Polylysine ester hydrochloride as well as preparation method and application thereof

Abstract

The invention discloses a polylysine ester hydrochloride and a preparation method and application thereof, and belongs to the field of chemical synthesis technology and biological application, the structural formula of the polylysine ester hydrochloride is as shown in # imgabs0 #, in the formula, R is selected from any one of alkyl groups with the carbon number smaller than 10, preferably methyl, ethyl, propyl, isopropyl, n-butyl or tertiary butyl; n ranges from 20 to 40; the preparation method of the polylysine ester hydrochloride provided by the invention is simple in steps and simple in subsequent treatment, and the prepared product polylysine ester hydrochloride has a certain antibacterial property and can be well applied to the fields of daily chemicals, foods, medicines, biology and the like.

Description

Technical Field

[0001] The present application specifically relates to a preparation method and application of polylysine ester hydrochloride, belonging to the fields of chemical synthesis technology and biological applications. Background Art

[0002] Polylysine ester hydrochloride is a derivative of amino acids, which is derived from the natural amino acid - lysine, and has excellent properties such as good film-forming property, solubilization effect, antioxidant property, and antibacterial property, and is widely used in the daily chemical and food fields. Further modification of polylysine hydrochloride to improve its efficacy and application scope in washing and care products is an important research direction at present. Polylysine hydrochloride has relatively high water solubility, and its oil solubility is increased by partial esterification to make it a new surfactant to expand its application in daily chemical products. At present, the industrial synthesis of esters in some washing and care products and cosmetics is carried out at high temperature using acid or base catalysts, which requires up to 150 - 240 °C. Such high-temperature conditions will produce inferior products (not suitable for skin application), which requires additional treatment and costs. The reaction temperature for the synthesis of polylysine ester hydrochloride is not high, and it shows excellent antibacterial properties against Staphylococcus aureus and Escherichia coli.

[0003] The preparation of polylysine ester hydrochloride belongs to the reaction of constructing ester bonds. In past research, H.C. Wancura et al. reported in The Canadian Journal of Chemical Engineering in 2019 that the soluble lipase of Thermomyces lanuginosus was used to catalyze the esterification of methanol. By changing the molar ratio of methanol to fatty acid and the lipase loading amount, the highest product yield could reach 85.1% at 35 °C. Zhang Xiaoqin mentioned in the Journal of Anqing Normal University in 1997 that under the protection of ethyl acetoacetate, dimethyl sulfoxide was used to catalyze the esterification reaction of amino acids with alkylating reagents, and high-yield amino acid esters without configuration conversion could be obtained by this method, but this experiment had disadvantages such as a long period and a complicated experimental process. The team of Chiara Salvitti et al. mentioned in Journal of the American Society for Mass Spectrometry in 2023 that the rapid evaporation of the H2O / CH3OH solvent mixture in the thin film formed by ESI microdroplet deposition accelerated the esterification reaction of amino acids catalyzed by H2SO4, and among them, L-tyrosine and L-phenylalanine with aromatic side chain substituents were the most reactive amino acids, and the absolute yield reached about 40 - 50%.

[0004] In 2014, the Kayo Terada team mentioned in ACS Polymers Au that hydrochloric acid was used as an acid catalyst. Hydrogen chloride not only served as an acid catalyst, but water also acted as a solvent that facilitated the solubility of lysine (Lys-OH), while the solubility of Lys-OH in EtOH was limited. The reaction formed an ester by using an excess of alcohol and removing the water produced from the system.

[0005] However, due to the relatively small proportion of hydrophilic groups in polylysine hydrochloride, its solubility in water is poor, and the esterification reaction is generally a reversible reaction. The esterification rate of the esterification reaction involving polylysine hydrochloride is not high. Therefore, it is necessary to find a green, economical, and controllable method for synthesizing polylysine ester hydrochloride. Summary of the Invention

[0006] Aiming at the problems in the prior art, the first aspect of the present invention aims to provide a polylysine ester hydrochloride.

