Antifungal compounds, methods of making and antifungal products thereof

Abstract

This application discloses an antifungal compound, its preparation method, and an antifungal product, relating to the field of antifungal materials technology. The disclosed antifungal compound comprises a quaternized polysaccharide derivative modified with a membrane-penetrating peptide. The antifungal compound of this application contains a quaternized polysaccharide derivative modified with a membrane-penetrating peptide, enabling it to effectively penetrate fungal biofilms and act on deep cells to achieve an antifungal effect.

Description

Technical Field

[0001] This application relates to the field of antifungal materials technology, and more particularly to an antifungal compound, its preparation method, and an antifungal product. Background Technology

[0002] Polysaccharides are widely sourced natural polymers. Among them, polysaccharide backbones derived from animals, plants, and microorganisms show broad application prospects in biomedicine, food science, and tissue engineering due to their excellent biocompatibility, biodegradability, and ease of chemical modification. Quaternization modification of polysaccharide backbones can significantly improve their water solubility and impart a higher positive charge density to the material. This enhanced cationic property allows them to effectively bind to the surface of microbial cells through electrostatic interactions, thus attracting widespread attention in the development of antibacterial materials.

[0003] However, despite the excellent performance of quaternized polysaccharides in inhibiting fungi, their effectiveness against certain pathogenic fungi that easily form biofilms (such as Candida albicans) remains challenging. Fungal biofilms are complex community structures encapsulated by extracellular polymers secreted by the fungi themselves. Their dense physical barriers severely limit the penetration of antimicrobial substances, and quaternized polysaccharides have limited penetration ability into mature fungal biofilms, making it difficult to effectively act on the deep cells within the biofilm. Summary of the Invention

[0004] The main objective of this application is to provide an antifungal compound, its preparation method, and an antifungal product. The antifungal compound in this application contains a quaternized polysaccharide derivative modified with a membrane-penetrating peptide, which can effectively penetrate the fungal biofilm and act on deep cells to achieve an antifungal effect.

[0005] To achieve the above objectives, embodiments of this application provide an antifungal compound comprising: a quaternized polysaccharide derivative modified with a membrane-penetrating peptide.

[0006] In one embodiment, the membrane-penetrating peptide-modified quaternized polysaccharide derivative is formed by the membrane-penetrating peptide being coupled to the quaternized polysaccharide backbone via a covalent bond or linker arm.

[0007] In one embodiment, the polysaccharide includes chitin and / or chitosan.

[0008] In one embodiment, the membrane-penetrating peptide includes at least one of: octaarginine, TAT, Penetratin, Transportan, MPG, Pep-1, pHLIP, and iRGD.

[0009] In one embodiment, the quaternized polysaccharide and the transmembrane peptide are respectively modified with complementary functional group pairs, wherein the complementary functional group pairs include any one of the following: Azide and alkynyl groups; Carboxyl and amino groups; Maleimide group and thiol group; Aldehyde and amino groups; Thiol group and thiol group.

[0010] In one embodiment, the degree of quaternization of the quaternized polysaccharide is 0.2 to 1.2.

[0011] In one embodiment, the weight-average molecular weight of the quaternized polysaccharide is 1 × 10⁻⁶. 3 ~8×10 5 g / mol.

[0012] In one embodiment, the degree of deacetylation of the quaternized polysaccharide is 20-99%.

[0013] In one embodiment, the grafting rate of the transmembrane peptide is 0.1 to 0.6.

[0014] In one embodiment, the molar ratio between the transmembrane peptide and the quaternized polysaccharide is 0.5 to 5:1.

[0015] To achieve the above objectives, this application provides a method for preparing an antifungal compound, comprising the following steps: A quaternized polysaccharide and a membrane-penetrating peptide are provided, wherein the quaternized polysaccharide and the membrane-penetrating peptide are respectively modified with complementary functional group pairs; The membrane-penetrating peptide is coupled to the quaternized polysaccharide backbone through a coupling reaction to obtain a membrane-penetrating peptide-modified quaternized polysaccharide derivative. Antifungal compounds were prepared based on the quaternized polysaccharide derivatives modified with the membrane-penetrating peptides.

[0016] In one embodiment, the coupling reaction includes any one of click chemistry, amidation, Michael addition, reductive amination, and disulfide exchange.

[0017] To achieve the above objectives, embodiments of this application provide an antifungal product, which includes the antifungal compound as described above, or an antifungal compound prepared by the antifungal compound preparation method described above.

[0018] This application provides an antifungal compound comprising: a quaternized polysaccharide derivative modified with a membrane-penetrating peptide. Because membrane-penetrating peptides possess unique cell-penetrating capabilities, they can effectively overcome the dense extracellular polymer barrier of fungal biofilms. Therefore, the antifungal compound of this application exhibits good penetration ability against mature fungal biofilms, solving the problem of quaternized polysaccharides being unable to enter the interior of biofilms. Furthermore, with the carrying effect of the membrane-penetrating peptide, the quaternized polysaccharide backbone can be delivered to the deep layers of the biofilm. Its high-density positive charge can electrostatically interact with the fungal cell membrane, disrupting the membrane structure integrity, thereby achieving highly efficient killing of fungal cells in a deeply encapsulated state. Attached Figure Description

