Difunctional chitosan oligosaccharide derivative based on phenylboronic acid and geraniol as well as preparation method and application of difunctional chitosan oligosaccharide derivative

Abstract

The invention discloses a bifunctional chitosan oligosaccharide derivative based on phenylboronic acid and geraniol as well as a preparation method and application of the bifunctional chitosan oligosaccharide derivative. Comprising the following steps: successfully covalently linking geraniol molecules to a chitosan oligosaccharide skeleton by adopting a bromide-mediated grafting technology, so that the water solubility of geraniol is remarkably improved. Through EDC / NHS mediated amidation reaction, a phenylboronic acid group is introduced into the geraniol-chitosan oligosaccharide derivative, and a novel derivative with dual functions is constructed. The derivative not only maintains good water solubility of chitosan oligosaccharide, but also significantly enhances antibacterial activity. Compared with geraniol-chitosan oligosaccharide derivatives, the phenylboronic acid functional modification enables the product to show excellent targeted recognition capability on bacteria, and efficient bacteriostasis can be realized at low concentration. The preparation method is simple and convenient in process, the raw materials are easy to obtain, the product is easy to purify, and a new idea is provided for functional modification of chitosan oligosaccharide. The multifunctional derivative has a wide application prospect in the fields of food preservation, medical antibiosis and the like, and has an important industrialization value.

Description

A bifunctional chitosan oligosaccharide derivative based on phenylboronic acid and geraniol, its preparation method and application Technical Field

[0001] This invention belongs to the field of bioactive substance modification technology, specifically relating to a bifunctional chitosan oligosaccharide derivative based on phenylboronic acid and geraniol, its preparation method and application. Background Technology

[0002] With the rapid development of the food industry, food preservation technology is facing significant challenges. Currently, traditional chemical preservatives are no longer sufficient to meet modern food safety requirements due to their potential health risks, making the development of novel natural preservatives imperative. Chitosan oligosaccharides, derived from natural chitin, have attracted widespread attention in the food preservation field as an environmentally friendly and safe biomaterial due to their good biocompatibility, biodegradability, and inherent broad-spectrum antibacterial properties. However, the antibacterial mechanism of natural chitosan oligosaccharides mainly relies on electrostatic interactions with microbial cell membranes, resulting in limited antibacterial efficacy and a lack of targeting, which greatly restricts their practical application.

[0003] To overcome these limitations, researchers have actively pursued chemical modifications of chitosan oligosaccharides. In existing technologies, the introduction of active groups such as geraniol to modify chitosan oligosaccharides has improved their water solubility and antibacterial activity to some extent. However, this antibacterial effect remains unsatisfactory. While increasing the concentration can enhance the antibacterial effect, the resulting biosafety concerns cannot be ignored. Therefore, improving antibacterial capabilities in a simple and safe manner is particularly important, and enhancing the bacterial targeting properties of antibacterial materials has become a significant research direction.

[0004] Phenylated acid, a typical boric acid derivative, has demonstrated unique application value in the control of foodborne pathogens. Studies have shown that phenylboronic acid can act as a molecular-level "targeting lens," its unique boric acid groups precisely recognizing the cis-diol structure in bacterial cell wall peptidoglycan / teichoic acid and forming a specific covalent bond with it. This "lock-and-strike" molecular recognition mechanism significantly enhances the targeting of ordinary antibacterial agents against pathogens, thereby precisely inhibiting foodborne pathogens such as Staphylococcus aureus and Escherichia coli through multiple pathways, including disrupting cell wall integrity, interfering with transmembrane transport, and inhibiting biofilm formation. Compared to ordinary antibacterial agents, phenylboronic acid's "guided" selective binding ability not only significantly improves antibacterial efficiency but also exhibits excellent biocompatibility due to its low affinity for mammalian cells. However, such "molecular targeting lenses" still face limitations in practical applications, such as limited water solubility and a narrow spectrum of action. Therefore, the development of a new generation of highly precise food antibacterial agents is urgently needed. Summary of the Invention

[0005] The purpose of this section is to outline some aspects of embodiments of the present invention and to briefly describe some preferred embodiments. Simplifications or omissions may be made in this section, as well as in the abstract and title of this application, to avoid obscuring the purpose of these documents; however, such simplifications or omissions should not be construed as limiting the scope of the invention.

