JP7111325B2 - Chondroitin Sulfate Derivatives Containing Reactive Groups - Google Patents

Description

TECHNICAL FIELD The present invention relates to a reactive group-containing chondroitin sulfate derivative, its production method, its utilization, and the like.

Chondroitin sulfate is known to have various physiological activities such as nerve cell elongation action and chondrocyte differentiation action. For this reason, chondroitin sulfate is desired to be applied and used in various fields such as regenerative medicine, highly functional pharmaceutical compounds, and treatment of joint diseases.

However, since chondroitin sulfate is a polymer, organic chemical artificial synthesis is impossible. Therefore, it is usually obtained by cutting and extracting from biological components by alkali treatment or the like. In this case, it is generally difficult to add the obtained chondroitin sulfate to other substances.

Non-Patent Document 1 describes that a salmon-derived chondroitin sulfate derivative into which a propargyl group was introduced was obtained by reacting salmon-derived proteoglycan (containing chondroitin sulfate) with propargyl alcohol in the presence of endo-β-xylosidase. has been reported. It has also been reported that the chondroitin sulfate derivative is added to other substances using a propargyl group. However, the enzymes used in this technique are difficult to extract and purify, and in particular require complicated steps for purification, making it difficult to obtain the required amount. In addition, in order to obtain the necessary amount of the raw material (chondroitin sulfate-containing proteoglycan) for this technique, it is necessary to use fish such as salmon as the raw material, so it is difficult to obtain mammalian chondroitin sulfate chains by this method. be.

In Non-Patent Document 2, by introducing a compound in which a long-chain alkyl group is added to a xylose-serine structure into a cell, a glycosaminoglycan chain in which the glycosaminoglycan chain extends from the compound as a starting point is introduced into the cell. It has been reported that saminoglycan derivatives have been produced. However, the molecular weight of the glycosaminoglycan chain in the obtained glycosaminoglycan derivative is very low (approximately 1000 or less). Moreover, the glycosaminoglycan derivative obtained by this technique cannot be easily added to other substances.

Tetrahedron Letters 47 (2006) 7455-7458. Carbohydrate Research 361 (2012) 33-40.

INDUSTRIAL APPLICABILITY The present invention enables addition to other substances more easily and efficiently, and a method that enables more easily and efficiently producing a mammalian chondroitin sulfate derivative having a chondroitin sulfate chain with a higher molecular weight. and the mammalian chondroitin sulfate derivative.

As a result of intensive research in view of the above problems, the present inventors found that the above problems can be solved by culturing cells containing a reactive group-containing xylose derivative represented by general formula (2). That is, the present invention includes the following aspects.

Section 1. General formula (2):

[wherein: n represents an integer of 0 or 1 to 10; R ² are the same or different and represent a hydrogen atom or an acyl group. Y ¹ represents reactive group 1. ]
Including the step of culturing cells containing a compound represented by
General formula (3):

[wherein: n represents an integer of 0 or 1 to 10; R ² are the same or different and represent a hydrogen atom or an acyl group. X indicates a chondroitin sulfate chain. Y ¹ represents reactive group 1. ]
A method for producing a compound represented by

Section 2. Item 2. The production method according to Item ¹ , wherein Y1 is an ethynyl group, a vinyl group, an azide group, an epoxy group, an aldehyde group, an oxylamino group, a thiol group, an isocyanate group, or an isothiocyanate.

Item 3. Item 3. The production method according to Item 1 or 2, wherein Y ¹ is an ethynyl group.

Section 4. Item 4. The production method according to any one of Items 1 to 3, wherein n is 0 or an integer of 1 to 4.

Item 5. Item 5. The production method according to any one of items 1 to 4, wherein said R ² is a hydrogen atom.

Item 6. Item 6. The production method according to any one of Items 1 to 5, wherein the cells are fibroblasts.

Item 7. Item 7. The production method according to any one of Items 1 to 6, wherein the chondroitin sulfate chain has a molecular weight of 1000 or more.

