A compound having a conjugated structure containing an alpha, beta-unsaturated aldehyde and applications thereof

Abstract

The application relates to the technical field of biological medicine, in particular to a compound with a conjugated structure containing an alpha, beta-unsaturated aldehyde and application thereof. The compound can covalently modify Sortase A enzyme of propionibacterium acnes through alpha, beta-unsaturated aldehyde, inhibit transpeptidation catalysis and adhesion of gram-positive bacteria (for example, methicillin-resistant staphylococcus aureus, streptococcus mutans and propionibacterium acnes), block anchoring of membrane proteins, inhibit growth of gram-positive bacteria or kill bacteria; conjugated groups with different electric effects and steric effects are screened to adjust the covalent modification efficiency of alpha, beta-unsaturated aldehyde and Sortase A enzyme, strengthen the activity of the compound in inhibiting Sortase A enzyme, thereby improve the antibacterial activity of the compound, and further make the compound with the conjugated structure containing the alpha, beta-unsaturated aldehyde in the application be applied to the fields of biological medicine and cosmetics as an antibacterial component.

Description

Technical Field

[0001] This application relates to the field of biomedical technology, and in particular to a compound with a conjugated structure containing α,β-unsaturated aldehydes and its applications. Background Technology

[0002] The widespread use and even abuse of antibiotics has led to a year-on-year increase in the detection rate of drug-resistant Gram-positive bacteria, including those resistant to macrolides, carbapenems, and tetracyclines, highlighting the growing problem of drug resistance in Gram-positive bacteria. Even more alarming is the emergence of multidrug-resistant bacteria such as methicillin-resistant Staphylococcus aureus, vancomycin-resistant Staphylococcus aureus, penicillin-resistant Streptococcus pneumoniae, linezolid-resistant Gram-positive bacteria, and daptomycin-resistant Gram-positive bacteria, posing numerous serious challenges to clinical infection control and treatment. Discovering potential new antibacterial targets and developing novel antibacterial drugs remain key to solving the treatment dilemma of drug-resistant Gram-positive bacteria.

[0003] Sortase A is a membrane-bound transpeptidase found on the surface of Gram-positive bacteria. It possesses membrane-bound thiol transpeptidase catalytic function and is primarily responsible for anchoring more than 20 proteins synthesized in the cytoplasm to the cell wall of Gram-positive bacteria. Sortase A plays a crucial role in bacterial survival and pathogenicity, and is a key virulence factor for Gram-positive pathogens, contributing to host cell adhesion, immune evasion, and the secretion of toxic proteins. Therefore, inhibiting Sortase A can prevent bacteria from attaching to specific tissues or organs, block the secretion of toxic proteins, avoid attacking host cells, and enhance the host's immune response. Furthermore, Sortase A is highly conserved in Gram-positive bacteria, and its localization on the cell membrane facilitates targeting. Using Sortase A as a target for anti-infection is an ideal target for developing antibacterial drugs to prevent the development of drug resistance.

[0004] Sortase A enzymes operate with a thiol group as their active site, cleaving between threonine and glycine residues to form an acylase intermediate. The active site, cysteine, forms a new bond with the carbonyl group of the target protein's threonine residue. Then, the free amino group of the linker intermediate acts as a nucleophile, attacking and decomposing it, thereby directly coupling the toxin to a specific site. Based on the biological principles of bacterial Sortase A enzymes, it is promising to explore the irreversible covalent modification of the active site of bacterial Sortase A enzymes with small molecule compounds to inhibit bacterial transpeptidase catalysis and adhesion, blocking the anchoring of various membrane proteins to achieve antibacterial effects. Researching covalently inhibiting drugs that suppress bacterial Sortase A enzymes may be an effective way to improve existing anti-Gram-positive bacterial infections and address bacterial drug resistance. Using small molecule compounds to covalently modify the surface of Propionibacterium acnes to inhibit the biological activity of Sortase A enzyme and control the over-colonization of Gram-positive bacteria is a promising approach. Based on this, discovering small molecule compounds that irreversibly covalently modify and inhibit Sortase A enzymes on bacterial surfaces may be an effective means to improve existing treatments for Gram-positive bacterial infections and an effective way to solve the dilemma of treating drug-resistant bacteria.

[0005] After more than a century of development, covalent inhibitors have been approved for use in a wide range of disease areas, including antibacterial, antiviral, antitumor, and antidiabetic applications. For example, covalent modification of penicillin to inhibit peptidopeptidase has yielded excellent antibacterial and anti-infective effects. Compared to non-covalent inhibitors, covalent inhibitors often possess unique advantages. They bind much more firmly to target proteins than their non-covalent counterparts, exhibiting higher affinity and a relatively longer duration of action. Therefore, they can effectively enhance the biological activity of drug molecules and address the problem of drug resistance.

[0006] Patent 201811631165.3 discloses the application of a food-grade plant-derived compound antibacterial agent, including cinnamaldehyde, eugenol and trans-o-methoxycinnamaldehyde, in cosmetics. However, the raw materials are limited to plant sources, and the structure, target of action, structure-activity relationship of the compounds are not explained.

