JP2006083380A - Cyclic glucan derivative having hydrophobic group and method for producing the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new cyclic glucan derivative having a highly polymerized cyclic glucan as a basic skeleton, useful for various purposes, such as a pharmaceutical, a reagent, a surfactant, a composition for a cosmetic, a composition for a food, and a film for a magnetic recording medium, having good solubility in oils, having excellent surface active characteristics, and having excellent properties as a host molecule for molecular recognition, and to provide a method for producing the same. <P>SOLUTION: This new cyclic glucan derivative is structured so that a hydrophobic group is combined with the highly polymerized cyclic glucan. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a cyclic glucan derivative having a hydrophobic group in the side chain of a highly polymerized cyclic glucan and a method for producing the same. The present invention also relates to a pharmaceutical, a surfactant, a cosmetic composition, a food composition and a magnetic recording medium film containing the cyclic glucan derivative.

  Cyclodextrins, which are glucans having only a cyclic structure composed of α-1,4-glucoside bonds, are known as existing glucans having a cyclic structure. These cyclodextrins are generally synthesized by allowing cyclodextrin glucanotransferase (CGTase) to act on starch, and the degree of polymerization is usually 6 to 8. Kobayashi (1993) Denpun Kagaku: vol. 40 103-116 reports that a glucan having only a cyclic structure composed of α-1,4-glucoside bonds having a degree of polymerization higher than this is synthesized by CGTase. The degree of polymerization is 13. Such cyclodextrins are well known in that they can include low molecular weight active substances by utilizing the cyclic molecular properties. However, cyclodextrins are generally insoluble in oil-based hydrocarbon solvents and are limited in use to surfactants, pharmaceuticals in the form of dispersions, cosmetic compositions and food compositions. It is.

  In order to change the properties of these compounds, it is known to substitute a hydroxyl group of a cyclodextrin having a polymerization degree of 6 to 8 with an alkyl group or a fluorinated alkyl group (see, for example, Patent Documents 1 and 2). However, these low-polymerization cyclodextrins have a very low degree of freedom in steric structure in aqueous solution, and can only take a planar cyclic structure, so the number of molecules that can be included in the ring is limited, and the dispersion form Applications to pharmaceutical and cosmetic compositions are limited.

In this way, a wide variety of compounds with low to high molecular weight, having good solubility in oils, excellent surface active properties, and changing the cyclic steric structure according to the structure of the inclusion compound There is still no new cyclic glucan derivative based on a highly polymerized cyclic glucan that has excellent properties as a molecular recognition host molecule that enables the inclusion of proteins, and it has not existed in the pharmaceutical, chemical, food, health, and electronics industries. Very important and its development is craved.
Japanese Patent No. 2749565 JP-A-9-241303

  The object of the present invention is to have good solubility in oils and excellent surface active properties useful for uses such as pharmaceuticals, reagents, surfactants, cosmetic compositions, food compositions and films for magnetic recording media. And has excellent properties as a molecular recognition host molecule that enables the inclusion of a wide variety of low to high molecular weight compounds by changing the cyclic steric structure according to the structure of the compound to be included. It is an object to provide a novel cyclic glucan derivative having a cyclic glucan having a polymerization degree as a basic skeleton and a method for producing the same.

  As a result of intensive research, the present inventors have found that the alcoholic hydroxyl group of a cyclic glucan having a high degree of polymerization can be substituted with a tosyl group, and that this tosyl group can be exchanged with fatty acid sodium. By providing the cyclic glucan derivative of the present invention in which a hydrophobic group is introduced as a side chain into the cyclic glucan, the above-mentioned problems have been solved.

  The present inventors have also found that the cyclic glucan derivative significantly reduces the surface tension of water, and completed the present invention.

  The present inventors further found that the composition containing the cyclic glucan derivative is useful as a pharmaceutical, a surfactant, a cosmetic composition, a food composition, and a film for magnetic recording media, and completed the present invention. did.

  The inventor has also found a new diagnosis, treatment, and differentiation method using the composition containing the cyclic glucan derivative, and has completed the present invention.

Thus, the present invention provides the following.
(1) It has a main chain composed of a cyclic glucan having a polymerization degree of 14 or more and one or more side chains composed of a hydrophobic group, and the side chain is bonded to the main chain via a linking group. Cyclic glucan derivative.
(2) The term wherein the hydrophobic group is a C _{3 to} C ₂₀ linear or branched alkyl group, a C _{3 to} C ₂₀ linear or branched fluorinated alkyl group, or a tosyl group The cyclic glucan derivative according to (1).
(3) The term wherein the hydrophobic group is a C _{3 to} C ₂₀ linear fluorohydroalkyl group having a fluorine substitution rate of at least 50% or a C _{3 to} C ₂₀ linear perfluoroalkyl group ( The cyclic glucan derivative according to 1).
(4) The cyclic glucan derivative according to claim 1, wherein the hydrophilic portion consists only of a cyclic glucan having a polymerization degree of 14 or more.
(5) The above-mentioned cyclic glucan is a glucan having one cyclic structure having a polymerization degree of 14 or more constituted by α-1,4-glucoside bonds in the molecule,
(I) a glucan having an acyclic structure in addition to a cyclic structure composed only of α-1,4-glucoside bonds,
(Ii) a glucan having an acyclic structure in addition to a cyclic structure composed of an α-1,4-glucoside bond and at least one α-1,6-glucoside bond,
(Iii) a glucan having only a cyclic structure composed only of α-1,4-glucoside bonds, and (iv) an α-1,4-glucoside bond and at least one α-1,6-glucoside bond. A glucan having only a cyclic structure,
The cyclic glucan derivative according to item (1), selected from the group consisting of:
(6) The cyclic glucan derivative according to item (1), wherein the side chain is bonded to the cyclic glucan through an amide bond, an ether bond, or an ester bond.
(7) The cyclic glucan derivative according to item (1), wherein at least one of the alcoholic hydroxyl groups of the cyclic glucan is bonded to a fluorine-substituted or unsubstituted fatty acid by an ester bond.
(8) Cyclic glucan derivative represented by the following structural formula:

Here, each of the n monomer units is independent, each of the n R ¹ independently represents -LR ⁴ or R ⁵ , and each of the n R ² is independently- L—R ⁴ or R ⁵ , and n R ^{3 s} independently represent —LR ⁴ or R ⁵ , provided that at least one —LR ⁴ is present in one molecule;
Each R ⁴ is independently a C _{3 to} C ₂₀ linear or branched alkyl group, or a C _{3 to} C ₂₀ linear or branched fluorinated alkyl group;
Each L is independently —NHCO—, —O—, —S—, —OSO ₂ —, —SCO— or —OCO—;
Each R ⁵ is independently a hydroxyl group or a tosyl group;
n is a cyclic glucan derivative having a molecular weight of 14 to 5,000.
(9) The cyclic glucan derivative according to item (8), wherein all of n R ² and n R ³ are hydroxyl groups.
(10) A method for producing a cyclic glucan derivative,
Introducing a tosyl group into a cyclic glucan having a polymerization degree of 14 or more to obtain a tosylated derivative, and reacting the tosylated derivative with a substituted or unsubstituted fatty acid ester to obtain an ester derivative;
Including the method.
(11) The following steps: a) A step of reacting a linear α-1,4-glucan or a saccharide containing these with a D enzyme to obtain a highly polymerized cyclic glucan;
b) In an alkaline aqueous solution, the high polymerization degree cyclic glucan reacts with p-toluenesulfonyl chloride, and at least one of the alcoholic hydroxyl groups of the high polymerization degree cyclic glucan reacts with the p-toluenesulfonyl chloride. Contacting to give a tosyl group-containing cyclic glucan derivative in which a tosyl group is bonded to a cyclic glucan having a high degree of polymerization;
c) A cyclic group in which a hydrophobic group is bonded by bringing the tosyl group-containing cyclic glucan derivative into contact with a fluorine-substituted or unsubstituted fatty acid sodium under conditions where the tosyl group and the fatty acid sodium can exchange with each other. Obtaining a glucan derivative;
A process for producing a cyclic glucan derivative.
(12) In the step b), at least 3% or more of all the alcoholic hydroxyl groups of the high polymerization degree cyclic glucan and the p-toluenesulfonyl chloride are substituted with tosyl groups. The method according to item (11), wherein the contact is performed under reaction conditions.
(13) In the step c), the tosyl group-containing cyclic glucan derivative and the fatty acid sodium are brought into contact under reaction conditions where the exchange rate between the tosyl group and the fatty acid sodium is at least 50%. The method according to 11).
(14) A medicament comprising the cyclic glucan derivative according to item (1) or a mixture thereof.
(15) A surfactant comprising the cyclic glucan derivative according to item (1) or a mixture thereof.
(16) A cosmetic composition comprising the cyclic glucan derivative according to item (1) or a mixture thereof.
(17) A film for a magnetic recording medium, comprising the cyclic glucan derivative according to item (1) or a mixture thereof.
(18) In a magnetic recording medium comprising one or more magnetic layers on a nonmagnetic support, the magnetic layer or a protective layer provided on the magnetic layer is provided on the surface of the magnetic recording medium according to item (17). A magnetic recording medium having a film formed thereon.

  According to the present invention, the solubility in oils is good, the surface active characteristics are excellent, and the cyclic steric structure is changed according to the structure of the compound to be included, so that a wide variety of low to high molecular weights can be obtained. It is possible to provide a novel cyclic glucan derivative based on a highly polymerized cyclic glucan having a superior property as a molecular recognition host molecule that enables inclusion of various compounds as a basic skeleton, and a method for producing the same.

  The composition containing a cyclic glucan derivative in which a hydrophobic group of the present invention is bonded to a cyclic glucan having a high polymerization degree is useful as a pharmaceutical, a surfactant, a cosmetic composition, a food composition, and a film for a magnetic recording medium.

  The present invention will be described below. Throughout this specification, it should be understood that the singular forms also include the plural concept unless specifically stated otherwise. In addition, it is to be understood that the terms used in the present specification are used in the meaning normally used in the art unless otherwise specified.

(the term)
Listed below are definitions of terms particularly used in the present specification.

  In the present specification, “glucan” means a general term for polysaccharides composed of D-glucose. The “glucan” of the present invention includes storage polysaccharides such as starch, glycogen, laminaran, cellulose, lichenin, dextran, and nigeran, structural polysaccharides, and metabolic byproducts.

  In the present specification, the “high degree of polymerization glucan” means a higher degree of polymerization than α-, β-, or γ-cyclodextrin, which is a conventional cyclic glucan having a degree of polymerization of 6 to 8, and α-1,4-glucoside. It means a glucan having one cyclic structure formed by a bond in the molecule. In the present invention, a preferable high-polymerization degree cyclic glucan is a glucan having one cyclic structure in the molecule having a polymerization degree of 14 or more constituted by an α-1,4-glucoside bond. As a typical example, (i) α A glucan having an acyclic structure in addition to a cyclic structure composed of only a -1,4-glucoside bond, (ii) an α-1,4-glucoside bond and at least one α-1,6-glucoside bond A glucan having an acyclic structure in addition to the constituted cyclic structure, (iii) a glucan having only a cyclic structure composed only of α-1,4-glucoside bonds, or (iv) an α-1,4-glucoside bond And a glucan having only a cyclic structure composed of at least one α-1,6-glucoside bond.

  In the present specification, the “monomer unit” or “glucose monomer unit” means a monomer unit composed of glucose or a glucose derivative, which constitutes a high polymerization degree cyclic glucan.

  In the present specification, the “hydrophilic portion containing a cyclic glucan having a polymerization degree of 14 or more” means a partial structure in which a substituent other than a hydrophobic group is introduced into a high polymerization degree cyclic glucan as necessary. That is, it means a portion other than the side chain comprising the hydrophobic group of the cyclic glucan derivative of the present invention. Examples of substituents introduced into the cyclic glucan as necessary in this hydrophilic site include, but are not limited to, hydrophilic receptors, ligands, dyes and the like. Examples of receptors include antibodies, enzymes, Fab fragments, receptor proteins such as lectins, nucleic acids and the like. Examples of ligands include, for example, antigens, enzyme substrates, hormones, haptens, vitamins, alkaloids, nucleic acids, monosaccharides, polysaccharides and the like. These substituents are introduced into any position of the cyclic glucan by a known method.

  In the present specification, the “hydrophobic group” means all functional groups that, when introduced as a side chain of the above-mentioned highly-polymerized cyclic glucan, reduce the surface tension of water and exhibit surface activity. . As the hydrophobic group of the present invention, as a tosyl group, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, cycloalkenyl, substituted cycloalkenyl, alkynyl, substituted alkynyl, Examples include, but are not limited to, alkoxy, substituted alkoxy, carbocyclic groups, and substituted carbocyclic groups. These functional groups are defined in detail in the following (Organic Chemistry) section.

  In the present specification, the “cyclic glucan derivative” is a compound obtained by binding the hydrophobic group defined above to a hydrophilic site containing a high-polymerization cyclic glucan or a cyclic glucan having a polymerization degree of 14 or more as defined above. It means everything. The term “cyclic glucan derivative” of the present invention is used herein interchangeably with the term “high-polymerization cyclic glucan after transesterification”.

  In the present specification, the “tosyl group-containing cyclic glucan derivative” means all compounds obtained by binding a tosyl group in the group of hydrophobic groups defined above to a cyclic glucan having a high polymerization degree. The term “tosyl group-containing cyclic glucan derivative” of the present invention is used interchangeably herein with the term “tosylated high-polymerization cyclic glucan”.

  As used herein, “(hydrophobic group bound to high-polymerization cyclic glucan)” means derivatization selected from the group consisting of an amide bond, an ether bond and an ester bond. These derivatizations can be appropriately selected by those skilled in the art depending on the application. If necessary, one of the derivatization methods is usually a method used for starch modification (Biochemical Experimental Method 19, “Starch and Related Sugar Experimental Method”: Nakamura et al. 1986, pages 273-303).

