JPH1171407A - Hydrophilic chitin derivative and preparation thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a hydrophilic chitin derivative having a fundamental chemical structure of chitin not largely damaged, the original chitin biocompatibility, moreover possible to be water-soluble despite of the high mol.wt., and to provide a process for preparing such hydrophilic chitin derivative. SOLUTION: This chitin derivative is expressed by the formula (wherein n1+n2+n3>5, and n1/(n1+n2+n3)>0.2; and R1 is a polyoxyalkylene group having an average degree of polymn. of 10-300). The chitin derivative is prepd. by reacting a polyalkylene compd. having at the one end an aldehyde group with chitin or chitosan to synthesize a Schiff base, reducing it pref. after having neutralized the reaction solution, and then acetylating it.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention provides a chitin derivative which has high biocompatibility and safety, and is excellent in water solubility, aqueous gel properties, and water swellability, and a method for producing the same.

[0002]

2. Description of the Related Art Chitin is a polymer composed mainly of N-acetyl-D-glucosamine linked by an ether bond at the β-1,4 position. In the chemical formula shown below, n3 = 0, n1 / (n1 + n2 + n3)>
0.2, that is, a polymer comprising only N-acetyl-D-glucosamine units and its deacetyl derivative units and having a degree of deacetylation of 20% or less (a degree of deacetylation of 8%).
Those with more than 0% are called chitosan. )

Chitin, a polymer having such a chemical structure, is a substance obtained from raw materials such as shells such as crab, shrimp, and squid bone, and is derived from natural products and has a leukocyte migratory property and a wound healing effect. It is a compound having useful properties such as physiological activity. However, chitin is practically insoluble in water and various organic solvents except for a special organic solvent and a strong alkaline liquid, and thus has a very limited use. Chitin having a degree of acetylation of 50% (the ratio of acetylamino groups to amino groups is 50/50) is known to be water-soluble, but its use is limited to special products such as molded articles.

[0004] As a technique for improving the above-mentioned disadvantages of chitin, and in particular for improving the hydrophilicity of chitin and expanding its use, hydrophilic chitin having a polyoxyethylene group introduced into the molecule, and chitin There is a method of introducing a polyoxyethylene group into a molecule of the above, which is disclosed in the following prior art documents.

(1) A method in which a polyoxyalkylene group is introduced by adding 10 or less alkylene oxide to each amino group and hydroxyl group in the presence of an alkali to at least one of the amino groups and hydroxyl groups of all glucosamine units. (2) A method of reacting alkali chitin with polyoxyethylene halohydrin to introduce a polyoxyethylene group into chitin. (Japanese Patent Application Laid-Open No. Sho 64-14201) (3) Chitin in which at least 5 alkylene oxides are added to each amino group and hydroxyl group to at least one of the amino groups and hydroxyl groups of all glucosamine units. (Japanese Patent Laid-Open No. 5
(4) A biocompatible and biologically inactive conjugate comprising chitin and PEG. (JP-A-7-278203) (5) The amino group of glucosamine unit is produced by simultaneously reacting the amino group of glucosamine produced by hydrolyzing chitin with polyoxyethylene glycol having one end aldehyde and sodium cyanoborohydride. A method of introducing one polyoxyalkylene group into (J. Polyme
r Sci. Polymer Chem. Ed. , 2
2, 341-352 (1984))

[0006]

However, the water-soluble chitosan derivatives disclosed in the above-mentioned known documents (1), (2) and (3) have three active hydrogens based on a hydroxyl group and an amino group of glucosamine. And derivatives in which a polyoxyalkylene group is bonded.Such a derivative certainly improves hydrophilicity, but the degree of denaturation, that is, the degree of change in the basic chemical structure is large, and the improvement in hydrophilicity and accompanying Although the moisturizing property and the like are good, useful properties such as leukocyte migration property and wound healing effect specific to chitin are impaired to a large extent.
Further, although (1), (2) and (3) also include a chitin or chitosan derivative with a small degree of modification in which one polyoxyalkylene group is bonded to an amino group, repetition of the alkylene oxide is described. Since the number is 10 or less, such derivatives are not sufficiently hydrophilic.

The hydrophilic chitosan or chitin derivative disclosed in the above-mentioned known document (4) is a compound in which polyoxyethylene glycol and chitin or chitosan are bonded by an amide bond or the like, and the polyoxyethylene glycol is bound at both ends. Reactions are disclosed. Such materials have increased hydrophilicity but produce only gel-like compounds and are therefore of very limited use.