[0006] In view of the problems existing in the above and / or prior art, the present invention is proposed.

[0007] Therefore, the purpose of this invention is to overcome the shortcomings of the prior art and provide a bifunctional chitosan oligosaccharide derivative based on phenylboronic acid and geraniol, its preparation method and application.

[0008] To solve the above-mentioned technical problems, the present invention provides the following technical solution: the bifunctional chitosan oligosaccharide derivative based on phenylboronic acid and geraniol has the chemical structural formula shown in Formula A. Wherein, R is H or geraniol, and the structural formula of the geraniol is shown in Formula B: When R is geranyl, it is connected to formula A at position X of formula B via an oxygen atom or a nitrogen atom.

[0009] Another objective of this invention is to overcome the shortcomings of the prior art and provide a method for preparing a bifunctional chitosan oligosaccharide derivative based on phenylboronic acid and geraniol.

[0010] To solve the above-mentioned technical problems, the present invention provides the following technical solution: 4-carboxyphenylboronic acid is dissolved in an organic solvent, an activator is added and stirred to obtain a first solution; geraniol-chitosan oligosaccharide derivative is dissolved in deionized water to obtain a second solution; the first solution is added dropwise to the second solution, reacted at room temperature in the dark, dialyzed, filtered, and freeze-dried to obtain phenylboronic acid-functionalized geraniol-chitosan oligosaccharide derivative.

[0011] In a preferred embodiment of the preparation method described in this invention, the molar ratio of 4-carboxyphenylboronic acid to the activator is 1:1.2.

[0012] In a preferred embodiment of the preparation method described in this invention, the organic solvent is methanol.

[0013] In a preferred embodiment of the preparation method described in this invention, the added activator is a combination of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS).

[0014] As a preferred embodiment of the preparation method described in this invention, the method for preparing the geraniol-chitosan oligosaccharide derivative includes: dissolving geraniol bromide in N,N-dimethylformamide to obtain a first solution; dissolving chitosan oligosaccharide in dimethyl sulfoxide to obtain a second solution; adding the first solution dropwise to the second solution, adding triethylamine, stirring the reaction at room temperature in the dark, adjusting the pH of the system to neutral after the reaction, reacting in a water bath, adding acetone, collecting the precipitate by centrifugation, Soxhlet extraction, and vacuum drying to obtain the geraniol-chitosan oligosaccharide derivative.

[0015] As a preferred embodiment of the preparation method described in this invention, the method for preparing geranylace includes: dissolving geraniol and pyridine in anhydrous diethyl ether, stirring and cooling under an ice-water bath to obtain a first solution; dissolving phosphorus tribromide in anhydrous diethyl ether to obtain a second solution, adding the second solution dropwise to the first solution and stirring to mix; transferring the mixed solution to a separatory funnel, taking the upper layer, washing, removing water, filtering, and rotary evaporating to obtain geranylace.

[0016] In a preferred embodiment of the preparation method described in this invention, the washing involves washing the upper layer three times with a 5% sodium bicarbonate solution, deionized water, and a saturated sodium chloride solution, respectively.

[0017] Another objective of this invention is to overcome the shortcomings of the prior art and provide an application of a bifunctional chitosan oligosaccharide derivative based on phenylboronic acid and geraniol.

[0018] In a preferred embodiment of the application described in this invention, the derivative is a water-soluble material, wherein phenylboronic acid molecules and geraniol essential oil molecules are grafted onto chitosan oligosaccharide molecules. In a preferred embodiment of the application described in this invention, the phenylboronic acid-functionalized geraniol-chitosan oligosaccharide derivative has higher water solubility than the monomeric geraniol molecule.

