WO2003070960A1 - Processes for producing chondroitin or chondroitin derivative - Google Patents

Abstract

A process for producing chondroitin or a chondroitin derivative which is a synthesis method easier than the conventional industrial process for production by extraction from biomaterials and in which the reaction product can be easily isolated from a reaction mixture and purified. The chondroitin or chondroitin derivative collected is industrially useful as a material for cosmetics, medicines, medical materials, etc. The process, which is for producing a chondroitin or chondroitin derivative, is characterized by causing a hyaluronic acid decomposition enzyme to act on an oxazoline derivative.

Description

 Description Method for producing chondroitin or chondroitin derivative

Technical field

 The present invention relates to a method for producing chondroitin or a chondroitin derivative using a hyaluronic acid-degrading enzyme. More specifically, the present invention relates to a method for producing chondroitin or a chondroitin derivative by enzymatically polymerizing a chondrosin oxazoline derivative as a monomer substrate using a mammalian hyaluronidase as a hyaluronic acid degrading enzyme as a catalyst. Background art

 Chondroitin is an unbranched high-molecular polysaccharide in which disaccharides of D-glucuronic acid and N-acetylgalactosamine are alternately linked in a linear chain. Chondroitin or chondroitin derivatives can be widely used as cosmetics, pharmaceuticals or medical materials.

 It is known that chondroitin is contained in the corneal skin of bovine eyes and the like, and is also obtained by desulfation of chondroitin sulfate A or C which is present in large amounts in cartilage tissue. As a method for producing chondroitin, an extraction method from skin with a relatively high content of slime is generally used, but it is economically disadvantageous because many steps are required to increase the purity. As described above, it can be produced by desulfurization of chondroitin sulfate A or C, but it is difficult to say that chondroitin sulfate is an advantageous method because it needs to be extracted and purified from cartilage tissue and the like.

 However, there has been a problem that the development of an enzymatic chemical synthesis method that can replace the conventional method of producing chondroitin from biomaterials has not been achieved until now, even after many trials.

The present inventors have conducted intensive studies on the development of a novel method for producing chondroitin or a chondroitin derivative which is highly useful even at a practical level, and as a result, the enzymatic method using hyaluronidase has been used. Chondroitin or chondroitin For the first time, a new process for the production of benzoquinone derivatives.

 More specifically, the present inventors have found that in the production of chondroitin or chondroitin derivatives by enzymatic chemistry, hyaluronidase, which is originally known as an enzyme that degrades hyaluronic acid, is used as an enzyme polymerization catalyst, and a chondroxin oxazoline derivative is used as a monomer When the substrate was used, it was found that a monomer substrate was enzymatically polymerized to produce high-molecular-weight chondroitin or a chondroitin derivative at a high yield, and the present invention was completed. Disclosure of the invention

 That is, the present invention has the following configuration.

 (1) A process for producing chondroitin or a chondroitin derivative, which comprises reacting a hyaluronic acid-decomposing enzyme with an oxazoline derivative represented by the following general formula (I).

-General formula (I)

(In the above formula, R represents hydrogen, an alkyl group, an optionally substituted alkyl group, a phenyl group, or an optionally placed phenyl group.)

 (2) The above oxazoline derivative is 2-methyl- [1,2-dideoxy-3-0- (sodium-D-darcopyranosyl oxalate)] — a—D—galactobranano [2,1–1d ] The production method according to the above (1), which is 2-oxazole.

 (3) The method according to the above (1) or (2), wherein the hyaluronic acid degrading enzyme is mammalian-derived hyaluronidase.

 (4) The method according to the above (3), wherein the mammal-derived hyaluronidase is a testicle-derived hyaluronidase or a sheep testis-derived hyaluronidase.

(5) A hyaluronic acid-degrading enzyme was produced on the oxazoline derivative at a pH of 5 to 10. The production method according to any one of claims 1 to 4, wherein the method is used.

 (6) The method according to any one of the above (1) to (4), wherein a hyaluronic acid degrading enzyme is allowed to act on the oxazoline derivative at a concentration of the oxazoline derivative of 0.1% by weight or more. '

(7) The method according to any one of the above (1) to (4), wherein a hyaluronic acid degrading enzyme is allowed to act on the oxazoline derivative at a temperature of 5 to 60 ° C. BRIEF DESCRIPTION OF THE FIGURES

 1 shows a synthesis scheme of a substrate monomer.

¹ shows a ¹ H NMR spectrum of produced chondroitin.

¹³ shows the ¹³ C NMR spectrum of the produced chondroitin.

The ¹³ C NMR spectrum (enlarged view of the sugar skeleton in FIG. 3) of the produced chondroitin is shown.

 The change with time of the residual ratio of the substrate monomer in the reaction solution is shown.

 1 shows a scheme of synthesis of a substrate monomer and an enzymatic polymerization reaction.

 The change with time of the residual ratio of the substrate monomer (14) is shown.

 The change with time of the residual ratio of the substrate monomer (15) is shown.

 The change over time in the residual ratio of the substrate monomer (16) is shown.

0: ¹ H NMR spectrum of chondroitin derivative (17) is shown. 1: ¹³ C NMR spectrum of chondroitin derivative (17). 2: Changes over time in the chondroitin derivative synthesis reaction. BEST MODE FOR CARRYING OUT THE INVENTION

 Hereinafter, the present invention will be described in more detail.

The synthesis procedure is described below using the chondrosin oxazoline derivative (6), which is one of the substrate monomers used in the present invention, as an example. Figure 1 shows the synthesis scheme. That is, methyl (2,3,4-tri-0-acetyl-] 3-D-darcopyranosyl trichloroacetimidate) peronate is converted to a sugar donor, benzyl 2-azido-4,6-0-benzylidene-2. -Doxy-0-D-galactopyranoside as a sugar acceptor JP02 / 11576

4 Benzyl 2-azido-4,6-0-benzylidene-2- by reacting in methane with trimethylsilyl trifluoromethanesulfonate (TMSOTf) as an activator at -20 ° C for 1 hour in an argon atmosphere Deoxy-3-0- (methyl 2,3,4-tri-0-acetyl_i3-D-darcopyranosyl ester) -i3-D-galactopyranoside (1) was synthesized. (1) was dissolved in thioacetic acid and reacted at room temperature in a dry atmosphere for 24 hours to give benzyl 2-acetamido-4,6-0-benzylidene-2-deoxy-3-0- (methyl2 , 3,4-Tri-0-acetyl- / 3-D-darcopyranosyl peroneto)-/ 3-D-galactopyranoside (2) was synthesized. (2) was dissolved in acetic acid and water, stirred at 80 ° C for 1 hour, and benzyl 2-acetoamide-2-deoxy-3--0- (methyl 2,3,4-tri-0-acetyl -j3- D-Darcopyranosyl peroneto)-) 3-D-galactopyranoside was obtained. Subsequently, acetic anhydride is added dropwise at 0 ° C in dehydrated pyridine and reacted at room temperature for 3 hours to obtain benzyl 2-acetoamide-4,6-di-0-acetyl-2-deoxy-3-0- (Methyl 2,3,4-tri-0-acetyl-) 3-D-Darcopyranosyl-N-)-) 3-D-galactopyranoside (3) was obtained. (3) was subjected to deprotection of the benzyl group by catalytic hydrogen reduction using 10% palladium hydroxide carbon as a catalyst in dehydrated methanol in a hydrogen atmosphere, followed by the action of acetic anhydride in dehydrated pyridine to give 2- Acetamide-4,6-di-0-acetyl-2-deoxy-3-0- (methyl 2,3,4-tri-0-acetyl-j3-D-darcopyranosyl )-D-galactopyranosyl acetate (4) was obtained. Subsequently, TMSOTi is allowed to act on (4) at room temperature under an argon atmosphere in dehydrated dichloromethane for 2 hours, and then triethylamine is caused to act at 0 ° C, whereby 2-methyl- [4,6-di- -0-Acetyl-1, 2-dideoxy-3-0- (methyl 2,3,4-tri- 0-acetyl -j3 -D -Darcopyranosyl ester)-CK-D-Galactopyrano]- [2, l, d] -2-oxazoline (5) was obtained. (5) was treated with sodium methoxide in dehydrated methanol to deprotect all acetyl groups, and the reaction solution was concentrated under reduced pressure and dried to dryness. This was taken up in a carbonate buffer (50 mM, pH 10.6). The methyl ester is deprotected by stirring for 1 hour, and the desired substrate monomer, 2-methyl-Π, 2-dideoxy-3-0- (sodidium i8-D-darcopyranosyl peronate) -α-D -Galactovirano]-[2,1, d]-2-oxazoline (6) was obtained. The chondrosin oxazoline derivative thus obtained is suitably used as a substrate monomer of a hyaluronan degrading enzyme as a polymerization catalyst. Substrate model during enzyme reaction The nomer concentration is 0.1% by weight or more, preferably 1% by weight or more, from a practical viewpoint.

 The reaction pH is preferably 5 to 10 and preferably 6.5 to 9.5 in consideration of the reactivity of the enzyme and the stability of the substrate monomer. The reaction temperature is from 5 ° C to 60 ° C, preferably from 20 ° C to 40 ° C.

 As the hyaluronic acid-degrading enzyme to be used, mammalian-derived hyaluronidase is preferable, and specifically, a testis-derived or sheep that is classified as end-i3-acetylhexosamidase (EC 3.2.1.35). Testicular hyaluronidase and the like are suitable, and the enzyme can be used in the form of an immobilized enzyme in which the enzyme is immobilized on a suitable carrier. Either batch reaction or continuous reaction is employed.