[0007] Currently, there is an urgent need for a small molecule compound that can serve as a covalent inhibitor targeting Sortase A enzyme, covalently modifying Sortase A enzyme to inhibit bacterial bioactivity. Summary of the Invention

[0008] The purpose of this application is to overcome the shortcomings of the prior art and provide a compound containing α,β-unsaturated aldehydes with a conjugated structure and its applications. The compound of this application can covalently modify bacterial Sortase A enzyme with α,β-unsaturated aldehydes, inhibiting bacterial transpeptide catalysis and adhesion, blocking membrane protein anchoring, inhibiting bacterial growth or killing bacteria, and can be used as an antibacterial ingredient in the fields of biomedicine and cosmetics.

[0009] To achieve the above objectives, the technical solution adopted in this application is as follows:

[0010] This application provides a compound having a conjugated structure of α,β-unsaturated aldehyde, the compound comprising at least one of the compounds with structures shown in formulas (1) to (8):

[0011]

[0012] Each of R1-R8 is independently selected from at least one of substituted or unsubstituted H, C1-C8 alkyl, C1-C8 alkoxy, cyano, and halogen.

[0013] Preferably, the alkyl group in C1-C8 alkyl (alkyl with 1 to 8 carbon atoms) refers to a free radical of a saturated or unsaturated aliphatic group, including straight-chain alkyl, straight-chain alkenyl, straight-chain alkynyl, branched-chain alkyl, branched-chain alkenyl, branched-chain alkynyl, cycloalkyl, cycloalkenyl, and cycloalkynyl.

[0014] For example, C1-C8 alkyl groups include C1-C8 saturated alkyl groups, C2-C8 alkenyl groups, and C2-C8 alkynyl groups. More preferably, straight-chain alkyl groups with 1 to 6 carbon atoms, branched alkyl groups with 1 to 6 carbon atoms, cyclic alkyl groups with 3 to 6 carbon atoms, straight-chain alkenyl groups with 2 to 6 carbon atoms, branched alkenyl groups with 1 to 6 carbon atoms, and cyclic alkenyl groups with 3 to 6 carbon atoms are selected. Examples of alkyl groups include: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, cyclopentyl, and cyclohexyl. Examples of alkenyl groups include: vinyl, allyl, isopropenyl, pentenyl, cyclohexenyl, cycloheptenyl, and cyclooctenyl. Examples of alkynyl groups include: ethynyl, propynyl, isopropynyl, pentyynyl, and cyclohexynyl.

[0015] The term “substituted or unsubstituted C1-C8 alkyl” includes substituted C1-C8 alkyl and unsubstituted C1-C8 alkyl; wherein a substituted C1-C8 alkyl means that one or more substituents replace hydrogen on one or more carbons of an alkyl chain, and such substituents are selected from at least one of C1-C5 alkyl, C1-C3 alkoxy, carboxyl, sulfonic acid, and sulfonyl groups.

[0016] More preferably, alkoxy groups with 1 to 6 carbon atoms are selected as examples of alkoxy groups, specifically including: methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, isopentoxy, cyclopentoxy, and cyclohexyloxy.

[0017] The term “substituted or unsubstituted C1-C8 alkoxy” includes substituted C1-C8 alkoxy and unsubstituted C1-C8 alkoxy; wherein a substituted C1-C8 alkoxy means that one or more substituents replace hydrogen on one or more carbons of the alkoxy chain, and such substituents are selected from at least one of C1-C5 alkyl, C1-C3 alkoxy, carboxyl, sulfonic acid, and sulfonyl groups.

[0018] The compounds of the present application with the structures shown in the above general formulas (1) to (8) can covalently modify Sortase A enzyme to inhibit the biological activity of bacteria and can inhibit Gram-positive bacteria.

[0019] As a preferred embodiment of the α,β-unsaturated aldehyde compound having a conjugated structure described in this application, each of R1-R8 is independently selected from at least one of -CH3, -CH2CH3, -OCH3, -OCH2CH3, -CHCH2, -CCH, -CN, -C6H5, F, Cl, and Br.

[0020] Through extensive research and experimentation, the inventors of this application have discovered that the small molecule compound containing α,β-unsaturated aldehydes with a conjugated structure provided in this application irreversibly covalently modifies bacterial Sortase A enzyme, thereby inhibiting the growth of drug-resistant Gram-positive bacteria or killing bacteria. By screening for conjugated groups with different electronic and steric effects, the covalent modification rate of the "α,β-unsaturated aldehyde" warhead group of the covalent inhibitor on bacterial Sortase A enzyme is adjusted, enhancing the inhibition of bacterial Sortase A enzyme activity and controlling the colonization and growth of drug-resistant Gram-positive bacteria.

[0021] As a preferred embodiment of the α,β-unsaturated aldehyde compound having a conjugated structure described in this application, the compound comprises any one or more of the following structures:

[0022]

[0023] The α,β-unsaturated aldehyde compound with a conjugated structure described in this application is used as an inhibitor to covalently modify the Sortase A enzyme of Propionibacterium acnes or Streptococcus mutans, thereby inhibiting the biological activity of Gram-positive bacteria. Conjugated groups with different electronic and steric effects are screened to adjust the covalent modification efficiency of the α,β-unsaturated aldehyde with Sortase A enzyme, enhancing its inhibitory activity on Sortase A enzyme and thus improving the antibacterial activity of the compound.

