Method for preparing chitosan conjugate

Abstract

A method of making a chitosan conjugate, the method comprising: contacting chitosan, a dicarboxylic acid, and 4-(4, 6-dimethoxy-1, 3, 5-triazin-2-yl)-4-methyl-morpholinium chloride (DMTMM) in an aqueous solvent, thereby forming a reaction mixture comprising a chitosan conjugate wherein the chitosan conjugate comprises a repeating unit of formula I, or a conjugate salt thereof, wherein R1 is-(C = O) (CH2) mCO2H; and m is an integer selected from 1 to 12.

Description

Technical Field

[0001] This disclosure relates to a method for preparing chitosan conjugates. Background Technology

[0002] Polysaccharides are biopolymers composed of monosaccharide units linked by glycosidic bonds. The composition of these monosaccharide units determines the physical and chemical properties of the resulting polysaccharide, which may include, for example, starch, cellulose, glycogen, and dextran, and may possess different inherent properties. Polysaccharides possess numerous chemical functional groups, such as hydroxyl, amino, carboxyl, and sulfate groups. These reactive groups can chemically modify polysaccharides, enhancing their physicochemical properties, such as hydrophobicity, water solubility, and stimuli responsiveness. This broadens the applications of such modified polysaccharides, particularly in personal care, medical, agricultural, food packaging, and paper / textile fields. Chemical modifications such as carboxymethylation, (de)acetylation, sulfation, and phosphorylation are common methods for polysaccharide modification. Alternatively, integrating functional macromolecules provides further methods for chemically modifying polysaccharides, allowing them to acquire the characteristics of the target macromolecule. This can be achieved through cross-linking, complexation, and bioconjugation.

[0003] Bioconjugation is becoming increasingly prevalent in the functionalization of polysaccharides. This chemical strategy leverages functional groups present in polysaccharides, which can be covalently linked using bifunctional / multifunctional crosslinking agents or coupling agents to generate novel derivatives. Functional groups present on polysaccharides (e.g., primary amines, thiol groups, and carbonyl groups) are common targets for bioconjugation. While bioconjugation of polysaccharides is typically carried out in organic solvents due to the water insolubility of biopolymers, water-based bioconjugation is also possible by modifying aqueous-water-soluble polysaccharides using water-soluble crosslinking agents or coupling agents.

[0004] Chitosan is a common polysaccharide with functional groups that are easily chemically modified. It is a copolymer of randomly distributed β-(1-4)-linked D-glucosamine units (deacetylated) and N-acetyl-D-glucosamine units (acetylated) produced by the deacetylation of chitin, which exposes the primary amino groups on the glucosamine backbone. This chemical characteristic allows for various chemical modifications and applications in paper, coatings, food, pharmaceuticals, and textiles.

[0005] The water solubility of chitosan is primarily influenced by its molecular weight and degree of deacetylation. Typical water-soluble forms of chitosan exist as oligosaccharides, which are oligomers of glucosamine units with a degree of polymerization less than 50-55 and a molecular weight less than 10,000 amu. Natural chitosan with a molecular weight greater than 10,000 amu is only soluble in water in the presence of organic acids (e.g., acetic acid, lactic acid, and citric acid) or inorganic acids (e.g., hydrochloric acid and phosphoric acid). In acidic environments, the amino groups on the glucosamine backbone of chitosan are protonated, generating a cationic charge on the polysaccharide, thus dissolving it in water. Conversely, the removal of acidic components renders chitosan insoluble in water due to the amino groups transitioning from a cationic to an uncharged state. While natural chitosan is also insoluble in many organic solvents, such as dimethylformamide (DMSO), acetone, ethanol, and dichloromethane, limiting the application of chitosan to aqueous solutions in the presence of acidic additives may result in an acidic odor in the final product or require the removal of acidic components. Therefore, there is a need to develop a novel chitosan conjugate with improved water solubility to expand its application range.

[0006] Chitosan possesses reactive functional groups suitable for chemical modification. The primary amino groups on the deacetylated glucosamine units of the polymer backbone can be chemically modified with molecules containing carboxyl groups via amidation, forming covalent bonds that allow for persistent linkage using coupling agents and coupling additives. The EDC-NHS carbodiimide coupling reaction is a well-established chemical strategy for the formation of bioconjugations of water-based amides. This conjugation utilizes the water-soluble coupling agent 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and optionally N-hydroxysuccinimide (NHS). During the reaction, the carboxylic acid group is initially activated by EDC to form an O-acylurea intermediate, which is reactive to primary amines. However, the O-acylurea intermediate is highly unstable due to rapid hydrolysis by water. Introducing NHS into the system stabilizes the activated carboxyl groups and makes their reaction with amines much faster than with water, resulting in higher yields of amide conjugations. The EDC / NHS reaction produces byproducts, including N-acylurea, O-acylurea, and regenerated carboxylic acids. Replacing NHS with sodium N-hydroxysuccinimide sulfonate (Sulfo-NHS) can improve amidation efficiency by extending the half-life and improving the solubility of amine-reactive esters in aqueous solution. In some cases, a water-miscible organic solvent is added to the reaction to reduce undesirable hydrolysis.