 The reaction is an aqueous solvent or an aqueous solvent such as methanol, ethanol, alcohols such as n-propanol, polyols such as glycerin and polyethylene glycol, dimethyl sulfoxide, dimethylformamide, ethyl acetate, dioxane, and adversely affect the reaction. It proceeds even under the condition that various inorganic salts or PH buffer etc. which do not affect the environment are appropriately added.

 The substrate monomer is not limited to the 2-methyl-chondrosinoxazoline derivative (6), but is a compound of the general formula (I) having a chondrosinoxazoline basic structure in which a polymerization reaction by hyaluronidase proceeds. Mouth synoxazoline derivatives are included in the present invention.

 For example, an alkyl group such as ethyl, propyl, and butyl, a phenyl group, a halogen-substituted alkyl group, a halogen-substituted phenyl group, and the like can be used in place of hydrogen or methyl for R in the general formula (I).

 The substrate monomer is not particularly limited as long as it is in the form of a free acid, a metal salt such as sodium or potassium, an ammonium salt, a triethylamine salt, or the like. The resulting chondroitin or chondroitin derivative depends on the form of the glucuronic acid salt of the substrate monomer, and includes free acids or forms such as metal salts such as sodium and potassium, ammonium salts, and triethylamine salts.

When a patch reaction is started under the above conditions, the reaction cannot be completely defined by the conditions, but the reaction is completed within several hours to several days. After completion of the reaction, centrifuge the reaction solution, ultrafiltrate, High purity chondroitin or chondroitin derivatives can be isolated and purified by combining known purification means such as dense filtration, various adsorption columns, solvent precipitation, and chromatographic separation. The chondroitin or chondroitin derivative thus obtained can be widely used as cosmetics, pharmaceuticals, medical materials and the like.

 Example

 Hereinafter, the details of the present invention will be described with reference to examples, but the present invention is not limited to the following examples.

 Example 1

 Benzyl 2-azide-4.6-0-benzylidene-2-deoxy-3-0- (methyl 2,3,4-tri-0-acetyl-β-D-darco pyranosyl ester)-<3- Synthesis of D-galact viranoside (1)

 In a light-shielded two-necked flask, benzyl 2-azido-4,6-0-benzylidene-2-deoxy-β-D-galactopyranoside (190 mg, 0.488 mmol) and methyl (2,3,4-tri-0- Dissolve acetyl-D-dalcoviranosyl trichloroacetimidate) lone (280 mg, 0.585 mmol) in dehydrated dichloromethane (5.00 ml), add molecular sieves 4A (MS4A; 1.00 g), and add argon gas. The mixture was stirred at -20 ° C for 30 minutes. A solution of trimethylsilyl trifluoromethanesulfonate (TMSOTf; 0.106 ml, 0.585 ol) diluted with dehydrated dichloromethane (1.00 ml) was added dropwise at −20 ° C., and the mixture was 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 with a saturated aqueous solution of sodium hydrogencarbonate and brine, and the organic layer was dried over magnesium sulfate. I let it. After drying, magnesium sulfate was removed by cerite filtration, and the filtrate was distilled off under reduced pressure. The resulting residue was purified by flash silica gel chromatography (elution solvent: hexane / ethyl acetate = 3: 1: 1) to give white crystalline benzyl 2-azido-4, 6-0-benzylide. 2-Doxy-3-0- (methyl 2,3,4-tri-0-acetyl-; 3-D-Dalcopyranosyl ester) -D-galactopyranoside (270 mg, 0.386 mol, yield) Rate 79%). The analysis data is as follows.

[a] -10. (c = l.0, CHCh)

_{Ή NMR (400MHz, CDC1 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 ,., 2. = 7.52Hz,} J 2 .. 3. = 8.52Hz), 4.99 (1H, d, PhCH 2, J = ll.60Hz), 4.92 (1H, d, Η-Γ, J ,. ₂ · = 7.52Hz), 4.69 (1H, d, PCH2 _> J = ll.60Hz), 4.37-4.29 (3H, m, H-6a, Hl, H-4), 4.09-4.02 (2H, m, H-6b, H - . 5 '), 3.88 (1H, dd, H-2, J ,. 2 = 8.04Hz, J 2 3 -10.5Hz), 3.72 (3H, s, COOCHs), 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 ₃ 0! ₄ )

 Actual 700 ・ 2357 / z (+ 0.5ppm)

 Example 2

 Benzyl 2-acetamide-4,6-0-benzylidene-2-deoxy-3-0- (methyl 2,3.4-tri-0-acetyl-6-D-darcopyranosyl) Synthesis of i8-D-galactoviranoside (2)

 In an eggplant flask, benzyl 2-azido-4,6-0-benzylidene-2-deoxy-3--0- (methyl 2,3,4-tri-0-acetyl-jS-D-darcopyranosylperone (1) -j3-D-Galactopyranoside (250 mg, 0.358 mmol) was dissolved in thioacetic acid (2.50 ml) and reacted at room temperature under 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 flash silica gel chromatography (elution solvent: toluene / ethyl acetate = 1: 0-1: 3) to give a white crystalline benzyl 2-acetate. Toamide-4,6-0-benzylidene-2-deoxy-3-0- (methyl 2,3,4-tri-0-acetyl-j3-D-darcopyranosyl peroneto) -j3-D-galacttopyranoside (220 mg, 0.307 mmol, 86% yield). The analysis is as follows.

[a] _D -9.0 ° (c = l.0, CHC)

_{^{: H NMR (400MHz, CDC1 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''Hl), 5.01 (1H, t, H-2') , _2- =) _2- , ₃ = 8.29Hz), 4.96-4, 91 (2H, m, PhCH ₂₎ Η-Γ), 4.79 (1H, dd, H-3, J = 11.5Hz, J ₃ , -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} = l.51Hz 'J ea 6 b -ll.80Hz), 4. 01 (1H, d, H-5 ', J ,., = 9.54 Hz), 3.69 (3H, s, C00CH ₃ ), 3.52-3.46 (2H, m, H-2, H-5), 2.02-2.01 (9H, m, CH ₃ C0), 1.92 (3H, s, CH ₃ C0 bandits)

High resolution FAB MS: Calculated value [M + H] ⁺ = 716.2554 m / z (C ₃₅ H, ₂ N0 _{J 5} )

 Found 716: 2554 m / z (+ 0.Oppm)

 Example 3

 Benzyl 2-acetoamide-4,6-di-0-acetyl-2-deoxy-3-0- (methyl 2,3,4-tri-0-acetyl- -D-darco pyranosyl ester)-/ Synthesis of 3 -D-galactopyranoside (3)

 In an eggplant flask, benzyl 2-acetoamide-4,6-0-benzylidene-2-deoxy-3-0- (methyl 2,3,4-tri-0-acetyl-j3-D-darcopyranosyl G) -D-Galactopyranoside (960 mg, 1.34 mmol) was dissolved in acetic acid (12.0 ml) and water (3.00 ml), and the mixture was stirred with 801: for 1 hour. After the completion of the reaction, the reaction solution was distilled off under reduced pressure, and the residue was purified by flash chromatography on silica gel (elution solvent: octaform / ethanol = 10: 1) to give benzyl '2-acetoamide. -2-Doxy-3-0- (methyl 2,3,4-tri -0-acetyl -j3 -D-darcopyranosyl peronet)-j3 -D-galactopyranoside (760 mg, 1.1 mmol , 90% yield). Next, the obtained compound (760 mg, 1.21 mmol) is dissolved in pyridine (10.0 ml), acetic anhydride (0.467 ml, .84 ramol) is added dropwise at 0 ° C, and the mixture is dried at room temperature for 3 hours at room temperature. Reacted. After completion of the reaction, the reaction solution was distilled off under reduced pressure, the residue was diluted with chloroform, and washed sequentially with 4% (w / v) aqueous potassium hydrogen sulfate, saturated aqueous sodium hydrogen carbonate, and saturated saline. The organic layer was dried with magnesium sulfate. After drying, magnesium sulfate was removed by celite filtration, and the filtrate was distilled off under reduced pressure. The resulting residue was purified by flash silica gel chromatography (elution solvent: hexane / ethyl acetate = 1: 2-1: 4) to give white crystalline benzyl 2-acetoamide-4,6-di-. 0-Acetyl-2-deoxy-3-0- (methyl 2,3,4-tri-0-acetyl-0-D-darcopyranosyl peroneto)-; 3-D-galactopyranoside (700mg , 0.984IHDIO1, yield 8).

 The analysis data is as follows.