[0019] Figure 1 This is a flowchart illustrating the method for preparing the antifungal compound involved in the embodiments of this application; Figure 2 The Fourier transform infrared spectrum of the sample involved in the embodiments of this application; Figure 3 This is a schematic diagram illustrating the determination results of the minimum inhibitory concentration (MIC) against Candida albicans in the samples involved in the embodiments of this application. Figure 1 ; Figure 4 This is a schematic diagram illustrating the determination results of the minimum inhibitory concentration (MIC) against Candida albicans in the samples involved in the embodiments of this application. Figure 2 ; Figure 5 This is a schematic diagram illustrating the determination results of the minimum inhibitory concentration (MIC) against Candida albicans in the samples involved in the embodiments of this application. Figure 3 ; Figure 6 This is a schematic diagram illustrating the measurement results of the sample's inhibition of Candida albicans biofilm formation in the embodiments of this application. Figure 1 ; Figure 7 This is a schematic diagram illustrating the measurement results of the sample's inhibition of Candida albicans biofilm formation in the embodiments of this application. Figure 2 ; Figure 8 The cell viability rate after incubation of the samples involved in the embodiments of this application with L929 cells; Figure 9 These are fluorescence images of live and dead cells of Candida albicans before and after treatment with QCS5 and QCS5-R8 in the embodiments of this application.

[0020] The realization of the purpose, functional features and advantages of this application will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions in the embodiments of this application will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0022] The following detailed description, with appropriate reference to the accompanying drawings, discloses embodiments of the antifungal compounds, their preparation methods, and antifungal products of this application. However, unnecessary details may be omitted. For example, detailed descriptions of well-known matters and repetitive descriptions of essentially identical structures may be omitted. This is to avoid unnecessarily lengthy descriptions and to facilitate understanding by those skilled in the art. Furthermore, the accompanying drawings and the following description are provided for the purpose of providing a full understanding of this application by those skilled in the art and are not intended to limit the subject matter of the claims.

[0023] The "range" disclosed in this application is defined by a lower limit and an upper limit. A given range is defined by selecting a lower limit and an upper limit, which define the boundaries of a particular range. Ranges defined in this way can include or exclude endpoints and can be arbitrarily combined; that is, any lower limit can be combined with any upper limit to form a range. For example, if ranges of 60~120 and 80~110 are listed for a specific parameter, it is also expected that ranges of 60~110 and 80~120 are also included. Furthermore, if minimum range values ​​of 1 and 2 are listed, and if maximum range values ​​of 3, 4, and 5 are listed, then the following ranges are all expected: 1~3, 1~4, 1~5, 2~3, 2~4, and 2~5. In this application, unless otherwise stated, the numerical range "a~b" represents a shortened representation of any combination of real numbers between a and b, where a and b are real numbers. For example, the numerical range "0~5" indicates that all real numbers between "0~5" have been listed in this article; "0~5" is simply a shortened representation of these numerical combinations. Furthermore, when a parameter is stated as an integer ≥2, it is equivalent to disclosing that the parameter is, for example, an integer such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, etc.

[0024] Unless otherwise specified, all embodiments and optional embodiments of this application can be combined to form new technical solutions.

[0025] Unless otherwise specified, all steps of this application may be performed sequentially or randomly, preferably sequentially. For example, if the method includes steps (a) and (b), it means that the method may include steps (a) and (b) performed sequentially, or it may include steps (b) and (a) performed sequentially. For example, if the method may also include step (c), it means that step (c) may be added to the method in any order. For example, the method may include steps (a), (b), and (c), or it may include steps (a), (c), and (b), or it may include steps (c), (a), and (b), etc.

[0026] To make the above-mentioned objectives, features, and advantages of this application more apparent and understandable, the technical solution of this application is further described below in conjunction with the accompanying drawings and embodiments. However, this application is not limited to the listed embodiments, but should also include any other well-known modifications within the scope of the claims made in this application.

[0027] To better understand the technical solution of this application, a detailed description will be provided below in conjunction with the accompanying drawings and specific implementation methods.

[0028] In conventional techniques, although quaternized polysaccharides perform well in inhibiting fungal growth, their ability to penetrate mature fungal biofilms is limited when dealing with certain pathogenic fungi that easily form biofilms (such as Candida albicans), making it difficult to effectively act on the deep cells inside the biofilm.

[0029] This application provides an antifungal compound comprising: a quaternized polysaccharide derivative modified with a membrane-penetrating peptide. Because membrane-penetrating peptides possess unique cell-penetrating capabilities, they can effectively overcome the dense extracellular polymer barrier of fungal biofilms. Therefore, the antifungal compound of this application exhibits good penetration ability against mature fungal biofilms, solving the problem of quaternized polysaccharides being unable to enter the interior of biofilms. Furthermore, with the carrying effect of the membrane-penetrating peptide, the quaternized polysaccharide backbone can be delivered to the deep layers of the biofilm. Its high-density positive charge can electrostatically interact with the fungal cell membrane, disrupting the membrane structure integrity, thereby achieving highly efficient killing of fungal cells in a deeply encapsulated state.

[0030] The first embodiment of this application provides an antifungal compound comprising: a quaternized polysaccharide derivative modified with a membrane-penetrating peptide.

[0031] In one feasible embodiment, the membrane-penetrating peptide-modified quaternized polysaccharide derivative is formed by the membrane-penetrating peptide being coupled to the quaternized polysaccharide backbone via a covalent bond or linker arm.

[0032] Optionally, the membrane-penetrating peptide-modified quaternized polysaccharide derivative is a membrane-penetrating peptide-coupled quaternized polysaccharide derivative.

[0033] In one feasible embodiment, the polysaccharide includes: chitin (QC) and / or chitosan (QCS).

