JP4763997B2 - Chondroitin 4-sulfate with clear structure and method for producing the same - Google Patents

Description

The present invention relates to a method for enzymatically producing chondroitin 4-sulfate having a well-defined structure using hyaluronic acid-degrading enzyme, and a chondroitin 4-sulfate having a well-defined structure to be obtained. More specifically, a method and a method for producing chondroitin 4-sulfate by enzymatically polymerizing hyaluronic acid-degrading mammalian hyaluronidase as a catalyst and using an oxazoline derivative represented by the following general formula (I) as a monomer substrate: And a well-defined chondroitin 4-sulfate.

A large amount of chondroitin sulfate used in pharmaceuticals, cosmetics, health foods, food additives, etc. is contained in shark and whale cartilage, and is industrially produced by extraction from these. In addition, a production method by extraction from the trachea of cattle or pigs has been put into practical use. However, since these methods are based on extraction from natural products, there are many contaminants, and most products have a low chondroitin sulfate content. In addition, in order to obtain a high-purity product for pharmaceuticals or the like, more purification steps are required.
In addition, it is known that chondroitin sulfate derived from natural products has a non-uniform distribution of sulfate groups in the molecule. In general chondroitin sulfate, chondroitin 4-sulfate structure in which sulfate group is bonded to the 4-position of N-acetylgalactosamine and chondroitin 6-sulfate structure in position 6 are present in the same chain. It depends on. In addition, chondroitin sulfate in which a sulfate group is bonded to the 2-position or 3-position of D-glucuronic acid, and chondroitin polysulfate having a plurality of sulfate groups in a disaccharide unit constituting chondroitin sulfate are also present.

  On the other hand, chondroitin sulfate, which is a kind of glycosaminoglycan, exists as a proteoglycan covalently bound to protein in the living body and plays an important role as a physiologically active substance. The position of the sulfate group and the degree of sulfation of chondroitin sulfate vary greatly depending on the tissue and its developmental stage, and it is speculated that the degree and pattern of sulfation greatly affects the function of chondroitin sulfate. However, due to the structural diversity derived from sulfate groups, it has been difficult to elucidate the function of chondroitin sulfate at the molecular level, and it has not progressed slowly.

  Chondroitin 4-sulfate (also known as chondroitin sulfate A) is an unbranched polymer polysaccharide in which D-glucuronic acid and N-acetylgalactosamine disaccharide with sulfate group bonded at the 4-position are linked alternately in a straight chain. . In general, naturally-occurring chondroitin sulfate has a non-uniform distribution of sulfate groups in the molecule as described above, and even in the research reagent sold as chondroitin sulfate A, the 6th position of N-acetylgalactosamine is present in the molecule. It is known that it has 10% or more of chondroitin 6-sulfate structure to which sulfate group is bonded. Therefore, it was insufficient for elucidating the function of the sulfate group of chondroitin sulfate, and development of a method for synthesizing chondroitin sulfate with a clear structure in which a sulfate group always exists at a specific position for each disaccharide repeating unit was expected. .

However, it is apparent that, in principle, it is impossible to synthesize chondroitin sulfate with a well-defined structure by extraction from natural materials, which is a conventional method for producing chondroitin sulfate. On the other hand, Patent Document 1 discloses a method for synthesizing chondroitin sulfate by chemical modification of polysaccharides obtained by other methods. However, chondroitin 4-sulfate structure has a maximum of 71% (7% without sulfate group). , Both sulfated at positions 4 and 6 are 18%), chondroitin 6-sulfate structure is at most 71% (no sulfate group is 7%, both positions 4 and 6 are sulfated) Sulfuric acid structure is 18%), and it is hard to say that chondroitin sulfate synthesis method has clear structure. Non-Patent Document 1 also includes an example in which an oligosaccharide having a structure of chondroitin sulfate E in which both the 4-position and 6-position of N-acetylgalactosamine are sulfated by organic synthesis from a monosaccharide derivative is performed. Although it is known, it is a compound that has a molecular weight that is at most as low as that of tetrasaccharides and that has a reducing end structure that differs from that of nature.
Accordingly, there has been a problem that development of the synthesis of chondroitin sulfate with a well-defined structure has not been achieved so far even after many trials.
USP 2003/0100534 A1 Sarah E. Tully et al. J. Am. Chem. Soc., 126, 7736 (2004)

  In view of the above problems, an object of the present invention is to provide a method for producing chondroitin sulfate having a clear structure and high molecular weight that is highly useful even at a practical level.

As a result of intensive studies to achieve the above object, the present inventors have found for the first time a novel method for producing chondroitin 4-sulfate with a well-defined structure by an enzymatic method using hyaluronic acid-degrading enzyme.
More specifically, the inventors of the present invention studied the production of chondroitin 4-sulfate with a well-defined structure by an enzymatic method, using hyaluronidase, which is originally known as an enzyme that degrades hyaluronic acid, as an enzyme polymerization catalyst, When the chondrocin oxazoline derivative represented by (I) is used as a monomer substrate, the monomer substrate is enzymatically polymerized to produce chondroitin 4-sulfate having a high molecular weight and a well-defined structure in a high yield. It came to be completed.

