WO1999001480A1

WO1999001480A1 - Hydrophilic chitin derivatives and method of manufacturing the same

Info

Publication number: WO1999001480A1
Application number: PCT/US1998/013744
Authority: WO
Inventors: Masastoshi Sugimoto; Yoshihiro Shigemasa
Original assignee: National Starch And Chemical Investment Holding Corporation
Priority date: 1997-07-03
Filing date: 1998-07-02
Publication date: 1999-01-14
Also published as: AU8282598A

Abstract

Hydrophilic chitin derivatives formed by ether bond of the unit of N-acetyl-D-glucosamine and its derivatives at β-1, 4 sites; wherein the chitin derivatives are expressed by formula (1) in which n1+n2+n3 > 5, n1/(n1+n2+n3) > 0.2 and R1 is poly(oxyalkylene) group having an average degree of polymerization from 10 to 300.

Description

HYDROPHILIC CHITIN DERIVATIVES AND METHOD OF MANUFACTURING THE SAME

The present invention relates to chitin derivatives having high biocompatibility and safety as well as being excellent in water solubility, waterborne gelling property and water swelling property and also relates to a method of manufacturing such chitin derivatives.

Chitin is a polymer mainly formed by either bond of the unit of N-acetyl-D- glucosamine at β-1 , 4 sites according to Formula 3 where n3=0, n1/(n1 +n2+n3) >0.2, that is, the polymer consisting solely of N-acetyl-D-glucosamine unit and its deacetylated unit and having a degree of deacetylation of not more than 20% (in a broader concept, the substance having a degree of deacetylation of not less than 80% is defined as "chitosan").

Chitin having such the chemical structure is a substance of natural origin obtained from such natural raw material as shrimp or crab shell, squid bone or the like. Chitin is a chemical compound having such useful properties as leukocyte floating effect and wound healing effect and so on. On the other hand, chitin is substantially insoluble in water and various organic solvents, except some special organic solvents and strong alkaline liquids, although it is known that chitin having 50% acetylation (ratio of acetylamino and amino groups: 50/50) is soluble in water. For this reason, industrial applications of chitin have been considerably limited to such special products as molded products.

In view of the above, the prior art has proposed various improvements for expanding the applications of chitin mainly by the rendering chitin more hydrophilic through incorporation of poly(oxyethylene) group in the molecule thereof while overcoming the defect of chitin and a method of manufacturing hydrophilic chitin by incorporating poly(oxyethylene) group onto the chitin molecule. These are disclosed specifically in the following publications.

(1 ) A method of incorporating poly(oxyethylene) group by adding not more than 10 alkylene oxides to at least one amino group or hydroxy group in the every glucosamine unit in the presence of alkali (Japanese Patent Kokai S63 (1988)-14714). (2) A method of incorporating poly(oxyethylene) group onto chitin by reacting alkaline chitin with poly(oxyethylene) halohydrin (Japanese Patent Kokai S64 (1989)- 14201 ).

(3) Chitin in which not more than 5 alkylene oxides have been added to at least one amino group or hydroxyl group in the every glucosamine unit (Japanese Patent

Kokai H5 (1993)-139939).

(4) A biocompatible and biologically inactive compound prepared by binding chitosan and PEG (Japanese Patent Kokai H7 (1995)-278203).

(5) A method of incorporating one poly(oxyalkylene) group onto an amino group in the unit of glucosamine, which was produced through hydrolysis of chitin, by simultaneously reacting the amino group with poly(oxyalkylene) glycol having an aldehyde group at one terminal end and sodium cyanoborohydride (J. Polymer Sci. Polymer Chem. Ed., 22, 341-352 (1984)).

However, the water-soluble chitin and chitosan derivatives disclosed in the above- mentioned publications (1 ), (2) and (3) also include those in which the poly(oxyalkylene) group is bonded with all of the three active hydrogens based on amino group and hydroxyl groups in the glucosamine unit. Although such derivatives indeed have improved hydrophilicity, they also have experienced a significant degree of denaturation, i.e., significant degree of change in its fundamental chemical structure. As a result, while improved in the hydrophilicity and other properties associated therewith such as moisture retentivity, these derivatives suffer a significant degree of deterioration in the leukocyte floating effect and wound healing effect which are unique and useful properties of chitin. the publications (1 ), (2) and (3) also describe further chitin or chitosan derivatives having only a small degree of denaturation resulting from bonding one poly(oxyalkylene) group to the amino group. However, in these derivatives, the number of repeated alkylene oxide units is within 10, but the hydrophilicity remains insufficient. The publication (4) describes the hydrophilic chitosan and chitin derivatives in which poly(oxyethylene) glycol is bonded to chitin or chitosan via an amide bonding or the like and that the reaction takes place at both terminals of poly(oxyethylene) glycol. While

- this substance has achieved enhancement of hydrophilicity, the substance is given to get only gel so that use of this substance is significantly limited to a narrow range.