[0034] Optionally, chitin and chitosan, as natural amino polysaccharides, inherently possess certain antifungal activities. Chitosan's antifungal activity primarily stems from its free amino groups. Under slightly acidic conditions, these amino groups protonate to form positively charged ammonium ions, making them polycationic electrolytes. These positive charges can electrostatically adsorb onto negatively charged components on the fungal cell membrane surface (such as phospholipids, proteins, and teichoic acid), leading to cell membrane structural disorder, increased permeability, leakage of intracellular electrolytes and proteins, and ultimately, fungal cell death. Chitin, as an insoluble fiber or nanocrystal, can encapsulate fungal hyphae or spores through physical entanglement and adsorption, interfering with their uptake and attachment of nutrients, thereby inhibiting fungal growth and reproduction.

[0035] Optionally, quaternization modification of the chitin or chitosan backbone introduces hydrophilic quaternary ammonium groups, solving the solubility bottleneck of chitin and endowing it with strong polycationic properties; at the same time, it also enhances the persistence of the positive charge of chitosan. This makes quaternized chitin and quaternized chitosan high-performance materials that combine water solubility, strong positive charge, and highly efficient antifungal activity.

[0036] In one feasible embodiment, the membrane-penetrating peptide includes at least one of: octaarginine (R8), TAT (trans-transcription activator membrane-penetrating peptide), Penetratin, Transportan, MPG, Pep-1, pHLIP, and iRGD.

[0037] Optionally, fungal biofilms (such as Candida albicans biofilms) are dense three-dimensional structures composed of extracellular polymers with small pore sizes and negative charges, making them susceptible to interception by traditional antibacterial macromolecules on their surface. R8 and TAT are rich in arginine (guanidinium groups), which can form bidentate hydrogen bonds, resulting in strong but reversible binding with anionic components such as phosphate and sulfate groups in the cell membrane and extracellular polymers. This dynamic "binding-dissociation" process endows them with an internalization ability like a "shuttle," enabling them to actively carry quaternized polysaccharides across the dense barrier of the biofilm to the fungal cells deep within the biofilm. Furthermore, R8 and TAT can carry quaternized polysaccharides into the cell interior through endocytosis or direct membrane penetration. Once inside the cell, the positive charge of the quaternized polysaccharides can interfere with mitochondrial function and disrupt nucleic acid metabolism, thereby killing fungi from within.

[0038] For example, the general structural formulas of membrane-penetrating peptide-modified quaternized polysaccharide derivatives include: QC-R8, QCS-R8, QC-TAT, and QCS-TAT.

[0039] Optionally, the membrane-penetrating peptide and the quaternized polysaccharide are coupled via a coupling reaction to generate a membrane-penetrating peptide-modified quaternized polysaccharide derivative, wherein the coupling reaction includes any one of the following: click chemistry reaction, amidation reaction, Michael addition reaction, reductive amination reaction, and disulfide bond exchange reaction.

[0040] In one feasible embodiment, the quaternized polysaccharide and the membrane-penetrating peptide are respectively modified with complementary functional group pairs, which include any one of the following: azide and alkynyl; carboxyl and amino; maleimide and thiol; aldehyde and amino; thiol and thiol.

[0041] Optionally, if a membrane-penetrating peptide-modified quaternized polysaccharide derivative is generated by amidation reaction, then a carboxyl group needs to be modified on one of the membrane-penetrating peptide and the quaternized polysaccharide, while an amino group needs to be modified on the other.

[0042] Optionally, if a membrane-penetrating peptide-modified quaternized polysaccharide derivative is generated via Michael addition reaction, then maleimide groups need to be modified on one of the membrane-penetrating peptides and the quaternized polysaccharide, while thiol groups are modified on the other.

[0043] Optionally, if a membrane-penetrating peptide-modified quaternized polysaccharide derivative is generated by a reductive amination reaction, then an aldehyde group needs to be modified on one of the membrane-penetrating peptide and the quaternized polysaccharide, while an amino group needs to be modified on the other.

[0044] Optionally, if a transmembrane peptide-modified quaternized polysaccharide derivative is generated through a disulfide bond exchange reaction, then one of the transmembrane peptide and the quaternized polysaccharide needs to be modified with a thiol group, while the other is modified with a disulfide bond structure (thiol group).

[0045] Optionally, if the membrane-penetrating peptide and the quaternized polysaccharide are reacted through a click chemical reaction to generate a membrane-penetrating peptide-modified quaternized polysaccharide derivative, the quaternized polysaccharide can be an azide-quaternized polysaccharide, and the membrane-penetrating peptide can be a terminally alkyne-modified membrane-penetrating peptide.

[0046] Optionally, the terminally alkyne-terminated transmembrane peptides include R8-Pra and / or TAT-Pra.

[0047] Optionally, membrane-penetrating peptides contain multiple active groups such as amino and carboxyl groups. If other reaction methods are used, the membrane-penetrating peptides may randomly attach to the polysaccharide backbone at multiple sites, causing some of the membrane-penetrating peptides to lose their penetrating activity. Click chemistry (azide-alkynyl cycloaddition) is an orthogonal reaction; the alkynyl group only reacts with the azide group and does not react at all with the natural functional groups such as amino and guanidine groups on the membrane-penetrating peptide. Therefore, by using terminally alkyne-modified membrane-penetrating peptides, it is possible to ensure that the membrane-penetrating peptide is fixed in an "upright, outward" manner, maximizing the exposure of its active penetrating domain. At the same time, click chemistry can be carried out under physiological conditions (room temperature, aqueous phase, neutral pH), which can protect the tertiary structure and membrane-penetrating activity of the membrane-penetrating peptide and avoid peptide chain denaturation or inactivation that may occur in traditional organic synthesis.

[0048] Alternatively, the quaternized polysaccharide can be azidolated to enable click chemistry. By introducing an azido group first, the membrane-penetrating peptide (terminal alkyneation) can only undergo a click reaction with the azido group at its terminal. This "terminal-to-backbone" linkage ensures that each membrane-penetrating peptide molecule is fixed to the polysaccharide in a defined, outwardly extended conformation, maximizing its activity for penetrating biological membranes.

