Manufacturing method for high-performance chitosan materials

Abstract

This invention provides a method for producing high-performance chitosan materials that have stable solubility. [Solution] The method includes an acetylation step in which the chitosan is N-acetylated using acetic anhydride in an aqueous acetic acid solution in which acetic acid is added to water with a molar error of ±25% relative to the chitosan.

Description

[Technical Field]

[0001] This invention relates to a method for producing a high-performance chitosan material with stable solubility. [Background technology]

[0002] Chitin is an aminopolysaccharide composed of N-acetylglucosamine linked by β1,4 bonds, found in the shells of crustaceans, the exoskeletons of insects, the backbones of squid, and mushrooms. Due to its excellent biological functions, chitin is being considered for use in a wide range of fields, including food, cosmetics, pharmaceuticals, and medical materials. However, its insolubility in water and organic solvents is a problem in its practical applications.

[0003] Chitosan, obtained by the deacetylation of chitin, can be dissolved in acidic aqueous solutions. Specifically, as described in Patent Document 1, chitosan is obtained by a solid-liquid reaction in which insoluble chitin is deacetylated by heating in a concentrated alkali. The N-acetylglucosamine units, which are monosaccharides that make up chitin, are deacetylated into glucosamine units, and the amino group of these glucosamine units becomes positively charged, making it possible to dissolve in acidic aqueous solutions. Furthermore, chitosan retains undeacetylated N-acetyl groups; in other words, chitosan is an aminopolysaccharide copolymerized from N-acetylglucosamine and glucosamine. The proportion of N-acetyl groups in chitosan is expressed as the degree of acetylation and is known to greatly affect the solubility of chitosan. For example, chitosan with a controlled degree of acetylation of approximately 50% is in demand as a water-soluble cationic polymer derived from natural polymers. [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] Japanese Patent Publication No. 2002-325835 (page 3) [Overview of the project] [Problems that the invention aims to solve]

[0005] However, the alkali treatment of chitin in Patent Document 1 is a solid-liquid reaction, which makes it difficult to control the degree of acetylation of the chitosan obtained by deacetylation, and also results in an uneven distribution of N-acetyl groups within the molecule, leading to problems with the stability of the chitosan's solubility.

[0006] This invention was made in view of these problems, and aims to provide a method for producing a high-performance chitosan material that can produce a high-performance chitosan material with stable solubility. [Means for solving the problem]

[0007] To solve the aforementioned problems, the method for producing the high-performance chitosan material of the present invention is as follows: The present invention is characterized by having an acetylation step in which the chitosan is N-acetylated using acetic anhydride in an aqueous acetic acid solution in which acetic acid is added to water with a molar error of ±25% relative to the chitosan. According to this characteristic, by performing N-acetylation of chitosan using acetic anhydride in an aqueous acetic acid solution in which acetic acid is added to water with a molar error of ±25% relative to chitosan, competition between the N-acetylation reaction and the hydrolysis reaction can be suppressed, and the reaction efficiency of the N-acetylation reaction can be increased. As a result, a high-performance chitosan material can be obtained that has a controlled degree of acetylation and stable solubility with a uniform distribution of N-acetyl groups within the molecule.

[0008] The acetylation of the acetylated chitosan obtained in the aforementioned acetylation step is characterized by being controlled to a degree of 30-60%. This characteristic allows for increased solubility of acetylated chitosan.

[0009] The present invention is characterized by having a substitution step in which the amino groups of the acetylated chitosan obtained in the acetylation step are converted into functional substituents. According to this feature, since the acetylation reaction can proceed in a homogeneous system in which acetylated chitosan with a controlled degree of acetylation and a uniform distribution of N-acetyl groups within the molecule is dispersed, a highly functional chitosan material with improved functionality due to the substituent can be obtained.

[0010] The substituent is characterized by being a guanidino group. According to this feature, since the guanidino group is positively charged even in the neutral range, the solubility and functionality of the highly functional chitosan material in the neutral range can be enhanced.