The technique described in the known document (5) aims at selectively binding polyoxyethylene glycol to the amino group of glucosamine. It is produced by simultaneously reacting sodium cyanoborohydride with chitosan in an aqueous solution, and does not reliably react with amino groups, but has a high probability of becoming a simple mixture with polyoxyethylene glycol.
Moreover, there is no description of the molecular weight of the used chitosan or the confirmation of whether the product is water-soluble or the like. Further, it does not examine the properties of chitosan as chitin by acetylation.

It is an object of the present invention to provide a chitin that does not significantly impair its basic chemical structure, retains its useful properties such as leukocyte chemotaxis and wound healing effect, and has a high molecular weight. An object of the present invention is to provide a chitin derivative that can be made water-soluble, and a production method capable of reliably synthesizing such a hydrophilic chitin derivative with little contamination of impurities such as polyethylene glycol.

[0010]

The present invention relates to a hydrophilic chitin derivative comprising N-acetyl-D-glucosamine and its derivative unit linked by an ether bond at the β-1,4 position, N1 + n
2 + n3> 5, n1 / (n1 + n2 + n3)> 0.2, in which R ₁ to provide a hydrophilic chitin derivatives is an average polymerization degree of 10 to 300 polyoxyalkylene group (POA).

[0011]

Embedded image

In the formula (3), the units of N-acetyl-D-glucosamine and its derivatives constituting the chitin of the present invention may be bonded in blocks or randomly. Note that n3 is not particularly limited as long as the desired characteristics of the present invention can be obtained, and is a numerical value larger than 1. However, if it is too large, the degree of denaturation of chitin increases, which is not preferable. Also, n1
, N2 and n3 are all average values.

By satisfying the condition of n1 + n2 + n3> 5, it becomes a compound which can sufficiently exhibit the original properties of chitin. The upper limit of n1 + n2 + n3 is not particularly limited, but the hydrophilic chitin derivative of the present invention is produced using natural chitin as a raw material, and the molecular weight of the natural product is substantially the upper limit. In particular, n1 + n2 + n3>
In the case of No. 8, the water-insoluble chitin can be made into a derivative which can be dissolved in distilled water or physiological saline with almost no change in its properties, and the use thereof can be expanded.

By satisfying the condition that the ratio of acetylated D-glucosamine units is n1 / (n1 + n2 + n3)> 0.2, the basic structure of chitin is not greatly altered or modified, and therefore chitin specific to chitin. Hydrophilicity is improved while maintaining physiological activities such as leukocyte migration and wound healing effects. When this value is 0.2 or less, the substance is classified as chitosan, and its properties appear strongly.

Furthermore, when the average degree of polymerization of the polyoxyalkylene group which binds to an amino group and imparts hydrophilicity is 10 or less, the hydrophilicity is poor.
Chitin cannot be given sufficient hydrophilicity. When the average degree of polymerization of the polyoxyalkylene group is 300 or more, the solution viscosity of the obtained water-soluble chitin is undesirably too high. When a polyoxyalkylene group having such a degree of polymerization is used, the hydrophilicity is improved even if the portion where the polyoxyalkylene group is bonded to the amino group among the D-glucosamine units is improved, and high molecular weight chitin is made water-soluble. Can also. That is, a highly hydrophilic chitin derivative which maintains the basic chemical structure of chitin and retains its original properties of biological activity and biocompatibility can be obtained. Here, the average degree of polymerization means a number average molecular weight determined by a terminal group determination method.

In the hydrophilic chitin derivative according to the first aspect, the polyoxyalkylene group (POA) is preferably a polyoxyethylene group (PEG). The polyoxyethylene group is a group having high hydrophilicity and is not decomposed in digestive organs of mammals and the like, and is suitable for imparting hydrophilicity to chitin. The other end of the polyoxyalkylene group bonded to chitin is preferably a hydroxyl group or an alkyloxy group having 1 to 4 carbon atoms.

As compared with the polyoxypropylene group, the polyoxyethylene group has a higher hydrophilicity, and a water-soluble chitosan derivative can be obtained even with a small polyoxyalkylene group content.

The content of POA in the hydrophilic chitin derivative of the present invention is not limited as long as chitin has a predetermined hydrophilicity and the properties of chitin are exhibited. The characteristics of chitosan can be confirmed, and when it is 80% by weight, the characteristics of chitosan are strongly recognized, which is preferable.