Item 8. Item 8. The production method according to any one of Items 1 to 7, wherein the chondroitin sulfate chain is a mammalian chondroitin sulfate chain.

Item 9. Item 9. The production method according to any one of Items 1 to 8, wherein the chondroitin sulfate chain is a human chondroitin sulfate chain.

Item 10. General formula (3):

[wherein: n represents an integer of 0 or 1 to 10; R ² are the same or different and represent a hydrogen atom or an acyl group. X indicates a mammalian chondroitin sulfate chain. Y ¹ represents reactive group 1. ]
A compound represented by

Item 11. General formula (2):

[wherein: n represents an integer of 0 or 1 to 10; R ² are the same or different and represent a hydrogen atom or an acyl group. Y ¹ represents reactive group 1. ]
A compound represented by

Item 12. A xylose donor and general formula (1):

[wherein: n represents an integer of 0 or 1 to 10; Y ¹ represents reactive group 1. ]
Including the step of reacting a compound represented by in the presence of xylanase,
General formula (2):

[wherein: n represents an integer of 0 or 1 to 10; R ² are the same or different and represent a hydrogen atom or an acyl group. Y ¹ represents reactive group 1. ]
A method for producing a compound represented by

Item 13. General formula (5):

[wherein: n represents an integer of 0 or 1 to 10; R ¹ represents a group derived from the substance to be modified. R ² are the same or different and represent a hydrogen atom or an acyl group. X indicates a mammalian chondroitin sulfate chain. Z indicates a cross-linking portion. ]
A compound represented by

Item 14. Item 14. The compound according to Item 13, wherein said Z is a 1,2,3-triazole ring.

Item 15. General formula (3):

[wherein: n represents an integer of 0 or 1 to 10; R ² are the same or different and represent a hydrogen atom or an acyl group. X indicates a mammalian chondroitin sulfate chain. Y ¹ represents reactive group 1. ]
A compound represented by the general formula (4):

[In the formula: R ¹ represents a group derived from a substance to be modified. Y ² represents reactive group 2. ]
Including the step of reacting with a compound represented by
General formula (5):

[wherein: n represents an integer of 0 or 1 to 10; R ¹ represents a group derived from the substance to be modified. R ² are the same or different and represent a hydrogen atom or an acyl group. X indicates a mammalian chondroitin sulfate chain. Z indicates a cross-linking portion. ]
A method for producing a compound represented by

INDUSTRIAL APPLICABILITY According to the present invention, addition to other substances can be performed more easily and efficiently, and mammalian chondroitin sulfate derivatives having a chondroitin sulfate chain with a higher molecular weight can be more simply and efficiently produced. and the mammalian-type chondroitin sulfate derivative.

Furthermore, according to the present invention, it is also possible to provide a substance modified with mammalian chondroitin sulfate and a production method thereof as a raw material for the production method, a production method thereof, and a utilization aspect of mammalian chondroitin sulfate derivatives.

2 shows a chromatogram obtained in Example 3. FIG. 2 shows a chromatogram obtained in Example 4. FIG.

As used herein, the expressions "contain" and "include" include the concepts "contain", "include", "consist essentially of" and "consist only of".

1. In one aspect of the present invention, a reactive group-containing chondroitin sulfate derivative has the general formula (3):

A compound represented by and a method for producing the same. These are described below.

n represents 0 or an integer of 1 to 10;

n is preferably 0 or an integer of 1 to 6, more preferably 0 or an integer of 1 to 4, still more preferably an integer of 1 to 3, still more preferably 1 or 2, and more 1 is more preferable.

R ² are the same or different and represent a hydrogen atom or an acyl group.

Examples of the acyl group include, but are not limited to, acetyl group, glycolyl group, levulinoyl group, chloroacetyl group, bromoacetyl group, trichloroacetyl group, trifluoroacetyl group, benzoyl group, pivaloyl group, toroc group, and the like. can. Among these, an acetyl group or a glycolyl group is preferred, and an acetyl group is more preferred.