[0019] As a preferred embodiment of the application described in this invention, the phenylboronic acid-functionalized geraniol-chitosan oligosaccharide derivative has higher antibacterial activity compared to the monomeric chitosan oligosaccharide molecule.

[0020] As a preferred embodiment of the application described in this invention, the phenylboronic acid-functionalized geraniol-chitosan oligosaccharide derivative has higher bacterial targeting than the geraniol-chitosan oligosaccharide derivative.

[0021] The beneficial effects of the present invention are as follows: (1) The present invention provides a bifunctional chitosan oligosaccharide derivative based on phenylboronic acid and geraniol. By performing bromide-mediated bromination reaction and EDC / NHS-mediated amide reaction, respectively, the grafting of alcohol essential oil molecules (geraniol) and aromatic boric acid molecules (phenylboronic acid) onto chitosan oligosaccharide molecules is realized, resulting in a water-soluble material.

[0022] (2) The present invention successfully functionalizes chitosan oligosaccharide by chemical modification. Compared with unmodified chitosan oligosaccharide molecules, phenylboronic acid-functionalized geraniol-chitosan oligosaccharide derivatives have higher antibacterial and bacterial targeting properties.

[0023] (3) The preparation method of the present invention is simple, the raw materials are cheap and readily available and the products are easy to purify, which expands the application range of chitosan oligosaccharide and gives it better application prospects in multiple fields such as food and biomedicine. Attached Figure Description

[0024] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Among them: Figure 1 is a synthetic route diagram of phenylboronic acid functionalized geraniol-chitosan oligosaccharide derivative in the present invention.

[0025] Figure 2 shows the infrared spectra of chitosan oligosaccharide, the geraniol-chitosan oligosaccharide derivative prepared in Example 1 of this invention, and the phenylboronic acid-functionalized geraniol-chitosan oligosaccharide derivative prepared in this invention.

[0026] Figure 3 shows the 1H NMR spectra of chitosan oligosaccharide, geraniol-chitosan oligosaccharide derivatives prepared in Example 1 of this invention, and phenylboronic acid-functionalized geraniol-chitosan oligosaccharide derivatives.

[0027] Figure 4 shows the antibacterial effects of chitosan oligosaccharide, geraniol-chitosan oligosaccharide derivatives prepared in this invention, and phenylboronic acid-functionalized geraniol-chitosan oligosaccharide derivatives on Escherichia coli.

[0028] Figure 5 shows the antibacterial effects of chitosan oligosaccharide, geraniol-chitosan oligosaccharide derivatives prepared in this invention, and phenylboronic acid-functionalized geraniol-chitosan oligosaccharide derivatives on Staphylococcus aureus.

[0029] Figure 6 shows scanning electron microscope images of physiological saline, geraniol-chitosan oligosaccharide derivatives prepared in this invention, and phenylboronic acid-functionalized geraniol-chitosan oligosaccharide derivatives on Escherichia coli.

[0030] Figure 7 shows scanning electron microscope images of physiological saline, geraniol-chitosan oligosaccharide derivatives prepared in this invention, and phenylboronic acid-functionalized geraniol-chitosan oligosaccharide derivatives against Staphylococcus aureus.

[0031] Figure 8 shows the thermogravimetric curves of chitosan oligosaccharide, geraniol-chitosan oligosaccharide derivatives prepared in this invention, and phenylboronic acid-functionalized geraniol-chitosan oligosaccharide derivatives. Detailed Implementation

[0032] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the examples in the specification.

[0033] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0034] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0035] Unless otherwise specified, all raw materials used in this invention are commercially available.

