JP2009073861A - Polyγ-glutamic acid derivatives containing cyclodextrins - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a poly-γ-glutamic acid (PGA) derivative having a cyclodextrin introduced therein. <P>SOLUTION: The present invention provides a poly-γ-glutamic acid (PGA) derivative having a cyclodextrin introduced therein. Preferably, the cyclodextrin is bound to about 1% to 30% of the side-chain carboxyl groups in PGA. It becomes possible to impart properties inherent in cyclodextrin (e.g., an effect of stabilizing an unstable substance, an effect of masking of unpleasant odor, unpleasant taste or the like) to PGA. Therefore, the PGA derivative can be widely used in various applications including biotechnology. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a poly-γ-glutamic acid derivative containing cyclodextrins.

  Poly-γ-glutamic acid, which is excellent in biodegradability and biocompatibility and has moisture retention, has attracted attention as a material for cosmetics, foods and the like. Hereinafter, in the present specification, poly-γ-glutamic acid is also referred to as “PGA”. Since PGA is a main component contained in the stickiness of natto, it not only has high biocompatibility, but also its safety is guaranteed by a long eating experience. In addition, even when polymers having the same molecular weight are compared, the PGA aqueous solution has a low viscosity and is industrially easy to handle.

  Japanese Patent Laid-Open No. 2002-233391 (Patent Document 1) discloses a Bacillus strain that produces high molecular weight poly-γ-glutamic acid.

  International Publication No. WO2004-7593 (Patent Document 2) discloses poly-γ-glutamic acid having an average molecular weight of 5,000 kDa or more.

However, it is clear that not only the biodegradability, biocompatibility, and moisture retention described above, but the use of PGA is further expanded if the function of PGA is further enhanced.
JP 2002-233391 A International Publication No. WO2004-7593

  The present invention is intended to further enhance the functionality of PGA in consideration of the above-described conventional technology, and aims to provide a PGA derivative having high performance by introducing a special structure into PGA. To do.

  As a result of extensive research, the present inventors have found that a high-performance PGA derivative can be provided by introducing cyclodextrins into PGA, and based on this, the present invention has been completed.

  Specifically, according to the present invention, the following PGA derivatives and the like are provided.

  (1) A poly-γ-glutamic acid derivative to which cyclodextrins are bound.

  (2) The derivative according to item 1, wherein the cyclodextrins are α-cyclodextrin, β-cyclodextrin, or γ-cyclodextrin.

  (3) The derivative according to Item 1, wherein the poly-γ-glutamic acid has a weight average molecular weight of 10,000 or more.

  (4) The derivative according to Item 1, wherein cyclodextrins are bound to 0.1% to 30% of the side chain carboxy group of the poly-γ-glutamic acid.

  According to the present invention, a PGA derivative into which cyclodextrins are introduced is obtained.

  According to the present invention, since the cyclodextrin molecules having inclusion ability are restrained in movement by the main chain of PGA, it can be expected that the inclusion ability is improved.

  Cyclodextrins are known to have a three-dimensional structure like a bucket with a bottom. In this three-dimensional structure, many hydrocarbons exist in the portion corresponding to the inside of the bucket and become hydrophobic, and many hydroxyl groups exist in the portion corresponding to the outside of the bucket and become hydrophilic. According to the present invention, since cyclodextrins having such a special hydrophobic partial structure and hydrophilic partial structure are introduced into PGA having very high hydrophilicity, there is a drawback due to the high hydrophilicity of PGA. Is resolved. Specifically, for example, it is possible to use a hydrophobic substance that is inherently immiscible with PGA together with the PGA derivative.

  Specifically, for example, cyclodextrins are known to be able to stabilize various unstable physiologically active substances (prostaglandins and the like). That is, it can be stabilized by inclusion of a substance unstable to light, ultraviolet rays and heat, or a substance which is easily oxidized or hydrolyzed with cyclodextrins.

  Cyclodextrins can mask unpleasant odors, unpleasant tastes, and the like by inclusion. For this reason, it can be used for various fragrances and foods.

  Cyclodextrins can be solubilized by inclusion of a substance that is hardly soluble in a solvent (for example, water).

  According to the present invention, a PGA derivative having both of these effects specific to cyclodextrins is provided.

  Hereinafter, the present invention will be described in detail.

