WO1999001479A1

WO1999001479A1 - Chitosan derivatives and methods of manufacturing the same

Info

Publication number: WO1999001479A1
Application number: PCT/US1998/013739
Authority: WO
Inventors: Masatoshi 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: AU8282298A

Abstract

Chitosan derivatives formed by glycoside bond of D-glucosamine and its derivatives modified with poly(oxyalkylene) oxide at β-1,4 sites, in which the chitosan derivatives are expressed by Formula (1) where: 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

CHITOSAN DERIVATIVES AND METHODS OF MANUFACTURING

THE SAME The present invention relates to chitosan derivatives having good physiological and biocompatibility with medical safety as well as high water- solubility and relates also to a method of manufacturing such chitosan derivatives.

Chitosan is a polymer formed mainly by glycoside bond of 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 of D-glucosamine and its N-acetyl derivatives and having a degree of N-acetylation of not more than 20% (in a broader concept, the chitosan having a degree of N-acetylation of not more than 20% is referred to as "chitin or derivatives thereof.) Chitosan is a substance of natural origin obtained through deacetylation of chitin which in turn is obtained from such natural raw material as shrimp or crab shell, squid bone or the like. Chitosan is a chemical compound having such useful properties as anti-bacterial property, physiological activity and so on. On the other hand, although chitosan may be rendered water-soluble in aqueous acidic solution as its amino group forms ammonium salt with counter ion in the presence of an acid in the solution, chitosan is not soluble in aqueous neutral or alkaline solution. Further, chitosan is substantially insoluble in organic solvents. For this reason, industrial application of chitosan has been limited. In view of the above, the prior art has proposed various improvements for expanding the applicable scope of chitosan mainly through improvement of hydrophilicity thereof, namely by making chitosan rendered water-soluble through introduction of poly(oxyalkylene) group in the molecule, and a method of manufacturing such water-soluble chitosan derivatives modified with poly(oxyalkylene) group. These are disclosed specifically in the following publications.

(1) A method of introducing poly(oxyethylene) group by adding not more than 10 alkylene oxides to at least one amino group or hydroxyl group in the glucosamine unit in the presence of an alkali (Japanese Patent Kokai S63 (1988)-14714).

(2) Chitosan in which not more than 5 alkylene oxides are added to at least one amino group or hydroxyl group in the glucosamine unit (Japanese Patent Kokai H5 (1993)-139939). (3) A biocompatible but biologically inactive complex prepared by binding chitosan to polyethylene glycol (Japanese Patent Kokai H7 (1995)- 278203).

(4) A method of manufacturing modified chitosan which comprises introducing one poly(oxyalkylene) group into amino group in the glucosamine unit of chitosan, being available through hydrolysis of chitin, by reacting chitosan with poly(oxyethylene) glycol having one terminal aldehyde group and sodium cyanoborohydride at a time (J. Polymer Sci. Polymer Chem. Ed., 22, 341-352 (1984)).

However, the water-soluble chitosan derivatives disclosed in the above publications (1) and (2) include those in which the poly(oxyalkylene) group is bound with all of the three active hydrogens based on amino groups and hydroxyl group in the glucosamine unit. Then, 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 from significant degree of deterioration in the physiological activity and biocompatibility which are unique and useful properties of chitosan.

Thus, either in case of the reaction of adding alkylene oxides or in case of reacting halohydrin such as ethylene chlorohydrin in the presence of an alkali, the reaction takes place predominantly in the amino group of chitosan and then takes place also in the hydroxyl group, so that denaturation based on the hydroxyl group cannot be prevented either. On the physiological activity and the biological activity unique to chitosan, not only the amino group but also the hydroxyl group plays an important role. Hence, the higher the conversion rate of these amino and hydroxyl groups, the greater the degree of loss that occurs in these properties.

The publications (1) and (2) also describe chitosan derivatives having only a small degree of denaturation resulting from binding one poly(oxyalkylene) group to amino group. However, in these chitosan derivatives, the number of alkylene oxide unit repeated is not more than 10 so that these chitosan derivatives cannot be said to have achieved a sufficient improvement in the hydrophilicity.

