JPS5930163B2 - Method for producing N-acylated chitosan - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing N-acyl chitosan, particularly a method for producing reactive chitosan derivatives by reacting chitosan with acylating agents having various functional groups. be.

Chitosan is produced by repeatedly heating chitin, which is a component of the outer skin of shellfish such as shrimp and crabs, to a temperature of 60°C or higher with an alkaline aqueous solution with a concentration of 30 to 50%, such as an aqueous sodium hydroxide solution. It is a substance obtained, and β-(1-4) with D-glucosamine as the basic unit
It is a conjugated polysaccharide.

This substance has a chemical structure similar to that of cellulose, but differs in that it has an amino group, whereas cellulose has a hydroxyl group at the 2nd-position carbon atom of the basic unit.

By the way, the amino group bonded to the carbon atom at the 2-position of chitosan has a higher reactivity with an acylating agent than the hydroxyl group bonded to the carbon atoms at the 3- and 6-position. It was found that since it is large and preferentially acylated to form an amide bond, various derivatives can be obtained using this bond.

The present inventors have conducted intensive research to develop a method for obtaining various N-acylated derivatives by utilizing the reactivity of the amino group of xatone. After formation, an N-acylated product can be obtained by dispersing this in an inert solvent and reacting with an acylating agent; in this case, if an acylating agent having a functional group is used, any functional group can be introduced. Since the amide bond thus formed has excellent chemical stability, the present invention was completed based on this finding.

That is, according to the present invention, chitosan is dissolved in an acid, and the resulting solution is then brought into contact with an alkaline solution to form powder and granules.
N-acylated chitosan can be produced by coagulating it into a fiber or film, dispersing the coagulated product in an inert solvent, and then reacting it with an acylating agent.

At this time, the acylating agent is a functional group such as a halogen atom,
If one having a carboxyl group, carbonyl group, unsaturated group, etc. is used, a reactive N-acylated chitosan can be obtained.

As mentioned above, the chitosan used in the present invention is a substance obtained by deacetylating chitin by heating it with a concentrated alkali.
The amino group value measured by colloid titration using polyvinyl potassium sulfate aqueous solution is 4.37 to 6.20.
(milliequivalent/chitosan dry weight f) is preferable.

In the method of the invention, chitosan is first dissolved in an aqueous solution of an acid such as acetic acid, hydrochloric acid, phosphoric acid, or the like. The acidity of this acidic aqueous solution is not particularly important, as long as it forms a chitosan solution. Usually, for example, 0.5 to 15.0% by weight acetic acid aqueous solution, 0.4 to 7.2% by weight hydrochloric acid aqueous solution, 0.
An acidic aqueous solution such as a 4 to 10.0% by weight phosphoric acid aqueous solution is used. Although the chitosan solution may be used as it is for the next alkaline contact treatment, it is advantageous to add a nonionic surfactant and use it after dispersing and emulsifying it. The acidic aqueous solution or emulsion of chitosan is then, for example, 0.1 to 5
Chitosan is precipitated by contacting with an alkaline aqueous solution of % by weight and coagulated into powder or fibers.

In this case, it is desirable to pour the chitosan solution into the alkaline aqueous solution while stirring vigorously. Further, chitosan can be easily coagulated in the form of a film by applying a chitosan solution onto a flat plate such as glass, drying the plate, and immersing the plate in an alkaline aqueous solution. As the alkaline aqueous solution used for precipitation and coagulation of chitosan, for example, a sodium hydroxide aqueous solution, a potassium hydroxide aqueous solution, or aqueous ammonia is conveniently used.

The powdery or fibrous chitosan coagulated by the alkaline aqueous solution is separated, usually washed with water, dried, and then dispersed in an inert solvent.