[0049] In one feasible embodiment, the degree of quaternization substitution of the quaternized polysaccharide is 0.2 to 1.2. For example, the degree of quaternization substitution of the quaternized polysaccharide is 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, etc. In this embodiment, by controlling the degree of quaternization substitution, an optimal balance is found between the solubility, bactericidal activity, linkage efficiency, and biosafety of the polysaccharide derivative, enabling the bactericidal polysaccharide and the penetrating peptide to synergistically enhance each other.

[0050] In one feasible embodiment, the weight-average molecular weight of the quaternized polysaccharide is 1 × 10⁻⁶. 3 ~8×10 5 g / mol; for example, the weight-average molecular weight of quaternized polysaccharides is 1 × 10⁻⁶ g / mol. 3 g / mol, 5×10 3 g / mol, 1×10 4 g / mol, 5×10 4 g / mol, 1×10 5 g / mol, 2×10 5 g / mol, 3×10 5 g / mol, 4×10 5 g / mol, 5×10 5 g / mol, 6×10 5 g / mol, 7×10 5 g / mol, 8×10 5g / mol, etc. Weight-average molecular weight determines the chain length and hydrodynamic volume of the polysaccharide backbone; in the final product of membrane-penetrating peptide modification, it mainly affects penetration ability, in vivo circulation time, and dosage form selection. If the weight-average molecular weight is too low, the efficiency of the membrane-penetrating peptide decreases; if the weight-average molecular weight is too high, the polysaccharide derivative faces severe steric hindrance when moving in the dense extracellular polymeric network, making it difficult to penetrate deep into the biological membrane even with the traction of the membrane-penetrating peptide.

[0051] In one feasible embodiment, the degree of deacetylation of the quaternized polysaccharide is 20-99%. For example, the degree of deacetylation of the quaternized polysaccharide is 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, etc. By maintaining the degree of deacetylation within a suitable range, on the one hand, a sufficient number of free amino groups on the polysaccharide backbone are ensured as active sites for subsequent quaternization reactions, thereby introducing sufficient quaternary ammonium groups to endow the polysaccharide derivative with a high density of positive charge and excellent water solubility, ensuring antifungal activity; on the other hand, a moderate degree of deacetylation retains an appropriate amount of acetylation units, allowing the polysaccharide derivative to still be recognized and gradually degraded by lysozyme in vivo, avoiding the risk of long-term retention caused by slow degradation due to excessively high deacetylation, and ultimately achieving a synergistic balance between highly efficient bactericidal activity and good biodegradability.

[0052] In one feasible embodiment, the grafting rate of the membrane-penetrating peptide is 0.1–0.6. For example, the grafting rate of the membrane-penetrating peptide is 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, etc. By controlling the grafting rate of the membrane-penetrating peptide, on the one hand, it ensures that a sufficient number of membrane-penetrating peptides synergistically act on the fungal biofilm to generate a highly efficient penetration driving force, enabling the quaternized polysaccharide backbone to successfully reach the deep layers of the biofilm; on the other hand, it avoids that an excessively high grafting rate would cause the membrane-penetrating peptides to become too crowded on the polysaccharide backbone, mutually obscuring the active domains or failing to fully extend due to steric hindrance, thereby preserving the independent penetration activity of each membrane-penetrating peptide molecule.

[0053] In one feasible embodiment, the molar ratio between the membrane-penetrating peptide and the quaternized polysaccharide is 0.5 to 5:1. For example, the molar ratio between the membrane-penetrating peptide and the quaternized polysaccharide is 0.5:1, 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1, etc. By maintaining the molar ratio between the membrane-penetrating peptide and the quaternized polysaccharide within a suitable range, it is ensured that a sufficient number of membrane-penetrating peptides are covalently linked to the polysaccharide backbone, enabling them to generate a synergistic penetration effect and efficiently drive the complex to cross the dense barrier of the fungal biofilm. On the other hand, it avoids excessive crowding of the polysaccharide backbone surface due to an excessively high proportion of membrane-penetrating peptides, preventing the membrane-penetrating peptide molecules from interfering with each other or losing activity due to insufficient extension caused by steric hindrance. At the same time, it also prevents unreacted free membrane-penetrating peptide residues from increasing potential toxicity and preparation costs.

[0054] Optionally, the molar ratio between the membrane-penetrating peptide and the quaternized polysaccharide is 1 to 3:1.

[0055] In this embodiment, the antifungal compound includes a quaternized polysaccharide derivative modified with a membrane-penetrating peptide. Because membrane-penetrating peptides possess unique cell-penetrating capabilities, they can effectively overcome the dense extracellular polymer barrier of fungal biofilms. Therefore, the antifungal compound of this embodiment exhibits good penetration ability against mature fungal biofilms, solving the problem of quaternized polysaccharides being unable to enter the biofilm. Furthermore, with the carrying capacity of the membrane-penetrating peptide, the quaternized polysaccharide backbone can be delivered to the deep layers of the biofilm. Its high-density positive charge can electrostatically interact with the fungal cell membrane, disrupting the membrane structure and thus achieving highly efficient killing of fungal cells in a deeply encapsulated state.

[0056] The second embodiment of this application provides a method for preparing an antifungal compound, used to prepare the antifungal compound as described above, referring to... Figure 1 The method includes the following steps: Step S10 provides quaternized polysaccharides and membrane-penetrating peptides.

[0057] Among them, the quaternized polysaccharides and membrane-penetrating peptides are modified with complementary functional groups.