The publication (5) is to selectively react poly(oxyethylene) glycol with the amino group of D-glucosamine unit. However, since this reaction is to be performed by reacting chitin with poly(oxyethylene) glycol having aldehyde group at one terminal thereof and sodium cyanoborohydride in an aqueous medium at a time, the reaction with the amino group does not take place in a reliable manner. Then, it is very likely that the resultant reaction mixture will be a mere mixture containing poly(oxyethylene) glycol. Moreover, this publication provides no report at all about the molecular weight of chitosan used therein or whether the resultant product has water solubility or not. Further, this publication doesn't disclose that the product is acetylated so as to study its properties as chitin.

In view of the above-described state of the art, an object of the present invention is to provide hydrophilic chitin derivatives which have not undergone significant change in the fundamental chemical structure of chitin thus retaining the unique and useful properties of chitin such as leukocyte floating effect and wound healing effect together with water solubility even when the chitin has a high molecular weight. Another object of the invention is to provide a method by which such chitin derivatives may be synthesized while minimizing the incorporation of impurities therein such as poly(oxyethylene glycol or the like.

For accomplishing the above-described objects, the invention provides hydrophilic chitin derivatives formed by either bond of the unit of N-acetyl-d-glucosamine and its derivatives at β-1 , 4 sites, wherein the chitin derivatives are expressed by chemical

Formula 3 in which n1 +n2+n3>5, n1/(n1 +n2+n3) >0.2 and Rj is poly(oxyalkylene) group having an average degree of polymerization from 10 to 300.

In Formula 3 above, the unit of N-acetyl-D-glucosamine and its derivatives which together constitute the chitin derivatives of the invention may be either a block-like bonded unit or a randomly bonded unit. Incidentally, the value of n3 is not particularly limited in the present invention, as long as the target properties of the product may be obtained, and this value is set to be not less that '1 '. However, too great a value will be undesirable since this will lose the unique properties of chitin with an enlarged degree of its denaturation. Also, n1 , n2 and n3 all are average values.

As the derivatives suffice the condition: n1 +n2+n3>5, they may provide the original useful properties of chitin sufficiently. The upper limit of the value of n1 +n2+n3 is not particularly limited in the present invention. However, as the hydrophilic chitin derivatives of this invention are obtained from natural chitin as a raw material, the molecular weight of the natural product becomes the substantial upper limit. Especially, when n1+n2+n3>8, the water-insoluble chitin may be rendered a derivative which may be dissolved even in distilled water or physiological saline with hardly changing the original properties of chitin. Therefore, the application can be advantageously expanded. Further, if the ratio of the N-acetyl D-glucosamine unit satisfies the condition: n1/(n1+n2+n3) >0.2, the fundamental chemical structure of chitin is not significantly changed or modified, so that the hydrophilicty may be enhanced while retaining the original and unique properties of chitin such as physiological activity of, e.g., leukocyte floating effect and wound healing effect. Conversely, if the ratio is 0.2 or less, the derivatives will exhibit more conspicuously the properties of a substance as classified in chitosan.

Furthermore, if the average degree of polymerization of poly(oxyalkylene) group which bonds to amino group of the glucosamine unit is below 10, the modification with the poly(oxyalkylene) group will not be enough to give the hydrophilicity to chitin. Conversely, if the average degree of polymerization of the poly(oxyalkylene) group exceeds 300, this will unfavorably result in excessively enhancing viscosity of the water-soluble chitin solution obtained. With the above-identified setting, even if only few of the D-glucosamine - units have their poly(oxyalkylene) group bonded with the amino group, the hydrophilicity may still be improved, so that the resultant high-molecular-weight chitin may be rendered water soluble. As a result, the fundamental chemical structure of chitin may be maintained, and the resultant chitin derivatives may have high hydrophilicity while retaining the physiological activity and biocompatibility which are the original properties of chitin. Incidentally, the 'average degree of polymerization' here means the numeric average molecular weight as determined by the terminal-group quantity determination method.

In the hydrophilic chitin derivatives set forth in Claim 1 , it is preferred that the poly(oxyalkylene) (POA) group may be poly(oxyethylene) (PEG) group. PEG group is highly hydrophilic and suitable for affording hydrophilicity to chitin without in-vivo digestion. It is preferably that other terminal end of PEG group which was bonded to chitin is hydroxy group or alkoxy group containing 1 to 4 carbon atoms.