[0007] Although the EDC / (sulfonyl-)NHS carbodiimide chemical process is a well-established and widely used method for modifying chitosan in aqueous reactions, it has several limitations. Typically, the chitosan used as a starting material exists at a low molecular weight and is water-soluble. On the other hand, reactions with water-insoluble chitosan (molecular weight > 100,000 amu) require the addition of acid to aid in the dissolution of chitosan in water, and the reaction also needs to be maintained at pH 4.5–7.5 for efficient carboxyl activation and conjugation. Purification of the crude product typically involves dialysis to remove byproducts followed by lyophilization, which takes several days or more, making large-scale production impractical. Furthermore, both NHS and sulfonyl-NHS are very expensive (US$5,000–6,000 / kg and US$5,500–10,000 / kg, respectively). They are more suitable for small-scale reactions, especially in peptide and protein modification.

[0008] Besides EDC / (sulfonyl-)NHS, the formation of water-based amide bonds can also be mediated by 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholinium chloride (DMTMM) because it is inexpensive and has good reactivity in water.

[0009] Nevertheless, there is still a need in the art to develop an improved method for preparing chitosan conjugates to overcome at least some of the drawbacks known in the art. Summary of the Invention

[0010] This article discloses a simple method for preparing chitosan conjugates via aqueous acylation. Amidation involves using DMTMM in an aqueous solvent at room temperature to conjugate the dicarboxylic acid linker adipic acid to the amino groups on the glucosamine backbone of chitosan. Figure 1 The conjugation process can be carried out at room temperature with simple stirring within 4 hours. The degree of conjugation can be easily controlled by changing the stoichiometry of reagents, reaction time, and reagent concentration. Purification of the chitosan conjugate can be achieved by solvent precipitation followed by drying to remove residual solvent. The entire preparation process can be completed within 1–1.5 days. Advantageously, the resulting chitosan-adipic acid conjugate does not exhibit cross-linking between chitosan chains. The conjugate also exhibits good solubility in acidic and neutral aqueous solutions, but remains insoluble in alkaline solutions.

[0011] In a first aspect, this document provides a method for preparing chitosan conjugates, the method comprising: contacting chitosan, dicarboxylic acid, and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholinium chloride (DMTMM) in an aqueous solvent to form a reaction mixture comprising a chitosan conjugate, wherein the chitosan conjugate comprises repeating units of formula I.

[0012]

[0013]

[0014] or its conjugate salt, wherein

[0015] R 1 It is -(C=O)(CH2) m CO2H; and

[0016] m is an integer selected from 1 to 12.

[0017] In some embodiments, dicarboxylic acid and DMTMM are present in an aqueous solvent at a molar ratio of 2-6:1.

[0018] In some embodiments, dicarboxylic acid and DMTMM are present in an aqueous solvent at a molar ratio of 2.5-3.5:1.

[0019] In some embodiments, chitosan, dicarboxylic acid, DMTMM, and an aqueous solvent are contacted at a mass ratio of 0.5-2.5:0.5-3:0.5-2:93-98.5, respectively.

[0020] In some embodiments, chitosan, dicarboxylic acid, DMTMM, and an aqueous solvent are contacted in a mass ratio of about 2: about 2.4: about 1.6: about 94.

[0021] In some embodiments, the contact involves contacting chitosan, dicarboxylic acid, and DMTMM at 20-40°C for 1-20 hours.

[0022] In some embodiments, the contacting step includes contacting chitosan, dicarboxylic acid, and DMTMM at about 25°C for about 4 hours.

[0023] In some embodiments, contacting includes contacting chitosan and dicarboxylic acid in an aqueous solvent to form a first solution, and contacting the first solution with DMTMM.

[0024] In some embodiments, contacting includes contacting the chitosan dispersion, the dicarboxylic acid dispersion, and the DMTMM dispersion to form a reaction mixture, wherein the chitosan dispersion comprises chitosan and an aqueous solvent, the dicarboxylic acid dispersion comprises dicarboxylic acid and an aqueous solvent, and the DMTMM dispersion comprises DMTMM and an aqueous solvent.

[0025] In some embodiments, the contacting step includes contacting the chitosan dispersion, dicarboxylic acid dispersion, and DMTMM dispersion at 20-40°C for 1-20 hours.

[0026] In some embodiments, the contacting step includes contacting the chitosan dispersion, dicarboxylic acid dispersion, and DMTMM dispersion at about 25°C for about 4 hours.

[0027] In some implementations, m is 2-4.

[0028] In some implementations, m is 4.

[0029] In some embodiments, the chitosan conjugate further comprises repeating units of Formula II and optionally repeating units of Formula III.

[0030]

[0031] In some embodiments, the repeating units of Formula I account for 20-40% based on the total number of repeating units of Formula I, Formula II, and optionally Formula III in the chitosan conjugate.

[0032] In some embodiments, the chitosan conjugate has a weight-average molecular weight of about 300,000 amu.

[0033] In some embodiments, the chitosan conjugate has a polydispersity index (PDI) of about 1.52.

[0034] In some embodiments, the method further includes the steps of contacting the reaction mixture with an organic solvent to precipitate the chitosan conjugate and collecting the precipitated chitosan conjugate.

[0035] In some embodiments, the organic solvent is selected from the group consisting of alcohols, ketones, and mixtures thereof. Attached Figure Description

[0036] In the accompanying drawings, the same reference numerals denote the same elements or elements that are functionally similar. The drawings include graphs of certain embodiments to further illustrate and clarify the above and other aspects, advantages, and features of this disclosure. It should be understood that these drawings depict exemplary embodiments and are therefore not intended to limit the scope of this disclosure. The methods described herein will be described and explained with further features and details using the accompanying drawings.