The present invention relates to a hydrophilic chitin derivative comprising N-acetyl-D-glucosamine or a derivative thereof bonded by an ether bond at the β-1,4 position and having a polyoxyalkylene group bonded to a nitrogen atom. The present invention also relates to a method for producing a hydrophilic chitin derivative, wherein, in the case of claim 3, a dissolving step of dissolving chitosan in an aqueous solution of an acid, wherein the chitosan and one aldehyde group in a molecule are used. Having an average degree of polymerization of 1
A Schiff base forming step of reacting a polyoxyalkylene compound of 0 to 300 to form a Schiff base, a reduction step of reducing the Schiff base, and an acetylation step of acetylating at least a part of unreacted amino groups. It is characterized by including. All unreacted amino groups may be acetylated.

The invention according to claim 4 provides a dissolving step of dissolving or swelling chitin in a solvent, using chitin or chitin in which a part of an acetyl group is hydrolyzed and deacetylated as a raw material, A polyoxyalkylene compound having an average degree of polymerization of 10 to 300 in which the alkylene oxide having one aldehyde group therein is reacted to form a Schiff base, and a reduction step of reducing the Schiff base is included. It is characterized by the following. This compound has a structure in which the amino group is already acetylated, and it is not necessary to specifically acetylate the compound.
In a preferred embodiment, an acetylation step of acetylating at least a part of unreacted amino groups is further provided in the same manner as in the production method using water-soluble chitosan as a starting material.

The reaction of forming a Schiff base between a polyoxyalkylene compound having an aldehyde group and chitin occurs in a homogeneous system, that is, a solution system or a heterogeneous system, that is, a swollen system. The reaction may be performed by dissolving in a solvent, or may be performed by swelling with an organic solvent or the like. In particular, chitin having a degree of acetylation of 50% is soluble in water and is suitable as a raw material of the present invention.

In a preferred embodiment, the method for producing hydrophilic chitin of the present invention further comprises a neutralization step of neutralizing a solution or a swelling solution containing the Schiff base, after the Schiff base formation step. Due to the presence of such a neutralization step, the equilibrium of the Schiff base formation reaction shifts in the direction of larger Schiff base formation, and the loss can be reduced when a reducing agent is used in the next reduction step. is there. That is, for example, when sodium cyanoborohydride is used as the reducing agent, the reducing agent has a higher reactivity with hydrogen ions than the reactivity with the Schiff base, so that the neutralizing step is not provided. By comparison, the reaction with the acid used to dissolve chitin and chitosan results in higher consumption.

According to the present invention, a polyoxyalkylene group can be surely introduced into an amino group of a D-glucosamine unit produced by hydrolyzing a part of a chitin skeleton. The fundamental difference from the above prior art (5) is the presence of a Schiff base forming step for reliably forming a Schiff base and an acetylation step. Without the step of reliably forming the Schiff base, the aldehyde group is reduced faster than the Schiff base in the reduction step, and the original polyoxyalkylene glycol is formed, lowering the yield of the desired hydrophilic chitin derivative. A big problem arises. Furthermore, when a reducing agent is used in the reduction step, there is also a problem that the reducing agent is consumed in the reduction of the aldehyde group and the reaction efficiency is reduced. Further, by providing the acetylation step, even when chitosan is used as a raw material, the obtained hydrophilic chitin derivative has a specific leukocyte migration property, a wound healing effect and the like.

The introduction of polyoxyalkylene groups into chitin, which is partially deacetylated and inherently water-soluble, has the advantage of further improving the stability of the solution.

For the above-mentioned reason, in the method for producing a hydrophilic chitin derivative of the present invention, the obtained chitin derivative is represented by the above-mentioned chemical formula (Formula 3), and n 1 + n 2 + n 3
> 5, particularly preferably n1 + n2 + n3> 8, n1 /
(N1 + n2 + n3)> 0.2, that it R ₁ is an average polymerization degree of 10 to 300 polyoxyalkylene group of the alkylene oxide, and the polyoxyalkylene group is a polyoxyethylene group preferable.

In a preferred embodiment of the method for producing a hydrophilic chitin derivative of the present invention, a washing step of washing the obtained hydrophilic chitosan derivative or hydrophilic chitin derivative with an organic solvent is provided. This washing step
It may be provided before the acetylation step and used for purifying the chitosan derivative, or may be provided after the acetylation step to purify the hydrophilic chitin derivative, but is preferably used for the purification of the chitosan derivative.