That is, the present invention provides a chondroitin 4-sulfate having a well-defined structure characterized by using a oxazoline derivative represented by the following general formula (I) as a substrate monomer and (1) allowing a hyaluronic acid-degrading enzyme to act on the substrate monomer. (2) The method for producing chondroitin 4-sulfate having a well-defined structure according to (1), wherein the hyaluronic acid-degrading enzyme is a mammal-derived hyaluronidase, (3) the hyaluronic acid-degrading enzyme is bovine testicular-derived hyaluronidase or (2) The method for producing chondroitin 4-sulfate with a clear structure described in (2), characterized in that it is hyaluronidase derived from ovine testicles, and (4) the pH is adjusted to 6 to 9 when the hyaluronic acid-degrading enzyme is allowed to act on the substrate monomer. A method for producing chondroitin 4-sulfate having a clear structure according to any one of (1) to (3), (5) ) 100% of the sulfate group is bonded to the 4-position of the N-acetylgalactosamine unit produced by any of the methods (1) to (4), and other sulfate groups including the D-glucuronic acid unit Is a well-defined chondroitin 4-sulfate, which is not bound.

  The enzymatic chemical chondroitin 4-sulfate production method of the present invention is a simple synthesis method compared with the extraction method from biomaterials that have been used industrially in the past, and the product from the reaction solution Isolation and purification are also easy. In addition, chondroitin sulfate obtained by extraction from biomaterials has a non-uniform distribution of sulfate groups in the molecule, whereas chondroitin 4-sulfate of the present invention has a sulfate group at the 4-position of the N-acetylgalactosamine unit. Is chondroitin 4-sulfate with a well-defined structure that is 100% bonded and has no other sulfate groups, including the D-glucuronic acid unit, and can be used as a standard substance for chondroitin 4-sulfate. Furthermore, it can be a useful tool for elucidating the function of chondroitin sulfate at the molecular level.

Hereinafter, the present invention will be described in more detail.
An example of the procedure for synthesizing the substrate monomer used in the present invention is described below, taking the synthesis of chondrosin oxazoline derivative (1) in the synthesis scheme shown in FIG. 1 as an example.
That is, methyl (2,3,4-tri-O-acetyl-α-D-glucopyranosyl trichloroacetimidate) uronate (3) was converted to a sugar donor, benzyl 2-azido-4,6-O-benzylidene-2- By reacting deoxy-β-D-galactopyranoside (4) as a sugar acceptor and using trimethylsilyl trifluoromethanesulfonate (TMSOTf) as an activator in dichloromethane at −20 ° C. under an argon atmosphere, Benzyl 2-azido-4,6-O-benzylidene-2-deoxy-3-O- (methyl 2,3,4-tri-O-acetyl-β-D-glucopyranosyluronate) -β-D -Synthesize galactopyranoside (5). 5 is dissolved in thioacetic acid and reacted at room temperature in a dry atmosphere to give benzyl 2-acetamido-4,6-O-benzylidene-2-deoxy-3-O- (methyl 2,3,4-tri-O -Acetyl-β-D-glucopyranosyluronate) -β-D-galactopyranoside (6) is synthesized. 6 is dissolved in a mixed solvent of acetic acid and water, stirred at 80 ° C., and benzyl 2-acetamido-2-deoxy-3-O- (methyl 2,3,4-tri-O-acetyl-β-D-glucose is added. Pyranosyluronate) -β-D-galactopyranoside is obtained. Subsequently, the reaction product obtained by adding acetyl chloride dropwise at -40 ° C. in pyridine under argon atmosphere was purified, dissolved in dimethylformamide, sulfur trioxide trimethylamine complex was added, and the mixture was heated to 50 ° C. under argon atmosphere. Triethylammonium benzyl 2-acetamido-6-O-acetyl-2-deoxy-3-O- (methyl 2,3,4-tri-O-acetyl-β-D-glucopyranosyluronate) -4-O-sulfonate-β-D-galactopyranoside (7) is obtained. 7 is deprotected by catalytic hydrogen reduction in dehydrated methanol under hydrogen atmosphere using 10% palladium hydroxide carbon as a catalyst, followed by triethylamine, diaminopyridine and tosyl chloride in dehydrated dichloromethane under argon atmosphere. In addition, 2-methyl- [triethylammonium 6-O-acetyl-1,2-dideoxy-3-O- (methyl 2,3,4-tri-O-acetyl-β-D-glucopyr Nosyluronate) -4-O-sulfonate-α-D-galactopyrano]-[2,1-d] -2-oxazoline (8) is obtained. Subsequently, an aqueous solution of sodium hydroxide in methanol is added dropwise and stirred at 0 ° C., followed by purification to purify the target substrate monomer 2-methyl- [sodium 1,2-dideoxy-3-O- (sodium β- D-glucopyranosyluronate) -4-O-sulfonate-α-D-galactopyrano]-[2,1-d] -2-oxazoline (1) can be obtained.

