alkyne cycloaddition reaction (Chem. Rev. 2021, 121, 7122-7154).

[0100] The floating bond intersecting with ring B means that ring B can be connected to the substrate by any available ring member (e.g., by any available carbide ring member).

[0101] Preferred R 10 For H.

[0102] Preferably, the substrate has a compound of formula (I) as defined herein covalently connected thereto by a linker portion as shown in formula (VIIIa).

[0103] The substrates include materials known for use in medical devices and industrial environments, with the aim of providing antimicrobial materials, particularly reducing or preventing biofilm formation (biofouling) on ​​the materials.

[0104] Suitable substrates include polymers; metals; and inorganic substrates such as glass and ceramics.

[0105] As used herein, the term metal includes its alloys. Suitable metals include titanium and its alloys such as titanium-nickel alloys, chromium-cobalt alloys, aluminum, stainless steel, and precious metals such as gold, silver, and platinum. Preferred metals include titanium and its alloys such as titanium-nickel alloys, chromium-cobalt alloys, aluminum, and stainless steel. As mentioned above, metals with an oxide layer, such as aluminum, are convenient because their surface hydroxyl groups can be used to attach alkynyl groups.

[0106] Inorganic substrates include glasses and ceramics such as alumina and zirconium oxide. Glass is a preferred inorganic substrate and can be prepared by silanization (i.e., incorporation of silanol groups) followed by modification with alkyne groups (e.g., using silyl halides or silyl ethers described below) to prepare coupling agents.

[0107] Suitable polymers for use as substrates include polyethylene (PE); polyurethane (PU); polyamide; polyethylene terephthalate (PET); synthetic rubber (SR); polystyrene (PS); polyacrylates such as polyacrylic acid (PAA), polymethyl methacrylate (PMMA), poly(ethyl cyanoacrylate), and polyacrylamide; polyacrylonitrile (PAN); polyetheretherketone (PEEK), polylactic acid (PLA), silicone, polysaccharides, and polyglycolic acid (PGA). Preferred polymers include polyacrylates and PU.

[0108] The substrate can be provided as a coating on other materials.

[0109] Suitable substrates include any material on which an alkyne moiety can be carried, which can react with the azide moiety of the coupling compound in a so-called click reaction, thereby covalently attaching the compound of formula (I) and the linker to the substrate. The alkyne can be terminal or internal, preferably terminal. A diagram of this reaction for formula (VIIIa) is provided below.

[0110]

[0111] As illustrated above, the carbon atom of the alkyne group is incorporated into the triazole ring of formulas (VIIIa) and (VIIIb). For the avoidance of doubt, the substrate structures in formulas (VIIIa) and (VIIIb) will correspond to the non-alkyne portion of the substrate structure before reaction with the conjugate of this invention.

[0112] The alkyne moiety can be incorporated into / on the substrate using methods known in the art and the methods described in the embodiments herein for polyurethane (PU) and glass. Therefore, the substrate material may need to be adapted (or functionalized to include) the alkyne moiety.

[0113] For polymer substrates, monomers containing one or more alkyne-containing side chains can be incorporated into the polymer structure, and / or the side chains can be reacted with alkyne-containing compounds to introduce the alkyne moiety.

[0114] For example, alkyne-containing compounds can be attached to a side chain via ester or amide bonds. This method can be used to introduce the alkyne moiety into a substrate formed from a polyacrylate (e.g., polyacrylic acid).

[0115] When the substrate is formed from a metal or alloy containing a surface oxide layer (e.g., titanium, aluminum, or alloys thereof), the surface hydroxyl groups on the oxide layer can react with alkyne-containing compounds to introduce alkyne moieties. The surface oxide layer typically contains surface hydroxyl groups when hydrated.

[0116] Glass substrates typically contain surface silanol groups, which can also react with alkyne-containing compounds to introduce alkyne moieties.

[0117] Metallic substrates (such as noble metals like gold, silver, and platinum) can be functionalized to include amino groups. These amino groups can then be reacted with alkyne-containing compounds to introduce an alkyne moiety.

[0118] Examples of alkyne-containing compounds suitable for reaction with polymer side chains or substrate surfaces containing silyl alcohols, hydroxyl groups, and / or alkyne ether compounds containing one or more alkoxy groups and one or more alkyne-containing groups are silyl halides (e.g., silyl chlorides) and / or silyl ether compounds containing one or more alkoxy groups and one or more alkyne-containing groups.

[0119] An example of a suitable silyl ether compound is (CAS No. 870987-68-1).

[0120] The alkyne moiety can also be introduced stepwise into polymer side chains or substrate surfaces containing silanols, hydroxyl groups, and / or amino groups. This involves first reacting with a silyl halide or silyl ether compound containing a functional group (e.g., an amino group), which can then react with an alkyne-containing compound. For example, (aminopropyl)trimethoxysilane can react with a suitable polymer side chain or a substrate containing surface hydroxyl, silanol, or amino groups to provide a fixed amino group. The fixed amino group can then be reacted with the alkyne-containing moiety (e.g., 5-hexyneic acid) via an amidation reaction to fix the alkyne group.

[0121] Alkyne-containing compounds, such as alkyne-containing thiols that form self-assembled monolayers (SAMs) on substrates (such as precious metals, such as gold, silver, and platinum), can also be used to incorporate alkyne moieties.

[0122] PU is a preferred substrate, and alkyne-functionalized diols can be used as chain extenders, providing modified PU surfaces with available alkyne groups for click reactions with the conjugates of the present invention.

[0123] The PU may contain an aromatic or aliphatic diol monomer containing one or more alkyne groups (e.g., acetylene). Suitable diols include 2,2-di(prop-2-ynyl)-propane-1,3-diol ((DPPD)) and 3,5-bis(hydroxymethyl)-1-propynylbenzene (PBM), with PBM being preferred.

[0124] Preferably, the chain length between the carbon marked with # in formula (VIIIa) or (VIIIb) and the substrate surface, or between the carbon marked with # and the polymer backbone (e.g., when the substrate is a polymer containing one or more alkyne moieties and / or has been modified to contain one or more alkyne moieties), is 25 atoms or less, more preferably 20 atoms or less, and even more preferably 10 atoms or less.

[0125] This chain length indicator includes the shortest atomic distance between the carbon marked with # and the substrate surface, or between the carbon marked with # and the polymer backbone, for example when the substrate is a polymer containing one or more alkyne moieties as side chains and / or has been modified to contain one or more alkyne moieties as side chains. The chain typically includes atoms in compounds used to introduce alkyne groups onto the substrate surface before reacting with the conjugate of this invention, atoms in side chains containing one or more alkyne groups, or atoms in side chains modified to contain one or more alkyne groups (when the substrate is a polymer containing one or more alkyne moieties as side chains and / or has been modified to contain one or more alkyne moieties as side chains). Hydrogen atoms are not considered atoms in this chain. Carbons marked with # are not considered part of the chain for calculating this chain length. When the alkyne moieties are attached to the substrate via surface-exposed hydroxyl or silanol groups, or via amino groups bonded to the substrate surface (e.g., using silyl chloride or silyl ether compounds as described above), the oxygen atoms of the hydroxyl or silanol groups or the nitrogen atoms of the amino groups are included in the chain length calculation. If a SAM is used to attach an alkyne portion to the substrate surface, the atoms in the alkyne-containing compound that form the SAM and are bonded to the substrate surface are included in the chain length calculation.

[0126] Preferably, the distance between the carbon marked with # in formula (VIIIa) or (VIIIb) and the substrate surface is 30 angstroms or less, preferably 25 angstroms or less, more preferably 20 angstroms or less, and more preferably 10 angstroms or less. This distance can be the distance between the carbon marked with # in formula (VIIIa) or (VIIIb) and the substrate surface when the substrate is exposed to aqueous conditions. Typically, the connecting portion shown in formula (VIIIa) or (VIIIb) will lift the compound of formula (I) away from the surface when exposed to aqueous conditions.

[0127] Preferably, the derivatives of the coupling (i.e., the coupling once covalently attached to the surface) are uniformly distributed on the surface (e.g., at the micrometer scale).

[0128] Therefore, it is preferable that the alkyne groups are uniformly distributed on the substrate surface (e.g., at the micrometer scale) before the click reaction to connect the couplings of the present invention.

[0129] The distribution of the coupling derivative on the substrate surface after the click reaction and / or the distribution of alkyne groups on the surface before the click reaction can be assessed by time-of-flight secondary ion mass spectrometry.

[0130] In another aspect, the present invention provides a method for preparing a substrate having a compound of formula (I) covalently linked thereto, the method comprising reacting a coupling compound as defined herein with a substrate having an alkyne group on its surface under conditions allowing a cycloaddition reaction between the alkyne and an azide group on the coupling compound. The present invention also provides a substrate having a coupling compound derivative covalently linked thereto, which can be obtained by (or through) this method.

[0131] The substrates described herein may be medical devices or coatings on medical devices, or may be molded into medical devices or coatings on medical devices.