[0024] As a preferred embodiment of the α,β-unsaturated aldehyde compound having a conjugated structure described in this application, the compound further includes a complex of 3-phenylpropenal, 3-(3-methoxyphenyl)propenal, 3-(o-methoxyphenyl)propenal and C10-C18 alkanes.

[0025] Preferably, the mass ratio of 3-phenylpropenal, 3-(3-methoxyphenyl)propenal, 3-(o-methoxyphenyl)propenal and C10-C18 alkanes is 60-80:2-10:2-10:10-20.

[0026] Another objective of this application is to provide the use of the above-mentioned α,β-unsaturated aldehyde compounds with conjugated structures in the preparation of covalent inhibitors targeting bacterial Sortase A enzyme.

[0027] Interaction analysis of the compound with bacterial sorting enzyme A molecule revealed that the α,β-unsaturated aldehyde component of the compound was modified onto the bacterial sorting enzyme A molecular chain through Michael addition reaction and acetal reaction to form a covalent compound.

[0028] In some specific embodiments, the compounds of this application also exhibit good inhibitory activity against other proteins with thiol groups as active sites.

[0029] The compounds in this application have advantages such as long interaction time with the target and strong efficacy, which provide new ideas and applications for drug development targeting protein targets with thiol groups as active centers, and have important drug research value.

[0030] Another object of this application is to provide the use of the above-mentioned α,β-unsaturated aldehyde compounds having conjugated structures in the preparation of reagents that inhibit the activity of bacterial Sortase A enzyme.

[0031] Another objective of this application is to provide an antibacterial drug comprising the above-mentioned compounds having a conjugated α,β-unsaturated aldehyde structure.

[0032] Another objective of this application is to provide the application of the above-mentioned α,β-unsaturated aldehyde compounds with conjugated structures as covalent inhibitors in the inhibition of Gram-positive bacteria.

[0033] The compounds with conjugated α,β-unsaturated aldehydes provided in this application exhibit good inhibitory activity against drug-resistant Gram-positive bacteria.

[0034] As a preferred embodiment of the application described in this application, the Gram-positive bacteria include at least one of methicillin-resistant Staphylococcus aureus, methicillin-resistant Staphylococcus epidermidis, Streptococcus mutans, Propionibacterium acnes, and Streptococcus agalactiae.

[0035] This application discloses a compound with a conjugated α,β-unsaturated aldehyde that covalently modifies the Sortase A enzyme of *Propionibacterium acnes* as an inhibitor, thereby inhibiting the bioactivity of *Propionibacterium acnes*. Conjugated groups with different electronic and steric effects are screened to regulate the covalent modification efficiency of the α,β-unsaturated aldehyde with Sortase A enzyme, enhancing its inhibitory activity on Sortase A enzyme and thus improving the antibacterial activity of the compound.

[0036] Another object of this application is to provide the use of the above-mentioned α,β-unsaturated aldehyde compounds having conjugated structures in the preparation of acne-removing cosmetic compositions.

[0037] Preferably, in the acne-removing cosmetic composition, the amount of the compound having a conjugated α,β-unsaturated aldehyde is at least 0.01%.

[0038] In some specific embodiments, the cosmetic includes one of gel, cream, and lotion. The cosmetic mentioned in this application can be a common form of cosmetic.

[0039] This application has been experimentally verified to show that applying the above-mentioned α,β-unsaturated aldehyde compound with a conjugated structure to an acne-removing cosmetic composition can effectively inhibit Propionibacterium acnes, thereby achieving the effect of removing acne.

[0040] In some specific embodiments, this application also provides an acne-removing cosmetic, which includes the above-mentioned compounds having a conjugated α,β-unsaturated aldehyde.

[0041] Compared with the prior art, this application has the following beneficial effects:

[0042] This application provides a compound with a conjugated structure containing α,β-unsaturated aldehydes and its applications. The compound can covalently modify the Sortase A enzyme of Gram-positive bacteria with α,β-unsaturated aldehydes, inhibiting bacterial transpeptidation and adhesion (e.g., methicillin-resistant Staphylococcus aureus, Streptococcus mutans, Propionibacterium acnes), blocking membrane protein anchoring, and inhibiting or killing the growth of Propionibacterium acnes or Streptococcus mutans. This application also screens conjugated groups with different electronic and steric effects to regulate the covalent modification efficiency of α,β-unsaturated aldehydes with Sortase A enzymes, enhancing their inhibitory activity on Sortase A enzymes, thereby improving the antibacterial activity of the compound. This allows the compound with a conjugated structure containing α,β-unsaturated aldehydes to be used as an antibacterial ingredient in the fields of biomedicine and cosmetics. Attached Figure Description

[0043] Figure 1 The high-resolution mass spectrum of the product of the reaction between covalent inhibitor (1) and cysteine;

[0044] Figure 2 LC-MS-MS image of covalent inhibitor (1) reacting with coenzyme A;

[0045] Figure 3 The high-resolution mass spectrum of hydrogen-deuterium exchange after the reaction of the covalent inhibitor (6) with Sortase A enzyme;

[0046] Figure 4 The results of DNA and RNA leakage induced by the covalent inhibitor (4) in methicillin-resistant Staphylococcus epidermidis;

[0047] Figure 5 The growth curve of Propionibacterium acnes inhibited by the covalent inhibitor (6);

[0048] Figure 6 Morphological analysis diagram of the covalent inhibitor (6) inhibiting Propionibacterium acnes;

[0049] Figure 7 The effect of covalent inhibitor (6) on intracellular oligosaccharide metabolism in Propionibacterium acnes. Detailed Implementation

[0050] To better illustrate the purpose, technical solution, and advantages of this application, the following description will be provided in conjunction with the accompanying drawings and specific embodiments.