  The substrate monomer is not particularly limited as long as it is in the form of free acid, metal salt such as sodium and potassium, ammonium salt, triethylamine salt and the like. The chondroitin 4-sulfate produced depends on the salt form of the substrate monomer, and includes free acids or forms such as metal salts such as sodium and potassium, ammonium salts, and triethylamine salts.

  The chondrosin oxazoline derivative thus obtained is suitably used as a substrate monomer for hyaluronic acid decomposing enzyme as a polymerization catalyst. The substrate monomer concentration during the enzyme reaction is 0.1% or more, preferably 1% or more, from the practical viewpoint. The reaction pH is preferably 5 to 10, preferably 6 to 9. The reaction temperature is preferably 0 ° C. to 50 ° C., usually 5 ° C. to 40 ° C.

  As the hyaluronic acid-degrading enzyme to be used, mammal-derived hyaluronidase is preferable, specifically, bovine testicle-derived or sheep testicle-derived hyaluronidase classified into endo-β-N-acetylhexosamidase (EC 3.2.1.35). It is also possible to use the enzyme in the form of an immobilized enzyme in which the enzyme is immobilized on an appropriate carrier. Either a batch reaction or a continuous reaction system is adopted.

  The reaction is an aqueous solvent or an alcohol such as methanol, ethanol or n-propanol, a polyol such as glycerin or polyethylene glycol, dimethyl sulfoxide, dimethylformamide, ethyl acetate, dioxane, various types that do not adversely affect the reaction. The process proceeds even under conditions appropriately added with inorganic salts or pH buffering agents.

When a batch reaction is started under the above conditions, the reaction can be completed in a few hours to a few days, although it cannot be defined unconditionally depending on the conditions. After the reaction is completed, the reaction solution is isolated and purified by combining known purification means such as centrifugation, ultrafiltration, microfiltration, various adsorption columns, solvent precipitation, and chromatographic separation. I can do it.
Regarding the molecular weight, it is possible to obtain chondroitin 4-sulfate having various molecular weights by controlling the conditions during the polymerization reaction of the substrate monomer by the enzyme catalyst. Further, the molecular weight can be reduced using an enzyme that degrades chondroitin 4-sulfate.

Chondroitin 4-sulfate is obtained by a polymerization reaction using a hyaluronic acid-degrading enzyme using a chondrocin oxazoline derivative represented by the general formula (I) as a substrate monomer, whereas a chondroitin represented by the following general formula (II) Even when the reaction using the synoxazoline derivative is carried out in the same manner, the polymerization product is not observed.
When the chondrocin oxazoline derivative represented by the general formula (II) is polymerized in the same manner as the substrate monomer of the general formula (I), chondroitin 6-sulfate should be obtained.
Therefore, it is considered impossible to synthesize chondroitin 6-sulfate by changing only the position of the sulfate group of the chondrosin oxazoline derivative, which is a substrate monomer, in the same manner as in the present invention.

  The details of the present invention will be described in the following examples, but the present invention is not limited to the following examples.

Benzyl 2-azido-4,6-O-benzylidene-2-deoxy-3-O- (methyl 2,3,4-tri-O-acetyl-β-D-glucopyranosyluronate) -β-D -Synthesis of galactopyranoside (5) benzyl 2-azido-4,6-O-benzylidene-2-deoxy-β-D-galactopyranoside (190 mg, 0.488 mmol) and methyl (2,3,4- Tri-O-acetyl-α-D-glucopyranosyl trichloroacetimidate) uronate (280 mg, 0.585 mmol) was dissolved in dehydrated dichloromethane (5.00 ml), and molecular sieves 4A (MS4A; 1.00 g) was added under an argon atmosphere. And stirred at −20 ° C. for 30 minutes. A solution obtained by diluting trimethylsilyl trifluoromethanesulfonate (TMSOTf; 0.106 ml, 0.585 mmol) with dehydrated dichloromethane (1.00 ml) was added dropwise at −20 ° C. and stirred for 1 hour. After completion of the reaction, triethylamine (0.100 ml) was added, MS4A was removed by celite filtration, the filtrate was diluted with chloroform, washed successively with saturated aqueous sodium hydrogen carbonate solution and saturated brine, and the organic layer was dried over magnesium sulfate. . After drying, magnesium sulfate was removed by Celite filtration, and the filtrate was distilled off under reduced pressure. The obtained residue was purified by silica gel chromatography (eluent: n-hexane / ethyl acetate = 3 / 1-1 / 1), and white crystalline benzyl 2-azido-4,6-O-benzylidene-2 -Deoxy-3-O- (methyl 2,3,4-tri-O-acetyl-β-D-glucopyranosyluronate) -β-D-galactopyranoside (270 mg, 0.386 mmol, yield 79 %).
The analysis data is as follows.
[α] _D -10 ° (c = 1.0, CHCl ₃ )
¹ H NMR (400 MHz, CDCl ₃ , TMS): δ (ppm); 7.54-7.52 (2H, m, aromatic), 7.38-7.31 (8H, m, aromatic), 5.56 (1H, s, PhCH), 5.26 5.22 (2H, m, H-3 ', H-4'), 5.06 (1H, dd, H-2 ', J _{1', 2 '} = 7.52Hz, J _{2', 3 '} = 8.52Hz), 4.99 _{(1H, d, PhCH 2,} J = 11.60Hz), 4.92 (1H, d, H-1 ', J 1', 2 '= 7.52Hz), 4.69 (1H, d, PhCH 2, J = 11.60Hz) , 4.37-4.29 (3H, m, H-6a, H-1, H-4), 4.09-4.02 (2H, m, H-6b, H-5 '), 3.88 (1H, dd, H-2, J _1,2 = 8.04Hz, J _2,3 = 10.5Hz), 3.72 (3H, s, COOCH ₃ ), 3.49 (1H, dd, H-3, J _2,3 = 10.50Hz, J _3,4 = 3.52Hz), 3.37 (1H, s, H-5), 2.06-2.01 (9H, m, Ac)
High resolution FAB MS: Calculated value [M + H] ⁺ = 700.2354 m / z (C ₃₃ H ₃₈ N ₃ O ₁₄ )
Actual value 700.2357 m / z (+ 0.5ppm)