  In this specification, the term “fluorine substitution rate” means the percentage (percentage) of substitution with fluorine atoms with respect to the total number of hydrogen atoms in the alkyl group before fluorine substitution.

(organic chemistry)
As used herein, “alcohol” refers to an organic compound in which one or more hydrogen atoms of an aliphatic hydrocarbon are substituted with a hydroxyl group.

As used herein, “alkyl” refers to a monovalent group formed by loss of one hydrogen atom from an aliphatic hydrocarbon (alkane) such as methane, ethane, or propane, and is generally represented by C _n H _{2n + 1} −. Where n is a positive integer. Alkyl can be linear or branched. In the present specification, the “substituted alkyl” refers to an alkyl in which H of the alkyl is substituted with a substituent specified below. Specific examples of the hydrophobic group of the cyclic glucan derivative of the present invention include C3-C4 alkyl, C3-C5 alkyl, C3-C6 alkyl, C3-C7 alkyl, C3-C8 alkyl, C3-C9 alkyl, C3-C10 alkyl, C3-C11 alkyl or C3-C12 alkyl, C3-C13 alkyl, C3-C14 alkyl, C3-C15 alkyl, C3-C16 alkyl, C3-C17 alkyl, C3-C18 alkyl, C3-C19 alkyl, C3-C20 alkyl, C3-C4 substituted alkyl, C3-C5 substituted alkyl, C3-C6 substituted alkyl, C3-C7 substituted alkyl, C3-C8 substituted alkyl, C3-C9 substituted alkyl, C3- C10 substituted alkyl, C3-C11 substituted alkyl or C3 C12 substituted alkyl, C3-C13 substituted alkyl, C3-C14 substituted alkyl, C3-C15 substituted alkyl, C3-C16 substituted alkyl, C3-C17 substituted alkyl, C3-C18 substituted Substituted alkyl, C3-C19 substituted alkyl, C3-C20 substituted alkyl. Here, for example, C3-C10 alkyl means linear or branched alkyl having 3 to 10 carbon atoms, and includes n-propyl (CH ₃ CH ₂ CH ₂ —), isopropyl ((CH ₃ ) ₂ CH -), n- butyl _{(CH 3 CH 2 CH 2 CH} 2 -), n- pentyl _{(CH 3 CH 2 CH 2 CH} 2 CH 2 -), n- hexyl _{(CH 3 CH 2 CH 2 CH} 2 CH ₂ CH ₂ -), n- heptyl _{(CH 3 CH 2 CH 2 CH} 2 CH 2 CH 2 CH 2 -), n- octyl _{(CH 3 CH 2 CH 2 CH} 2 CH 2 CH 2 CH 2 CH 2 -), n- nonyl _{(CH 3 CH 2 CH 2 CH} 2 CH 2 CH 2 CH 2 CH 2 CH 2 -), n- decyl _{(CH 3 CH 2 CH 2 CH} 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH _{2 -), - C (CH} 3) 2 CH 2 CH 2 CH (CH 3) 2, -CH 2 CH (CH 3) such as ₂ are exemplified. In addition, for example, C3-C10 substituted alkyl refers to C3-C10 alkyl, in which one or more hydrogen atoms are substituted with a substituent.

  In the present specification, “fluorinated alkyl (group)” means an alkyl group in which at least one hydrogen atom in the alkyl group defined above is substituted with a fluorine atom, and particularly a hydrogen atom constituting the alkyl group. Alkyl groups that are all substituted with fluorine atoms are referred to as “perfluoroalkyl groups” or “perfluoroalkyl groups”. In contrast, alkyl groups in which some hydrogen atoms are replaced with fluorine atoms and hydrogen atoms remain. Is referred to as “fluorohydroalkyl group” or “hydrofluoroalkyl group”.

  As used herein, “cycloalkyl” refers to alkyl having a cyclic structure. “Substituted cycloalkyl” refers to a cycloalkyl in which H of the cycloalkyl is substituted by the substituent specified below. Specific examples of the hydrophobic group of the cyclic glucan derivative of the present invention include C3-C4 cycloalkyl, C3-C5 cycloalkyl, C3-C6 cycloalkyl, C3-C7 cycloalkyl, C3-C8 cycloalkyl, C3-C9 cycloalkyl. Alkyl, C3-C10 cycloalkyl, C3-C11 cycloalkyl, C3-C12 cycloalkyl, C3-C13 cycloalkyl, C3-C14 cycloalkyl, C3-C15 cycloalkyl, C3-C16 cycloalkyl, C3-C17 cycloalkyl, C3-C18 cycloalkyl, C3-C19 cycloalkyl, C3-C20 cycloalkyl, C3-C4 substituted cycloalkyl, C3-C5 substituted cycloalkyl, C3-C6 substituted cycloalkyl, C3-C7 substituted Cycloalkyl, C3 C8 substituted cycloalkyl, C3-C9 substituted cycloalkyl, C3-C10 substituted cycloalkyl, C3-C11 substituted cycloalkyl or C3-C12 substituted cycloalkyl, C3-C13 substituted cycloalkyl Alkyl, C3-C14 substituted cycloalkyl, C3-C15 substituted cycloalkyl, C3-C16 substituted cycloalkyl, C3-C17 substituted cycloalkyl, C3-C18 substituted cycloalkyl, C3-C19 It can be a substituted cycloalkyl, a C3-C20 substituted cycloalkyl. For example, cycloalkyl is exemplified by cyclopropyl, cyclohexyl and the like.

As used herein, “alkenyl” refers to a monovalent group formed by loss of one hydrogen atom from an aliphatic hydrocarbon having one double bond in the molecule, such as ethylene and propylene. _n H _2n-1 − (where n is a positive integer of 2 or more). “Substituted alkenyl” refers to alkenyl in which H of alkenyl is substituted by a substituent specified below. Specific examples of the hydrophobic group of the cyclic glucan derivative of the present invention include C3-C4 alkenyl, C3-C5 alkenyl, C3-C6 alkenyl, C3-C7 alkenyl, C3-C8 alkenyl, C3-C9 alkenyl, C3-C10 alkenyl. C3-C11 alkenyl, C3-C12 alkenyl, C3-C13 alkenyl, C3-C14 alkenyl, C3-C15 alkenyl, C3-C16 alkenyl, C3-C17 alkenyl, C3-C18 alkenyl, C3-C19 alkenyl, C3-C20 alkenyl C3-C4 substituted alkenyl, C3-C5 substituted alkenyl, C3-C6 substituted alkenyl, C3-C7 substituted alkenyl, C3-C8 substituted alkenyl, C3-C9 substituted alkenyl, C3 ~ C10 substituted a Kenyl, C3-C11 substituted alkenyl, C3-C12 substituted alkenyl, C3-C13 substituted alkenyl, C3-C14 substituted alkenyl, C3-C15 substituted alkenyl, C3-C16 substituted alkenyl, It can be C3-C17 substituted alkenyl, C3-C18 substituted alkenyl, C3-C19 substituted alkenyl, C3-C20 substituted alkenyl. Here, for example, C3 to C10 alkyl means a linear or branched alkenyl containing 3 to 10 carbon atoms, and examples include allyl (CH ₂ ═CHCH ₂ —), CH ₃ CH═CH—, and the like. Is done. In addition, for example, C3-C10 substituted alkenyl refers to C3-C10 alkenyl, in which one or more hydrogen atoms are substituted with a substituent.

  As used herein, “cycloalkenyl” refers to alkenyl having a cyclic structure. “Substituted cycloalkenyl” refers to cycloalkenyl in which H of cycloalkenyl is substituted by the substituent specified below. Specific examples of the hydrophobic group of the cyclic glucan derivative of the present invention include C3-C4 cycloalkenyl, C3-C5 cycloalkenyl, C3-C6 cycloalkenyl, C3-C7 cycloalkenyl, C3-C8 cycloalkenyl, C3-C9 cyclo Alkenyl, C3-C10 cycloalkenyl, C3-C11 cycloalkenyl, C3-C12 cycloalkenyl, C3-C13 cycloalkenyl, C3-C14 cycloalkenyl, C3-C15 cycloalkenyl, C3-C16 cycloalkenyl, C3-C17 cycloalkenyl, C3-C18 cycloalkenyl, C3-C19 cycloalkenyl, C3-C20 cycloalkenyl, C3-C4 substituted cycloalkenyl, C3-C5 substituted cycloalkenyl, C3-C6 substituted cycloalkenyl, 3-C7 substituted cycloalkenyl, C3-C8 substituted cycloalkenyl, C3-C9 substituted cycloalkenyl, C3-C10 substituted cycloalkenyl, C3-C11 substituted cycloalkenyl or C3-C12 substituted Cycloalkenyl, C3-C13 substituted cycloalkenyl, C3-C14 substituted cycloalkenyl, C3-C15 substituted cycloalkenyl, C3-C16 substituted cycloalkenyl, C3-C17 substituted cycloalkenyl, C3 -C18 substituted cycloalkenyl, C3-C19 substituted cycloalkenyl, C3-C20 substituted cycloalkenyl. For example, preferable cycloalkenyl includes 1-cyclopentenyl, 2-cyclohexenyl and the like.

As used herein, “alkynyl” refers to a monovalent group formed by losing one hydrogen atom from an aliphatic hydrocarbon having one triple bond in the molecule, such as acetylene, and is generally C _n H _{2n −3} − (where n is a positive integer of 2 or more). “Substituted alkynyl” refers to alkynyl in which H of alkynyl is substituted by the substituent specified below. Specific examples of the hydrophobic group of the cyclic glucan derivative of the present invention include C3-C4 alkynyl, C3-C5 alkynyl, C3-C6 alkynyl, C3-C7 alkynyl, C3-C8 alkynyl, C3-C9 alkynyl, C3-C10 alkynyl. C3-C11 alkynyl, C3-C12 alkynyl, C3-C13 alkynyl, C3-C14 alkynyl, C3-C15 alkynyl, C3-C16 alkynyl, C3-C17 alkynyl, C3-C18 alkynyl, C3-C19 alkynyl, C3-C20 alkynyl C3-C4 substituted alkynyl, C3-C5 substituted alkynyl, C3-C6 substituted alkynyl, C3-C7 substituted alkynyl, C3-C8 substituted alkynyl, C3-C9 substituted alkynyl, C3 ~ C10 substituted a Quinyl, C3-C11 substituted alkynyl, C3-C12 substituted alkynyl, C3-C13 substituted alkynyl, C3-C14 substituted alkynyl, C3-C15 substituted alkynyl, C3-C16 substituted alkynyl, It can be C3-C17 substituted alkynyl, C3-C18 substituted alkynyl, C3-C19 substituted alkynyl, C3-C20 substituted alkynyl. Here, for example, C2-C10 alkynyl means, for example, straight-chain or branched alkynyl containing 2 to 10 carbon atoms, and includes ethynyl (CH≡C-), 1-propynyl (CH ₃ C≡C -) Etc. are exemplified. Further, for example, C2-C10 substituted alkynyl refers to C2-C10 alkynyl, in which one or more hydrogen atoms are substituted with a substituent.

As used herein, “alkoxy” refers to a monovalent group generated by loss of a hydrogen atom of a hydroxy group of an alcohol, and is generally represented by C _n H _{2n + 1} O— (where n is 1 or more). Is an integer). “Substituted alkoxy” refers to alkoxy in which H of alkoxy is substituted by a substituent specified below. Specific examples of the hydrophobic group of the cyclic glucan derivative of the present invention include C3-C4 alkoxy, C3-C5 alkoxy, C3-C6 alkoxy, C3-C7 alkoxy, C3-C8 alkoxy, C3-C9 alkoxy, C3-C10 alkoxy. C3-C11 alkoxy, C3-C12 alkoxy, C3-C13 alkoxy, C3-C14 alkoxy, C3-C15 alkoxy, C3-C16 alkoxy, C3-C17 alkoxy, C3-C18 alkoxy, C3-C19 alkoxy, C3-C20 alkoxy C3-C4 substituted alkoxy, C3-C5 substituted alkoxy, C3-C6 substituted alkoxy, C3-C7 substituted alkoxy, C3-C8 substituted alkoxy, C3-C9 substituted alkoxy, C3 ~ C10 substituted a Coxy, C3-C11 substituted alkoxy, C3-C12 substituted alkoxy, C3-C13 substituted alkoxy, C3-C14 substituted alkoxy, C3-C15 substituted alkoxy, C3-C16 substituted alkoxy, It can be C3-C17 substituted alkoxy, C3-C18 substituted alkoxy, C3-C19 substituted alkoxy, C3-C20 substituted alkoxy.

  In this specification, the “carbocyclic group” is a group containing a cyclic structure containing only carbon, and the above-mentioned “cycloalkyl”, “substituted cycloalkyl”, “cycloalkenyl”, “substituted cyclo A group other than “alkenyl”. The carbocyclic group can be aromatic or non-aromatic and can be monocyclic or polycyclic. The “substituted carbocyclic group” refers to a carbocyclic group in which H of the carbocyclic group is substituted with a substituent specified below. Specific examples include C3-C4 carbocyclic group, C3-C5 carbocyclic group, C3-C6 carbocyclic group, C3-C7 carbocyclic group, C3-C8 carbocyclic group, C3-C9 carbocyclic group, C3-C10. Carbocyclic group, C3-C11 carbocyclic group, C3-C12 carbocyclic group, C3-C4-substituted carbocyclic group, C3-C5-substituted carbocyclic group, C3-C6-substituted carbocyclic group, C3- C7 substituted carbocyclic group, C3-C8 substituted carbocyclic group, C3-C9 substituted carbocyclic group, C3-C10 substituted carbocyclic group, C3-C11 substituted carbocyclic group or C3- It can be a C12 substituted carbocyclic group. The carbocyclic group can also be a C4-C7 carbocyclic group or a C4-C7 substituted carbocyclic group. Examples of the carbocyclic group include those in which one hydrogen atom is deleted from the phenyl group. Here, the hydrogen deletion position may be any position chemically possible, and may be on an aromatic ring or a non-aromatic ring.