The publication (3) describes a hydrophilic chitosan in which the reaction between poly(oxyalkylene) glycol and chitosan took place at both terminals of poly(oxyethylene) glycol. While this substance has achieved enhancement of hydrophilicity, the product is provided in the form of gel but is not soluble in water. Indeed, the object of this reference is to prepare such gel-like substance.

The publication (4) has an object to selectively bind poly(oxyethylene) glycol to the amino group of glucosamine. However, as this reaction is to be performed by reacting poly(oxyethylene) glycol having aldehyde group at one terminal thereof and sodium cyanoborohydride with chitosan in aqueous solution at a time, the reaction with the ammo group does not take place in a reliable manner, highly possibly resulting in that the reaction mixture will be a mere mixture containing poly(oxyethylene) glycol Moreover, this publication provides no report at all about results of confirmation on the binding between PEG and chitosan, and it entirely lack any description whether or not the resultant product is soluble in water

Accordingly, an object of the present invention is to provide chitosan derivatives which do not have any significant change in the fundamental chemical structure of chitosan thus retaining the physiological activity and biocompatibility, i e unique and original properties of chitosan and which have water-solubility in case of tnose of a higner molecular weight and also which provide more improved nydrophi city and stability in case of a lower molecular weight, as well as a method by whicn such chitosan derivatives may be synthesized while minimizing a mixing ratio c impurities therein such as poly(oxyethylene) glycol or the like

For accomplishing the object above, the invention provides chitosan derivatives formed by glycoside bond of -acetyl-D-glucosamine and its derivatives modified at β-1 , 4 sites, in which the chitosan ceπvatives a^ra expressed by Formula 3 where, n1+n2+n3 ≥5, n1/(n1+n2+n3) ≤Q 2 and R, is poly(oxyalkylene) group having an average degree of polymerization from 10 to 300

The inventors have found out that water-solubility of chitosan having a high molecular weight can be significantly improved by binding poly(oxyalkylene) group having an average degree of polymerization from 10 to 300 only to some of ammo groups without denaturing hydroxyl group at all Thus, the present invention has been established

In Formula 3, the unit of D-glucosamine and its derivative which together constitute the chitosan derivatives of the invention may be either block-like bound unit or randomly bound unit Incidentally, the value of n3 is not particularly limited in the present invention, as long as the target properties of the invention may be obtained, and this value is set to be greater than '1' However, too great a value of n3 will be undesirable since this will lead to increased degree of denaturation of chitosan Also, n1 , n2 and n3 all are average values

Chitosan of the invention having a lower molecular weight whose total number of repeating units (n1+n2+n3) of D-glucosamine and its derivatives together constituting the hydrophilic chitosan is between 5 and 10 has naturally water-solubility Still, as this chitosan is modified into its derivatives having the above-described structure according to the present invention, their hydrophilic property may be enhanced so that other properties thereof such as stability in mixing with other compounds may be improved

On the other hand, if total number of repeating units (n1+n2+n3) exceeds 10, this chitosan derivatives can be dissolved only in aqueous acidic solution Provided with the above-construction of the present invention, however, such chitosan derivatives can be dissolved also in distilled water and physiological saline, and thus the applicable scope of the chitosan derivatives may be expanded Further, as the ratio (n1/(n1+n2+n3)) of the acetylated D-glucosamine unit is maintained to be not more than 20% of the total number, the fundamental chemical structure of chitosan may be maintained Conversely, if the ratio exceeds 20%, the chitosan derivatives will exhibit the original properties of chitin more conspicuously Moreover, average degree of polymerization of alkylene oxides in the poly(oxyalkylene) group being responsible for providing the hydrophilicity through binding to the amino group is set to exceed 10. Thereby, the content of poly(oxyalkyiene) group (POA) in the chitosan derivatives may be increased, so that the hydrophilicity of the chitosan derivatives can be increased. Even if, of the D-giucosamine units, the portion in which poly(oxylakylene) group is bound to the amino group is relatively smaller (i.e. the value of n3 is small), the hydrophilicity may still be improved, so that the resultant chitosan derivatives in a high molecular weight may be rendered water-soluble. As a result, the fundamental chemical structure of chitosan may be maintained, and the resultant chitosan may have high hydrophilicity while retaining the physiological activity and biocompatibility which are the original properties of chitosan.

Further, in the chitosan derivatives according to the present invention, the poly(oxyalkylene) compound has an average degree of polymerization from 10 to 300.