[0007] The technical solution adopted by the present invention is as follows:

[0008] A polylysine ester hydrochloride, the structural formula of which is shown as follows

[0009]

[0010] In the formula:

[0011] The R is selected from any one of alkyl groups with less than 10 carbon atoms, preferably methyl, ethyl, propyl, isopropyl, n-butyl or tert-butyl; the n is between 20 and 40.

[0012] The polylysine ester hydrochloride has a molecular weight between 3500 and 6000.

[0013] The second aspect of the present invention aims to provide a simple and efficient method for preparing polylysine ester hydrochloride.

[0014] A method for preparing polylysine ester hydrochloride, using polylysine hydrochloride and alcohol as starting materials, p-toluenesulfonic acid as an acid catalyst, and synthesizing polylysine ester hydrochloride in the presence of a solvent;

[0015]

[0016] In the formula: The R is selected from any one of alkyl groups with less than 10 carbon atoms, preferably methyl, ethyl, propyl, isopropyl, n-butyl or tert-butyl; the n is between 20 and 40.

[0017] The further settings are as follows:

[0018] The alcohol in the reaction is preferably any one of methanol, ethanol, propanol, isopropanol, n-butanol, and tert-butanol.

[0019] The solvent for the reaction is preferably any one of methanol, ethanol, propanol, isopropanol, n-butanol, and tert-butanol.

[0020] The molar ratio of the reaction is polylysine hydrochloride: alcohol = 1:30 - 50.

[0021] The acid catalyst for the reaction is p-toluenesulfonic acid, and the molar amount of the acid catalyst is 0.5 - 1.5 times that of polylysine.

[0022] The temperature of the reaction is 30 - 50 °C.

[0023] The time of the reaction is 12 - 20 h.

[0024] The purification method of the product is that after the reaction is completed, first rotary evaporate to remove about 2 / 3 volume of the solvent, then precipitate with anhydrous ether to obtain the solid of the crude esterification product, and then wash the solid with distilled water, dilute hydrochloric acid, and ethanol in sequence, and dry to obtain a white solid.

[0025] The third aspect object of the present invention is to provide an application of the polylysine ester hydrochloride prepared above in drugs and daily chemical products, specifically in the preparation of drugs with antibacterial activity and in washing and care products.

[0026] The beneficial effects of the present invention are as follows:

[0027] 1. The present invention provides a preparation method of polylysine ester hydrochloride, which solves the problems in the prior art that when using the esterification method to prepare polylysine ester hydrochloride, the yield is low due to the small proportion of hydrophilic groups in polylysine hydrochloride resulting in poor solubility in alcohol, and the esterification reaction is generally a reversible reaction, resulting in incomplete reaction, low product yield, large interference of by-products on subsequent treatment, and many steps required for purification. The preparation method of the present invention has simple steps and simple subsequent treatment, and can obtain high-purity products.

[0028] 2. The present invention is carried out at a relatively low reaction temperature, with mild reaction conditions, avoiding the safety hazards brought by high-temperature reactions, being easy to control the reaction, and at the same time, the reaction operation process and post-treatment are simple.

[0029] 3. The product polylysine ester hydrochloride prepared by the present invention has certain antibacterial properties and can have good applications in the fields of daily chemicals, food, medicine, biology, etc.;

[0030] 4. The process provided by the present invention is simple and the conditions are mild, which is more suitable for industrial production, obtaining high-yield and high-purity products while reducing the preparation cost, showing better reaction advantages and economic advantages.

[0031] The following further illustrates the present invention in conjunction with the drawings and specific embodiments. Brief Description of the Drawings

[0032] Figure 1 1H NMR spectrum of polylysine methyl ester hydrochloride prepared in Example 1 of the present invention.

[0033] Figure 2 1H NMR spectrum of polylysine n-butyl ester hydrochloride prepared in Example 2 of the present invention.

[0034] Figure 3 Comparison chart of infrared spectra between polylysine methyl ester hydrochloride prepared in Example 1 of the present invention and raw material polylysine hydrochloride.

[0035] Figure 4 Inhibitory effect diagrams of polylysine n-butyl ester hydrochloride, polylysine methyl ester hydrochloride and raw material polylysine hydrochloride with a concentration of 20 mg / mL on Staphylococcus aureus.