[0037] Figure 1 The chemical structure of the chitosan conjugate is shown.

[0038] Figure 2 A schematic diagram and exemplary process for the reaction and purification of chitosan-adipic acid conjugates synthesized via DMTMM coupling reaction are shown.

[0039] Figure 3 Fourier transform infrared (FTIR) spectra of adipic acid, chitosan, and the chitosan-adipic acid conjugate prepared by the DMTMM coupling reaction are shown.

[0040] Figure 4 The gel formed by the DMTMM coupling reaction in reaction EP1306-80b is shown.

[0041] Figure 5 Comparative FTIR spectra of chitosan-adipic acid conjugates obtained by optimization of DMTMM coupling reactions EP1306-80c, EP1306-81, EP1306-82a and EP1306-82b are shown.

[0042] Figure 6 Size exclusion chromatography (SEC) chromatograms are shown, displaying traces of chitosan and the chitosan-adipic acid conjugate prepared by DMTMM coupling reaction (0.5 M acetic acid, 30 °C).

[0043] Figure 7 A comparison is shown of unreacted chitosan, unreacted adipic acid, and chitosan-adipic acid conjugates synthesized via DMTMM coupling. 1 H-NMR spectrum (500MHz) (acetylated glucosamine units on chitosan and chitosan-adipic acid conjugate were not observed).

[0044] Figure 8 This illustrates the chitosan-adipic acid conjugate synthesized via DMTMM coupling. 1 H-NMR spectrum (500MHz) (acetylated glucosamine units on chitosan conjugates were not observed).

[0045] Figure 9 Comparative images show the solubility tests of chitosan-adipic acid conjugates prepared by DMTMM coupling reaction in acidic aqueous solution (pH=2), water (pH=7), and alkaline aqueous solution (pH=9 and 12).

[0046] Figure 10 An exemplary experimental setup is shown for a 1 kg reaction of chitosan-adipic acid conjugate synthesized by DMTMM coupling reaction.

[0047] Figure 11 Comparative FTIR spectra of chitosan-adipic acid conjugates at reaction scales of 100 g, 500 g, and 1 kg are shown.

[0048] Figure 12 Comparative FTIR spectra of chitosan-adipic acid conjugates separated at different reaction time points in the DMTMM coupling reaction are shown.

[0049] Figure 13 A proposed synthetic route for preparing chitosan-adipic acid conjugates via DMTMM coupling reaction is shown according to certain embodiments described herein. Detailed Implementation

[0050] definition

[0051] The definitions of terms used herein are intended to include the most current and generally accepted definition of each term in the field of biotechnology. Examples are provided where appropriate. These definitions apply to the terms used in this specification unless, in particular, the definition of those terms is limited individually or as part of a larger group.

[0052] In this specification, unless the context otherwise requires, the word "comprising" or its variations should be understood to mean including the stated whole or group of wholes, but not excluding any other whole or group of wholes. It should also be noted that in this disclosure, particularly in the claims and / or paragraphs, the term "comprising" and its grammatical variations may have the meaning given to it under U.S. patent law; for example, they may mean "comprising"; and the term "substantially consisting of" and its grammatical variations have the meaning given to it under U.S. patent law, for example, they allow elements not expressly listed, but exclude elements present in the prior art or affecting the essential or novel features of the invention.

[0053] Furthermore, in this specification and claims, unless the context otherwise requires, the word “comprising” or its grammatical variations shall be understood to mean including the said whole or group of wholes, but not excluding any other whole or group of wholes.

[0054] Unless otherwise expressly stated, the singular as used herein includes the plural (and vice versa). Furthermore, unless otherwise expressly stated, when the term "about" is used before a quantity, this teaching also includes the specific quantity itself. Unless otherwise shown or can be inferred, the term "about" as used herein refers to a change of ±10%, ±7%, ±5%, ±3%, ±1%, or ±0% from the nominal value.

[0055] This article provides a method for preparing chitosan conjugates, the method comprising: contacting chitosan, dicarboxylic acid, and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) in an aqueous solvent to form a reaction mixture comprising a chitosan conjugate, wherein the chitosan conjugate comprises repeating units of formula I.

[0056]

[0057] or its conjugate salt, wherein

[0058] R 1 It is -(C=O)(CH2) mCO2H; and

[0059] m is an integer selected from 1 to 12.

[0060] Figure 13 A schematic diagram of an exemplary reaction between chitosan and adipic acid according to certain embodiments described herein is provided.

[0061] Chitosan is a linear polysaccharide containing randomly distributed D-glucosamine repeating units (Formula II), and optionally, depending on the degree of deacetylation, contains acetyl-D-glucosamine repeating units (Formula III):

[0062]

[0063] Chitosan may contain 0.01-20%, 0.5-20%, 1-20%, 5-20%, 10-20%, 15-20%, 1-15%, 1-10%, 5-15%, or 5-10% repeating units of Formula III (based on the total number of repeating units of Formula II and Formula III in the chitosan). In some embodiments, repeating units of Formula III account for about 5% to about 10% of the repeating units in the chitosan.

[0064] Chitosan can have a weight-average molecular weight of 100,000-300,000 amu or 180,000-260,000 amu. The degree of deacetylation of chitosan can be 50-100%, 70-100%, 80-100%, or 80-95%.