The chitosan or hydrophilic chitin derivative to which POA is bound contains POA used as a raw material as an impurity, and it may be necessary to remove this depending on the use. In such a case, by washing with an organic solvent that dissolves the POA but has low solubility of the chitosan derivative or the chitin derivative, it is possible to effectively and easily remove the POA as an impurity, and to achieve high purity chitin. Derivatives can be obtained.

The hydrophilic chitin derivative of the present invention has a high hydrophilicity and can be made water-soluble even with a high molecular weight, and can be formed into various shapes when formed into an aqueous solution. By reacting with the agent, it is possible to obtain a molded article which is insoluble in water while maintaining its specific activity and high hydrophilicity.

[0029]

BEST MODE FOR CARRYING OUT THE INVENTION Chitin or chitosan used as a raw material in the present invention can be obtained by partially modifying the chemical structure of chitin that naturally exists as an organic skeletal substance such as arthropods and mollusks. . Specific examples of obtaining chitin include shells such as crab, shrimp, and okami,
Calcium carbonate is removed by using squid bone or the like as a raw material, pulverizing it, and treating it with hydrochloric acid. Then, when treated with caustic soda, proteins and other contaminants are removed, and chitin can be obtained. The substance used as a raw material in the present invention may be chitin, which is a natural product obtained as described above, a chitin derivative obtained by partially hydrolyzing and deacetylating chitin of a natural product, or acetylated chitin. Chitosan having almost all amino groups as amino groups.

The process for synthesizing the hydrophilic chitin derivative of the present invention from chitin or chitosan having an amino group is as follows. Dissolution step of dissolving chitosan in aqueous acid solution This is a step of dissolving commercially available chitosan in an aqueous acid solution. Acids that can be used in the dissolving step of dissolving this chitosan include inorganic acids such as hydrochloric acid and phosphoric acid, organic carboxylic acids such as formic acid, acetic acid, propionic acid, tartaric acid, malic acid and phthalic acid, and p-toluenesulfonic acid and the like. Examples thereof include organic sulfonic acids and the like.
A mixture of more than one species can be used. When dissolving water-soluble chitin, an aqueous solution of such an acid may be used.

In this step, a water-soluble organic solvent can be used if necessary, which is also a preferred embodiment. By using such an organic solvent, the solubility of chitosan and the solubility of the reactants can be adjusted and the solution viscosity can be reduced, and the following reaction can be efficiently performed. Examples of such organic solvents include alcohols such as methanol and ethanol, ketones such as acetone and MEK, cellosolves, ethers such as tetrahydrofuran and dioxane, and N-methylpyrrolidone and pyridine.
More than species are available. In particular, the use of methanol is preferable because of its large solubility and viscosity decreasing effect.

In the case of the invention described in claim 4,
In the dissolving step, unlike the case of the invention described in claim 3, chitin is dissolved or swelled. The solvent used in this step is water-soluble in the case of chitin having a degree of acetylation of about 50%, water is used, and in the case of dissolving other water-insoluble chitin, calcium chloride hydrate is used. Methanol saturated solution and saturated solution of lithium chloride in dimethylacetamide are suitable solvents. When the reaction is performed in a swollen state, use of an organic solvent such as dimethylsulfoxide (DMSO) or pyridine or a mixed solvent of these and water is preferable. .

An aqueous solution of chitosan obtained in a Schiff base forming step of forming a Schiff base by reacting the chitosan or chitin with a polyoxyalkylene compound having one aldehyde group in the molecule (the invention according to claim 3) Alternatively, this is a step of adding a polyoxyalkylene compound having an aldehyde at one end to a chitin solution or a swelling liquid (the invention according to claim 4) and reacting the mixture. This reaction is carried out at room temperature or under heated conditions.

As a method for synthesizing the aldehyde-modified polyoxyalkylene compound, any method known to those skilled in the art can be used, and in particular, dimethyl sulfoxide (DMS)
An O) -acetic acid oxidation method, a bromoacetaldehyde diethyl acetal substitution reaction method, and the like can be used because a commercially available polyoxyalkylene glycol can be used as a raw material.