[0051] In the following embodiments, unless otherwise specified, the experimental methods used are conventional methods, and the materials and reagents used are commercially available unless otherwise specified.

[0052] This application provides a compound with a conjugated structure containing α,β-unsaturated aldehydes, which can covalently bind to Sortase A enzyme and selectively inhibit the activity of bacterial Sortase A enzyme.

[0053] Example 1: A compound containing α,β-unsaturated aldehydes with a conjugated structure.

[0054] This embodiment involves the following compounds with conjugated structures containing α,β-unsaturated aldehydes, which are named covalent inhibitors (1) to (10), and specific information is shown in Table 1.

[0055] Table 1. Components of covalent inhibitors

[0056]

[0057]

[0058]

[0059] Example 2: Reaction of covalent inhibitors with thiol groups

[0060] A certain amount of covalent inhibitor (1) was dissolved in 10 times its mass of DMSO and then added to PBS solution for thorough dispersion. A certain amount of cysteine ​​was added, and the volume was adjusted to 1.0 mL with PBS solution. The final concentration of covalent inhibitor (1) was 150 μM, and the concentration of cysteine ​​was 80 μM. The mixture was then reacted in a shaker at 37 °C for 6 hours. After purification by precipitation and column chromatography, the mixture was dried under vacuum at 25 °C. After dissolution in DMSO, the mixture was detected by high-resolution mass spectrometry. The results are as follows: Figure 1 As shown in the figure. Analysis shows that the covalent inhibitor (1) reacts with two molecules of cysteine ​​in an acetal reaction, as shown in equation (I).

[0061]

[0062] A certain amount of covalent inhibitor (1) was dissolved in 10 times its mass of DMSO and then added to PBS solution for thorough dispersion. A certain amount of coenzyme A was added, and the volume was adjusted to 1.0 mL with PBS solution. The final concentration of covalent inhibitor (1) was 150 μM, and the concentration of cysteine ​​was 25.0 μM. The mixture was then incubated at 37°C in a shaker for 6 hours. After the reaction, the precipitate was collected by centrifugation with 100% acetonitrile, dissolved in DMSO, and analyzed by LC-MS-MS. The results are shown in [Figure number missing]. Figure 2 As shown in the figure. Analysis shows that the thiol group on coenzyme A undergoes a Michael addition reaction with the covalent inhibitor (1), as shown in equation (II).

[0063]

[0064] Example 3: Interaction analysis between covalent inhibitor and bacterial sorting enzyme A

[0065] The covalent inhibitor (1) was dissolved in DMSO and then dispersed thoroughly in PBS solution. An appropriate amount of the covalent inhibitor (1) solution and bacterial sorting enzyme A solution were added to a 1.5 mL EP tube, and the volume was adjusted to 100 μL with PBS solution. This resulted in a final covalent inhibitor (1) concentration of 60 μM and a bacterial sorting enzyme A concentration of 6 μM. The mixture was incubated at 37°C in a shaker for 6 hours, with DMSO of the same concentration used as a control. After the reaction, the reactants were placed in a 3000 Da ultrafiltration centrifuge tube, and ammonium acetate solution was added to a final volume of 500 μL. The tube was centrifuged at 14000 × g for 30 min at 4°C. After three centrifugations, the ultrafiltration tube was immediately inverted into another clean collection tube and centrifuged at 1000 × g for 2 min to obtain the sample. The sample was then subjected to high-resolution hydrogen-deuterium exchange mass spectrometry detection within 2 hours. The results are shown in [Figure 1]. Figure 3 The results showed two new protein detection peaks. One protein showed an increased molecular weight, which matched the molecular weight of the covalent inhibitor (1), while the other protein showed an increased molecular weight, which matched the molecular weight of the covalent inhibitor (1) minus one water molecule. This indicates that the covalent inhibitor (1) is modified onto the bacterial sorting enzyme A molecular chain via Michael addition or acetal reaction.

[0066] The covalent inhibitor (6) was dissolved in DMSO and then dispersed thoroughly in PBS solution. An appropriate amount of the covalent inhibitor (6) solution and bacterial Sortase A enzyme solution were added to a 1.5 mL EP tube, and the volume was adjusted to 100 μL with PBS solution. This resulted in a final covalent inhibitor (6) concentration of 60 μM and a bacterial Sortase A enzyme concentration of 6 μM. The mixture was incubated at 37°C in a shaker for 6 hours, with DMSO of the same concentration used as a control. After the reaction, the reactants were placed in a 3000 Da ultrafiltration centrifuge tube, and ammonium acetate solution was added to a final volume of 500 μL. The tube was centrifuged at 14000 × g for 30 min at 4°C, and centrifuged three times. Immediately after centrifugation, the ultrafiltration tube was inverted into another clean collection tube and centrifuged at 1000 × g for 2 min to obtain the sample. The sample was then subjected to high-resolution hydrogen-deuterium exchange mass spectrometry detection within 2 hours. The results are shown in [Figure 1]. Figure 3 The results showed the emergence of three new proteins with molecular weights of 19241.1016, 19259.2012, and 19278.5000, respectively, which were 114.2012, 132.3008, and 151.5996 larger than the molecular weights of sortase A (19126.9004). This indicates that the covalent inhibitor (6), containing α,β-unsaturated aldehydes, modifies the bacterial sortase A molecular chain through Michael addition and acetal reactions, forming a covalent compound.