[0065] DMTMM is an organotriazine commonly used for the activation of carboxylic acids and is marketed as a chloride or tetrafluoroborate. However, this disclosure covers all salts of DMTMM, including but not limited to chlorides, bromides, iodides, nitrates, tetrafluoroborates, and hexafluorophosphates. In some embodiments, DMTMM chloride is used in the methods described herein.

[0066] Dicarboxylic acids can be produced by the chemical formula HO₂C(CH₂). m CO2H represents the dicarboxylic acid, where m is an integer selected from 1-12, 1-11, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-6, or 3-5. In some embodiments, the dicarboxylic acid is malic acid (malonic acid), succinic acid (succinic acid), glutaric acid (glutaric acid), adipic acid (adipic acid), phytic acid (pimelic acid), succinic acid (octanoic acid), sebacic acid (sebacic acid), undecanoic acid, dodecanoic acid, brassic acid (tridecanoic acid), or combinations thereof. In some embodiments, the dicarboxylic acid is adipic acid (m is 4).

[0067] The concentration of chitosan in the aqueous solution relative to the total weight of chitosan, dicarboxylic acid, DMTMM, and the aqueous solution can be 0.5-2.5 wt%, 1-2.5 wt%, 1.5-2.5 wt%, 2-2.5 wt%, 0.5-2.0 wt%, 0.5-1.5 wt%, 0.5-1 wt%, 1.6-2.4 wt%, 1.7-2.3 wt%, 1.8-2.2 wt%, or 1.9-2 wt%. In some embodiments, the concentration of chitosan in the aqueous solution is approximately 2 wt% relative to the total weight of chitosan, dicarboxylic acid, DMTMM, and the aqueous solution.

[0068] The concentration of dicarboxylic acid in the aqueous solution relative to the total weight of chitosan, dicarboxylic acid, DMTMM, and the aqueous solution can be 1-3 wt%, 1.5-3 wt%, 2-3 wt%, 2.5-3 wt%, 1-2.5 wt%, 1-2 wt%, 1-1.5 wt%, 2-2.8 wt%, 2.1-2.7 wt%, 2.2-2.6 wt%, or 2.3-2.5 wt%. In some embodiments, the concentration of dicarboxylic acid in the aqueous solution relative to the total weight of chitosan, dicarboxylic acid, DMTMM, and the aqueous solution is approximately 2.4 wt%.

[0069] The concentration of DMTMM in the aqueous solution can be 1-3 wt%, 1.5-3 wt%, 2-3 wt%, 2.5-3 wt%, 1-2.5 wt%, 1-2 wt%, 1-1.5 wt%, 1.2-2 wt%, 1.3-1.9 wt%, 1.4-1.8 wt%, or 1.5-1.7 wt% relative to the total weight of chitosan, dicarboxylic acid, DMTMM, and the aqueous solution. In some embodiments, the concentration of DMTMM in the aqueous solution is approximately 1.6 wt% relative to the total weight of chitosan, dicarboxylic acid, DMTMM, and the aqueous solution.

[0070] Dicarboxylic acid and DMTMM can be present in an aqueous solvent in molar ratios of 2-6:1, 2-5.5:1, 2-5:1, 2-4.5:1, 2-4:1, 2-3.5:1, 2-3:1, 2-2.5:1, 2.5-6:1, 3-6:1, 3.5-6:1, 4-6:1, 4.5-6:1, 5-6:1, 5.5-6:1, or 2.5-3.5:1, respectively. In some embodiments, dicarboxylic acid and DMTMM are present in an aqueous solvent in molar ratios of about 2.5:about 1, about 3.5:about 1, or about 2.5-3.5:about 1.

[0071] The grafting rate can be controlled by adjusting the mass ratio of chitosan, dicarboxylic acid, DMTMM, and aqueous solvent. In some embodiments, chitosan, dicarboxylic acid, and aqueous solvent are contacted at mass ratios of 0.5-2.5:0.5-3:0.5-2:93-98.5 or 1.5-2:2-3:1.5-2:93-95, respectively. Advantageously, no intermolecular crosslinking between chitosan molecules is observed when chitosan, dicarboxylic acid, DMTMM, and aqueous solvent are contacted at mass ratios of about 2:about 2.4:about 1.6:about 94, respectively.

[0072] The coupling reaction between chitosan and dicarboxylic acid can be carried out in an aqueous solvent. In some embodiments, the aqueous solvent comprises or is composed of water. In other embodiments, the aqueous solvent comprises water and one or more organic solvents selected from the group consisting of alcohols (e.g., methanol, ethanol, isopropanol, etc.), ethers (e.g., tetrahydrofuran, dioxane, etc.), and ketones (e.g., acetone, methyl ethyl ketone, etc.). In some embodiments, the aqueous solvent is water.

[0073] Contacting chitosan, dicarboxylic acid, and DMTMM in an aqueous solvent may include contacting them at 20-40°C, 20-35°C, 20-30°C, or 20-25°C in an aqueous solvent. In some embodiments, contacting chitosan, dicarboxylic acid, and DMTMM in an aqueous solvent is performed at room temperature (e.g., about 25°C).