The polyoxyalkylene compound used in this step may have hydroxyl groups at both ends or only one end may have a hydroxyl group. Those having hydroxyl groups at both ends are polyoxyalkylene glycols, which are obtained by ring-opening addition polymerization of alkylene oxide to water or low molecular weight glycols such as ethylene glycol, and those having hydroxyl groups at one end are monoalcohols such as methanol. It is obtained by subjecting an alkylene oxide to ring-opening addition polymerization to phenol or phenol.

Reduction Step for Reducing the Schiff Base As the reducing agent used in this reduction step, sodium cyanoborohydride, sodium borohydride and the like can be used, and a noble metal catalyst such as Pt and a metal catalyst such as Raney nickel It is also possible to use catalytic reduction using When a reducing agent is used, sodium cyanoborohydride is most preferred from the viewpoint of reaction rate, yield, and the like.

Acetylation step of acetylating chitin or chitosan having a polyoxyalkylene group In this step, all or a part of the amino groups other than those to which the polyoxyalkylene group is bonded are acetylated to obtain a hydrophilic chitin derivative. . As the acetylating agent used in this step, all compounds generally used as acetylating agents can be used, and examples thereof include acetic anhydride and acetyl chloride. In the acetylation step, it is preferable to use an organic solvent generally used in the acetylation reaction, acetic acid, methanol, dioxane,
Pyridine, DMSO and the like are exemplified as suitable solvents, and these are used alone or as a mixture.

The acetylation reaction is preferably carried out in a homogeneous system by dissolving chitosan or chitin to which POA is bound, but it is also possible to carry out the reaction in a heterogeneous system in which these are precipitated or swollen. It is. The hydroxyl groups of chitosan and chitin may also be acetylated.However, this reaction uses a mixture ratio of the solvent used, particularly methanol, to control the compounding amount, and acetic anhydride for the amino group and the hydroxyl group. The acetylation reaction of the hydroxyl group of chitosan or chitin can be suppressed by controlling the equivalent ratio of the chitosan or chitin, and the acetylation reaction of the amino group can be preferentially performed.

When a strong acetylating agent such as acetyl chloride is used, the hydroxyl group of chitin may be acetylated, and the chemical structure of chitin may be modified to a large extent. By providing a step of treating with an alkaline solution such as a KOH / ethanol solution, it is possible to return to the original hydroxyl group, which is preferable.

By providing a neutralization step for neutralizing the solution containing the generated Schiff base prior to the reduction step, the equilibrium of the Schiff base formation reaction can be shifted in an advantageous direction. It is preferable that the reduction is efficiently performed in the reduction step.

When a POA having an average degree of polymerization of 13 or less is used, Schiff base formation easily occurs without a neutralization step, but when a POA having a high average molecular weight exceeding 13 is used, The presence of this neutralization step forms a sufficient Schiff base, and the presence of a neutralization step is particularly preferred.

Examples of the base used in the neutralization step include hydroxides of alkali metals, hydroxides of alkaline earth metals, amines, and quaternary ammonium hydroxides. These can be used as a mixture, and it is particularly preferable to use them as an aqueous solution.

The organic solvent usable in the washing step of washing the hydrophilic chitosan derivative and the hydrophilic chitin derivative according to the ninth aspect of the present invention with an organic solvent does not dissolve the hydrophilic chitin derivative as the target substance, and POA, especially P
A solvent having a property of dissolving EG, and having a low boiling point or a highly hydrophilic solvent is preferable. Specifically, ketones such as acetone and MEK, alcohols such as methanol, ethanol and isopropanol, and tetrahydrofuran and the like are preferable. Examples thereof include ethers and cellosolves such as ethyl cellosolve. All of these dissolve PEG, but have low solubility of chitosan derivatives.

[0044]

Embodiments of the present invention will be described below. In the following examples, chitosan was used as a starting material. However, synthesis can be performed in substantially the same manner using water-soluble chitin having a higher acetylation degree than chitosan. In addition, examples using polyoxyethylene glycol (PEG) and polyoxyethylene glycol monomethyl ether (Me-PEG) as the polyoxyalkylene compound (POA) will be described. PEG and Me-PEG used
Is as follows. PEG # 4000: Mw = 3000 (manufactured by Wako Pure Chemical Industries) Me-PEG Mn = 5000 (Aldrich Ch)
chemical) Me-PEG Mn = 2000 (Aldrich Ch)
(made by electronic company)

(Synthesis of POA having aldehyde group at one end) 5.0 g of POA is reacted with 15 ml of DMSO by adding chloroform in an amount corresponding to the POA used as shown in the following table (Table 1). Next, (Table 1)
An aldehyde group is formed by adding the amount of acetic anhydride shown in Table 1 and reacting it under the conditions shown in Table 1. In the following, POA having an aldehyde group at one end is PEG-CH
O may be abbreviated.