The PEG group has higher hydrophilicity than poly(oxypropylene group. Then, even with a low degree of substitution with POA group, the resultant chitin derivatives may be water soluble. Incidentally, the weight ratio of POA group present in the chitin derivatives according to the present invention is not particularly limited as far as the POA may provide chitin with a predetermined degree of hydrophilicity. Further, the upper limit of the ratio of POA is not also particularly limited as long as the original and unique properties of chitin may exhibit themselves. However, if the ratio is over 90 weight percent, one can readily recognize the properties of chitin. And, if the ratio is over 80 weight percent, the resultant product will exhibit the chitin properties more conspicuously. Hence, such ratios are desirable.

According to a further aspect of the present invention, there is provided a method of manufacturing hydrophilic chitin derivatives formed by either bond of the unit of N- acetyl-D-glucosamine and its derivatives at β-1 , 4 sites and including POA group bonded to a nitrogen atom of D-glucosamine unit.

Thus, the method according to Claim 3 comprises: dissolving the chitosan in

- aqueous acidic solution, forming a Schiff-base by reacting the chitosan with a POA compound having one aldehyde group in the molecule thereof and having an average degree of polymerization from 10 to 300, reducing the Schiff-base with a reducing agent, and acetylating at least some of intact amino groups of D-glucosamine units. In the last step, alternatively, all of the intact amino groups may be acetylated.

In the case of Claim 4, the method uses, as raw material, chitin or partially deacetylated chitin in which some acetyl groups thereof have been hydrolyzed, and the method comprises swelling or dissolving chitin or deacetylated chitin in a solvent, forming a Schiff-base by reacting the chitin with a POA compound having one aldehyde group in the molecule thereof and having an average degree of polymerization from 10 to 300 and reducing the Schiff-base. The compound has a structure in which the amino group has already been acetylated, so that the compound does not require any acetylating step. Like the method using the water-soluble chitosan as a starting material, it is preferred that this method also include a further step for acetylating at least some of intact amino groups.

The formation of Schiff-base between the POA compound having an aldehyde group and chitin may take place both in a homogenous phase like solution and in a heterogenous phase like swelling phase. Thus, this reaction may be effected by dissolving chitin in a special solvent or swelling chitin in, e.g., an organic solvent. Especially, chitin having a degree of acetylation 50% is water-soluble and hence suitable as the raw material for the present invention. For manufacturing hydrophilic chitin derivatives in this invention, the method further comprises preferably neutralizing the solution or swelling liquid containing the Schiff-base after its formation above. Addition of such neutralizing step becomes possible to give advantage to the equilibrium to the formation of Schiff-base. Also, when a reducing agent is used in the subsequent step, the waste of the reducing agent may be lowered Namely, when sodium cyanoborohydπde is employed as a reducing agent for instance, this reducing agent has higher reactivity with hydrogen ion than with the Schiff- base So, it results in wasting this reducing agent in more amount in its reaction with acid - used in dissolving the chitin or chitosan than in the case without said neutralizing step With the above-described methods of the present invention, it is possible to reliably incorporate the POA group into the ammo group of the D-glucosamine unit in the chitin derivative produced by partial deacetylation of chitin Essential difference of the present invention from those of the prior art (5) above consists in the step for the reliable formation of Schiff-base and the acetylation step Without reliable formation of the Schiff- base, the aldehyde group will be reduced faster than the Schiff-base, so that the original POA group would be recovered to a decrease of yield of the target hydrophilic chitin derivatives as a big problem Further, if a reducing agent is employed in the reducing step, there would occur another problem that this reducing agent would be consumed through the reduction of the aldehyde group, thus leading to a fall of the reaction efficiency Further, through introducing the acetylation step, even when chitosan was used as raw material, the obtained hydrophilic chitin derivatives will retain the original properties of chitin including leukocyte floating effect and wound healing effect

Incorporation of POA group into the chitin which is originally water soluble affords an advantage of further improvement in the stability of the solution For the reasons mentioned above also, in manufacturing the hydrophilic chitin derivatives, preferably, the chitin derivatives is expressed by Chemical Formula 3 where, n1 +n2+n3>5, or more preferably n1+n2+n3>8 and n1/(n1 +n2+n3) > 0 2 and Rj is poly(oxyalkylene) group having an average degree of polymerization from 10 to 300

Further, during manufacturing hydrophilic chitin derivatives, preferably there may be further provided a step of rinsing the resultant chitin derivatives with an organic solvent This rinsing step may be provided prior to the acetylation so as to be utilized for refining chitosan derivatives or may be provided after the acetylation so as to be utilized for refining the hydrophilic chitin derivatives However, it is preferred that this step be utilized for refining chitosan derivatives Namely, the chitosan derivatives of hydrophilic chitin derivatives having the bonded POA contains, as an impurity therein, the POA used as raw material. And, depending on the application of the derivatives, it is necessary to eliminate this impurity. - In such case, if the derivatives are rinsed by using such an organic solvent as dissolves POA well but hardly dissolves the chitosan or chitin derivatives, then, the elimination of the POA as impurity may be effected efficiently and easily, whereby the obtained chitin derivatives will be of higher purity.