  In this specification, “halogen” refers to a monovalent group of an element such as fluorine (F), chlorine (Cl), bromine (Br), iodine (I) belonging to Group 7B of the periodic table.

  In the present specification, the “hydroxyl group” refers to a group represented by —OH. In this specification, the term “hydroxyl group” may be used interchangeably with the term “hydroxy” or “hydroxyl”.

In this specification, “amide” is a group in which hydrogen of ammonia is substituted with an acid group (acyl group), and is preferably represented by —CONH ₂ .

  As used herein, “aryl” refers to a group formed by leaving one hydrogen atom bonded to an aromatic hydrocarbon ring, and is included in the present specification as a carbocyclic group.

  In the present specification, unless otherwise specified, substitution refers to replacement of one or more hydrogen atoms in an organic compound or substituent with another atom or atomic group. One hydrogen atom can be removed and substituted with a monovalent substituent, and two hydrogen atoms can be removed and substituted with a divalent substituent.

  When the hydrophobic group of the cyclic glucan derivative of the present invention is substituted with a substituent R, such substituent R is present in one or more, and each independently represents hydrogen, halogen, alkyl, cycloalkyl, alkenyl. , Cycloalkenyl, alkynyl, alkoxy and carbocyclic groups. Preferably, each R may be independently selected from the group consisting of hydrogen, halogen and alkyl. More preferably, the substituent R is fluorine.

(Description of Preferred Embodiment)
Hereinafter, preferred embodiments of the present invention will be described.

  In one aspect, the present invention provides a cyclic glucan derivative in which a hydrophobic group is bound to a high polymerization degree cyclic glucan. By introducing such a hydrophobic group, the solubility of the cyclic glucan derivative of the present invention in oil can be improved, and excellent surface active properties can be obtained. Since the cyclic glucan derivative of the present invention has a cyclic glucan moiety having a much higher degree of polymerization than cyclodextrins, the cyclic steric structure is changed in accordance with the structure of the compound to be included, and various kinds of low molecular weight to high molecular weight are obtained. It has excellent properties as a molecular recognition host molecule that enables inclusion of various compounds. Thereby, the cyclic glucan derivative of the present invention is useful for uses such as pharmaceuticals, surfactants, cosmetic compositions, food compositions, and magnetic recording medium films.

  Hereinafter, the structure, properties, and production method of the high polymerization degree cyclic glucan which is a raw material for producing the cyclic glucan derivative of the present invention will be described.

  The high-polymerization cyclic glucan used in the present invention is a glucan having one cyclic structure composed of at least 14 α-1,4-glucoside bonds in the molecule. Specifically, for example, FIG. And various glucans schematically shown in FIG. In FIG. 1, horizontal straight lines and curves indicate glucan chains connected by α-1,4-glucoside bonds, and vertical arrows indicate α-1,6-glucoside bonds (horizontal in the following schematic diagram). The same applies to straight lines and curves, and vertical arrows).

  As described above, the high-polymerization cyclic glucan used in the present invention includes a glucan having only a cyclic structure and a glucan having an acyclic structure in addition to the cyclic structure. And in this cyclic structure, the case where it is composed only of α-1,4-glucoside bond, and the case where it is composed of α-1,4-glucoside bond and at least one α-1,6-glucoside bond There is. A glucan having at least one α-1,6-glucoside bond in a cyclic structure or in a non-cyclic structure portion is generated when a glucan having a branched structure such as amylopectin is used as a substrate.

  A glucan having only a cyclic structure is treated with a glucoamylase that cleaves an α-1,4-glucoside bond and an α-1,6-glucoside bond from a non-reducing end in addition to the cyclic structure. can get. Alternatively, it can be obtained directly using a linear α-1,4-glucan or a glucan having a branched structure as a substrate.

The high polymerization degree cyclic glucan used in the present invention has the following properties.
(1) Degradation further occurs when glucoamylase (Toyobo Co., Ltd.), which is an exo-amylase that hydrolyzes α-1,4-glucoside bonds and α-1,6-glucoside bonds at the non-reducing end, is allowed to act. Ingredients (glucoamylase-resistant ingredients) remain. The component is not decomposed even when glucoamylase is allowed to act after dephosphorylating enzyme (Sigma) is allowed to act.
(2) The above-mentioned glucoamylase-resistant component is decomposed by isoamylase (Hayashibara Biochemical Laboratories Co., Ltd.) that hydrolyzes α-1,6-glucoside bonds in starch, and receives the action of glucoamylase. There is a case.
(3) The glucoamylase-resistant component is degraded by α-amylase which is an endo-amylase.

Among the highly polymerized cyclic glucans used in the present invention, a glucan having only a cyclic structure composed only of at least 14 α-1,4-glucoside bonds and having no α-1,6-glucoside bonds ( Hereinafter, the cyclic glucan having only the α-1,4-glucoside bond of the present invention) has the following properties.
(1) Neither a reducing end nor a non-reducing end can be detected.
(2) It is not degraded by β-amylase (Seikagaku Corporation) and glucoamylase (Toyobo Co., Ltd.), which are exo-type amylases that hydrolyze the α-1,4-glucoside bond at the non-reducing end.
(3) Combined use of isoamylase (Hayashibara Biochemical Laboratories Co., Ltd.) and pullulanase (Hayashibara Biochemical Laboratories Co., Ltd.) that hydrolyzes α-1,6-glucoside bonds in starch, or isoamylase, pullulanase and β -It is not decomposed even with amylase.
(4) It is completely decomposed by α-amylase (Nagase Seikagaku Corporation), which is an endo-type amylase that hydrolyzes α-1,4-glucoside bonds inside starch molecules.
(5) When hydrolyzed with bacterial saccharified α-amylase (Nagase Seikagaku Corporation) and analyzed by HPLC, only glucose, maltose, and some amount of maltotriose are obtained. That is, there is no bond other than the α-1,4-glucoside bond.

Among the highly polymerized cyclic glucans used in the present invention, a glucan having only a cyclic structure composed of at least 14 α-1,4-glucoside bonds and at least one α-1,6-glucoside bond (hereinafter referred to as “glucan”). The cyclic glucan having an α-1,6-glucoside bond of the present invention has the following properties.
(1) Neither a reducing end nor a non-reducing end can be detected.
(2) It is not degraded by β-amylase (Seikagaku Corporation) and glucoamylase (Toyobo Co., Ltd.), which are exo-type amylases that hydrolyze the α-1,4-glucoside bond at the non-reducing end.
(3) It is decomposed by isoamylase (Hayashibara Biochemical Laboratories Co., Ltd.), which hydrolyzes α-1,6-glucoside bond in starch, and receives the action of glucoamylase.
(4) It is decomposed by endo-type α-amylase (manufactured by Nagase Seikagaku Corporation) that hydrolyzes α-1,4-glucoside bonds in starch, and receives the action of glucoamylase. Further, it is known that the minimum limit dextrin when the same type α-amylase is allowed to act on a cyclic glucan having an α-1,6-glucoside bond is isomaltosyl maltose (IMM) (Yamamoto). , T. Handbook of amylase and related enzymes, Pergamon press, p40-45 (1988)). IMM is detected by treating the cyclic glucan having an α-1,6-glucoside bond of the present invention with an endo-type α-amylase.

  The number of glucose constituting the cyclic structure of the highly polymerized cyclic glucan used in the present invention is at least 14, preferably 17 or more, more preferably 22 or more, still more preferably 50 or more, The upper limit is about 5000. In the case of having an α-1,6-glucoside bond, the number thereof should be at least 1, usually 1 to 500, preferably 1 to 100.

  Quantification of the reducing end is described in Hizukuri et al. (1981) Carbohydr. Res: 94: 205-213, modified Park Johnson method, quantitation of non-reducing ends was determined by Hizukuri et al. (1978) Carbohydr. Res: 63: 261-264.

  The degradation by β-amylase and glucoamylase, which are exo-type amylases, or iso-amylase, pullulanase, or α-amylase, which is an endo-type amylase, can be achieved by, for example, converting the high-polymerization cyclic glucan used in the present invention to 0. Dissolve in distilled water to 1% (w / v), take 100 μl, add an appropriate amount of each of the above-mentioned degrading enzymes, and react at 30-45 ° C. for several hours. The reaction product can be analyzed by applying it to a sugar analysis system of DIONEX (liquid feeding system: DX300, detector: PAD-2, column: CarboPacPA100). Elution is performed, for example, under conditions of flow rate: 1 ml / min, NaOH concentration: 150 mM, sodium acetate concentration: 0 min-50 mM, 2 min-50 mM, 37 min-350 mM, 45 min-850 mM, 47 min-850 mM. The degree and the resulting sugar can be analyzed.

Detection of the glucoamylase-resistant component from the high-polymerization cyclic glucan used in the present invention can be performed as follows. For example, 100 mg of the highly polymerized cyclic glucan of the present invention is dissolved in 5 ml of distilled water, glucoamylase is added to a final concentration of 10 units / ml, and the reaction is carried out at 40 ° C. overnight. The reaction product is heated at 100 ° C. for 10 minutes, and insoluble materials are removed by centrifugation. Then, 10 times the amount of ethanol is added, and the remaining polysaccharide is recovered as a precipitate by centrifugation. The precipitate is further dissolved in 1 ml of distilled water, glucoamylase is added to a final concentration of 50 units / ml, reacted at 40 ° C. for 1 hour, heated at 100 ° C. for 10 minutes, and insolubles are removed by centrifugation. To do. A 10-fold amount of ethanol is added thereto to obtain a precipitate. In the case where the raw material of the glucan of the present invention is a raw material such as starch partially modified with a phosphate group, the obtained precipitate is mixed with 10 mM carbonate buffer (pH 9.4, 10 mM MgCl ₂ and 0.3 mM ZnCl _2). 20 units of phosphatase (derived from bovine, manufactured by Sigma) and reacted at 40 ° C. for 24 hours, and then 10 times the amount of ethanol is added to recover the precipitate. Dissolving in distilled water again, adding glucoamylase to a final concentration of 50 units / ml, reacting at 40 ° C. for 1 hour, adding 10 times the amount of ethanol, and obtaining a glucoamylase resistant component as a precipitate. it can.

  The quantification of the non-cyclic structure portion, the cyclic structure portion having an α-1,6-glucoside bond, and the cyclic structure portion having only an α-1,4-glucoside bond in the high-polymerization cyclic glucan of the present invention is as follows. Can be done as follows. After dissolving 10 mg of the sample highly polymerized cyclic glucan in 1 ml of DMSO, the sample is rapidly diluted with 8 ml of 100 mM sodium acetate buffer. Take 4 samples of this diluted solution of 900 μl, and add 100 μl of distilled water, glucoamylase solution, mixed solution of debranching enzyme and glucoamylase, and mixed solution of endo-type α-amylase and glucoamylase to each sample. In addition, the reaction is carried out at 40 ° C. for 4 hours. After completion of the reaction, the resulting glucose is measured using a commercially available glucose quantification kit, so that an acyclic structure portion in the sample glucan, a cyclic structure portion having an α-1,6-glucoside bond, and α-1, A cyclic structure portion having only a 4-glucoside bond can be determined by calculation. Details will be described in Examples.

  The degree of polymerization of the cyclic structure portion of the high polymerization degree cyclic glucan used in the present invention can be measured using chromatography. In general, cyclic polysaccharides are known to behave differently in chromatography than straight-chain polysaccharides with the same degree of polymerization, and this property is used to prove that they are cyclic and to determine the degree of polymerization of cyclic polysaccharides. Can be done. For example, the cyclic glucan having only the α-1,4-glucoside bond of the present invention obtained by reacting with the D enzyme is separated by the above-mentioned DONEEX sugar analysis system to obtain a single peak fraction. obtain. The obtained fraction is treated with, for example, 0.1N HCl at 100 ° C. for 30 minutes, and this cyclic structure portion is partially hydrolyzed, and then linear glucans having various polymerization degrees generated by the decomposition are obtained. The degree of polymerization can be determined using a DONEEX sugar analysis system. A detailed analysis method will be described in Examples.

  The high-polymerization cyclic glucan used in the present invention has a cyclic structure composed of linear α-1,4-glucan or a saccharide containing them and at least 14 α-1,4-glucoside bonds in the molecule. It can be obtained by reacting with an enzyme capable of producing a glucan having one thereof. Any enzyme can be used as long as it has such an activity. In the present invention, it is preferable to use D enzyme.

  The D enzyme was first discovered from potato but has been found to be present in various plants and microorganisms such as E. coli. This enzyme of microorganisms such as Escherichia coli is called amylomaltase or 4-α-glucanotransferase. Therefore, the enzyme D can be used regardless of its origin, even if the gene encoding these enzymes is expressed using a host such as E. coli. Here, although the purification method of D enzyme from potato and Escherichia coli is disclosed as an example, it is not limited thereto.

  A method for purifying D enzyme from potato is described in Takaha et al. Biol. Chem. vol. 268, 1391-1396 (1993). First, potato tubers are homogenized in an appropriate buffer containing 5 mM mercaptoethanol, centrifuged, passed through a 0.45 μm membrane, applied to a Q-Sepharose column, and containing, for example, 5 mM 2-mercaptoethanol. Wash with a buffer containing 150 mM NaCl in 20 mM Tris-HCl (pH 7.5) (buffer A). The D enzyme elutes in buffer A containing 450 mM NaCl. This was dialyzed against buffer A, a solution containing 500 mM ammonium sulfate was loaded onto a Phenyl Toyopearl 650M (Tosoh) column, and elution was carried out by changing the ammonium sulfate concentration in buffer A from 500 mM to 0 mM. The enzyme activity fraction is collected and dialyzed against buffer A. The dialysis internal solution was loaded on a PL-SAX column (Polymer Laboratory UK) equilibrated with buffer A, and the NaCl concentration in buffer A was changed to 150 mM-400 mM to elute, and the D enzyme activity fraction Collect. The D enzyme can be purified from potato by the above method. The measurement of enzyme activity is described in detail in the examples.