Benzyl 2-acetamido-4,6-O-benzylidene-2-deoxy-3-O- (methyl 2,3,4-tri-O-acetyl-β-D-glucopyranosyluronate) -β-D Synthesis of 2- galactopyranoside (6) Benzyl 2-azido-4,6-O-benzylidene-2-deoxy-3-O- (methyl 2,3,4-tri-O-acetyl-β-D-glucose Pyranosyluronate) -β-D-galactopyranoside (250 mg, 0.358 mmol) was dissolved in thioacetic acid (2.50 ml) and reacted at room temperature in a dry atmosphere for 24 hours. After completion of the reaction, the reaction solution was distilled off under reduced pressure, and the residue was purified by silica gel chromatography (eluent: toluene / ethyl acetate = 1 / 0-1 / 3) to give white crystalline benzyl 2-acetamide-4, 6-O-benzylidene-2-deoxy-3-O- (methyl 2,3,4-tri-O-acetyl-β-D-glucopyranosyluronate) -β-D-galactopyranoside (220 mg 0.307 mmol, yield 86%).
The analysis data is as follows.
[α] _D -9.0 ° (c = 1.0, CHCl ₃ )
¹ H NMR (400 MHz, CDCl ₃ , TMS): δ (ppm); 7.57-7.52 (2H, m, aromatic), 7.37-7.30 (8H, m, aromatic), 5.75 (1H, d, NH, J _{NH, 2} = 7.03Hz), 5.57 (1H, s, PhCH), 5.23-5.19 (3H, m, H-4 ', H-3', H-1), 5.01 (1H, t, H-2 ', J _{1 ', 2' = J 2} ', 3' = 8.29Hz), 4.96-4.91 (2H, m, PhCH 2, H-1 '), 4.79 (1H, dd, H-3, J 2,3 = 11.5 _{Hz, J 3,4 = 3.53Hz),} 4.56 (1H, d, PhCH 2, J = 12.10Hz), 4.38-4.35 (2H, m, H-6a, H-4), 4.10 (1H, dd, H -6b, J _5,6b = 1.51Hz, J _{6a, 6b} = 11.80Hz), 4.01 (1H, d, H-5 ', J _{4', 5 '} = 9.54Hz), 3.69 (3H, s, COOCH ₃ ), 3.52-3.46 (2H, m, H-2, H-5), 2.02-2.01 (9H, m, CH ₃ CO), 1.92 (3H, s, CH ₃ CONH)
High resolution FAB MS: Calculated value [M + H] ⁺ = 716.2554 m / z (C ₃₅ H ₄₂ NO ₁₅ )
Actual value 716.2554 m / z (+ 0.0ppm)