The hydrophilic chitin derivatives according to the present invention has high hydrophilicity and can be soluble in water even when the derivatives have a high molecular weight. And, when prepared as aqueous solution, this may be molded into a variety of shapes. After molding, if reacted with a multi-functional crosslinking agent, this molded product may become water-insoluble while retaining the unique activities and high hydrophilicity.

Chitin or chitosan employed as raw material in the present invention can be obtained by partially modifying the chemical structure of chitin which exists naturally in the form of organic skeletal substance of, e.g., Arthropoda or Mollusca. According to a typical method of obtaining chitin, shell of crab, shrimp, krill and so on as well as bone on squids are used as raw material. Thus, after this raw material is pulverized, the material is treated with hydrochloric acid to remove calcium carbonate therefrom. Subsequently, when this material is treated with caustic soda, impurities such as proteins and the like are removed to give the target chitin. Then, the substance employed as raw material in the present invention may be any of the natural chitin, deacetylated chitin derivatives through the partial deacetylation of the natural chitin and chitosan having D-glucosamine units in which most of the acetylated amino groups of chitin have been hydrolyzed in the amino groups.

Next, the process of synthesizing the hydrophilic chitin derivatives of the invention from chitin or chitosan having amino groups will be described.

(1 ) Step of dissolving chitosan in aqueous acidic solution:

In this step, commercially available chitosan is dissolved in aqueous solution. The acid usable in this step can be inorganic acid such as hdyrochloric acid, phosphoric acid or the like, organic carboxylic acid such as formic acid, acetic acid, propionic acid, tartaric acid, malic acid, phthalic acid or the like, or organic sulfonic acid - such as p-toluene sulfonic acid. These acids may be used singly or in combination of two or more members. Such aqueous acidic solution may be used for dissolving water- soluble chitin.

In this step, water-soluble organic solvent may be used, if necessary, and use of such solvent is preferred, too. By use of such organic solvent, the solubility of chitosan and reactants may be adjusted with decreasing the viscosity of the solution, so that the following reaction may take place efficiently.

The organic solvent can be one or more of alcohols such as methanol, ethanol or the like, ketones such as acetone, methyl ethyl ketone or the like, cellosolves, ethers such as tetrahydrofuran, dioxane, N-methylpyrrolidone, pyridine or the like. Among them, methanol is particularly preferred because of its good solubility and great effect of decreasing viscosity.

Incidentally, in the case of the invention set forth in Claim 4, chitin is dissolved or swollen in its dissolving step, unlike the corresponding step set forth in Claim 3.

Further, in the case of chitin having a degree of acetylation of about 50% which is water-soluble, water may be available as a solvent to be used in the step above. Alternatively, for dissolving water-insoluble chitin, the preferred solvents are methanol- saturated aqueous solution of calcium chloride hydrate and dimethyl acetamide-saturated aqueous solution of lithium chloride. In the case of reaction under the swollen condition, the preferred solvents are such organic solvents ads dimethyl sulfoxide (DMSO), pyridine or mixture of such organic solvent and water. (2) Schiff-base forming step by reacting chitosan or chitin with POA compound having one aldehyde group in the molecule thereof.

In this step, the POA compound having an aldehyde group at one terminal thereof is allowed to react with aqueous solution of chitosan (Claim 3) or solution or swelled liquid of chitin (Claim 4) obtained from the above-mentioned Step (1 ). This reaction is effected at room temperature or under heating.

The method of synthesizing the aldehydic POA can be nay of those well-know to

- one skilled in the art. Among them, however, the oxidization using DMSO-acetic acid or the substitution reaction of diethylacetal by bromo-acetaldehyde is particularly preferred for its convenience, in view that such syntheses allow use of commercially available poly(oxyalkylene) glycol as raw material.

The POA compounds to be used in this step may have hydroxyl groups at both terminals or a hydroxyl group at one terminal thereof. The POA compound having hydroxyl groups at both terminals is poly(oxyalkylene) glycol and may be obtained by ring- opening additional polymerization of alkylene oxide to water or low-molecular glycol such as ethylene glycol. The POA compounds having a hydroxyl group at one terminal thereof may be obtained by, e.g., ring-opening additional polymerization of alkylene oxide to mono-alcohol such as methanol or phenol. (3) Step for reducing the Schiff-base:

The reducing agent used in this reducing step can be sodium cyanoborohydride, sodium borohydride or the like. Alternatively, the reducing step may take place as hydrogenation over precious metal catalyst such as Pt or metal catalyst such as nickel or the like. In the case of using the reducing agent, the sodium cyanoborohydride is particularly preferred in view of the reaction rate, yield and so on.