If chitosan derivatives were prepared by binding the poly(oxyalkylene) compound having an average degree of polymerization not more than 10, such chitosan derivatives would have insufficient water-solubility. Conversely, if chitosan derivatives were obtained by binding a poly(oxyalkylene) compound having an average degree of polymerization exceeding 300, aqueous solution containing such chitosan derivatives in a high concentration would have excessive viscosity, which causes a problem in handling. Incidentally, use of a poly(oxyalkylene) compound having an average degree of polymerization not more than 13 is preferably used. Also, chitosan derivatives including a poly(oxyalkylene) group having an average degree of polymerization not more than 18 is more preferred since these derivatives may be rendered water-soluble in a reliable manner regardless of the kind of chitosan employed as raw material.

As alkylene oxides constituting the poly(oxyalkylene) group, either one or both of ethylene oxide and propylene oxide may be employed. And, in case of copolymers, these copolymers may be either a block copolymer or a random copolymer. It is preferable that other terminal of the poly(oxyalkylene) group bound to chitosan comprises a hydroxyl group or an alkoxy group of 1-4 carbon atoms. According to the present invention, preferable content of the poly(oxyalkylene) group in the chitosan derivatives is not less than 40 wt. %.

If the content of poly(oxyalkylene) group is below 40 wt. %, the chitosan derivatives with a high molecular weight will not have sufficient hydrophilicity and water-solublility. Incidentally, the content of poly(oxyalkylene) group is understood to refer to the weight ratio of poly(oxyalkylene) group relative to the entire chitosan derivatives. On the other hand, the upper limit of the content of poly(oxyalkylene) group is not particularly limited in the present invention, as long as the resultant chitosan derivatives achieve the intended properties of the present invention. If the content is 90 wt. %, one can clearly recognize the properties of chitosan. If it is set to 80 wt. %, the properties of chitosan will exhibit themselves more strongly.

In the chitosan derivatives of the present invention, preferable poly(oxyalkylene) group is poly(oxyethylene) group. The poly(oxyethylene) group has a higher hydrophilicity than poly(oxypropylene) group. Then, even with a less content of poly(oxyalkylene) group present therein, the water-soluble chitosan derivatives can be produced.

According to a further aspect of the present invention relating to Claim 4, there is provided a method of manufacturing chitosan derivatives formed by glycoside bond of D-glucosamine and its derivatives modified at β- glucosamine and its derivatives modified at β-1 ,4 sites, in which the chitosan derivatives are manufactured by the steps of: dissolving chitosan in aqueous acidic solution, forming the Schiff-base by reacting the chitosan with a poly(oxyalkylene) compound having one terminal aldehyde group in the molecule and reducing the Schiff-base with a reducing agent.

According to the method of the present invention, it is possible to reliably bind the poly(oxyalkylene) group to the amino group of the chitosan. Unlike further conceivable methods using other functional groups such as isocyanate group or carbodiimide group, the above-mentioned method eliminates the necessity of protecting the hydroxy group without denaturation of the chemical structure due to the reaction of unprotected hydroxyl group. Therefore, the poly(oxyalkylene) group may be selectively bound to the amino group alone.

Essential difference between the method of the present invention and the prior art (4) above consists in providing a step for reliably forming the Schiff-base. Without this step in the subsequent reducing step, the aldehyde group will be reduced faster than the Schiff-base group, so that the original poly(oxyalkylene) compound would be reproduced in a large amount, thus lowering yield of the target chitosan derivatives. Further, if a reducing agent is employed in the reducing step, there would occur another problem that this reducing agent would be wasted during reducing the aldehyde group, thus leading to lowering in the reaction efficiency. y

In the manufacturing method according to the present invention, it is preferred that this method is further accompanied by a step for neutralizing aqueous solution containing the Schiff-base which was formed in the foregoing step. By such a neutralizing step, it becomes advantageously possible to shit the point of equilibrium of the Schiff-base forming reaction to conditions where a larger amount of Schiff-base may be formed. Also, when a reducing agent is used in the subsequent reducing step, waste of the reducing agent maybe favorably lowered. When sodium cyanoborohydride is employed as a reducing agent for instance, this reducing agent has a greater reactivity with hydrodgen ion than with the Schiff-base. Then, in comparison with a case without the neutralizing step, this reducing agent will be much more wasted in its reaction with the acid used for dissolving chitosan.