The reaction mixture obtained by the above reaction was poured into 100 ml of diethyl ether and reprecipitated.
Filtered using a filter. The resulting precipitate was dissolved in chloroform, reprecipitated in diethyl ether,
The product was purified by repeating the cycle of filtration with an o2 filter 2 to 4 times, then dried under vacuum and subjected to a reaction with chitosan.

The method for quantifying aldehyde groups in PEG-CHO is as follows. PE equivalent to about 1 μmol
1.5 ml of an aqueous solution containing G-CHO is collected in a test tube. Me-PEG-CHO with Mn = 2000,
Assuming that the conversion of OH to CHO is 0.5,
1 μmol is 4 mg. 0 to 0.075 mg / ml of glutaraldehyde as a standard sample for preparing a calibration curve
An aqueous solution having a concentration of (equivalent to 0 to 1 μmol / ml) is prepared, and 1.5 ml is collected in a test tube. To the above test tube, 2 ml of a 0.5 M sodium carbonate aqueous solution containing 1.52 mM potassium ferricyanide is added, and the test tube is stoppered, and then immersed in boiling water for 15 minutes. After cooling to room temperature, the absorbance at 420 nm is measured using a spectrophotometer. The number of moles of PEG-CHO was determined from the calibration curve, and the C of OH was calculated from the number of moles of the original PEG-CHO collected.
The conversion to HO was calculated. The results are shown in Table 1.

[0048]

[Table 1]

(Synthesis of water-soluble chitosan)
C (chitosan (manufactured by Kyowa Technos); number average molecular weight 28
000, degree of deacetylation 86%) 0.25 g in 10 ml
Of 2% aqueous acetic acid and 5 ml of methanol and stirred to dissolve. The PEG-C obtained in the above (Synthesis of POA having one terminal aldehyde group) was added to this solution.
An aqueous solution of HO is added and stirred for 30 minutes at room temperature to form a Schiff base. CH in this reaction
The O / NH ₂ (chitosan) equivalent ratio is 0.3 to
It is a numerical value appropriately selected in the range of 1.0.

The pH of the resulting solution was adjusted to 6.5 using a 1N aqueous solution of sodium hydroxide, and the mixture was further stirred at room temperature for 60 minutes to further reduce the equilibrium reaction of Schiff base formation. By moving to the production side, the Schiff base was sufficiently produced.

Next, 4 ml of an aqueous solution of sodium cyanoborohydride (NaCNBH ₃ ) was added dropwise over 20 minutes. The amount of sodium cyanoborohydride used is Na
The CNBH ₃ / CHO ratio was set to be 10. After completion of the dropwise addition, the reaction mixture was further stirred at room temperature for 18 hours to complete the reaction, thereby obtaining a solution containing chitosan in which POA was bonded to a nitrogen atom.

The solution containing chitosan in which POA was bonded to a nitrogen atom obtained above was placed in a dialysis membrane having a molecular weight of 12,000 (manufactured by Wako Pure Chemical Industries, Ltd.), and dialyzed with 1 liter of 0.05N sodium hydroxide solution. Then, dialysis was performed using 1 liter of deionized water until the pH of the external solution became 8.5 or less. At this time, the water-soluble chitosan is contained in the solution side, and the non-water-soluble chitosan precipitates.
A solution containing this chitosan derivative is applied at 37,000 G for 15 minutes.
Centrifugation was performed to separate into a precipitate and a supernatant, and the precipitate was washed with distilled water.

The supernatant was kept at 40 to 45 ° C.
The solution was evaporated to a concentration and concentrated, and then freeze-dried. The obtained substance is immersed in 100 ml of acetone,
After leaving overnight, the mixture was stirred at room temperature for 4 hours, and then filtered twice using a glass filter. The operation was repeated twice, and impurities such as by-products such as PEG were removed using acetone and diethyl ether. After that, the product was dried under reduced pressure.