Brief Description of the Drawings

[0011] [Figure 1] It is a diagram showing a method for producing a highly functional chitosan material in an embodiment of the present invention. (a) is a diagram showing an acetylation step based on a chemical reaction formula, and (b) is a diagram showing a substitution step based on a chemical reaction formula. [Figure 2] It is a diagram showing an example of a 1H NMR spectrum of acetylated chitosan (O-CS, DA45%) measured by 1H NMR analysis.

Modes for Carrying Out the Invention

[0012] Modes for carrying out a method for producing a highly functional chitosan material according to the present invention will be described below based on examples. Hereinafter, in the examples, modes for producing acetylated chitosan and guanidylated chitosan as highly functional chitosan materials will be described as examples.

Examples

[0013] As shown in FIG. 1, the method for producing a highly functional chitosan material in this example (hereinafter, sometimes referred to as "this production method") has an acetylation step (see FIG. 1(a)) in which N-acetylation of chitosan is carried out using acetic anhydride in an aqueous acetic acid solution obtained by adding acetic acid with a molar amount error of ±25% with respect to chitosan to water, and acetylated chitosan as a highly functional chitosan material can be obtained.

[0014] Furthermore, if the amount of acetic acid in the aqueous acetic acid solution is insufficient, it can lead to undissolved chitosan, and if the amount of acetic acid is excessive, it can reduce the reaction efficiency. Therefore, the reaction efficiency of the acetylation reaction can be increased by keeping the error in the amount of chitosan and acetic acid within ±25%, preferably within ±15%, and more preferably within ±5%. The inventors have also confirmed that the reaction efficiency of the acetylation reaction is maximized when there is no error in the amount of chitosan and acetic acid, that is, by adding the same amount of acetic acid as chitosan to water, and keeping the amount of acetic acid in the aqueous acetic acid solution to the minimum necessary.

[0015] Furthermore, this manufacturing method includes a substitution step (see Figure 1(b)) in which the amino group (-NH2) of the acetylated chitosan obtained in the acetylation step is converted to a guanidino group (-NH-(NH=C)-NH2) as a functional substituent, making it possible to synthesize guanidylated chitosan as a high-performance chitosan material.

[0016] In Figure 1, the structural formulas of acetylated chitosan and guanidylated chitosan show the introduced N-acetyl group (-NH-COCH3) with elliptical shading, and the structural formula of guanidylated chitosan shows the substituted guanidino group with triangular shading.

[0017] Hereinafter, the degree of acetylation (DA) will be defined as the proportion of N-acetyl groups in the acetylated chitosan obtained by the acetylation step of this manufacturing method (m / n+m × 100 (%)) and the proportion of N-acetyl groups in the guanidylated chitosan obtained by the substitution step (m / n+m+l × 100 (%)). The degree of guanidylation (DG) will be defined as the proportion of guanidino groups in the guanidylated chitosan obtained by the substitution step (l / n+m+l × 100 (%)). Note that the DA of the acetylated chitosan and guanidylated chitosan before and after the substitution reaction are the same (see Figure 1(b)). In this example, DA and DG were measured from the C / N ratio obtained by CHN elemental analysis (Perkin Elmer 2400II).

[0018] The acetylated chitosan obtained by the acetylation step of this production method can enhance its solubility in the neutral range and the distribution of N-acetyl groups within the molecule can be made uniform by controlling the DA to 30-60%, more preferably 40-60%, and even more preferably 45-55%. The inventors have confirmed this. The distribution of N-acetyl groups within the molecule can be measured by w 1H NMR analysis (see Figure 2). Specifically, 1 the amount of GlcNAc-GlcN (glucosamine unit) sequence and GlcNAc-GlcNAc sequence can be evaluated from the area ratio of the peak at the 1-position of GlcNAc (N-acetylglucosamine unit) in the 1 1H NMR spectrum obtained by 1H NMR analysis. Since the ratio of GlcNAc adjacent to GlcNAc and GlcN is generally consistent with DA from the integration ratio, it can be determined that it is a random sequence, that is, the distribution of N-acetyl groups within the molecule is uniform.