  Takaha et al., J., supra. Biol. Chem. vol. 268, 1391-1396 (1993) includes a cDNA sequence of potato D enzyme (p. 1394, FIG. 3), preparation of a recombinant plasmid of D enzyme (p. 1392), expression of the recombinant plasmid in E. coli, And the purification of D enzyme from recombinant E. coli has been disclosed, and D enzymes produced by recombinant methods can of course be used. A method for purifying D enzyme from E. coli is, for example, firstly cultivating E. coli TG-1 strain, which is a D enzyme production strain, at 37 ° C. to the logarithmic growth phase using LB liquid medium, and then maltose at a final concentration of 1% ( w / v), and further cultured at 37 ° C. for 2 hours. The bacterial cells collected by centrifugation are suspended in the buffer A and subjected to sonication and centrifugation to obtain a bacterial cell extract. Next, for example, a Q-Sepharose Fast Flow (Pharmacia) column equilibrated with buffer A is loaded, elution is carried out by changing the NaCl concentration in buffer A from 0 mM to 500 mM, and the D enzyme activity fraction is obtained. Gather. To the active fraction, ammonium sulfate was added to a final concentration of 1 M, left to stand, insoluble precipitates were removed by centrifugation, and the supernatant was equilibrated with buffer A containing 1 M ammonium sulfate Phenyl Toyopearl 650 M (Tosoh) Load into the column. Elution is performed by changing the ammonium sulfate concentration in buffer A from 1 M to 0 mM, and the D enzyme activity fraction is collected. After this fraction is dialyzed against buffer A, it is loaded onto a Resource Q column (Pharmacia) equilibrated with buffer A, and the NaCl concentration in buffer A is changed from 0 mM to 500 mM. Elution to purify the D enzyme.

  The D enzyme can be purified as described above. However, if endo-type amylases acting on α-1,4-glucoside bonds in the starch molecule are not detected, it is a crude enzyme in any of the above purification steps. However, it can be used for the synthesis of cyclic glucan having a high polymerization degree used in the present invention.

  In addition, the enzyme used in the present invention may be used for the reaction regardless of whether it is a purified enzyme or a crude enzyme, and the reaction may be batch or continuous. As the immobilization method, a carrier bonding method (for example, a covalent bonding method, an ionic bonding method, or a physical adsorption method), a crosslinking method, or a comprehensive method (lattice type or microcapsule type) can be used.

  When only the cyclic glucan having only α-1,4-glucoside bond of the present invention is to be obtained, only α-1,4-glucoside bond such as amylose, starch debris, phosphorylase-synthesized amylose, maltooligosaccharide, etc. The above-mentioned enzyme that can be used for the high-polymerization cyclic glucan of the present invention, for example, the D enzyme, is allowed to act on a linear α-1,4-glucan comprising

  In the case of using a sugar having an α-1,6-branched structure such as amylopectin, such as amylopectin, glycogen, starch, waxy starch, high amylose starch, soluble starch, dextrin, starch hydrolysate, and phosphorylase, When only the cyclic glucan having only the α-1,4-glucoside bond of the present invention is to be obtained, the enzyme that can be used for the high polymerization degree cyclic glucan of the present invention, for example, the D enzyme is reacted directly with the raw material. Can be manufactured. Alternatively, the saccharide is highly polymerized according to the present invention in the presence or absence of an enzyme that cleaves an α-1,6-glucoside bond but does not cleave an α-1,4-glucoside bond, such as isoamylase or pullulanase. It can be produced by reacting with the above-mentioned enzymes that can be used for cyclic glucan, such as the D enzyme.

  For example, as shown in FIG. 2, after the amylose (12) having the reducing end (11) is reacted with the D enzyme, the cyclic glucan (13) having only the α-1,4-glucoside bond is prepared. Then, glucoamylase is added to hydrolyze the non-cyclic glucan sequentially from the non-reducing end. Next, ethanol is added to recover the cyclic glucan as a precipitate, followed by lyophilization to obtain a cyclic glucan (13) having only an α-1,4-glucoside bond.

  Alternatively, an amylopectin (14) having a reducing end (11), an isoamylase that cleaves an α-1,6-glucoside bond, and a D enzyme are reacted at the same time to form a cyclic structure having only the α-1,4-glucoside bond. After preparing glucan (13), glucoamylase is added and hydrolysis is performed sequentially from the non-reducing end. Next, ethanol is added to recover the cyclic glucan as a precipitate, followed by lyophilization to obtain a cyclic glucan (13) having only an α-1,4-glucoside bond.

  Alternatively, by reacting the D enzyme with the amylopectin (14) having the reducing end (11), the cyclic glucan (13) having only the α-1,4-glucoside bond and the α-1,4-glucoside bond only. A cyclic structure composed of a glucan (18) having a cyclic structure and an acyclic structure portion, at least 14 α-1,4-glucoside bonds and at least one α-1,6-glucoside bond Is prepared and then reacted with glucoamylase and isoamylase. Furthermore, pullulanase can be reacted. Subsequently, ethanol is added to precipitate cyclic glucan, and this precipitate is recovered and then freeze-dried to obtain cyclic glucan (13) having only α-1,4-glucoside bonds.

  Further, the glucan having one cyclic structure in the molecule composed of at least 14 α-1,4-glucoside bonds and at least one α-1,6-glucoside bond of the present invention (hereinafter referred to as the present invention). α-1,6-glucoside-linked glucan) is α-1,6 such as amylopectin, glycogen, starch, waxy starch, high amylose starch, soluble starch, dextrin, starch hydrolyzate, phosphorylase, and enzymatically synthesized amylopectin. -The above-mentioned enzyme that can be used in the glucan of the present invention, for example, D enzyme, can be allowed to act on a raw material having a branched structure.

  For example, as shown in FIG. 3, D enzyme is reacted with amylopectin (14) having a reducing end (11), and the above-mentioned at least 14 α-1,4-glucoside bonds and at least one α-1 are reacted. , 6-glucoside bond, glucan (15) having one cyclic structure in the molecule, cyclic glucan (13) having only α-1,4-glucoside bond, and α-1,4-glucoside bond After preparing a glucan (18) having a cyclic structure composed solely of a cyclic structure and an acyclic structure portion, the above-described at least 14 α-1,4-glucoside bonds and at least one of the above-mentioned are obtained by gel filtration chromatography. A glucan (15) having one cyclic structure composed of an α-1,6-glucoside bond in the molecule can be separated. To this glucan (15), glucoamylase is added to sequentially hydrolyze the acyclic structure from the non-reducing end. After hydrolysis, ethanol is added to recover the cyclic glucan as a precipitate, followed by lyophilization to obtain glucan (16) formed only by a cyclic structure having at least one α-1,6-glucoside bond.

  Examples of the linear α-1,4-glucan that can be used in the present invention or a saccharide having the same include malto-oligosaccharides such as maltotriose, maltotetraose, maltopentaose, amylose, amylopectin, glycogen, starch, waxy starch , High amylose starch, soluble starch, dextrin, starch residue, partially hydrolyzed starch, enzymatically synthesized starch using phosphorylase, and derivatives thereof. These may be used alone or in combination. Here, the starch branch material refers to a product obtained by enzymatically cleaving all or part of the bonds other than α-1,4-glucoside bonds in starch. Further, the partial starch hydrolyzate refers to a product obtained by enzymatically or chemically cleaving a part of α-1,4-glucoside bond in starch. For example, the degree of polymerization is about 100 or more. Amylopectin, amylose having a degree of polymerization of about 22 or more can be used as a raw material.

  Moreover, it has one linear structure in a molecule | numerator comprised by the linear (alpha) -1, 4- glucan which can be used for this invention, or a saccharide | sugar which has this, and at least 14 (alpha) -1, 4- glucoside bond. The reaction with an enzyme capable of producing glucan can be carried out in the presence of phosphorylase and glucose-1-phosphate. Phosphorylase catalyzes the elongation reaction of α-1,4-glucan chains when glucose-1-phosphate is present in excess. For this reason, phosphorylase and glucose-1-phosphate coexist when the α-1,4-glucan chain of the raw material does not have a sufficient length to undergo the cyclization reaction of the enzyme. Thus, the yield of the glucan of the present invention can be increased.

  Furthermore, a derivative of the above starch or a partially decomposed product of starch can also be used as a raw material. For example, at least one of the alcoholic hydroxyl groups of the starch is ionic bond, etherified (carboxymethylated, hydroxyalkylated, etc.), esterified (phosphorylated, acetylated, sulfated, etc.), crosslinked or grafted. Derivatives etc. can also be used. Furthermore, a mixture of two or more of these may be used as a raw material.

  For the reaction between the raw material and the enzyme that can be used in the present invention, any conditions can be used as long as the conditions such as pH and temperature at which the highly polymerized cyclic glucan of the present invention is generated.

  Taking D enzyme as an example, the pH of the reaction is usually from 3 to 10, preferably from 4 to 9, more preferably from 6 to 8, from the viewpoints of reaction rate, efficiency, enzyme stability, and the like. The temperature is preferably about 10 ° C. to 90 ° C., preferably about 20 ° C. to 60 ° C., more preferably 30 ° C. to 40 ° C. in terms of reaction rate, efficiency, enzyme stability, and the like. When an enzyme obtained from a thermostable microorganism is used, it can be used at a high temperature of 50 ° C to 90 ° C. The concentration of the raw material (substrate concentration) can also be determined in consideration of the degree of polymerization of the substrate used and the reaction conditions. In general, from the viewpoint of 0.1% to 30%, reaction rate, efficiency, ease of handling of the substrate solution, etc., preferably 0.1% to 10%, more preferably 0.5% from the viewpoint of solubility. 5%. The amount of the enzyme to be used is determined in relation to the reaction time and the concentration of the substrate, and it is usually preferable to select the amount of the enzyme so that the reaction is completed in about 1 to 48 hours. 50 to 10,000 units, preferably 70 to 2,500 units, more preferably 400 to 2,000 units.

  The high-polymerization cyclic glucan of the present invention can be separated and purified by applying a method known per se. For example, the highly polymerized cyclic glucan of the present invention can be separated and purified by precipitation with a solvent, separation with a membrane, separation by chromatography, etc. after the above reaction is completed. Preferably, the reaction solution is heated or purified as it is. The cyclic glucan having only the α-1,4-glucoside bond of the present invention is obtained by adding exo-type β-amylase or glucoamylase and decomposing the remaining linear α-1,4-glucan. . When a branched polysaccharide such as starch is used as a raw material, an exo-type β-amylase or glucoamylase and an enzyme that cleaves an α-1,6-glucoside bond are used in combination to form a cyclic α-1,4. -React so that only glucan remains. Thereafter, separation and purification means such as precipitation using a solvent, membrane separation, and chromatographic separation can be applied.

  The high-polymerization cyclic glucan thus obtained may be used as it is as a hydrophilic site, or may be used as a hydrophilic site by further introducing a hydrophilic substituent, if necessary. When a hydrophilic substituent is introduced, the position at which the substituent is introduced can be any position of the high polymerization degree cyclic glucan. For example, it is possible to introduce a hydrophilic substituent to one of the hydroxyl groups at the 2-position, 3-position or 6-position of any of the glucose monomer units constituting the cyclic glucan. Hydrophilic substituents may be introduced into two of the hydroxyl groups at the 2-position, 3-position or 6-position, and hydrophilic substituents may be introduced to all hydroxyl groups at the 2-position, 3-position and 6-position. May be.

  However, when a hydrophilic substituent is introduced into all hydroxyl groups at the 2nd, 3rd and 6th positions of all glucose units constituting the cyclic glucan, the hydrophobic group is not introduced. Accordingly, the hydrophilic substituent is introduced at a position other than the position where the introduction of the hydrophobic group is planned.

  The cyclic glucan derivative of the present invention can be obtained by chemically bonding a hydrophobic group to the hydrophilic portion containing the high polymerization degree cyclic glucan thus obtained. The cyclic glucan derivative of the present invention is preferably, for example, specifically represented by the following structural formula:

It can be expressed as
Here, n monomers in the above formula are selected independently of each other. That is, it is not required that n single glucose derivative monomers are polymerized. A homopolymer structure in which n kinds of monomers are polymerized may be used, but a structure in which two kinds of monomers are copolymerized may be used. Alternatively, a structure in which three or more types of monomers are copolymerized may be used.

n pieces of ^{R 1} each independently represents an ^{-L-R 4} or ^{R 5,} n pieces of ^{R 2} each independently represent a ^{-L-R 4} or ^{R 5,} n pieces of ^{R 3} is Each independently represents -LR ⁴ or R ⁵ . However, at least one -LR ⁴ is present in one molecule. In one embodiment, the number of groups -LR ⁴ present is 2 or more, preferably 5 or more, more preferably 10 or more, in one molecule. The number of groups -LR ⁴ is preferably 1% or more of the total hydroxyl groups, more preferably 2% or more of the total hydroxyl groups, as a ratio of the high polymerization degree cyclic glucan to the total hydroxyl groups. More preferably, it is 3% or more. Further, it is preferably 50% or less of the total hydroxyl groups, more preferably 20% or less of the total hydroxyl groups, and further preferably 10% or less of the total hydroxyl groups.

R ⁴ is each independently a C _{3 to} C ₂₀ linear or branched alkyl group, or a C _{3 to} C ₂₀ linear or branched fluorinated alkyl group.