[0036] Figure 5 Inhibitory effect diagrams of polylysine n-butyl ester hydrochloride, polylysine methyl ester hydrochloride and raw material polylysine hydrochloride with a concentration of 10 mg / mL on Staphylococcus aureus.

[0037] Figure 6 Inhibitory effect diagrams of polylysine n-butyl ester hydrochloride, polylysine methyl ester hydrochloride with different concentrations and raw material polylysine hydrochloride in the present invention on Escherichia coli.

[0038] Figures 4 - 6 Wherein: a: polylysine methyl ester hydrochloride at 10 mg / L; b: polylysine methyl ester hydrochloride at 20 mg / L; c: polylysine hydrochloride at 10 mg / L; d: polylysine hydrochloride at 20 mg / L; e: polylysine n-butyl ester hydrochloride at 10 mg / L; f: polylysine n-butyl ester hydrochloride at 20 mg / L.

[0039] Figure 7 Effect diagrams of different hair conditioners in the present invention on hair gloss (g is the comparison before and after treatment with hair conditioner I containing 3% by mass concentration of polylysine n-butyl ester hydrochloride in Group I; h is the comparison before and after treatment with hair conditioner II containing 3% by mass concentration of polylysine hydrochloride in Group II; i is the comparison before and after treatment with hair conditioner III as the blank control group in Group III).

[0040] Figure 8 Effect diagrams of different hair conditioners in the present invention on hair fluffiness (j is the comparison before and after treatment with hair conditioner I containing 3% by mass concentration of polylysine n-butyl ester hydrochloride in Group I; k is the comparison before and after treatment with hair conditioner II containing 3% by mass concentration of polylysine hydrochloride in Group II; l is the comparison before and after treatment with hair conditioner III as the blank control group in Group III). Detailed Description of the Invention

[0041] Combination Figures 1 - 8 Specific embodiments of the present invention will be described in detail below, but the claims of the present invention are not limited in any way. The raw materials and equipment involved in the embodiments are known products or commercially available products unless otherwise specifically described.

[0042] Example 1: Preparation of polylysine methyl ester hydrochloride

[0043] In a 100 mL three-necked flask, 24.27 mL (0.6 mol, 20 equivalents) of anhydrous methanol was added, and 5.481 g (30.00 mmol, 1 equivalent) of polylysine hydrochloride was added and dissolved by ultrasound. 7.747 g (45 mmol, 1.5 equivalents) of p-toluenesulfonic acid was slowly added dropwise to 24.3 mL of anhydrous methanol, and the mixture was stirred and reacted at 40 °C for 20 h. After the reaction was completed, about 2 / 3 of the solvent was first removed by rotary evaporation, and the crude esterification product solid was precipitated with 40 mL of anhydrous ether. Then the solid was washed successively with distilled water, 50 mL of 1 mol / L dilute hydrochloric acid, and 100 mL of ethanol, and dried to obtain 9.44 g of a white solid with an esterification rate of 84.8%.

[0044] Product confirmation:

[0045] The 1H NMR spectrum of the polylysine methyl ester hydrochloride prepared in Example 1 of the present invention is as shown in Figure 1 shown.

[0046] The comparison of the infrared spectra of the polylysine methyl ester hydrochloride prepared in Example 1 of the present invention and the raw material polylysine hydrochloride is as shown in Figure 3 shown: By comparing the infrared spectra, there are differences in the absorption peaks between the product and the polylysine raw material: a strong alkane C-H stretching vibration absorption peak appears at 3000 cm -1 ~2800 cm -1 ; the appearance of split peaks at 1380 cm -1 and 1350 cm -1 indicates the presence of methyl; the C=O stretching vibration absorption peak at 1640 cm -1 ; the appearance of doublets at 1116.4 cm -1 and 1020.1 cm -1 indicates the presence of an ester group. The formation of polylysine methyl ester hydrochloride is proved by the comparison of the NMR and infrared spectra.

[0047] Substitution example:

[0048] According to the preparation method of Example 1, the difference is that: the types of solvents, reaction temperature, reaction time, reaction feed ratio and other conditions are adjusted, and other conditions remain unchanged, and their effects on the reaction are tested respectively.