(4) Step for acetylating chitin or chitosan having POA group or chitosan: In this step, some or all of amino groups except for the amino groups to which the polyoxyalkylene group is bonded are acetylated, whereby hydrophilic chitin derivatives may be obtained. The acetylating agent to be employed in this step can be any of those compounds which are commonly used as acetylating agent, such as acetic anhydride, acetyl chloride or the like. In this acetylating step, it is preferred that organic solvents commonly employed for acetylation may be used. Specifically, the preferred organic solvents are acetic acid, methanol, dioxane, pyridine, DMSO and so on,. And, these may be used singly or in combination.

Further, it is preferred that the acetylation take place in a homogenous phase in

- which chitosan or chitin having bonded to POA is dissolved therein. Alternatively, this reaction may take place in a heterogeneous phase in which such a substance as above is precipitated or swollen therein. Also, while there is a possibility to deacetylate the hydroxyl group of chitosan or chitin, such acetylation of hydroxyl group of chitosan or chitin may be restricted by controlling a mixing ratio of the acetic anhydrate relative to amino group and hydroxyl group, so that the acetylation of amino group will occur more predominantly.

Also, if such a strong acetylating agent as acetyl chloride is employed, there is a possibility to deacetylate the hydroxyl group of chitin thereby, thus causing excessive modification to the fundamental chemical structure of chitin. In such a case, there should be provided, subsequent to the acetylating step, a step of treating with alkaline solution such as KOH/ethanol solution. With this additional step, the acetylated group may be rendered back advantageously to the hydroxyl group.

Incidentally, if a step for neutralizing the solution including the Schiff-base is set prior to the reduction step, the point of equilibrium of the Schiff-base forming reaction can be shifted to the advantageous direction, so that the subsequent reduction may take place more efficiently.

Formation of the Schiff-base takes place without neturalizing step when POA in an average degree of polymerization of not more than 13 is used, but when POA in an average of polymerization exceeding 13 is used the formation of Schiff-base is accelerated sufficiently by this neutralizing step, which is particularly preferable. The base used in the neutralizing step can be any one or combination of hydroxide of alkaline metal, hydroxide of alkaline earth metal, amines, quaternary ammonium hydroxide and so on. And, it is particularly preferred that this or these bases will be used in aqueous solution. Preferably, the organic solvent which can be used for washing the hydrophilic chitosan derivatives or hydrophilic chitin derivatives as set forth in Claim 9 of the present invention is incapable of dissolving the objective hydrophilic chitin derivatives but has a ^■ solubility to POA, particularly PEG Specifically, the solvent preferably having a low boiling point or high hydrophilicity illustratively includes ketones such as acetone, MEK or the like, alcohols such as methanol, ethanol, isopropanol, ethers such as tetrahydrofuran, cellosolves such as ethyl cellosolve All of these solvents can dissolve well POA such as the poly(oxyethylene) glycol (PEG), but hardly dissolve the target chitin derivatives

Example

Practical embodiments of the present invention will be described below In the following examples, chitosan was employed as a starting material However, even if water-soluble chitin having a higher degree of acetylation than chitosan is employed, the synthesis may be effected in substantially identical manners Further, as POA, PEG and poly(oxyethylene) glycol monomethyl ether (Me-PEG) were employed The employed PEG #4000 Mw=3000 (Wako Pure Chemicals Co , Ltd ), Me-PEG Mn=5000 (Aldπch Chemical Co , Ltd ), Me-PEG Mn=2000 (Aldπch Chemical Co , Ltd )

Synthesis of POA having aldehyde group at one terminal POA 5 0 g was allowed to react with DMSO 15 ml together with chloroform in amount in accordance with the POA used in Table 1 below

Next, an amount of acetic anhydride (the amount shown also in Table 1) was added thereto and this mixture was allowed to react under the conditions shown in Table 1 , whereby the aldehyde group is formed In the following description, this POA having aldehyde group at one terminal will be referred to simply as PEG-CHO when appropriate

The reaction mixture was introduced into 100 ml of diethyl ether to be re- precipitated therein And, the precipitate was filtered out by using No 2 filter The resultant precipitate was then refined by 2 to 4 repeated cycles of the above-mentioned steps consisting of dissolution in chloroform, re-precipitation in diethyl ether, and filtration through No. 2 filter. Thereafter, this refined substance was dried in vacuum and then subjected to reaction with chitosan.