[0132] Preferably, the substrate having the aforementioned coupling derivative covalently attached thereto has a contact angle of less than 90°, more preferably 80° or less, and even more preferably 70° or less. The contact angle can be determined by the seat drop method. The applied droplet can be distilled water, and a suitable droplet volume can be 3-10 μL, for example, about 5 μL. The droplet shape can be analyzed 10-30 seconds, for example, about 10 seconds, after deposition on the surface. The droplet shape can be analyzed under ambient conditions (e.g., at 18-25°C and 50-90% relative humidity). Commercially available instruments and software can be used to deposit and evaluate the droplet shape (e.g., the DSA100 instrument and DSA3 software from Krüss GmbH (Hamburg, Germany)).

[0133] An exemplary method for determining contact angle may include: extracting a substrate containing a covalently linked coupling derivative overnight in acetone / ethanol, followed by careful washing with EtOH. Droplet deposition and shape imaging can then be evaluated using a DSA100 instrument and DSA3 software (Krüss GmbH, Hamburg, Germany). A droplet volume of 5 μL can be deposited using ultrapure deionized water (resistivity >18.2 MΩ cm), and the droplet shape is measured 10 seconds after surface deposition. Analysis can be performed at ambient temperature and humidity. The contact angle can be reported as the average of at least six measurements.

[0134] On the other hand, a method is provided for attaching the coupling compound of the present invention to a substrate comprising one or more alkyne moieties on its surface, the method comprising reacting the coupling compound with a substrate comprising one or more alkyne moieties. Suitable substrates comprising one or more alkyne moieties are as described above. In this respect, the substrate starting material will comprise one or more alkyne groups, which subsequently react with an azide to form a triazole ring as shown in the structure of formula (VIIIa) or (VIIIb) above. As described above, the reaction is an azide-alkyne cycloaddition reaction, such as the Huisgen azide-alkyne cycloaddition reaction. Preferably, the reaction is a copper-catalyzed azide-alkyne cycloaddition reaction (CuAAC) or a strain-promoted azide-alkyne cycloaddition reaction (SPAAC), more preferably CuAAC. These are examples of “click” reactions, and suitable reaction conditions are known. Suitable reaction conditions are described, for example, in the examples. The present invention also provides a substrate having a coupling compound derivative covalently attached thereto, which can be obtained by (or through) this method.

[0135] The substrates of this invention can be medical devices or coatings on medical devices, or molded into medical devices or coatings on medical devices. Suitable medical devices include implantable devices (including orthopedic implants such as hip and knee implants, and dental implants), surgical and other medical devices, and disposable devices such as syringes. Of particular interest are stents, catheters, tubing, fasteners, pins and sutures, dressings, patches, prosthetic liners, and sutures; PU catheters are particularly preferred devices according to this invention.

[0136] Methods for preparing couplings

[0137] Another aspect of the present invention provides a method (method A) for preparing a conjugate of the compound of formula (I) and a linker portion described herein, the method comprising:

[0138] Coupled with compound of formula (I) to be labeled An amide bond is formed between the carbon atom and the N-terminal amino group of the compound of formula (I). A, Z, and m are as defined elsewhere in this document.

[0139] The coupling step may include activating the carboxylic acid group in the compound of formula (IX) with a suitable peptide coupling agent. Suitable peptide coupling agents are known in the art, such as HBTU.

[0140] Compound (IX) can be prepared by a method comprising the following steps: [The steps are described in the original text, but the provided text is incomplete and cannot be accurately translated.]

[0141] Transformed into compound of formula (XI)

[0142] The compound of formula (XI) is converted into the compound of formula (XII). ,as well as Compound (XII) is reacted with 5-7 membered cyclic anhydrides to give compound (IX).

[0143] Compound (X) can be converted into compound (XI) by activating the OH group to undergo nucleophilic substitution (e.g., converting the OH group into a better leaving group), followed by reacting the activated intermediate with an azide nucleophilic reagent source (such as sodium azide). Examples of leaving groups into which the OH group can be converted include toluenesulfonates and methanesulfonates. Preferably, compound (X) is ditoluenesulfonated and then converted into compound (XI).

[0144] Compounds of formula (XI) are preferably converted to compounds of formula (XII) using a reducing agent (e.g., triphenylphosphine, as by Staudinger reduction). This step conveniently produces asymmetric intermediates because once an azide moiety is reduced to an amine, the molecule migrates from the organic phase to the aqueous phase, where further reduction is impossible. The inventors have found that this ability to migrate from one phase to another depends on the length of the compound of formula (XI), thus limiting the lengths of "m" and the portion "A," thereby limiting the length of the linker / coupling as defined herein.

[0145] Compounds of formula (X) may preferably have an M of 600 g / mol or less. n More preferably, the number-average molecular weight of the compound of formula (X) is 500 g / mol or less, more preferably 400 g / mol or less, and even more preferably 300 g / mol or less. Preferably, the number-average molecular weight of the compound of formula (X) is... n 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 M n The upper and lower limits can be combined arbitrarily. Therefore, the compound of formula (X) preferably has an M of 100 to 600 g / mol. n More preferably 100 to 500 g / mol, more preferably 120 to 400 g / mol, more preferably 150 to 300 g / mol, more preferably 175 to 250 g / mol, and most preferably 180 to 225 g / mol, for example about 200 g / mol.

[0146] The dispersion (also known as polydispersity index) value of the compound of formula (X) can be from 1.000 to 1.15, preferably from 1.001 to 1.05 or 1.10, more preferably from 1.001 to 1.01, and even more preferably from 1.001 to 1.007 or 1.001 to 1.005.

[0147] Preferably, the compound of formula (X) is polyethylene glycol, polytetrahydrofuran or an oligomer thereof, more preferably PEG or an oligomer thereof.

[0148] Any 5-7 membered cyclic anhydride can be used to convert a compound of formula (XII) into a compound of formula (IX). In this context, "5-7 membered" refers to the number of atoms in the ring containing the anhydride functional group -C(O)-OC(O)-. Other ring members may be selected from oxygen and carbon atoms, and the ring containing the anhydride functional group may be saturated or unsaturated. Cyclic anhydrides may contain one or more substituents, such as those defined above for the Z group. Preferably, cyclic anhydrides may have a structure Where Z is as defined above with respect to formula (V). Suitable examples of cyclic anhydrides include hydroxyglycolic anhydride, succinic anhydride, glutaric anhydride, adipic anhydride, methylsuccinic anhydride, phthalic anhydride, maleic anhydride, 1,2-cis-cyclohexanedicarboxylic anhydride, high-phthalic anhydride, octenylsuccinic anhydride, S-acetylmercaptosuccinic anhydride, itaconic anhydride, citraconic anhydride, dimethylmaleic anhydride, cis-aconitine anhydride, 2-(2'-carboxyethyl)maleic anhydride, and 1-methyl-2-(2'-carboxyethyl)maleic anhydride. Preferably, the cyclic anhydride is glycolic anhydride.

[0149] On the other hand, a method (method B) is provided for preparing a conjugate of a compound of formula (I) as defined herein with a linker portion, the method comprising taking a compound of formula (X) and...

[0150] Where A is absent or is an organic group containing carbon, oxygen and hydrogen atoms (preferably a polyoxyethylene [more preferably PEG or PTHF] or a single repeating unit or oligomer thereof); m is an integer from 2 to 4, preferably 2 or 4; And the M of compound (X) n 600 g / mol or less; Transformed into compound of formula (XI)

[0151] To convert compound (XI) into compound (XII) ,as well as Reacting compound (XII) with a 5-7 membered ring yields compound (IX).

[0152] The compound of formula (IX) is coupled with the compound of formula (I) to form a compound labeled in formula (IX). An amide bond is formed between the carbon atom and the N-terminal amino group of the compound of formula (I).

[0153] To avoid any doubt, the above text regarding the M of compound (X) disclosed in method A... n The same applies to this aspect of the invention as to the dispersion value. The details above regarding the coupling steps between formulas (IX) and (I) disclosed in method A, as well as the steps for converting compound (X) to compound (XI), converting compound (XI) to compound (XII), and the properties of cyclic anhydrides, also apply to this aspect of the invention.

[0154] To avoid any doubt, the definitions disclosed above regarding formula (V) (including but not limited to preferred definitions of Z and A, number-average molecular weight, and polydispersity index) are as follows: After making the necessary modifications (mutatis mutandis) The methods applicable to this invention, as well as the couplings obtained or obtainable by the methods of this invention (including methods A and B), are applicable to the present invention.

[0155] On the other hand, a coupling of a compound of formula (I) with a linker portion is provided, which is obtainable (or acquired) by the methods of the present invention (including methods A and B). The coupling obtained (or acquired) by the methods of the present invention (including methods A and B) can be attached to a substrate containing one or more alkyne moieties on its surface by reacting the coupling with such a substrate. As described above, this reaction is an azide-alkyne cycloaddition reaction. The present invention also provides a substrate having a coupling derivative covalently linked thereto, which can be obtained (or by) this method.

