CN115975070B - Preparation method of acetylated hyaluronate - Google Patents
Preparation method of acetylated hyaluronate Download PDFInfo
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- CN115975070B CN115975070B CN202211637766.1A CN202211637766A CN115975070B CN 115975070 B CN115975070 B CN 115975070B CN 202211637766 A CN202211637766 A CN 202211637766A CN 115975070 B CN115975070 B CN 115975070B
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- hyaluronate
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- acetylated
- activating reagent
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- WCDDVEOXEIYWFB-VXORFPGASA-N (2s,3s,4r,5r,6r)-3-[(2s,3r,5s,6r)-3-acetamido-5-hydroxy-6-(hydroxymethyl)oxan-2-yl]oxy-4,5,6-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@@H]1C[C@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](C(O)=O)O[C@@H](O)[C@H](O)[C@H]1O WCDDVEOXEIYWFB-VXORFPGASA-N 0.000 title claims abstract description 77
- 229940014041 hyaluronate Drugs 0.000 title claims abstract description 77
- 238000002360 preparation method Methods 0.000 title claims abstract description 49
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims abstract description 91
- 238000006243 chemical reaction Methods 0.000 claims abstract description 53
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 51
- 230000003213 activating effect Effects 0.000 claims abstract description 43
- 238000000034 method Methods 0.000 claims abstract description 28
- 239000003960 organic solvent Substances 0.000 claims abstract description 24
- 230000035484 reaction time Effects 0.000 claims abstract description 9
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 64
- 239000007787 solid Substances 0.000 claims description 57
- VHYFNPMBLIVWCW-UHFFFAOYSA-N 4-Dimethylaminopyridine Chemical compound CN(C)C1=CC=NC=C1 VHYFNPMBLIVWCW-UHFFFAOYSA-N 0.000 claims description 56
- 239000011259 mixed solution Substances 0.000 claims description 54
- 239000012043 crude product Substances 0.000 claims description 42
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 40
- 150000007530 organic bases Chemical class 0.000 claims description 40
- 238000002156 mixing Methods 0.000 claims description 30
- 239000000243 solution Substances 0.000 claims description 30
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims description 24
- 239000000047 product Substances 0.000 claims description 23
- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical class CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 claims description 22
- 229920002674 hyaluronan Polymers 0.000 claims description 20
- 229960003160 hyaluronic acid Drugs 0.000 claims description 20
- SJRJJKPEHAURKC-UHFFFAOYSA-N N-Methylmorpholine Chemical compound CN1CCOCC1 SJRJJKPEHAURKC-UHFFFAOYSA-N 0.000 claims description 18
- 238000001035 drying Methods 0.000 claims description 16
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 15
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 claims description 12
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 claims description 12
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 claims description 12
- 239000011777 magnesium Substances 0.000 claims description 12
- 229910052749 magnesium Inorganic materials 0.000 claims description 12
- 239000011591 potassium Substances 0.000 claims description 12
- 229910052700 potassium Inorganic materials 0.000 claims description 12
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 11
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 9
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 claims description 8
- -1 7-azabenzotriazole-1-yl Chemical group 0.000 claims description 6
- GKQLYSROISKDLL-UHFFFAOYSA-N EEDQ Chemical compound C1=CC=C2N(C(=O)OCC)C(OCC)C=CC2=C1 GKQLYSROISKDLL-UHFFFAOYSA-N 0.000 claims description 6
- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 claims description 5
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 claims description 4
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 claims description 4
- SECXISVLQFMRJM-UHFFFAOYSA-N N-Methylpyrrolidone Chemical compound CN1CCCC1=O SECXISVLQFMRJM-UHFFFAOYSA-N 0.000 claims description 4
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 claims description 4
- 150000003863 ammonium salts Chemical class 0.000 claims description 4
- 239000011575 calcium Substances 0.000 claims description 4
- 229910052791 calcium Inorganic materials 0.000 claims description 4
- 239000011734 sodium Substances 0.000 claims description 4
- 229910052708 sodium Inorganic materials 0.000 claims description 4
- 239000011701 zinc Substances 0.000 claims description 4
- 229910052725 zinc Inorganic materials 0.000 claims description 4
- BDNKZNFMNDZQMI-UHFFFAOYSA-N 1,3-diisopropylcarbodiimide Chemical compound CC(C)N=C=NC(C)C BDNKZNFMNDZQMI-UHFFFAOYSA-N 0.000 claims description 3
- BZLVMXJERCGZMT-UHFFFAOYSA-N Methyl tert-butyl ether Chemical compound COC(C)(C)C BZLVMXJERCGZMT-UHFFFAOYSA-N 0.000 claims description 3
- 238000004090 dissolution Methods 0.000 claims description 2
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 abstract description 38
- 238000003756 stirring Methods 0.000 abstract description 28
- 238000001914 filtration Methods 0.000 abstract description 17
- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid Substances OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 abstract description 14
- 230000021736 acetylation Effects 0.000 abstract description 8
- 238000006640 acetylation reaction Methods 0.000 abstract description 8
- 230000000694 effects Effects 0.000 abstract description 8
- 238000009776 industrial production Methods 0.000 abstract description 6
- 238000003912 environmental pollution Methods 0.000 abstract description 5
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 abstract description 3
- 239000003814 drug Substances 0.000 abstract description 3
- 238000004519 manufacturing process Methods 0.000 abstract description 3
- 231100000331 toxic Toxicity 0.000 abstract description 2
- 230000002588 toxic effect Effects 0.000 abstract description 2
- 239000002351 wastewater Substances 0.000 abstract description 2
- 229920002385 Sodium hyaluronate Polymers 0.000 description 82
- 229940010747 sodium hyaluronate Drugs 0.000 description 82