[0019] In addition, the inventors have confirmed that the solubility of the acetylated chitosan obtained by the acetylation step of this production method varies greatly depending on the difference in molecular weight (M n ). The molecular weight (M n ) of the chitosan used in this production method is preferably less than 1.0×10 5 , and more preferably less than 1.0×10 4 .

[0020] In addition, the guanidylated chitosan obtained by the substitution step of this production method can exhibit solubility and functional expression in the neutral range due to its property of being positively charged (pKa = 12.5) even in the neutral range of the guanidino group. However, the inventors have confirmed that the solubility varies greatly depending on the differences in DA, DG, and molecular weight (M n ). For example, the guanidylated chitosan with DA; 11.2%, DG; 56.4%, and M n ; 6.2×10 3 (M w / M n = 1.6) precipitates in water and does not dissolve sufficiently even in an acetic acid aqueous solution, forming a suspension.

[0021] In this manufacturing method, the DA is controlled by N-acetylation using acetic anhydride during the acetylation step, and an acetylated chitosan with a uniform distribution of N-acetyl groups within the molecule is obtained. As a result, the solubility of the acetylated chitosan is enhanced in the substitution step using this acetylated chitosan. Thus, because the substitution reaction can proceed in a homogeneous system in which the acetylated chitosan is dispersed by DA control, it becomes possible to synthesize guanidylated chitosan with controlled DG. Furthermore, compared to cases where the substitution reaction is carried out using unacetylated chitosan or acetylated chitosan with a non-uniform distribution of N-acetyl groups within the molecule, the DG of the synthesized guanidylated chitosan can be increased.

[0022] The following manufacturing method is used for DA and M n Acetylated chitosan and DA, DG, M with different viscosities n Guanidylated chitosans with different viscosity values ​​will be synthesized. n The measurement was performed by gel permeation chromatography analysis.

[0023] First, regarding the acetylation step of this manufacturing method, four types of chitosan shown in Table 1 (O-CS (chitosan obtained by removing lactic acid from SKII manufactured by Koyo Chemical Co., Ltd. through dialysis and freeze-drying), L-CS (FL-80 manufactured by Koyo Chemical Co., Ltd.), M-CS (FM-80 manufactured by Koyo Chemical Co., Ltd.), H-CS (FH-80 manufactured by Koyo Chemical Co., Ltd.)) were dissolved in an aqueous acetic acid solution with the minimum necessary amount of acetic acid, and N-acetylation was performed using acetic anhydride (011-00276 manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.). Specifically, 10.0 g (62.1 mmol) of chitosan and 3.26 g (62.1 mmol) of acetic acid were added to a predetermined amount of purified water in equal amounts, stirred at room temperature for 3 hours, then a predetermined amount of acetic anhydride was added, stirred overnight at 30°C to react, and then dialysis and freeze-drying were performed to obtain solid acetylated chitosan. Furthermore, a stirrer (AS ONE Corporation, MSA-1N) was used for stirring. In order to reduce the viscosity of each chitosan solution to a level that could be stirred with the stirrer, the amount of purified water used was 250 mL for O-CS and L-CS, 500 mL for M-CS, and 800 mL for H-CS.

[0024] [Table 1]

[0025] Tables 2-5 show the DA of acetylated chitosan obtained when the equivalent amount of acetic anhydride used in the acetylation process is varied for the four types of chitosan (O-CS, L-CS, M-CS, H-CS) shown in Table 1. Note that the equivalent amount of acetic anhydride referred to here is the equivalent amount of acetic anhydride calculated for chitosan with 100% deacetylation.