[0036] The chemical formula of pyridine used in this invention is shown in Formula C. Example 1 This example provides a method for preparing a bifunctional chitosan oligosaccharide derivative based on phenylboronic acid and geraniol, comprising: 1) Preparing geraniol bromide: 0.05 mol of geraniol is added to a three-necked flask containing 30 mL of anhydrous diethyl ether and mixed thoroughly. An appropriate amount of pyridine is then added as a catalyst. 0.025 mol of phosphorus tribromide is slowly added dropwise to the solution, and the reaction is stirred for 30 min after the addition is complete. The entire reaction is carried out in an ice bath.

[0037] After the reaction was complete, the solution was poured into a separating funnel. The precipitate was purified with anhydrous diethyl ether and the precipitate was combined with the precipitate in the separating funnel. The liquid mixture was then washed several times with 5% (w / w) sodium bicarbonate, deionized water, and saturated sodium chloride solution, respectively. The liquid was dried over anhydrous magnesium sulfate, filtered, and then evaporated under vacuum to obtain geranylide bromide.

[0038] 2) Geraniol-chitosan oligosaccharide derivatives were prepared using chitosan oligosaccharides with a molecular weight of 1000 Da and a degree of polymerization (n) of 4–7. The molar ratio of geraniol bromide to chitosan oligosaccharide was 2:1. Chitosan oligosaccharide (2 mmol) was dissolved in dimethyl sulfoxide, and a certain amount of triethylamine catalyst was added dropwise, stirring until the solution was clear and transparent. Geraniol bromide (4 mmol) was dissolved in N,N-dimethylformamide. Under magnetic stirring, the geraniol bromide solution was added dropwise to the chitosan oligosaccharide solution using a constant pressure funnel, and the reaction was carried out at room temperature for 12 h. After the reaction was completed, excess acetone was added to terminate the reaction, and the precipitate was collected by centrifugation. The precipitate was washed repeatedly with acetone several times, and then dried under vacuum at 45 °C for 6 h to obtain crude geraniol-chitosan oligosaccharide. The crude product was extracted with petroleum ether using a Soxhlet extractor for 24 h, dialyzed for 72 h, filtered, and freeze-dried to obtain the geraniol-chitosan oligosaccharide derivatives.

[0039] 3) Preparation of phenylboronic acid-functionalized geraniol-chitosan oligosaccharide derivative: The molar ratio of geraniol-chitosan oligosaccharide derivative and 4-carboxyphenylboronic acid was 1:1. The geraniol-chitosan oligosaccharide derivative was dissolved in deionized water. 4-Carboxyphenylboronic acid (1 mmol), EDC (1.2 mmol), and NHS (1.2 mmol) were dissolved in methanol and stirred at room temperature in the dark for 30 min. The two solutions were mixed under magnetic stirring and reacted at room temperature in the dark for 24 h. After the reaction was completed, the sample was dialyzed with deionized water for 72 h. The phenylboronic acid-functionalized geraniol-chitosan oligosaccharide derivative was obtained by freeze-drying.

[0040] Figure 1 is a synthetic route diagram of phenylboronic acid functionalized geraniol-chitosan oligosaccharide derivative in this invention.

[0041] Figure 2a shows the infrared spectrum of chitosan oligosaccharide, 3451 cm⁻¹. -1 The absorption peaks around 2938 cm⁻¹ are the superposition absorptions of the stretching vibrations of NH and OH. -1 Stretching vibrations of CH in -CH2 and residual acetyl groups; 1644 cm⁻¹, 1519 cm⁻¹, and 1383 cm⁻¹. -1 These are the stretching vibrations of C=O in the residual N-acetyl group (amide I), the in-plane bending vibrations of NH in NH3 (amide II), and the stretching vibrations of CN (amide III).

[0042] Figure 2b shows the infrared spectrum of the geraniol-chitosan oligosaccharide derivative, 1516 cm⁻¹. -1 The amino stretching vibrations on both sides decreased significantly, and the C=O stretching vibration in chitosan oligosaccharides shifted to 1625 cm⁻¹. -1 Furthermore, the absorption peak broadened, possibly because after geraniol was introduced into chitosan oligosaccharide, the stretching vibration of C=C in geraniol coincided with the stretching vibration of C=O in chitosan oligosaccharide. Infrared results indicate that geraniol may have successfully bound to the amino group of chitosan oligosaccharide.