[0074] Contacting chitosan, dicarboxylic acid, and DMTMM in an aqueous solvent may include contacting them in an aqueous solvent for 1-20 hours, 1-16 hours, 1-15 hours, 1-14 hours, 1-13 hours, 1-12 hours, 1-11 hours, 1-10 hours, 1-9 hours, 1-8 hours, 1-7 hours, 1-6 hours, 1-5 hours, 1-4 hours, 1-3 hours, 1-2 hours, 2-6 hours, or 3-5 hours. In some embodiments, the contacting of chitosan, dicarboxylic acid, and DMTMM in an aqueous solvent is approximately 4 hours. In some embodiments, the contacting of chitosan, dicarboxylic acid, and DMTMM in an aqueous solvent is less than 1 hour, less than 2 hours, less than 3 hours, less than 4 hours, less than 5 hours, less than 6 hours, less than 7 hours, less than 8 hours, less than 9 hours, less than 10 hours, less than 11 hours, less than 12 hours, less than 13 hours, or less than 14 hours.

[0075] The order of addition of chitosan, dicarboxylic acid, DMTMM, and the aqueous solvent can vary. This disclosure considers all possible orders of addition, including simultaneous and / or sequential addition of each / all materials. In some embodiments, the dicarboxylic acid is first mixed with the aqueous solvent, followed by the addition of chitosan, and then the addition of DMTMM. In some embodiments, the dicarboxylic acid dispersion is mixed with a chitosan dispersion, followed by a DMTMM dispersion, wherein the dicarboxylic acid dispersion comprises dicarboxylic acid and an aqueous solvent, the chitosan dispersion comprises chitosan and an aqueous solvent, and the DMTMM dispersion comprises DMTMM and an aqueous solvent.

[0076] The method described herein may also include purification and / or separation of the chitosan conjugate. In some embodiments, the method further includes contacting the reaction mixture with an organic solvent to precipitate the chitosan conjugate, collecting the precipitated chitosan conjugate, and optionally washing the precipitated chitosan conjugate with an organic solvent. The organic solvent used to precipitate the chitosan conjugate may be one or more solvents selected from the group consisting of alcohols (e.g., methanol, ethanol, isopropanol, etc.), ethers (e.g., tetrahydrofuran, dioxane, etc.), and ketones (e.g., acetone, methyl ethyl ketone, etc.). The organic solvent used to wash the precipitated chitosan conjugate may be one or more solvents selected from the group consisting of alcohols (e.g., methanol, ethanol, isopropanol, etc.), ethers (e.g., tetrahydrofuran, dioxane, etc.), and ketones (e.g., acetone, methyl ethyl ketone, etc.).

[0077] In some embodiments, the chitosan conjugate further comprises a D-glucosamine repeating unit (Formula II) and optionally an acetyl-D-glucosamine repeating unit (Formula III):

[0078]

[0079]

[0080] The repeating units of Formula I may account for 0.5-50%, 1-50%, 5-50%, 5-45%, 1-45%, 1-40%, 5-35%, 6-35%, 6-34%, 7-34%, 7-30%, 7-25%, 7-20%, 7-15%, 7-10%, 7-9%, 10-35%, 15-35%, 20-35%, 25-35%, 26-35%, 27-35%, 28-35%, 25-34%, or 28-34% of the repeating units of Formula I, Formula II, and Formula III (based on the total number of repeating units of Formula I, Formula II, and Formula III). In some embodiments, the repeating units of Formula I account for approximately 28-34% of the repeating units of the chitosan conjugate. In some embodiments, the repeating unit of Formula I accounts for about 25% to about 34% or about 28% to about 34% of the repeating units in the chitosan conjugate.

[0081] The chitosan conjugate may contain 30-70%, 35-70%, 40-70%, 45-70%, 50-70%, 55-70%, 60-70%, 65-70%, 30-65%, 30-60%, 30-55%, 30-50%, 30-45%, 30-40%, 30-35%, or 51-70% of repeating units of Formula II (based on the total number of repeating units of Formula I, Formula II, and Formula III). In some embodiments, repeating units of Formula II account for about 51% to about 70% of the repeating units in the chitosan conjugate.

[0082] The chitosan conjugate may contain 0.01-20%, 0.5-20%, 1-20%, 5-20%, 10-20%, 15-20%, 1-15%, 1-10%, 5-15%, or 5-10% of repeating units of Formula III (based on the total number of repeating units of Formula I, Formula II, and Formula III). In some embodiments, repeating units of Formula III account for about 5% to about 10% of the repeating units in the chitosan conjugate.

[0083] Figure 1 An exemplary chitosan conjugate prepared according to the method described herein is shown, wherein R 1 As defined in this article, x can be 3-7, y can be 0.5-5, z can be 0-2, and n can be 10-500.

[0084] Chitosan conjugates can have capacities of 10,000-400,000 amu, 50,000-400,000 amu, 100,000-400,000 amu, 150,000-400,000 amu, 200,000-400,000 amu, 250,000-400,000 amu, 300,000-400,000 amu, and 350,000-400,000 amu. The weight-average molecular weights are 100,000-350,000 amu, 100,000-300,000 amu, 100,000-250,000 amu, 100,000-200,000 amu, 100,000-150,000 amu, 150,000-400,000 amu, 200,000-350,000 amu, or 250,000-300,000 amu. In some embodiments, the chitosan conjugate has a weight-average molecular weight of about 260,000 amu.