L is independently —NHCO—, —O—, —S—, —OSO ₂ —, —SCO— or —OCO—.

R ⁵ is each independently a hydroxyl group or a tosyl group.

  n is 14 to 5000.

  In one embodiment, the compound can be a compound of the following formula:

Here, L represents a linking group, and specific examples include, for example, L is —NHCO—, —O—, —S—, —OSO ₂ —, —SCO— or —OCO—, and R ⁴ is hydrophobic. Specific examples include, for example, R ⁴ is a C _{3 to} C ₂₀ linear or branched alkyl group, or a C _{3 to} C ₂₀ linear or branched fluorinated alkyl group. , R ⁵ is a hydroxyl group or a tosyl group, and n is 14 to 5000. The ratio of m to n is preferably 1 to 50%, more preferably 2 to 20%, and still more preferably 3 to 10%.

In a preferred embodiment, C _{3 to} C ₂₀ linear or branched alkyl group, C _{3 to} C ₂₀ linear or branched fluorinated group as the hydrophobic group bonded to the high-polymerization cyclic glucan. Alkyl groups or mixtures thereof are used. Thereby, good surface activity can be obtained reliably. Furthermore, when an alkyl group or a fluorinated alkyl group is bonded to a cyclic glucan having a high degree of polymerization, in consideration of steric hindrance during the bonding, these alkyl groups or fluorinated alkyl groups are linear regardless of the chain length. It is preferable that

In a more preferred embodiment, the hydrophobic group attached to the high degree of polymerization glucan is a C _{3 to} C ₂₀ linear fluorohydroalkyl group having a fluorine substitution rate of at least 50% or a C _{3 to} C ₂₀ straight chain. A chain perfluoroalkyl group is used. Thereby, particularly excellent surface activity can be obtained.

  In a preferred embodiment, the hydrophobic group bond to the high-polymerization cyclic glucan is selected from the group consisting of an amide bond, an ether bond and an ester bond. Thus, by adopting a relatively stable chemical bond, it is bonded during use in applications such as pharmaceuticals, reagents, surfactants, cosmetic compositions, food compositions and magnetic recording media films. The function and activity of the cyclic glucan derivative of the present invention can be maintained in a stable state without being decomposed. Among the above bonds, it is preferable that at least one of the alcoholic hydroxyl groups possessed by the high-polymerization cyclic glucan is derivatized by an ester bond with a fluorine-substituted or unsubstituted fatty acid.

(Side chain introduction method)
The highly polymerized cyclic glucan derivative of the present invention introduces a tosyl group into a hydroxyl group of glucose, for example, a highly polymerized cyclic glucan obtained by the above-described method, and the introduced tosyl group is substituted with a (substituted or unsubstituted) fatty acid ester. Can be obtained by transesterification.

  In one aspect of the present invention, a cyclic glucan derivative in which a hydrophobic group of the present invention is bonded to a high-polymerization degree cyclic glucan is obtained by combining a high-polymerization degree cyclic glucan synthesized by the above-described method in an alkaline aqueous solution with p-toluene chloride. A tosyl group in which a tosyl group is bonded to a high polymerization degree cyclic glucan by contacting sulfonyl with at least one alcoholic hydroxyl group possessed by the high polymerization degree cyclic glucan and p-toluenesulfonyl chloride. Containing cyclic glucan derivative (hereinafter, this step is referred to as tosylation of high-polymerization cyclic glucan; see schemes 1 and 2 in FIG. 11), this tosyl group-containing cyclic glucan derivative, fluorine-substituted or unsubstituted fatty acid sodium, Is contacted under conditions where the tosyl group and the fatty acid sodium can undergo an exchange reaction (hereinafter, this step is referred to as “tosyl group”). That transesterification of fatty acid sodium; see Scheme 3 and 4 in FIG. 14) can be obtained by.

  More specifically, the tosylation of the high polymerization degree cyclic glucan is performed as follows. First, the high polymerization degree cyclic glucan obtained by the above-described method is dissolved in an alkaline aqueous solution such as an aqueous NaOH solution, and p-toluenesulfonyl chloride (abbreviation: p-TsCl) is added to the aqueous solution. At this time, since p-TsCl is not dissolved in the alkaline aqueous solution, it is considered that only p-TsCl included in the hydrophobic environment in the ring of the high polymerization degree cyclic glucan is involved in the reaction. Therefore, since the stirring state affects the formation of the inclusion complex, it is considered that the progress of the reaction is greatly affected. Other factors that influence the progress of the reaction include the stirring temperature, the stirring time, and the concentration of the reaction solution. The reason why the concentration of the reaction solution affects the progress of the reaction is that the aqueous alkali solution traps HCl generated during the tosylation reaction to promote the reaction. A person skilled in the art can obtain a desired degree of tosylation according to the application by freely changing the stirring state, the stirring temperature, the stirring time, and the concentration of the reaction solution. To purify the target tosylated highly polymerized cyclic glucan, the aqueous alkali solution containing the target product is neutralized with dilute hydrochloric acid, and the NaCl salt, which is a by-product of the tosylation reaction, is removed by gel filtration chromatography or dialysis. It is carried out by removing under reduced pressure by evaporation and vacuum drying.

Confirmation of tosylation can be carried out by the UV spectrum method, focusing on the absorption based on the S═O group peculiar to the tosyl group (FIG. 12). The degree of tosylation, that is, the ratio of all hydroxyl groups of the highly polymerized cyclic glucan substituted with tosyl groups can be easily calculated by ¹ H-NMR spectroscopy. It can be calculated by the ratio of the proton integral calculation value of (a + b) and the proton integral calculation value of d in the ¹ H-NMR spectrum of the tosylated high-polymerization cyclic glucan in FIG.

  The transesterification reaction between the tosyl group of the tosylated highly polymerized cyclic glucan obtained above and sodium fatty acid is more specifically performed as follows. Tosylated highly polymerized cyclic glucan is dissolved in a suitable solvent (for example, dimethyl sulfoxide (abbreviation: DMSO)), fatty acid sodium or sodium fluorinated fatty acid is added to the solution, and nitrogen is added at a temperature of about 70 to 90 ° C. It is carried out by stirring for several hours under an air stream. Purification of the final product after transesterification is performed by first adding a solvent such as acetone to the reaction solution, precipitating the final product, dissolving the precipitate in water, and dialyzing it for an appropriate time. The reaction is carried out by removing the fatty acid sodium salt or fluorinated fatty acid sodium salt and the by-product sodium p-toluenesulfonate (abbreviation: TsNa) and lyophilizing.

The transesterification can be confirmed by the IR spectrum method by paying attention to the CH stretching vibration peculiar to the alkyl group of the fatty acid and the C═O stretching vibration peculiar to the ester group (FIG. 15). In addition, the degree of transesterification, that is, the proportion of all hydroxyl groups of the highly polymerized cyclic glucan substituted with fatty acid or fluorinated fatty acid can be easily calculated by ¹ H-NMR spectroscopy. It can be calculated by the ratio between the proton integral calculated value of (d + e) and the proton integral calculated value of c in the ¹ H-NMR spectrum diagram of the highly polymerized cyclic glucan after transesterification in FIG.

  The surface activity of the highly polymerized cyclic glucan after tosylation and the highly polymerized cyclic glucan after transesterification is evaluated by surface tension measurement using the Wilhelmy plate method by dispersing or dissolving them in ultrapure water. (FIG. 17).

  The physical properties of the dispersion in water of the highly polymerized cyclic glucan after transesterification can be examined by dynamic light scattering (FIG. 18).

  Moreover, the visual observation of the dispersion in water of the high polymerization degree glucan after transesterification can be performed using a transmission electron microscope (FIG. 19).

(Surfactant)
Since the cyclic glucan derivative of the present invention has a hydrophilic cyclic glucan moiety and a hydrophobic side chain, it can further exhibit the performance as a surfactant while maintaining the performance as the cyclic glucan. From the viewpoint of performance as a surfactant, the polymerization degree of the glucan of the cyclic glucan derivative is preferably 14 to 5000, and more preferably 17 to 100. More preferably, it is 22-50. Further, the number of hydrophobic groups is preferably such that 1 to 50% of the glucose residues present in the cyclic glucan derivative have a hydrophobic group introduced therein, more preferably 2 to 20%, and more preferably 3 to 10%. % Is more preferable. As a kind of hydrophobic group, an alkyl group or a substituted alkyl group is preferable, and alkyl or perfluoroalkyl is more preferable. A linear alkyl group or a linear perfluoroalkyl group is more preferable. The chain length of the alkyl group or the substituted alkyl group is preferably C _{3 to} C ₂₀ , more preferably C _{3 to} C ₁₂ , and still more preferably C _{3 to} C ₈ .

  The cyclic glucan derivative of the present invention may be used alone as a surfactant or may be combined with other surfactants.

  For example, the surfactant comprising the cyclic glucan derivative of the present invention has the following characteristic advantages.

  1) Since the hydrophilic part has a large molecular weight, micelles can be formed with several molecules. On the other hand, a huge micelle having a diameter of several hundreds of nanometers can be formed. This can be used as nanoparticles or as an emulsifier.

  2) Since the cyclic glucan of the present invention has many hydroxyl groups into which substituents can be introduced, the number of hydrophobic groups that can be introduced is large. For this reason, a series of surfactants with a wide balance between the hydrophilic group and the hydrophobic group (HLB value) can be obtained simply by changing the substitution degree of the hydrophobic group.

  3) A surfactant having a large molecular weight in the hydrophilic portion, and even if a hydrophilic group is further introduced into the hydrophilic portion, the properties as a surfactant can be maintained. Therefore, a surfactant having molecular recognition ability can be obtained by introducing a receptor, a ligand or the like as a hydrophilic group. By using this surfactant dispersed in a solvent as micelles or bubbles, it can be accumulated at a position where a substance binding to a receptor or a ligand exists. This property can be applied, for example, as an inspection technique or a diagnostic technique.

  4) The cyclic glucan derivative of the present invention has a molecular recognition ability of changing the cyclic steric structure according to the structure of the compound to be included, and enabling inclusion of a wide variety of compounds having a low molecular weight to a high molecular weight. . For this reason, a surfactant having an inclusion ability is obtained. Furthermore, an element that recognizes a specific substance can be manufactured by using the cyclic glucan derivative of the present invention as an LB film.

  5) Degradation of the cyclic glucan derivative of the present invention with an enzyme such as α-amylase and destruction of the hydrophilic part makes it possible to control the function of the surfactant (emulsification / control of the micelle state).

  6) The above also means that biodegradable surfactants can be synthesized. This is an environmentally favorable property. This is because many surfactants are difficult to decompose and often cause environmental problems. On the other hand, this property is also a preferable property for use as a pharmaceutical / cosmetic product.

(Medicine, cosmetics, etc., and treatment and prevention using the same)
In another aspect, the present invention relates to pharmaceuticals (eg, pharmaceuticals such as vaccines, health foods, residual proteins or lipids having reduced antigenicity) and cosmetics. The medicament and cosmetic may further comprise a pharmaceutically acceptable carrier and the like. Examples of the pharmaceutically acceptable carrier contained in the medicament of the present invention include any substance known in the art.

  Such suitable formulation materials or pharmaceutically acceptable carriers include antioxidants, preservatives, colorants, flavors, and diluents, emulsifiers, suspending agents, solvents, fillers, bulking agents, buffers. Examples include, but are not limited to, agents, delivery vehicles, diluents, excipients and / or pharmaceutical adjuvants. Typically, the medicament of the present invention is a composition comprising isolated pluripotent stem cells, or a variant or derivative thereof, together with one or more physiologically acceptable carriers, excipients or diluents. It is administered in the form of For example, a suitable vehicle can be water for injection, physiological solution, or artificial cerebrospinal fluid, which can be supplemented with other materials common to compositions for parenteral delivery. .

  Acceptable carriers, excipients or stabilizers used herein are non-toxic to the recipient and are preferably inert at the dosages and concentrations used and Phosphate, citrate, or other organic acids; ascorbic acid, α-tocopherol; low molecular weight polypeptides; proteins (eg, serum albumin, gelatin or immunoglobulin); hydrophilic polymers (eg, polyvinyl Pyrrolidone); amino acids (eg, glycine, glutamine, asparagine, arginine or lysine); monosaccharides, disaccharides and other carbohydrates (including glucose, mannose, or dextrin); chelating agents (eg, EDTA); sugar alcohols (eg, EDTA) Mannitol or sorbitol ; Salt-forming counterions (such as sodium); and / or non-ionic surface-active agents (e.g., Tween, Pluronic (pluronic) or polyethylene glycol (PEG)).

  Exemplary suitable carriers include neutral buffered saline or saline mixed with serum albumin. Preferably, the product is formulated as a lyophilizer using a suitable excipient (eg, sucrose). Other standard carriers, diluents and excipients may be included as desired. Other exemplary compositions include pH 7.0-8.5 Tris buffer or pH 4.0-5.5 acetate buffer, which may further include sorbitol or a suitable substitute thereof. .

  The medicament of the present invention can be administered orally or parenterally. Alternatively, the medicament of the present invention can be administered intravenously or subcutaneously. When administered systemically, the medicament used in the present invention may be in the form of a pharmaceutically acceptable aqueous solution free of pyrogens. Such a pharmaceutically acceptable composition can be easily prepared by those skilled in the art by considering pH, isotonicity, stability and the like. In this specification, the administration method includes oral administration, parenteral administration (for example, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, mucosal administration, intrarectal administration, intravaginal administration, local administration to the affected area, Skin administration, etc.). Formulations for such administration can be provided in any dosage form. Examples of such a pharmaceutical form include a liquid, an injection, and a sustained release agent.