In that case, the coagulated chitosan is separated, washed thoroughly with water without drying to remove anolekali, washed with a water-soluble solvent such as alcohol to replace the water, and then washed with an inert solvent to be dispersed. Alternatively, the chitosan may be dispersed in an inert solvent by immersing it in the solvent to replace the solvent. The inert solvent is used in the next step of the acylation reaction.
A solvent that does not show any activity towards the acylating agent or reaction product and has no effect on the acylation reaction, and is generally used for aprotic amino compounds, aliphatic ether compounds,
Aromatic hydrocarbon compounds and aliphatic chlorine compounds are included, and typical examples include dimethylformamide, dimethylacetamide, N-methylpyrrolidone, benzene, toluene, xylene, diethyl ether, diisopropyl ether, di-n- Mention may be made of butyl ether, dioxane, tetrahydrofuran, chloroform, and carbon tetrachloride. These solvents may be used alone or in combination of two or more, and alcohols may also be used in combination. Preferred examples of these alcohols include methanol, ethanol, n-propanol, and isopropanol. When lower alcohols are present in an inert solvent, after the above-mentioned chitosan is precipitated with an alkaline aqueous solution and coagulated, the powdery or fibrous material is washed with water, replaced with alcohol, and then directly poured into the inert solvent and dispersed. Not only is this extremely convenient, but it also has the effect of suppressing acylation of the hydroxyl group of chitosan. Next, the chitosan dispersed in the inert solvent is acylated using a predetermined acylating agent.

In the method of the present invention, a carboxylic acid anhydride or a carboxylic acid chloride is advantageously used as the acylating agent, and representative examples thereof include acetic anhydride, monochloroacetic anhydride, dichloroacetic anhydride, propionic anhydride, etc. acid, butyric anhydride, acrylic anhydride, methacrylic anhydride, benzoic anhydride, acetyl chloride, acroyl chloride, methacryloyl chloride, and the like. An inert solvent dispersion of chitosan in the form of powder, fiber, tape or film is
There is no particular limit to the dispersoid concentration as long as stirring is not hindered and a smooth acylation reaction is possible.

The acylation reaction is preferably carried out by adding an acylating agent to the chitosan dispersion and stirring under moderate temperature conditions. The amount of the acylating agent used varies depending on the desired degree of acylation, but in reactions targeting amino groups, it is
About 0.5 to 15 times the mole amount is used. When using two or more types of acylating agents, their total amount should be within the above molar ratio range. In that case, two or more types may be mixed and subjected to the reaction, or one component may be added and subjected to the reaction. After completion, other acylated components can be added and reacted to control the degree of acylation of the desired specific component.

The reaction can also be carried out under heating, for example at 30°C.
It is advantageous to carry out the reaction for 10 to several tens of hours under the following mild temperature conditions.

When obtaining acylated chitosan by reacting an acylating agent with chitosan, the acylating agent usually reacts with the amino groups and hydroxyl groups in chitosan, forming amide bonds and ester bonds, respectively, to form acylated products. When an aprotic amide compound is used as an inert solvent, or a mixed solvent of this compound and another inert solvent is used, the acylation of the hydroxyl group of chitosan is suppressed and the N-acylated product is selectively produced. Obtainable.

The degree of substitution of acyl groups in acylated chitosan containing N-acylation produced by the method of the present invention can be easily calculated by elemental analysis and determining the composition ratio of nitrogen element to carbon element (N/C value). can.

For example, the degree of substitution for chloroacetylated chitosan, ie, the degree of acetylation (DS), is calculated by the following formula. DS-{0.5831/(N/C)}-3 (The number 0.5831 in the formula is determined by the type of acylating agent.

) Moreover, the acylation of acylated chitosan can be confirmed by infrared absorption spectrum.