(PGA)
Poly-γ-glutamic acid (PGA) refers to a compound obtained by polymerization reaction of the amino group of D, L-glutamic acid and the carboxy group at the γ position. PGA can also be used in the present invention as a salt thereof. For example, a sodium salt of PGA can be suitably used.

  The molecular weight of PGA is not particularly limited. However, in view of physical properties, the weight average molecular weight is preferably 10,000 or more, and more preferably 100,000 or more. More preferably, it is 500,000 or more, more preferably 1 million or more, particularly preferably 1.5 million or more, and particularly preferably 2 million or more. Further, in terms of synthesis difficulty, the weight average molecular weight is preferably 13 million or less, and more preferably 10 million or less.

  For example, Patent Document 1 (Japanese Patent Application Laid-Open No. 2002-233391) describes PGA having a weight average molecular weight of about 13 million, and this PGA can be suitably used in the present invention.

  In addition, as described in the conventional technical column of Patent Document 1, a PGA having a molecular weight of 100,000 to 2,000,000 has been conventionally used, and a PGA having this molecular weight range can be used in the present invention. is there.

  The PGA used in the present invention can be produced by any known production method. For example, the method described in Patent Document 1 can be used.

  The PGA is a completely different compound from α-polyglutamic acid. In α-polyglutamic acid, a carboxyl group at the α-position is polycondensed with an amino group to constitute the main chain of the polymer. Alpha polyglutamic acid does not exist in nature and can be obtained by chemical synthesis. Since glutamic acid has two carboxyl groups, there is a very high possibility that branching will occur when attempting to polymerize chemically, and it is extremely difficult to synthesize a polymer that does not contain branching. The cost is high and it is extremely difficult to use industrially. Therefore, α-polyglutamic acid is commercially available as a reagent and used for various studies, but at present, it is hardly used industrially.

  On the other hand, in PGA (γ polyglutamic acid), the carboxyl group at the γ position is polycondensed with an amino group to constitute the main chain of the polymer. PGA is a natural polymer contained in the stringing component of natto and is synthesized by an enzyme with high substrate specificity, so its molecular structure is linear. PGA has been confirmed to be highly safe based on a long dietary experience (natto). The γ-type polyglutamic acid is a completely different substance from the α-type even in the same polyglutamic acid, and its reactivity, properties and functions are completely different.

(Cyclodextrins)
In the present invention, cyclodextrins are compounds in which a plurality of glucoses are bonded to form a large cyclic structure. Examples thereof include those in which 6, 7 or 8 glucose forms a cyclic structure (α, β, γ-cyclodextrin). One or more of the hydroxyl groups of the cyclodextrin may be substituted with a substituent (for example, a lower acyl group). Preferable examples of the lower acyl group include a linear or branched alkylcarbonyl group. The lower acyl group preferably has 6 or less carbon atoms, more preferably 4 or less. The lower acyl group has 1 or more carbon atoms, and in one embodiment, 2 or more carbon atoms. Specific examples include an acetyl group and a propionyl group.

(Synthesis method)
The method for synthesizing the compound of the present invention is not particularly limited. In a preferred embodiment, PGA into which cyclodextrins are introduced is obtained by reacting PGA with cyclodextrins in the presence of an esterification catalyst. It is done.

  Here, the number of moles of cyclodextrins used is appropriately selected according to the desired amount to be introduced to PGA. Roughly 0.1% or more, preferably 1% or more, more preferably 3% or more, still more preferably 5% or more, more preferably 10% or more with respect to the number of moles of the side chain carboxy group of PGA before the reaction. Is particularly preferred. Moreover, it is preferable to set it as 150% or less, 100% or less is more preferable, and 70% or less is further more preferable. 50% or less is more preferable. In one embodiment, it may be 40% or less, and in another embodiment, it may be 30% or less. If the amount to be introduced is too small, it is difficult to obtain the effect of introducing cyclodextrins in the resulting PGA derivative. When the amount to be introduced is too large, the performance as PGA in the obtained PGA derivative tends to be lost.

  In the method described in the examples described later, since the amount of CD in the obtained PGA derivative is smaller than the amount of cyclodextrins used, in such a case, the CD during the synthesis reaction is reduced. It is preferable to determine the amount of cyclodextrins used in the reaction in consideration of a decrease in the amount.