[0043] Figure 2c shows the infrared spectrum of the phenylboronic acid-functionalized geraniol-chitosan oligosaccharide derivative. Compared with the geraniol-chitosan oligosaccharide derivative, the phenylboronic acid-functionalized geraniol-chitosan oligosaccharide derivative has a larger infrared spectrum at 3451 cm⁻¹. -1 and 1664 cm -1 The peak at 1546 cm⁻¹ corresponds to the stretching vibrations of the OH group in the hydroxyl group and the C=O bond in the amide bond. The increased peak intensity here is due to the increased hydroxyl and C=O content resulting from the introduction of PBA. -1 and 715 cm -1 A new peak appears at 1350 cm⁻¹, which is the characteristic absorption band of the benzene ring, corresponding to the bending vibrations of C=C and CH₄ in the benzene ring; at 1350 cm⁻¹... -1 A characteristic peak of -B(OH)2 caused by BO stretching vibration also appeared. Infrared results indicate that phenylboronic acid may have successfully bound to the amino group of chitosan oligosaccharide.

[0044] Figure 3a shows the 1H NMR spectrum of chitosan oligosaccharide. The solvent peak corresponding to heavy water is located at 4.84 ppm, the most characteristic peak of the glucosamine ring is located at 4.57 ppm, and the proton peaks of other sugar rings are located at 3.07-3.99 ppm.

[0045] Figure 3b shows the 1H NMR spectrum of the geraniol-chitosan oligosaccharide derivative in this embodiment. The geraniol-chitosan oligosaccharide derivative exhibits distinct new peaks between 1.59 and 1.76 ppm. The appearance of these new peaks is attributed to the introduction of -CH2- and -CH3 by grafting geraniol.

[0046] Figure 3c shows the 1H NMR spectrum of the phenylboronic acid-functionalized geraniol-chitosan oligosaccharide derivative in this embodiment. The phenylboronic acid-functionalized geraniol-chitosan oligosaccharide derivative exhibits a distinct new peak in the range of 7.71-8.01 ppm. The appearance of these new peaks is attributed to the introduction of benzene ring proton peaks by grafting 4-carboxyphenylboronic acid.

[0047] Example 2 This example is used to determine the solubility of the derivatives prepared in this invention, including: dissolving 2 g of chitosan oligosaccharide, 0.5 g of geraniol-chitosan oligosaccharide derivative and 0.5 g of phenylboronic acid-functionalized geraniol-chitosan oligosaccharide derivative in 5 ml of deionized water, stirring at room temperature for 12 h, filtering, freeze-drying the filtrate, weighing the mass of the obtained solid, and determining the solubility in water; mixing geraniol and water at a volume ratio of 1:4 and observing the layering.

[0048] Geraniol, chitosan oligosaccharide, 4-carboxyphenylboronic acid, and the chitosan oligosaccharide derivative in Example 1 were designated as Ger, COS, PBA, COS-Ger, and PBA-COS-Ger, respectively. The solubility measurement results of each sample are shown in Table 1.

[0049] Table 1. Results of solubility determination of samples in water

[0050] The above solvent properties test results show that the derivative prepared by simultaneously grafting geraniol and phenylboronic acid onto chitosan oligosaccharide molecules in this invention is soluble in water, overcoming the defect in the prior art that geraniol and phenylboronic acid are insoluble in water. After modification with phenylboronic acid, the water solubility of the chitosan oligosaccharide derivative is improved to some extent.

[0051] Example 3 This example is used to determine the antibacterial effect of the derivative prepared by the present invention. Specifically: 1. Experimental strains The experimental strains selected in this example are the most common Gram-negative bacteria (Escherichia coli) and Gram-positive bacteria (Staphylococcus aureus).