By acylation, absorption bands in the 1630-1670 (177!-1 region (゛amide I) and 1530-1555cfn-1 region (゛amide)) based on the amide group derived from the amino group, and the hydroxyl group are acylated. The carbonyl of the ester bond formed by (〉C-0: 1720
Absorption bands in the ~1750C7rL-1 region are observed, and the intensity of these absorption bands increases in proportion to the increase in the degree of acylation of the respective amino and hydroxyl groups. This means that the acylation of chitosan involves a reaction in which the amino group at the 2-position of the glucosamine unit, which is a component of chitosan, is acylated to form an amide group, and the hydroxyl group at the 6-position and the hydroxyl group at the 3-position are acylated. This indicates that the reactions that form the ester bond usually occur simultaneously, and the ratio of the degree of acylation of the hydroxyl group to the degree of acylation of the amino group is determined by the absorption intensity of the ester carbonyl group relative to the absorption intensity D of amide I, D, 67O. D
The value of the ratio (4) 1750/Dl67O of l75O can be estimated. According to the method of the present invention, reactive groups such as chloroacetyl group, acryloyl group, methacryloyl group, etc. can be introduced into chitosan as desired, and the characteristics of each reactive group can be utilized. For example, substances useful as intermediates for ion exchange resins, chelate resins, catalyst-supporting resins, etc., or as resins for enzyme immobilization and gels for chromatography can be obtained. The present invention will be explained in more detail with reference to Examples below.

Example 1 Chitosan with amino group value 5.92 meq/7 0.207
was dissolved in 5% acetic acid aqueous solution 107, and surfactant Span-60 (sorbitan monostearate) 1.
An emulsion was prepared by adding 30 d of 0% benzene solution.

This emulsion was added at once to 200 ml of a 2.5% aqueous sodium hydroxide solution while stirring vigorously, continued stirring for 40 minutes, allowed to stand for 1 hour, and then filtered through p.
Insoluble matter was collected. The insoluble precipitate is removed by washing with alcohol to remove the containing active agent (Span-60).
Washing with water was repeated until the mixture became neutral. The precipitate was then washed with methanol and then with dimethylacetamide, and excess solvent was removed by filtration. Chitosan beads were obtained as a precipitate wetted with dimethylacetamide.
Dimethylacetamide 107! A dispersion was prepared by adding 2.2V of chitosan beads moistened with a solvent to Ll, and anhydrous monochloroacetic acid was added while stirring the dispersion. acid 0.917
was added, and the reaction was carried out at room temperature for 26 hours.

The product was separated from the solvent by filtration, washed with methanol,
Subsequently, washing with water was repeated until the mixture became neutral, and then the mixture was replaced with methanol and then with benzene, and freeze-drying was performed. The dry weight of the product was 0.1587. The degree of chloroacetylation was 1.17, the chlorine content was 15.4%, and the absorbance ratio Dl75O/Dl67O was 0.21. In addition, using dimethylformamide instead of dimethylacetamide, chitosan beads were dispersed in 10 ml of dimethylformamide, and monochloroacetic anhydride was reacted for 47 hours to obtain a monochloroacetylation degree of 1.22, a chlorine content of 15.5%, an absorbance ratio of D,
75O/Dl67O75SO. 42 reaction products were obtained. Example 2 Chitosan beads prepared in Example 1 2.2
0.083f7 of monochloroacetic anhydride was added to a solution in which 7 (solvent-containing) was dispersed in 10d of dimethylacetamide, and the reaction was carried out at room temperature for 22 hours.

After that, 0.969% of acetic anhydride was added to the reaction solution. 26 by adding
Allowed time to react. The reactive product was separated from the solvent by filtration, washed with methanol, followed by repeated washing with water, replaced with methanol, then with benzene, and freeze-dried. The dry weight of the product is 0.132y, and the degree of substitution of its chloroacetyl and acetyl groups is 1.08.
Chlorine content is 8.7%, absorbance ratio Dl75O/Dl67O
was 0.12. Similarly, 0.87 g of benzoic anhydride was added to chitosan beads dispersed in dimethylacetamide.
After adding y and reacting for 23 hours, the degree of substitution of the benzoyl group in the reaction product was 0.48, and strong absorption based on the amide group was observed in the infrared absorption spectrum at 1650 (177!-1 and 1540Cf1L-1). However, the absorption near 1720CT!L-1 due to the carbonyl group of the ester bond is extremely weak, confirming that the benzoyl group is selectively bonded to the amino group.

Example 3 To a solution of 0.21 t of chitosan dissolved in a 5% acetic acid aqueous solution, 30 ml of a 2% Span-60 benzene solution was added and shaken to prepare an emulsion.