Triethylammonium benzyl 2-acetamido-6-O-acetyl-2-deoxy-3-O- (methyl 2,3,4-tri-O-acetyl-β-D-glucopyranosyluronate) -4-O Of 2-Sulfonate-β-D-Galactopyranoside (7) Benzyl 2-acetamido-4,6-O-benzylidene-2-deoxy-3-O- (methyl 2,3,4-tri-O-acetyl -β-D-glucopyranosyluronate) -β-D-galactopyranoside (696 mg, 0.972 mmol) was dissolved in a mixed solvent of acetic acid (16.0 ml) and water (4.00 ml), Stir for hours. After completion of the reaction, the reaction solution was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography (elution solvent: chloroform / methanol = 20 / 1-15 / 1) to give benzyl 2-acetamido-2-deoxy-3- O- (Methyl 2,3,4-tri-O-acetyl-β-D-glucopyranosyluronate) -β-D-galactopyranoside (505 mg, 0.806 mmol, 87% yield) was obtained. . Next, the obtained compound (505 mg, 0.806 mmol) was dissolved in pyridine (10.0 ml), and acetyl chloride (0.070 ml, 0.967 mmol) was added dropwise at −40 ° C. in an argon atmosphere, followed by reaction for 1.5 hours. Further, acetyl chloride (0.035 ml, 0.484 mmol) was added dropwise and reacted for 1.5 hours. After completion of the reaction, methanol (2.00 ml) was added dropwise at −40 ° C. under an argon atmosphere, the reaction solution was distilled off under reduced pressure, and the resulting residue was subjected to silica gel chromatography (elution solvent: hexane / ethyl acetate = 1 / 3−). 0/1). Next, the obtained compound was dissolved in dimethylformamide (10.0 ml), sulfur trioxide trimethylamine complex (0.895 g, 6.432 mmol) was added, and the mixture was reacted at 50 ° C. overnight under an argon atmosphere. After completion of the reaction, methanol (2.00 ml) was added, the reaction solution was distilled off under reduced pressure, and the resulting residue was subjected to silica gel chromatography (eluent: chloroform / methanol 20 / 1-10 / 1, triethylamine 0.5% (v / v )), And colorless amorphous triethylammonium benzyl 2-acetamido-6-O-acetyl-2-deoxy-3-O- (methyl 2,3,4-tri-O-acetyl-β-D -Glucopyranosyluronate) -4-O-sulfonate-β-D-galactopyranoside (669 mg, 0.785 mmol, yield 80%) was obtained.
The analysis data is as follows.
[α] _D ²² -26 ° (c = 1.0, CHCl ₃ )
¹ H NMR (400 MHz, CDCl ₃ , TMS): δ (ppm); 9.50 (1H, s, NHCH ₂ CH ₃ ), 7.35-.7.28 (5H, m, aromatic), 6.20 (1H, br, NHCOOCH ₃ ) , 5.23 (1H, t, H-3 ', J _{2', 3 '} = J _{3', 4 '} = 9.03 Hz), 5.16 (1H, t, H-4', J _{3 ', 4'} = J _{4 ', 5'} = 9.03 Hz), 5.01-4.98 (2H, m, H-2 ', H-1'), 4.83 (1H, d, PhCH ₂ , J = 11.54 Hz), 4.79 (1H, s, H -4), 4.70 (1H, d, H-1, J _1,2 = 7.53Hz), 4.59 (1H, d, PhCH ₂ , J = 11.54Hz), 4.49-4.43 (2H, m, H-6) , 4.27 (1H, d, H-3, J _2,3 = 9.54 Hz), 4.14 (1H, d, H-5 ', J _{4', 5 '} = 9.54 Hz), 3.91-3.87 (2H, m, H-2, H-5), 3.73 (3H, s, COOCH ₃ ), 3.18 (2H, q, NHCH ₂ CH ₃ , J = 7.03 Hz), 2.08-1.94 (15H, m, CH ₃ CO), 1.35 (3H, t, NHCH ₂ CH ₃ , J = 7.03 Hz)
High resolution FAB MS: Calculated [M + H] ⁺ = 851.3120 m / z (C ₃₆ H ₅₅ N ₂ O ₁₉ S)
Actual value 851.3112 m / z (-1.0ppm)