[0058] In one feasible embodiment, a quaternized polysaccharide and a membrane-penetrating peptide are provided, wherein the quaternized polysaccharide and the membrane-penetrating peptide are respectively modified with complementary functional group pairs to achieve a coupling reaction between the two.

[0059] Optionally, the complementary functional group pair includes any one of the following: azide and alkynyl; carboxyl and amino; maleimide and thiol; aldehyde and amino; thiol and thiol.

[0060] Optionally, the membrane-penetrating peptide includes at least one of: octaarginine, TAT, Penetratin, Transportan, MPG, Pep-1, pHLIP, and iRGD.

[0061] Optionally, if the membrane-penetrating peptide-modified quaternized polysaccharide derivative is prepared by click chemistry, a terminally alkyne-modified membrane-penetrating peptide can be provided, and the quaternized polysaccharide can be azidated to obtain an azidated quaternized polysaccharide.

[0062] Optionally, the quaternized polysaccharide can be modified by azidation through a diazo transfer reaction to obtain azidated quaternized polysaccharide.

[0063] Alternatively, a diazo transfer reagent (such as imidazole-1-sulfonyl azide, trifluoromethanesulfonyl azide, etc.) can be used to react with the primary amino groups on the quaternized polysaccharide backbone to convert these amino groups into azide groups.

[0064] Step S20: The membrane-penetrating peptide is coupled to the quaternized polysaccharide backbone through a coupling reaction to obtain a membrane-penetrating peptide-modified quaternized polysaccharide derivative.

[0065] Step S30: Prepare antifungal compounds based on membrane-penetrating peptide-modified quaternized polysaccharide derivatives.

[0066] In one feasible embodiment, a membrane-penetrating peptide is coupled to a quaternized polysaccharide backbone via a coupling reaction to obtain a membrane-penetrating peptide-modified quaternized polysaccharide derivative, thereby preparing an antifungal compound based on the membrane-penetrating peptide-modified quaternized polysaccharide derivative.

[0067] Optionally, the coupling reaction includes any one of the following: click chemistry, amidation, Michael addition, reductive amination, and disulfide exchange reaction.

[0068] Optionally, the coupling chemical bonds in the membrane-penetrating peptide-modified quaternized polysaccharide derivatives include: single bonds, amide bonds (-CO-NH-), thioether bonds (-S-), disulfide bonds (-SS-), secondary amine bonds (-CH2-NH-), or linking groups containing 1,2,3-triazole rings.

[0069] Optionally, if the membrane-penetrating peptide and the quaternized polysaccharide are reacted through a click chemical reaction to generate a membrane-penetrating peptide-modified quaternized polysaccharide derivative, the quaternized polysaccharide can be an azide-quaternized polysaccharide, and the membrane-penetrating peptide can be a terminally alkyne-modified membrane-penetrating peptide.

[0070] Optionally, the click chemical reaction is a copper-catalyzed azido-alkynyl cycloaddition reaction; the reaction is carried out in a copper salt / reducing agent catalytic system, where the copper salt provides the catalyst source and the reducing agent is used to reduce divalent copper to monovalent copper in situ to initiate the catalytic cycle.

[0071] Optionally, to optimize reaction efficiency and product purity, the catalytic system may also include ligands (such as nitrogen-containing ligands) to stabilize monovalent copper and accelerate the reaction, as well as free radical or side reaction inhibitors to suppress by-products or oxidative degradation that may occur during the reaction.

[0072] Optionally, the click reaction is carried out under mild conditions, and the reaction system is an aqueous system or a mixture of water and organic solvents to ensure that the reactants are fully dissolved and to maintain the bioactivity of the transmembrane peptides.

[0073] In one feasible embodiment, the reaction temperature of the click chemical reaction is 0~60 ℃, for example, 0 ℃, 5 ℃, 10 ℃, 15 ℃, 20 ℃, 25 ℃, 30 ℃, 35 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃, etc.

[0074] In one feasible implementation, the reaction time of the click chemical reaction is 1 to 72 h, for example, 1 h, 5 h, 10 h, 20 h, 30 h, 40 h, 50 h, 60 h, 70 h, 72 h, etc.

[0075] Optionally, the product obtained from the coupling reaction is purified to obtain a membrane-penetrating peptide-modified quaternized polysaccharide derivative.

[0076] Optionally, the purification process includes at least one of complexation copper removal, dialysis, ultrafiltration, precipitation separation, washing, freeze drying, and spray drying.

[0077] Exemplarily, the steps for preparing octaargine-modified quaternized chitosan via amidation include: accurately weighing 1.0 g of quaternized chitosan and dissolving it in 50 mL of MES buffer (0.1 M, pH 5.5) to prepare a homogeneous solution with a concentration of 20 mg / mL. Subsequently, 0.15 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl) and 0.09 g of N-hydroxysuccinimide (NHS) are added sequentially to the solution, wherein the molar ratio between the amino group of the quaternized chitosan, EDC, and NHS is controlled at 1:2:2, and the mixture is activated at 25 °C with stirring in the dark for 2 h. After activation, a pre-prepared octaargine solution is slowly added dropwise, wherein the octaargine solution is prepared by dissolving 0.5 g of Fmoc-R8-COOH with an N-terminal Fmoc protecting group in 10 mL of deionized water; the pH of the reaction system is adjusted to 7.2 using 0.1 M NaOH solution. The reaction was carried out under constant temperature and magnetic stirring at 25°C for 24 h. After the reaction, a 20% (v / v) piperidine solution was added to the system, and the reaction was carried out at room temperature for 2 h to completely remove the Fmoc protecting group. The final reaction mixture was transferred to a dialysis bag with a molecular weight cutoff (MWCO) of 3500 Da, and dialyzed continuously in deionized water in the dark for 72 h, with the deionized water being replaced every 8 h to remove unreacted small molecules and inorganic salts. Finally, the dialysate was pre-frozen at -80°C for 12 h and then freeze-dried in a freeze dryer for 48 h to obtain pure octameric arginine-quaternized chitosan peptide polysaccharide modified with amide bond acid. It was a pale yellow flocculent solid with a yield of approximately 78%.