[0156] peptide synthesis

[0157] The compound of formula (I) used in the couplings of this invention can be synthesized in any convenient manner. Typically, any reactive groups present (e.g., amino, thiol, and / or carboxyl groups) will be protected throughout the synthesis. Therefore, the final step in the synthesis will be the deprotection of the protected derivative of this invention.

[0158] When constructing peptides, one can, in principle, start from either the C-terminus or the N-terminus, although the C-terminus initiation step is preferred.

[0159] Peptide synthesis methods are well known in the art, but for the purposes of this invention, synthesis on a solid support, which is well known in the art, may be particularly convenient.

[0160] A variety of amino acid protecting groups are known to be available, and suitable amine protecting groups may include benzyloxycarbonyl (also known as Z), tert-butyloxycarbonyl (also known as Boc), 4-methoxy-2,3,6-trimethylbenzenesulfonyl (Mtr), and 9-fluorenylmethoxycarbonyl (also known as Fmoc). It should be understood that when constructing a peptide starting from the C-terminus, an amine protecting group is present on the α-amino group of each newly added residue, and this group needs to be selectively removed before the next coupling step.

[0161] Used carboxyl protecting groups include, for example, easily cleavable ester groups such as benzyl (Bzl), p-nitrobenzyl (ONb), pentachlorophenyl (OPClP), pentafluorophenyl (OPfp), or tert-butyl (OtBu), as well as coupling groups on the solid support, such as methyl groups attached to polystyrene.

[0162] Thiol protecting groups include p-methoxybenzyl (Mob), triphenylmethyl (Trt), and acetaminomethyl (Acm).

[0163] Several methods exist for removing amine and carboxyl protecting groups. However, these methods must be consistent with the synthetic strategy employed. The side-chain protecting group must be stable under the conditions used to remove the temporary α-amino protecting group prior to the next coupling step.

[0164] Amine protecting groups such as Boc and carboxyl protecting groups such as tBu can be removed simultaneously by acid treatment (e.g., with trifluoroacetic acid). Thiol protecting groups such as Trt can be selectively removed using oxidizing agents (e.g., iodine).

[0165] The invention will now be further described with reference to the following non-limiting embodiments and the accompanying drawings.

[0166] Figure 1 Top: Alkynyl-functionalized diols used in the PU material of Example 1. Bottom: Schematic diagram of AMP functionalization of PU films containing alkynyl monomers mediated by click chemistry.

[0167] Figure 2 The structures of modified AMC and AMC-109 prepared in Example 1.

[0168] Figure 3 As described in Example 1, with Staphylococcus epidermidis ( S. epidermidis CLSM images of the plastic film after incubation. Left: Control PU6 film. Right: PU6 film coated with AMC-25-04, with a much lower bacterial load.

[0169] Figure 4 As described in Example 1, fluorescence micrographs of the PU-coated catheters. The top image shows the catheter segment coated with PU6. The bottom image shows the interaction with Staphylococcus epidermidis (…). S. epidermidisAfter incubation and staining together, the ducts were coated with PU6 and covalently linked AMC-25-04.

[0170] Figure 5 The green columns (first and third from the left) represent the control experiment of azide-coupled D-mannose binding to the glass surface, and the blue columns (second and fourth from the left) represent the experiment of AMC-25-04 binding to the glass surface in *E. coli* (WT). The significance was statistically analyzed using a Student's t-test, where ns indicates no significance. p<0.05， p<0.01， p<0.005 and p < 0.001. The bar chart shows the mean and standard error of the median GRs measured before (“initial GR”) and after (“final GR”) the transition time in a single experiment.

[0171] Figure 6 and Figure 7 Mass spectrum of the conjugate prepared in Example 3. The number average molecular weight of the PEG starting material was 200 g / mol. Figure 6 ) and 400 g / mol ( Figure 7 ).

[0172] Figure 8 The antimicrobial effect of the coated substrate (right) prepared in Example 4 compared with the control (left) was evaluated using the standard shake-flask test ASTM E2149-13a and the Certika data evaluation method.

[0173] Example 1 – Biocompatible polyurethane coating incorporating surface-anchored antimicrobial peptides

[0174] like Figure 1 As shown, polyurethane surfaces with alkyne moieties are functionalized with peptide-linker-azide conjugates via a CuAAC (copper-catalyzed azido-alkyne cycloaddition) reaction, commonly referred to as a click reaction. The linker is a short polyethylene glycol (PEG) moiety, which is monodisperse (n = 3) or polydisperse, and the PEG-linker has an azide moiety at one end while the other end is modified with a carboxylic acid to attach to the N-terminus of the antimicrobial peptide.

[0175] Materials and Methods

[0176] peptide synthesis

[0177] The peptides were prepared by Amicoat A / S and used as is, with a purity exceeding 95%. In summary, AMC-109 was prepared according to the literature procedure (Svenson J, Stensen W, Brandsdal BO, Haug BE, Monrad J, Svendsen JS. Biochemistry. 2008, 47, 3777-88 DOI: 10.1021 / bi7019904).

[0178] The peptides were prepared using the standard Fmoc protocol and coupled to PEG-linkers on resin or in solution.

[0179] According to Jiang et al. (ACS chemical neuroscience. 2018, 9, 100-6 DOI: 10.1021 / acschemneuro.7b00111), PEG200 amino-azido derivatives prepared from PEG200 are reacted with diethylene glycol anhydride according to patent (US2009 / 0203584A1, p15) to provide bifunctional polydisperse PEG-connectors with azido and carboxylic acid ends.

[0180]

[0181] The linker (1.0 eq.) was subsequently coupled with AMC-109 (1.0 eq.) using HBTU (1.2 eq.) and TEA (4.8 eq.) to provide the final product (AMC-25-04). The crude peptide was purified by preparative HPLC and lyophilized to obtain the TFA salt. Purity (HPLC): >95%.

[0182] A small amount of monodisperse AMC-25-04 was also prepared to optimize the CuAAC reaction in solution.

[0183] The peptides were purified by reversed-phase HPLC on a Supelco Ascentis C18 column (10 μm, 21.2 × 100 mm) using a mixture of water and acetonitrile (both containing 0.1% TFA) as the eluent. The purity of the peptides was analyzed by RP-HPLC using a Supelco Ascentis Express C18 column (2.7 μm, 3.0 × 100 mm) and positive ion electrospray mass spectrometry on a Thermo Orbitrap mass spectrometer.

[0184] Synthesis of AMC-08-89 (comparative example).

[0185] The peptide was synthesized on 2-chlorotriphenylmethyl resin using a standard solid-phase Fmoc protocol, employing HBTU and DIPEA as activators, except for the final coupling, where Boc-Trp(Boc)-OH was used to ensure the N-terminus remained protected after cleavage from the resin. The fully protected peptide Boc-Trp(Boc)-Arg(Pbf)-Trp(Boc)-Arg(Pbf)-Phe-Gly-OH was released from the resin using acetic acid:trifluoroethanol:DCM (1:1:8). A PEG-linker 2-[2-(2-azidoethoxy)ethoxy]ethylamine toluenesulfonate (IRIS Biotech PEG4980) was coupled to the C-terminus of the protected peptide using HBTU and DIPEA, followed by final and complete deprotection using TFA:H2O:triisopropylsilane (95:2.5:2.5). The crude peptide was purified by preparative HPLC and lyophilized to obtain the TFA salt. Purity (HPLC): 87%. ESI-MS: (MH+, C51H70N18O8 calculated value: 1063.57, measured value: 1063.5700). The structure of AMC-08-89 is WRWRFG-NHCH2CH2(OCH2CH2)2-N3.

[0186] Synthesis of AMC-25-01 (comparative example).

[0187] This peptide was synthesized on Rink Amide AM resin using a standard solid-phase Fmoc procedure (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)). A linker was introduced into the peptide by coupling 11-azido-3,6,9-trioxaundecanoic acid (TCI, A2293) to the N-terminus of the Rink amide-binding peptide using HCTU and DIPEA as activators. The crude peptide was purified by preparative HPLC and lyophilized to obtain the TFA salt. Purity (HPLC): 92%. ESI-MS: (MH+, C 51 H 69 N 17 O9 calculated value: 1064.55, measured value: 1064.5549). The structure of AMC-25-01 is N3-(CH2CH2O)3-CH2C(O)-FRWRW-NH2.