- YWIVKILSMZOHHF-QJZPQSOGSA-N sodium;(2s,3s,4s,5r,6r)-6-[(2s,3r,4r,5s,6r)-3-acetamido-2-[(2s,3s,4r,5r,6r)-6-[(2r,3r,4r,5s,6r)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2- Chemical compound [Na+].CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 YWIVKILSMZOHHF-QJZPQSOGSA-N 0.000 description 82
- 229960000549 4-dimethylaminophenol Drugs 0.000 description 25
- 230000004913 activation Effects 0.000 description 11
- 125000000218 acetic acid group Chemical group C(C)(=O)* 0.000 description 10
- 230000000052 comparative effect Effects 0.000 description 10
- VJVOFLWZDWLHNR-MRCUWXFGSA-N icosan-9-yl (z)-docos-13-enoate Chemical compound CCCCCCCCCCCC(CCCCCCCC)OC(=O)CCCCCCCCCCC\C=C/CCCCCCCC VJVOFLWZDWLHNR-MRCUWXFGSA-N 0.000 description 8
- 230000003020 moisturizing effect Effects 0.000 description 7
- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide Substances CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 description 5
- 238000006555 catalytic reaction Methods 0.000 description 5
- 238000005886 esterification reaction Methods 0.000 description 5
- 239000007821 HATU Substances 0.000 description 4
- 230000009286 beneficial effect Effects 0.000 description 4
- WETWJCDKMRHUPV-UHFFFAOYSA-N acetyl chloride Chemical compound CC(Cl)=O WETWJCDKMRHUPV-UHFFFAOYSA-N 0.000 description 3
- 239000012346 acetyl chloride Substances 0.000 description 3
- 238000005917 acylation reaction Methods 0.000 description 3
- 239000003513 alkali Substances 0.000 description 3
- 239000003054 catalyst Substances 0.000 description 3
- 230000001050 lubricating effect Effects 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 150000001242 acetic acid derivatives Chemical class 0.000 description 2
- 230000032683 aging Effects 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 239000012295 chemical reaction liquid Substances 0.000 description 2
- 230000032050 esterification Effects 0.000 description 2
- 230000004060 metabolic process Effects 0.000 description 2
- 239000002574 poison Substances 0.000 description 2
- 231100000614 poison Toxicity 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 238000007086 side reaction Methods 0.000 description 2
- 230000037394 skin elasticity Effects 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- FDKXTQMXEQVLRF-ZHACJKMWSA-N (E)-dacarbazine Chemical compound CN(C)\N=N\c1[nH]cnc1C(N)=O FDKXTQMXEQVLRF-ZHACJKMWSA-N 0.000 description 1
- VZSRBBMJRBPUNF-UHFFFAOYSA-N 2-(2,3-dihydro-1H-inden-2-ylamino)-N-[3-oxo-3-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)propyl]pyrimidine-5-carboxamide Chemical compound C1C(CC2=CC=CC=C12)NC1=NC=C(C=N1)C(=O)NCCC(N1CC2=C(CC1)NN=N2)=O VZSRBBMJRBPUNF-UHFFFAOYSA-N 0.000 description 1
- MSWZFWKMSRAUBD-IVMDWMLBSA-N 2-amino-2-deoxy-D-glucopyranose Chemical compound N[C@H]1C(O)O[C@H](CO)[C@@H](O)[C@@H]1O MSWZFWKMSRAUBD-IVMDWMLBSA-N 0.000 description 1
- AEMOLEFTQBMNLQ-AQKNRBDQSA-N D-glucopyranuronic acid Chemical compound OC1O[C@H](C(O)=O)[C@@H](O)[C@H](O)[C@H]1O AEMOLEFTQBMNLQ-AQKNRBDQSA-N 0.000 description 1
- IAJILQKETJEXLJ-UHFFFAOYSA-N Galacturonsaeure Natural products O=CC(O)C(O)C(O)C(O)C(O)=O IAJILQKETJEXLJ-UHFFFAOYSA-N 0.000 description 1
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 1
- 229940124532 absorption promoter Drugs 0.000 description 1
- WJGAPUXHSQQWQF-UHFFFAOYSA-N acetic acid;hydrochloride Chemical compound Cl.CC(O)=O WJGAPUXHSQQWQF-UHFFFAOYSA-N 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- MSWZFWKMSRAUBD-UHFFFAOYSA-N beta-D-galactosamine Natural products NC1C(O)OC(CO)C(O)C1O MSWZFWKMSRAUBD-UHFFFAOYSA-N 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
- 150000002016 disaccharides Chemical class 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003792 electrolyte Substances 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 229960002442 glucosamine Drugs 0.000 description 1
- 229940097043 glucuronic acid Drugs 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 239000005457 ice water Substances 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 239000011261 inert gas Substances 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000000314 lubricant Substances 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 239000012046 mixed solvent Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 201000008482 osteoarthritis Diseases 0.000 description 1
- 206010033675 panniculitis Diseases 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000004537 pulping Methods 0.000 description 1
- 230000008591 skin barrier function Effects 0.000 description 1
- PNGLEYLFMHGIQO-UHFFFAOYSA-M sodium;3-(n-ethyl-3-methoxyanilino)-2-hydroxypropane-1-sulfonate;dihydrate Chemical compound O.O.[Na+].[O-]S(=O)(=O)CC(O)CN(CC)C1=CC=CC(OC)=C1 PNGLEYLFMHGIQO-UHFFFAOYSA-M 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000002798 spectrophotometry method Methods 0.000 description 1
- 238000001694 spray drying Methods 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 210000004304 subcutaneous tissue Anatomy 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 230000002792 vascular Effects 0.000 description 1
- 230000029663 wound healing Effects 0.000 description 1
- 230000037303 wrinkles Effects 0.000 description 1
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Abstract
The invention relates to a preparation method of acetylated hyaluronate, which belongs to the technical field of biological medicine, and comprises the steps of dissolving hyaluronate in a first organic solvent to obtain a first reaction solution; dissolving acetic acid in a second organic solvent, and adding an activating reagent to perform preactivation to obtain a second reaction solution; and adding the second reaction solution into the first reaction solution, stirring and reacting, slowly adding the reacted solution into a third organic solvent, stirring, filtering and the like, and obtaining the acetylated hyaluronate. The invention does not use the reagents of acetic anhydride and concentrated sulfuric acid which are easy to be toxic in the preparation process, and the used acetic acid is only small, so that the production cost is reduced to the greatest extent, the preparation reagent is easy to obtain, the invention is more suitable for industrial production, reduces the discharge amount of wastewater, reduces the environmental pollution, and improves the yield and quality of the acetylated hyaluronate. The method pre-activates acetic acid, increases carboxyl activity, promotes reaction efficiency, shortens reaction time and improves acetylation degree.