[0026] [Table 2]

[0027] [Table 3]

[0028] [Table 4]

[0029] [Table 5]

[0030] As shown in Tables 2-5, it was confirmed that acetylated chitosan with a DA of 30% or more could be obtained with 0.3 eq equivalents of acetic anhydride, and with 0.5 eq equivalents, with a DA of 45% or more. For example, if chitosan is acetylated using acetic anhydride in a 10% aqueous acetic acid solution where a large excess of acid is present, it is presumed that this will lead to a decrease in the nucleophilicity of the amino group, and approximately 3 eq equivalents of acetic anhydride would be required to achieve a DA of 30%. In contrast, as in this example, by adding the same amount of acetic acid as chitosan to purified water, and minimizing the amount of acetic acid in the aqueous acetic acid solution, the reaction efficiency of the acetylation reaction is increased, and it was confirmed that the distribution of N-acetyl groups within the molecule of acetylated chitosan becomes uniform (see Figure 2). Furthermore, the molar concentration of the 10% aqueous acetic acid solution is 1.7 mol / L, and the molar concentration of the aqueous acetic acid solution in this example is approximately 0.25 mol / L, meaning that the amount of acetic acid is about 1 / 7.

[0031] Next, regarding the substitution step of this manufacturing method, the amino groups of each acetylated chitosan obtained from the four types of chitosan (O-CS, L-CS, M-CS, H-CS) shown in Tables 2-5 above, in which DA is controlled and the intramolecular distribution of N-acetyl groups is uniform, are substituted with guanidino groups to synthesize guanidylated chitosan with controlled DG. Specifically, acetylated chitosan is added to purified water and stirred well, then 1-amidinopyrazole hydrochloride (Tokyo Chemical Industry Co., Ltd., A2055) and triethylamine (NEt3, Fujifilm Wako Co., Ltd., 202-02646) are added and stirred at room temperature for one week to react, and then dialysis and freeze-drying are performed to obtain guanidylated chitosan (O-GCS, L-GCS, M-GCS, H-GCS).

[0032] Tables 6-9 show samples of guanidylated chitosan (O-GCS, L-GCS, M-GCS, H-GCS) with controlled DA and DG. Note that the DG 0% samples in Tables 6-9 (O-1, O-5, O-9, L-1, L-3, L-5, M-1, M-3, M-5, H-1, H-3) are samples of chitosan (O-CS, L-CS, M-CS, H-CS) that have not undergone guanidylation. Furthermore, to reduce the viscosity of each chitosan solution that has not undergone guanidylation to a level that allows for stirring with a stirrer, the amount of purified water used was 2 mL for O-CS, 8 mL for L-CS, 24 mL for M-CS, and 36 mL for H-CS.

[0033] [Table 6]

[0034] [Table 7]

[0035] [Table 8]

[0036] [Table 9]

[0037] Furthermore, the DG control of guanidylated chitosan (O-GCS, L-GCS, M-GCS, H-GCS) was performed by changing the amount of 1-amidinopyrazole hydrochloride relative to acetylated chitosan. Specifically, for O-GCS, it was confirmed that the DG could be controlled to approximately 15%, 30%, and 45% by using 0.9 g (6.2 mmol), 1.8 g (12.4 mmol), and 4.5 g (31.0 mmol) of 1-amidinopyrazole hydrochloride per 1.0 g of acetylated chitosan of each DA (see Table 6).

[0038] Furthermore, for L-GCS, M-GCS, and H-GCS, by using 3.6 g (24.8 mmol) of 1-amidinopyrazole hydrochloride per 1.0 g of acetylated chitosan of each DA, the DA and M n It was confirmed that DG is controlled accordingly (see Tables 7-9).

[0039] Furthermore, since the substitution reaction of the amino group of chitosan in the substitution step is a solid-liquid reaction, the conventional limit was around 23% DG. However, in this example, by increasing the solubility of acetylated chitosan, the substitution reaction can proceed in a homogeneous system in which the acetylated chitosan is dispersed, and it was confirmed that the upper limit can be raised to around 55% DG.