2-Methyl- [triethylammonium 6-O-acetyl-1,2-dideoxy-3-O- (methyl 2,3,4-tri-O-acetyl-β-D-glucopyranosyluronate) -4 Synthesis of -O-sulfonate-α-D-galactopyrano]-[2,1-d] -2-oxazoline (8) Triethylammonium benzyl 2-acetamido-6-O-acetyl-2-deoxy-3-O- ( Methyl 2,3,4-tri-O-acetyl-β-D-glucopyranosyluronate) -4-O-sulfonate-β-D-galactopyranoside (213 mg, 0.250 mmol) in methanol (20.0 ml 10% palladium hydroxide on carbon (133 mg) was added, and the mixture was stirred at room temperature under a hydrogen atmosphere for 2 hours. After completion of the reaction, the reaction solution was filtered through Celite to remove 10% palladium hydroxide carbon, and the filtrate was distilled off under reduced pressure. The obtained residue was dried overnight under reduced pressure, and the obtained residue was dissolved in dehydrated dichloromethane (10.0 ml), and triethylamine (0.060 ml, 0.230 mmol), dimethylaminopyridine (30.0 mg, 0.138 mmol), Tosyl chloride (94.0 mg, 0.276 mmol) was added, and the mixture was stirred at room temperature for 9 hours under an argon atmosphere. Further, triethylamine (0.060 ml, 0.230 mmol), dimethylaminopyridine (30.0 mg, 0.138 mmol), tosyl chloride were added at room temperature. (94.0 mg, 0.276 mmol) was added and allowed to react overnight at room temperature. After completion of the reaction, the reaction solution was distilled off under reduced pressure, and the resulting residue was subjected to silica gel chromatography (eluent: chloroform / methanol = 20/1, triethylamine 0.5%), followed by Sephadex LH-20 (Amersham Biosciences) And purified by silica gel chromatography (eluent: chloroform / methanol = 20/1, triethylamine 0.5%) and colorless amorphous. Of 2-methyl- [triethylammonium 6-O-acetyl-1,2-dideoxy-3-O- (methyl 2,3,4-tri-O-acetyl-β-D-glucopyranosyluronate)- 4-O-sulfonate-α-D-galactopyrano]-[2,1-d] -2-oxazoline (100 mg, 0.135 mmol, 54% yield) was obtained.
The analysis data is as follows.
[α] _D ²⁴ + 4.6 ° (c = 0.68, CHCl ₃ )
¹ H NMR (400 MHz, CDCl ₃ , TMS): δ (ppm); 9.45 (1H, s, NHCH ₂ CH ₃ ), 5.93 (1H, d, H-1, J _1,2 = 6.53 Hz), 5.27 ( 1H, t, H-3 ', J _{2', 3 '} = J _{3', 4 '} = 9.03 Hz), 5.20 (1H, t, H-4', J _{3 ', 4'} = J _{4 ', 5 '} = 9.03 Hz), 5.15 (1H, d, H-1', J _{1 ', 2'} = 7.53Hz), 4.97 (H, dd, H-2 ', J _{1', 2 '} = 7.53Hz, J _{2 ', 3'} = 9.03 Hz), 4.81 (1H, dd, H-4, J _3,4 = 4.52 Hz, J _4,5 = 3.01 Hz), 4.48 (1H, dd, H-6a, J _{5, 6a} = 3.51 Hz, J _{6a, 6b} = 12.55 Hz), 4.39 (1H, dd, H-6b, J _5,6b = 9.04 Hz, J _{6a, 6b} = 12.55 Hz), 4.24 (1H, ddd, H-5 _{, J 4,5 = 3.01 Hz, J} 5,6a = 3.51 Hz, J 5,6b = 9.04 Hz), 4.20 (1H, dd, H-3, J 2,3 = 5.52 Hz, J 3,4 = 4.52 Hz), 4.11 (1H, d, H-5 ', J _{4', 5 '} = 9.03 Hz), 4.07 (1H, dd, H-2, J _1,2 = 6.53 Hz, J _2,3 = 5.52 Hz ), 3.75 (3H, s, COOCH ₃ ), 3.19 (2H, q, NHCH ₂ CH ₃ , J = 7.03 Hz), 2.07-2.00 (15H, m, CH ₃ CO), 1.38 (3H, t, NHCH ₂ (CH ₃ , J = 7.03 Hz)
High resolution FAB MS: Calculated [M + H] ⁺ = 743.2544 m / z (C ₂₉ H ₄₇ N ₂ O ₁₈ S)
Actual value 743.2529 m / z (-2.1ppm)

2-Methyl- [sodium 1,2-dideoxy-3-O- (sodium β-D-glucopyranosyluronate) -4-O-sulfonate-α-D-galactopyrano]-[2,1-d] -2-Oxazoline (1) synthesis
2-Methyl- [triethylammonium 6-O-acetyl-1,2-dideoxy-3-O- (methyl 2,3,4-tri-O-acetyl-β-D-glucopyranosyluronate) -4 -O-sulfonate-α-D-galactopyrano]-[2,1-d] -2-oxazoline (75 mg, 0.101 mmol) was dissolved in methanol (3.00 ml), and 1N aqueous sodium hydroxide solution (0.303 mL, 0.303 mmol) was dissolved. ) Was added dropwise at 0 ° C. and stirred for 1 hour. After completion of the reaction, the reaction solution was distilled off under reduced pressure and lyophilized. Next, after dissolving in water and adding DOWEX 50W-X8 (manufactured by Dow Chemical) to adjust the pH of the solution to about 8, DOWEX 50W-X8 is removed by filtration, freeze-dried again, Methyl- [sodium 1,2-dideoxy-3-O- (sodium β-D-glucopyranosyluronate) -4-O-sulfonate-α-D-galactopyrano]-[2,1-d] -2 -Oxazoline (41 mg, purity 92%) was obtained.
The analysis data is as follows.
¹ H NMR (400 MHz, D ₂ O, Acetone): δ (ppm); 5.89 (1H, d, H-1, J _1,2 = 6.02 Hz), 4.53 (1H, d, H-1 ′, J _{1 ', 2'} = 8.03 Hz), 3.92-3.88 (3H, m, H-3, H-6a, H-5), 3.65-3.57 (2H, m, H-2, H-6b), 3.50 (1H , d, H-5 ', J _{4', 5 '} = 9.04 Hz), 3.36-3.30 (2H, m, H-4', H-3 '), 3.19 (1H, dd, H-2', J _{1 ', 2'} = 8.03 Hz, J _{2 ', 3'} = 8.53 Hz), 1.84 (3H, s, CH ₃ C of oxazoline)
High resolution FAB MS: Calculated value [M + Na] ⁺ = 526.0219 m / z (C ₁₄ H ₁₉ NNa ₃ O ₁₄ S)
Actual value 526.0288 m / z (+ 1.6ppm)