Description
Technical Field
The invention relates to the technical field of biological medicine, in particular to a preparation method of acetylated hyaluronate.
Background
Hyaluronic acid, also known as hyaluronic acid, is an acidic linear polysaccharide formed by repeated arrangement of disaccharides of glucuronic acid and glucosamine. The professor Meyer et al, university of columbia in the united states in 1934, first isolated this material from bulls eye vitreous. Hyaluronic acid shows various important physiological functions in the body by virtue of unique molecular structure and physicochemical properties, such as lubricating joints, regulating permeability of vascular walls, regulating protein, water electrolyte diffusion and operation, promoting wound healing and the like. Particularly, hyaluronic acid has a special water-retaining effect, is a substance with better water-retaining property in the nature which is found at present, and is called an ideal natural moisturizing factor. Hyaluronic acid is a multifunctional matrix, and hyaluronic acid (hyaluronic acid) HA is widely distributed in various parts of the human body. Wherein the skin also contains a large amount of hyaluronic acid. The maturation and aging process of human skin also changes along with the content and metabolism of hyaluronic acid, and the hyaluronic acid can improve the nutrition metabolism of the skin, make the skin tender, smooth, remove wrinkles, increase elasticity, prevent aging, and is a good percutaneous absorption promoter while keeping moisture.
The acetylated hyaluronate has the effects of moisturizing, repairing skin barrier, increasing skin elasticity and the like more effectively than the traditional hyaluronate. The acetyl is added on the hyaluronic acid structure, so that the biological activity difference is obvious, the solution has good elasticity and moisturizing and lubricating functions, is widely applied to moisturizing and beautifying cosmetics, and is a lubricant for moisturizing and lubricating the salary agent and bone joints in ophthalmic surgery, the moisturizing performance is greatly enhanced, and the solution is easier to be absorbed by human bodies. Can be absorbed by skin when being used externally, increases the content of subcutaneous tissue, can effectively increase skin elasticity, achieves the effect of deep moisturizing, can supplement the deficiency in vivo when being taken orally, has remarkable effects on beautifying, preventing and treating osteoarthritis, and has good application prospect in the aspects of medicines and health care products at present.
At present, the preparation method of the acetylated hyaluronate has certain defects. For example, CN109206537a discloses an acetylated sodium hyaluronate, which is prepared by performing an acylation reaction of hyaluronic acid or a salt thereof in a mixed solvent of acetic acid and acetic anhydride under the catalysis of concentrated sulfuric acid, adding a reaction liquid flow into water after the reaction is finished to precipitate, filtering and washing with a large amount of water to obtain the acetylated hyaluronic acid; CN114133419B discloses a low molecular weight acetylated hyaluronate, a preparation method and application thereof, under the protection of inert gas, adding low molecular weight hyaluronic acid or its salt into a mixed solution of acetic acid and acetic anhydride, adding concentrated sulfuric acid catalyst for acylation reaction; after the reaction is finished, adding the reaction solution into an ether solvent, crystallizing and filtering, washing, adjusting the pH value by alkali liquor, and drying to obtain the catalyst. The controlled acetic anhydride and the concentrated sulfuric acid are used in the reaction process of the two, the operation is complex, the environmental pollution is high, the spray drying is needed, the cost is high, and the industrial production is not facilitated. CN 113045686B discloses a method for preparing acetylated hyaluronic acid, which adopts an organic base to activate and treat hyaluronic acid, then adopts acetyl chloride to activate and treat hyaluronic acid, and finally prepares acetylated hyaluronic acid salt, although acetic anhydride, concentrated sulfuric acid and other easily toxic reagents are not used, the product yield is lower; and the catalyst is used singly for catalysis, so that the catalysis effect is poor.
At present, great difficulties are encountered in improving yield, quality, reducing cost and reducing environmental pollution while obtaining the acetylated hyaluronate, so that it is necessary to develop a new preparation method of the acetylated hyaluronate.
Disclosure of Invention
The invention aims to provide a preparation method of acetylated hyaluronate, which can improve the yield and quality of products, reduce the cost, and simultaneously avoid the use of easily-toxic raw materials such as acetic anhydride, sulfuric acid and the like, and solve the problems of low product yield, high cost, large environmental pollution, easily-toxic raw materials and difficult obtainment in the prior art. To achieve the purpose, the invention adopts the following technical scheme:
the invention provides a preparation method of acetylated hyaluronate, which comprises the following steps:
step one: dissolving hyaluronate in a first organic solvent to obtain a first mixed solution;
step two: mixing acetic acid with a first organic solvent and an activating reagent/organic base to obtain a second mixed solution;
step three: uniformly mixing the second mixed solution with the first mixed solution, and reacting at a certain temperature to obtain a reaction solution;
step four: mixing the reaction solution in the third step with a second organic solvent to separate out a solid crude product;
step five: purifying the solid crude product by using a second organic solvent, and drying to obtain an acetylated hyaluronate solid pure product; or the operation sequence of the first step and the second step can be exchanged successively.
In the process of preparing the acetylated hyaluronate, acetic acid is dissolved in a second organic solvent, and an activating reagent is added for preactivation, so that the generated second reaction liquid and hyaluronate are subjected to esterification reaction, the acetylated hyaluronate is produced, the reagents of acetic anhydride and concentrated sulfuric acid which are easy to poison are not used in the preparation process, the used acetic acid is only small, the cost is reduced, the preparation reagent is easy to obtain, the method is more suitable for industrial production, and the pollution to the environment is reduced. The quality of the obtained acetylated hyaluronate is better by only using the preactivated acetic acid.