In the invention set forth in Claim 4, preferable are chitosan derivatives having poly(oxyalkylene) group expressed by Formula 3 where, n1+n2+n3>5, n1/(n1+n2+n3) ≤0.2.

In the method of the invention of manufacturing chitosan derivatives it is preferred that preferable content of the poly(oxyalkylene) group is not less that 40 weight percent and also that the poly(oxyalkylene group is poly(oxyethylene) group.

Further, the method of manufacturing chitosan derivatives preferably comprises washing the resultant chitosan derivatives with organic solvent.

Thus, the chitosan derivatives contains, as impurities, the poly(oxyalkylene) compound used as raw material. And, depending on the application of the derivatives, it is necessary to eliminate the impurities. In such a case, if the chitosan derivatives are washed with such an organic solvent enabling to dissolve the poly(oxyalkylene) compound as impurities maybe effected efficiently and easily, whereby the chitosan derivatives will be prepared with a higher purity.

Chitosan employed in the present invention can be prepared by deacetyiating chitin, for example, by treating chitin with an alkali. Chitin, being raw material for preparing chitosan, exists naturally in the substance of organic skeleton of, e.g., Arthropoda or Mollusca and may be prepared therefrom. According to a typical method of preparing chitin, shell of crab, shrimp or krill and squid cartilage are used as raw material. After this raw material is pulverized, the powdery material is treated with hydrochloric acid to remove calcium carbonate. Subsequently, when the resultant mixture is treated with sodium hydroxide, protein and other impurities are removed, whereby the target chitin is obtained.

The process for synthesizing the chitosan derivatives of the invention from chitosan will be described below. (1 ) Step of dissolving chitosan in aqueous acidic solution:

In this step, commercially available chitosan is dissolved in aqueous acidic solution.

Examples of acids usable in this step are inorganic acids such as hydrochloric acid, phosphoric acid or the like, organic carboxylic acids such as formic acid, acetic acid, propionic acid, tartaric acid, malic acid, phthalic acid or the like, and organic suifonic acids such as p-toluenesulfonic acid.

These acids may be used singly or in combination of two or more members.

In this step, water-soluble organic solvents may be preferably used, depending upon the necessity. By use of such organic solvents, the solubility of chitosan and reactants may be adjusted and further the viscosity of the solution during the step may be lowered, so that the following reaction may take place efficiently. Examples of these organic solvents can be one or more kinds of alcohols such as methanol, ethanol or the like, ketones such as acetone, MEK or the like, cellosolves, ethers such as tetrahydrofuran, dioxane, N- methylpyrrolidone, pyridine and so on. Among them, the methanol is particularly preferred because of its good solubility and great effect of lowering viscosity and so on.

(2) Step for forming the Schiff-base by reacting chitosan with a poly(oxyalkylene) compound having one terminal aldehyde group in the molecule: A poly(oxyalkylene compound having one terminal addehyde group

(hereinafter referred to as "POA-aldehyde") is allowed to react with chitosan (in aqueous solution) obtained in Step (1) above. This reaction is effected at room temperature or under heating.

The POA-aldehyde can be prepared according to any of those well- known to one skilled in the art. Among them, however, oxidation with dimethyl sulfoxide-acetic acid or substitution reaction with bromoacetoaldehde diethyl acetal is particularly preferred for convenience, since such methods allows use of commercially availbe poly(oxyalkylene) glycol as raw material.

The POA used in this step may have hydroxyl groups at both terminals or hydroxyl group at one terminal thereof. The POA having hydroxyl groups at both terminals can be prepared by ring-opening addition polymerization of alkylene oxide to water or low-molecular-weight glycol such as ethylene glycol. The POA having hydroxyl group at one terminal thereof can be prepared by, e.g., ring-opening addition polymerization of alkylene oxide to monohydric alcohol like methanol or phenol. Incidentally, although poly(oxyalkylene) glycol generally refers to compounds having two functional groups, here the poly(oxyalkylene) glycol is understood to refer also to such multi-functional compounds having more than three functional groups. Even if two or more terminal hydroxy groups remain, this will not affect the target properties at all.