R ² is preferably a hydrogen atom, an acetyl group, or a glycolyl group, more preferably a hydrogen atom or an acetyl group, still more preferably a hydrogen atom.

X indicates a chondroitin sulfate chain.

The chondroitin sulfate chain is not particularly limited. A chain containing a repeating structure with at least one sugar Q selected from the group consisting of sulfated amino sugars in which hydroxyl groups are sulfated. Chondroitin sulfate chains usually contain a linker structure. Typically, the chondroitin sulfate chain includes a group represented by WL- (in the formula, W represents a structure containing a repeating structure of sugar P and sugar Q. L represents a linker structure.) mentioned. W includes, for example, a repeating structure of sugar P and sugar Q as a main chain, and may have a branched chain. L is usually a structure in which glucuronic acid, galactose and galactose are linked in this order.

The structure of the chondroitin sulfate chain, particularly the structure containing the repeating structure of sugar P and sugar Q, shows different types depending on the organism species that produce it. The type of chondroitin sulfate chain is preferably mammalian type, more preferably human type, monkey type, mouse type, rat type, etc., and still more preferably human type.

Although the molecular weight of the chondroitin sulfate chain is not particularly limited, it is, for example, 1,000 or more, preferably 2,000 or more, more preferably 5,000 or more, and still more preferably 10,000 or more. The upper limit is, for example, 100,000 and 50,000.

Y ¹ represents reactive group 1.

The reactive group 1 is not particularly limited as long as it is a group capable of forming a cross-linking portion (Z) described later by reacting with another group (reactive group 2 (Y ² ) described later). Examples of the reactive group 1 include an ethynyl group, a vinyl group, an azide group, an epoxy group, an aldehyde group, an oxylamino group, a thiol group, an isocyanate group, and an isothiocyanate group.

Y ¹ is preferably an ethynyl group.

The compound represented by the general formula (3) has the general formula (2):

It can be produced by a method comprising the step of culturing cells containing the compound represented by (step 2).

In general formula (2), n, R ² and Y ¹ are the same as above.

The cells used in step 2 are not particularly limited as long as they produce chondroitin sulfate chains. Cells are derived from preferably mammals, more preferably humans, monkeys, mice, rats and the like, and still more preferably humans. By using cells of these organisms, compounds represented by general formula (3) having mammalian chondroitin sulfate chains can be produced simply and efficiently. Cell types include, for example, fibroblasts, epithelial cells, vascular endothelial cells, nerve cells, hepatocytes, keratinocytes, muscle cells, epidermal cells, blood cells, hematopoietic stem cells/progenitor cells, and gametes (sperm, eggs). , fertilized eggs, endocrine cells, ES cells, iPS cells, tissue stem cells, cancer cells, etc., preferably fibroblasts, and more preferably skin fibroblasts. By using fibroblasts, it is believed that the compound represented by general formula (3) can be produced more efficiently.

Cells containing the compound represented by general formula (2) in step 2 can be obtained by adding the compound represented by general formula (2) to the cell culture medium. The final concentration in the medium when added can be, for example, 0.1 to 10 mmol/L, preferably 0.5 to 5 mmol/L, more preferably 1 to 3 mmol/L. Further, the density of the cells when adding the compound represented by the general formula (2) is preferably as high as possible. It is preferably 80-100%, more preferably 90-100%.

The culture period in step 2 is not particularly limited, but is, for example, 1 to 10 days, preferably 3 to 7 days.

After culturing in step 2, the means for recovering the compound represented by general formula (3) is not particularly limited, but it can be conveniently recovered from the medium. The medium may be subjected to further purification steps. Examples of purification means include alcohol precipitation, enzymatic proteolysis (enzyme treatment), protein precipitation, and the like.