  As the solvent for the reaction between cyclodextrins and PGA, for example, water can be used.

  The reaction between cyclodextrins and PGA can be performed using a condensing agent known to be usable for the reaction between carboxylic acid and alcohol. Although it is possible to carry out the reaction without using a condensing agent, it is extremely preferable to use a condensing agent in view of reaction efficiency and the like. Since the reaction in the production method of the present invention is preferably performed in an aqueous solvent, the condensing agent is preferably a water-soluble condensing agent. Examples of the water-soluble condensing agent include a water-soluble carbodiimide, a triazine-type dehydrating condensing agent, and the like, preferably a water-soluble carbodiimide. From these points, particularly preferred examples of the condensing agent used in the production method of the present invention include 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) which is a water-soluble carbodiimide.

  The amount of the condensing agent (for example, EDC) to be used is preferably 1 mol or more, preferably 2 mol or more, more preferably 2.5 mol or more with respect to 1 mol of cyclodextrins. . Moreover, it is preferable to set it as 10 mol or less with respect to 1 mol of cyclodextrins, it is more preferable to set it as 8 mol or less, and it is further more preferable to set it as 6 mol or less.

  The reaction can also be carried out using an active auxiliary agent together with a condensing agent. The activity assistant is to increase the activity of the condensing agent. Examples of the water-soluble carbodiimide active aid include N-hydroxysuccinimide (NHS). The amount of the active aid used is preferably 0.01 mol or more, preferably 0.1 mol or more, and 0.5 mol or more with respect to 1 mol of the condensing agent. More preferred. Moreover, it is preferable to set it as 10 mol or less with respect to 1 mol of condensing agents, it is more preferable to set it as 5 mol or less, and it is more preferable to set it as 3 mol or less. When NHS is used as an activity aid, it is preferably used in an amount of about 0.1 to 3 mol per 1 mol of water-soluble carbodiimide.

  Moreover, as a catalyst at the time of reaction of cyclodextrins and PGA, it is possible to use the catalyst known that it can be used for reaction of carboxylic acid and alcohol as needed. About the usage-amount of a catalyst, it can determine suitably according to the kind and performance of each catalyst.

  The temperature during the reaction is not particularly limited. It may be room temperature or may be heated. However, if the temperature is too low, the reaction takes a very long time, so it is preferable to carry out the reaction at room temperature or above room temperature. Specifically, it is preferably 10 ° C. or higher, more preferably 15 ° C. or higher, and further preferably 20 ° C. or higher. Moreover, Preferably it is 100 degrees C or less, More preferably, it is 50 degrees C or less. If it is too high, the PGA tends to decompose. Therefore, it is preferable to carry out at around room temperature.

  The reaction time is preferably 30 minutes or more, more preferably 1 hour or more, still more preferably 2 hours or more, still more preferably 6 hours or more, and particularly preferably 12 hours or more. It is. However, in order to shorten the length of the entire process, it is preferably 7 days or less, more preferably 4 days or less, and still more preferably 2 days or less.

  It is known that EDC activates a carboxyl group under conditions of weakly acidic to basic pH, and an active intermediate thereof reacts with an amino group or a hydroxyl group to form an amide bond or an ester bond. Examples of the use of EDC are described in Biomaterials 17 765-773 (1996), or Journal of Applied Polymer Science 90 747-753 (2003). By applying the reaction conditions described in these documents, the reaction can be performed efficiently. Accordingly, the pH during the reaction is preferably 6 or more, more preferably 7 or more. Moreover, Preferably it is 11 or less, More preferably, it is 10 or less.

  After the reaction is completed, the derivative of the present invention can be obtained by neutralization, removal of the remaining reaction materials (condensation agent, active auxiliary agent, etc.), removal of the solvent and purification, as necessary. In the neutralization of the aqueous solution after the reaction, an alkali such as sodium hydroxide, potassium hydroxide, sodium bicarbonate, arginine or the like can be used.