(3) Step for reducing the Schiff-base: Reducing agents used in this step illustratively include sodium cyanoborohydride, sodium borohydride and the like. Alternatively, hydrogenation can be performed over precious metal catalyst such as Pt or metal catalyst such as Raney nickel or the like. In the case of using reducing agents, the sodium cyanoborohydride is particularly preferred in view of reaction rates, yields and so on.

If a neutralizing step of the solution including a Schiff-base is provided prior to the reducing step, the point of equilibrium of the Schiff-base forming reaction may be shifted to the advantageous direction, so that the reduction in the subsequent step may take lace more efficiently. In case of using a poly(oxyalkylene) compound with an average degree of polymerization below 13, the Schiff-base formation takes place well without neutralizing step. However, in case of using a high-molecular-weight poly(oxyalkylene) compound with an average degree of polymerization not less than 13, this neutralizing step may advantageously assure reliable binding to chitosan via the Schiff-base formation.

Examples of the bases used in the neutralizing step are alkali metal hydroxides, alkaline earth metal hydroxides, amines, quaternary ammonium hydroxide and the like. These bases can be used singly or in combination of two or more members. And it is particularly preferred that these bases are used in aqueous solution.

The solvent which can be used for refining the chitosan derivatives of the present invention has preferably a low boiling point or high hydrophilicity. Specifically, examples of the solvent are ketones such as acetone, MEK or the like, alcohols such as methanol, ethanol, isopropanol or the like, ethers such as tetrahydrofuran, cellosolve such as ethyl cellosolve, etc. All of these solvents can dissolve well poly(oxyalkylene) compound such as poly(oxyethylene) glycol (PEG), but hardly dissolve the chitosan derivatives.

Examples

Next, practical embodiments of the present invention will be illustratively shown in the following Examples. In the following Examples, PEG and poly(oxyethylene) glycol monomethyl ether (Me-PEG) were employed as POA. The employed PEG and Me-PEG are specifically identified below.

PEG #4000; Mw = 3000 (Wako Pure Chemical Co., Ltd.) Me-PEG Mn = 5000 (Aldrich Chemical Co., Ltd.) Me-PEG Mn = 2000 (Aldrich Chemical Co., Ltd.)

Me-PEG Mn = 550 (Aldrich Chemical Co., Ltd.)

Synthesis of POA having aldehyde group at one terminal

In an argon atmosphere, POA (5.0 g) was dissolved in DMSO (15 ml) containing chloroform in the amount adjusted in accordance with the POA shown in Table 1 below, and the resultant solution was provided for the following reaction.

Next, acetic anhydride in 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 was 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 poured into 100 ml of diethyl ether for reprecipitation. And, the precipitate was filtered out by using No. 2 filter. The resultant precipitate was then refined by two to four repeated cycles of the steps above consisting of dissolving in chloroform, reprecipitaiton in diethyl ether and filtration with No. 2 filter. Thereafter, this refined substance was dried in vacuo and provided for reactions with chitosan or chitin.

Incidentally, determination of the aldehyde group present in PEG- CHO was conducted in the manner described below.

Into a test tube was collected 1.5 ml of aqueous solution containing PEG-CHO equivalent to 1 μ mol approximately was collected in a test tube. Provided that the PEG-CHO was Me-PEG-CHO (Mn = 2000) and the conversion ratio from OH to CHO was 0.5, then 1 μ mol corresponds to 4 mg.

As a standard sample for obtaining calibration curves, aqueous solution containing 0 to 0.075 mg/ml of glutaraldehyde was prepared and 1.5 ml of this sample was collected in a test tube.

Into the above-described test tube, 0.5 M aqueous solution of sodium carbonate including 1.52 mM of potassium ferricyanide was added each by a volume of 2 ml. Thereafter, this test tube was sealed and kept submerged for 15 minutes in boiling water. After the tube was cooled to room temperature, absorbence at 420 nm was measured using a spectrophotomerter. From the resultant calibration curve, mole number of PEG-CHO was firstly obtained, and the conversion ratio from OH to CHO was calculated based on the mole number of the sampled original PEG-CHO. The result is 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 ten ip. 9 0.56

Me-PEG (Mn = 550) 0 4.65 5 50 3 1.07

PEG #4000 4 0.41 1.2 room temp. 30 0.38

Synthesis of Chitosan Derivatives

To a mixture of 10 ml of 2% acetic acid and 5 ml of methanol was added 0.25 g of Flonac C (chitosan manufactured by Kyowa Technos Co., Ltd., numeric average molecular weight: 28000, the degree of deacetylation: 86%) and the resultant mixture was dissolved under stirring.