[} _D- 20. (c = l.0, CHC1 ₃ )

Melting point: 173-174 ° C _{Ή NM (400MHz, CDC1 3,} TMS): δ (ppm); 7.37-7.29 (5H, m, aromatic), 5. 64 (. 1H, d, already _{J NH 2 = 7.03Hz), 5.39} (1H, d , H-4, J _3. „= 3.01Hz), 5.22-5. 1

4 (2H, m, H- 4 ', H-3'), 5.02 (1H, d, Hl, J ,. 2 = 8.03Hz), 4.97 (1H, t, H-2 ',. 2 · = J ₂ .. ₃ · = 8.53Hz), 4.89 (1H, d, PhCH ₂₎ J = ll.80Hz), 4.70-4.6

5 (2H, m, Η-Γ, H-3), 4.58 (1H, d, PhCH ₂₎ J = ll.80Hz), 4.17 (1H, dd, H-6a, J s.ea = 6.28Hz _{, J 6 a 6 b -l 1.29Hz} ), 4.08 (1H, dd, H -.. 6a, J 5, 6 a = 6.28Hz, J 6 o 6 b -ll.29Hz) '3.98 (1H, d, _{H-5 ', J 4 ..} s. = 9.5 other), 3.87 (1H, t, H-5, J 5. = J 5. = 6.28Hz), 3.75 (3H, s, C0OCH 3), 3.46 ( 1H, ddd, H-2, J lt 2 = 8. 03Hz, J NH, 2 = 7.03Hz, J 2. 3 = 9.28Hz), 2.09-2.00 (15H, m, CH 3 CO), 1.89 (3H, s, CH ₃ CONH)

High resolution FAB MS: calcd [M + H] + = 712.2453 m / z (C 32 H 42 N0 17)

 Found 712.2453 m / z (+ 0.Oppm)

 Example 4

 2-acetoamide-4,6-di-0-acetyl-2-deoxy-3-0- (methyl 2,3.4-tri-0-acetyl-<3 _D_darcopyranosyl peronet)-D -Synthesis of galactobiranosyl acetate (4)

In an eggplant flask, benzyl 2-acetamide-4,6-di-0-acetyl-2-deoxy-3-0- (methyl 2,3,4-tri-0-acetyl-j3-D-darcopyranosine Dissolve ゥ / ゥ -D-galactopyranoside (700 nig, 0.984 mmol) in methanol (20.0 ml), add 10% palladium hydroxide carbon (200 mg), and hydrogen atmosphere at room temperature for 3 hours Stirred. After completion of the reaction, the reaction solution was filtered through celite to remove 10% palladium hydroxide and carbon, and the filtrate was distilled off under reduced pressure. The obtained residue was dried under reduced pressure overnight. The obtained white amorphous substance was dissolved in pyridine (10.0 ml), and acetic anhydride (0.315 ml, 3.23 mmol) was added dropwise at 0 ° C and dried. The reaction was performed for 3 hours at room temperature under an atmosphere. After completion of the reaction, the reaction solution was distilled off under reduced pressure, and the residue was diluted with chloroform.Then, washed sequentially with 4% (w / v) aqueous sodium hydrogen sulfate, saturated aqueous sodium hydrogen carbonate, and saturated saline. The organic layer was dried with magnesium sulfate. After drying, magnesium sulfate was removed by celite filtration, and the filtrate was distilled off under reduced pressure. The resulting residue was purified by flash silica gel chromatography (elution solvent: ethyl acetate) to give a colorless, amorphous 2-acetate. Mid-4,6-di-0-acetyl-2-deoxy-3-0- (methyl 2,3,4-tri-0-acetyl_3-D-darcopyranosyl ゥ mouth) -D -Galactopyranosyl acetate (680 mg, 1.03 mmol, a / j3 = 1/1) was obtained quantitatively.

 The analysis data is as follows.

JH NMR (400 band z, CDC, TMS): <5 (ppm); 6.34-6.32 (2H, m, H-1, Ma), 5.89 () H, d, H-1 β, J ,. ₂ -8.53Hz), 5. 78 (] H, d, NH ^, J 2. NH = 8. 03Hz), 5. 49 (1H, d, H-4/3, J 3. 4 = 3.01Hz) , 5.29-5.13 (7H, m, H-4a, H-3, β, H-4'aβ, H-2 '), 5.02 (1H, dd, H-2'β, 1 , ₂ · = 8.03 Hz, J ₂ .. ₃ = 8.53 Hz), 4.86 (1H, d, Η-Γ a,. _2. = 8. 03 Hz), 4.77 (1H, d, Η- _{Γ β, 1. 2 · =} 8. 03 Hz), 4. 55 (1H, ddd, H-2, 1 ,, 2 = 3. 52Hz, J 2. NH = 7.03Hz, J 2. 3 = 8. 53Hz), 4. 38 (1H, dd, H -.. 3/3, J 2 3 = 10.79Hz, J 3 = 3.01Hz), 4. 24-3.97 (11H, m, H-3, H-6 a) 3, H-5 ' β, -2 β, H-5 K / 3), 3.76 (6H, s, C00CH 3), 2. 19-2. 02

(36H, m, CH3CO), 1.93-1.90 (6H, d, CH 3 C0NH)

High resolution FAB MS: calcd ^{[M + H] + = 664} · 2089 m / z (C 27 H 38 N0 18)

 Found 664.2092 m / z (+ 0.4ppm)

 Example 5

 2-methyl- [4.6-di-0-acetyl-1,2-dideoxy-3--0- (methyl 2,3,4-tri-0-acetyl-0-D-darcopyranosyl perone G) -a-D-Galactovirano 1- [2. Synthesis of 1-dl-2-oxazoline (5)

In a two-necked flask, under an argon atmosphere, 2-acetamide-4,6-di-0-acetyl-2-deoxy-3-0- (methyl 2,3,4-tri -0-acetyl- β-D-Dalco pyranosyl acetate) -D-galactopyranosyl acetate (200 mg, 0.301 ol) was dissolved in dehydrated dichloromethane, TMSOTf (0.110 ml, 0.603 ol) was added dropwise at 0 ° C, and the mixture was added at room temperature. Stirred for 12 hours. After completion of the reaction, triethylamine (0.200 ml) was added at 0 ° C, and the mixture was stirred at room temperature for 30 minutes. The reaction solution was distilled off under reduced pressure, and the obtained residue was purified by flash silica gel chromatography (elution solvent: hexane / ethyl acetate =]: 2-2: 5) to give colorless amorphous 2-methyl- [4,6-di-0-acetyl-1,2-dideoxy-3-0- (methyl 2,3,4-tri-0-acetyl-j3-D-darcopyranosyl peronet) -a -D-galactovirano]-[2, trid] -2-oxazoline (161 mg, 0.268 ol, yield 89) was obtained. The analysis data is as follows.

 [a] +34. (c = l.0, CHCla)

_{'Η NMR (400MHz, CDC1 3} , TMS):. Δ (ppm); 5.93 (1H, d, Hl, J ,. 2 = 6.53Hz), 5.40 (1H, d, H-4, J 3 4 = 3.51 Hz), 5.30-5.20 (2H, m, H-3 ', H-4'), 5.07 (1H, d, Η-Γ, Ja .. ₂ · -8.03Hz), 4.99 (1H, t, H- 2,, J ₁ .. _2. = J ₂ .. _3. = 8.03Hz), 4.18-4.12 (3H, m, H-6, H-5), 4.08 (1H, d, H -5 ', J ₄ .. _5. = 9.53 Hz),

3.93 (1H, dd, H- 3, J 2. 3 = 6.53Hz, J 3. = 3.51Hz), 3.83 (1H, t, H-2, J ,. 2 = J 2, = 6.53Hz), 3.76 (3H, s, C00CH ₃ ), 2.08-2.02 (18H, m, CH ₃ C0, CH ₃ C of oxa zol ine)

High resolution FAB MS:. Calcd ^{[M + H] + = 604} 1877 m / z (C 25 H 34 N0 16)

 Found 604, 1879 m / z (+ 0.3ppm)

 Example 6

 2-Methyl-2-doxy-3-0- (sodium β-D-darcopyranosylperone-)--D-galactovirano]-[2.1-dl-2-oxazoline (6) Synthesis of

 In a two-necked flask, under an argon atmosphere, 2-methyl- [4,6-di-0-acetyl-1,2-dideoxy-3--0- (methyl-13,4-tri-0-acetyl-j8 -D-Darcopyranosyl ester)-a-D-galactobirano]-[2, trid]-2-oxazoline (240mg, 0.398 marl ol) dissolved in methanol (1.00ml) and sodium methoxide Of methanol (0.500 ml, 0.0795 mmol) was added and stirred at room temperature for 30 minutes. After completion of the reaction, the reaction solution was distilled off under reduced pressure and freeze-dried. This was dissolved in a carbonate buffer (50 mM, H 10.6, 3.98 ml), stirred at room temperature for 1 hour, and lyophilized to give 2-methyl- [1,2-dideoxy-3-0 -(Sodium / 3-D-Darcopyranosylone) -Q! -D-Galactopyrano]-[2, trid] -2-oxazoline (160 mg, purity 82%) was obtained. 'The analysis data is as follows.

l NMR (400 band z, D ₂₀ , Acetone): δ (ppm); 5.92 (1H, d, Hl, 1: _{2, 2} = 703Hz),

4.47 (1H, d, Η- Γ, J 2. = 8.03Hz), 4.00 (1H, d, H-4, J 3. 4 = 3.52Hz), 3. 80-3.73 (2H, m, H-2 , H-5), 3.64-3.52 (4H, m, H-3, H-6, H-5 '), 3.34-3.32 (2H, 1, H-4', H-3 '), 3 . 21 (1H, dd, H -2 ', J. 2. = 8. 03Hz, J 2 .. 3. = 9. 03Hz), 1.84 (3H, s, CH 3 C of oxazoline) High resolution FAB MS: Calculated value [M + H] ⁺ = 402.1012 m / z (C, ^ UNaCh!)