[0049] Table 1: Effect comparison table of different embodiment schemes

[0050]

[0051] As shown in Table 1, the types of solvents, reaction temperature, reaction time, and reaction feed ratio all have significant effects on the esterification rate of the reaction. Among them: the technical solution of Example 1 is the best, and the highest esterification rate is 84.8%.

[0052] Example 2: Preparation of polylysine n-butyl ester hydrochloride

[0053] In a 250 mL three-necked flask, 54.6 mL (0.6 mol, 20 equivalents) of anhydrous n-butanol was added, and 5.481 g (30.00 mmol, 1 equivalent) of polylysine hydrochloride was added for ultrasonic dissolution. 7.749 g (45 mmol, 1.5 equivalents) of p-toluenesulfonic acid was slowly added dropwise to 54.6 mL of anhydrous n-butanol, stirred, and reacted at 40 °C for 20 h. After the reaction was completed, about 2 / 3 of the volume of the solvent was first removed by rotary evaporation, and then precipitated with 40 mL of anhydrous ether to obtain a solid of crude esterification product. The solid was then washed successively with distilled water, 50 mL of 1 mol / L dilute hydrochloric acid, and 100 mL of ethanol, and dried to obtain 4.806 g of a white solid with an esterification rate of 83.3%.

[0054] Product confirmation:

[0055] The 1H NMR spectrum of the polylysine n-butyl ester hydrochloride prepared in Example 2 of the present invention is as Figure 2 shown.

[0056] Application Example 1

[0057] The antibacterial properties of the polylysine methyl ester hydrochloride prepared in Example 1 and the polylysine n-butyl ester hydrochloride prepared in Example 2 were tested against Staphylococcus aureus and Escherichia coli, respectively.

[0058] The test method is as follows:

[0059] 1. Preparation of antibacterial material samples for testing

[0060] Accurately weigh the samples and dilute them to 10 mg / mL and 20 mg / mL, and test their inhibitory effects on Staphylococcus aureus and Escherichia coli, respectively.

[0061] 2. Preparation of bacterial suspension

[0062] Take each test strain, inoculate it on a fresh slant medium, incubate at 35 °C for 16 - 20 hours, elute the bacterial lawn with an appropriate amount of nutrient broth medium, compare the turbidity with a No. 0.5 McFarland turbidimeter, and dilute appropriately to a concentration of about 5×10 6 CFU / mL for standby.

[0063] 3. Determination of antibacterial rate

[0064] 3.1 Inoculation

[0065] After turning on the ultra-clean bench fan for 30 minutes, perform aseptic operation. Aspirate 100 μL (5×10 6 CFU / mL) of each test bacterial solution and add it to a sterile test tube. Separately add 0.5 mL of bacteriostatic material dilutions with concentrations of 10 mg / mL and 20 mg / mL, and count after 4 hours of action.

[0066] 3.2 Counting and Culturing

[0067] Perform aseptic operation. Separately aspirate 1 mL of each dilution-level bacterial solution and add it to a sterile petri dish. Take the nutrient agar medium that has been melted and kept at a constant temperature of 46°C, add 15 mL to each petri dish and mix well. Cover the dish and wait for it to solidify. After solidification, invert the petri dish and culture it in a constant-temperature incubator at 37°C (Staphylococcus aureus and Escherichia coli are cultured at 36 ± 1°C) overnight. After culturing, calculate the number of bacterial colonies in each dilution-level petri dish. Dip a sterile cotton swab into the bacterial solution and rotate it on the tube wall to squeeze out the excess bacterial solution. Among them, the solvent DMSO is used as a blank control.

[0068] Use a cotton swab to evenly spread and inoculate 1 mL of each dilution-level bacterial solution on the surface of 15 mL of solid nutrient agar medium in the petri dish 3 times. Rotate the petri dish 60° each time to ensure that the bacterial solution evenly covers the entire agar surface. Finally, spread it along the edge of the petri dish for one week to remove possible residual bacterial solution accumulation. Place the petri dish with the applied medicine at room temperature for 5 minutes to allow the bacterial solution to fully adsorb on the agar surface. Then stick the filter paper soaked with the medicine on the agar surface that has been inoculated with the test bacteria. The filter paper is evenly stuck on the agar surface, and the distance between the papers should be no less than 24 mm, and the distance from the paper to the edge of the petri dish should be no less than 15 mm. After spreading, culture it in a constant-temperature incubator at 37°C for 18 h, and then observe the size of the inhibition zone.