To this solution, the aqueous solution of PEG-CHO obtained ads above in the synthesis of POA having aldehyde group at one terminal thereof was added and this mixture was reacted under stirring for 30 minutes at room temperature to give a Schiff-base. Equivalent ratios of CHO/NH₂ (chitosan) in this reaction were 0.1 , 0.2, 0.3, 0.5 and 1.0, respectively.

With 1 M aqueous solution of sodium hydroxide, the solution obtained as above was adjusted to pH 6.5 and then 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. Additionally, the neutralization would decrease hydrogen ion (H⁺) which consumes sodium cyanoborohydride in the subsequent step.

Then, 4 ml of aqueous solution of sodium cyanoborohydride (NaCNBH₃) was dropwise added into the solution above for 20 minutes. The amount of sodium cyanoborohydride used was set so that the NaCNBH₃/CHO ratio may be 10. After completing the addition, the reaction mixture was further stirred for 18 hours at room temperature so as to complete the reaction therein. Thus, there was obtained a solution containing a chitosan derivative having poly(oxyalkylene) group bound to the amino group of glucosamine unit in chitosan.

The resultant solution containing the chitosan derivative having poly(oxyalkylene) group bound to the amino group of chitosan was put in a dialysis membrane tube (cut-off value: molecular weight 12000; Wako Pure Chemical Co., Ltd.) and dialyzed against 0.05 M aqueous sodium hydroxide, and then the dialysis was continued with 1 liter of deionized water until the solution outside the tube had a pH value below 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 had not yet been rendered water-soluble was collected as precipitate. Then the liquid containing this chitosan derivative was centrifuged at 37,000 G for 15 minutes. The precipitate was separated from the supernatant and washed away with distilled water. The supernatant was concentrated at 40 to 45°C to a volume fo 10 ml and freeze-dried. The resultant substance was sub merged in 1 ^'00 ml of acetone and allowed to stand overnight. It was stirred again at room temperature for four hours and filtered with a glass filter. The submerging and filtration were repeated for two cycles. Further, the product was refined by eliminating therefrom such by-product impurities as PEG or the like with acetone. After washing with diethyl ether, it was dried under reduced pressure.

When precipitate was formed, this precipitate was filtered out. similarly to the substance obtained from the supernatant, the precipitate was submerged in 100 ml of acetone and allowed to stand overnight. Then, this was stirred for four hours and twice filtered with a glass filter. The precipitate was refined by eliminating therefrom such by-product impurities as PEG or the like with acetone. After washing with diethyl ether, the product was dried under reduced pressure. In the above-described synthesis, Me-PEG (Mn = 5000) was employed. Examples 1 through 3 included those in which the PEG concentration ranged between 0.60 and 0.68. Examples 4 through 8 included those in which the PEG concentration ranged between 0.52 and 0.82 (Me- PEG, Mn = 2000). Example 9 included that in which the PEG concentration was 0.43 (Me-PEG, Mn = 550). Example 10 included that in which PEG#4000 was used and the PEG concentration was 0.72.

Further, Comparative Examples 1 and 2 included those in which Me- PEG (Mn = 2000) was used and the PEG concentration was 0.37 (twice repeated). And, Comparative Examples 3 and 4 included those in which Me- PEG (Mn = 550) was employed and the PEG concentration was 0.09 and 0.14, respectively.

Comparative Examples 5 - 6

Comparative Examples 5 and 6 included those samples in which ethylene glycol was bound to the amino group.

In these samples, 0.5 g of the same chitosan (Flonac C) was employed in Examples 1 through 10 was dissolved in 20 ml of 2% aqueous acetic acid solution, to which 10 ml of methanol was added to lower the viscosity of the solution. As a mono-aldehyde derivative of ethylene glycol, 0.045 g and 0.15 g of glycol aldehyde were used in Comparative Examples 5 and 6, respectively. Further, the reaction equivalent ratios of CHO/NH₂ in these cases were 0.3 and 1.0, respectively.