Alcohol precipitation treatment can be performed according to a known method. Typically, the medium after the culture in step 2 is concentrated as necessary, mixed with 1 to 5 times (preferably 2 to 4 times) alcohol, and left for a certain period of time. to form a precipitate, followed by centrifugation to collect the resulting pellet.

The alcohol is not particularly limited as long as it can precipitate chondroitin sulfate. Examples of alcohols include ethanol, isopropanol, etc. Among these, ethanol is preferred from the viewpoint of lower toxicity. One type of alcohol may be used alone, or two or more types may be used in combination.

The alcohol preferably contains a salt. The salt is not particularly limited and includes common salts used for alcohol precipitation, such as sodium chloride, sodium acetate, ammonium acetate, lithium chloride, magnesium chloride and the like. Salts may be used singly or in combination of two or more. The concentration of the salt in the alcohol is not particularly limited, and may be a normal salt concentration employed in alcohol precipitation, such as the saturation concentration for alcohol in the case of sodium chloride.

The temperature when left to stand to form a precipitate is not particularly limited as long as the proteoglycan can be precipitated. The temperature can be, for example, about -80°C to room temperature, preferably about 0 to 10°C.

The time for standing to form a precipitate is not particularly limited as long as the proteoglycan can be precipitated, and is appropriately set according to the temperature. The time can be, for example, 8-48 hours, preferably 16-32 hours, provided that the temperature is between 0 and 10°C.

Enzyme treatment is not particularly limited as long as it is treatment with an enzyme capable of decomposing protein components in the medium. Enzymes to be used include, for example, known proteolytic enzymes such as actinase, chymotrypsin, subtilisin, pepsin, cathepsin, HIV protease, thermolysin, papain, caspase, and collagenase.

The protein precipitation treatment is not particularly limited as long as it is a treatment capable of denaturing and precipitating proteins (including peptides). Typical examples include TCA (trichloroacetic acid) precipitation treatment, ammonium sulfate precipitation treatment, acetone precipitation treatment, TCA/acetone precipitation treatment, organic solvent (phenol, chloroform, mixed solvent thereof, etc.) extraction treatment, and the like.

2. Raw materials for the reactive group-containing chondroitin sulfate derivative Although the compound represented by the general formula (2) has an appropriate degree of lipid solubility, Since it has a structure that has little adverse effect on elongation, it is thought that by using it, it can be efficiently taken up into cells and the chondroitin sulfate chain can be efficiently elongated.

Accordingly, in one aspect of the present invention, the general formula (2):

A compound represented by and a method for producing the same.

In general formula (2), n, R ² and Y ¹ are the same as above.

The compound represented by general formula (2) can be produced by various methods, but preferably a xylose donor and general formula (1):

It can be produced by a method comprising a step (step 1) of reacting a compound represented by in the presence of xylanase.

In general formula ( ¹ ), n and Y1 are the same as above.

The xylose donor used in step 1 is not particularly limited as long as it is xylose or a xylose-containing polysaccharide or xylose derivative capable of liberating xylose by enzymatic degradation by xylase. Xylose-containing polysaccharides include, for example, xylooligosaccharides. Examples of xylose derivatives include PNP-xylose and the like. Xylose donors preferably include xylooligosaccharides.

The xylanase used in step 1 is an enzyme classified under EC number EC 3.2.1.8, and is not particularly limited in this respect. As the xylanase, those derived from various organisms can be used. Examples thereof include xylanases derived from filamentous fungi such as organisms belonging to the genus Trichoderma (preferably Trichoderma longibrachiatum (ressei)) and organisms belonging to the genus Aspergillus (preferably Aspergillus niger). Among these, xylanase is preferably derived from the genus Trichoderma, more preferably derived from Trichoderma longibrachiatum (ressei), from the viewpoint that the compound represented by general formula (2) can be produced more efficiently. A single xylanase may be used alone, or two or more xylanases may be used in combination.