(Synthesis of hydrophilic chitin derivative) 0.5 g of each of the chitosan derivatives obtained in the above (Synthesis of water-soluble chitosan) was sampled, and the resultant was subjected to 10 ml of acetic acid.
And 40 ml of methanol, and acetic anhydride (Ac ₂ O) was added so that Ac ₂ O / NH ₂ = 1.4 to 8 and stirred at room temperature for 24 hours to perform acetylation. .

After the completion of the acetylation reaction, the pH of the reaction mixture was adjusted to 11 or more using a 1N aqueous sodium hydroxide solution, and then methanol was removed by evaporation at room temperature. The obtained solution was purified by dialysis using distilled water, and then freeze-dried to obtain a hydrophilic chitin derivative which is the object of the present invention.

Regarding the solubility of the hydrophilic chitin derivative obtained in the above Examples in water, the obtained dried chitosan derivative was dissolved in distilled water and phosphate buffered saline (PB).
S; pH = 7.2) and hydrophilic chitin 5 mg / 1
It was evaluated by the solubility when immersed for 4 days at a ratio that resulted in a ml solvent. The evaluation results of the water solubility are shown in Table 2 below. ：: dissolved △: swollen gel ×: precipitated (insoluble)

[0057]

[Table 2]

(Comparative Example) The same chitosan (Flona) used in (Synthesis of water-soluble chitosan) of the example was used.
c C) 0.116 g was dissolved in 10 ml of a 2% aqueous acetic acid solution, and 10 ml of methanol was added to reduce the viscosity. To this solution, the Me-PEG (M) described in the above (Synthesis of POA having aldehyde group at one terminal) was used.
4.003 g of POA having one terminal aldehyde group (CHO / OH equivalent ratio = 0.64) using n = 5000)
A solution obtained by dissolving 0.243 g of NaCNBH ₃ in 10 ml of distilled water was gradually added dropwise. The chemical equivalent ratio of the reaction is
CHO / NH ₂ (chitosan) = 0.99, NaCNBH
_{3 /} CHO = 7.5. After completion of the dropwise addition, at room temperature 18
After stirring for a time to complete the reaction, the reaction mixture was sealed in a dialysis tube having a molecular weight of 12000, and 0.05N
Dialysis was performed for 4 hours using 1 liter of an aqueous sodium hydroxide solution. At this time, generation of a large amount of precipitate was observed in the dialysis tube. Next, the external solution was exchanged for 1 liter of deionized water to perform dialysis. When the pH of the external solution reached 6.7, the contents of the dialysis tube were taken out, and the contents were removed at 37,000 G for 20 minutes.
The precipitate and the supernatant were separated by centrifugation for minutes and each was freeze-dried. When 50 ml of acetone was added to the dried product obtained by drying the supernatant, the whole was dissolved. Therefore, it is considered that the substance contained in the supernatant is not a chitosan derivative in which chitosan and PEG are bound. The dried product of the precipitate was immersed in 50 ml of acetone, allowed to stand overnight, and then washed twice with a glass filter. After the obtained film-like substance was vacuum-dried, ¹ H-NMR was measured by the method described below. As a result, this precipitate was chitosan, and the POA introduction rate (PEG concentration) to this chitosan was 0%. .24 or less. The chitosan was not acetylated because it was substantially insoluble in the solvent.

The results of evaluating the water solubility of the above-mentioned hydrophilic chitin derivatives are shown in (Table 2). The PE in Table 2 was used.
G concentration was determined by the methods described below ¹ H-N
It is calculated by the following formula based on the data obtained by MR.

From these results, it is apparent that use of a polyoxyalkylene compound having a high molecular weight can provide a chitin derivative having high water solubility or hydrophilicity even if the degree of substitution of chitin with POA is low. Also, from the results of the comparative examples, when the POA having an aldehyde group and the reducing agent are simultaneously introduced and the Schiff base forming step and the reducing step are not separated, the reducing agent is converted to water or acetic acid before the Schiff base is formed. It can be seen that the reaction caused the rate of introduction of PEG into chitosan to be significantly reduced. Further, it is clear that unreacted POA such as PEG can be effectively removed from the chitosan derivative by washing the organic solvent using acetone as an example. POA can hardly be removed only by dialysis.

( ¹ H-NMR sample preparation method) Chitosan derivative (80 mg) was added to 0.8 ml of DCl / D ₂ O (20
wt% solution) under ice-cooling. 0.8 ml of D ₂ O or DC for highly water-soluble chitosan derivatives
0.8 m with one drop of 1 / D ₂ O (20 wt% solution)
1 D ₂ O. The obtained sample solution was subjected to ¹ H-NMR measurement at room temperature or 80 ° C.
The chemical shift (δ) of the obtained peak and the position of the proton corresponding to the peak are shown in (Table 3) and the chemical formula (Formula 4). As a standard substance, 3- (trimethylsilyl)
Sodium propane sulfonate was used.