Other operations were made in the same manner as in Examples 1- 10 to give the chitosan derivative having the structure of ethylene glycol bound to the amino group.

Comparative Example 7

In 10 ml of 2% aqueous acetic acid was dissolved 0.116 g of the same chitosan (Flonac C) as employed in Examples 1 through 10 above, and the resultant solution was mixed with 10 ml of methanol to reduce the viscosity of the solution. Into this solution was gradually added a solution 4.003 g of the POA (equivalent ratio CHO/OH = 0.64) having one terminal aldehyde group using the Me-PEG (Mn=5000) above-described (in Synthesis of POA having aldehyde group at one terminal) and 0.243 g of NsCNBH₃ in 10 ml of distilled water. Chemical equivalent ratios in the reaction were as follows: CHO/NH₂ (chitosan) = 0.99 and NaCNBH₃/CHO = 7.5, respectively.

After finishing the addition, the mixture was stirred at room temperature for 18 hours to complete the reaction therein and then introduced and sealed in a dialysis membrane tube having a molecular weight cutoff value of 12000. The dialysis was conducted for 4 hours against 1 liter of 0.05 N aqueous sodium hydroxide solution. At this point, a large amount of precipitate was observed inside the dialysis membrane 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 content inside the dialysis membrane tube was taken out and then subjected to a centrifugation at 37.000G for 20 minutes to separate the precipitate from the supernatant. Then, each of them was freeze-dried. Since dried substance from the supernatant was dissolved in 50 ml of acetone, it was considered that the substance contained in the supernatant was not the chitosan derivative in which chitosan was bound to PEG.

On the other hand, the dried product from the precipitate was submerged in 50 ml of acetone and left overnight. Thereafter, this was subjected to two cycles of washing with acetone and filtration with a glass filter. The resultant film-like substance was then dried in vauo and H-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 less than 0.24.

Then, the water-solubility of the chitosan derivatives obtained in the above-described Examples and Comparative Examples was determined. Specifically, the solubility thereof relative to normal water was evaluated at the time of dialysis against 0.05 N aqueous sodium hydroxide solution during the synthesis. Whereas, the solubility in 0.01 M phosphate-buffered saline (PBS: pH = 7.2) was evaluated as the solubility determined when the dried chitosan derivatives obtained were soaked into PBS for four days at a concentration of 5 mg/ml. The result of these evaluations were denoted with the following marks.

O: Dissolved

Δ: Swelled in gel

X: Precipitated without dissolution

Table 2

From the results above, the following will be readily understood. Namely, when POA having a high molecular weight is employed, water- soluble chitosan derivatives may be obtained even if the degree of substitution to amino group of chitosan is low. Also, when the ratio of poly(oxyalkylene) group is not less than 40 wt. %, water-soluble chitosan derivatives may be obtained.

Further, from the result of Comparative Example 7, the following may be understood. Namely, when the Schiff-base formation and reduction are effected by charging the POA having aldehyde group and reducing agent at one time, the reducing agent reacts with aldehyde group which should act with the amino group, so that the degree of substitution of PEG into chitosan is significantly lowered. Further, by washing with acetone as an example of organic solvent, by-product such as intact POA (e.g. intact PEG) or the like may be effectively eliminated. Incidentally, POA cannot be eliminated by means of dialysis against water alone.

Binding PEG to the amino group of chitosan is apparent from the above-described solubility to water. This will be apparent also from the result of measuring NMR spectra. Namely, the ¹H-NMR measurement results revealed, as shown in Table 3 below, that the peak of methylene of PEG as raw material and the peak of methylene of PEG bound to chitosan are located at different positions from each other.

Method of adjusting a sample for ¹H-NMR measurement

80 mg of chitosan derivative was dissolved in 0.8 ml of DC1/D₂0 (20 wt. % solution) with ice cooling. The highly water-soluble chitosan derivative may be dissolved in 0.8 ml of D₂0 of 0.8 ml of D₂0 containing one drop of DC1/D₂0 (20 wt. %). On the resultant sample solution, ¹H-NMR measurement was conducted at room temperature or at 80°C. The position of the peak of the chemical shift (δ) and the position of proton corresponding thereto are shown in Table 3 and represented by Formula 4.