[0069] 4. Data Processing

[0070] Bacteriostatic rate = ((D1(mm) - D0(mm)) / D1(mm)) * 100% (where: D1 is the diameter of the inhibition zone in the blank group = 3.5 mm, D0 is the diameter of the inhibition zone of the sample)

[0071] The test results of antibacterial performance are shown in Table 2 as follows:

[0072] Table 2: Bacteriostatic Test Effects of Raw Materials and Products

[0073]

[0074] As shown in Table 2:

[0075] The raw material polylysine hydrochloride and the products polylysine methyl ester hydrochloride and polylysine butyl ester hydrochloride all have certain antibacterial abilities. The antibacterial abilities of the products polylysine methyl ester hydrochloride and polylysine butyl ester hydrochloride against Escherichia coli are stronger than those against Staphylococcus aureus. Their inhibitory effects on both increase with the increase in concentration, and the antibacterial effects are not lower than those of the polylysine hydrochloride raw material.

[0076] Application Example 2

[0077] (1) HLB value determination

[0078] The polylysine methyl ester hydrochloride prepared in Example 1, the polylysine butyl ester hydrochloride prepared in Example 2, and the raw material polylysine hydrochloride were respectively subjected to HLB value determination:

[0079] The polylysine methyl ester hydrochloride prepared in Example 1, the polylysine butyl ester hydrochloride prepared in Example 2, and the raw material polylysine hydrochloride were respectively configured into aqueous solutions with a mass fraction of 1%. Then, they were placed in large test tubes and slowly heated with stirring in a glycerol bath. When the transparency of the solution decreased and became turbid, the temperature in the test tube was the cloud point of the surfactant. Then, their HLB values were calculated respectively. Through calculation, the HLB value of polylysine hydrochloride was approximately 17.54, while the HLB value of the polylysine methyl ester hydrochloride prepared in Example 1 was approximately 5.39, and the HLB value of the polylysine butyl ester hydrochloride prepared in Example 2 was approximately 4.74. Thus, it can be seen that the HLB values of the polylysine methyl ester (butyl ester) hydrochloride prepared in the present invention are in the water-in-oil (W / O) type and can be added as surfactants to hair care products.

[0080] (2) Application of polylysine butyl ester hydrochloride in shampoo

[0081] The product polylysine butyl ester hydrochloride prepared in Example 2 and the raw material polylysine hydrochloride were formulated into solutions I, II, and III (blank control) according to the requirements of QB / T1975-2013 "Hair Conditioner", and relevant performance tests were carried out:

[0082] Table 3: Hair conditioner formula table

[0083]

[0084]

[0085] The test method is as follows:

[0086] 1. Cleaning and Pretreatment: Wash three hair bundles twice with a 5% sodium dodecyl sulfate solution by mass to remove surface impurities. Then place them in a constant temperature (25°C) and constant humidity (80%) room to dry, and divide them into three groups, numbered I, II, and III respectively. Wash the three groups of hair bundles numbered I, II, and III twice with the above-prepared hair conditioners I, II, and III solutions respectively, and then place them in a constant temperature (25°C) and constant humidity (80%) room to dry.

[0087] 2. Initial Evaluation: Conduct blank tests on the gloss, fluffiness, and handle feel of the three groups of hair numbered I, II, and III. Take photos to record the gloss and fluffiness of the three groups of hair after drying, and invite 10 non-experimental personnel to observe the taken photos and conduct a touch test on the three groups of hair bundles, and score their gloss, fluffiness, and handle feel. The score range is 0 - 10 points, and take the average value (the higher the performance, the higher the score).

[0088] 3. Cleaning and Drying: Immerse the three groups of hair bundles processed in step 1 in deionized water at 30°C for about 1 minute, wash them in a cycle 3 times, and then squeeze out the excess water by hand. Repeat this step 2 times. Finally, dry the cleaned hair bundles under constant temperature and humidity conditions overnight.