As one embodiment of the invention, the activating reagent is one or more of DIC (N, N ' -diisopropylcarbodiimide), EDCI [1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride ], HATU [ N, N, N ', N ' -tetramethyl-O- (7-azabenzotriazol-1-yl) hexafluorophosphoric acid urea ], HBTU (benzotriazol-N, N, N ', N ' -tetramethylurea hexafluorophosphate), EEDQ (2-ethoxy-1-ethoxycarbonyl-1, 2-dihydroquinoline), and the organic base is one or more of DIEA (N, N-diisopropylethylamine), TEA (triethylamine), DMAP (4-dimethylaminopyridine) and NMM (N-methylmorpholine).
As a more specific embodiment of the invention, the activating reagent/organic base is DIC/DMAP, and the selection of the activating reagent/organic base is beneficial to improving the esterification efficiency, improving the yield and reducing the cost.
As an embodiment of the present invention, the mass ratio of acetic acid to hyaluronate is 0.1:1 to 0.5:1. too little acetic acid and insufficient acetylation; the amount of acetic acid is excessive, the acetylation degree is basically unchanged, and the cost is increased. The mass ratio range is beneficial to reducing the cost while ensuring the acetylation degree. The mass ratio of the activating reagent to the hyaluronate is 0.1: 1-2: 1, preferably 0.5:1 to 1.5:1, a step of; the mass ratio of the organic base to the hyaluronate is 0.1: 1-2: 1, preferably 0.5:1 to 1.5:1, a step of; the mass ratio range is beneficial to reducing the cost while ensuring the acetylation degree.
As an embodiment of the present invention, the first organic solvent includes one or more of DCM (dichloromethane), DMF (N, N-dimethylformamide), toluene, N-methylpyrrolidone; the mass ratio of the hyaluronate to the first organic solvent for dissolution is 1: 3-1: 10. the consumption of the first organic solvent is too small, and the product is not easy to stir, so that the reaction affects the reaction yield under heterogeneous conditions; when the first organic solvent is used in an excessive amount, the cost is increased. The mass ratio is selected to reduce the cost while ensuring homogeneous reaction.
As one embodiment of the invention, the reaction temperature is 25-60 ℃ and the reaction time is 4-24 hours; preferably, the reaction temperature is 35-50 ℃ and the reaction time is 5-7 h. The reaction temperature is 45 ℃, which is the preferable reaction temperature, and the quality and the yield of the obtained acetylated hyaluronate are optimal.
As an embodiment of the present invention, the second organic solvent includes one or more of ethanol, methyl t-butyl ether, methanol, diethyl ether, preferably ethanol.
As one embodiment of the present invention, the hyaluronate has a molecular weight of 1 to 2000kDa. The preparation method is suitable for hyaluronate with various molecular weights and has wide application range.
As an embodiment of the present invention, the hyaluronate includes 1 or a combination of at least 2 of sodium, potassium, magnesium, calcium, zinc, or ammonium salts of hyaluronic acid. The preparation method used by the invention is suitable for various hyaluronate and has wide application range.
Compared with the prior art, the invention has the following beneficial effects:
1. according to the invention, the acetic acid is dissolved in the second organic solvent, an activating reagent/organic alkali is added for pre-activation, and esterification reaction is carried out with hyaluronate, so that acetylated hyaluronate is obtained, no reagents of acetic anhydride and concentrated sulfuric acid which are easy to poison are used in the preparation process, the used acetic acid is small, the production cost is reduced to the greatest extent, the preparation reagent is easy to obtain, the preparation method is more suitable for industrial production, the discharge amount of waste water is reduced, and the pollution to the environment is reduced. By utilizing the preactivated acetic acid, the yield and the acetylation degree of the acetylated hyaluronate are improved, and the quality of the obtained product is better. The yield of the invention is more than 95%, and further more than 98%; the acetyl content reaches more than 24 percent, and further reaches more than 28 percent.
2. The method pre-activates acetic acid, increases carboxyl activity, promotes reaction efficiency, shortens reaction time and improves acetylation degree. The selected activating reagent/organic alkali has the advantages of low cost and high esterification efficiency.
3. The preparation method is suitable for various hyaluronate and various molecular weights, and has wide application range and high use value.
Detailed Description
The present invention is further described below with reference to examples, but embodiments of the present invention are not limited thereto.
A process for preparing acetylated hyaluronate, as used in the examples, comprising hyaluronate starting materials of various molecular weights, all supplied by the biosciences company of tao, tokyo.
The equipment or other reagents/materials used in the examples are commercially available.
The yield of the acetylated hyaluronate can be simply converted from the mass ratio of the obtained acetylated hyaluronate to the starting hyaluronate.
The acetyl content of the acetylated hyaluronate is determined by ultraviolet spectrophotometry.
Example 1
In this embodiment, a preparation method of acetylated sodium hyaluronate is provided, and the preparation method includes the following steps:
step one: sodium hyaluronate and DMF are mixed according to the mass ratio of 1:6, mixing to obtain a first mixed solution; the molecular weight of the sodium hyaluronate is 1kDa;
step two: acetic acid and sodium hyaluronate are mixed according to the mass ratio of 0.2:1 is mixed with DMF, and an activating reagent DIC/organic base DMAP is added for pre-activation, wherein the mass ratio of the adding mass of the activating reagent DIC to the mass ratio of sodium hyaluronate is 0.6:1, wherein the mass ratio of the organic base DMAP to the sodium hyaluronate is 0.6:1, obtaining a second mixed solution;
step three: uniformly mixing the second mixed solution with the first mixed solution, and stirring and reacting for 6 hours at 45 ℃ to obtain a reaction solution;
step four: slowly adding the reaction solution in the third step into 20 times of ethanol, stirring, and suction filtering to separate out a solid crude product;
step five: and purifying the solid crude product by using ethanol, and drying the solid crude product in vacuum at 45 ℃ for 8 hours to obtain an acetylated sodium hyaluronate solid pure product.