[0040] Next, Table 10 shows the results of solubility evaluations in the neutral range for acetylated chitosan and guanidylated chitosan samples O-1~O-12, L-1~L-6, M-1~M-6, and H-1~H-4, which have controlled DA and DG as shown in Tables 6~9 above. For solubility evaluation, 15 mg of each acetylated chitosan and guanidylated chitosan sample was added to 3 mL of purified water and stirred at room temperature for 5 hours using a stirrer. Samples that were uniformly dispersed but suspended were rated as △, and those that were not uniformly dispersed and precipitated were rated as ×. Furthermore, for samples that were uniformly dispersed and completely dissolved, an additional 5 mg of each sample was added and stirred at room temperature for 5 hours, repeating this process until the concentration reached 30 mg / mL. The concentration at which turbidity was observed (mg / mL) was used as the solubility evaluation.

[0041] [Table 10]

[0042] As shown in Table 10, solubility evaluation in the neutral range confirmed that, regardless of DA, solubility in purified water decreased when DG was between 10% and 30% (O-2, O-3, O-6, O-10, L-6, M-6). This suggests that the guanidino group, in small amounts, contributed to insolubilization through hydrogen bonding.

[0043] Furthermore, samples O-4 and L-2, whose DA was controlled to approximately 30-40% and DG to 40-50%, showed improved solubility in purified water. This suggests that the increased solubility of acetylated chitosan controlled to approximately 30-40% DA allowed for the synthesis of guanidylated chitosan with a high DG, and the increased amount of guanidino groups led to a positive charge-induced improvement in solubility, resulting in more stable solubility.

[0044] Furthermore, it was confirmed that O-6, O-7, O-8, O-10, O-11, O-12, and L-4, which have a DA of approximately 50%, exhibit sufficient solubility in the neutral range, although their solubility decreases upon guanidylation compared to O-5, O-9, and L-3, which have a DA of approximately 50% and a DG of 0%.

[0045] Furthermore, for M-GCS samples M-2, M-4, M-6 and H-GCS samples H-2, H-4, regardless of DA or DG, the molecular weight of chitosan (M n Because the molecular weight (M) of the chitosan used in this manufacturing method is large (see Table 1), it is presumed that the guanidino group forms strong hydrogen bonds at multiple points, making it difficult to dissolve. n ) is 1.0 × 10 5 It is preferable that it be smaller than 1.0 × 10 4 It is even preferable if it is smaller than that.

[0046] As explained above, in the acetylation step of this manufacturing method, by performing N-acetylation of chitosan using acetic anhydride in an aqueous acetic acid solution (water with the same amount of acetic acid as chitosan), competition between the N-acetylation reaction and the hydrolysis reaction can be suppressed, and the reaction efficiency of the N-acetylation reaction can be increased. As a result, acetylated chitosan can be obtained as a high-performance chitosan material with controlled DA and stable solubility, where the distribution of N-acetyl groups within the molecule is uniform.

[0047] Furthermore, the solubility of acetylated chitosan can be increased by controlling the DA of the acetylated chitosan obtained in the acetylation process to 30-60%. In particular, the solubility in the neutral range can be further increased by controlling the DA of acetylated chitosan to 45-55%. Also, the molecular weight (M) of acetylated chitosan n ) is 1.0 × 10 5 It is preferable that it be smaller than 1.0 × 10 4 It is even preferable if it is smaller than that.

[0048] Furthermore, by including a substitution step in which the amino groups of the acetylated chitosan obtained in the acetylation step are converted into functional substituents, the degree of acetylation can be controlled, and the substitution reaction can proceed in a homogeneous system in which acetylated chitosan with a uniform distribution of N-acetyl groups within the molecule is dispersed. This makes it possible to obtain a high-performance chitosan material with enhanced functionality due to substituents.

[0049] Furthermore, in the substitution process, because the functional substituent converted from the amino group of acetylated chitosan is a guanidino group, the guanidino group remains positively charged even in the neutral range. This enhances the solubility and functionality of guanidylated chitosan in the neutral range as a high-performance chitosan material.