As a standard substance, 3-(trimethylsilyl)propanesulfonic acid sodium salt 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 CCCH (acetyi group) 2.0-2.2

P-6 -OCH₂ (PEG) 3.6- 3.7

P-7 -OCH₂ (Me-PEG) 3.3-3.4

P-8 -NHCH₂ (PEG bound to chitosan) 2.6-2.8

Note: Numerals 1-6 denote positions of C for the unit saccharide of chitosan.

(R:H. COCHa.-PEG)

Calculation of PEG concentrations based on the ¹H-NMR measurement was conducted by mathematical equation 1.

fc, G concentration = — —

Mw(NAG) -a+ w(G) ^• (l-a)+Mw(PEG) wherein Mw (NAG) molecular weight of N-acetyl glucosamine unit Mw (G) molecular weight of glucosamine unit Mw (PEG) molecular weight of PEG bound degree of acetylation of chitosan (0 14 in case of Flonac C) degree of substitution of PEG (mathematical equation 2)

[ PEG-H] x =

C raw material PEG-H 1

wherein

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

[(20C0 -15)/44j x 4 + 3 = 183

(where 15 molecular weight for CH₃ and 44 molecular weight of CH₂-CH₂-0), and

[PEG-H] value determined by mathematical equation 3 based on peak intensity of the respective peaks obtained from the ¹H-NMR measurement

Incidentally [P-1] - [P-8] denote the peak intensity of the peaks identifieα in

Table 3 [PEG-H] =

{ [ P-2] -f [ P-33 4 [P-4] [P-7] } { [P-6] t [P-8] }

[ P-1 ]

The chitosan derivatives being highly water-soluble according to the present invention may be used as raw material for cosmetics, pharmaceutical or medical products which are useable in direct contact with human body or administered directly into human body or in cleaners such as shampoo liquid

Claims

What is claimed:

1. Chitosan derivatives formed by glycoside bond of D- glucosamine and its derivatives modified with poly(oxyalkylene) oxide at ╬▓-1,4 sites, in which the chitosan derivatives are expressed by Formula 1 where, n1+n2+n3ΓëÑ5, n1/(n1+n2+n3)Γëñ0.2 and R, is poly(oxyalkylene) group having an average degree of polymerization from 10 to 300.

2. The chitosan derivatives according to Claim 1 , in which the derivatives includes the po!y(oxyaikylene) group at a ratio of not less than 40 wt. %.

3. The cnitosan derivatives according to Claim 1 or 2, in which the poly(oxyalkylene) group is poly(oxyethylene) group.

4. The chitosan derivatives formed by glycoside bond of D- glucosamine and its derivatives modified with poly(oxyalkylene) oxide at ╬▓-1 ,4 sites and including pciy(oxyalkylene) group bound to the amino group of D- glucosamine unit, in which the chitosan derivatives are manufactured by the steps of: dissolving chitosan in aqueous acidic solution, forming its Schiff- base by reacting the chitosan with a poly(oxyalkylene) compound having one terminal aldehyde group in the molecule and reducing the Schiff-base with a reducing agent.

5. A method of manufacturing chitosan derivatives formed by glycoside bond of D-glucosamine and its derivatives modified at ╬▓-1 ,4 sites and including poly(oxyalkylene) group bound to the amino group of D- glucosamine unit, which comprises the steps of dissolving chitosan in aqueous acidic solution, forming its Schiff-base by reacting the chitosan with a poly(oxyalkylene) compound having one terminal aldehyde group in the molecule and reducing the Schiff-base with a reducing agent.

6. The method of manufacturing chitosan derivatives according to Claim 5, in which the chitosan derivatives are expressed by Formula 2 where, n1+n2+n3ΓëÑ5, n1/(n1+n2+n3) Γëñ0.2 and R, is poiy(oxyalkylene) group having an average degree of polymerization from 10 to 300.

7. The method according to Claim 5 or 6, in which the derivatives includes the poiy(oxyalkylene) group at a ratio of not less than 40 wt %.

8. The method according to Claim 5 or 6, in which the poly(oxyaikylene) group is poly(oxyethylene) group.

9. The method according to any one of Claims 5 through 8, comprising a further step of washing the resultant chitosan derivatives with organic solvent.