The relevant indexes of the obtained sodium hyaluronate are shown in Table 1 in detail.
Example 2
In this embodiment, a preparation method of acetylated sodium hyaluronate is provided, and the preparation method includes the following steps:
step one: sodium hyaluronate and DMF are mixed according to the mass ratio of 1:6, mixing to obtain a first mixed solution; the molecular weight of the sodium hyaluronate is 20kDa;
step two: acetic acid and sodium hyaluronate are mixed according to the mass ratio of 0.2:1 is mixed with DMF, and an activating reagent DIC/organic base DMAP is added for pre-activation, wherein the mass ratio of the adding mass of the activating reagent DIC to the mass ratio of sodium hyaluronate is 0.6:1, wherein the mass ratio of the organic base DMAP to the sodium hyaluronate is 0.6:1, obtaining a second mixed solution;
step three: uniformly mixing the second mixed solution with the first mixed solution, and stirring and reacting for 6 hours at 45 ℃ to obtain a reaction solution;
step four: slowly adding the reaction solution in the third step into 20 times of ethanol, stirring, and suction filtering to separate out a solid crude product;
step five: and purifying the solid crude product by using ethanol, and drying the solid crude product in vacuum at 45 ℃ for 8 hours to obtain an acetylated sodium hyaluronate solid pure product.
The relevant indexes of the obtained sodium hyaluronate are shown in Table 1 in detail.
Example 3
In this embodiment, a preparation method of acetylated sodium hyaluronate is provided, and the preparation method includes the following steps:
step one: sodium hyaluronate and DMF are mixed according to the mass ratio of 1:6, mixing to obtain a first mixed solution; the molecular weight of the sodium hyaluronate is 500kDa;
step two: acetic acid and sodium hyaluronate are mixed according to the mass ratio of 0.2:1 is mixed with DMF, and an activating reagent DIC/organic base DMAP is added for pre-activation, wherein the mass ratio of the adding mass of the activating reagent DIC to the mass ratio of sodium hyaluronate is 0.6:1, wherein the mass ratio of the organic base DMAP to the sodium hyaluronate is 0.6:1, obtaining a second mixed solution;
step three: uniformly mixing the second mixed solution with the first mixed solution, and stirring and reacting for 6 hours at 45 ℃ to obtain a reaction solution;
step four: slowly adding the reaction solution in the third step into 20 times of ethanol, stirring, and suction filtering to separate out a solid crude product;
step five: and purifying the solid crude product by using ethanol, and drying the solid crude product in vacuum at 45 ℃ for 8 hours to obtain an acetylated sodium hyaluronate solid pure product.
The relevant indexes of the obtained sodium hyaluronate are shown in Table 1 in detail.
Example 4
In this embodiment, a preparation method of acetylated sodium hyaluronate is provided, and the preparation method includes the following steps:
step one: sodium hyaluronate and DMF are mixed according to the mass ratio of 1:6, mixing to obtain a first mixed solution; the molecular weight of the sodium hyaluronate is 2000kDa;
step two: acetic acid and sodium hyaluronate are mixed according to the mass ratio of 0.2:1 is mixed with DMF, and an activating reagent DIC/organic base DMAP is added for pre-activation, wherein the mass ratio of the adding mass of the activating reagent DIC to the mass ratio of sodium hyaluronate is 0.6:1, wherein the mass ratio of the organic base DMAP to the sodium hyaluronate is 0.6:1, obtaining a second mixed solution;
step three: uniformly mixing the second mixed solution with the first mixed solution, and stirring and reacting for 6 hours at 45 ℃ to obtain a reaction solution;
step four: slowly adding the reaction solution in the third step into 20 times of ethanol, stirring, and suction filtering to separate out a solid crude product;
step five: and purifying the solid crude product by using ethanol, and drying the solid crude product in vacuum at 45 ℃ for 8 hours to obtain an acetylated sodium hyaluronate solid pure product.
The relevant indexes of the obtained sodium hyaluronate are shown in Table 1 in detail.
Comparative example 1
Comparative example referring to the method disclosed in CN110981991, acetic anhydride is acylated with sodium hyaluronate under the catalysis of concentrated sulfuric acid, and the relevant indexes of the obtained acetylated sodium hyaluronate are shown in table 1 in detail.
Step one: sodium hyaluronate and acetic anhydride are mixed according to the mass ratio of 1:13.5, mixing, and carrying out acylation reaction for 6h at 45 ℃ in concentrated sulfuric acid; wherein the molecular weight of the sodium hyaluronate is 500kDa; the adding amount of the concentrated sulfuric acid is 33% of the mass of the sodium hyaluronate;
step two: slowly adding the reaction solution obtained after the reaction is finished into 20 times of ethanol, stirring, and suction filtering to separate out a solid crude product;
step three: purifying the solid crude product by ethanol, and drying the solid crude product in vacuum at 45 ℃ for 8 hours to obtain the acetylated sodium hyaluronate.
The related indexes of the obtained sodium hyaluronate are shown in Table 1 in detail
Table 1 index of sodium hyaluronate obtained in examples 1 to 4 and comparative example 1
| # | Molecular weight/kDa | Yield/% | Acetyl content/% |
| Example 1 | 1 | 98.9 | 28.6 |
| Example 2 | 20 | 96.8 | 28.3 |
| Example 3 | 500 | 97.6 | 27.5 |
| Example 4 | 2000 | 98.2 | 28.7 |
| Comparative example 1 | 500 | 90.0 | 21.4 |
As can be seen from Table 1, according to the preparation method provided by the invention, acetic acid is pre-activated and then is subjected to esterification reaction with sodium hyaluronate, and reagents acetic anhydride and sulfuric acid which are easy to prepare are not used in the preparation process, so that the yield can be up to more than 96%, the environmental protection pressure is reduced, and the material cost is saved; the acetic acid is only small, compared with the large amount of acetic anhydride in the comparative example 1, the invention is more suitable for industrialization, has less environmental pollution and lower cost, and the acetyl content of the prepared acetylated sodium hyaluronate is above 27 percent, and meets the acetyl content within the range of 23-29 percent, so that the product quality is better.