  The medicament of the present invention may be a physiologically acceptable carrier, excipient or stabilizer (Japanese Pharmacopoeia 14th edition or its latest edition, Remington's Pharmaceutical Sciences, 18th Edition, AR, as required). Gennaro, ed., Mack Publishing Company, 1990, etc.) and a composition comprising a cyclic glucan derivative of the present invention having the desired degree of purity, in the form of a lyophilized cake or aqueous solution. Can be prepared and stored.

  The amount of the composition containing the cyclic glucan derivative used in the treatment method of the present invention is the purpose of use, the target disease (type, severity, etc.), the patient's age, weight, sex, medical history, cell morphology or type. It can be easily determined by those skilled in the art in consideration of the above. The frequency with which the treatment method of the present invention is applied to a subject (or patient) also depends on the purpose of use, target disease (type, severity, etc.), patient age, weight, sex, medical history, treatment course, etc. In view of this, it can be easily determined by those skilled in the art. Examples of the frequency include administration every day to once every several months (for example, once a week to once a month). It is preferable to administer once a week to once a month while observing the course.

  When the present invention is used as a cosmetic product, the cosmetic product can also be prepared while complying with the regulations stipulated by the authorities.

(Health and food)
The present invention can also be used in the health / food field. In such a case, the points to be noted when used as an oral medicine as described above should be considered as necessary. In particular, when used as a functional food / health food such as a specific health food, it is preferable to treat it according to a medicine.

(Magnetic recording medium)
The present invention can also be used as a lubricant applied to the outermost layer of a magnetic disk in response to the recent increase in recording density of magnetic recording media. The magnetic recording medium is used as an external memory such as an electronic computer or a word processor, and examples thereof include a hard disk device and a floppy (registered trademark) disk. The size of the disc is 1 to 14 inches depending on the application. In the case of a hard disk device, a large recording capacity is secured by stacking one or several disks. Such a disk is mainly composed of a substrate (metal alloy such as Al and Cu, ceramics such as glass, or organic polymer materials such as polycarbonate and polystyrene), magnetic layers (Fe, Co, Ni, etc.). Alloy or oxide thereof), a protective layer (carbon, SiC, SiO2, etc.), and a film containing the fluorine compound of the present invention. If necessary, a new layer (for example, Ni, Cr, or Zn-based complex) may be provided between the substrate and the magnetic layer, or between the magnetic layer and the protective layer, for the purpose of improving the adhesion. Oxide, phosphorus metal complex, etc.) may be provided.

  The film of the present invention formed on the protective layer of the magnetic recording medium preferably has a thickness of 30 nm or less. This is because if it is too thick, the coefficient of static friction acting between the head and the disk may increase. If the thickness is less than 1 nm, a sufficient effect cannot be obtained.

  In order to form such a thin film, a method in which the film forming material is not directly applied but dissolved in an appropriate solvent and formed by dip coating is preferable because the thickness of the film can be easily controlled. Further, although the post-treatment method differs depending on the boiling point and heat of vaporization of the solvent used, it is necessary to remove the solvent by drying at room temperature, slight heating, normal pressure, or reduced pressure. At this time, if a solvent having a large heat of vaporization is used, moisture in the air may be taken into the film. Therefore, a solvent having a small heat of vaporization is desirable.

  Hereinafter, the cyclic glucan derivative in which the hydrophobic group of the present invention is bonded to a cyclic glucan having a high degree of polymerization will be specifically described with reference to examples. However, the scope of the present invention is not limited to the following examples. Absent.

(Example 1: Preparation of D enzyme)
Takaha et al. Biol. Chem. vol. 268, 1391-1396 (1993), and the D enzyme was purified. First, potato tubers were homogenized in 20 mM Tris-HCl (pH 7.5) buffer (buffer A) containing 5 mM 2-mercaptoethanol, centrifuged, passed through a 0.45 μm membrane, and Q- The column was applied to a Sepharose column (16 × 100 mm Pharmacia) and washed with buffer A containing 150 mM NaCl. D enzyme was eluted in buffer A containing 450 mM NaCl. After elution, it was dialyzed against buffer A, and ammonium sulfate was added so that the final concentration was 500 mM. This solution was loaded onto a Phenyl Toyopearl 650M (Tosoh) column (10 × 100 mm), and elution was carried out by changing the ammonium sulfate concentration in Buffer A from 500 mM to 0 mM. D Enzyme activity fractions are collected, dialyzed against buffer A, dialysate concentrated using Amicon Centricon 30 microconcentrator, applied to PL-SAX HPLC column (Polymer Laboratory UK), buffer Elution was performed with a 150-400 mM NaCl linear gradient in A, and the active fractions were collected and concentrated with the Amicon Centricon 30 microconcentrator.

  The enzyme activity was measured by reacting 100 μl of a reaction mixture containing 100 mM Tris-HCl (pH 7.0), 5 mM 2-mercaptoethanol, 1% (w / v) maltotriose, and enzyme at 37 ° C. for 10 minutes. The reaction solution was heated in boiling water for 3 minutes to stop the reaction, and glucose released by the reaction was quantified by a method using glucose oxidase (Barham et al. (1972) Analyst 97: 142). The amount of enzyme that produces 1 μmol of glucose per minute was defined as 1 unit.

(Example 2: Preparation of high polymerization degree cyclic glucan having only α-1,4-glucoside bond of the present invention)
After completely dissolving 500 mg of commercially available amylose (average molecular weight 30,000) in 10 ml of 1N-NaOH, 10 ml of 1M Tris-HCl buffer (pH 7.5), 20 ml of distilled water and 10 ml of 1N-hydrochloric acid were added in order, and the amylose solution was added. I made it. To this amylose solution, 200 units of potato-derived D enzyme was added and reacted at 30 ° C. for 24 hours.

  After centrifuging the reaction solution, the supernatant was treated at 100 ° C. for 5 minutes, and centrifuged again to remove the denatured enzyme protein. About 100 units of β-amylase and 100 units of glucoamylase were added to the supernatant and reacted at 50 ° C. for 3 hours, and then 10 times the amount of ethanol was added to cause precipitation. This precipitate was freeze-dried to obtain about 400 mg of powder.

(Example 3: Preparation of cyclic glucan having a high degree of polymerization having only α-1,4-glucoside bonds of the present invention)
After completely dissolving 500 mg of commercially available soluble starch in 10 ml of 1N-NaOH, 10 ml of 1M Tris-HCl buffer (pH 7.5), 20 ml of distilled water, and 10 ml of 1N-hydrochloric acid were sequentially added to form a starch solution. To this starch solution, 10 units of commercially available isoamylase and 200 units of potato-derived D enzyme were added and reacted at 30 ° C. for 24 hours.

  After centrifuging the reaction solution, the supernatant was treated at 100 ° C. for 5 minutes, and centrifuged again to remove the denatured enzyme protein. About 100 units of β-amylase and 100 units of glucoamylase were added to the supernatant and reacted at 50 ° C. for 3 hours, and then 10 times the amount of ethanol was added to precipitate cyclic amylose. This precipitate was freeze-dried to obtain about 150 mg of a cyclic glucan powder having only α-1,4-glucoside bonds.

(Example 4: Preparation of a highly-polymerized cyclic glucan having only α-1,4-glucoside bonds of the present invention)
After completely dissolving 20 mg of commercially available amylose (average molecular weight 320,000) in 1 ml of DMSO, 9 ml of 20 mM citrate buffer (pH 7.0) containing 68 units of D enzyme purified in Example 1 was added, The reaction was carried out at 30 ° C. for 10 minutes, 20 minutes, 30 minutes, 2 hours, 6 hours, and 18 hours. The reaction solution was heated at 100 ° C. for 10 minutes, and then the enzyme protein denatured by centrifugation was removed. 5 units of glucoamylase was added to 1 ml of the supernatant and reacted at 40 ° C. for 4 hours to remove non-cyclic amylose. The reaction solution was heated again at 100 ° C. for 10 minutes, the enzyme protein denatured by centrifugation was removed, and 10 times the amount of ethanol was added to 250 μl of the supernatant to precipitate cyclic glucan.

(Example 5: Confirmation that it is a high polymerization degree cyclic glucan having only α-1,4-glucoside bond)
(1) Quantification of reducing end and non-reducing end The quantification of the reducing end of the powder obtained in Example 2 was determined by Hizukuri et al. (1981) Carbohydr. Res: 94: 205-213. Quantification of non-reducing ends is described by Hizukuri et al. (1978) Carbohydr. Res: 63: 261-264. As a result, neither reducing end nor non-reducing end could be detected.

(2) Digestion with exo-type enzyme and α-1,6-glucoside bond-degrading enzyme After dissolving the powder obtained in Example 2 in distilled water to 0.1% (w / v), this substance One unit of each of the following amylolytic enzymes shown below was added to 100 μl of an aqueous solution and reacted at 40 ° C. for 2 hours. This reaction product was analyzed by a sugar analysis system (liquid feeding system: DX300, detector: PAD-2, column: CarboPacPA100) manufactured by DIONEX. Elution: flow rate: 1 ml / min, NaOH concentration: 150 mM, sodium acetate concentration: 0 min-50 mM, 2 min-50 mM, 37 min-350 mM (Gradient curve No. 3), 45 min-850 mM (Gradient curve No. 7) , 47 min-850 mM. The result is shown in FIG.

  As shown in FIG. 4, the powder obtained in Example 2 is β-amylase (Seikagaku Corporation), which is an exo-amylase that hydrolyzes α-1,4-glucoside bonds at the non-reducing end of starch molecules. ) And glucoamylase (Toyobo Co., Ltd.). Also, a combination of isoamylase (Hayashibara Biochemical Laboratories Co., Ltd.) and pullulanase (Hayashibara Biochemical Laboratories Co., Ltd.), which hydrolyzes α-1,6-glucoside bonds in starch, or isoamylase, pullulanase and β- It was not decomposed even in combination with amylase. However, this powder was completely decomposed by α-amylase (Nagase Seikagaku Corporation), which is an endo-type amylase that hydrolyzes α-1,4-glucoside bonds inside starch molecules.

(3) Digestion with endo-type enzyme The powder obtained in Example 2 was dissolved in distilled water so as to be 0.1% (w / v), and then the bacterial saccharified α-amylase (Nagase Bioscience) was added to 100 μl of this aqueous solution. Chemical Industry Co., Ltd.) 1 unit was added and reacted at 40 ° C. for 2 hours. The reaction was analyzed by HPLC and only glucose, maltose, and some amount of maltotriose were obtained. From this, it was proved that the powder obtained in Example 2 had no bonds other than α-1,4-glucoside bond, and the obtained powder was only α-1,4-glucoside bond. It was found to be a cyclic glucan having a high polymerization degree.

  As a result of the same confirmation, it was found that the powders obtained in Examples 3 and 4 were also highly-polymerized cyclic glucans having only α-1,4-glucoside bonds.

(Example 6: Measurement of degree of polymerization of cyclic glucan having a high degree of polymerization having only α-1,4-glucoside bonds)
In general, it is known that cyclic polysaccharides behave differently from linear polysaccharides having the same degree of polymerization in various chromatography. This property was used to prove that it was cyclic and to determine the degree of polymerization of the cyclic polysaccharide.

  (1) Purification of cyclic glucan having only α-1,4-glucoside bond having a specific degree of polymerization The substance obtained in Example 2 is cyclic having only α-1,4-glucoside bond having various degrees of polymerization. Since it is considered to be a mixture of glucans, the cyclic glucan having the above specific degree of polymerization was purified.

  FIG. 5-1 shows an elution pattern when 100 μg of the substance obtained in Example 2 is analyzed by a sugar analysis system (equipment and elution conditions are the same as those described above) manufactured by DIONEX. FIG. 5-2 shows an elution pattern of linear α-1,4-glucan under the same elution conditions.

  The earliest detectable peak shown in FIG. 5A is A, and the subsequent peaks are B, C, D... L, respectively, and the G to L peaks are fractionated and purified. .

(2) Analysis of acid hydrolyzate The separated G to L peaks were each hydrolyzed with 0.1 N HCl at 100 ° C. for 30 minutes. This condition is a condition for partial hydrolysis. The degradation product was analyzed with a sugar analysis system (equipment and elution conditions are the same as in Example 5) of DIONEX. FIG. 6 shows these results.

  The fraction of peak G was subjected to partial hydrolysis with acid, and was decomposed into glucose and linear α-1,4-glucan having a polymerization degree of 2 to 23.

  The peak G before acid decomposition is eluted around the polymerization degree 20 of the linear α-1,4-glucan, and the decomposition product is eluted at a higher polymerization degree position than that before decomposition. A phenomenon was found. This phenomenon is also found in other H to L peaks, which is considered to suggest that the G to L peak is a cyclic glucan having only α-1,4-glucoside bonds.

  The linear α-1,4-glucan having the highest degree of polymerization detected in the aforementioned partial hydrolysis experiment represents the degree of polymerization of a cyclic glucan having only α-1,4-glucoside bonds at the respective peaks. it is conceivable that. Therefore, it was found that the polymerization degrees of the peaks G, H, I, J, K, and L were 23, 24, 25, 26, 27, and 28, respectively. Further, if estimated from this result, the polymerization degree of peak A, which is a cyclic glucan having only α-1,4-glucoside bonds having the minimum polymerization degree, is considered to be 17.

  On the other hand, the degree of polymerization of a cyclic glucan having only α-1,4-glucoside bonds having a higher degree of polymerization can be analyzed by gel filtration chromatography. In Example 4, the powder of cyclic glucan having only α-1,4-glucoside bonds obtained in each reaction time was dissolved in 250 ul of distilled water, and the total amount thereof was Superose 6 (φ1 cm × 30 cm, manufactured by Pharmacia). And Superose 30 (φ1 cm × 30 cm, manufactured by Pharmacia) were loaded onto a column and eluted with a 150 mM sodium acetate aqueous solution.