 Obtained value 402.1018 m / z (+ 1.4ρρπι)

 Example 7

 Synthesis of chondroitin by enzyme-catalyzed polymerization

Substrate monomer, 2-methyl- [], 2-dideoxy-3-0- (sodium-D-glucopyranosylone) -a-D-galactovirano]-[2, trid] -2 -oxazoline (5.OOmg, 12.5 mol) was dissolved in phosphate buffer (50 mM, H7.5, 1251), and hyaluronidase from sheep testis (ICN Biochemicals, Lot No. 6830B, 0.500 mg of 2160 units / mg, hereinafter referred to as H-0TH) was added, and the reaction was carried out at 30 for 24 hours. After completion of the reaction, the reaction solution was heated in a hot water bath at 90 ° C for 3 minutes to inactivate the enzyme, then an excess amount of THF was added, and the resulting precipitate was collected by centrifugation. . The obtained precipitate was dissolved again in pure water to obtain an aqueous solution of a polymerization product. The yield and molecular weight of the polymerization product relative to the substrate monomer were determined by the GPC measurement of the obtained aqueous solution, yield = 50, _Mn = 2100, and _Mw = 2500. In the GPC measurement, the molecular weight was calculated using a calibration curve created using sodium hyaluronate ( _Mn = 800, 2000, 4000) as the molecular weight index. Of chondroitin sodium salt (Seikagaku Corporation, Μπ = 4000, Μw = 5000) prepared by desulfating chondroitin sulfate . The GPC measurement conditions are shown below.

GPC conditions

 Detector Differential detector

 Column Shodex Ohpak SB-803HQ

 Column temperature

 Mobile phase 0.1M sodium nitrate

 0.5ml / min

From the above aqueous solution, the produced chondroitin was purified using Sephadex G-10 size exclusion chromatography. FIG. 2 shows the ¹ H NMR spectrum of the obtained chondroitin, and FIGS. 3 and 4 (enlarged views of the sugar skeleton in FIG. 3) show the ¹³ C R spectrum. These spectra were consistent with those of naturally occurring chondroitin. The peak assignments of the Awakening R vector are as follows.

_{! H NMR (400MHz, D 2} 0, Acetone): δ (ppm); 4.31 (2Η, bs, Hl, Η-Γ), 3. 92 (1H, bs, H-4), 3.80 (1H, bt, H-2), 3.63-3.50 (6H, m, H-3, H-4, H-5 ', H-6, H-5), 3.41 (1H, bt, H-3'), 3.17 (1H, bt, H-2 '), 1.82 (1H, s, CH 3 C0)

¹³ C NMR (100 MHz, D ₂₀ , Acetone): δ (ppm); 174.55 (C-6 ′), 173.91 (CH ₃ CO), 103.90 (C-Γ), 100.43 (C-), 79.87 (C -3), 79.26 (C-4 '), 75.82 (C-5'), 74.53 (C-5 '), 73.26 (C-3'), 71.98 (C-2 '), 67.30 (C-4 ), 60.63 (C-6), 50.56 (C-2) '22.04 (CH ₃ C0)

 Example 8

 Synthesis of chondroitin using various enzymes

 Sheep testis-derived hyaluronidase (ICN Biochemicals, Lot No. 9303B, 560 units / mg, hereinafter referred to as 0TH) as a catalytic enzyme in the polymerization reaction, bovine testis-derived hyaluronidase (SIGMA, Lot No. .30K7049, 330units / mg, hereinafter referred to as BTH), H-0TH, or bovine testis-derived hyaluronidase (SIGMA, Lot No.38H7026, IOIOUnits / mg, hereinafter referred to as H-BTH) Table 1 shows the results of the same reaction and analysis as in Example 7 except that the reaction time was changed to 23 hours or 40 hours. Chondroitin synthesis was possible using any of the hyaluronidases.

 table 1

 Yield and molecular weight of formed chondroitin Reaction conditions Result Enzyme time Chondroitin M n M w

 (h) Yield (%)

0TH 2 3 3 5 2 5 0 0 3 2 4 0

BTH 4 0 1 0 2 8 2 0 3 5 6 0

H-0TH 2 3 5 0 2 0 5 0 2 4 6 0

H-BTH 4 0 2 9 2 5 7 0 3 3 8 0 Example 9

 Effect of Reaction ΐH on Chondroitin Synthesis

 In the polymerization reaction, three types of hyaluronidases (OTH, BTH or H-0TH) were used as the catalytic enzymes, and the reaction time was as shown in Table 2 and the phosphate buffer of pH shown in Table 2 was used. Table 2 shows the results of the same reaction and analysis as in Example 7 except that the reaction pH was changed. Table 3, Table 4, and FIG. 5 show the change over time of the residual ratio of the substrate monomer in the reaction mixture at pH 7.0 and 8.0 using 0TH or BTH as the hyaluronidase. The residual ratio of the substrate monomer was calculated from the area ratio of peaks appearing at a retention time of 7.3 to 7.4 minutes under the following HPLC conditions by collecting 30 l of the reaction solution at regular intervals.

HPLC conditions

 Detection UV 220nm

 Column Shodex Asahipak NH2P-50 4E

 Column temperature 30

 Mobile phase Phosphate buffer (10 mM, pH 7.0) 30¾ + acetonitrile 70%

 0.5ml / min

From the results shown in Tables 3, 4 and 5, it was clarified that the system in which 0TH or ΒΤΗ was added significantly accelerated the monomer consumption as compared to the system in which 0TH or ΒΤΗ was not added, and chondrosinoxazolyl was added. The derivatives were recognized by both enzymes, indicating that the oxazoline ring was undergoing ring opening.

Product Chondroitin Yield and Molecular Weight Reaction Conditions & J

Table Τ-Ι Time Chondroitin M n M w

 (h) Yield (%)

0TH 6.0 .0 丄 0 1 4 6 0 1 6 0 0

 One o

 7.01 U2 2 9 0 2 9 2 0

71 .Ο 2 3 3 5 2 5 0 n 3 2 4 o Ο ο. Λ U 4 7 2 8 2 5 6 n 3 4 5 o ο ο r-

ο 5 6 1 2 3 0 0 ri 3 9 o o

BTH 6.03 0 1 8 7 0 2 3 6 0

 7.01 2 8 2 2 8 0 3 3 7 0

7.5 4 0 1 0 2 8 2 0 3 5 6 0

8.04 7 1 1 2 8 7 0 3 7 8 0

8.5 5 6 7 2 4 6 0 2 9 9 0

H-0TH 7.0.0 9 4 7 1 9 1 0 2 2 3 0

 7.5 2 3 5 0 2 0 5 0 2 4 6 0

8.03 3 4 9 2 1 9 0 2 6 9 0

8.5 5 4 8 3 2 2 3 8 0 3 1 3 0

Table 3

 Residual rate of substrate monomer at pH 7.0 Residual rate of substrate monomer (%)

(h) No enzyme added 0TH BTH

0 1 0 0 1 0 0 1 0 0

3 42 2 6 3 7

7 1 3 2 8

1 2 9 1 Table 4

 Residual rate of substrate monomer at pH 8.0 Residual rate of substrate monomer (%) Reaction time

 (h) No enzyme added 0TH .. BTH

0 1 0 0 1 0 0 1 0 0

3 8 5 7 3 7 9

6 7 5 5 7 6 2

9.5 6 4 44 5 1

1 6.5 5 2 9 3 7

1 8 4 7

2 8 3 5 1 0 2 0

4 7 1 6 4 7 Example 10

 Effect of enzyme concentration on chondroitin synthesis

 Table 5 shows the results of the same reaction and analysis as in Example 7, except that the amount of the catalytic enzyme H-0TH and the reaction time were changed to the conditions described in Table 5 in the polymerization reaction.

 Table 5

 The yield and molecular weight of the produced chondroitin

Example 1 1

 Effect of reaction temperature on chondroitin synthesis

Table 6 shows the results of the same reaction and analysis as in Example 7, except that the reaction temperature and the reaction time in the polymerization reaction were changed to the conditions described in Table 6. Table 6

 Product chondroitin yield and molecule:

実施例 1 2

Example 1 2

 Effect of substrate monomer concentration on chondroitin synthesis

 In the polymerization reaction, the substrate monomer, 2-methyl- [1,2-dideoxy-3-0- (sodium β-D-darcopyranosylone)]-a;-D-galactopyrano]-[ Table 7 shows the results of the same reaction and analysis as in Example 7, except that the amount of 2,1, d] -2 -oxazoline added and the reaction time were changed to the conditions shown in Table 7.

 Table 7

 Yield and molecular weight of formed chondroitin Reaction conditions Result Time Chondroitin M n M w Amount added (mg) (h) Yield (%)

1 0 2 4 4 6 2 0 4 0 2 4 1 0

5 2 3 5 0 2 0 5 0 2 4 6 0

2.5 2 4 4 0 2 0 8 0 2 5 2 0 Example 13

 Benzyl 4, 6-di-0-acetyl-2-azido-2-deoxy-3-0- (methyl 2.3.4-tri-0-acetyl-0-D-Darco pyranosylperonate)-0-D- Synthesis of galactopyranoside (7)

 Benzyl 2-azido-4,6-0-benzylidene-2-dex-3-0- (methyl 2,3,4-tri-0-acetyl-j3-D-Darcopyranosi obtained in Example 1 Dissolve D-galactopyranoside (1) (555 mg, 0.793 t ol) in 80 acetic acid aqueous solution (15 ml) and react with 80 ^ for 1 hour to obtain 4, 6-0-benzylidene. The group was deprotected. Subsequently, the reaction solution was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography (eluting solvent: hexane / ethyl acetate = 1: 3) to give benzyl 2-azido-2-deoxy-3-0. -(Methyl 2,3,4-tri-0-acetyl-) 3-D-Darcopyranosyl lipate-j3-D-galactopyranoside (453 mg, 0.741 bandol, 93% yield) Was. Next, the obtained compound (453 mg, 0.741 mmol) was dissolved in pyridine (10 ml), and acetic anhydride (0.431 ml, 4.44 bandol) was added dropwise at 0 ° C under a dry atmosphere, and the reaction was carried out at room temperature for 4 hours. I let it. After completion of the reaction, the reaction solution was distilled off under reduced pressure, the residue was diluted with chloroform, washed successively with 4% (w / v) aqueous sodium hydrogen sulfate, saturated aqueous sodium hydrogen carbonate, and saturated saline, and then washed with an organic solvent. The layer was dried with 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 (elution solvent: hexane / ethyl acetate = 1: 1) to give benzyl 4,6-di-0-acetyl-2-azide-2- white crystalline benzyl. Deoxy-3-0- (methyl 2,3,4-tri-0-acetyl-i3-D-darcopyranosyl oxalate)-/ 3-D-galactopyranoside (7) (478 mg, 0.687 mmol, yield 93%). The analysis data is as follows.