[0078] Exemplarily, the steps for preparing octaargine-modified quaternized chitosan via thiol-maleimide Michael addition include: accurately weighing 0.8 g of pre-synthesized maleimide-modified quaternized chitosan (Mal-QC, maleimide substitution degree approximately 15%), dissolving it in 40 mL of deoxyphosphate buffer (PBS, 0.1 M, pH 6.5), and purging with high-purity nitrogen for 30 minutes to prevent subsequent thiol oxidation. Separately, accurately weighing 0.2 g of cysteine-terminated octaargine (Cys-R8, containing free thiol groups as detected by Ellman's reagent) and dissolving it in 10 mL of the same deoxyphosphate buffer. Under nitrogen protection and at room temperature (25 °C), the Cys-R8 solution is slowly added dropwise to the Mal-QC solution using a constant-pressure dropping funnel, with the addition time controlled at 30 min; after the addition is complete, the system is kept at 25 °C and magnetically stirred for 6 h in the dark. Due to the high specificity and conversion rate of this reaction, the reaction solution was directly transferred to a dialysis bag with a molecular weight cutoff of 3500 Da after the reaction, and dialyzed with deionized water for 48 h. Subsequently, it was freeze-dried at -50 ℃ and a vacuum degree of less than 10 Pa for 36 h to finally obtain octameric arginine-modified quaternized chitosan peptide polysaccharide linked by thioether bonds, which was a white powder solid with a mass of about 0.85 g.

[0079] Exemplarily, the steps for preparing responsive octaargine-modified quaternized chitosan via disulfide bond oxidative crosslinking include: accurately weighing 1.0 g of thiolized quaternized chitosan and dissolving it in 50 mL of Tris-HCl buffer (0.05 M, pH 8.0) to prepare a homogeneous polymer solution. Subsequently, 0.3 g of cysteine-terminated octaargine (Cys-R8) is added to the system. After the short peptide is completely dissolved and mixed evenly, sterile air is continuously and slowly introduced into the reaction system at a flow rate of approximately 50 mL / min to provide a mild oxidative environment. The reaction is carried out under constant temperature water bath conditions of 30 °C with continuous magnetic stirring for 12 h, promoting oxidative crosslinking between the thiol groups of the QC-SH side chain and the thiol groups of the Cys-R8 terminal group to form stable disulfide bonds. After the reaction is completed, the pH of the system is neutralized to 7.0 using 0.1 M HCl solution to terminate the reaction. The solution was placed in a dialysis bag with a molecular weight cutoff of 3500 Da and dialyzed with deionized water at 4 °C in the dark for 72 h. After sterile filtration and freeze-drying, the dialysate yielded a disulfide-modified octameric arginine-quaternized chitosan peptide polysaccharide derivative with tumor microenvironment reduction responsiveness. It was a sponge-like solid with a mass of approximately 1.1 g.

[0080] In this embodiment, the antifungal compound is prepared from a quaternized polysaccharide derivative modified with a membrane-penetrating peptide. Because membrane-penetrating peptides possess unique cell-penetrating capabilities, they can effectively overcome the dense extracellular polymer barrier of fungal biofilms. Therefore, the antifungal compound of this embodiment exhibits good penetration ability against mature fungal biofilms, solving the problem of quaternized polysaccharides being unable to enter the interior of biofilms. Furthermore, with the carrying capacity of the membrane-penetrating peptide, the quaternized polysaccharide backbone can be delivered to the deep layers of the biofilm. Its high-density positive charge can electrostatically interact with the fungal cell membrane, disrupting the membrane structure and thus achieving highly efficient killing of fungal cells in a deeply encapsulated state.

[0081] The third embodiment of this application provides an antifungal product comprising the antifungal compound described above.

[0082] Optionally, antifungal products are used to inhibit fungal growth and / or kill fungi.

[0083] Alternatively, fungi include Candida albicans.

[0084] Optionally, antifungal products are used to inhibit fungal biofilm formation and / or remove mature fungal biofilms.

[0085] Optionally, antifungal products include at least one of the following: antifungal coatings, dressings, gels, lotions, sprays, membrane materials, granules, and compound formulations.

[0086] Compared with conventional technologies, the beneficial effects of the antifungal products provided in the embodiments of the present invention are the same as those of the antifungal compounds provided in the above embodiments, and other technical features of the antifungal products are the same as those disclosed in the methods of the above embodiments, and will not be repeated here.

[0087] In order to enable those skilled in the art to clearly understand the details and operations of the above embodiments of this application, and to demonstrate the significant improvement in performance of the embodiments of this application, the above technical solutions are illustrated below through multiple embodiments.