[0062]

[Table 3] (Note) 1 to 6 indicate the position of C to which H is bonded.

[0063]

Embedded image

The calculation of the PEG concentration based on the ¹ H-NMR measurement results was performed by the following equation.

[0065]

(Equation 1)

Here, each symbol is as follows. Mw (NAG): molecular weight of N-acetylglucosamine unit Mw (G): molecular weight of glucosamine unit Mw (PEG): molecular weight of bound PEG a: acetylation rate of chitosan (0.14 in Flonac C) x: PEG Introduction rate (Equation 2)

[0067]

(Equation 2)

Here, [raw material PEG-H]: the H number per PEG molecule used as the raw material is shown. For example, M
In the case of e-PEG (Mn = 2000), [(2000−15) / 44] × 4 + 3 = 183. (The molecular weight of 15: CH3, 44: CH2-CH2-O is shown.) [PEG-H] is a numerical value obtained by (Equation 3) based on the integrated value of each peak obtained by ¹ H-NMR measurement. . [P-1] to [P-8] indicate the integrated values of the peaks belonging to (Table 3).

[0069]

(Equation 3)

The degree of acetylation of the hydrophilic chitin derivative was determined by calculating the integrated value [P-
5] and (Equation 4).

[0071]

(Equation 4)

Claims (9)

[Claims] 1. A hydrophilic chitin derivative composed of N-acetyl-D-glucosamine and a derivative thereof bonded by an ether bond at the β-1,4 position, and represented by the following formula: , N1 + n2 + n3> 5, n1 / (n1
+ N2 + n3)> is 0.2, R ₁ is an average polymerization degree of 10
A hydrophilic chitin derivative, which is a polyoxyalkylene group of at least 300. Embedded image 2. The hydrophilic chitin derivative according to claim 1, wherein the polyoxyalkylene group is a polyoxyethylene group. 3. Production of a hydrophilic chitin derivative comprising N-acetyl-D-glucosamine and a derivative thereof bonded by an ether bond at the β-1,4 position and having a polyoxyalkylene group bonded to a nitrogen atom. A method for dissolving chitosan in an aqueous solution of an acid, wherein the chitosan and an average degree of polymerization having one aldehyde group in a molecule are included.
A Schiff base forming step of reacting a polyoxyalkylene compound of 0 to 300 to form a Schiff base, a reduction step of reducing the Schiff base, and an acetylation step of acetylating at least a part of unreacted amino groups. For producing a hydrophilic chitin derivative containing the same. 4. Production of a hydrophilic chitin derivative comprising N-acetyl-D-glucosamine and a derivative thereof bonded by an ether bond at the β-1,4 position and having a polyoxyalkylene group bonded to a nitrogen atom. A method of swelling or dissolving chitin or deacetylated chitin with a solvent, a polyoxyalkylene having an average degree of polymerization of 10 to 300 having chitin and one aldehyde group in a molecule. A method for producing a hydrophilic chitin derivative, comprising: a step of forming a Schiff base by reacting a compound to form a Schiff base; and a step of reducing the Schiff base. 5. The method for producing a hydrophilic chitin derivative according to claim 3, further comprising a neutralizing step of neutralizing a solution containing the Schiff base, subsequent to the Schiff base forming step. 6. The method for producing a hydrophilic chitin derivative according to claim 4, further comprising an acetylation step of acetylating at least a part of unreacted amino groups. 7. The chitin derivative is represented by the formula (Formula 2), wherein n1 + n2 + n3> 5, and n1 / (n1 + n2 + n).
3)> 0.2 The method of manufacturing a hydrophilic chitin derivative according to any one of claims 3 to 6 R ₁ is a polyoxyalkylene group having a polymerization degree of 10 to 300.. Embedded image 8. The method for producing a hydrophilic chitin derivative according to claim 3, wherein the polyoxyalkylene group is a polyoxyethylene group. 9. The method for producing a hydrophilic chitin derivative according to claim 3, further comprising a washing step of washing the hydrophilic chitosan derivative or the hydrophilic chitin derivative with an organic solvent.