[a] _D + 0.12 ° (c = l.0, CHC1 ₃ )

m 144-145 ° C

Marauder R (400MHz, CDCh, TMS) δ (ppm); 7.39-7.31 (5H, m, aromatic), 5.33 (1H, d, H— 4, J _3. , = 3.01 Hz), 5.23-5.21 (2H, m, H—4,, H-3 '), 4.99-4.93

(2H, m, H-2 ', PhCH ₂ ), 4.80 (1H, d, Η-Γ, J! .. _2. = 8.03 Hz), 4.70 (1H, d, PhCH ₂ , J = 12.05 Hz), 4.29 (1H, d, Hl, J ,, ₂ = 8.03 Hz), 4.17 (1H, dd, H-6a, J = 5.52 Hz, J _{6 a} .e _b = ll.54 Hz), 4.08 (1H , dd, H-6a, J = 7.02 Hz, J _{6 a 6b = ll.54 Hz), 3.98 (} 1H, d, H-5 ', J 4 .. 5. = 10.04 Hz), 3.75 (3H, s, COOCH 3), 3.73-3.64 (2H, m, H- 5, H-2), 3.53 (1H, dd, H-3, J 2. 3 = 10.54 Hz, J 3. 4 = 3. 01 Hz), 2.11-2.01 (15H, m, CH 3 CO)

High resolution FAB MS: calcd ^{[M + H] + = 696.2252} m / z (C 3 .H 38 N 3 0 ₆₎

 Found 696.2249 m / z (-0.4 ppm)

 Example 14

 4. 6-Di-0-Acetyl-2-deoxy-3-0- (methyl 2,3,4-tri-0-Acetyl- -D-Dalcopyranosyl ゥ mouth)-2-Propanamide- Synthesis of D-galactopyranosyl acetate (8)

Benzyl 4, 6-di-0-acetyl-2 diazide-2-deoxy-3-0-(methyl 2,3,4-tri-0-acetyl-j8-D -Darco pyrano silperonate)-β-D -Dissolve galactopyranoside (7) (100 mg, 0.144 mmol) in methanol (10 ml), add 10% palladium hydroxide carbon (50 mg), and react at room temperature under a hydrogen atmosphere for 4 hours. The azide group was converted to an amino group, and at the same time, the benzyl group was deprotected. After completion of the reaction, 10% palladium hydroxide carbon was removed by filtering the reaction solution through celite, and the filtrate was distilled off under reduced pressure. The obtained residue was dissolved in methanol (10 ml), and triethylamine (1 ml) and then propionic anhydride (0.180 ml, 1.44 mmol) were added under a dry atmosphere at 01 :, and the mixture was reacted at room temperature for 2 hours. . After completion of the reaction, pyridine (1 ml) was added, the reaction solution was distilled off under reduced pressure, and the obtained residue was dried overnight under reduced pressure. After drying, the obtained white amorphous substance was dissolved in pyridine (10 ml), acetic anhydride (0.084 ml, 0.863 mmol) was added dropwise at 0 ° C under a dry atmosphere, and the mixture was reacted at room temperature for 3 hours. After completion of the reaction, the reaction solution was distilled off under reduced pressure, and the residue was diluted with chloroform.Then, washed sequentially with 4% (w / V) aqueous potassium hydrogen sulfate, saturated aqueous sodium hydrogen carbonate, and saturated saline, and then organically. The layer was dried with magnesium sulfate. After drying, magnesium sulfate was removed by cerite filtration, and the filtrate was distilled off under reduced pressure. The obtained residue was purified by silica gel chromatography (elution solvent: hexane / ethyl acetate = 1: 4) to give a colorless amorphous 4,6-di-0-acetyl-2-deoxy-3- 0- (Methyl 2,3,4-tri-0-acetyl- -D-darcopyranosyl peronet) -2-propanamide-D-galactobyranosyl acetate (8) (37.Omg, 0.0546 mmol, yield 3, / β = 5/1). Analysis The evening is as follows.

 'Η NMR (400 MHz, CDC, TMS) δ (ppm); 6.34 (1H, d, H-la, 51 Hz),

6. 16 (1H, d, ΝΗ , J ,. 2 -8.03Hz), 5. 90 (1H, d, Hl β, J ,. 2 = 8. 52 Hz), 5. 6 2 (1H, d, _{.. NHiS, J 2 NH =} 8. 52 Hz), 5. 27 (1H, s, H-4 a), 5. 25-5 13 (3H, m, H - 3 ', H-4', H -2 'a), 4.85 (1H, d, Hl' a,-. ₂ = 8.04 Hz), 4.57 (1 H, ddd, H-2, J ,, ^^. 51 Hz, J ₂ . _{NH = 8.03 Hz, J 2.} 3 = 8. 53 Hz), 4. 23-4. 19 (2H, m, H-3, H-6aa), 4. 11-4.07 (2H, m, H-5 ', H-6b), 4.00 (1H, dd, H -5, J 5, 6 a = 6. 53 Hz, J 5. 6 b = ll. 54 Hz), 3.76 (3H, s, C00CH 3), 2.19-2.01 (2 OH, m, CH ₃ C0, CH ₃ CH ₂ C0NH), 1.09 (3H, t, CH ₃ CH ₂ C0NH, J-8.03 Hz)

High resolution FAB MS: Calculated value [M + H] + = 678.2245 m / z (C ₂₈ H _4, N0 ₁₈ )

 Found 678.2248 m / z (+0.4 ppm)

 Example 15

 2-Ethyl- [4,6-di-0-acetyl-1, 2-di-deoxy-3-0- (methyl 2,3.4-tri-0-acetyl-3 -D-darcopyranosyl peronet Synthesis of) -hi-D-galactopyrano [2.1 d] -2 -year-old oxazoline (11)

 4,6-Di-0-acetyl-2-deoxy-3-0- (methyl 2,3,4-tri-0-acetyl-3-D-darcopyranosyl ゥ mouth) -D-galactobyranosyl acetate (8) (100 mg, 0.148 ramol) is dissolved in dehydrated dichloromethane, and trimethylsilyl trifluoromethanesulfonate (TMSOTf; 0.0404 ml, 0.degree. (221) was added dropwise and the mixture was stirred at room temperature for 18 hours. After completion of the reaction, triethylamine (0.1 ml) was added at 0T, and the mixture was stirred at room temperature for 30 minutes, and the reaction solution was distilled off under reduced pressure. The obtained residue was purified by silica gel chromatography (elution solvent: hexane / ethyl acetate = 1: 2-2: 5, triethylamine 0.5%) to give a colorless amorphous 2-ethyl- [4, 6-di-0-acetyl-1,2-dioxy-3-0- (methyl 2,3,4-tri-0-acetyl-D-darcopyranosyl peronet)-a-D-galact [Topirano] [2, tri d] -2-oxazoline (11) (75.0 mg, 0.121 mmol, yield 8) was obtained. The analysis data is as follows.

_{[a) D + 20 ° (} c = 0.75, CHC1 3)

Marauder R (400MH2, CDC, TMS) δ (ppm); 5.93 (1H, d, Hl, J: ₂ = 7.02 Hz), 5. 38 (1H, d, H - .. 4, J 3, = 3.51 Hz, J 4 5 = 2.00 Hz), 5.31-5.20 (2H, m, H-3 ', H- 4'), 5.09 (1H, d, Η-Γ, J,-, ₂ = 7.52 Hz), 5.00 (1H, dd, H-2 ', J, _2. = 7.52 Hz, J' = 8.53 Hz), 4.16-4.11 (3H, m, H-6 , H-5), 4.05 (1H, d, H-5 ', J 4 -. 5 · = 9.53 Hz), 3.94 (1H, dd, H-3, J 2, 3 = 7.02 Hz, J ₃ , ₄ = 3.51 Hz), 3.83 (1 H, t, H-2, J ,. ₂ = J _2. = 7.02 Hz), 3.76 (3H, s, COOCH ₃ ), 2.32 (2H , a, CH ₃ CH

₂ of oxazoline, J = 7.53 Hz), 2.08-2.02 (15H, m, CH ₃ C0), 1.18 (3H, t, CH ₃ CH ₂ of oxazol ine, J = 7.53 Hz)

High resolution FAB MS: Calculated value [M + H] ⁺ = 618.2034 m / z (C ₂₆ H ₃₆ N0 ₁₆ )

 Found 618.2035 m / z (+0.1 ppm)

 Example 16

 2-Ethyl-[1,2-di-doxy-3-0- (sodium β-D-darcopyranosyl peronet)--D-galactopyrano] [2, tri d]-2-oxazoline ( 14 )

 2-Ethyl- [4,6-di-0-acetyl-1,2-di-deoxy-3-0- (methyl 2,3,4-tri-0-acetyl -j8-D-Darcopyranosyl ゥ(Ronetto)-a-D-force 'lactopyrano] [2, 1-d]-2-year-old xazoline (11) (75 mg, 0.121 mmol) is dissolved in methanol (1.21 ml), and then' argon Under an atmosphere, a methanol solution of sodium methoxide (2.34 mg, 0.0121 mmol) was added with O: at 0 ° C., and the mixture was stirred at 0 ° C. for 30 minutes and then at room temperature for 30 minutes to remove all 0-acetyl groups. . After completion of the reaction, the reaction solution was dried under reduced pressure. Subsequently, the compound was dissolved in a carbonate buffer (50 mM, pH 10.6, 1.21 ml), and stirred at room temperature for 1 hour to deprotect the methyl ester. After completion of the reaction, the reaction solution is freeze-dried as it is, and 2-ethyl- [1,2-di-doxy-3-0- (sodidium_D-darcopyranosyl ester) -α-D-galactovirano] [2, d] -2-oxazoline (14) (56 mg, purity 93%) was obtained. The analysis data is as follows.