[0089] 4. Post-treatment Evaluation: Take photos of the three groups of hair bundles processed in step 3 to record their gloss, and conduct a frizzy test on them (i.e., use the same force to comb the hair bundles 10 times and then take photos to record their fluffiness). Again, invite 10 non-experimental personnel to observe the photos taken in this step and conduct a touch test on them, and score the gloss, fluffiness, and handle feel of the three groups of hair bundles. The score range is 0 - 10 points, and take the average value. (The higher the performance, the higher the score)

[0090] 5. Data Processing: List the scores corresponding to the three indicators as follows:

[0091] Table 4: Scores of the gloss, fluffiness, and handle feel of the hair after sample treatment

[0092]

[0093] As Figure 7 、 Figure 8 and shown in Table 4:

[0094] Three groups of data show that in terms of glossiness and handfeel, the score change values of sample group Ⅰ (hair treated with conditioner Ⅰ), sample group Ⅱ (hair treated with conditioner Ⅱ) and blank control group Ⅲ (hair treated with conditioner Ⅲ) decrease in turn. This indicates that both conditioner Ⅰ containing polylysine n-butyl ester hydrochloride and conditioner Ⅱ containing polylysine hydrochloride can improve the touch of hair bundles and make the hair look more shiny, and the former shows stronger performance in these two aspects. In terms of fluffiness, the opposite effect appears, indicating that conditioner Ⅰ with a formulation containing polylysine n-butyl ester hydrochloride can better maintain the fluffiness of hair bundles and reduce frizz.

[0095] Summary:

[0096] The present invention provides a preparation method of polylysine ester hydrochloride, which has the advantages of mild conditions and easy scale-up production, solves the problems of low yield and many steps required for purification in the prior art when preparing polylysine ester hydrochloride by an esterification method. The preparation method of the present invention has simple steps and simple subsequent treatment. According to the requirements of actual applications, products with different esterification rates can be obtained by adjusting the reaction conditions. And because polylysine ester hydrochloride has excellent antibacterial properties, it can be well used in the field of surfactants and in the fields of medicine and biological applications.

Claims

1. A polylysine ester hydrochloride, the structural formula of which is as follows: Where: The R is selected from any one of the alkyl groups with less than 10 carbon atoms, and the n is between 20 and 40.

2. A polylysine ester hydrochloride according to claim 1, characterized in that: The R is selected from methyl, ethyl, propyl, isopropyl, n-butyl or tert-butyl.

3. A polylysine ester hydrochloride according to claim 1, characterized in that: The molecular weight of the polylysine ester hydrochloride is between 3500 and 6000.

4. A method for preparing the polylysine ester hydrochloride according to claim 1, characterized in that: Using polylysine hydrochloride and alcohol as starting materials and p-toluenesulfonic acid as acid catalyst, polylysine ester hydrochloride is synthesized in the presence of a solvent. In the formula: R is selected from any one of the alkyl groups with less than 10 carbon atoms, and n is between 20 and 40.

5. The method for preparing a polylysine ester hydrochloride according to claim 4, characterized in that: The alcohol in the reaction is selected from any one of methanol, ethanol, propanol, isopropanol, n-butanol and tert-butanol.

6. The method for preparing polylysine ester hydrochloride according to claim 4, characterized in that: The solvent of the reaction is selected from any one of methanol, ethanol, propanol, isopropanol, n-butanol and tert-butanol.

7. The method for preparing polylysine ester hydrochloride according to claim 4, characterized in that: The molar ratio of the reaction is polylysine hydrochloride:alcohol=1:30-50.

8. The method for preparing polylysine ester hydrochloride according to claim 4, characterized in that: The acid catalyst of the reaction is p-toluenesulfonic acid, and the molar amount of the acid catalyst is 0.5-1.5 times that of polylysine.

9. The method for preparing polylysine ester hydrochloride according to claim 4, characterized in that: The reaction temperature is 30-50°C, and the reaction time is 12-20h.

10. Use of the polylysine ester hydrochloride of claim 1 in the preparation of antimicrobial active drugs and cleaning products.

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