[0188] In vitro antibacterial assessment

[0189] The effect of peptides on Staphylococcus aureus was determined by microdilution method. Staphylococcus aureus ATCC29213), Escherichia coli ( Escherichia coli ATCC 25922) and Staphylococcus epidermidis ( Staphylococcus epidermidis The minimum inhibitory concentration (MIC) of RP62a was determined. In short, 2 × 10⁻⁶ colonies were prepared using overnight colonies. 8 CFU / mL stock solution. Further dilute the bacterial solution 1:100 in MHB to produce the final inoculum (2 × 10⁻⁶). 6 The peptides were dissolved in DMSO to a concentration of 20 mg / mL and serially diluted to the appropriate concentration (128 to 2 μg / mL) in microtiter plates (10 μL per well). Bacterial inoculum (90 μL) was added to each well, and the plates were incubated at 37°C for 20 h to obtain satisfactory growth. Bacterial growth was analyzed by visual inspection and OD measurement at 600 nm. The MIC was defined as the lowest antimicrobial concentration that inhibited visible growth of the studied bacteria (with a 50% reduction in OD as the cutoff). Each peptide concentration was analyzed in duplicate. DMSO (0.6%) in the growth medium was used as a negative control, and DMS-free medium was used as a blank. Gentamicin sulfate and AMC-109 were used as positive controls.

[0190] Polyurethane Synthesis

[0191] Polytetrahydrofuran (PTHF) (CAS 25190-06-1, M) w = 950 - 1050 g / mol and M w= 2825 -2976 g / mol) and diisocyanates: isophorone diisocyanate (IPDI, CAS 4098-71-9), toluene diisocyanate (TDI, CAS 584-84-9), 4,4'-methylene bis(phenyl isocyanate) (MPI, CAS 101-68-8) and hexamethylene diisocyanate (HDI, CAS 822-06-0) were purchased from Sigma-Aldrich (Sweden). Glyceryl ethoxylate-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 were also supplied by Sigma-Aldrich (Sweden). Dimethyltin neodecanoate catalyst was supplied by Avison GmbH (Germany). PTHF was dried under vacuum at 60°C for 16 hours before use, while glycerol ethoxylate-co-propoxylate triol and diisocyanate were used as is. 3,5-bis(hydroxymethyl)-1-propynylbenzene (PBM) and 2,2-di(prop-2-ynyl)propane-1,3-diol (DPPD) chain extenders were obtained from Amicoat A / S and stored under dry conditions in a desiccator before use.

[0192] Flexible block thermoplastic polyurethane

[0193] For the synthesis of the prepolymer, dry PTHF (~0.005 mol) was dissolved in DMF (40 mL) and added to a three-necked round-bottom flask (100 mL) equipped with a burette, condenser, and airtight mechanical stirrer. The flask was placed in an 80°C oil bath, purged with nitrogen, and stirred with a Teflon blade. Then, diisocyanate (MDI, TDI, or HDI) was added to the flask at an NCO:OH ratio of 2:1. Dimethyltin neodecanoate catalyst (0.4 w% based on total monomer mass) dissolved in DMF (10 mL) was added, and the reaction proceeded until the theoretical isocyanate content was reached, followed by ATR FT-IR at 2270 cm⁻¹. -1The disappearance of the isocyanate absorption peak was used to determine the polymer composition. For the final polymer synthesis, 0.005 mol (NCO:OH molar ratio 1:1) of PBM was dissolved in DMF (10 mL) and added to the prepolymer. Polymerization was carried out at 80°C under nitrogen purging until the final polymer reached a stable molecular weight as determined by size exclusion chromatography (SEC). The reaction mixture was precipitated in ice-cold Milli-q water, washed three times with water and once with ethanol. The finished polymer was dried under vacuum at 70°C until it reached a stable quality (usually 20-24 hours). Films (≈25 μm) used for characterization and functionalization were prepared on PET substrates (Kodak ESTAR, Rochester, USA). The PU polymer was dissolved in BHT-stabilized (0.025%) tetrahydrofuran (THF, VWR Chemicals, France) at a concentration of 1 w / v%, and films were prepared using a 200 μm film coater, followed by solvent evaporation overnight at room temperature. Thicker films (≈250 μm) were prepared using a 10% w / v% PU THF solution on a polytetrafluoroethylene (PTFE) foil and a 500 μm film coater. After preparation, the 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 small pieces.

[0194] Quartz crystal microbalance with dissipation monitoring (QCM-D)

[0195] The time-resolved peptide coupling during the click reaction was assessed using QCM-D. Immobilization measurements were performed using an E1 QCM-D instrument from Q-sense (Gothenburg, Sweden). The mass of the coupled peptide (ng / cm³) was also measured. -2 The frequency shift during the coupling process is calculated using the Sauerbrey relation (Equation 1), where... C f It is a sensor-specific constant (17.7). nr = 3, which is the number of odd overtones used for calculation. Δ f It is the change in resonant frequency:

[0196] The gold (Au) coated sensor surface (Q-Sense, Gothenburg, Sweden) was washed for 10 min at 80°C with an alkaline piranha solution containing 5:1:1 Milli-q water, NH3 (24%, Sigma-Aldrich, Sweden), and H2O2 (30%, Sigma-Aldrich, Sweden), followed by a thorough wash with large amounts of Milli-q water and drying under N2. To prepare the coated sensor surface, the polymer was dissolved in THF (1% w / v) and applied to the clean sensor surface by spin coating (50 μL, 2000 rpm, 1 min). After the THF solvent evaporated, the sensor surface was mounted in the instrument, and Milli-q water (0.1 mL / min) was pumped over the surface. After 20 min, AMC-peptide (1 μM), CuSO4 (10 μM), and ascorbate (20 μM) were injected, and mass changes during surface coupling were monitored. The coupling agents were removed by washing with Milli-q water after 16 hours. The AMC-109 peptide lacks an azide functional group and was therefore used as a control.

[0197] CuAAC-reaction

[0198] in solution

[0199] Acetonitrile, trifluoroacetic acid, sodium ascorbate, and copper sulfate pentahydrate (CuSO4·5H2O) were purchased from Sigma Aldrich, Sweden. PBM and AMC-25-04 were supplied by Amicoat AS (Norway). Optimal coupling conditions were explored using different concentrations of ascorbic acid and copper sulfate. For a typical solution reaction, 250 μL of an aqueous solution of PBM (3.2 mM) was mixed with 250 μL of the peptide (3.2 mM) also dissolved in deionized water. Different amounts of ascorbic acid (0.16–20 mM) and copper sulfate (0.032–3.2 mM) were each added as aqueous solutions, 250 μL each, to achieve a final volume of 1 mL. The reaction was monitored for 40 hours at ambient temperature using a Perkin Elmar Flexar HPLC system equipped with a Flexar binary pump. Compounds were separated over 60 minutes by gradient elution (from 25% to 40% acetonitrile) using a mixture of acetonitrile and deionized water (each solvent containing 0.1% TFA). 10 μL of the reaction mixture was injected into a Quasar C18 column (250 mm × 4.6 mm inner diameter, 5 μm particle size) heated to 30°C, using a Flexar Peltier LC system placed in a Flexar Peltier column oven. The signal was detected and processed using a Flexar PDA Plus detector equipped with a 10 mm flow cell. Data were collected at 254 nm and 280 nm wavelengths, with the primary processed data derived from the 254 nm reading.

[0200] The formation of solution click products was also validated and quantified by MS. Samples were separated by LC (Acquity UPLC IClass, Waters) using an Acquity UPLC CSH C18 1.7 μm column at 70°C. The gradient used was from 95:5 to 5:95 of 0.1% formic acid 0.05% TFA aqueous solution: 0.1% formic acid 0.05% TFA acetonitrile, completed within 2 minutes at a flow rate of 0.8 mL / min. Analytes were detected by electrospray ionization (ESI) positive ion mode mass spectrometry (MS, G2-S QTOF, Waters Corporation), and data were evaluated using MassLynx 2.1 (Waters Corporation) software.

[0201] On alkyne-functionalized PU

[0202] PU films were prepared and placed in Eppendorf tubes. The PU films were first washed in EtOH for 5 minutes, followed by washing with Milli-Q water (3 × 5 minutes). Then, Cu(II) solution, ascorbic acid solution, and peptide solution were added to the tubes. In preliminary studies, Cu concentrations varied between 2–1000 nM, ascorbic acid concentrations between 4–2000 nM, and peptide concentrations between 0.05–10 μM. After a controlled reaction time at ambient room temperature (RT), the reactants were removed, and the PU films were washed with Milli-Q water (3 × 5 minutes). The films were then allowed to dry at room temperature (RT) for 1 hour and then placed in a refrigerator until chemical, physical, or biological testing was performed. For most subsequent coating experiments, a peptide:CuSO4:ascorbic acid molar ratio of 1:2:10 was used, and overnight incubation was performed to ensure good coupling. The presence of peptides on the prepared surfaces was confirmed using XPS, Tof-SIMS, static contact angle measurements, and QCM-D.