Synthesis of chondroitin 4-sulfate by enzyme-catalyzed polymerization 2-Methyl- [sodium 1,2-dideoxy-3-O- (sodium β-D-glucopyranosyluronate) -4-O-sulfonate- α-D-galactopyrano]-[2,1-d] -2-oxazoline (5.00 mg, 9.93 μmol) is dissolved in phosphate buffer (50 mM, pH 7.5, 99 μl), and sheep testis-derived hyaluronidase (SIGMA) , Product number: H6254, 3720 units / mg, hereinafter referred to as OTH) was added at 0.500 mg, and the reaction was carried out at 30 ° C. for 1.5 hours. After completion of the reaction, the reaction solution was heated in a 90 ° C. water bath for 3 minutes to inactivate the enzyme, and then the yield and molecular weight of the polymerization product were calculated by size exclusion chromatography (SEC) measurement. . The yield calculated from the area ratio of the polymerization product and the hydrolyzate of the substrate monomer was 79%, and a calibration curve prepared using hyaluronic acid sodium salt (M _n = 800, 2000, 4000, 50000) as the molecular weight marker was used. The calculated molecular weight was M _n = 7200. The SEC measurement conditions are shown below.
SEC condition Detector: Differential detector Column: Shodex OHpak SB-804HQ
Column temperature: 40 ° C
Mobile phase: 0.1M sodium nitrate Flow rate: 0.5ml / min
From the above reaction solution, the polymerized product is collected under the same conditions as in SEC measurement, and then desorbed by size exclusion chromatography (mobile phase: pure water) using Sephadex G-10 (Amersham Biosciences). Salting was performed to purify chondroitin 4-sulfate. FIG. 2 shows the ¹ H NMR spectrum of the obtained chondroitin 4-sulfate, and FIG. 3 shows the ¹³ C NMR spectrum. From these spectra, the polymerization product is chondroitin 4-sulfate in which the substrate monomer is position- and stereoselective ring-opening polyaddition, and further, the sulfate group is 100% at the 4-position of the N-acetylgalactosamine unit of the disaccharide repeating unit. It was found to be chondroitin 4-sulfate with a well-defined structure that is bound and has no other sulfate groups, including the D-glucuronic acid unit.
The assignment of peaks in the NMR spectrum is as follows.
¹ H NMR (400 MHz, D ₂ O, Acetone): δ (ppm); 4.56 (1H, s, H-4), 4.38 (1H, d, H-1, J _1,2 = 6.52 Hz), 4.27 ( 1H, d, H-1 ', J _{1', 2 '} = 7.04 Hz), 3.85-3.83 (2H, m, H-2, H-3), 3.63-3.57 (4H, m, H-6, H -5, H-4 '), 3.47 (1H, d, H-5', J _{4 ', 5'} = 9.04 Hz), 3.39 (1H, t, H-3 ', J _{2', 3 '} = J _{3 ', 4'} = 7.04 Hz), 3.18 (1H, t, H-2 ', J _{1', 2 '} = J _{2', 3 '} = 7.04 Hz), 1.84 (3H, m, CH ₃ CO)
¹³ C NMR (100 MHz, D ₂ O, Acetone): δ (ppm); 174.58 (C-6 ′), 174.07 (CH ₃ CO), 103.35 (C-1 ′), 100.49 (C-1), 79.98 ( C-4 '), 76.28 (C-5'), 76.16 (C-4), 75.21 (C-3), 74.14 (C-5), 73.09 (C-3 '), 71.82 (C-2') , 60.58 (C-6), 51.06 (C-2), 22.09 (CH ₃ CO)
Comparative Example 1

NMR analysis of a commercially available chondroitin sulfate A sodium salt (manufactured by Seikagaku Corporation, derived from whale cartilage, SSG) was performed. According to the reagent information of this product, “chondroitin sulfate A is a mucopolysaccharide mainly composed of chondroitin 4-sulfate structure and also containing chondroitin-6 sulfate structure. In addition, the product is highly purified by Schiller's chromatographic method. ”In addition, it is described as“ Molar ratio (4-sulfuric acid / 6-sulfuric acid) 80/20 Enzymatic analysis ”as a general property. ing. ^{The 1} H NMR spectrum is shown in FIG. 4, and the ¹³ C NMR spectrum is shown in FIG. From these spectra, this product also has a structure in which a sulfate group is bonded to the 6-position in addition to the sulfate group bonded to the 4-position of the N-acetylgalactosamine unit of the disaccharide repeating unit. It was confirmed.
The assignment of peaks in the NMR spectrum is as follows.
¹ H NMR (400 MHz, D ₂ O, Acetone): δ (ppm); 4.56 (1H, s, H-4), 4.36 (1H, br, H-1), 4.28 (1H, br, H-1 ' ), 4.02-3.99 (2H, br, H-4, H-6 of 6-sulfated unit), 3.83 (2H, m, H-2, H-3), 3.63-3.60 (4H, m, H-6 , H-5, H-4 '), 3.48 (1H, br, H-5'), 3.39 (1H, br, H-3 '), 3.18 (1H, br, H-2'), 1.84-1.83 (3H, m, CH ₃ CO).
¹³ C NMR (100 MHz, D ₂ O, acetone) d (ppm); 174.51 (C-6 '), 174.01 (CH ₃ CO), 103.25 (C-1'), 100.37 (C-1), 79.90 ( C-4 '), 76.12 (C-5'), 75.88 (C-4), 75.13 (C-3), 74.05 (C-5), 73.02 (C-3 '), 71.75 (C-2') , 60.51 (C-6), 51.01 (C-2), 22.05 (CH ₃ CO)
Comparative Example 2

Verification of whether it is possible to synthesize chondroitin 6-sulfate by enzyme-catalyzed polymerization The following compound (III) was synthesized and subjected to the same reaction and analysis as in Example 6 except that this was used as a substrate monomer. No polymerization product was observed.