  As a result, as shown in FIG. 7, in the sample having a reaction time of D enzyme of 10 minutes, the average molecular weight of the produced cyclic glucan having only α-1,4-glucoside bond was about 70,000. . As the reaction time of the D enzyme becomes longer, the molecular weight of the cyclic glucan having only the α-1,4-glucoside bond produced is decreasing. However, after the reaction time of 6 hours, the average molecular weight did not change from about 15,000, and no further reduction in molecular weight occurred. The molecular weight of cyclic glucan is a value calculated using enzyme-synthesized amylose as a standard.

  Thus, the degree of polymerization of the cyclic glucan having only α-1,4-glucoside bonds produced by the D enzyme ranges from 17 to several hundreds depending on the degree of polymerization of amylose used as a substrate, the amount of D enzyme, and the reaction time. It was found that it can be controlled arbitrarily.

(Example 7: Preparation of glucan of the present invention)
After suspending 40 mg of commercially available waxy corn starch in 2 ml of DMSO (dimethyl sulfoxide), 18 ml of 20 mM citrate buffer (pH 7.0) containing 680 units of the D enzyme purified in Example 1 was added, and 30 ° C. For 40 hours.

  After heating the reaction solution at 100 ° C. for 10 minutes, the denatured enzyme protein was removed by centrifugation. Ten times the amount of ethanol was added to the supernatant to precipitate glucan. Next, the obtained precipitate was freeze-dried to obtain about 40 mg of glucan powder of the present invention. This powder is composed of the glucan having only the α-1,4-glucoside bond of the present invention, the glucan having the α-1,6-glucoside bond of the present invention, and the α-1,4-glucoside bond of the present invention alone. It was a mixture of a glucan having a structured cyclic structure and an acyclic structure portion.

(Example 8: Preparation of glucan having at least one α-1,6-glucoside bond of the present invention in a cyclic structure part or an acyclic structure part)
The precipitate obtained in Example 7 was fractionated by gel filtration chromatography. The precipitate was dissolved in 250 μl of distilled water, and the whole amount was loaded onto a column connected with Superose 6 (φ1 cm × 30 cm, Pharmacia) and Superdex 30 (φ1 cm × 30 cm, Pharmacia). Subsequently, elution was performed using a 150 mM sodium acetate aqueous solution. As shown in FIG. 8, the amylopectin eluted in the void volume is reduced in molecular weight by the reaction of the D enzyme, and the peak I having an average molecular weight of about 30,000 and the peak II having an average molecular weight of about 3,000. Two components were produced. Among them, the peak II having a molecular weight of about 3000 was a cyclic glucan mainly having only α-1,4-glucoside bonds. On the other hand, Peak I having an average molecular weight of about 30,000 was a glucan having an α-1,6-glucoside bond. The fraction of peak I was collected, and 10 times the amount of ethanol was added thereto to precipitate glucan having an α-1,6-glucoside bond. The precipitate was collected by centrifugation and then lyophilized. Thus, 20 mg of glucan having an α-1,6-glucoside bond was obtained. The molecular weight of cyclic glucan is a value calculated using enzyme-synthesized amylose as a standard.

(Example 9: Preparation of cyclic glucan having a high degree of polymerization having only α-1,4-glucoside bonds of the present invention)
20 mg of maltohexaose and 200 mg of glucose monophosphate were dissolved in 10 ml of 100 mM citrate buffer (pH 7.0) containing 5 mM adenosine monophosphate, and 20 units of D enzyme and phosphorylase a prepared in Example 1 were further dissolved. (Sigma) 1 mg was added and reacted at 30 ° C. for 2 hours.

  After centrifuging the reaction solution, the supernatant was treated at 100 ° C. for 5 minutes and centrifuged again to remove the denatured enzyme protein. 50 units of glucoamylase was added to the supernatant to decompose linear amylose into glucose, and 10 times the amount of ethanol was added to precipitate cyclic amylose. This precipitate contained about 30 mg of cyclic glucan having only α-1,4-glucoside bonds. Furthermore, the glucose monophosphate which exists in this precipitation was removed by performing gel filtration chromatography.

(Example 10: Confirmation that the substance of Example 8 contains a cyclic structure portion having at least one α-1,6-glucoside bond)
Glucoamylase is an enzyme that hydrolyzes α-1,4-glucoside bonds sequentially from the non-reducing end of glucans such as starch. Although slow, it is known that α-1,6-glucoside bonds can be hydrolyzed from the non-reducing end. As shown in FIG. 9, amylose and amylopectin that do not have a cyclic structure are completely degraded to glucose (17) by glucoamylase. However, in the glucans (15) and (13) having a cyclic structure in the molecule, only the non-cyclic structure part is degraded by glucoamylase, and the cyclic structure part is a substance that is not degraded by glucoamylase (hereinafter referred to as glucoamylase). It remains as a resistant component). Furthermore, this glucoamylase-resistant component has a cyclic glucan (16) having an α-1,6-glucoside bond and a cyclic glucan (13) having only an α-1,4-glucoside bond, depending on the sensitivity to starch debranching enzyme. Can be classified. That is, the glucoamylase resistant component that is not decomposed by the combined use of the debranching enzyme and glucoamylase is considered to be a cyclic glucan (13) having only α-1,4-glucoside bonds. On the other hand, the glucoamylase resistant component decomposed by the combined use of the debranching enzyme and glucoamylase is considered to be a cyclic glucan (16) having an α-1,6-glucoside bond. However, the cyclic glucan (13) having only α-1,4-glucoside bond, which is not decomposed by the combined use of the debranching enzyme and glucoamylase, is completely decomposed to glucose by using endo-type α-amylase and glucoamylase in combination. Can be done. By utilizing these properties, the acyclic structure part in the sample glucan, the cyclic structure part having an α-1,6-glucoside bond, and the cyclic structure part having only an α-1,4-glucoside bond are quantified. It becomes possible to do.

  Using this method, the cyclic structures of glucan and amylopectin obtained in Example 8 were quantified. Table 1 shows the results of quantification of the cyclic structures of glucan and amylopectin obtained in Example 8 using this method.

  10 mg of the substance obtained in Example 8 or 10 mg of amylopectin was dissolved in 1 ml of DMSO, and then quickly diluted with 8 ml of 100 mM sodium acetate buffer. 900 μl of this diluted solution was dispensed into four tubes. Next, 100 μl each of (1) distilled water, (2) glucoamylase solution, (3) mixed solution of debranching enzyme and glucoamylase, and (4) mixed solution of endo-type α-amylase and glucoamylase is added to each tube. In addition, the mixture was reacted at 40 ° C. for 4 hours. After completion of the reaction, the produced glucose was measured using a commercially available glucose determination kit. And the non-cyclic structure part in a sample glucan, the cyclic structure part which has (alpha) -1, 6- glucoside bond, and the cyclic structure part which has only (alpha) -1, 4- glucoside bond were calculated | required with the following formula, respectively.

  Here, c, x, y, and z are the amounts of glucose generated from the reaction solutions (1), (2), (3), and (4), respectively. From this result, it was found that the substance obtained in Example 8 contained glucan having a cyclic structure containing at least one α-1,6-glucoside bond.

(Example 11: Measurement of the degree of polymerization of the cyclic structure portion of the substance of Example 8)
After dissolving 10 mg of the substance obtained in Example 8 in 1 ml of DMSO, 1 ml of 1M sodium acetate buffer (pH 5.5), 8 ml of distilled water, and 200 units of glucoamylase were added and reacted at 40 ° C. for 1 hour. I let you. After heating at 100 ° C. for 10 minutes, the denatured enzyme was removed by centrifugation. After adding 10 times ethanol to the supernatant to precipitate the polysaccharide, the precipitate was dried. The obtained polysaccharide was dissolved in 1 ml of distilled water. 50 units of glucoamylase was added to this solution and reacted at 40 ° C. for 1 hour. After the reaction solution was heated at 100 ° C. for 10 minutes, the denatured enzyme was removed by centrifugation. Ten times the amount of ethanol was added to the supernatant, and the resulting precipitate was dried to obtain 1.1 mg of a powder of glucoamylase resistant component (cyclic structure portion).

  The glucoamylase-resistant component obtained as described above is dissolved in distilled water so as to be 0.4% (w / v), and then a sugar analysis system (liquid feeding system: DX300, detector: manufactured by Dionex). PAD-2, column: CarboPacPA100). For elution, flow rate: 1 ml / min, NaOH concentration: 150 mM, sodium acetate concentration: 0 min-50 mM, 2 min-50 mM, 37 min-350 mM (Gradient curve No. 3), 45 min-850 mM (Gradient curve No. 7). ), 47 min-850 mM.

  As a marker for comparing the degree of polymerization, linear α-1,4-glucan and glucan having only α-1,4-glucoside bond obtained in Example 2 were also analyzed under the same conditions.

  As shown in FIG. 10, the obtained glucoamylase-resistant component has a clean separation pattern as obtained in a linear α-1,4-glucan and a glucan having only α-1,4-glucoside bonds. It was not obtained. This suggests that this glucoamylase resistant component contains various numbers of α-1,6-glucoside bonds. However, the peak (arrow) considered to be the smallest corresponds to the position of the polymerization degree 15 of the linear α-1,4-glucan, and as shown in Example 6, the glucan having a cyclic structure is Considering that it elutes faster than linear glucan having the same degree of polymerization, the degree of polymerization of the cyclic structure part having at least one α-1,6 bond, which is considered to be the smallest, is considered to be at least 15. It was.

(Example 12: Preparation of amylomaltase from Thermus aquaticus ATCC 33923 strain)
Terada et al., Appl. Environ. Microbiol. vol. Thermus aquaticus ATCC 33923-derived amylomaltase was prepared according to the method of 65,910-915 (1999). Escherichia coli TG-1 transformed with the expression plasmid pFQG8 in Escherichia coli of amylomaltase derived from Thermus aquaticus ATCC 33923 strain was added to 1 liter of LB medium (1% tryptone, 0.5% yeast extra) containing ampicillin at a final concentration of 100 μg / ml. And 1% NaCl, pH 7.5) at 37 ° C. for 18 hours, and centrifuged to collect the cells. The obtained bacterial cells were treated with 300 ml of 10 mM KH ₂ PO ₄ -Na ₂ HPO ₄ (pH 7.5) (buffer B).
Washed twice and then dispersed in 60 ml buffer B. The bacterial cells were crushed by ultrasonic waves. This cell disruption solution was heated at 70 ° C. for 30 minutes, and the centrifuged supernatant was used as a amylomaltase solution derived from Thermus aquaticus ATCC 33923 strain.

  The activity of amylomaltase is determined by quantifying glucose produced when amylomaltase is allowed to act on 10% (w / v) maltotriose at 70 ° C. for 10 minutes in a 50 mM sodium acetate buffer (pH 5.5). It was measured. The amount of enzyme that liberates 1 μmol of glucose per minute was defined as 1 unit, and the activity unit was calculated.

(Example 13: Preparation of highly-polymerized cyclic glucan having only α-1,4-glucoside bond by amylomaltase derived from Thermus aquaticus ATCC 33923 strain)
A completely dissolved amylose solution was prepared by adding 125 g of commercially available amylose (average molecular weight 30,000) in 1 liter of 1N NaOH. This amylose solution was added to 25 liters of water, and after adjusting the pH to 5.5 with HCl, 920 units of Thermus aquaticus ATCC 33923-derived amylomaltase was added and reacted at 60 ° C. for 4 hours. The pH of the reaction solution was adjusted to 3 with HCl, and the reaction was stopped by heating at 90 ° C. for 30 minutes. After adjusting the pH of the reaction solution to 5 with NaOH, 8,000 units of β-amylase was added and reacted at 50 ° C. for 15 hours. Ethanol was added to the permeate obtained by filtering the reaction solution through a 0.45 μm filter so that the final concentration was 75% (v / v), whereby a high polymerization degree cyclic glucan was collected as a precipitate by filtration. The obtained precipitate was dissolved in water and dried by lyophilization to obtain about 40 g of a highly polymerized cyclic glucan having only α-1,4-glucoside bonds. When the high polymerization degree cyclic glucan thus obtained was analyzed by a sugar analysis system manufactured by DIONEX shown in Example 4, it was a mixture of high polymerization degree cyclic glucans having a polymerization degree of 22 or more. This cyclic glucan having a high polymerization degree is dissolved in distilled water so as to be 1% (w / v), and then ethanol is added so that the final concentration is 40% (v / v). Cyclic glucan was removed as a precipitate by filtration. Ethanol was added to the filtrate to a final concentration of 75% (v / v), and the precipitate was collected by filtration. The obtained precipitate was dissolved in water and then dried by lyophilization to obtain about 30 g of a highly polymerized cyclic glucan having a polymerization degree of 22-50.

(Example 14: Preparation of a tosyl group-containing cyclic glucan derivative in which the tosyl group of the present invention is bonded to a cyclic glucan having a high degree of polymerization)
1 g of a highly polymerized cyclic glucan (abbreviation: CA) having a degree of polymerization of 22 to 50 having only an α-1,4-glucoside bond obtained by the same method as in Example 13 was charged into an eggplant-shaped flask. 20 ml of 4N NaOH aqueous solution was added, dissolved, and 1 g of p-toluenesulfonyl chloride (abbreviation: p-TsCl) was added to the aqueous solution while stirring (see Schemes 1 and 2 in FIG. 11). After stirring at 0 ° C. for 2 hours, unreacted p-TsCl was removed by filtration, and the aqueous solution was neutralized with an aqueous HCl solution. The NaCl salt, a byproduct of this tosylation reaction, was removed by dialysis overnight and the water was lyophilized to give a white solid (1.0 g, 84% yield).

It was confirmed by the UV spectrum method that the tosylation proceeded because there was an absorption based on the S═O group peculiar to the tosyl group (FIG. 12). Further, from the ratio of the proton integral calculated value of (a + b) and the proton integral calculated value of d in the ¹ H-NMR spectrum of the tosylated highly polymerized cyclic glucan in FIG. The ratio of glucan substituted with tosyl group out of all hydroxyl groups was calculated to be about 4.9%.