[0067] Example 4: MIC determination and DNA / RNA leakage determination of covalent inhibitors against methicillin-resistant Staphylococcus epidermidis.

[0068] 1. MIC determination of covalent inhibitors against Streptococcus mutans

[0069] MIC determination was performed using the method recommended by the Clinical and Laboratory Standards Institute (CLSI, 2017). In a biosafety cabinet, methicillin-resistant Staphylococcus epidermidis (ATCC 35984) was picked from corresponding standard culture plates and inoculated into 5.0 mL of sterilized CAMHB liquid medium. The plates were then incubated at 37°C in a water bath at 180 rpm until the logarithmic growth phase. Covalent inhibitors (1)-(5), (10) were dissolved in 4 times their mass of DMSO and diluted halfway with CAMHB liquid medium to prepare drug solutions of different concentrations. 100 μL of drug was added to each well of a 96-well plate, with six technical replicates for each concentration. The logarithmic growth phase bacterial culture was adjusted to a concentration of 2.0 × 10⁻⁶ by adding an appropriate amount of sterilized CAMHB liquid medium. 8 CFU / mL was diluted 1000 times and 100 μL was added to a 96-well plate to make the final drug concentration 20-800 μg / mL. DMSO of the same concentration was used as a control. The plates were incubated at 37℃ for 24 h and observed. The drug concentration at which bacteria were not visible to the naked eye was the minimum inhibitory concentration. The results are shown in Table 2.

[0070] Table 2. MICs of covalent inhibitors against methicillin-resistant Staphylococcus epidermidis ATCC 35984

[0071] Serial Number name MIC value 1 Covalent inhibitors (1) 200.0 μg / mL 2 Covalent inhibitors (2) 230.0 μg / mL 3 Covalent inhibitors (3) 330.0 μg / mL 4 Covalent inhibitors (4) 220.0 μg / mL 5 Covalent inhibitors (5) 160.0 μg / mL 6 Covalent inhibitors (10) 220.0 μg / mL

[0072] 2. Determination of DNA and RNA leakage from methicillin-resistant Staphylococcus epidermidis by covalent inhibitors.

[0073] Inside the biosafety cabinet, methicillin-resistant Staphylococcus epidermidis (ATCC 35984) was picked from the corresponding standard culture plates and inoculated into 5.0 mL of sterilized CAMHB liquid medium. The plates were then incubated at 180 rpm in a 37°C water bath until the logarithmic growth phase. The covalent inhibitor (4) was dissolved in 4 times its mass of DMSO and diluted halfway with CAMHB liquid medium to prepare drug solutions of different concentrations. 200 μL of the drug was added to each well of a 12-well plate, with three technical replicates for each concentration. The logarithmic growth phase bacterial culture was adjusted to a concentration of 2.0 × 10⁻⁶ by adding an appropriate amount of sterilized CAMHB liquid medium. 8 CFU / mL was diluted 1000-fold and 200 μL was added to a 12-well plate to achieve a final drug concentration of 2MIC (400 μg / mL) and MIC (200 μg / mL). DMSO at the same concentration was used as a control. The plates were incubated at 37°C for 24 h. After centrifugation at 4°C and 4500 rpm for 10 min, the supernatant was collected, filtered through a 0.22 μm filter, and analyzed using a NanoDrop 2000. Results are shown below. Figure 4 As shown.

[0074] Example 5: MIC determination of covalent inhibitors against Streptococcus mutans

[0075] MIC determination was performed using the method recommended by the Clinical and Laboratory Standards Institute (CLSI, 2017). In a biosafety cabinet, *Streptococcus mutans* (ATCC 700610) strains from corresponding standard culture plates were picked and inoculated into 5.0 mL of sterilized BHI liquid medium. The plates were then incubated at 37°C in a water bath at 180 rpm until the logarithmic growth phase. Covalent inhibitors (1)-(5), (10) were dissolved in 4 times their mass of DMSO and diluted halfway with BHI liquid medium to prepare drug solutions of different concentrations. 100 μL of drug was added to each well of a 96-well plate, with six technical replicates for each concentration. The logarithmic growth phase bacterial culture was adjusted to a concentration of 2.0 × 10⁻⁶ by adding an appropriate amount of sterilized BHI liquid medium. 8 CFU / mL was diluted 1000 times and 100 μL was added to a 96-well plate to achieve a final drug concentration of 20–800 μg / mL. DMSO of the same concentration was used as a control. The plates were incubated at 37°C for 24 hours. The concentration at which bacteria were no longer visible to the naked eye was taken as the minimum inhibitory concentration (MIC). The results are shown in Table 3.