[0085] Chitosan conjugates may have a PDI of about 1.1-2, 1.2-2, 1.3-2, 1.4-2, 1.5-2, 1.6-2, 1.7-2, 1.8-2, 1.9-2, 1.1-2, 1.1-1.9, 1.1-1.8, 1.1-1.7, 1.1-1.6, 1.1-1.5, 1.1-1.4, 1.1-1.3, 1.1-1.2, 1.1-1.9, 1.2-1.8, 1.3-1.7, or 1.4-1.6. In some embodiments, the chitosan has a PDI of about 1.52.

[0086] Example

[0087] Example 1 - Preparation of chitosan-adipic acid conjugates via DMTMM coupling reaction on a 100-gram reaction scale

[0088] Chitosan (degree of deacetylation = 80-95%) and adipic acid were dissolved in water by stirring at room temperature for at least 30 minutes. DMTMM was added to the chitosan / adipic acid solution, and the mixture was stirred at room temperature for 4 hours. The composition of the reaction is summarized in Table 1. At the end of mixing, the formed chitosan-adipic acid conjugate was purified and precipitated from the ethanol. Acetone, isopropanol, or methanol are also suitable for the purification of the precipitate. The resulting residue was dried at room temperature, then dried at 50-60°C to obtain a white powder. Figure 2 Successful conjugation was detected by attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy. 1 H-NMR (500MHz, D2O) analysis of the chemical structure of the chitosan conjugate: δ (ppm) 1.51 (s, 4H, adipic acid [CH2CH2] backbone), 2.17 (s, 4H, adipic acid CH2 group), 3.02 (s, 1H, -CH-NH2 GlcN backbone), 3.49-3.88 (m, 5H, GlcN and conjugated GlcN backbone), 4.53 (m, 1H, glycoside with D2O).

[0089] Table 1. Weight percentage of reactants when preparing chitosan-adipic acid conjugates on a 100g reaction scale.

[0090]

[0091] 1.2 Description of the obtained chitosan-adipic acid conjugate

[0092] Figure 3 The FTIR spectra of adipic acid, chitosan, and purified chitosan-adipic acid conjugate are shown (acetylated glucosamine units on chitosan and the chitosan-adipic acid conjugate are not shown). The conjugate spectrum shows a peak at 1633 cm⁻¹. 1 The C=O stretch at 1541 cm- 1 The presence of amide NH bending at the site proves that adipic acid was successfully grafted onto chitosan.

[0093] With 1685 cm⁻¹ from unreacted adipic acid and chitosan, respectively 1 The C=O signal at 1590 cm⁻¹ and 1590 cm⁻¹ 1 Compared to the NH signal at the given location, the C=O stretching and amide NH bending signals from the conjugate indicate a shift of the corresponding peaks to lower wavenumbers. This signal shift suggests that the conjugate was formed from the starting material.

[0094] A one-pot reaction was carried out in EP1306-80b, EP1306-80c, EP1306-81, EP1306-82a, and EP1306-80b. Reaction EP1306-80b resulted in gel formation. Figure 4 In reaction EP1306-80c, when the DMTMM content decreased to 0.1%, the reaction remained a homogeneous solution. However, in the FTIR spectrum ( Figure 5 The purified product was at 1630-1660 cm⁻¹. -1 The absence of a free carboxyl group signal (C=O stretched, highlighted area) indicates unsuccessful conjugation (dashed line). In reaction EP1306-81, increasing adipic acid to 1.6% and DMTMM to 0.8% improved the conjugation, as shown in the FTIR spectrum (dashed line) at 1637 cm⁻¹. -1 The presence of free carboxyl group signals at the FTIR spectra confirms this. In reaction EP1306-82b, increasing the adipic acid content to 2.4% and doubling the DMTMM content to 1.6% yielded significant free carboxyl group signals in the FTIR spectrum (solid line). Reaction EP1306-82b was selected as the preferred condition for the preparation of chitosan-adipic acid conjugates.

[0095] The molecular weight distribution of the chitosan-adipic acid conjugate synthesized via reaction EP1306-82b was determined by size exclusion chromatography (SEC). The SEC instrument was calibrated using dextran standards. Chitosan and chitosan-adipic acid samples were injected into an Ultrahydrogel Linear column (Waters). TM In the sample, elution was performed at 30°C with 0.5M acetic acid at a flow rate of 1.0 mL / min. The eluted sample was detected by a refractive index (RI) detector. Figure 6 According to number average molecular weight (M) n ), weight-average molecular weight (M) w The molecular weight distribution is determined by the polydispersity index (PDI).

[0096] Table 2 summarizes the molecular weight distribution of chitosan and chitosan-adipic acid conjugates. According to M... nThe degree of polymerization (DP) of chitosan was estimated to be 1000, with chitosan composed of 800-950 D-glucosamine units and 50-200 acetyl-D-glucosamine units. Considering the number of D-glucosamine units, the number of primary amines was estimated to be 800-950. The conjugation of adipic acid with chitosan showed M... w An increase of 40,000 amu equates to 274 adipic acid molecules grafted onto each chitosan link. This increase in molecular weight also indicates the absence of observed inter-crosslinks between chitosan chains, which would typically result in a two-fold or greater increase in the molecular weight distribution of the conjugate and a bimodal or multimodal molecular weight distribution on the SEC trace. Assuming one of the carboxyl groups on the adipic acid molecule is activated by the DMTMM coupling agent, the conjugation efficiency is 36%, and the grafting rate is 28-34%.

[0097] Table 2. Molecular weight distribution of chitosan and chitosan-adipic acid conjugate prepared by DMTMM coupling reaction EP1306-82b.