NMR (400 band 匪, D ₂₀ , acetone) <5 (ppm); 5.91 (1H, d, H-1, J _{1 † 2} = 7.53 Hz), 4.47 (1H, d, Η-Γ, 1: ₂ = 8.03 Hz), 4.00 (1H, s, H-4), 3.78-3.73 (2H, m, H-2, H-5), 3.63-3.52 (4H, m, H-3, H- 6, H-5 '), 3.34-3.32 (2H, m, H- 4,, H-3'), 3.22 (1H, t, H-2 ', J. 2. = J 2 .. 3. = 8.03 Hz), 2.16 (2H, Q, CH ₃ CH ₂ of oxazoline, 1 = 1.53 Hz), 0.96 (3H, t, CH ₃ CH ₂ of oxazoline, J = 7.5

3 Hz) 1576

twenty three

High resolution FAB MS:. Calcd ^{[M + H] + = 416} 1169 m / z (!! 5 H 23 N0 Na) Found 416. 1161 m / z (-1.8 ppm )

 Example 17

 4, 6-di-0-acetyl-2-deoxy-3-0-(methyl 2.3.4-tri- 0-acetyl- -D- darcopyranosyl peronet)-1- (2-methylpropane Synthesis of amide) -D-galacttopyranosyl acetate (9)

Benzyl 4,6-di-0-acetyl-2-azide-2-dex-3-0- (methyl 2,3,4-tri-0-acetyl-;; 3-D- obtained in Example 13 Glucopyranosyl ゥ mouth)-3-D-galactopyranoside (7) (315 mg, 0.453 mmol) is dissolved in methanol (30 ml), and 10% palladium hydroxide carbon (150 mg) is added. By reacting at room temperature for 3.5 hours, the azide group was converted to an amino group, and at the same time, the benzyl group was deprotected. After completion of the reaction, 10% palladium hydroxide and carbon were removed by filtering the reaction solution through celite, and the filtrate was distilled off under reduced pressure. The obtained residue was dissolved in methanol (30 ml), and triethylamine (1 ml) and then isobutyric anhydride (0.240 ml, 1.44 mmol) were added at 0 under a dry atmosphere, followed by reaction at room temperature for 2 hours. After completion of the reaction, pyridine (3 ml) was added, and the reaction solution was dried under reduced pressure. After drying, the obtained white amorphous substance was dissolved in pyridine (30 ml), acetic anhydride (0.265 ml, 2.72 mmol) was added dropwise at 0 under a dry atmosphere, and the mixture was reacted at room temperature for 6 hours. After completion of the reaction, the reaction solution was distilled off under reduced pressure, and the residue was diluted with chloroform.Then, washed sequentially with 4% (w / v) aqueous potassium hydrogen sulfate, saturated aqueous sodium hydrogen carbonate, and saturated saline. The organic layer was dried with magnesium sulfate. After drying, magnesium sulfate was removed by filtration through celite, and the filtrate was distilled off under reduced pressure. The obtained residue was purified by silica gel chromatography (elution solvent: hexane / ethyl acetate = 1: 2- 1: 3) to give a colorless amorphous 4,6-di-0-acetyl-2-dexoxy. -3-0- (Methyl ϊ 3,4-tri-0-acetyl-; 3-D-Dalcopyranosyl ゥ mouth)-2-(2-Methylpropanamide)-D-galacttopyranosyl acetate (9) (129 mg, 0.187 t ol, yield 41%, α /] 3 = 20/3). The analysis data is as follows. Ή NMR (400 negation _{z, CDC1 3, TMS) δ} (ppm); 6.35 (1H, d, Hl, J ,. 2 = 3.51 Hz), 6. 10 (1H, d, Ma, JL 2 = 7.53 Hz) , 5.90 (1H, d, Hl β, Jj. 2 = 8.54 Hz), 5. 27 (1H, d, H-4a, J 3 .4 = 2.00 Hz), 5.26-5. 13 (3H, m, H -3 ', H-4' a, H-2 ' T / JP02 / 11576

24 a), 4.84 (1H, d, Η-Γ, J, .. 2 · = 8.53 Hz), 4.55 (1H, ddd, H-2a, J ,. 2 = 3.51 Hz, J 2, NH = 7.53 Hz _{, J 2 3 = 8.53 Hz)} , 4.25-4.20 (2H, m, H-3 a, H-Baa) 4. 11-4.07 (2H, m, H-5 ', H -. 6ba), 4.00 (1H , dd, H-5a, J 5. 6a -6.52 Hz, J = ll.04 Hz), 3.76 (3H, s, COOCHs), 2.27 (1H, m, (CH 3) 2 CHCONH), 2. 19- 1.95 (18H, m, CH ₃ CO), 1.10-1.04 (6H, m, (CH ₃ ) ₂ CHCONH)

High resolution FAB MS Calculated value [M + H] + = 692.2402 m / z (C ₂₉ H ₄₂ N0 ₁₈ )

 Actual 6922402 m / z (+0.1 pm)

 Example 18

 2-Isopropyl- [4,6-di-0-acetyl-1,2-di-doxy-3-0- (methyl 2,3.4-tri-0-acetyl- / 8-D-darcopyranosyl Net)--D-galactopyrano] d]-Synthesis of 2-oxazoline (12)

 4, 6-di-0-acetyl-2-deoxy-3-0- (methyl 2,3,4-tri-0-acetyl-/ 3-D-darcopyranosyl peroneto) -2- ( 2-Methylpropanamide) -D-galactopyranosyl acetate (9) (118 mg, 0.1 mmol) was dissolved in dehydrated dichloromethane, and TMSOTi (0.047 ml, 0.256 mmol) was added dropwise with Ot under an argon atmosphere. The mixture was stirred at room temperature for 15 hours. After completion of the reaction, triethylamine (0.1 ml) was added at 0 ° C, and the mixture was stirred at room temperature for 30 minutes. The reaction solution was distilled off under reduced pressure, and the obtained residue was purified by silica gel chromatography (elution solvent: hexane / ethyl acetate = 1: 1, triethylamine 0.5%) to give a colorless amorphous Isopropyl- [4,6-di-0-acetyl-1,2-di-deoxy-3-0- (methyl 2,3,4-tri-0-acetyl- / 3-D-darcopyranosyl perone-1 G) -α-D-galactopyrano] [2, trid] -2-oxazoline (12) (82. Omg, 0.130 mmol, yield 76) was obtained. The analysis data is as follows.

[a] + 17 ° (c = 0.82, CHC)

_{Ή NM (400MHz, CDC1 3,} TMS) <5 (ppm); 5.91 (1H, d, H- 1, J :. 2 = 7.03 Hz), 5. 36 (1H, t, H- 4, J 3, , = U. ₅ = 2.51 Hz), 5.31-5.19 (2H, m, H-3 ', H-4'), 5.0 9 (1H, d, Η-Γ, J, .. ₂ Hz), 5.00 (1H, t, H-2 ',-, ₂ = Ι ₂ , ₃ = 8.53 Hz), 4.15-4.09 (3 Η, m, H-6, H-5), 4.03 (1H, d, H-5 ','. s. = 9.54 Hz), 3. 94 (1H, dd, H-3, J 2, 3 = 7.03 Hz, J 3. 4 -2.51 Hz), 3.83 ( 1H, t, H-2, J ,. 2 = J 2. = 7.03 Hz), 3.75 (3H, s, C00CH 3), 2.58 (1H, m, (CH 3) 2 CHC0 negation), 2.08-2.02 (15H, m, CH3CO), 1.20-1.18 (6H, m, (CH ₃ ) ₂ CHC0NH)

High resolution FAB MS: Calculated value [M + H] ⁺ = 632.2190 m / z (C ₂₇ H ₃₈ N0 _{I 6} )

 Found 632.2186 m / z (-0.7 ppm)

 Example 19

 2-Isopropyl- [1,2-di-doxy-3-0- (sodium β-D-Darcopyranosylone) -ο! -D-Galactopyrano] [2, tri d] -2-oxazoli Synthesis of (15)

 2-Isopropyl- [4,6-di-0-acetyl-1,2-di-deoxy-3-0- (methyl 2,3,4-tri-0-acetyl-) 3-D-darcopyranosyl Mouth)-α-D-galactopyrano] [2, trid]-2-oxazoline (12) (82. Omg, 0.130 mmol) is dissolved in methanol (1, 29 ml) and placed under an argon atmosphere. Then, a methanol solution of sodium methoxide (2.5 mg, 0.013 mmol) was added at 0 ° C, and the mixture was stirred at 0 ° C for 1 hour and at room temperature for 1 hour. Then, sodium methoxide solution in methanol (2.5 mg, 2.5 mg, 0.013 mmol) was added. , 0.013 mmol) and reacted at room temperature for 1 hour to remove all 0-acetyl groups. After the completion of the reaction, the reaction solution was dried under reduced pressure. After drying, the compound was dissolved in carbonate buffer (50 mM, H 10.6, 1.3 ml) and stirred at room temperature for 1 hour to deprotect the methyl ester. After the completion of the reaction, the reaction solution is freeze-dried as it is, and 2-isopropyl- [1,2-di-doxy-3-0- (sodium 3-D-darcopyranosyl-peronet) -α-D-galactopyrano [2, tri d] -2- 2-year-old xazoline (15) (65 mg, purity 83%) was obtained. The analysis data is as follows.