Example 5
In this embodiment, a preparation method of acetylated sodium hyaluronate is provided, and the preparation method includes the following steps:
step one: sodium hyaluronate and DMF are mixed according to the mass ratio of 1:6, mixing to obtain a first mixed solution; the molecular weight of the sodium hyaluronate is 20kDa;
step two: acetic acid and sodium hyaluronate are mixed according to the mass ratio of 0.2:1 with DMF, adding an activating reagent EDCI/organic base TEA for pre-activation, wherein the mass ratio of the adding mass of the activating reagent DIC to the mass ratio of sodium hyaluronate is 0.6:1, wherein the mass ratio of the organic base DMAP to the sodium hyaluronate is 0.6:1, obtaining a second mixed solution;
step three: uniformly mixing the second mixed solution with the first mixed solution, and stirring and reacting for 6 hours at 45 ℃ to obtain a reaction solution;
step four: slowly adding the reaction solution in the third step into 20 times of ethanol, stirring, and suction filtering to separate out a solid crude product;
step five: and purifying the solid crude product by using ethanol, and drying the solid crude product in vacuum at 45 ℃ for 8 hours to obtain an acetylated sodium hyaluronate solid pure product.
The relevant indexes of the obtained sodium hyaluronate are shown in Table 2 in detail.
Example 6
In this embodiment, a preparation method of acetylated sodium hyaluronate is provided, and the preparation method includes the following steps:
step one: sodium hyaluronate and DMF are mixed according to the mass ratio of 1:6, mixing to obtain a first mixed solution; the molecular weight of the sodium hyaluronate is 20kDa;
step two: acetic acid and sodium hyaluronate are mixed according to the mass ratio of 0.2:1 with DMF, adding an activating reagent HATU/organic base DIEA for pre-activation, wherein the mass ratio of the adding mass of the activating reagent DIC to the mass ratio of sodium hyaluronate is 0.6:1, wherein the mass ratio of the organic base DMAP to the sodium hyaluronate is 0.6:1, obtaining a second mixed solution;
step three: uniformly mixing the second mixed solution with the first mixed solution, and stirring and reacting for 6 hours at 45 ℃ to obtain a reaction solution;
step four: slowly adding the reaction solution in the third step into 20 times of ethanol, stirring, and suction filtering to separate out a solid crude product;
step five: and purifying the solid crude product by using ethanol, and drying the solid crude product in vacuum at 45 ℃ for 8 hours to obtain an acetylated sodium hyaluronate solid pure product.
The relevant indexes of the obtained sodium hyaluronate are shown in Table 2 in detail.
Example 7
In this embodiment, a preparation method of acetylated sodium hyaluronate is provided, and the preparation method includes the following steps:
step one: sodium hyaluronate and DMF are mixed according to the mass ratio of 1:6, mixing to obtain a first mixed solution; the molecular weight of the sodium hyaluronate is 20kDa;
step two: acetic acid and sodium hyaluronate are mixed according to the mass ratio of 0.2:1 with DMF, adding an activating reagent HBTU/organic base NMM for pre-activation, wherein the mass ratio of the adding mass of the activating reagent DIC to the mass ratio of sodium hyaluronate is 0.6:1, wherein the mass ratio of the organic base DMAP to the sodium hyaluronate is 0.6:1, obtaining a second mixed solution;
step three: uniformly mixing the second mixed solution with the first mixed solution, and stirring and reacting for 6 hours at 45 ℃ to obtain a reaction solution;
step four: slowly adding the reaction solution in the third step into 20 times of ethanol, stirring, and suction filtering to separate out a solid crude product;
step five: and purifying the solid crude product by using ethanol, and drying the solid crude product in vacuum at 45 ℃ for 8 hours to obtain an acetylated sodium hyaluronate solid pure product.
The relevant indexes of the obtained sodium hyaluronate are shown in Table 2 in detail.
Example 8
In this embodiment, a preparation method of acetylated sodium hyaluronate is provided, and the preparation method includes the following steps:
step one: sodium hyaluronate and DMF are mixed according to the mass ratio of 1:6, mixing to obtain a first mixed solution; the molecular weight of the sodium hyaluronate is 20kDa;
step two: acetic acid and sodium hyaluronate are mixed according to the mass ratio of 0.2:1 is mixed with DMF, an activating reagent EEDQ/organic base DMAP is added for preactivation, and the mass ratio of the adding mass of the activating reagent DIC to the mass ratio of sodium hyaluronate is 0.6:1, wherein the mass ratio of the organic base DMAP to the sodium hyaluronate is 0.6:1, obtaining a second mixed solution;
step three: uniformly mixing the second mixed solution with the first mixed solution, and stirring and reacting for 6 hours at 45 ℃ to obtain a reaction solution;
step four: slowly adding the reaction solution in the third step into 20 times of ethanol, stirring, and suction filtering to separate out a solid crude product;
step five: and purifying the solid crude product by using ethanol, and drying the solid crude product in vacuum at 45 ℃ for 8 hours to obtain an acetylated sodium hyaluronate solid pure product.
The relevant indexes of the obtained sodium hyaluronate are shown in Table 2 in detail.