[0098]

[0099] Compared to unreacted chitosan and adipic acid, the NMR spectrum of the chitosan-adipic acid conjugate showed a shift to lower ppm. The peak shape of the conjugate at 3.49–3.88 ppm (N-glucosamine backbone) appeared different from the peak shape of the chitosan N-glucosamine backbone at 3.65–3.84 ppm. This change and shift in the NMR signal indicates that adipic acid is covalently linked to the chitosan in the conjugate (…). Figure 7 The resulting chitosan-adipic acid conjugate can be represented by formula IV, where x is 2-3, y is 1, z is 0.2, and n is 274 (the molar ratio of formula I is 23.81%-31.25%); or x is 2, y is 1, z is 0.7, and n is 274 (the molar ratio of formula I is 27%), and R 1 It is -(C=O)(CH2)4CO2H.

[0100]

[0101] The grafting degree of adipic acid to chitosan was determined by integrating the NMR spectrum of the conjugate. Figure 8The ratio of the peak area at 3.02 ppm (GlcN, H2 on chitosan) to the peak area at 2.17 ppm (conjugated adipic acid CH2 group) was 1:1.3384. After dividing the peak areas by their respective proton numbers, this ratio was calculated to be 1:0.33 (chitosan:adipic acid portion), equal to the ratio of NH2 group to conjugated adipic acid (3:1). The results indicate a grafting degree of 25% for adipic acid. The grafting degree determined by NMR spectroscopy was correlated with the estimated grafting degree calculated based on the molecular weight distribution from the SEC.

[0102] 1.3 Solubility Test

[0103] The developed chitosan-adipic acid conjugate exhibits advantageously improved water compatibility, which will be suitable for the development of stable water-based formulations. The obtained chitosan conjugate is soluble in water or aqueous solutions with pH < 7 at concentrations of 0.01–2.5% by weight or 0.01–0.1% by weight. The obtained chitosan conjugate remains insoluble in aqueous solutions with pH > 7. The conjugate is completely soluble in water and acidic aqueous solutions with pH = 2, but insoluble in alkaline aqueous solutions with pH = 9 and pH = 12. Figure 9 pH-switchable solubility indicates the presence of unconjugated amino groups on the chitosan backbone, which contributes to hydrophobicity due to reduced electrostatic charge at high pH.

[0104] Example 2 - Preparation of chitosan-adipic acid conjugates via DMTMM coupling reaction on a 500g reaction scale

[0105] The aqueous dispersion of chitosan (degree of deacetylation = 80-95%) was dissolved in the aqueous dispersion of adipic acid by stirring at room temperature for at least 30 minutes. An aqueous dispersion of DMTMM was added to the chitosan / adipic acid solution, and the mixture was stirred at room temperature for 4 hours using a top-mounted stirrer. Figure 10 The composition of the reaction is summarized in Table 3. The resulting chitosan-adipic acid conjugate was purified and precipitated from the ethanol. Acetone, isopropanol, or methanol are also suitable for purifying the precipitate. The resulting residue was dried at room temperature, then dried at 50-60°C to obtain a white powder.

[0106] Table 3. Weight percentage of reactants when preparing chitosan-adipic acid conjugates on a 500g reaction scale.

[0107]

[0108] Example 3 - Preparation of chitosan-adipic acid conjugates via DMTMM coupling reaction at a reaction scale of at least 1 kg

[0109] The aqueous dispersion of chitosan (degree of deacetylation = 80-95%) was dissolved in the aqueous dispersion of adipic acid by stirring at room temperature for at least 30 minutes. An aqueous dispersion of DMTMM was added to the chitosan / adipic acid solution, and the mixture was stirred at room temperature for 4 hours using a top-mounted stirrer. Figure 10 The composition of the reaction is summarized in Table 4. The resulting chitosan-adipic acid conjugate was purified and precipitated from the ethanol. Acetone, isopropanol, or methanol are also suitable for purifying the precipitate. The resulting residue was dried at room temperature, then dried at 50-60°C to obtain a white powder.

[0110] Table 4. Weight percentage of reactants when preparing chitosan-adipic acid conjugates on a reaction scale of at least 1 kg.

[0111]

[0112] 3.2 Description of the obtained chitosan-adipic acid conjugate

[0113] In small-scale reactions (i.e., in 100 g reactions), chitosan with molecular weights of 100,000-300,000 amu can be completely dissolved in adipic acid solution through simple mixing. However, the dissolution of chitosan powder in a 1 kg reaction scale requires several hours, which prolongs the total preparation time of the conjugate, making large-scale preparation impractical and inefficient. Adding all powdered components in the form of an aqueous dispersion ensures complete dissolution of chitosan. This procedure reduces the mixing time between chitosan and adipic acid from several hours to 30 minutes before adding DMTMM. The separated chitosan-adipic acid conjugates were characterized by ATR-FTIR. The characteristics were obtained by C=O stretching at 1633 cm⁻¹ and [the specific characteristic of the conjugate is missing from the original text]. 1 The signal of amide NH bending at the location ( Figure 11 The successful conjugation was demonstrated by the FTIR bands of the conjugates obtained from the reactions of 100 g and 500 g.