_{^ NMR (400MHz, D 2 0} , acetone) δ (ppm);.. 5.91 (1H, d, Hl,, Z = T.03 Hz), 4.46 (1H, d, Η-Γ, J 2 = 8.03 Hz .), 4.01 (1H, d , H-4, J 3 = 2.01 Hz), 3. 77-3.73 (2H, m, H-2, H-5), 3.65-3.52 (4H, m, H - 3 , H-6, H-5 '), 3.36-3.3 0 (2H, m, H-4', H-3 '), 3.23 (1H, t, H-2',. ₂ = J _2- . ₃ = 8.03 Hz), 2.4 5 (1H, m, (CH ₃ ) ₂ CHC0NH), 1.01-0.97 (6H, m, (CH ₃ ) ₂ CHC0NH)

High resolution FAB MS: calcd [M + H] + = 430. 1325 m / z (C, 6 H 25 Ν0 ,: Na)

 Found 430.1323 m / z (-0.5 ppm)

 Example 20

4.6-Di-0-acetyl-2-benzamido-2-deoxy-3--0- (methyl 2,3.4-tri-0-acetyl-] 3-D-Darcopyranosylone-D-galacttopyranosyl acetate Synthesis of (10) Benzyl 4,6-di-0-acetyl-2-azide-2-dex-3-3-0- (methyl 2,3,4-tri-0-acetyl -j3 -D_ obtained in Example 13 (Darcopyranosyl ester)-j3-D-Galactopyranoside (7) (300 mg, 0.431 iinol) is dissolved in methanol (30 ml), 10% palladium hydroxide carbon (150 mg) is added, and the mixture is heated under a hydrogen atmosphere at room temperature. The reaction was carried out for 3.5 hours to convert the azide group to an amino group, and at the same time, deprotection of the benzyl group was performed. After completion of the reaction, 10% palladium hydroxide and carbon were removed by filtering the reaction solution through celite, and the filtrate was distilled off under reduced pressure. The obtained residue was dissolved in methanol (30 ml), and triethylamine (1 ml) and benzoyl chloride (0.1 ml, 0.863 related) were added at 0 ° C under a dry atmosphere, and the mixture was reacted for 1 hour. After completion of the reaction, pyridine (3 ml) was added, the reaction solution was distilled off under reduced pressure, and the obtained residue was dried overnight under reduced pressure. After drying, the obtained white amorphous substance was dissolved in pyridine (30 ml), acetic anhydride (0.252 ml, 2.59 mmol) was added dropwise at 0 ° C under a dry atmosphere, and the mixture was reacted at room temperature for 3 hours. . After completion of the reaction, the reaction solution was distilled off under reduced pressure, and the residue was diluted with chloroform.Then, washed sequentially with 4% (w / v) aqueous potassium hydrogen sulfate, saturated aqueous sodium hydrogen carbonate, and saturated saline. The organic layer was dried with 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 (elution solvent: hexane / ethyl acetate = 1: 1-1: 2) to give a colorless amorphous 4,6-di-0-acetyl-2-benzamide. -2-Doxy-3-0- (methyl 2,3,4-tri -0-acetyl-] 3 -D-Dalcopyranosyl peroneto) -D-Galactopyranosyl acetate (10 ) (180 mg, 0.248 ol, yield 5, α / = 5/2). The analysis is as follows.

_{Έ NMR (400MHz, CDC1 3,} TMS) δ (ppm);. 7.72-7 37 (5H, m, aromatic), 6. 55 (. 1H, d, ΝΗα, J 2 NH = 7.53 Hz), 6.44 (1H , d, Hla, J ,, ₂ = 3.01 Hz), 6.16

(1H, d, Hl j3, U. 2 = 9.04 Hz), 5.37 (1H, d, H-4 a, J 3. 4 = 2.00 Hz), 5.23-5.

10 (3H, m, H-3 'a, H-4', H-2 'a), 4.87 (1H, d, Η-Γ,, ₂ = 8.03 Hz), 4.83 (1H, ddd, _{H-2 a, J x.} 2 -3.01 Hz, J 2. NH = 7. 53 Hz, J 2, 3 = 8.54 Hz),

4.37 (1H, dd, H- 3 a, J 2, 3 = 8. 54 Hz, J 3. 4 = 2.00 Hz), 4. 28 (1H, dd, H-6a a, J 5. 6 a = 5.52 . Hz, J = 11 · 04 Hz), 4. 14 (1H, dd,, H-6bo;., J 5 6 b = 7.03 Hz, J 6 a, 6 b 11.04 Hz), 4.06-4.00 (2H, dd, H-5 ', H-5 a), 3.62 (3H, s, C00C H ₃ ), 2.20-1.91 (18H, m, CH ₃ C0)

High resolution FAB MS: Calculated value [M + H] ⁺ = 726.2245 m / z (C ₃₂ H _4, N0 ₁₈ )

 Found 726.2248 m / z (+0.3 pm)

 Example 2 1

 2-phenyl- [4.6-di-0-acetyl-1,2-di-deoxy-3-0- (methyl ϊ3.4-tri-0-acetyl-0-D-darcopyranosyl peronet)-α -D-galactopyrano] [2, tri d] -2-oxazoline (13)

 4, 6-di-0-acetyl-2-benzamido-2-deoxy-3-0- (methyl I 3,4-tri-0-acetyl-] 3-D-darcopyranosyl lipate) -D -Dissolve the galactopyranosyl acetate (10) (180 mg, 0.248 benzyl) in dehydrated dichloromethane (9 ml), and add TMSOTi (0.0680 ml, 0.372 mmol) dropwise at 0 ° C under argon atmosphere. For 12 hours. After completion of the reaction, at 0, triethylamine (0.2 ml) was added, and the mixture was stirred at room temperature for 30 minutes. The reaction solution was evaporated under reduced pressure, and the obtained residue was purified by silica gel chromatography (eluting solvent: toluene / ethyl acetate = 3: 1-2: 1, triethylamine 0.5 0.5) to give a colorless amorphous -Phenyl- [4,6-di-0-acetyl-], 2-di-doxy-3-0- (methyl 1,3,4-tri- 0-acetyl-j3-D -Darcopyranosyl ester )--D -galactopyrano] [2, trid]-2-oxoxolin (13) (105 mg, 0.158 mmol, yield 6 «) was obtained.

[a] _D + 30 ° (c = l.0, CHC1 ₃ )

_{. l NMR (400MHz, CDC1 3} , TMS) δ (ppm); 7.96-7.43 (5H, m, aromatic), 6. 15 (1H, d, Hl, 2 = 6.02 Hz), 5.42 (1H, s, H -4), 5.33 (1H, t, H-3 ', J _2- ,

_{3 · = J 3 '..} = 8.29 Hz), 5.27-5 19 (2H, m, H-4', H - Γ), 5.04 (1H, t, H-2 ', I, ·, · -Ι ₂ .. ₃ = 8.29 Hz), 4.17 (3H, m, H-6, H-5), 4.11-4.02 (3H, m, H-5,, H-2, H-3) , 3.72 (3H, s, C00CH ₃ ), 2.10-2.04 (15H, m, CH ₃ C0)

High resolution FAB MS: calcd ^{[M + H] + = 666.2034} m / z (C 30 H 36 NO I 6)

 Found 666.2043 m / z (+1.3 ppm)

 Example 22

2-phenyl-2-di-doxy-3-0- (sodium β-D-darcopyranosylone)-α-1) -galactovirano] [2, tri (0-2-oxazoline (1 6) Synthesis of 2-Phenyl- [4,6-di-0-acetyl-1,2-di-deoxy-3-0- (methyl 2,3,4-tri-0-acetyl-] 3-D-Darcopyranosi [2, tri-d] -2-oxazoline (13) (105 mg, 0.158 mmol) was dissolved in methanol (1.58 ml), and dissolved in methanol (1.58 ml). A methanol solution of sodium methoxide (3.04 mg, 0.0158 ol) was added at 0 ° C, and the mixture was stirred at 0 ° C for 0.5 hour and at room temperature for 1 hour.Then, a methanol solution of sodium methoxide (3.04 mg, 0.0158 mmol) was added. Was added and the mixture was stirred at room temperature for 0.5 hour to deprotect all 0-acetyl groups. After completion of the reaction, the reaction solution was dried under reduced pressure. After drying, the compound was dissolved in carbonate buffer (50 mM, H 10.6, 1.58 ml), and the methyl ester was deprotected by stirring at room temperature for 1 hour. After completion of the reaction, the reaction solution is lyophilized to give 2-phenyl- 门, 2-di-doxy-3-0- (sodium β-D-darcopyranosyl peroneto) -a-D-galactopyrano. [2, 1-d] -2 -oxazoline (16) (60 mg, purity 87%) was obtained. The analysis data is as follows.