[0088] Example 1 A certain amount of quaternized polysaccharides, including quaternized chitosan (QCS) and quaternized chitin (QC), were weighed and dissolved in HCl solution. NaHCO3 was added, and the mixture was stirred vigorously for 30 min. Then, imidazole-1-sulfonyl azidohydrochloride and NaHCO3 were slowly added. CuSO4·5H2O was dissolved in water and methanol, and the resulting solution was added to the above reaction system. The mixture was stirred at room temperature for 24 h. After the reaction was completed, the reaction solution was placed in a dialysis bag with a molecular weight cutoff of 3500 Da and dialyzed in deionized water for more than one week until the dialysis was complete. The solution was then freeze-dried to obtain quaternized azidopolysaccharide intermediates, including quaternized chitosan azidopolysaccharide intermediate (QCS-N3) and quaternized chitin azidopolysaccharide intermediate (QC-N3). A certain amount of CuSO4·5H2O and sodium ascorbate were weighed and dissolved in deionized water. A certain amount of azido-quaternized polysaccharide intermediate and a certain amount of terminally alkydinated transmembrane peptides, including terminally alkydinated octameric arginine (R8-Pra), were added to the solution. THPTA (tris(3-hydroxypropyltriazolylmethyl)amine) ligand and aminoguanidine hydrochloride were added simultaneously, and the reaction was carried out at room temperature for 48 h. After the reaction was completed, MEDTA / Na solution was added to complex and remove copper, and stirring was continued for 1 h. The reaction solution was then placed in a dialysis bag with a molecular weight cutoff of 3500 Da and dialyzed in deionized water for more than one week until the dialysis was clean. The solution was then freeze-dried to obtain transmembrane peptide-modified azido-quaternized chitosan derivatives, including QC-R8 and QCS-R8.

[0089] The raw materials used in Example 1 above are shown in Tables 1 and 2 below: Table 1:

[0090] Table 2:

[0091] The molar ratios in Table 2 represent the reaction molar ratios of polysaccharide structural units and quaternizing reagents.

[0092] The transmembrane peptide-modified quaternized chitosan derivatives prepared by the method described in Example 1 above are shown in Table 3 below: Table 3:

[0093] The molar ratios in Table 3 represent the reaction molar ratios of the azido-quaternized polysaccharide intermediate and the terminally alkyne-modified transmembrane peptide.

[0094] The structural characterization of the samples involved in Example 1 was performed using Fourier transform infrared spectroscopy, and the results are as follows: Figure 2As shown, the horizontal axis represents wavenumber, and the vertical axis represents transmittance. The tested samples included CS3, QCS3, QCS3-N3, and QCS3-R8 (1:2). It can be seen that the infrared absorption characteristics of the samples changed after quaternization and transmembrane peptide grafting, indicating that the target derivative was successfully constructed.

[0095] Furthermore, the antifungal activity of the examples was evaluated using the minimum inhibitory concentration (MIC) method for Candida albicans, and the results are as follows: Figures 3-5 As shown, where, Figure 3 To assess the inhibitory effect of samples with different degrees of deacetylation on Candida albicans, the tested samples included: QC1, QCS3, QCS5, QC1-R8 (1:2), QCS3-R8 (1:2), and QCS5-R8 (1:2). Figure 3 It is known that derivatives with a high degree of deacetylation have superior antifungal activity. Figure 4 To assess the inhibitory effect of samples with different weight-average molecular weights on Candida albicans, the tested samples included: QCS5, QCS5... 2h QCS5 6h QCS5-R8(1:2), QCS5 2h -R8(1:2), QCS5 6h -R8(1:2), based on Figure 4 It can be seen that as the weight-average molecular weight of QCS decreases, the antifungal activity of QCS and its derivatives gradually weakens. Figure 5 The inhibitory effects of samples with different molar ratios on Candida albicans were evaluated, including: QCS5, QCS5-R8 (1:0.5), QCS5-R8 (1:1), and QCS5-R8 (1:2). Figure 5 It can be seen that as the molar ratio between QCS5-N3 and R8 increases from 1:0.5 to 1:2, the minimum inhibitory concentration of QCS5-R8 decreases from 100 μg / mL to 50 μg / mL, indicating that increasing the proportion of transmembrane peptide grafting can further enhance the antifungal activity of the material.

[0096] Furthermore, the ability of the above samples to inhibit the formation of Candida albicans biofilm was evaluated using the MTT assay. The tested samples included: QC1, QCS3, QCS5, and QCS6. 2h QCS5 6h The result is as follows Figure 6 As shown in the embodiments of this application, the inhibitory effect on the formation of Candida albicans biofilm gradually increases with the increase of the concentration of each sample, and the inhibitory effect of the high degree of deacetylation sample is better than that of the low degree of deacetylation or low molecular weight sample. The results of the ability of the samples modified with membrane-penetrating peptides to inhibit the formation of Candida albicans biofilm are as follows: Figure 7 As shown, the test samples include: QC1-R8(1:2), QCS3-R8(1:2), QCS5-R8(1:2), and QCS5. 2h -R8(1:0.5), QCS5 6h The biofilm inhibition capabilities of QC-R8 (1:2), QCS5-R8 (1:0.5), and QCS5-R8 (1:1) were further enhanced after modification with the membrane-penetrating peptide. Among them, QCS5-R8 (1:2) showed the best inhibitory effect, significantly inhibiting the formation of Candida albicans biofilm at 12.5 μg / mL.

[0097] Furthermore, in vitro cytotoxicity assays were performed on the above samples using cell viability testing. The experimental method included: culturing L929 cells in DMEM medium containing 10% FBS and 1% penicillin-streptomycin; sterilizing the samples with ultraviolet light; dissolving them in the medium; and diluting the cell suspension (1×10⁻⁶). 4 Cells (100 cells / well) were seeded into 96-well plates and cultured for 24 h. The culture medium was then discarded, and 100 μL of sample solutions of different concentrations were added, with sample-free culture medium serving as a control. After co-culturing for 24 h, the solution in each well was discarded, and 100 μL of culture medium containing 10% (v / v) CCK-8 was added. The cells were cultured at 37 ℃ for 2 h, and the absorbance of the solution was measured at 450 nm. Each group was repeated at least three times. Cell viability was calculated as follows: ; Wherein, A0, A1, and A2 are the absorbance values ​​of the blank group, the control group, and the experimental group, respectively.