[0050] Furthermore, in this manufacturing method, by adding the same amount of acetic acid as chitosan to purified water in the acetylation step, and minimizing the amount of acetic acid in the aqueous acetic acid solution, competition between the N-acetylation reaction and the hydrolysis reaction of chitosan in the aqueous acetic acid solution is suppressed, thereby increasing the reaction efficiency of the N-acetylation reaction. This allows for increased efficiency in the production of guanidylated chitosan with controlled DA. Moreover, since this manufacturing method does not involve adding additives such as methanol to suppress the hydrolysis reaction of chitosan, the reaction solution after the substitution reaction can be used directly as the product.

[0051] Furthermore, guanidylated chitosan has a molecular weight of 30-40% DA, 40-50% DG, and M n ) is 1.0 × 10 5Being smaller allows for more stable solubility in the neutral range.

[0052] Furthermore, guanidylated chitosan synthesized by this manufacturing method exhibits enhanced functionality beyond solubility in the neutral range, due to the positive charge of the guanidino group even in the neutral range. For example, the inventors have confirmed that guanidylated chitosan exhibits a greater ability to bind to proteins than chitosan.

[0053] Furthermore, the inventors have confirmed that guanidylated chitosan exhibits properties that make it more easily absorbed by cells than chitosan.

[0054] Although embodiments of the present invention have been described above with reference to the drawings, the specific configurations are not limited to these embodiments, and any changes or additions that do not depart from the spirit of the present invention are also included.

[0055] For example, in the above embodiment, an embodiment in which acetylated chitosan and guanidylated chitosan are synthesized by a method for producing high-performance chitosan materials has been described. However, the method is not limited to this, and by changing the substituent that is substituted for the amino group of acetylated chitosan in the substitution step, it is possible to apply this to the synthesis of high-performance chitosan materials with various functions. For example, when polyethylene glycol is grafted onto acetylated chitosan in the substitution step, it usually becomes insoluble. However, by increasing the DA in the acetylation step, the solubility of the synthesized polyethylene glycol-grafted chitosan can be improved.

[0056] Furthermore, "high-performance chitosan material" refers to a chitosan material that has at least improved solubility compared to regular chitosan, and also includes chitosan materials in which functional substituents have improved functions (properties) such as protein binding ability and intracellular presence. [Industrial applicability]

[0057] This invention has industrial applicability as a manufacturing method that can be used in a wide range of fields such as food, cosmetics, pharmaceuticals, and medical materials, and easily and stably produces high-performance chitosan materials with improved solubility by substituting the amino groups of chitosan. Furthermore, this invention can, for example, control the degree of acetylation (DA) in acetylated chitosan as a high-performance chitosan material and ensure a uniform distribution of N-acetyl groups within the molecule. In addition, it is possible to control the degree of acetylation (DA) and guanidylation (DG) in guanidylated chitosan as a high-performance chitosan material, and by synthesizing uniform guanidylated chitosan, not only is solubility in the neutral range improved, but functionality is also effectively enhanced, improving ease of binding to proteins and ease of uptake into cells, making it possible to use it in drug delivery systems for protein / peptide pharmaceuticals, thus having a wide range of applications.

Claims

1. A method for producing a high-performance chitosan material, characterized by comprising an acetylation step in which the chitosan is N-acetylated using acetic anhydride in an aqueous acetic acid solution in which acetic acid is added to water with a molar error of ±25% relative to the chitosan.

2. The method for producing a high-performance chitosan material according to claim 1, characterized in that the degree of acetylation of the acetylated chitosan obtained in the acetylation step is controlled to 30 to 60%.

3. A method for producing a high-performance chitosan material according to claim 1 or 2, characterized by comprising a substitution step in which the amino groups of the acetylated chitosan obtained in the acetylation step are converted into functional substituents.

4. The method for producing a high-performance chitosan material according to claim 3, characterized in that the substituent is a guanidino group.

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