[0076] Table 3. MICs of covalent inhibitors against Streptococcus mutans ATCC 700610

[0077] Serial Number name MIC value 1 Covalent inhibitors (1) 220.0 μg / mL 2 Covalent inhibitors (2) 400.0 μg / mL 3 Covalent inhibitors (3) 500.0 μg / mL 4 Covalent inhibitors (4) 380.0 μg / mL 5 Covalent inhibitors (5) 180.0 μg / mL 6 Covalent inhibitors (10) 280.0 μg / mL

[0078] Example 6: MIC determination and biological effects of covalent inhibitors inhibiting Propionibacterium acnes

[0079] 1. MIC determination of covalent inhibitors inhibiting Propionibacterium acnes

[0080] The MIC was determined using the microbroth dilution method recommended by the Clinical and Laboratory Standards Institute (CLSI, 2017). Propionibacterium acnes strains were taken from a -80°C freezer, thawed at room temperature, and 150 μL of the strain solution was evenly spread onto Clostridium enrichment plates in a biosafety cabinet. The plates were then inverted and placed in an anaerobic incubator at 37°C with an anaerobic bag for 48 h. Covalent inhibitors (4) to (9) were dissolved in 4 times their mass of DMSO and diluted halfway with BHI liquid medium to different concentrations. 100 μL of the drug was added to each well of a 96-well plate, with six technical replicates for each concentration. Bacterial growth was scraped from the plates using a sterile swab moistened with BHI liquid medium, suspended in BHI liquid medium, and 100 μL of the bacterial solution was added to each well of a 96-well plate after adjusting the bacterial concentration. This resulted in a final drug concentration of 5–240 μg / mL and a bacterial concentration of approximately 1.0 × 10⁻⁶. 5CFU / mL, with 0.5% DMSO as a control. Anaerobic incubation at 37℃ for 60–72 h was performed. The lowest concentration at which bacteria were not visible to the naked eye was defined as the MIC value. Results are shown in Table 4.

[0081] Table 4. MICs of covalent inhibitors against Propionibacterium acnes ATCC 6919

[0082] Serial Number name MIC value 1 Covalent inhibitors (7) 248.0 μg / mL 2 Covalent inhibitors (8) 80.0 μg / mL 3 Covalent inhibitors (9) 150.0 μg / mL 4 Covalent inhibitors (4) 45.0 μg / mL 5 Covalent inhibitors (5) 20.0 μg / mL 6 Covalent inhibitors (6) 60.0 μg / mL

[0083] 2. Determination of the growth curve of covalent inhibitors inhibiting Propionibacterium acnes.

[0084] Referring to the literature (Kim et al., 2021), the growth curve of *Propionibacterium acnes* inhibited by the covalent inhibitor was determined. *Propionibacterium acnes* was inoculated into BHI liquid medium until the logarithmic growth phase. The covalent inhibitor (6) was dissolved in 4 times its mass of DMSO and diluted to a certain concentration with BHI liquid medium. DMSO of the same concentration was used as a control. The bacterial culture and covalent inhibitor (6) were added to a six-well plate, and the volume was increased to 2.5 mL with BHI liquid medium. The final bacterial concentration was approximately 1.0 × 10⁻⁶. 5 The concentrations of covalent inhibitor (5) were 2.5MIC (150 μg / mL), 2MIC (120 μg / mL), MIC (60 μg / mL), and 1 / 2MIC (30 μg / mL). The bacteria were anaerobic at 37℃. The absorbance at 600 nm was monitored over 5 days to measure the growth of *Propionibacterium acnes* in the control group and the covalent inhibitor (6) treatment group. The results are shown in […]. Figure 5 .

[0085] 3. Morphological analysis of covalent inhibitors inhibiting Propionibacterium acnes

[0086] Morphological analysis of the covalent inhibitor's inhibition of Propionibacterium acnes was performed according to the literature (Zhou et al., 2020), and the experimental conditions were adjusted appropriately. Propionibacterium acnes was inoculated into BHI liquid medium until the logarithmic growth phase. The covalent inhibitor (6) was dissolved in 4 times its mass of DMSO and then diluted with BHI liquid medium to a certain concentration. DMSO of the same concentration was used as a control. The bacterial culture and covalent inhibitor (6) were added to a 50.0 mL centrifuge tube, and the volume was adjusted to 40.0 mL with BHI liquid medium. The final bacterial concentration was approximately 1.0 × 10⁻⁶. 7CFU / mL, the concentrations of covalent inhibitor (6) were 8MIC (480 μg / mL), 4MIC (240 μg / mL), and 2MIC (120 μg / mL). The bacteria were anaerobically cultured at 37℃ with shaking at 180 rpm for 8 h. After centrifugation at 4℃ and 4500 rpm for 10 min, the bacteria were collected, washed three times with PBS, and incubated overnight at 4℃ with 2.5% glutaraldehyde solution. The bacteria were then washed with 0.1 M phosphate buffer, centrifuged at 4℃ and 4500 rpm for 10 min, and collected for later use.

[0087] (1) Scanning electron microscopy analysis: The prepared bacteria were dehydrated with ethanol at concentrations of 30%, 50%, 70%, 90%, and 100%. The dehydrated samples were then freeze-dried and sputtered with gold. Morphological changes of *Propionibacterium acnes* were observed under a SEM (SU8020, Hitachi, Japan). The results are shown in [Figure number missing]. Figure 6 .