The xylanase may be a xylanase immobilized on a carrier. A known method can be adopted as a method for immobilizing xylanase on a carrier. The carrier is not particularly limited as long as it can immobilize the xylanase, and includes, for example, a carrier having a carboxyl group capable of forming an amide bond with an amino group contained in the xylanase. As a specific example, xylanase is immobilized on a carrier by forming an amide bond between the activated ester group of sepharose (carrier) in which the carboxyl group is esterified with N-hydroxysuccinimide (NHS) and the amino group of xylanase. can be done. Another specific example is a method of immobilization in which the carrier is activated with cyanogen bromide to form an amide bond with the amino group of xylanase.

The step 1 reaction is carried out in a suitable solvent. The solvent is not particularly limited as long as it can dissolve the xylose donor and the compound represented by the general formula (1) and does not deactivate the xylanase. Examples of such solvents include water, acetate buffers, phosphate buffers, citrate buffers, borate buffers, tartrate buffers, Tris buffers, phosphate buffered saline, etc., and are preferably include acetate buffers.

The reaction temperature in step 1 is not particularly limited as long as it does not deactivate the xylanase, and is appropriately selected according to the optimum reaction temperature of the xylanase. For example, it can be 30°C to 60°C, preferably 40°C to 60°C, more preferably 45°C to 55°C.

The reaction time of step 1 is not particularly limited as long as the enzymatic activity of xylanase can be sufficiently exhibited. For example, it can be 1-100 hours, preferably 12-48 hours, more preferably 18-30 hours.

The reaction pH in step 1 is not particularly limited as long as the enzymatic activity of xylanase is sufficiently exhibited. For example, it can be pH 3-10, preferably pH 4-6.

The molar ratio of the xylose donor and the compound represented by general formula (1) in the reaction of step 1 is not particularly limited as long as the enzymatic reaction by xylanase occurs. The compound represented by (1) can be, for example, 1 to 50 mol, preferably 10 to 20 mol.

After the reaction in step 1, the solution containing the compound represented by general formula (2) obtained by the reaction may be further subjected to a purification step. As purification means, known methods (liquid separation, distillation, chromatography, recrystallization, etc.) can be employed.

3. Modes of Use of Reactive Group-Containing Chondroitin Sulfate Derivatives has a reactive group of An ethynyl group is known to form a 1,2,3-triazole ring through a 1,3-dipolar cycloaddition reaction with an azide group. A vinyl group reacts with a thiol group to form a bond. Epoxy groups react with amino groups and thiol groups to form bonds. An aldehyde group reacts with an amino group to form a Schiff base, which upon reduction forms a bond. Oxylamino groups react with ketone groups and aldehyde groups to form oximes. An azide group is known to form a 1,2,3-triazole ring through a 1,3-dipolar cycloaddition reaction with an ethynyl group. If these properties of the reactive group are used, the compound represented by the general formula (3) and a group that reacts with the reactive group (for example, an azide group, an amino group, a thiol group, a hydroxy group, a ketone group, an aldehyde group), thereby providing a wide variety of substances modified with chondroitin sulfate chains.

Accordingly, in one aspect of the present invention, the general formula (5):

A compound represented by and a method for producing the same. This will be explained below.

In general formula (5), n, R ² and X are the same as above.

R ¹ represents a group derived from the substance to be modified. R ¹ is, for example, a group obtained by removing one or more atoms or atomic groups from a substance to be modified.

Either an organic substance or an inorganic substance can be used as the substance to be modified.