Table 2 index of sodium hyaluronate obtained in example 1 and examples 5 to 8
| # | Molecular weight-kDa | Activating reagent | Organic base | Reaction temperature (DEG C) | Yield/% | Acetyl content/% |
| Example 1 | 20 | DIC | DMAP | 45 | 98.9 | 28.6 |
| Example 5 | 20 | EDCI | TEA | 45 | 96.5 | 25.8 |
| Example 6 | 20 | HATU | DIEA | 45 | 95.6 | 26.7 |
| Example 7 | 20 | HBTU | NMM | 45 | 97.5 | 24.5 |
| Example 8 | 20 | EEDQ | DMAP | 45 | 96.7 | 27.1 |
As can be seen from Table 2, in the preparation method provided by the invention, the activating reagent DIC, EDCI, HATU, HBTU, EEDQ and the organic base DIEA, TEA, DMAP, NMM are used in combination with acetic acid, and the advantages of using the activating reagent and the organic base are high acetylation efficiency and less side reaction; the carboxyl of acetic acid is pre-activated by an activating reagent to form an active intermediate, the active intermediate and hydroxyl in sodium hyaluronate are easier to esterify under the catalysis of organic base, side reactions are fewer, and finally the yield and quality of acetylated hyaluronate are improved, wherein DIC is matched with DMAP to achieve the best use effect, the highest yield and the highest acetyl content of the obtained product.
Example 9
In this embodiment, a method for preparing acetylated potassium hyaluronate is provided, the method comprising the following steps:
step one: acetic acid and potassium hyaluronate are mixed according to the mass ratio of 0.1:1 is mixed with DCM, an activating reagent DIC/organic base DMAP is added for pre-activation, and the mass ratio of the adding amount of the activating reagent DIC to the potassium hyaluronate is 0.1:1, wherein the mass ratio of the adding amount of the organic base DMAP to the potassium hyaluronate is 0.1:1, obtaining a second mixed solution;
step two: the mass volume ratio of the potassium hyaluronate to the DCM is 1:3, mixing to obtain a first mixed solution; the molecular weight of the potassium hyaluronate is 1kDa;
step three: uniformly mixing the second mixed solution with the first mixed solution, and stirring at 60 ℃ for reaction for 4 hours to obtain a reaction solution;
step four: slowly adding the reaction solution in the third step into 20 times of ethanol, stirring, and suction filtering to separate out a solid crude product;
step five: and purifying the solid crude product by using ethanol, and drying the solid crude product in vacuum at 45 ℃ for 8 hours to obtain an acetylated potassium hyaluronate solid pure product.
The relevant indexes of the obtained acetylated potassium hyaluronate are shown in Table 3 in detail.
Example 10
In this embodiment, a preparation method of acetylated magnesium hyaluronate is provided, and the preparation method includes the following steps:
step one: the mass volume ratio of the magnesium hyaluronate to the toluene is 1:8, mixing to obtain a first mixed solution; the molecular weight of the magnesium hyaluronate is 1kDa;
step two: acetic acid and magnesium hyaluronate are mixed according to the mass ratio of 0.5:1 is mixed with toluene, an activating reagent DIC/organic base DMAP is added for pre-activation, and the mass ratio of the adding amount of the activating reagent DIC to the magnesium hyaluronate is 0.5:1, wherein the mass ratio of the adding amount of the organic base DMAP to the magnesium hyaluronate is 0.5:1, obtaining a second mixed solution;
step three: uniformly mixing the second mixed solution with the first mixed solution, and stirring and reacting for 24 hours at 25 ℃ to obtain a reaction solution;
step four: slowly adding the reaction solution in the third step into 20 times of ethanol, stirring, and suction filtering to separate out a solid crude product;
step five: and (3) purifying the solid crude products respectively by ethanol, and drying the solid crude products in vacuum at 45 ℃ for 8 hours to obtain the acetylated magnesium hyaluronate solid pure product.
The relevant indexes of the obtained acetylated magnesium hyaluronate are shown in Table 3 in detail.
Example 11
In this embodiment, a preparation method of acetylated zinc hyaluronate is provided, and the preparation method includes the following steps:
step one: zinc hyaluronate and N-methyl pyrrolidone are mixed according to the mass volume ratio of 1:10, mixing to obtain a first mixed solution; the molecular weight of the zinc hyaluronate is 1kDa;
step two: acetic acid and zinc hyaluronate are mixed according to the mass ratio of 0.4:1 is mixed with N-methyl pyrrolidone, an activating reagent DIC/organic base DMAP is added for pre-activation, and the mass ratio of the adding amount of the activating reagent DIC to the zinc hyaluronate is 1.5:1, wherein the mass ratio of the adding amount of the organic base DMAP to the zinc hyaluronate is 1.5:1, obtaining a second mixed solution;
step three: uniformly mixing the second mixed solution with the first mixed solution, and stirring and reacting for 5 hours at 50 ℃ to obtain a reaction solution;
step four: slowly adding the reaction solution in the third step into 20 times of ethanol, stirring, and suction filtering to separate out a solid crude product;
step five: and (3) purifying the solid crude products respectively by ethanol, and drying the solid crude products in vacuum at 45 ℃ for 8 hours to obtain the acetylated zinc hyaluronate solid pure product.
The relevant indexes of the obtained acetylated zinc hyaluronate are shown in Table 3 in detail.
Comparative example 2
This example refers to patent CN113045686a for a process for the preparation of acetylated hyaluronate, comprising the steps of:
step one: sodium hyaluronate with molecular weight of 1kDa and DMF are mixed according to the mass volume ratio of 1:6, mixing, namely activating sodium hyaluronate by using organic base triethylamine, wherein the mass ratio of the added triethylamine to the sodium hyaluronate is 0.6:1, obtaining a first mixed solution;
step two: acetyl chloride was added to the above mixture in an ice water bath, the mass ratio of chloride acetate to sodium hyaluronate was 0.26: stirring at 1, 45 ℃ for reaction for 6 hours;
step three: slowly adding the reacted liquid into 20 times of ethanol, stirring, and suction filtering to separate out a solid crude product;
step four: pulping the solid crude products with ethanol for 2 times respectively, and suction filtering to obtain solid pure products;
step five: and (5) drying the solid pure product at 45 ℃ in vacuum for 8 hours to obtain the acetylated sodium hyaluronate.