_{XH NMR (400MHz, D 2 0} , acetone) <5 (ppm); 7.77-7.34 (5H, m, aromatic), 6. 13 (1H, d, Hl, 2 = 7.03 Hz), 4.53 (1H, d, Η-Γ, J, .. ₂ = 7.53 Hz), 4.06

(1H, d, H-4 , J 3, 4 -2.51 Hz), 4.00 (1H, dd, H-2, J l 2 = 7.03 Hz, J 2. 3 = 7.53 Hz), 3.83 (1H, dd, _{. H-5, J 5 6a} = 7.53 Hz, J 5, 6b = ll.54 Hz), 3.74 (1H, dd, H -.. 3, J 2 = 7.53, J 3 = 7.53 Hz), 3.68-3.52 (3H, m, H-6a, H-6b, H-5 '), 3.45- 3.32 (2H, m, H-4', H-3 '), 3.27 (1H, t, H-2', J _{, '2 = J 2..} ' 3 = 7.53 Hz) High resolution FAB MS:..! calculated [M + H] + = 464.1169 m / z (C, gUO, Na) Found 464. 1173 m / z (+0.9 ppm)

 Example 2 3

 Synthesis of chondroitin derivatives by enzyme-catalyzed polymerization

Substrate monomer 2-ethyl- [1,2-di-doxy-3-0- (sodium β-D-Darco pyranosyl peronet) -a-D-galactovirano] [2, tri d]-2- Oxazoline (14) (5.OOmg, 12.ΟΜ ΠΙΟΙ), 2-isopropyl- [1,2-di-deoxy-3-0- (sodium β-D-Darcopyranosylone) -H-D- Galactopyrano] [2, 1-d] -2-oxazoline (15) (5.OOing, 11.6 mol), 2-phenyl- [1,2-di-deoxy 3-0- (sodium β-D -Darcopyranosyl peroneto)-0!-D -galactovirano] [2, tri d] -2-oxosazolin (16) (5.OOmg, 10.8 mol) 116 w K 108 1 2/11576

Dissolve in phosphate buffer solution (50 mM, pH 7.5) and add 0.5 mg of hyaluronidase (H-0TH; 2502 units / mg, Lot No. 8838E) from sheep testis, and react at 30 ° C. Went. At each time shown in Table 8, Table 9, and Table 10, 3.O 1 was collected from the reaction solution, subjected to HPLC analysis under the same conditions as in Example 9, and determined from the area ratio of the peak that appeared at a retention time of 6 minutes. The residual monomer ratio was determined. The changes over time are shown in Table 8, Table 9, Table 10, and FIGS. 7, 8, and 9 in comparison with the case where no enzyme was added. After completion of the reaction, the enzyme was inactivated by heating the reaction solution in a water bath at 90 ° C. for 3 minutes, then an excess amount of THF was added, and the resulting precipitate was collected by centrifugation. The obtained precipitate was dissolved again in pure water to obtain an aqueous solution of a polymerization product. Table 11 shows the yield and molecular weight of the polymerization product, based on the substrate monomer, determined by GPC measurement of the obtained aqueous solution. GPC measurement and calibration curve preparation were performed under the same conditions as in Example 7. From the aqueous solution of the polymerization product obtained from the reaction solution using the substrate monomers (14) and (15), the polymerization product was purified using Sephadex G-10 size exclusion chromatography. The ¹ H and ¹³ C NMR spectra of the product obtained from the substrate monomer (14) are shown in FIGS. 10 and 11, respectively. These spectra revealed that this product was a chondroitin derivative having the structure (17) in FIG. 6 (R = CH ₂ CH ₃ ).

 The assignment of the NMR spectrum is as follows.

JH Ryu _{R (400 MHz, D 2 0} , acetone) <5 (ppm); 4.38 (. 1H, d, H- 1, 2 = 8.53 H z), 4.34 (1H, d, Η-Γ, I, · _{. 2 · -8.03 Hz), 3.98} (1H, s, H-4), 3.88 (1H, t,

H-2, J ,. ₂ = 8.53 Hz), 3.69-3.52 (6H, m, H—3, H-4 ', H-5', H-6a, H-6b, H-5), 3.43 ( 1H, t, H-3 ' , J 2 .. 3. = 8.03 Hz), 3.22 (1H, t, H-2', J, .. 2 · = 8.03

Hz), 2.16 (2H, a, CH ₃ CH ₂ C0, J = 7.53 Hz), 0.97 (3H, t, CH ₃ CH ₂ C0, J-7.53 Hz)

^{! 3 C NMR (100 MHz,} D 2 0, acetone) δ (ppm); 179.54 (C-6 '), 175.35 (CH 3 CH 2 CO), 105.02 (C-Γ), 101. 1 (C- 1 ), 80.86 (C-1), 80.06 (C-4 '), 77.17 (C-5'), 75.69 (C-5), 74.38 (C-3 '), 73.26 (C-2'), 68.60 (C-4), 61.85 (C-6), 51.60 (C-2), 30.17 (CH ₃ CH ₂ C0), 9.96 (CH ₃ CH ₂ C0) JP02 / 11576

 30

Residual rate of substrate monomer (14) Reaction Residual rate (%) Reaction Residual rate (%)

 Time time

 (h) No enzyme added Enzyme added (h) No enzyme added Enzyme added

0. 0 1 0 0 1 0 0 8. 0 1 2 3

1.0 0 8 2 9.0 7 9 ―

2.09 4 1 2 7 5 2 5

3.0 5 6 2 2 4.3

4.09 0 2 4 6 5

5. 0 3 7 3 4 5 6

6.0 8 6 3 5 2. 1

9

 Residual rate of substrate monomer (15) Reaction Residual rate (%) Reaction Residual rate (%) Time Time

 (h) No enzyme added Enzyme added (h) No enzyme added Enzyme added

0. 0 1 0 0 1 0 0 2 5 8 0 6 9

2.0 ― 9 5 3 5 6 9 6 0

2.3 9 5 4 9 6 1 5 1

4.09 2 9 3 8 3 4 0 3 2

6.09 3 8 9 1 2 3 2 4 1 5

9.091 8 6 1 6 8 9.8 6.6

1 2 8 9 8 2

¾ 1 0

 Residual rate of substrate monomer (16), Residual rate (%) Reaction Residual rate (%) Time Time

 (h) Add enzyme without enzyme Add enzyme (h) Add enzyme without enzyme Add enzyme

0. 0 1 0 0 1 0 0 9 0 8 0 7 7

1 6 9 2 1 6 1 6 0 6 3

1 7 9 4 2 3 9 5 5 5 9

4 8 8 9 8 5 Table 11

 Yield and molecular weight of formed chondroitin derivatives

Example 2 4

 Time course of chondroitin derivative synthesis reaction

Substrate monomer, 2-ethyl- [1,2-di-doxy-3-0- (sodium β-D-Darcopyranosyl lipate)-α-D-galactovirano] [2, 1-d] -2- Oxazoline (14) (15.Omg, 36.mol) was dissolved in 3601 phosphate buffer (50 mM, H7.5), and sheep testis-derived hyaluronidase (H-0TH; 2502 units / mg, Lot No. 8838E) was added and reacted at 30 ° C. At each time shown in Table 12, samples of reaction solution 201 were collected and heated in a hot water bath at 90 ° C for 3 minutes to inactivate the enzyme, and then the same conditions as in Example 23 were used. HPLC and GPC measurements were performed at to determine the residual ratio of the substrate monomer, the yield of the polymerization product relative to the substrate monomer, and the molecular weight of the polymerization product. The results are shown in Table 12 and FIG. Table 1 2

 Reaction time change

産業上の利用可能性

Industrial applicability

 The method for producing chondroitin or a chondroitin derivative according to the present invention is a simpler synthesis method than an extraction method from a biomaterial which has been conventionally used industrially, and a product from a reaction solution. And a method for producing chondroitin or a chondroitin derivative which can be easily isolated and purified. The collected chondroitin or chondroitin derivative is industrially useful as a material for cosmetics, pharmaceuticals and medical materials.

Claims

The scope of the claims 1. A method for producing chondroitin or a chondroitin derivative, which comprises reacting a hyaluronic acid degrading enzyme with an oxazoline derivative represented by the following general formula (I). -General formula (I)

(In the above formula, R represents hydrogen, an alkyl group, an optionally substituted alkyl group, a phenyl group, or an optionally substituted phenyl group.)  2. The oxazoline derivative is 2-methyl- [1,2-dideoxy-3—0— (sodiumu D-darcopyranosyl oxalate)]-α-D-galactobirano [2,1-d ] The production method according to claim 1, which is 2-oxoxolin.  3. The method according to claim 1, wherein the hyaluronic acid-degrading enzyme is a mammalian hyaluronidase.  4. The method according to claim 3, wherein the mammal-derived hyaluronidase is a testicular hyaluronidase or a sheep testis-derived hyaluronidase.  5. The method according to any one of claims 1 to 4, wherein the oxazoline derivative is allowed to act on a hyaluronic acid-decomposing enzyme at a pH of 5 to 10.  6. The method according to any one of claims 1 to 4, wherein a hyaluronic acid degrading enzyme is allowed to act on the oxazoline derivative at a concentration of the oxazoline derivative of 0.1% by weight or more.  7. The method according to any one of claims 1 to 4, wherein the oxazoline derivative is allowed to act on a hyaluronic acid degrading enzyme at a temperature of 5 to 60 ° C.