[0052] 2. Procedure: Streak Escherichia coli and Staphylococcus aureus separately on LB agar plates and incubate the plates at 37°C for 24 hours. Pick a single colony from the plate and inoculate it into LB liquid medium. Incubate on a shaker until the logarithmic growth phase. Then, dilute the bacterial culture to 10⁻⁶. 5 CFU / mL available for use.

[0053] Prepare a liquid culture medium with a sample concentration of 1 mg / mL in a test tube. Add 5 μL of the above bacterial suspension to the test tube, and use LB liquid medium without the sample as a blank control. Incubate the test tube containing the culture medium in a shaker for 5 hours. Then, perform serial dilutions, take 100 μL of the culture medium from the test tube with the appropriate concentration, spread it on LB solid medium, and incubate at 37°C for 24 hours. Observe and photograph the results.

[0054] Figure 4 shows the antibacterial experiment results for *E. coli*, where a, b, c, and d correspond to the blank control, chitosan oligosaccharide, and the geraniol-chitosan oligosaccharide derivative and phenylboronic acid-functionalized geraniol-chitosan oligosaccharide derivative from Example 1, respectively. As can be seen from the figure, the unmodified chitosan oligosaccharide has a certain antibacterial effect compared to the blank control. After grafting geraniol, the number of colonies on the plates corresponding to the geraniol-chitosan oligosaccharide derivative was significantly reduced. After grafting phenylboronic acid, the antibacterial effect of the derivative was significantly improved again.

[0055] Figure 5 shows the antibacterial experiment results of Staphylococcus aureus, where a, b, c, and d correspond to the blank control, chitosan oligosaccharide, and the geraniol-chitosan oligosaccharide derivative and phenylboronic acid-functionalized geraniol-chitosan oligosaccharide derivative of Example 1, respectively. It can be seen that both chitosan oligosaccharide and its derivatives exhibit antibacterial activity. Furthermore, compared to Escherichia coli, the geraniol-chitosan oligosaccharide derivative and the phenylboronic acid-functionalized geraniol-chitosan oligosaccharide derivative showed stronger antibacterial effects against Staphylococcus aureus. This phenomenon may be related to the cell wall structural characteristics of Staphylococcus aureus or the specific mechanism of action of the derivatives.

[0056] Example 4 This example is used to determine the bacterial targeting of the derivatives prepared in this invention. Specifically: 1. Experimental bacterial species The experimental bacterial species selected in this example are the most common Gram-negative bacteria (Escherichia coli) and Gram-positive bacteria (Staphylococcus aureus).

[0057] 2. Procedure: The bacterial targeting of the derivatives was determined by cold field emission scanning electron microscopy. *Escherichia coli* and *Staphylococcus aureus* were streaked onto LB solid medium and incubated at 37°C for 24 hours. Single colonies were picked from the plates and inoculated into LB liquid medium, cultured in a shaker until the logarithmic growth phase, and then the bacterial concentration was diluted to 10⁻⁶. 5 CFU / mL available for use.

[0058] Two mL of 1 mg / mL geraniol-chitosan oligosaccharide derivative and phenylboronic acid-functionalized geraniol-chitosan oligosaccharide derivative sample solutions were co-cultured with two mL of bacterial culture at 37°C for 4 hours. After centrifugation, the supernatant was discarded, and the precipitate was washed repeatedly with PBS. Then, 2.5% glutaraldehyde was added and the mixture was fixed overnight at 4°C. After centrifugation, the precipitate was washed three times with PBS and then dehydrated using a gradient concentration of ethanol solutions (30%, 50%, 70%, 80%, 90%, 95%, 100%). After dehydration, a small amount of anhydrous ethanol was added, mixed well, and 10 μL of the suspension was dropped onto a silicon wafer. The wafer was allowed to air dry at room temperature, then mounted on conductive adhesive, sputtered with gold, and photographed.