[0203] In vitro antibacterial evaluation of peptide coating materials

[0204] Microscopic images of peptide-coated surfaces

[0205] Using overnight cultured Staphylococcus epidermidis ( Staphylococcus epidermidis RP62a) and Staphylococcus aureus ( S.aureus ATCC 29213) colonies, prepared in 0.5% NaCl at a concentration of 0.5% McFarland (1×10⁻⁶). 8 A CFU / mL solution was prepared and further diluted to 10 in TSB (Sigma Aldrich, Missouri, USA) containing 1% glucose. 5CFU / mL. 50 μL of bacterial solution was applied to the test material and incubated in an incubator at 37°C for 24 hours. After incubation, the test material was rinsed once with PBS, placed in wells, and shaken on a shaker for 5 minutes (100 rpm). The PBS was replaced, and the plate was shaken again for 5 minutes. The film was left in fresh PBS until staining and imaging. Bacterial activity was assessed by staining with the LIVE / DEAD® BacLight™ Bacterial Activity Kit. Attached bacteria and formed biofilms were observed using a Leica LSM800 confocal microscope and a Zeiss AxioObserver fluorescence microscope. Two replicates of each treatment were analyzed at two different locations (150 × 150 μm). The entire experiment was repeated once with fresh McFarland bacterial solution. Data presented are the overall mean of the two experiments (± 95% confidence interval). Significant differences between the control and peptide-functionalized surfaces were assessed using the Mann-Whitney nonparametric unpaired u-test. ImageJ software was used to evaluate the image. First, the red and green channels were segmented, then the image was converted to a binary image, and grain analysis was performed with a cutoff value of 5 pixels. 2 (pixels) 2 The circularity is 0.02-1.0. A threshold is set so that orange bacteria appear in the survival (green) channel.

[0206] Microscopic images of peptide-coated catheters

[0207] Staphylococcus epidermidis ( S. epidermidis Overnight cultures of RP62a were diluted 1:100 in TSB containing 1% glucose. The tubes were cut into 1 cm segments, further horizontally divided, and immersed in the bacterial solution. The coated tubes were incubated overnight, rinsed in PBS, and stained with the LIVE / DEAD® BacLight™ Bacterial Viability Kit (Thermo Scientific, Massachusetts, USA). Images were acquired using a ZEISS Axio Zoom.V16 fluorescence microscope.

[0208] Results and Discussion

[0209] Table 1 shows the intrinsic antimicrobial effects of the peptides, expressed as minimum inhibitory concentrations (MICs). AMC-109 exhibited high activity against all tested bacterial strains.

[0210] AMC-109 is coupled with the corresponding linker molecule to obtain AMC-25-04 ( Figure 2 It is worth noting that the MIC values ​​of AMC-25-04, whether monodisperse or polydisperse derived from PEG 200, provide the same antibacterial effect.

[0211] Table 1. Minimum inhibitory concentrations of the evaluated peptides 1 (MIC).

[0212]

[0213] ¹(μg / mL), ²Literature value comes from (Bagheri M, Beyermann M, Dathe M. Immobilization reduces the activity of surface-bound cationic antimicrobial peptides with noinfluence upon the activity spectrum. Antimicrobial Agents Chemotherapy.2009, 53, 1132-41)

[0214] Adding a adapter to AMC-109 significantly reduced its antimicrobial activity. However, given the high initial antimicrobial activity of AMC-109, AMC-24-04 remained highly active, with MIC values ​​ranging from 16 to 64 μg / mL against the included bacterial strains.

[0215] As shown in Table 1, after adding the adapter, the couplings AMC-08-89 and AMC-25-01 showed no activity.

[0216] Click chemistry in solution

[0217] Under optimized reaction conditions, the formation of PBM-peptide conjugates from AMC-25-04 solution was rapid (<10 min), and these conditions were also applied to 2D polymer form to link peptides to polymerized PBMs. Covalent linkage of the peptides to the polymer was confirmed by XPS, Tof-SIMS, and static contact angle measurements, and the reaction conditions and quantification were investigated using QCM-D.

[0218] PU material

[0219] Many different PU materials were considered and evaluated, and the material referred to in this paper as PU6 was selected for coupling and testing. PU6 is a flexible thermoplastic polymer, the composition of which is shown in Table 2.

[0220] Table 2. Composition of PU6

[0221] 1 3-Propanediol, 2 Glyceryl ethoxylate-co-propoxylate triol

[0222] Polymerization and characterization of PU6

[0223] The polymerization conditions were optimized using infrared (IR) spectroscopy to track the disappearance of isocyanate groups during the synthesis of the prepolymer and the final polymer, and the final PU film was characterized. The conversion rate during the polymerization reaction was further investigated using SEC to monitor the change in molecular weight over polymerization time. Both Fourier transform infrared spectroscopy (FT-IR) and SEC analysis showed that a polymer with an acceptable molecular weight was generated after 1 hour of polymerization. The molecular weights (number average and weight average) of the polymers are shown in Table 3.

[0224] Table 3. Molecular weight

[0225] a All values ​​are the average of two measurements. b All values ​​are the average of four measurements, with an error of 95% confidence interval.

[0226] Coatings for PU6 can be rapidly prepared by dissolving the polymer in, for example, THF, and then applying it using a coater or spraying process. This coating preparation process does not alter the mechanical properties of the polymer.

[0227] Peptide conjugation with PU6

[0228] QCM-D was used to accurately quantify attachment kinetics and final peptide surface density. In a typical QCM-D experiment, a rapid increase in the relevant mass was observed after the injection of the peptide and coupling reagent. This rapid increase was followed by a slow and steady increase. The increase in mass may originate from the physisorbed component, the covalently bound peptide, or both. The sample chamber was washed with 2 mL of buffer after different time intervals. The washing step removed approximately 30% of the attached peptides, but a stable level of attached peptides was observed after washing. In addition to the standard single-wash step, an enhanced washing procedure was performed, namely rinsing with 3 × 5 mL of buffer followed by washing with 2 mL of 1:1 Milli-Q water:DMSO. The results showed that this enhanced washing did not remove more peptides, and a single washing step was sufficient (data not shown).

[0229] The AMC-109 peptide lacks an azide functional group and therefore should not be anchored to the PU surface via click chemistry. The peptide was removed after a single washing step, indicating that its adsorption on the PU is non-covalent.

[0230] QCM-D results indicate that AMC-25-04 undergoes covalent bonding under click conditions. This is also an important observation for the study of the bioactivity of the prepared material, as immobilization experiments clearly show that AMC-109 adsorbs onto the PU6 surface but is easily removed after a single wash; therefore, the potential release of the adsorbed peptide is unlikely to interfere with biological studies.

[0231] In vitro antibacterial assessment

[0232] To evaluate the antibacterial efficacy of the developed PU6, the coated and control polymers were inoculated with the biofilm-producing bacteria Staphylococcus epidermidis (Staphylococcus epidermidis). Staphylococcus epidermidis RP62A. As described in the Materials and Methods section, PU films with controlled film thickness were prepared on plastic films using a coater. After incubation, the PU films were rinsed once with PBS and stained with LIVE / DEAD™ BacLight™ and examined by confocal microscopy. Figure 3 As shown.

[0233] The control surface showed an established biofilm of live bacteria after incubation. This finding contrasts with PU6 coated with AMC 25-04, which showed very sparse bacterial colonization, with the observed bacteria being predominantly dead. The significant difference in colonization between the materials indicates that the optimized AMC 25-04 peptides retain activity upon covalent linkage, and suggests that the anchoring points allow the peptides to maintain a biologically active conformation. Failure to ensure sufficient rotational and structural degrees of freedom has been shown to result in surfaces that attract bacteria. The PEG spacer arms in AMC-25-04 may also interfere with bacterial attachment and adhesion, and facilitate biofilm removal during washing.

[0234] To explore the versatility of this functional antimicrobial layer, a formulation was also evaluated using a commercially available lumbar drainage catheter, the exterior of which was coated and inoculated with bacteria. The catheter was coated with PU6-AMC 25-04, and catheters coated with the same PU6 membrane without attached peptides were also included as negative controls. The coated catheters were cut into 1 cm segments or longitudinally in half and inoculated with Staphylococcus epidermidis (S. epidermis). S. epidermidis Incubate the catheters in overnight culture (diluted 1:100 with fresh TSB containing glucose). Incubate the catheters at 37°C for 24 hours. After incubation, rinse the catheter samples once with PBS and stain with LIVE / DEAD™ BacLight™. Examine the samples by fluorescence microscopy. Figure 4 As shown.

[0235] from Figure 4 As can be seen, the duct appears green in the image due to autofluorescence, but... Figure 4Bacterial biofilms were clearly visible on the control catheters in the top three panels. As shown in the three images below, no colonization or biofilm formation was observed on the exterior of the peptide-coated catheters. Slight bacterial growth occurred inside the AMC 25-04-coated catheters (especially at the ends) due to the absence of a peptide coating. The control catheters were heavily colonized by bacteria, forming a thick biofilm. The biofilm consisted primarily of live (green) bacteria, but dead bacteria were also observed in the images of the control catheters. Quantitative analysis was not possible due to the 3D morphology of the coated catheters. Preliminary antimicrobial assessment demonstrates how effective peptide coatings can be for enhancing the resistance of flexible PU to bacterial biofilm formation.