(Linker)
In the present invention, the cyclodextrins are preferably directly bonded to the side chain carboxyl group of PGA. However, if necessary, the cyclodextrins may be bonded to the carboxy group of PGA via a linker. Good. As the linker, any low molecular weight compound having a functional group capable of binding to a hydroxyl group of cyclodextrin and a functional group capable of binding to a carboxyl group of PGA can be used. The molecular weight of such a low molecular compound is preferably 500 or less, and more preferably 300 or less. Moreover, it is preferable that it is 50 or more. The main chain of the linker is preferably an alkylene group. That is, as the linker, for example, any compound having a functional group capable of binding to a hydroxyl group of cyclodextrin and a functional group capable of binding to a carboxyl group of PGA to lower alkylene (for example, alkylene having 1 to 5 carbon atoms) Can be preferably used.

(Product)
When a linker is not used, the derivative obtained by the above-described method has a structure in which cyclodextrins are covalently bonded to a part of the side chain carboxyl group of PGA by an ester bond.

  Although it is possible to bind cyclodextrins to all of the carboxyl groups of PGA, binding to only a part thereof is effective in that the performance of PGA is not impaired. The number of moles of cyclodextrins to be introduced can be controlled by adjusting the blending amount and reaction time when the cyclodextrins and PGA are reacted. The number of moles of cyclodextrins to be introduced can be appropriately designed according to the application. 0.001% or more is preferable, 0.1% or more is more preferable, 1% or more is further preferable, and 3% or more is particularly preferable with respect to the number of moles of the side chain carboxyl group of PGA before the reaction. Moreover, it is preferable to set it as 80% or less, 60% or less is more preferable, and 40% or less is further more preferable. In one embodiment, it may be 20% or less, and in another embodiment, it may be 10% or less. When the amount to be introduced is too small, it is difficult to obtain the effect of introducing cyclodextrins. When the amount to be introduced is too large, the performance as PGA in the obtained PGA derivative tends to be lost.

  The glucose unit constituting the cyclodextrin has a secondary hydroxyl group at the 2nd and 3rd positions and a primary hydroxyl group at the 6th position. The primary hydroxyl group reacts with the side chain carboxyl group of PGA.

  Since cyclodextrins have many glucose units, and each glucose unit has a primary hydroxyl group at the 6-position, theoretically, multiple hydroxyl groups of one cyclodextrin molecule react with PGA. As a result, it is considered that cyclodextrin crosslinks between PGA molecules and gels PGA. However, in Examples 1 to 3 described later, such gelation does not occur, and under the conditions as in these Examples, there is a reaction in which only one hydroxyl group of one cyclodextrin binds to PGA. It was found to occur preferentially.

  On the other hand, when a linker is used, the PGA derivative has a structure in which one end of the linker is bonded to a part of the side chain carboxyl group of PGA and the other end of the linker is bonded to a hydroxyl group of cyclodextrins. . The bond between the side chain carboxyl group of PGA and the linker may be an ester bond or another bond (for example, an amide bond). Moreover, arbitrary coupling | bonding can be employ | adopted for the coupling | bonding of a linker and the hydroxyl group of cyclodextrins.

(Use)
The derivatives of the present invention are expected to be applied in a wide range of fields. That is, the hydrophilicity is controlled while maintaining the biocompatibility of PGA, and the action effect peculiar to cyclodextrin is added, so that it can be used as various materials in various fields such as pharmaceuticals, cosmetics and foods. It becomes possible.

  Below, the Example which introduce | transduces cyclodextrins into PGA is demonstrated.

(Preparation of PGA)
According to the method described in International Publication WO2004-7593, a sodium salt of PGA having a molecular weight of about 2 million (PGANa) was obtained. This was used in the following experiment.

Example 1
A PGANa salt aqueous solution was prepared by dissolving 3 mmol of PGANa salt (0.451 g) as a glutamic acid monomer in 70 mL of deionized water. Β-cyclodextrin (β-CD) 0.3 mmol (0.341 g) was added and dissolved in the aqueous solution, N-hydroxysuccinimide (NHS) 0.9 mmol (0.104 g) was added, and then 1 The reaction was initiated by adding 0.9 mmol (0.172 g) of ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride (EDC). All the reactions were carried out by stirring at room temperature, and the reaction time was 24 hours. After completion of the reaction, 30 g of ion exchange resin swollen in the reaction solution (manufactured by Organo, Amberlite IR-120H) was added to 1 g of EDC used in the reaction, and the remaining amine was neutralized by stirring for 12 hours. The crude purification was performed. Sodium bicarbonate was added to the crude purified product aqueous solution to neutralize the crude purified product, and the aqueous solution was purified by dialysis using a dialysis membrane having a molecular weight fraction of 2000 (water was exchanged four times). Then, the product was obtained by freeze-drying the contents. The yield based on the amount of PGA and β-cyclodextrin used (theoretical yield assuming that all of the β-cyclodextrin used in the charge was introduced into the charged PGA) was 55%.