[0088] (2) Scanning electron microscopy analysis: The prepared bacteria were fixed with 1% osmium tetroxide solution for 2 hours, washed three times with 0.1M phosphate buffer, and then dehydrated in a gradient of ethanol solutions (30, 50, 70, 80, 90, 95, 100%). The samples were then treated with pure acetone for 20 minutes. The samples were embedded overnight at 70°C in a mixture of embedding medium and acetone, and then sectioned using a LEICA EMUC7 ultramicrotome. The sections were stained with lead citrate solution and uranyl acetate 50% ethanol saturated solution for 5–10 minutes. After drying, the sections were observed under a transmission electron microscope (HITACHI H-7650). The results are shown in the figure below. Figure 6 .

[0089] 4. Analysis of the effects of covalent inhibitors on intracellular oligosaccharide metabolism in Propionibacterium acnes

[0090] Bacterial oligosaccharide metabolism was analyzed using a GC-MS-based strategy (Booth et al., 2015), with appropriate adjustments made. *Propionibacterium acnes* was inoculated into BHI liquid medium until the logarithmic growth phase. The covalent inhibitor (6) was dissolved in 4 times its mass of DMSO and then diluted to a specific concentration using BHI liquid medium. DMSO of the same concentration served as a control. The bacterial culture and covalent inhibitor (6) were added to a 50.0 mL centrifuge tube, and the volume was adjusted to 40.0 mL with BHI liquid medium, resulting in a final bacterial concentration of approximately 1.0 × 10⁻⁶. 7 CFU / mL, covalent inhibitor (6) concentration was MIC (60 μg / mL), anaerobic culture was performed at 37℃ with shaking at 180 rpm for 8 h. Bacteria were collected by centrifugation at 4℃, 4500 rpm for 10 min, and washed twice with PBS at 4℃. Bacteria were resuspended to OD. 600The value was 0.20. 20.0 mL of bacterial culture was centrifuged at 4500 rpm for 10 min to collect the bacteria. Then, 1.0 mL of pre-cooled methanol was added, and the metabolites were extracted three times using an ultrasonic cell disruptor on ice (50% power, 2 s supernatant, 3 s pause). After centrifugation at 4500 rpm for 10 min, the supernatant was collected, and ribitol internal standard was added. The metabolites were then concentrated in a vacuum drying oven. The metabolites from the control and MIC groups were subjected to MSTFA derivatization according to the methods reported in the literature (Chen j et al., 2020; Tang et al., 2021). Gas chromatography-mass spectrometry (GaC-MS) was performed within 48 h (Agilent 7890A GC equipped with an Agilent 5975CVL MSD detector) to analyze the changes in intracellular oligosaccharide metabolism of Propionibacterium acnes under the influence of covalent inhibitors. The results are shown in [Figure 1]. Figure 7 As shown.

[0091] Experimental Example 1: Application of Covalent Inhibitors in Acne Treatment Gels

[0092] According to the requirements of the "Cosmetic Safety Technical Specifications" and referring to the inclusion of cinnamon oil, p-hydroxyacetophenone, phenoxyethanol, and other raw materials in the "List of Used Cosmetic Raw Materials (2021 Edition)", the covalent inhibitors (1), (3), and (5) are added as acne-removing active ingredients to the acne gel. The formulation of the acne gel containing covalent inhibitors of this invention is shown in Table 5:

[0093] Table 5. Formulas of Acne Treatment Gels

[0094]

[0095]

[0096] Preparation process: Add item A to the aqueous phase pot, heat to 85℃, and disperse evenly; add item B to the oil phase pot, heat to 85℃, and disperse evenly; add item C to the mixing pot and disperse evenly. First, transfer item A to the emulsification pot, then transfer item B, and homogenize for 5 minutes. Maintain temperature to defoam. Cool to 45℃, add item C, and disperse evenly. After vacuum defoaming, unload and package.

[0097] Results: Evaluation of the acne-fighting efficacy of acne-fighting gels containing covalent inhibitors:

[0098] The acne-fighting efficacy of the above-mentioned acne-fighting gel formulation was evaluated according to the WS / T 650-2019 method for evaluating antibacterial and bacteriostatic effects. The acne-fighting gel was diluted halfway with BHI liquid culture medium to prepare solutions of different concentrations. 300 μL of each solution was added to a 1.5 mL sterile centrifuge tube. Bacterial growth was scraped from the agar plate using a sterile swab moistened with BHI liquid culture medium, and the bacterial suspension was resuspended in BHI liquid culture medium. After adjusting the bacterial concentration, 300 μL of the suspension was added to the centrifuge tube and dispersed evenly to achieve a final acne-fighting product concentration of 0.075–2.40% and a bacterial concentration of approximately 1.0 × 10⁻⁶. 5 CFU / mL. Take 200 μL of the mixed liquid and spread it evenly on Clostridium enrichment plates. Incubate anaerobicly at 37℃ for 60-72 h and observe the number of Propionibacterium acnes colonies. Six technical replicates were performed for each concentration. The results are shown in Table 6.

[0099] Table 6 shows the results of acne-removing gels containing covalent inhibitors inhibiting Propionibacterium acnes (+ indicates ≥5 colonies, - indicates ≤3 colonies).