Incidentally, the determination of the quantity of aldehyde group present in PEG-CHO was conducted in the manner described below. 1.5 ml of aqueous solution containing PEG-CHO by an amount corresponding to

1 μ mol approximately was collected in a test tube. Provided that the PEG-CHO is Me- PEG-CHO of Mn=2000 and the conversion ratio from OH to CHO is 0.5, then 1 μ mol corresponds to 4 mg. As a standard sample for obtaining analytical curves, aqueous solution containing 0 to 0.075 mg/ml (corresponding to 0 to 1 μ mol/ml) of glutaraldehyde was prepared and 1.5 ml of this sample was collected in a test tube.

Into the above-described test tube, 0.05M aqueous solution of sodium carbonate including 1.52mM of sodium ferricyanide was added in repeated steps by an amount of 2 ml in each step. Thereafter, this test tube was sealed and kept submerged for 15 minutes in boiling water. After the test tube was cooled to the room temperature, absorbency at 420 nm was measured using a spectrophotometer. From the obtained analytical curve, the mole number of PEG-CHO was obtained and the conversion ratio from OH to CHO was calculated based on the mole number of the sampled original PEG-CHO. Result in shown in Table 1.

Table 1

POA Reaction Condition Conversion of -OH to -CHO

Chloroform Ac₂0 Ratio of Temperature Time (Ratio of -CHO/lnitial - OH) ml ml Ac₂0/-OH °C hr

Me-PEG (Mn = 5000) 5 2.05 20 room temp. 12 0.83 Me-PEG (Mn = 2000) 1 0.25 10 room temp. 9 0.56 PEG #4000 4 0.41 1.2 room temp. 30 0.38

r c~ m ro

Synthesis of Water Soluble Chitosan

0.25 g of Flonac C (chitosan (Kyowa Technos Co., Ltd.): number average molecular weight: 28,000, deacetylation degree: 85%) was added to 10 ml of 2% aqueous -solution of acetic acid and 5 ml of methanol and dissolved therein under stirring.

To this solution, the aqueous solution of PEG-CHO obtained as above (in the synthesis of POA having aldehdye group at one terminal thereof) was added and this mixture was reacted under stirring for 30 minutes at room temperature, whereby Schiff- base was formed. The equivalent ratios of CHO/NH₂ (chitosan) in the above reaction were those conveniently selected within the range of 0.3 to 1.0.

Using 1 N aqueous solution of sodium hydroxide, pH of the solution obtained as above was adjusted to 6.5 and then this solution was further stirred for 60 minutes at room temperature, so as to shift the Schiff-base forming reaction, being an equilibrium reaction, toward the Schiff-base forming side, so that a sufficient amount of Schiff-base could be produced.

Next, 4 ml of aqueous solution of sodium cyanoborohydride (NaCNBH₃) was dripped into the solution over 20 minutes. The amount of sodium cyanoborohydride used was set so that the NaCNBH₃/CHO ratio might be 10. After completion of dripping, the reaction mixture was further stirred for 18 hours at room temperature so as to complete the reaction therein. With this, there was obtained a solution including chitosan derivatives having POA therein bonded to nitrogen atom. The above-obtained solution including the chitosan derivative having POA bonded to nitrogen atom was then placed within a dialysis tube having 12,000 molecular weight cut-off value and dialyzed therethrough with using 1 L of 0.05 N aqueous solution of sodium hydroxide; and then the dialysis was continued with using 1 L of deionized water until the solution outside the tube had a pH value not more than 8.5. At this point, some of the chitosan which had been rendered water-soluble was included in the solution, while the other portion thereof which was not yet rendered water-soluble was collected as precipitate. Thereafter, the liquid containing this chitosan derivative was subjected to a centrifugal separation under 37,000 G for 15 minutes to separate between precipitate and supernatant. Then, the precipitate was washed away with distilled water.

The supernatant was evaporated with keeping at 40 to 45°C to 10 ml and then yophilized. The resultant substance was submerged in 100 ml of acetone, allowed to stand overnight, stirred again for 4 hours at room temperature and filtrated through a glass filter. The submerging and filtration were repeated by two cycles. Further, the product was refined by washing therefrom such by-product impurities as PEG or the like with acetone and diethyl ether and then dried in vacuo.