[0059] Figure 6 shows the electron microscopy results of Staphylococcus aureus, where a, b, and c correspond to the electron microscopy images after co-culturing with physiological saline, geraniol-chitosan oligosaccharide derivative (Example 1), and phenylboronic acid-functionalized geraniol-chitosan oligosaccharide derivative, respectively. Normal Staphylococcus aureus exhibits intact, smooth spherical shapes, clustered in chains. After treatment with geraniol-chitosan oligosaccharide derivative, the surface shrinks and deforms. Treatment with phenylboronic acid-functionalized geraniol-chitosan oligosaccharide derivative exacerbates surface shrinkage and even destroys bacterial structure, causing membrane rupture and extracellular leakage. Only a small number of the treated bacteria retain their original shape; most of the structure is completely lysed, effectively inhibiting bacterial growth and reproduction.

[0060] Figure 7 shows the electron microscopy results of *E. coli*, where a, b, and c correspond to the electron microscopy images after co-culturing with physiological saline, the geraniol-chitosan oligosaccharide derivative of Example 1, and the phenylboronic acid-functionalized geraniol-chitosan oligosaccharide derivative, respectively. Normal *E. coli* are rod-shaped with a rough surface. After co-culturing with the geraniol-chitosan oligosaccharide derivative, the treated bacteria exhibited abnormalities, showing aggregation, shriveling, membrane depression, deformation, and some obvious damage. After co-culturing with the phenylboronic acid-functionalized geraniol-chitosan oligosaccharide derivative, the abnormalities were more pronounced, and the material was clearly visible adhering to the bacterial surface.

[0061] Observation of the bacterial surface after treatment with geraniol-chitosan oligosaccharide derivatives and phenylboronic acid-functionalized geraniol-chitosan oligosaccharide derivatives revealed that, compared with the geraniol-chitosan oligosaccharide derivative treatment group, the bacterial cell surface of the phenylboronic acid-functionalized geraniol-chitosan oligosaccharide derivative treatment group was coated with more particles. This confirms that after grafting phenylboronic acid, the derivative's ability to recognize bacteria is enhanced due to the special structure of phenylboronic acid. Since the bacterial cell membrane is covered with a large amount of lipopolysaccharide (LPS) / peptidoglycan (PGN), the dihydroxyl-containing phenylboronic acid can interact with LPS / PGN to form tight borate ester bonds, which can enhance the targeting ability of the derivative and thus improve its antibacterial activity.

[0062] Example 5 This example is used to investigate the thermal stability of the derivatives prepared by the present invention. Specifically, the thermal stability of the derivatives is determined by thermogravimetric analysis. 2-3 mg of sample is taken and the weight change of the sample between 25 and 550 °C is measured in a thermogravimetric analyzer.

[0063] Thermogravimetric analysis results are shown in Figure 6, where a, b, and c correspond to the thermogravimetric curves of chitosan oligosaccharide, the geraniol-chitosan oligosaccharide derivative of Example 1, and the phenylboronic acid-functionalized geraniol-chitosan oligosaccharide derivative, respectively. The derivatives of chitosan oligosaccharide, the geraniol-chitosan oligosaccharide derivative, and the phenylboronic acid-functionalized geraniol-chitosan oligosaccharide derivative reached their maximum weight loss temperatures at 222℃, 282℃, and 256℃, respectively, indicating that the derivatives prepared in this invention have better thermal stability compared to chitosan oligosaccharide.