Substances to be modified include, for example, substances that can serve as scaffold materials for regenerative medicine inside and outside the body, specifically extracellular matrix components (eg, collagen, fibronectin, laminin, matrigel, etc.), gelatin, fibrin, fibroin, sericin, cotton, and cellulose. , methyl cellulose, agar, agarose, alginate, chitin, chitosan, biodegradable polymers (e.g. polyglycolic acid, polylactic acid, etc.), biodegradable polymers (e.g. polystyrene, polyurethane, nylon, etc.), oxide ceramics (for example, alumina, zirconia, etc.), calcium phosphate-based ceramics (for example, hydroxyapatite, etc.), titanium, stainless steel, etc., and composite materials thereof. By modifying these with chondroitin sulfate chains, physiological activities of chondroitin sulfate chains (nerve cell elongation action, chondrocyte differentiation action, etc.) can be imparted. By using the obtained scaffold material, cell growth, differentiation, proliferation, etc., tissue repair, etc. can be further promoted.

Other substances to be modified include phenol resin, epoxy resin, melamine resin, urea resin, unsaturated polyester resin, alkyd resin, polyurethane, thermosetting polyimide, polyethylene, polypropylene, polyvinyl chloride, polyvinylidene chloride, and polystyrene. , polyvinyl acetate, polytetrafluoroethylene, acrylonitrile butanediene styrene resin, styrene acrylonitrile copolymer, acrylic resin, polyamide, nylon, polyacetal, polycarbonate, modified polyphenylene ether, polybutylene terephthalate, polyethylene terephthalate, polyphenylene sulfide, polytetrafluoro Plastic materials such as plastics such as ethylene, polysulfone, polyethersulfone, amorphous polyarylate, liquid crystal polymer, polyetheretherketone, thermoplastic polyimide, or polyamideimide, and materials obtained by processing these plastics can be used. Examples of materials obtained by processing plastic include plastic cell culture vessels, medical tubes, and the like.

In addition, as a modification target substance, fiber materials such as cotton, wool, silk, hemp, viscose fiber, cuprammonium fiber, acetate fiber, promix fiber, nylon fiber, vinylon fiber, polyvinylidene chloride synthetic fiber , polyvinyl chloride-based synthetic fiber, polyester-based synthetic fiber, polyacrylonitrile-based synthetic fiber, polyethylene-based synthetic fiber, polypropylene-based synthetic fiber, polyurethane-based synthetic fiber, polyclar-based synthetic fiber, carbon fiber, or fibers themselves such as feathers, or Examples include materials processed from these fibers. Examples of materials obtained by processing the fibers include clothing, bedding, band-aids, masks, bandages, and nursing care products. Although the clothing is not particularly limited, clothing that can come into direct contact with the skin is preferable. Such clothing includes, for example, hats, underwear, gloves, socks, stockings, mufflers, stoles, underwear, diapers (for babies, nursing care), and the like. The bedding is not particularly limited, but preferably includes those that can come in direct contact with the skin. Examples of such bedding include sheets, duvet covers, pillowcases, and the like.

Other examples of substances to be modified include pharmaceutical compounds. Modification of a pharmaceutical compound with a chondroitin sulfate chain can improve the stability of the pharmaceutical compound.

Z indicates a cross-linking portion. The cross-linking portion is a linking portion formed by reacting with a reactive group 2 (Y ² ), which will be described later, and has a different structure depending on the type of reactive group 1 and reactive group 2 .

Z preferably includes a 1,2,3-triazole ring.

The compound represented by general formula (5) is, for example, general formula (3):

A compound represented by the general formula (4):

It can be produced by a method including a step of reacting with a compound represented by (step 3).

In general formula (3), n, R ² , X and Y ¹ are the same as above.

In general formula (4), R ¹ is the same as above.

Y ² represents reactive group 2. The reactive group 2 is not particularly limited as long as it is a group capable of forming the above-described cross-linking portion (Z) by reacting with another group (the above-described reactive group 1 (Y ¹ )). Examples of the reactive group 2 include an azide group, an amino group, a thiol group, a hydroxy group, a ketone group, an aldehyde group and an ethynyl group.

^Y2 is preferably an azide group.

The reaction in step 3 is carried out under appropriate conditions depending on the types of reactive groups 1 and 2. The reaction when the reactive group 1 is an ethynyl group and the reactive group 2 is an azide group will be described in detail below.