Table 3 index of acetylated hyaluronate obtained in example 1, examples 9 to 11 and comparative example 2
As can be seen from Table 3, the data of the preparation methods provided in examples 1, 9, 10 and 11 and comparative example 2 show that the yields of the preparation methods are over 95%, the acetyl content is higher than that of comparative example 2, the heat release is serious when acetyl chloride is added in comparative example 2, the low temperature is controlled during feeding, and the preparation method is not easy for industrial production.
In conclusion, the preparation method provided by the invention is suitable for various hyaluronate, the yield is over 95%, the acetyl content is higher, and the preparation method is suitable for industrial production and has great use value.
Claims (19)
1. A method for preparing an acetylated hyaluronate, characterized by: the preparation method comprises the following steps:
step one: dissolving hyaluronate in a first organic solvent to obtain a first mixed solution;
step two: mixing acetic acid with a first organic solvent and an activating reagent/organic base to obtain a second mixed solution;
the activating reagent is one or more of N, N ' -diisopropylcarbodiimide, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, N, N, N ', N ' -tetramethyl-O- (7-azabenzotriazole-1-yl) hexafluorophosphate urea, benzotriazole-N, N, N ', N ' -tetramethylurea hexafluorophosphate and 2-ethoxy-1-ethoxycarbonyl-1, 2-dihydroquinoline, and the organic base is one or more of N, N-diisopropylethylamine, triethylamine, 4-dimethylaminopyridine and N-methylmorpholine;
the mass ratio of the activating reagent to the hyaluronate is 0.1: 1-2: 1, a step of;
the mass ratio of the organic base to the hyaluronate is 0.1: 1-2: 1, a step of; the mass ratio of the acetic acid to the hyaluronate is 0.1:1 to 0.5:1, a step of;
step three: uniformly mixing the second mixed solution with the first mixed solution, and reacting at 25-60 ℃ to obtain a reaction solution;
step four: mixing the reaction solution in the third step with a second organic solvent to separate out a solid crude product;
step five: purifying the solid crude product by using a second organic solvent, and drying to obtain an acetylated hyaluronate solid pure product; or (b)
The operation sequence of the first step and the second step can be exchanged successively.
2. The method according to claim 1, characterized in that: the activating reagent/organic base is N, N' -diisopropylcarbodiimide/4-dimethylaminopyridine.
3. The method according to any one of claims 1-2, characterized in that: the first organic solvent comprises one or more of dichloromethane, N-dimethylformamide, toluene and N-methylpyrrolidone; the mass ratio of the hyaluronate to the first organic solvent for dissolution is 1: 3-1: 10.
4. the method according to claim 1, characterized in that: the reaction temperature is 25-60 ℃ and the reaction time is 4-24 h.
5. The method according to claim 4, wherein: the reaction temperature is 25-60 ℃ and the reaction time is 5-7 h.
6. The method according to claim 4, wherein: the reaction temperature is 35-50 ℃ and the reaction time is 4-24 h.
7. The method according to claim 4, wherein: the reaction temperature is 35-50 ℃ and the reaction time is 5-7 h.
8. The method according to claim 4, wherein: the reaction temperature is 45 ℃, and the reaction time is 6 hours.
9. The method according to any one of claims 1-2 or 4-8, wherein: the second organic solvent comprises one or more of ethanol, methyl tertiary butyl ether, methanol and diethyl ether.
10. A method according to claim 3, characterized in that: the second organic solvent comprises one or more of ethanol, methyl tertiary butyl ether, methanol and diethyl ether.
11. The method according to claim 9, wherein: the second organic solvent is ethanol.
12. The method according to claim 10, wherein: the second organic solvent is ethanol.
13. The method according to claim 1, characterized in that: the molecular weight of the hyaluronate is 1-2000 kDa.
14. The method according to any one of claims 1-2 or 4-8 or 10-13, characterized in that: the hyaluronate comprises 1 or a combination of at least 2 of the sodium, potassium, magnesium, calcium, zinc, or ammonium salts of hyaluronic acid.
15. A method according to claim 3, characterized in that: the hyaluronate comprises 1 or a combination of at least 2 of the sodium, potassium, magnesium, calcium, zinc, or ammonium salts of hyaluronic acid.
16. The method according to claim 9, wherein: the hyaluronate comprises 1 or a combination of at least 2 of the sodium, potassium, magnesium, calcium, zinc, or ammonium salts of hyaluronic acid.
17. The method according to claim 1, characterized in that: the mass ratio of the activating reagent to the hyaluronate is 0.1: 1-2: 1, a step of; the mass ratio of the organic base to the hyaluronate is 0.5:1 to 1.5:1.
18. the method according to claim 1, characterized in that: the mass ratio of the activating reagent to the hyaluronate is 0.5:1 to 1.5:1, a step of; the mass ratio of the organic base to the hyaluronate is 0.1: 1-2: 1.
19. the method according to claim 1, characterized in that: the mass ratio of the activating reagent to the hyaluronate is 0.5:1 to 1.5:1, a step of; the mass ratio of the organic base to the hyaluronate is 0.5:1 to 1.5:1.
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| JPH069707A (en) * | 1992-04-21 | 1994-01-18 | Shiseido Co Ltd | Highly acetylated hyaluronic acid and production thereof |
| CN109206537A (en) * | 2018-10-10 | 2019-01-15 | 华熙福瑞达生物医药有限公司 | A kind of preparation method and applications of acetylation Sodium Hyaluronate |
| CN114133419A (en) * | 2021-12-21 | 2022-03-04 | 南京乐韬生物科技有限公司 | Low-molecular-weight acetylated hyaluronate and preparation method and application thereof |
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| JPH069707A (en) * | 1992-04-21 | 1994-01-18 | Shiseido Co Ltd | Highly acetylated hyaluronic acid and production thereof |
| CN109206537A (en) * | 2018-10-10 | 2019-01-15 | 华熙福瑞达生物医药有限公司 | A kind of preparation method and applications of acetylation Sodium Hyaluronate |
| CN114133419A (en) * | 2021-12-21 | 2022-03-04 | 南京乐韬生物科技有限公司 | Low-molecular-weight acetylated hyaluronate and preparation method and application thereof |
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