[0064] In summary, this invention achieved the grafting of alcohol essential oil molecules (geraniol) and aromatic boric acid molecules (phenylboronic acid) onto chitosan oligosaccharide molecules through bromide-mediated bromination and EDC / NHS-mediated amide reactions, respectively, resulting in water-soluble materials. The functionalization of chitosan oligosaccharides was successfully achieved through chemical modification. Compared to unmodified chitosan oligosaccharide molecules, geraniol-chitosan oligosaccharide derivatives and phenylboronic acid-functionalized geraniol-chitosan oligosaccharide derivatives exhibit higher thermal stability. Compared to chitosan oligosaccharide molecules and geraniol-chitosan oligosaccharide derivatives, phenylboronic acid-functionalized geraniol-chitosan oligosaccharide derivatives exhibit higher antibacterial activity.

[0065] The preparation method of this invention is simple, the raw materials are inexpensive and readily available, and the product is easy to purify, which expands the application range of cyclodextrin and gives it better application prospects in multiple fields such as food and biomedicine.

[0066] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention 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 the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A bifunctional chitosan oligosaccharide derivative based on phenylboronic acid and geraniol, characterized in that: The bifunctional chitosan oligosaccharide derivative based on phenylboronic acid and geraniol has the chemical structural formula shown in Formula A: Wherein, R is H or geraniol, and the structural formula of the geraniol is shown in Formula B: When R is geranyl, it is connected to formula A at position X of formula B via an oxygen atom or a nitrogen atom.

2. A method for preparing a bifunctional chitosan oligosaccharide derivative based on phenylboronic acid and geraniol, characterized in that: The process involves dissolving 4-carboxyphenylboronic acid in an organic solvent, adding an activator and stirring to obtain a first solution; dissolving a geraniol-chitosan oligosaccharide derivative in deionized water to obtain a second solution; adding the first solution dropwise into the second solution, reacting at room temperature in the dark, and then dialysis, filtering, and freeze-drying to obtain a bifunctional chitosan oligosaccharide derivative based on phenylboronic acid and geraniol.

3. The preparation method according to claim 2, characterized in that: The molar ratio of 4-carboxyphenylboronic acid to the activator is 1:1 - 1.

5.

4. The preparation method according to claim 2, characterized in that: The organic solvent is methanol.

5. The preparation method according to claim 2, characterized in that: The activator is a combination of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS).

6. The preparation method according to claim 2, characterized in that: The preparation method of the geraniol-chitosan oligosaccharide derivative includes dissolving geraniol bromide in N,N-dimethylformamide to obtain a first solution; and dissolving chitosan oligosaccharide in dimethyl sulfoxide to obtain a second solution. The first solution was added dropwise to the second solution, and triethylamine was added. The mixture was stirred and reacted at room temperature in the dark. After the reaction, the pH of the system was adjusted to neutral. After the reaction in a water bath, acetone was added, and the precipitate was collected by centrifugation. The precipitate was extracted by Soxhlet extraction and dried under vacuum to obtain geraniol-chitosan oligosaccharide derivative.

7. The preparation method according to claim 6, characterized in that: The method for preparing geranylide bromide includes dissolving geraniol and pyridine in anhydrous diethyl ether, stirring and cooling in an ice-water bath to obtain a first solution; dissolving phosphorus tribromide in anhydrous diethyl ether to obtain a second solution, adding the second solution dropwise to the first solution and stirring to mix; transferring the mixed solution to a separatory funnel, taking the upper layer, washing, removing water, filtering, and rotary evaporating to obtain geranylide bromide.

8. The preparation method according to claim 7, characterized in that: The washing process involves washing the upper layer three times with a 5% sodium bicarbonate solution, deionized water, and a saturated sodium chloride solution, respectively.

9. An application of a bifunctional chitosan oligosaccharide derivative based on phenylboronic acid and geraniol, characterized in that: The bifunctional chitosan oligosaccharide derivative is a water-soluble material, wherein phenylboronic acid molecules and geraniol essential oil molecules are grafted onto chitosan oligosaccharide molecules.

10. The application as described in claim 9, characterized in that: The phenylboronic acid-functionalized geraniol-chitosan oligosaccharide derivative exhibits higher antibacterial activity compared to the geraniol-chitosan oligosaccharide derivative.

Images