This emulsion was added to 200 ml of a 2.5% aqueous sodium hydroxide solution while stirring vigorously, and the mixture was stirred for an additional hour and then allowed to stand for an hour. This was filtered through P to collect the precipitate, washed with ethanol to remove the activator, and then washed with water repeatedly until the pH of the washing solution reached 6.0. After that, it was washed with ethanol again and replaced with tetrahydrofuran, and the result was 6.85? of wet chitosan beads were obtained. After soaking 2.757 of these wet chitosan beads in tetrahydrofuran and replacing the solvent, 20ml of tetrahydrofuran was added.
0.79 y of monochloroacetic anhydride was added to the chitosan bead dispersion suspended in water, and the mixture was reacted for 66 hours.

After the reaction, the precipitate was separated, washed with methanol and water repeatedly, then replaced with methanol and benzene, and freeze-dried. The dry weight of the reaction product obtained was 0.1
467, degree of chloroacetylation 1.67, chlorine content 17.
9%, and the absorbance ratio Dl75O/Dl67O was 0.62. Example 4 After soaking 4.107 of the wet chitosan beads prepared in Example 3 in dioxane and replacing the solvent, dioxane 20WLI! 0.907% of monochloroacetic anhydride was added to the dispersion, and the mixture was reacted for 66 hours.

After the reaction, the precipitate was washed with methanol and repeatedly washed with water, then replaced with methanol, then benzene, and freeze-dried. The dry weight of the reaction product obtained was 0.171y1
The degree of chloroacetylation was 1.43, the chlorine content was 1.27%, and the absorbance ratio Dl75O/Dl67O was 0.64. Example 5 Chitosan (value of amino group 5.92 meq/i) 4.0
r was dissolved in 200d of 10% acetic acid aqueous solution to prepare a 2% chitosan solution.

15 ml of a 2% benzene solution of span 60 was added to 5.0 y of this chitosan solution to form an emulsion.

Add 2.5% of this emulsion solution while stirring vigorously.
The chitosan was poured into 200 d of aqueous sodium hydroxide solution and stirred for 30 minutes to solidify the chitosan. The precipitate was collected, washed with methanol, and then washed with water repeatedly until it became neutral. After washing the chitosan beads with water, the adsorbed water was replaced with methanol, and the beads were further replaced with benzene, and then dispersed in 20 ml of benzene. Alcohol was added to this dispersion, and monochloroacetic anhydride was added to cause a reaction, and the amount of the reaction product obtained, the degree of chloroacetylation, the chlorine content, and the absorbance ratio Dl75O/Dl67O were examined. The reaction was carried out in the same manner by changing the type and amount of alcohol added, and the results are shown in Table 1 below along with the conditions of the reaction system. Note that the results obtained when no alcohol was added and the amount of monochloroacetic anhydride used was varied widely are also summarized in the table. For comparison, unlike the method of the present invention, 1 y of a 2% span 60 benzene solution was added to 5.0 y of a 2% chitosan solution.
Add 5 ml of it to emulsify it, add 7.0 d of 5% aqueous sodium hydroxide solution, and directly add a solution of 1.767 ml of monochloroacetic anhydride dissolved in 5.0 WLI of benzene to the reaction mixture containing the coagulated and precipitated chitosan. After stirring for a period of time, the mixture was allowed to stand for one day, and then the precipitate was separated, washed, and dried to obtain a solid having a particle size of 0.0557, and its infrared absorption spectrum showed a pattern similar to that of chitosan. In addition, we attempted to separate the precipitate after applying a benzene solution of acrylic anhydride in a similar manner, but the product swelled significantly in the aqueous medium, and almost no N-acylation of chitosan occurred. I found out that it wasn't. In addition, instead of the chitosan precipitated by the method of the present invention, chitosan powder obtained by saponifying chitin (particle size 80 to 150 mesh, amino group value 5.92 meq/y by colloid titration) 0
．． When 036y was dispersed in benzene and 1.60y of monochloroacetic anhydride was applied for 24 hours, the yield of the product was 0.036y.
It was 0407.