Reaction time using OTH or bovine testis-derived hyaluronidase (manufactured by SIGMA, product number: H3884, 1010 units / mg, hereinafter referred to as BTH) as a catalytic enzyme in the synthetic polymerization reaction of chondroitin 4-sulfate using various enzymes Table 1 shows the results of the same reaction and analysis as in Example 6 except that OTH was 1.5 hours and BTH was 48 hours.

Effect of reaction pH in chondroitin 4-sulfate synthesis In the polymerization reaction, the reaction pH was changed by using OTH as a catalytic enzyme, the reaction time shown in Table 2, and using a phosphate buffer solution having the pH shown in Table 2. Table 2 shows the results of the same reaction and analysis as in Example 6 except that.

Influence of enzyme concentration in chondroitin 4-sulfate synthesis In the polymerization reaction, the same reaction and analysis as in Example 6 were performed, except that the addition amount of OTH as a catalytic enzyme and the reaction time were changed to the conditions shown in Table 3. The results are shown in Table 3.

Effect of reaction temperature in chondroitin 4-sulfate synthesis Table 4 shows the results of the same reaction and analysis as in Example 6 except that the reaction temperature and reaction time were changed to the conditions shown in Table 4 in the polymerization reaction. .

Effect of substrate monomer concentration on chondroitin 4-sulfate synthesis In the polymerization reaction, substrate monomer 2-methyl- [sodium 1,2-dideoxy-3-O- (sodium β-D-glucopyranosyluronate) -4 -O-sulfonate-α-D-galactopyrano]-[2,1-d] -2-oxazoline and the same reaction as in Example 6 except that the addition amount and reaction time were changed to the conditions shown in Table 5. Table 5 shows the results of the analysis.

  The chondroitin 4-sulfate of the present invention can be used as cosmetics, pharmaceuticals, medical materials and the like. Moreover, since it is chondroitin 4-sulfate with a clear structure, it can be used as a standard substance for chondroitin 4-sulfate. Furthermore, it can be a useful tool for elucidating the function of chondroitin sulfate at the molecular level.

A synthesis scheme is shown. ¹ shows the ¹ H NMR spectrum of synthetic chondroitin 4-sulfate. The ¹³ C NMR spectrum of synthetic chondroitin 4-sulfate is shown. ¹ H NMR spectrum of commercial chondroitin sulfate A (4-sulfuric acid / 6-sulfuric acid = 80/20) is shown. The ¹³ C NMR spectrum of commercial chondroitin sulfate A (4-sulfuric acid / 6-sulfuric acid = 80/20) is shown.

Claims (10)

A method for producing chondroitin 4-sulfate having a clear structure, wherein a hyaluronic acid-degrading enzyme is allowed to act on an oxazoline derivative represented by the following general formula (I).
  The method for producing chondroitin 4-sulfate according to claim 1, wherein the hyaluronic acid-degrading enzyme is a hyaluronidase derived from a mammal.   The method for producing chondroitin 4-sulfate according to claim 1 or 2, wherein the hyaluronic acid-degrading enzyme is bovine testis-derived hyaluronidase or sheep testicle-derived hyaluronidase.   The method for producing chondroitin 4-sulfate according to any one of claims 1 to 3, wherein the hyaluronic acid-degrading enzyme is ovine testicle-derived hyaluronidase. A method for producing chondroitin 4-sulfate according to any one of claims 1 to 4,
A pH is adjusted to 6-9 in making the said hyaluronic acid decomposing enzyme act, The manufacturing method characterized by the above-mentioned. A method for producing chondroitin 4-sulfate according to any one of claims 1 to 5,
The method further comprises the step of producing an oxazoline derivative represented by the general formula (I) by reacting an oxazoline derivative represented by the following general formula (IV) with an aqueous sodium hydroxide solution in methanol: .
A method for producing chondroitin 4-sulfate according to any one of claims 1 to 6,
A production method in which the hyaluronic acid-degrading enzyme is allowed to act at 0 to 50 ° C. A method for producing chondroitin 4-sulfate according to any one of claims 1 to 7,
The manufacturing method whose number average molecular weight of the said chondroitin 4-sulfuric acid is 4000 or more. Chondroitin 4-sulfate obtained by the production method according to any one of claims 1 to 8 , wherein the number average molecular weight is 4000 or more. The chondroitin 4 according to claim 9 , wherein the sulfate group is 100% bonded to the 4-position of the N-acetylgalactosamine unit, and the other structure including the D-glucuronic acid unit is free of other sulfate groups. -Sulfuric acid.