[0100]

[0101] Experimental Example 2: Application of Covalent Inhibitors in Acne-Clearing and Moisturizing Face Cream

[0102] According to the requirements of the "Cosmetic Safety Technical Specifications" and referring to the inclusion of cinnamon oil, preservatives, fragrances, and other raw materials in the "List of Used Cosmetic Raw Materials (2021 Edition)", covalent inhibitors (7), (9), and (6) are added as acne-removing active ingredients to an anti-wrinkle and moisturizing face cream. The formula of the acne-removing and moisturizing face cream containing covalent inhibitors of this invention is shown in Table 7:

[0103] Table 7. Formulas for Acne-Clearing and Moisturizing Face Creams

[0104]

[0105] Preparation process: Add item A to the aqueous phase pot, disperse evenly, and heat to 85℃; add item B to the oil phase pot, heat to 85℃, and disperse evenly; add item C to the mixing pot, and disperse evenly. First, transfer item A to the emulsification pot, then transfer item B, and homogenize for 20 minutes. Cool to 45℃, add item C, and disperse evenly. After vacuum defoaming, unload and package.

[0106] Experimental Example 3: Application of Covalent Inhibitors in Acne Treatment Lotions

[0107] According to the requirements of the "Cosmetic Safety Technical Specifications" and referring to the inclusion of cinnamon oil, p-hydroxyacetophenone, azelaic acid, and other raw materials in the "List of Used Cosmetic Raw Materials (2021 Edition)", the covalent inhibitors (8), (4), and (6) are added as acne-removing active ingredients to an anti-wrinkle lotion. The formula of the acne-removing lotion containing covalent inhibitors of this invention is shown in Table 8:

[0108] Table 8. Formulas of Acne-Clearing Lotions Containing Covalent Inhibitors

[0109]

[0110]

[0111] Preparation process: Add item A to the aqueous phase pot, heat to 85℃, and disperse evenly; add item B to the oil phase pot, heat to 85℃, and disperse evenly; add item C to the mixing pot and disperse evenly. First, transfer item A to the emulsification pot, then transfer item B, and homogenize for 20 minutes. Cool to 45℃, add item C, and disperse evenly. After vacuum defoaming, unload and package.

[0112] Results: Evaluation of the acne-fighting efficacy of acne treatment products containing covalent inhibitors.

[0113] The acne-fighting efficacy of acne treatment products containing covalent inhibitors was evaluated according to the WS / T 650-2019 method for evaluating antibacterial and bacteriostatic effects. The acne treatment product was diluted halfway with BHI liquid medium to prepare solutions of different concentrations, and 300 μL of each solution was added to a 1.5 mL sterile centrifuge tube. Bacterial growth was scraped from the agar plate using a sterile swab moistened with BHI liquid medium, and the culture was resuspended in BHI liquid medium. After adjusting the bacterial concentration, 300 μL of the culture was added to the centrifuge tube and dispersed evenly to achieve a final acne treatment product concentration of 0.075–2.40% and a bacterial concentration of approximately 1.0 × 10⁻⁶. 5 CFU / mL. Take 200 μL of the mixed liquid and spread it evenly on Clostridium enrichment plates. Incubate anaerobicly at 37℃ for 72 h and observe the number of Propionibacterium acnes colonies. Six technical replicates were performed for each concentration. The results are shown in Table 9.

[0114] Table 9. Results of acne treatment products containing covalent inhibitors inhibiting Propionibacterium acnes (+ indicates ≥5 colonies, - indicates ≤3 colonies).

[0115]

[0116]

[0117] In summary, by adding the compound of this application to acne treatment products, the α,β-unsaturated aldehyde in the compound covalently modifies the Sortase A enzyme of Propionibacterium acnes, effectively inhibiting the growth of Propionibacterium acnes. This application also screens conjugated groups with different electronic and steric effects to regulate the covalent modification efficiency of α,β-unsaturated aldehyde and Sortase A enzyme, enhance its inhibitory activity on Sortase A enzyme, thereby improving the antibacterial activity of the compound. Consequently, the compound of this application with a conjugated structure containing α,β-unsaturated aldehyde can be used as an antibacterial ingredient in the fields of biomedicine and cosmetics.

[0118] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application and are not intended to limit the scope of protection of this application. Although this application has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of this application without departing from the substance and scope of the technical solutions of this application.

Claims

1. The application of a compound having a conjugated α,β-unsaturated aldehyde, characterized in that, The compound with the conjugated α,β-unsaturated aldehyde is used to prepare a covalent inhibitor targeting bacterial Sortase A enzyme, to prepare a reagent to inhibit bacterial Sortase A enzyme activity, to prepare an acne-removing cosmetic composition, or as a covalent inhibitor to inhibit Gram-positive bacteria. The compounds having a conjugated α,β-unsaturated aldehyde include any of the following structures: 。 2. The application according to claim 1, characterized in that, Covalent inhibitors targeting bacterial Sortase A enzyme, reagents for preparing agents that inhibit bacterial Sortase A enzyme activity, and covalent inhibitors for preparing acne-removing cosmetic compositions or inhibiting Gram-positive bacteria include complexes of 3-phenylpropenal, 3-(3-methoxyphenyl)propenal, 3-(o-methoxyphenyl)propenal, and C10-C18 alkanes.

3. The application according to claim 1, characterized in that, The Gram-positive bacteria include at least one of methicillin-resistant Staphylococcus aureus, methicillin-resistant Staphylococcus epidermidis, Streptococcus mutans, Propionibacterium acnes, and Streptococcus agalactiae.

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