[0236] in conclusion

[0237] We developed a novel functionalized polyurethane polymer that allows for the click-covalent bonding of short antimicrobial peptides to combat bacterial biofilm formation. Azide-functionalized antimicrobial peptides were successfully bonded to the coating in a rapid and uniform manner via cycloaddition, resulting in a uniform peptide surface. The repulsive properties of the peptide coating material resist Staphylococcus epidermidis (S. epidermis) on the coated plastic film. S. epidermidis The biofilm was validated and further demonstrated on coated commercial catheters.

[0238] Example 2 - Bacterial growth analysis of antimicrobial peptides covalently linked to glass

[0239] method

[0240] Synthesis of AMC-25-04

[0241] Such as Jiang et al. ( supra The amino-azido derivative of PEG200 was prepared and reacted with diethylene glycol anhydride to obtain an azide-PEG-COOH linker. One equivalent (EQUIV) of azide-PEG-COOH was coupled with one EQUIV of AMC-109 (Amicoat A / S, Norway) using 1.2 EQUIV of HBTU and 4.8 EQUIV of TEA to obtain AMC-25-04. The crude peptide was purified by preparative HPLC and lyophilized to obtain the TFA salt. HPLC purity was >95%. The obtained product was monodisperse.

[0242] Microfluidic channel assembly and silanization

[0243] By using an inner diameter (ID) of 0.8 × 0.8 mm 2Microfluidic channels were prepared by mounting square glass capillaries (VitroCom, USA) onto microscope slides using UV-curable adhesive (NOA 68, Norland Products Inc., USA). The capillaries were connected at both ends using gel loading tips (VWR). The channels (and the coverslip glass surfaces used for fluorescence microscopy and TOF-SIMS analysis) were first soaked overnight in Hellmanex III cleaning solution (2%, Hellma GmbH, Germany), followed by immersion in sulfuric acid (2 M, Sigma-Aldrich 99.9%) for 1 hour, and then thoroughly rinsed with water (Milli-Q, Merck Life Science). The cleaned glass was rinsed with ethanol (99.5%, Solveco, Sweden) and then silanized by immersion in a 10% solution of O-(propynoxy)-N-(triethoxysilylpropyl)carbamate (90%, ABCR, Germany) and ethanol (99.5%, Solveco, Sweden) for one hour.

[0244] Click chemical modification of glass surfaces

[0245] Silanized capillary tubes and coverslips via copper-catalyzed alkyne-azide cycloaddition reaction (Click Chemistry) (Kolb, HC, Finn, MG & Sharpless, KB) Angewandte Chemie International Edition 40, 2004–2021 (2001)), modified with AMC-25-04, α-mannose-PEG3-azide (>95%, Sigma-Aldrich), or azide-fluorine 488 (>90%, Sigma-Aldrich). Alkynyl silane-modified glass was immersed in a click reaction solution for 10 minutes to couple with different azide molecules. The reaction solution contained 33 µM azide reactant diluted in PBS buffer (pH 7.4), 17 mM guanidine hydrochloride (Sigma-Aldrich), 75 µM CuSO4 (Sigma-Aldrich), 250 µM tris(3-hydroxypropyltriazolylmethyl)amine (THPTA, Tokyo Chemical Industry Co., Ltd.), and 500 µM ascorbic acid (Merk).

[0246] Bacteria and Culture Media

[0247] Escherichia coli ( E. coli A variant of the wild-type (WT) strain MG1655 was used in all experiments. Transformation with plasmid pBE1-mGPFmut2 resulted in the creation of a variant of WT *E. coli*. E. coliThe bacteria exhibited green fluorescence (GFP) and kanamycin resistance. They were stored in a deep-frozen glycerol stock and streaked weekly on LB agar. For live-cell microscopy, single colonies were selected from the plates, inoculated into LB medium supplemented with 50 μg / ml kanamycin, and grown overnight at 37°C. The overnight culture was gently centrifuged to remove aggregated bacteria. The OD was then typically... 600 The supernatant with a concentration of approximately 0.1 was diluted 1:1 in fresh LB medium supplemented with kanamycin and grown at 37°C until 0.35 < OD. 600 <0.6.

[0248] Live cell microscopy

[0249] The microfluidic channel was mounted on 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 PumpSystems) via polypropylene tubing. A pipette tip was secured to the tubing using UV-curable adhesive (NOA 68, Norland Products Inc., USA), serving as an adapter between the tubing and the pipette tip connected 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 images were acquired at 2 frames per second (2 fps) using a microscope camera (Axiocam 305Colour, Carl Zeiss Microscopy). Bacteria in LB medium (see above) were removed directly from the incubator, transferred to a syringe, and injected into the channel. Initially, the flow rate was 100 µl / min for 10 minutes to allow the system to equilibrate, followed by a lower flow rate of 20 µl / min to promote bacterial binding. The syringe was then replaced with a new syringe containing LB medium or LB medium supplemented with 100 µM AMPs, and injected at a flow rate of 100 µl / min for approximately 3 hours.

[0250] Image Analysis and Segmentation

[0251] Image analysis was performed using an automated workflow written in Matlab (MATLAB version: 9.13.0.2049777 [R2022b], TheMathWorks Inc., USA), which invoked the functionality of the ImageProcessing Toolbox. For growth rate (GR) analysis, the effective frame rate was reduced from 2 fps to 0.1 fps by averaging every 20 consecutive frames, thus excluding analyses that did not incorporate bacteria. For analysis of the minute movements of bacteria around their long axis, the images were analyzed at the original frame rate over 2 minutes.

[0252] The image undergoes non-uniform illumination correction and background subtraction. A mask is constructed from the processed image by subtracting a user-defined constant, performing two rounds of median filtering, label-based watershed segmentation, and applying shape / size criteria. This mask is used to extract attributes of individual bacteria, with length measured along the major axis of the best-fit ellipse center. If bacteria in subsequent frames have overlapping coverage areas and their length changes by less than 25%, their trajectories are connected. To address the issue of bacteria temporarily "disappearing" from the segmentation mask due to focus drift, if a bacterium reappears with an unchanged morphology after a brief period of invisibility, it is still considered a member of the same trajectory. Otherwise, a new trajectory begins when a bacterium splits or appears in a new location.

[0253] Growth rate and mean binding time analysis

[0254] Using Matlab (MATLAB version: 9.13.0.2049777 [R2022b], The MathWorks Inc., USA), the Curve Fitting Toolbox and the Statistics and Machine Learning Toolbox were invoked to extract the population growth rate from the trajectory of growing cells. GR (t) ,), cell length ( L(t) ) and overall average combination time ( T ½ The evolution of the bacterial growth rate (GR) was analyzed. Instantaneous GR was determined by linear regression of size data within a 5-minute range before and after each time point in the trajectory. Trajectories shorter than 10 minutes, and the 5-minute intervals before and after each trajectory (which are susceptible to noise), were excluded from the analysis. The experimental time axis was divided into 20-minute intervals, and the average GR of each bacterium within each time interval was calculated.

[0255] result

[0256] Microfluidic channels coated with antimicrobial peptides via click reaction

[0257] AMC-109 is a synthetic tripeptide AMP that has been shown to be effective against common bacterial and fungal pathogens. To create a pharmacophore-coated AMP surface, we prepared peptide AMC-25-04 by attaching an azide-terminated short polyethylene glycol PEG linker to the N-terminus of AMC-109, which has a cyclic anhydride.

[0258] Bacterial binding and growth experiments were performed using glass microfluidic channels. The capillary inner wall was modified with silane exhibiting alkyne functional groups, which allow orthogonal binding of azide-functionalized molecules via a copper-catalyzed click reaction. Fluorescence microscopy showed that, in the presence of all reagents required for the click reaction, the conjugated fluorophores of the azide readily bound to the silane-modified surface, but binding was very sparse if copper ions were omitted, confirming the reactivity of the silane modification (data not shown).

[0259] Two types of molecules were attached to the glass surface via click chemistry. Azide-conjugated D-mannose was used as a control. (Wild-type Escherichia coli) E. coli It can easily bind to D-mannose-coated surfaces via its type 1 pili. AMP-coated surfaces are prepared by linking AMC-25-04.

[0260] Bacterial response to antimicrobial peptide-coated surfaces

[0261] Wild-type Escherichia coli was found. E. coli It readily binds to surfaces coated with AMC-25-04. Immediately after binding, it is more effective against WT E. coli growing on mannose-coated surfaces. E. coli The initial growth rate (GR) decreased by 26%. However, GRs increased to control levels over time. Growth rate (GR) was calculated using the slope of the length-time curve at each time point, and the average values ​​obtained for each bacterium were binned.