(Example 15: Preparation of tosyl group-containing cyclic glucan derivatives having different degrees of substitution)
The reaction was performed under the same tosylation reaction conditions as in Example 14 except that the reaction temperature, time, and stirring rate were changed. The results are shown in Table 2. Thus, the substitution degree of the tosyl group could be changed by changing the reaction conditions.
(Example 16: Preparation of an alkyl group-containing cyclic glucan derivative in which an alkyl group of the present invention is bonded to a cyclic glucan having a high degree of polymerization)
The transesterification reaction between the tosyl group of the tosylated highly polymerized cyclic glucan obtained in Example 14 and sodium fatty acid was performed as follows (see schemes 3 and 4 in FIG. 14). First, 1 g of tosylated highly polymerized cyclic glucan (CA-tosylate) and 1 g of sodium laurate were dissolved in DMSO and stirred at a temperature of 80 ° C. under a nitrogen stream for 5 hours. The reaction solution was then added with vigorously stirred acetone and the resulting precipitate was filtered, dissolved in water, and dialyzed overnight to give unreacted sodium laurate and by-product p- Sodium toluenesulfonate was removed and lyophilized to give a white solid (0.4 g, 80% yield).

By IR spectrum measurement (KBr method) of this white solid, it was confirmed that transesterification proceeded because peaks based on CH stretching vibration peculiar to alkyl groups of fatty acids and C = O stretching vibration peculiar to ester groups appeared. (FIG. 15). Further, from the ratio between the proton integral calculated value of (d + e) and the proton integral calculated value of c in the ¹ H-NMR spectrum diagram of the high polymerization degree cyclic glucan (CA-laurate) after transesterification in FIG. In other words, the proportion of all hydroxyl groups of the highly polymerized cyclic glucan substituted with fatty acid was calculated as 3.3%.

  High-degree-of-polymerization cyclic glucan (CA) before tosylation, high-degree-of-polymerization cyclic glucan (CA-tosylate) after tosylation, and laurylated high-degree-of-polymerization cyclic glucan (CA-laurate) after transesterification, respectively, in ultrapure water Dispersion or dissolution was performed, and surface tension measurement was performed using the Wilhelmy plate method (FIG. 17). From FIG. 17, the high polymerization degree cyclic glucan (CA) before tosylation does not change the surface tension of water at all even when the concentration is increased, whereas the laurylation high degree polymerization cyclic glucan (CA-) after transesterification. In the case of laurate), it was found that the surface tension greatly decreased as the concentration increased. From this result, it was suggested that the laurylated highly polymerized cyclic glucan (CA-laurate) after transesterification has excellent surface activity. In addition, the high-polymerization cyclic glucan (CA-tosylate) after tosylation is less decreased in surface tension than the laurylated high-polymerization cyclic glucan (CA-laurate) after transesterification, but has surface activity. It was suggested.

  FIG. 18 shows the results of examining the physical properties of a dispersion (0.6 mM) in water of a 0.4 wt% solution of laurylated highly polymerized cyclic glucan (CA-laurate) after transesterification. From FIG. 18, the presence of a micelle-like molecular assembly having a diameter of 6 to 20 nm was confirmed.

  Further, the dispersion in water of the 0.4% by weight solution of laurylated highly polymerized cyclic glucan after transesterification was visually observed using a transmission electron microscope (freeze replica method) (FIG. 19). As a result, a plurality of particles having a size matching the dynamic light scattering data were confirmed.

  The dispersion in water of a 4.0% by weight laurylated highly polymerized cyclic glucan after transesterification was visually observed using a transmission electron microscope (FIG. 20). As a result, spherical micelles having a diameter of 70 to 200 nm were confirmed.

(Example 17: Preparation of an alkyl group-containing cyclic glucan derivative in which an alkyl group of the present invention is bonded to a cyclic glucan having a high degree of polymerization)
A cyclic glucan containing a myristic acid group was obtained in the same manner as in Example 16 except that sodium myristic acid was used instead of sodium laurate in Example 16.

(Example 18: Preparation method of fluorinated alkyl group-containing cyclic glucan derivative in which the fluorinated alkyl group of the present invention is bonded to cyclic glucan having a high degree of polymerization)
The fluorinated alkyl group-containing cyclic glucan derivative of the present invention was obtained in the same manner as in Example 16 except that sodium perfluorobutyrate was used instead of sodium laurate in Example 16.

(Example 19: Complex formation of an alkyl group-containing cyclic glucan derivative of the present invention and a guest compound)
The CA-laurate solution dispersed in water at a concentration of 0.4% by weight, iodine solution (0.1 wt% KI, 0.01 _wt% I 2, 0.004 N HCl) was added dropwise, the brown A solution was obtained. This indicated that iodine was included in the CA portion of CA-laurate, indicating that CA-laurate has the ability to form a complex with the guest compound.

  According to the present invention, the solubility in oils is good, the surface active characteristics are excellent, and the cyclic steric structure is changed according to the structure of the compound to be included, so that a wide variety of low to high molecular weights can be obtained. It is possible to provide a novel cyclic glucan derivative based on a highly polymerized cyclic glucan having a superior property as a molecular recognition host molecule that enables inclusion of various compounds as a basic skeleton, and a method for producing the same.

  The composition containing a cyclic glucan derivative in which a hydrophobic group of the present invention is bonded to a cyclic glucan having a high polymerization degree is useful as a pharmaceutical, a surfactant, a cosmetic composition, a food composition, and a film for a magnetic recording medium.

It is a schematic diagram of the high polymerization degree cyclic glucan used by this invention. It is a schematic diagram which shows a series of processes which make D enzyme act on amylose or amylopectin, and obtain the high polymerization degree cyclic glucan which has only (alpha) -1, 4- glucoside bond. It is a schematic diagram showing a series of processes for obtaining a glucan formed only by a cyclic structure having at least one α-1,6-glucoside bond by reacting amylopectin with a D enzyme. It is an elution pattern when the highly polymerized cyclic glucan having only the α-1,4-glucoside bond of the present invention is digested with various glycolytic enzymes. 5-1 is an elution pattern of a high polymerization degree cyclic glucan having only α-1,4-glucoside bonds obtained in Example 2, and 5-2 is a linear α-1,4-glucan. This is an elution pattern. Each peak-like number indicates the degree of polymerization. It is the elution pattern of the partial hydrolysis of each peak GL and a non-decomposition product. Each peak-like number indicates the degree of polymerization. It is the elution pattern by gel filtration of the highly polymerized cyclic glucan having only α-1,4-glucoside bonds obtained in Example 4. It is the elution pattern by gel filtration of waxy corn starch before making D enzyme act and after making it act. The acyclic structure portion, the cyclic structure portion having at least one α-1,6-glucoside bond, and the cyclic structure portion having only the α-1,4-glucoside bond in the glucan obtained in Example 8 are quantified. It is a schematic diagram which shows a process. A cyclic structure part of glucan having at least one α-1,6-glucoside bond obtained in Example 8, a linear α-1,4-glucan, and α-1,4 obtained in Example 2 -Elution pattern of cyclic glucan having only glucoside bonds. Each peak-like number indicates the degree of polymerization. The synthetic schematic diagram (Scheme 1) and synthetic scheme (Scheme 2) of the tosylated highly polymerized cyclic glucan (CA-tosylate) of the present invention are shown. The UV spectrum figure of CA of the present invention and CA-tosylate is shown. The ¹ H-NMR spectrum diagram of CA-tosylate of the present invention. The synthetic schematic diagram (Scheme 3) and synthetic scheme (Scheme 4) of the laurylated high polymerization degree cyclic glucan (CA-laurate) of this invention are shown. The IR spectrum figure of CA-tosylate of this invention and CA-laurate is shown. The ¹ H-NMR spectrum diagram of CA-laurate of the present invention. The comparison of the surface tension measurement data about each aqueous solution of CA of this invention, CA-tosylate, and CA-laurate is shown. The dynamic light-scattering data of the dispersion (0.6 mM) in the water of CA-laurate of this invention are shown. The transmission electron microscope (freeze replica method) photograph of the dispersion (0.4 weight%) in the water of CA-laurate of this invention is shown. The transmission electron micrograph of the dispersion (4.0 weight%) in the water of CA-laurate of this invention is shown.

Explanation of symbols

11 reducing end 12 amylose 13 cyclic glucan having only α-1,4-glucoside bond 14 amylopectin 15 composed of at least 14 α-1,4-glucoside bonds and at least one α-1,6-glucoside bond Glucan having one cyclic structure in the molecule 16 Glucan formed only by a cyclic structure constituted by at least one α-1,6-glucoside bond 17 Glucose 18 constituted by α-1,4-glucoside bond only Having a cyclic structure and a non-cyclic structure moiety

Claims (18)

Cyclic glucan having a hydrophilic part containing a cyclic glucan having a degree of polymerization of 14 or more and one or more side chains comprising a hydrophobic group, wherein the side chain is bonded to the main chain via a linking group Derivative. The hydrophobic group is a C _{3 to} C ₂₀ linear or branched alkyl group, a C _{3 to} C ₂₀ linear or branched fluorinated alkyl group, or a tosyl group. The cyclic glucan derivative described. The hydrophobic group is a C ₃ -C linear fluorohydrocarbon alkyl group having ₂₀ or straight-chain perfluoroalkyl group of C ₃ -C _20, with at least 50% degree of fluorine substitution, according to claim 1 Cyclic glucan derivative.   The cyclic glucan derivative according to claim 1, wherein the hydrophilic portion consists only of a cyclic glucan having a polymerization degree of 14 or more. The cyclic glucan is a glucan having one cyclic structure with a polymerization degree of 14 or more constituted by α-1,4-glucoside bonds in the molecule,
(I) a glucan having an acyclic structure in addition to a cyclic structure composed only of α-1,4-glucoside bonds,
(Ii) a glucan having an acyclic structure in addition to a cyclic structure composed of an α-1,4-glucoside bond and at least one α-1,6-glucoside bond,
(Iii) a glucan having only a cyclic structure composed only of α-1,4-glucoside bonds, and (iv) an α-1,4-glucoside bond and at least one α-1,6-glucoside bond. A glucan having only a cyclic structure,
The cyclic glucan derivative according to claim 1, which is selected from the group consisting of: The cyclic glucan derivative according to claim 1, wherein the side chain is bonded to the cyclic glucan by an amide bond, an ether bond, or an ester bond. The cyclic glucan derivative according to claim 1, wherein at least one of the alcoholic hydroxyl groups of the cyclic glucan is bonded to a fluorine-substituted or unsubstituted fatty acid by an ester bond. Cyclic glucan derivatives represented by the following structural formula:
Here, each of the n monomer units is independent, each of the n R ¹ independently represents -LR ⁴ or R ⁵ , and each of the n R ² is independently- L—R ⁴ or R ⁵ , and n R ^{3 s} independently represent —LR ⁴ or R ⁵ , provided that at least one —LR ⁴ is present in one molecule;
Each R ⁴ is independently a C _{3 to} C ₂₀ linear or branched alkyl group, or a C _{3 to} C ₂₀ linear or branched fluorinated alkyl group;
Each L is independently —NHCO—, —O—, —S—, —OSO ₂ —, —SCO— or —OCO—;
Each R ⁵ is independently a hydroxyl group or a tosyl group;
n is a cyclic glucan derivative having a molecular weight of 14 to 5,000. The cyclic glucan derivative according to claim 8, wherein all of n R ² and n R ³ are hydroxyl groups. A method for producing a cyclic glucan derivative, comprising:
Introducing a tosyl group into a cyclic glucan having a polymerization degree of 14 or more to obtain a tosylated derivative, and reacting the tosylated derivative with a substituted or unsubstituted fatty acid ester to obtain an ester derivative;
Including the method. The following steps:
a) a step of reacting D-enzyme with a linear α-1,4-glucan or a saccharide containing these to obtain a highly polymerized cyclic glucan;
b) In an alkaline aqueous solution, the high polymerization degree cyclic glucan reacts with p-toluenesulfonyl chloride, and at least one of the alcoholic hydroxyl groups of the high polymerization degree cyclic glucan reacts with the p-toluenesulfonyl chloride. Contacting to give a tosyl group-containing cyclic glucan derivative in which a tosyl group is bonded to a cyclic glucan having a high degree of polymerization;
c) A cyclic group in which a hydrophobic group is bonded by bringing the tosyl group-containing cyclic glucan derivative into contact with a fluorine-substituted or unsubstituted fatty acid sodium under conditions where the tosyl group and the fatty acid sodium can exchange with each other. Obtaining a glucan derivative;
A process for producing a cyclic glucan derivative. In the step b), the high polymerization degree cyclic glucan and the p-toluenesulfonyl chloride are subjected to reaction conditions in which at least 3% or more of all alcoholic hydroxyl groups of the high polymerization degree cyclic glucan are substituted with tosyl groups. The method according to claim 11, which is contacted at The said step c) WHEREIN: The said tosyl group containing cyclic glucan derivative and the said fatty acid sodium are contacted on the reaction conditions from which the exchange rate of the said tosyl group and the said fatty acid sodium will be at least 50%. the method of. A pharmaceutical comprising the cyclic glucan derivative according to claim 1 or a mixture thereof. A surfactant comprising the cyclic glucan derivative according to claim 1 or a mixture thereof. A cosmetic composition comprising the cyclic glucan derivative according to claim 1 or a mixture thereof. A film for a magnetic recording medium comprising the cyclic glucan derivative according to claim 1 or a mixture thereof. The magnetic recording medium comprising one or more magnetic layers on a nonmagnetic support, and the film according to claim 17 is formed on the surface of the magnetic layer or a protective layer provided on the magnetic layer. Magnetic recording medium.