Synthesis of Hydrophilic Chitin Derivatives 0.5 g each of the chitosan derivative obtained as above (in the synthesis of water- soluble chitosan) was sampled. And, this was dissolved in a solution of 10 ml of 1 % aqueous acetic acid and 40 ml of methanol, and then acetic anhydride (Ac₂0) was added in the ratio of: AcO/NH₂ = 1.4 to 8. This mixture was stirred for 24 hours at the room temperature for acetylation. Upon completion of acetylation, the reaction product obtained as above was adjusted to pH 11 or higher with 1 N aqueous solution of sodium hydroxide. Then, the methanol was eliminated through evaporation at room temperature. The resultant mixture was then refined through dialysis against distilled water. Thereafter, this was lyophilized, whereby the target hydrophilic chitin derivative of the present invention were obtained. Then, the water-solubility of the chitin derivative obtained in the above-described embodiment was determined. Specifically, the water-solubility of the resultant dry chitin derivatives was assayed by soaking them in each of distilled water and 0.01 M phosphate buffered saline (PBS: pH = 7.2) at a concentration of 5 mg/ml. Results of water-solubility assays are shown in Table 2 with the following marks: O: complete dissolution

Δ: swollen gel

X: precipitation (no dissolution) TABLE 2

10 PEG concentration means weight ratio of PEG in chitosan derivative.

cz r~ m ro

Comparative Example

In this comparative example, 0.116 g of the same chitosan (Flonac C) as employed in Example was dissolved in 10 ml of 2% aqueous acetic acid, to which 10 ml of -methanol was added to reduce the viscosity of the solution. Into this solution, a solution of 4.003 g of the POA (CHO/OH equivalent ratio = 0.64) having aldehyde group at one terminal using the Me-PEG (MN=5000) described hereinbefore (synthesis of POA having aldehyde group at one terminal) and 0.243 g of NACNBH₃ in 10 ml of distilled water was gradually dripped. The equivalent ratios of the reaction were: CHO/NH₂ (chitosan) = 0.99 and NaCNBH₃/CHO = 7.5, respectively.

After completion of dripping, the mixture was stirred at room temperature for 18 hours to complete the reactions, sealed within a dialysis tube having a molecular weight cutoff value of 12,000 and dialysis was conducted for 4 hours using 0.05N aqueous sodium hydroxide solution 1 liter. At this point, formation of a great amount of precipitate was observed inside the dialysis tube. Then, the outer liquid was replaced by 1 liter of deionized water and further dialysis was conducted. When the outer liquid had a pH value of 6.7, the substance inside the dialysis tube was taken out and then subjected to a centrifugal separation at 37,000G for 20 minutes to separate the precipitate from supernatant. Then, each of them was lyophilized. When 50 ml of acetone was poured over the dried substance obtained by drying the supernatant, the entire substance was dissolved therein. Accordingly, it is believed that the substance contained in the supernatant was not chitosan derivative in which the chitosan and PEG were bonded to each other.

On the other hand, the dried substance obtained by drying the precipitate was submerged in 50 mi of acetone and left overnight. Thereafter, this was rinsed two times by filtration using a glass filter. The resultant film-like substance was then dried in vacuo and ¹H-NMR was measured. The result showed that this substance was chitosan and moreover that the ratio of POA introduced into this chitosan (i.e. PEG concentration) was smaller than 0.24. As this chitosan was substantially insoluble in solvents, no acetylation was effected.

Result of evaluating water solubility of the above-described hydrophilic chitin derivative is shown in Table 2.

Incidentally, the PEG concentration (weight ratio of PEG in chitosan derivative bonded to PEG) in this Table 2 was determined by the following method, i.e. by using an expression to be described later based on data obtained by ¹H-NMR.

From the results above, the following will be readily understood. Namely, when a POA compound having a high molecular weight is employed, highly water-soluble or hydrophlic chitin derivatives may be obtained even if the degree of substitution of POA in chitin may be low.

Further, from the result of the comparative examples, the following may be understood. Namely, when the Schiff-base formation and reduction are, without separation, effected by adding the POA compound having aldehyde group and a reducing agent at one time, the reducing agent reacts with water or acetic acid before the Schiff- base is formed, so that the degree of substitution of PEG introduced into chitosan is significantly reduced. Further, by rinsing with acetone as an example of organic solvent, the POA compound such as intact PEG or the like may be effectively eliminated. Incidentially, POA can hardly be eliminated by means of dialysis against water.

Method of Preparing a Sample for ¹H-NMR Measurement

80 mg of chitin derivative was dissolved in 0.8 ml of DC1/D₂0 (20 wt. % solution) under ice-cooling. As for the chitin derivative having high water-solubility, this may be dissolved in 0.8 ml of D₂0 or 0.8 ml of D₂0 added with a drop of DC1/D₂0 (20 wt. %). On the resultant sample solution, H-NMR measurement was conducted at room temperature or at 80°C. The positions of the obtained peak of the chemical shift (δ) and proton corresponding thereto are shown in Table 3 and by Formula 4. As a standard substance, sodium 3-(trimethylsilyl) propanesulfate was employed. Table 3 peak mark proton δ (ppm)

P-1 1-H 4.5-5.0

P-2 2-H 3.1 -3.3

P-3 3, 4, 6'-H 3.8-3.9

P-4 5, 6-H 3.7-3.8

P-5 CH₃ (acetyl group) 2.0-2.2

P-6 CH₂ (PEG) 3.6-3.7

P-7 CH₃ (Me-PEG) 3.3-3.4

P-8 CH₂ (PEG bonded to chitosan) 2.6-2.8

Note: Numerals 1-6 denote positions of C bonded to H.