[0262] Example 3 - Synthesis of AMC-109 couplings containing PEG 200 and PEG 400 connectors

[0263] O,O' -Bis(toluenesulfonyloxy)polyethylene glycol (56-1, 57-1)

[0264] Polyethylene glycol with a number average molecular weight (Mn) of 200 was dissolved in 60 mL of DCM and cooled on an ice bath. KOH (6.73 g, 0.12 mol, 8.0 eq.) and p-toluenesulfonyl chloride (8.6 g, 0.045 mol, 3.0 eq.) were added, and the mixture was stirred overnight at room temperature. The reaction was stopped by adding 40 mL of ice water to the solution, and the mixture was extracted with DCM (30 mL x 3). The organic phase was washed with brine, dried over MgSO4, filtered, and concentrated under vacuum. The crude product was purified by rapid chromatography to remove excess p-toluenesulfonyl chloride. Product 56-1 (7.249 g, 95%) was given as a colorless oil. 1 H NMR (400 MHz, CDCl3) δ 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).

[0265] The synthesis of 57-1 was carried out using the same protocol as 56-1, employing polyethylene glycol (3.0 g, 0.0075 mol, 1.0 eq, Sigma Aldrich, product number 202398) with a number average molecular weight (Mn) of 400. The crude product was purified by rapid chromatography (DCM / MeOH) to obtain product 57-1 (4.516 g, 85%) as a pale yellow oil. 1 H NMR (400 MHz, CDCl3) δ7.84 – 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).

[0266] O,O' -Bis(2-azidoethyl)polyethylene glycol (56-2, 57-2)

[0267] 56-1 (12.68 g, 0.0249 mol, 1.0 equivalent) was dissolved in 50 mL of DMF, and NaN3 (4.867 g, 0.0748 mol, 3.0 equivalent) was slowly added with stirring. The solution was heated to 80-90°C and refluxed, stirred overnight. The reaction was terminated by adding 50 mL of ice water, and extracted with Et2O (50 mL x 3). The organic phase was dried over MgSO4, filtered, and concentrated under vacuum. Residual DMF was removed by lyophilization. Product 56-2 (5.560 g, 89%) was obtained as a colorless oil and used directly in the next synthesis without purification. 1 H NMR (400 MHz, CDCl3) δ 3.74 – 3.63 (m, 14H), 3.39 (td, J = 5.1, 2.4Hz, 4H).

[0268] The synthesis of 57-2 followed the same protocol as 56-2, using 57-1 (4.516 g, 0.0064 mol, 1.0 eq.), but with slight modifications in the post-processing. The crude product was extracted with EtOAc:MeOH (10:1, 30 mL x 3). Since all DMF was removed under vacuum, lyophilization was not required. Product 57-2 (2.187 g, 76%) was obtained as a colorless oil and was used directly without further purification. 1 H NMR (400 MHz, CDCl3) δ 3.70 – 3.61 (m, 30H), 3.39 (t, J =5.1 Hz, 4H).

[0269] O -(2-aminoethyl)- O′ -(2-azidoethyl)polyethylene glycol (56-3, 57-3)

[0270] 56-2 (2.161 g, 1.0 eq.) was dissolved in 20 mL of Et₂O. 20 mL of 1M HCl and triphenylphosphine (2.263 g, 0.0086 mol, 1.0 eq.) were added to this solution. The reaction mixture was stirred overnight at high speed at room temperature. Triphenylphosphine oxide (TPPO) formed as a white solid, which was removed by filtration. The aqueous phase was washed with EtOAc to remove trace amounts of TPPO and starting material. The aqueous phase was cooled in an ice bath, and KOH was added until pH > 12. The product was extracted from the aqueous phase with DCM (20 mL x 5), dried over MgSO₄, filtered, and concentrated under vacuum. Product 56-3 (1.590 g, 82%) was obtained as a colorless oil and used directly in the next synthesis without purification. 1 H NMR (400 MHz, CDCl3) δ 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).

[0271] The synthesis of 57-3 was carried out using the same protocol as that of 56-3, using 57-2 (2.126 g, 0.0047 mol, 1.0 eq.) to obtain product 57-3 (1.382 g, 69%) as a colorless oil, which was used directly without further purification. 1 H NMR (400 MHz, CDCl3) δ 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).

[0272] O -(2-azidoethyl)- O -[2-(diethylene glycol-amino)ethyl]polyethylene glycol (56-4, 57-4)

[0273] To 50 mL of DCM solution containing 56-3 (5.322 g, 0.0238 mol, 1.0 eq.), diethylene glycol anhydride (5.515 g, 0.047 mol, 2.0 eq.) and DMAP (0.533 g, 0.0047 mol, 0.2 eq.) were added, and the reaction was stirred overnight. After confirming the reaction was complete by MS, ethylenediamine (2.856 g, 0.047 mol, 2.0 eq.) was added, and the mixture was stirred overnight. Then, 30 mL of DCM and 20 mL of 1M HCl were added, and the organic phase was washed with 1M HCl (20 mL x 3). The collected aqueous phase was extracted with DCM (20 mL x 5). The organic phase was dried over MgSO4, filtered, and concentrated under vacuum. Product 56-4 (5.703 g, 70%) was obtained as a light pink oily substance and was used directly without further purification. 1 H NMR (400 MHz, CDCl3) δ 8.80 (s, 1H), 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).

[0274] The synthesis of 57-4 was carried out using the same protocol as that of 56-4, using 57-3 (1.382 g, 0.0033 mol, 1.0 eq.) to obtain product 57-4 (1.589 g, 90%) as a light pink oily substance, which was used directly without further purification. 1 H NMR (400 MHz, CDCl3) δ 7.65 (s, 1H), 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).

[0275] Combined with AMC-109 O -(2-azidoethyl)- O -[2-(diethylene glycol-amino)ethyl]polyethylene glycol (56-5, 57-5)

[0276] To a 25 mL DMF solution containing 56-4 (505 mg, 1.485 mmol, 1.3 eq.), HBTU (519 mg, 1.370 mmol, 1.2 eq.) and triethylamine (554 mg, 5.482 mmol, 4.8 eq.) were added. The reaction mixture was stirred at room temperature for 15 min, and then AMC-109 (900 mg, 1.1421 mmol, 1.0 eq.) was added. The reaction mixture was stirred at room temperature and monitored by MS. Unreacted AMC-109 was observed, so 505 mg of 56-4 and equivalent amounts of HBTU and triethylamine were added again, and the mixture was stirred for 2 days until the reaction was complete. 100 mL of H2O was added, and the mixture was extracted with EtOAc (3 x 150 mL). The combined organic phases were then washed with H2O (5 x 50 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude product yield was 1.5 g. The crude product was then processed via C... 18 Purification by reversed-phase high-performance liquid chromatography (RP-HPLC) yielded 56-5 fractions of varying dispersions as white powders. High-resolution ESI (positive ion + VE mode) mass spectra were perfectly matched to the molecular formula and used to determine the dispersion of each purified fraction.

[0277] Following the same protocol as 56-5, AMC-109 (500 mg, 1.0 eq.) at a scale of 0.635 mmol and 57-4 (446 mg, 0.825 mmol, 1.3 eq.) were used, with slight modifications. Since the reaction was completed after 2 days, no additional 57-4 was needed, and trace amounts of DMF were removed by lyophilization. The crude product was obtained via C... 18 Purification by reversed-phase high-performance liquid chromatography (RP-HPLC) yielded 57-5 (385 mg, 46%) as a white powder. The high-resolution ESI (positive ion + VE mode) mass spectrum perfectly matched the molecular formula.

[0278] The mass spectra of 56-5 and 57-5 are as follows: Figure 6 and Figure 7 As shown.

[0279] ESI-MS

[0280] The following instruments are used to provide Figure 6 and Figure 7 Mass spectrum in: Thermo Scientific Orbitrap Exploris 120 mass spectrometer equipped with an electrospray ionization source (positive ion mode). Thermo Scientific Vanquish Ultra-High Performance Liquid Chromatography System Chromatographic column: Accucore Vanquish C18+ 50x2.1, particle size 1.5µ. Eluent was an acetonitrile-water mixture containing 0.1% trifluoroacetic acid.

[0281] Example 4 – Preparation and Testing of Polyacrylate-Coated Cellulose Materials Covalently Linked with AMC-25-04

[0282] AMC-25-04 was prepared according to the method described in the foregoing examples.

[0283] The preparation methods for nonwoven cellulose materials are as follows: either commercial grade viscose fiber (80 g / m²) is wet-spun with carboxymethyl cellulose, or cotton / linen / hemp / wood pulp is used with the blown fiber technique described in J. Appl. Polym. Sci. 2020, DOI: 10.1002 / APP.48339.

[0284] Cellulose substrates were coated with polyacrylic acid via plasma polymerization of acrylic monomers (parameters: flow rate: 3 sccm, pressure: 20–25 mtorr, frequency: 135 kHz, power: 10 W, time: 2 min). The resulting crosslinked coating was approximately 20 nm thick and uniformly distributed. Approximately 12% of the -COOH functional groups were retained for modification to link peptides.

[0285] Prior to using the click chemical linking of the AMC-25-04 peptide as described in the previous examples, the carboxyl groups on the surface were converted to acetylene esters using a standard coupling reaction.