The step 3 reaction is a 1,3-dipolar addition reaction. The reaction can be carried out in the presence or absence of a solvent, but when carried out in the presence of a solvent, the solvent is not particularly limited as long as it does not adversely affect the reaction. For example, distilled water, methanol, tetrahydrofuran, dioxane, dimethylsulfoxide, N,N-dimethylformamide and the like can be used. It is preferred to carry out the reaction preferably in the presence of a suitable catalyst. Catalysts include, for example, copper (I) catalysts. When using a copper (I) catalyst, for example, a method of introducing divalent copper such as copper sulfate and a reducing agent (e.g., hydroquinone, sodium ascorbate) into the system and reacting monovalent copper can be mentioned. be done.

The reaction temperature in step 3 is not particularly limited as long as it is below the boiling point of the solvent, but may be, for example, 15 to 80°C, preferably 25 to 50°C, more preferably 32 to 42°C.

The reaction time in step 3 is not particularly limited, but can be, for example, 1 to 100 hours, preferably 24 to 60 hours.

The molar ratio of the compound represented by general formula (3) and the compound represented by general formula (4) in the reaction of step 3 is not particularly limited, but for example, represented by general formula (3) It can be 1 mol to 5 mol, preferably 1 mol to 3 mol, of the compound represented by the general formula (4) per 1 mol of the compound.

After the reaction in step 3, the solution containing the compound represented by general formula (5) obtained by the reaction may be further subjected to a purification step. As purification means, known methods (liquid separation, distillation, chromatography, recrystallization, etc.) can be employed.

EXAMPLES The present invention will be described in detail below based on examples, but the present invention is not limited by these examples.

Example 1. Synthesis of propargyl group-containing xylose derivatives Xylooligosaccharides (10 wt %, 1.5 mL, about 0.53 mmol) were added to sodium acetate buffer (pH = 5.0, 50 mM, 1 mL), xylanase (Sumizym X (Shinnihon Chemical Industry Co., Ltd., (derived from Trichoderma longibrachiatum (ressei))))) and 0.5 mL of propargyl alcohol were added to a reaction solution and incubated at 50°C for 24 hours. After completion of the reaction, the syrup obtained by concentration under reduced pressure was subjected to flash silica gel chromatography (Fuji Silysia 300 mesh, l = 17 cm), and β-propargyl xylose was extracted with an eluent (CHCl ₃ : MeOH = 10: 1). (12 mg, 0.59 mmol) was obtained.
1H ^- NMR ₍ D2O)
4.54 (d, 1 H, J = 7.6 Hz), 4.36 (t, 1 H), 3.82 (d, 1 H,), 3.62 (dd, 1 H), 3.42-3.26 (m), 3.18 (t, 1 H), 2.81 (br-d).

Example 2. Synthesis of Alkynyl Group-Containing Human Chondroitin Sulfate Derivative Normal human skin fibroblasts (KURABO, P-7: 7th passage) were cultured on a Petri dish until conferred. β-propargyl xylose dissolved in dimethylsulfoxide was added to the medium of each petri dish (14 plates) so that the final concentration in the medium was 2 mmol/L. After administration, the cells were cultured in a CO ₂ incubator (37°C) for 5 days, after which the medium was collected and lyophilized. The resulting residue was dissolved in distilled water (10 mL), 3 volumes of sodium chloride-saturated ethanol (30 mL) was added thereto, and the mixture was allowed to stand at 4°C for 24 hours. After centrifugation (15000 rpm, 15 minutes, 4°C), the supernatant was discarded to collect the precipitate. The precipitate was dissolved in 100 mM TrisHCl buffer containing calcium chloride (50 mM), pH = 8.0 (15 mL), actinase (5 mg) was added, and the reaction was allowed to proceed at 37°C for 3 days. . Then, trichloroacetic acid (500 mg) was added at 0°C to stop the reaction. The reaction solution was centrifuged (15000 rpm, 15 minutes, 4°C) and the supernatant was collected. Three volumes of sodium chloride-saturated ethanol (45 mL) was added to the supernatant for ethanol precipitation to obtain an alkynyl group-containing human chondroitin sulfate.