The degree of chloroacetylation of this product is 0.71. Chlorine content is 8.5%, absorbance ratio Dl75O/Dl67O is 0.82
It was hot. Therefore, it was found that this reaction product was locally chloroacetylated, unlike the reaction product obtained by the method of the present invention. Example 6 The chitosan beads prepared by the method of Example 5 were immersed in dimethylacetamide to replace the solvent, then dispersed in 20d of dimethylacetamide, 1.1y of acrylic anhydride was added, and the mixture was reacted with stirring for 48 hours.

The yield of the product is quantitative, the degree of substitution of acryloyl group is 1.30, and the absorbance ratio Dl728/Dl665 is 0.6.
It was 7. Example 7 A solution of chitosan 4.07 dissolved in 200 d of a 10% aqueous acetic acid solution was applied onto a glass plate, and after drying, the plate was immersed in a 0.5% aqueous sodium hydroxide solution to coagulate a chitosan film.

The coagulated film was peeled off from the glass plate, washed with water, and then immersed in methanol and benzene. The film (thickness 19~
24μm) is suspended in 20ml of benzene, and 1.67ml of monochloroacetic anhydride is added thereto to carry out the reaction.
Films showing strong absorption of amide I and amide at 1670CTrL-1 and 1545 (V7!-1, respectively) and absorption of carbonyl of ester bond at 1750-1 were obtained.For comparison, films were obtained that were not based on the method of the present invention. When the prepared chitosan film was dried and then immersed in benzene and treated with 1.6y monochloroacetic anhydride for 38 hours, it showed weak absorption at 1670C1rL-1 and 1545U-1, and a weak absorption at 1750C1n-1. It is clear that a film with only weak absorption was obtained, and acylation is extremely unlikely to occur.The attached drawings show infrared absorption spectra of each film obtained by the above method, and a indicates This is the absorption spectrum of a chitosan film treated with monochloroacetic anhydride regardless of the method, and the absorption spectrum of an untreated chitosan film is shown as a dotted line for comparison.

b is an absorption spectrum of the chloroacetylated chitosan film obtained by the method of the present invention. As is clear from both spectra, a has almost the same absorption tendency as the original chitosan film, whereas b has three sharp peaks due to chloroacetylation. Example 8 A chitosan film prepared from a chitosan solution according to the method of Example 7 was suspended in benzene 100", and acrylic anhydride was added thereto at 0.55 V and reacted for 42 hours.

After the reaction, it was washed with benzene, immersed in methanol for 10 minutes, washed with water repeatedly, and then dried. The product is a tough, transparent film, 1662C! RL-1 and 1550CI! The strong absorption of amide group in L-1 is also 1
728c! Absorption of ester group was observed at n-1. Using methacrylic anhydride instead of acrylic anhydride and treating in the same manner, a similarly tough and transparent film was obtained.

That film is. 1660cm-1 and 1550c7n-
1 shows strong absorption of amide group, 1750c! The absorption based on the ester bond near n-1 was extremely weak.

[Brief explanation of the drawing]

The figure shows infrared absorption spectra of a chitosan film and its monochloroacetic anhydride-treated film, in which a is obtained by a method other than the present invention, b is obtained by a method of the present invention, and a dotted line is that of an untreated chitosan film.

Claims (1)

[Claims] 1. Chitosan is dissolved in an acid, and this solution is brought into contact with an alkaline solution to coagulate it into powder, fibrous or film form,
A method for producing N-acylated chitosan, which comprises dispersing this coagulated material in an inert solvent and reacting it with an acylating agent. 2. The manufacturing method according to claim 1, wherein the acylating agent is one or more selected from acetic anhydride, monochloroacetic anhydride, acrylic anhydride, and methacrylic anhydride. 3. Claim 1, wherein the inert solvent is one or more selected from aprotic amide compounds, aromatic hydrocarbon compounds, aliphatic chlorine compounds, and aliphatic ether compounds. manufacturing method. 4. The manufacturing method according to claim 3, wherein the aprotic amide compound is dimethylformamide, dimethylacetamide or N-methylpyrrolidone. 5. The manufacturing method according to claim 3 or 4, wherein the inert solvent is a mixed solvent containing alcohol.