  The synthesis was confirmed from the nuclear magnetic resonance spectrum. As a result, the peak of hydrogen bonded to the carbon of the cyclodextrin residue (for example, the peak of hydrogen bonded to the carbon at position 1 of the glucose unit: 5.04 ppm) is larger than that before binding to PGA. Since it was remarkably broadened, it was confirmed that cyclodextrin was bound to the side chain of PGA.

  As a result of examining NMR of the obtained derivative for the cyclodextrin introduction rate, the integrated value of the peak (4.12 ppm) of hydrogen bonded to the β-position carbon in the PGA residue and the glucose of each βCD residue From the ratio of the integrated value of the hydrogen peak (3.5 to 4.0 ppm) bonded to the 2nd to 6th carbons of the unit, about 3.6% of the carboxyl groups in the side chain of PGA It was confirmed that cyclodextrin was introduced.

(Example 2)
The amount of β-cyclodextrin (β-CD) used is 0.6 mmol (0.681 g), and the amount of N-hydroxysuccinimide (NHS) is 1.8 mmol (0.207 g). The experiment was performed in the same manner as in Example 1 except that the amount of -ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride (EDC) used was 1.8 mmol (0.346 g). As a result, the yield based on the amount of PGA and β-cyclodextrin used (based on the theoretical yield assuming that all β-cyclodextrin used in the charging was introduced into the charged PGA) was 38%. The β-CD introduction rate was 6.4%.

(Example 3)
The amount of β-cyclodextrin (β-CD) used is 0.9 mmol (1.022 g), and the amount of N-hydroxysuccinimide (NHS) is 2.7 mmol (0.311 g). The experiment was conducted in the same manner as in Example 1 except that the amount of use of -ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride (EDC) was 2.7 mmol (0.518 g). As a result, the yield based on the amount of PGA and β-cyclodextrin used (based on the theoretical yield assuming that all β-cyclodextrin used in the charging was introduced into the charged PGA) was 38%. The β-CD introduction rate was 9.6%.

  The following table summarizes the conditions and results of Examples 1-3.

なお、上記実施例１〜３においては、ゲル化が起こらなかったので、これらの生成物においては、β−ＣＤの複数の水酸基がＰＧＡのカルボキシル基と反応して架橋する現象は起こらなかったことが理解される。すなわち、β−ＣＤに存在する７つのグルコースユニットのうちの１つのグルコースユニットの６位の水酸基がＰＧＡのカルボキシル基と反応して結合したことがわかる。

In Examples 1 to 3, since gelation did not occur, in these products, the phenomenon that a plurality of hydroxyl groups of β-CD reacted with the carboxyl groups of PGA and cross-linked did not occur. Is understood. That is, it can be seen that the hydroxyl group at the 6-position of one glucose unit among the seven glucose units present in β-CD reacts with and binds to the carboxyl group of PGA.

  As mentioned above, although this invention has been illustrated using preferable embodiment of this invention, this invention should not be limited and limited to this embodiment. It is understood that the scope of the present invention should be construed only by the claims. Those skilled in the art can understand the scope equivalent to the claims based on the description of the specific preferred embodiments of the present invention and the common general technical knowledge. Patents, patent applications, and documents cited herein should be incorporated by reference in their entirety, as if the contents themselves were specifically described herein. Understood.

  According to the present invention, a PGA derivative into which cyclodextrins are introduced is provided. For this reason, in various uses of biotechnology, various biological materials (for example, pharmaceuticals) can be included to express various effects.

Claims (4)

A poly-γ-glutamic acid derivative to which cyclodextrins are bound. The derivative of claim 1, wherein said cyclodextrins are alpha cyclodextrin, beta cyclodextrin, or gamma cyclodextrin. The derivative according to claim 1, wherein the poly-γ-glutamic acid has a weight average molecular weight of 10,000 or more. The derivative according to claim 1, wherein cyclodextrins are bound to 0.1% to 30% of the side chain carboxyl groups of the poly-γ-glutamic acid.