[0114] Example 4 - Stability of the DMTMM-mediated reaction in water

[0115] Aqueous dispersions of chitosan (degree of deacetylation = 80-95%) were dissolved in aqueous dispersions of adipic acid by stirring at room temperature for at least 30 minutes. Aqueous dispersions of DMTMM were added to the chitosan / adipic acid solution, and the mixture was stirred at room temperature for 16 hours using a top-mounted stirrer. Samples were collected at different time points to precipitate the crude product from ethanol, which was then dried at 50-60°C. The separated products were characterized by ATR-FTIR. The composition of the reaction is summarized in Table 5.

[0116] Table 5. Weight percentage of reactants used in stability studies of DMTMM-mediated reactions.

[0117]

[0118] 4.2 Description of chitosan-adipic acid conjugates separated from different reaction time points

[0119] The chitosan conjugates isolated from the reactions at 1 hour, 3 hours, 4 hours, and 16 hours were all white powders, indicating that no side reactions occurred with increasing reaction time. The FTIR spectra of the isolated products (…) Figure 12 This indicates that, after 1 hour of reaction, C=O stretching occurred at 1633 cm⁻¹ and at 1541 cm⁻¹. 1 The amide NH at the site bends and persists even 16 hours after the reaction has proceeded. FTIR bands are correlated across all time points, and no byproducts are observed in the spectrum, indicating that the reaction is stable in water for at least 16 hours.

[0120] exist Figure 13 The amidation reaction mechanism involving adipic acid, chitosan, and DMTMM is presented (byproducts are not shown). The molar equivalent of adipic acid can be 2–6 times that of DMTMM to ensure that only one carboxyl terminus of adipic acid is activated by DMTMM, forming a DMTMM-activated ester, while releasing N-methylmorpholinium (NMM) as a byproduct. The DMTMM-activated ester reacts with the amino group on chitosan to form a chitosan-adipic acid conjugate, in which 1-hydroxy-3,5-dimethoxytriazine is formed as a second byproduct. Both byproducts are soluble in water, acetone, or alcohols (selected from ethanol, isopropanol, or methanol) and can be removed together with unreacted adipic acid by solvent precipitation of the conjugate.

Claims

1. A method for preparing chitosan conjugates, the method comprising: Chitosan, dicarboxylic acid, and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) are contacted in an aqueous solvent to form a reaction mixture comprising the chitosan conjugate, wherein the chitosan conjugate comprises repeating units of Formula I. or its conjugate salt, wherein R 1 It is -(C=O)(CH2) m CO2H; and m is an integer selected from 1 to 12.

2. The method according to claim 1, wherein the dicarboxylic acid and DMTMM are present in the aqueous solvent in a molar ratio of 2-6:

1.

3. The method according to claim 1, wherein the dicarboxylic acid and DMTMM are present in the aqueous solvent at a molar ratio of 2.5-3.5:

1.

4. The method according to claim 1, wherein the chitosan, the dicarboxylic acid, the DMTMM and the aqueous solvent are in contact at a mass ratio of 0.5-2.5:0.5-3:0.5-2:93-98.

5.

5. The method according to claim 1, wherein the chitosan, the dicarboxylic acid, the DMTMM and the aqueous solvent are in contact at a mass ratio of about 2: about 2.4: about 1.6: about 94.

6. The method according to claim 1, wherein contact comprises contacting the chitosan, the dicarboxylic acid and the DMTMM at 20-40°C for 1-20 hours.

7. The method of claim 5, wherein the contacting step comprises contacting the chitosan, the dicarboxylic acid and the DMTMM at about 25°C for about 4 hours.

8. The method of claim 1, wherein contacting comprises contacting the chitosan and the dicarboxylic acid in the aqueous solvent to form a first solution, and contacting the first solution with DMTMM.

9. The method of claim 1, wherein contact comprises contacting the chitosan dispersion, the dicarboxylic acid dispersion, and the DMTMM dispersion to form a reaction mixture, wherein the chitosan dispersion comprises chitosan and the aqueous solvent, the dicarboxylic acid dispersion comprises dicarboxylic acid and the aqueous solvent, and the DMTMM dispersion comprises DMTMM and the aqueous solvent.

10. The method according to claim 9, wherein the contacting step comprises contacting the chitosan dispersion, the dicarboxylic acid dispersion and the DMTMM dispersion at 20-40°C for 1-20 hours.

11. The method of claim 9, wherein the contacting step comprises contacting the chitosan dispersion, the dicarboxylic acid dispersion, and the DMTMM dispersion at about 25°C for about 4 hours.

12. The method of claim 1, wherein m is 2-4.

13. The method of claim 7, wherein m is 4.

14. The method of claim 11, wherein m is 4.

15. The method of claim 1, wherein the chitosan conjugate further comprises repeating units of formula II and optionally repeating units of formula III.

16. The method according to claim 15, wherein, Based on the total number of repeating units of Formula I, repeating units of Formula II, and optionally repeating units of Formula III in the chitosan conjugate, the repeating units of Formula I account for 20-40%.

17. The method according to claim 1, wherein the chitosan conjugate has a weight-average molecular weight of about 300,000 amu.

18. The method of claim 1, wherein the chitosan conjugate has a polydispersity index (PDI) of about 1.

52.

19. The method of claim 1, further comprising the steps of contacting the reaction mixture with an organic solvent to precipitate the chitosan conjugate, and collecting the precipitated chitosan conjugate.

20. The method of claim 17, wherein the organic solvent is selected from the group consisting of alcohols, ketones, and mixtures thereof.

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