Example 3. Synthesis of Human Chondroitin Sulfate Modified Fluorescent Substance Synthesized alkynyl group-containing human chondroitin sulfate (500 μg) was dissolved in ultrapure water (500 μL). Azido-Alexa Fluor (registered trademark) 488 solution (1 mg / 400 μL DMSO solution, 10 μL), copper sulfate (10 mM, 30 μL), and sodium ascorbate (10 mM, 30 μL) were added to 50 μL of the resulting solution. ) was added and stirred for 2 days at 37° C. under a nitrogen atmosphere. The resulting reaction solution was applied to a reversed-phase column cartridge (Allteck _C18 ) and eluted in the order of H ₂ O and MeOH/H ₂ O ₌ 1/1. A sulfuric acid-modified fluorescent substance was obtained.

The resulting syrup was redissolved in ultrapure water (300 µL), and the solution was subjected to HPLC analysis. The HPLC analysis conditions were a flow rate of 0.5 mL/min, an eluent of 0.2 M NaCl, a column temperature of 40°C, a TOSOH TSKgel G5000PW _XL column, and detection using a fluorescence detector (excitation wavelength: 501 nm, fluorescence wavelength: 525 nm). went. A chromatogram is shown in FIG. As a result, the molecular weight was calculated to be about 22,000. The molecular weight was calculated using a pullulan standard (manufactured by Shodex, P-82).

Example 4. Confirmation of Chondroitin Sulfate The human chondroitin sulfate-modified fluorescent substance obtained in Example 3 was dissolved in ultrapure water (500 μL). To 50 μL of the obtained solution, add 60 μL of 0.4M Tris-HCl buffer pH=8.0 and 60 μL of 0.4M sodium acetate, and add 5 μL of chondroitinase ABC (2.5 mU/mL) and heat to 37°C. and reacted for 24 hours. The reaction solution was heated at 100° C. for 3 minutes to terminate the reaction. The reaction solution was centrifuged (15000 rpm, 15 minutes, 4° C.), and the supernatant was collected and subjected to HPLC analysis in the same manner as in Example 3. A chromatogram is shown in FIG. A comparison with FIG. 1 confirmed that digestion with chondroitinase ABC slowed the retention time and reduced the molecular weight. From this, it was confirmed that the product obtained in Example 2 was indeed chondroitin sulfate.

Claims (6)

General formula (2):

[wherein: n represents an integer of 0 or 1 to 10; R ² are the same or different and represent a hydrogen atom or an acyl group. Y1 represents ^an ethynyl group, vinyl group, azide group, epoxy group, aldehyde group, oxylamino group, thiol group, isocyanate group, or isothiocyanate. ]
Including the step of culturing fibroblasts containing a compound represented by
General formula (3):

[wherein: n represents an integer of 0 or 1 to 10; R ² are the same or different and represent a hydrogen atom or an acyl group. X represents a chondroitin sulfate chain with a molecular weight of 10,000 or more . Y1 represents ^an ethynyl group, vinyl group, azide group, epoxy group, aldehyde group, oxylamino group, thiol group, isocyanate group, or isothiocyanate. ]
A method for producing a compound represented by The production method according to claim 1, wherein said Y ¹ is an ethynyl group. 3. The production method according to claim 1, wherein said n is 0 or an integer of 1-4. The production method according to any one of claims 1 to 3, wherein said R ² is a hydrogen atom. The production method according to any one of claims 1 to 4 , wherein the chondroitin sulfate chain is a mammalian chondroitin sulfate chain. The production method according to any one of claims 1 to 5 , wherein the chondroitin sulfate chain is a human chondroitin sulfate chain.

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