[0286] The antimicrobial efficacy of the coated substrates was evaluated using the standard shake-flask test ASTM E2149-13a and Certika data assessment method. According to this technique, bacterial growth on the AMC-25-04 coated substrate was optically measured every 30 minutes over 48 hours and compared to a control coated only with polyacrylic acid but without AMC-25-04. Antimicrobial activity was calculated based on the time required for the coated substrate to reach an optical density (OD) of 0.2 relative to the control. A time difference (net initial OD) of 6 hours represented a reduction of ≥99.9% (3 logs) of bacteria on the substrate surface, while a net initial OD of 8 hours represented a reduction of 99.99% (4 logs). The AMC-25-04 sample showed a very significant delay of more than 9 hours relative to the control (see [link to relevant documentation]). Figure 8 ).

Claims

1. A coupling of a compound of formula (I) as defined herein with a connector of formula (V), The compound of formula (I) has the following structure: ； in, In any order, two of the AA (amino acid) moieties are cationic amino acids, and one of the AA is an amino acid having a lipophilic R group, wherein the R group has 14-27 non-hydrogen atoms; X is an N atom, which can be branched or unbranched C1-C. 10 Alkyl or aryl substitution, the group may contain up to two heteroatoms selected from N, O, and S; and Y is selected from the group consisting of R1-R2-R3, R1-R2-R2-R3, R2-R2-R1-R3, R1-R3, and R4; in: R1 is C, O, S, or N; R2 is C; Each of R1 and R2 may be substituted with a C1-C4 alkyl group or may not be substituted; R3 is a group containing 1 to 3 cyclic groups, each cyclic group having 5 or 6 non-hydrogen atoms; two or more cyclic groups may be fused; one or more rings may be substituted; R3 contains a maximum of 15 non-hydrogen atoms; and R4 is an aliphatic moiety having 2-20 non-hydrogen atoms, wherein the moiety is straight-chain, branched, or cyclic; Furthermore, the V-type connector portion has the following structure: ； in: Marked The carbon atom forms an amide bond with the N-terminal amino group of the compound of formula (I); A does not exist, or it is an organic group containing carbon, oxygen, and hydrogen atoms with a chain length of 3 to 40 atoms; m is an integer from 2 to 4; Z is selected from the group consisting of -(CR4R5)p-(O)q-(CR4R5)r-, -CR6=CR7-(CR4R5)s-, -(CR4R5)s-CR6=CR7-, -C(=CR8R9)-(CR4R5)t- and -(CR4R5)tC(=CR8R9-); in 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 R5 is independently chosen from H, halogen, C1-C 10 Alkyl, C2-C 10 Alkenyl, aryl, -(CH2) 1-6 COOH and -SC(O)-C1-C 10 Groups composed of alkyl groups; Alternatively, two R4 groups on adjacent -(CR4R5)- units may form a carbocyclic or heterocyclic ring together; Each R6 and R7 is independently selected from H, halogen, C1-C 10 Alkyl, C2-C 10 Alkenyl, aryl, -(CH2) 1-6 COOH and -SC(O)-C1-C 10 Groups composed of alkyl groups; Or R6 and R7 together form a carbon ring or heterocycle; Each R8 and R9 is independently selected from H, halogen, C1-C 10 Alkyl, C2-C 10 Alkenyl, aryl, -(CH2) 1-6 COOH and -SC(O)-C1-C 10 A group composed of alkyl groups.

2. The coupling compound according to claim 1, wherein the chain length of the A group is 3 to 25 atoms.

3. The coupling compound according to claim 2, wherein the chain length of the A group is 6 to 18 atoms.

4. The coupling compound according to any one of the preceding claims, wherein the A group is a polyether or a single repeating unit or oligomer thereof.

5. The coupling compound according to any one of the preceding claims, wherein the A group is a polyoxyethylene or a single repeating unit or oligomer thereof, wherein the polyoxyethylene is selected from polyethylene glycol or polytetrahydrofuran.

6. The coupling compound according to any one of the preceding claims, wherein the -N in the compound of formula (V) -A-(CH2) m Number-average molecular weight (M) of -NH- units n ) is 600 g / mol or less, where indicated Nitrogen (N) The nitrogen atom corresponding to the azide moiety is bonded to the A group, or, if A is absent, to the -(CH2) group. m - Group bonding.

7. The coupling according to claim 6, wherein the -N -A-(CH2) m The dispersion value of the -NH- unit is 1.000 to 1.

15.

8. The coupling compound according to any one of the preceding claims, wherein Z is -(CR4R5)p-(O)q-(CR4R5)r-.

9. The coupling according to any one of the preceding claims, wherein each R4 and R5 is independently selected from H, halogen, C1-C. 10 Alkyl, C2-C 10 Groups consisting of alkenyl and aryl groups; or two R4 groups on adjacent -(CR4R5)- units together forming a carbocyclic or heterocyclic ring.

10. The coupling according to claim 9, wherein each R4 and R5 is H.

11. The coupling compound according to any one of the preceding claims, 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 coupling compound according to any one of the preceding claims, wherein the compound of formula (I) is a compound of formula (II). ； in: AA1 is a cationic amino acid; AA2 is an amino acid having the lipophilic R group, wherein the R group has 14-27 non-hydrogen atoms; and X and Y are as defined in claim 1.

13. The coupling compound according to any one of the preceding claims, wherein the compound of formula (I) has the structural formula 。 14. A substrate having a derivative of the coupling agent as described in any one of the preceding claims covalently linked thereto.

15. The substrate according to claim 14, wherein the compound of formula (I) is covalently connected to the substrate via a linker as shown in formula (VIIIa) or (VIIIb): ， ； in, In formula (VIIIa) And the bond from ring B through which the wavy line passes in equation (VIIIb) represents the connection point with the substrate; Marked The carbon atom 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 one of claims 1-11; In formula (VIIIb), ring B is a cyclic group, such as an 8-10 membered cyclic group, optionally wherein ring B is fused with one or more other carbocyclic or heterocyclic rings, and wherein ring B is optionally substituted, preferably substituted by one or more substituents independently selected from halogen, hydroxyl, C1-C6 alkoxy or benzyl. And R 10 It is hydrogen or an organic group containing 1 to 30 non-hydrogen atoms, preferably R. 10 Choose freely H, C1-C 10 Alkyl, C1-C 10 alkenyl, C1-C 10 Alkoxy group, -C(O)Cl-C 10 Alkyl group, -C(O)OC1-C 10 The group consisting of alkyl, carbocyclic, and heterocyclic groups, wherein the alkyl, alkenyl, and alkoxy groups are optionally selected independently by one or more halogens, hydroxyl groups, amino groups, (C1-C2) 10 alkyl)amino and di(C1-C) 10 Substituents of alkyl)amino groups, more preferably R 10 For H.

16. The substrate of claim 14 or 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 its alloys, chromium-cobalt alloys, aluminum, stainless steel, and precious metals such as gold, silver, and platinum, and / or The polymers mentioned are selected from polyethylene (PE); polyurethane (PU); polyamide; polyethylene terephthalate (PET); synthetic rubber (SR); polystyrene (PS); polyacrylates such as polyacrylic acid (PAA), polymethyl methacrylate (PMMA), poly(ethyl cyanoacrylate) and polyacrylamide; polyacrylonitrile (PAN); polyetheretherketone (PEEK), polylactic acid (PLA), silicone, polysaccharides and polyglycolic acid (PGA).

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

19. The substrate according to any one of claims 15-18, wherein the chain length between the carbon atom marked with # in formula (VIIIa) or (VIIIb) and the substrate surface, or when the substrate is a polymer, is 25 atoms or less between the carbon atom marked with # and the backbone of the polymer.

20. The substrate according to any one of claims 15-19, wherein the distance between the carbon atom marked with # in formula (VIIIa) or (VIIIb) and the surface of the substrate is 30 angstroms or less.

21. A method for preparing a substrate having a compound of formula (I) covalently linked thereto, the method comprising reacting a coupling compound as defined in any one of claims 1-13 with a substrate having an alkyne group on its surface under conditions allowing a cycloaddition reaction between the alkyne and the azide group of the coupling compound.

22. A substrate having a derivative of a coupling agent as defined in any one of claims 1-13 covalently linked thereto, said substrate being obtainable by the method of claim 21.

23. A method for preparing the conjugate according to any one of claims 1-13, the method comprising: [The method is described in the original text, but the translation is incomplete.] ； Coupled with compound of formula (I) to be labeled The carbon atom forms an amide bond with the N-terminal amino group of the compound of formula (I). Wherein A, Z and m are as defined in any one of claims 1-11.

24. The method of claim 23, wherein the compound of formula (IX) is prepared by a method comprising the step of: preparing the compound of formula (X) ， Transformed into compound of formula (XI) ， The compound of formula (XI) is converted into the compound of formula (XII). ， The compound of formula (XII) is then reacted with a 5-7 membered cyclic anhydride to obtain the compound of formula (IX).

25. A conjugate comprising a compound of formula (I), said conjugate being obtainable by the method of claim 23 or 24.

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