(R:H. COCHJ. -PEG)

The calculations of the PEG concentrations based on the ¹H-NMR measurement results were conducted by the following equation.

PEG concentration^ Mw(PEG) - x Mw(NAG) ■ a+Mw(G) ■ (1-a) + Mw(PEG) - x

wherein each mark means the following significance: Mw (NAG): Molecular weight of N-acetyl glucosamine unit Mw (G): Molecular weight of glucosamine unit Mw (PEG): Molecular weight of bonded PEG a: Degree of acetylation of chitosan (0.14 in Flonac C) x: Ratio of introduced PEG (Mathematical equation 2) x = [PEG-H]

[raw material PEG-H]

wherein each mark means the following significance:

[Raw material PEG-H]: H number per one molecule of PEG used a raw material. For instance, in the case of Me-PEG (Mn=2000):

[(2000-15)/44] x 4 + 3 = 183

(15: CH₃, 44: Molecular weight of CH₂-CH₂-0); and [PEG-H]: Value determined by the following Mathematical Equation 3 based on an integrated value of the respective peaks obtained from the ¹H-NMR measurement.

Incidentally, [P-1] - [P-8] denote the integrated values of the peaks identified in Table 3.

[PEG-H] =

{ [P-2J + [P-3] + [P-4] + [P-7] } + { [P-6] + [P-8] } ,₆

[P-1]

Further, the degree of acetylation of the hydrophilic chitin derivatives is as obtained from Equation 4 using the peak intensity [P-5] of the peak of P-5 in Table 3

degree of acetylation = [P-5] /3 _{x 10u} (%)

[P-1]

Claims

What is claimed:

1. Hydrophilic chitin derivatives formed by ether bond Of the unit of N-acetyl-D- glucosamine and its derivatives at ╬▓-1 , 4 sites; wherein the chitin derivatives are -expressed by Formula 1 in which n1 +n2+n3>5, n1/(n1 +n2+n3) >0.2 and R, is poly(oxyalkylene) group having an average degree of polymerization from 10 to 300.

2. The hydrophilic chitin derivatives according to Claim 1 , wherein the poly(oxyalkylene) group is poly(oxyethylene) group.

3. A method of manufacturing hydrophilic chitin derivatives formed by ether bond of the unit of N-acetyl-D-glucosamine and its derivatives at ╬▓-1 , 4 sites containing a poly(oxyalkylene) group bonded to a nitrogen atom of D-glucosamine unit, which comprises dissolving chitosan in aqueous acidic solution, forming a Schiff-base by reacting the chitosan with a poly(oxyalkylene) compound having one aldehyde group in the molecule thereof and having an average degree of polymerization from 10 to 300, reducing the Schiff-base with a reducing agent and acetylating at least some of intact amino groups in the modified chitosan derivatives.

4. A method of manufacturing hydrophilic chitin derivatives formed by ether bond of the unit N-acetyl-D-glucosamine and their derivatives at ╬▓-1 , 4 sites containing a poly(oxyalkylene) group bonded to a nitrogen atom of D-glucosamine unit which ccr^rises swelling or dissolving chitin having a small amount of glucosamine unit or deacetylated chitin in a solvent, forming a Schiff-base by reacting the chitin with a poly(oxyalkylene) compound having one aldehyde group in the molecule thereof and having an average degree of polymerization from 10 to 300 and reducing the Schiff-base.

5. The method of manufactuing hydrophilic chitin derivatives according to Claim 3 or

4, which further comprises neutralizing the solution containing the Schiff-base obtained by he precedent step.

6. The method of manufacturing hydrophilic chitin derivatives according to Claim 3 or

4, which further comprises acetylating at least some of intact amino groups of D- glucosamine unit.

7. The method of manufacturing hydrophilic chitin derivatives according to any one of Claims 3 through 6, wherein the chitin derivatives are expressed by Formula 2 in which n1+n2+n3>5, n1/(n1+n2+n3) > 0.2 and R, is poly(oxyalkylene) group having an average degree of polymerization from 10 to 300.

8. The method according to any one of Claims 3 through 7, wherein the poly(oxyalkylene) group is poly(oxyethylene) group.

-9. The method according to any one of Claims 3 through 8, which further comprises rinsing the resultant hydrophilic chitosan derivatives or hydrophilic chitin derivatives with organic solvent.