[0098] Experimental results are as follows Figure 8 The figure shows the cell viability of L929 cells after incubation with samples of different concentrations. Figure 8 (a) The test samples include: QC1, QCS3, QCS5, QC1-R8(1:2), QCS3-R8(1:2), and QCS5-R8(1:2); Figure 8 (b) Test samples include: QCS5, QCS5 2h QCS5 6h QCS5-R8(1:2), QCS5 2h -R8(1:2), QCS5 6h -R8(1:2); Figure 8(c) Test samples included: QCS5, QCS5-R8 (1:0.5), QCS5-R8 (1:1), and QCS5-R8 (1:2). At all test concentrations (100–1000 μg / mL), QC1, QCS3, QCS5, and QCS5... 2h and QCS5 6h Both the samples and L929 cells showed high cell compatibility, with cell viability exceeding 80%. For the QCS-R8 derivative, cell viability remained at 50% even at a concentration of 1000 μg / mL. Combined with the corresponding MIC and MFC values ​​of the samples, this indicates that the QCS-R8 derivative exhibits good antibacterial activity within the HC50 range and within its cell-safe concentration range.

[0099] Furthermore, using the Calcein-AM / PI live / dead cell double staining kit combined with an inverted laser confocal microscope, the changes in cell membrane permeability of Candida albicans after treatment with QCS5 and QCS5-R8 were observed, respectively. Freshly cultured Candida albicans were collected and diluted in RPMI-MOPS buffered medium to a concentration of 1 × 10⁻⁶. 6 Working bacterial suspension at CFU / mL. 1 mL of bacterial suspension and 1 mL of sample solution were simultaneously added to a confocal culture dish and incubated at 37 °C for 24 h. 1 mL of RPMI-MOPS buffered medium was used as a control instead of 1 mL of sample solution. After incubation, the medium was discarded, the cells were washed with PBS, stained with Calcein-AM in the dark for 20 min, and then stained with PI for 5 min. After washing to remove excess dye, fluorescence distribution was observed using an inverted laser confocal microscope. Calcein-AM could freely penetrate the cell membrane of living cells, was hydrolyzed by intracellular esterases, and accumulated in large quantities within the cell, emitting green fluorescence. The non-membrane-permeable PI could only pass through damaged cell membranes and bind to nucleic acids, emitting red fluorescence. Results are as follows: Figure 9 As shown, the Candida albicans in the control group all exhibited green fluorescence. Most of the Candida albicans treated with QCS5 emitted red fluorescence, with the red fluorescence being more pronounced in the QCS5-R8 treatment group. This indicates that QCS5 and QCS5-R8 altered the permeability of the fungal cell membrane.

[0100] The above are merely preferred embodiments of this application and do not limit the patent scope of this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the patent protection scope of this application.

Claims

1. An antifungal compound, characterized in that, The antifungal compounds include: membrane-penetrating peptide-modified quaternized polysaccharide derivatives.

2. The antifungal compound according to claim 1, characterized in that, The membrane-penetrating peptide-modified quaternized polysaccharide derivative is formed by the membrane-penetrating peptide being coupled to the quaternized polysaccharide backbone via covalent bonds or linkers.

3. The antifungal compound according to claim 2, characterized in that, The polysaccharide includes: chitin and / or chitosan; And / or, the membrane-penetrating peptide comprises at least one of: octaarginine, TAT, Penetratin, Transportan, MPG, Pep-1, pHLIP, and iRGD.

4. The antifungal compound according to claim 2, characterized in that, The quaternized polysaccharide and the transmembrane peptide are respectively modified with complementary functional group pairs, wherein the complementary functional group pairs include any one of the following: Azide and alkynyl groups; Carboxyl and amino groups; Maleimide group and thiol group; Aldehyde and amino groups; Thiol group and thiol group.

5. The antifungal compound according to claim 2, characterized in that, The degree of quaternization of the quaternized polysaccharide is 0.2 to 1.2; and / or the quaternary ammonium modified polysaccharide has a weight average molecular weight of 1 x 10 3 ~ 8 x 10 5 g / mol; And / or, the degree of deacetylation of the quaternized polysaccharide is 20-99%.

6. The antifungal compound according to claim 2, characterized in that, The grafting rate of the membrane-penetrating peptide is 0.1–0.

6.

7. The antifungal compound according to claim 2, characterized in that, The molar ratio between the membrane-penetrating peptide and the quaternized polysaccharide is 0.5 to 5:

1.

8. A method for preparing an antifungal compound, characterized in that, The method is applied to the preparation of the antifungal compound as described in any one of claims 1 to 7, and the method comprises the following steps: A quaternized polysaccharide and a membrane-penetrating peptide are provided, wherein the quaternized polysaccharide and the membrane-penetrating peptide are respectively modified with complementary functional group pairs; The membrane-penetrating peptide is coupled to the quaternized polysaccharide backbone through a coupling reaction to obtain a membrane-penetrating peptide-modified quaternized polysaccharide derivative. Antifungal compounds were prepared based on the quaternized polysaccharide derivatives modified with the membrane-penetrating peptides.

9. The method as described in claim 8, characterized in that, The coupling reaction includes any one of the following: click chemistry, amidation, Michael addition, reductive amination, and disulfide exchange reaction.

10. An antifungal product, characterized in that, The antifungal product includes the antifungal compound as described in any one of claims 1 to 7, or the antifungal compound prepared by the method described in any one of claims 8 or 9.

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