CN115975070A - Preparation method of acetylated hyaluronate - Google Patents
Preparation method of acetylated hyaluronate Download PDFInfo
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- CN115975070A CN115975070A CN202211637766.1A CN202211637766A CN115975070A CN 115975070 A CN115975070 A CN 115975070A CN 202211637766 A CN202211637766 A CN 202211637766A CN 115975070 A CN115975070 A CN 115975070A
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- hyaluronate
- acetylated
- organic solvent
- mass ratio
- mixed solution
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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 64
- 229940014041 hyaluronate Drugs 0.000 title claims abstract description 57
- 238000002360 preparation method Methods 0.000 title claims abstract description 32
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims abstract description 94
- 238000006243 chemical reaction Methods 0.000 claims abstract description 50
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 42
- 238000000034 method Methods 0.000 claims abstract description 41
- 230000003213 activating effect Effects 0.000 claims abstract description 40
- 239000003960 organic solvent Substances 0.000 claims abstract description 21
- 230000035484 reaction time Effects 0.000 claims abstract description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 60
- 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
- 238000002156 mixing Methods 0.000 claims description 44
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 41
- 150000007530 organic bases Chemical class 0.000 claims description 38
- 239000000243 solution Substances 0.000 claims description 31
- 239000012265 solid product Substances 0.000 claims description 30
- 229960000549 4-dimethylaminophenol Drugs 0.000 claims description 27
- 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 25
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims description 23
- 239000007787 solid Substances 0.000 claims description 23
- 229920002674 hyaluronan Polymers 0.000 claims description 18
- 229960003160 hyaluronic acid Drugs 0.000 claims description 18
- 239000012043 crude product Substances 0.000 claims description 17
- SJRJJKPEHAURKC-UHFFFAOYSA-N N-Methylmorpholine Chemical compound CN1CCOCC1 SJRJJKPEHAURKC-UHFFFAOYSA-N 0.000 claims description 16
- 239000000047 product Substances 0.000 claims description 15
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 claims description 12
- 238000001035 drying Methods 0.000 claims description 12
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 8
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 claims description 7
- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide Substances CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 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
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-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
- 239000007821 HATU Substances 0.000 claims description 5
- BZLVMXJERCGZMT-UHFFFAOYSA-N Methyl tert-butyl ether Chemical compound COC(C)(C)C BZLVMXJERCGZMT-UHFFFAOYSA-N 0.000 claims description 4
- SECXISVLQFMRJM-UHFFFAOYSA-N N-Methylpyrrolidone Chemical compound CN1CCCC1=O SECXISVLQFMRJM-UHFFFAOYSA-N 0.000 claims description 4
- 239000003513 alkali Substances 0.000 claims description 3
- 239000003795 chemical substances by application Substances 0.000 claims description 3
- 150000003863 ammonium salts Chemical class 0.000 claims description 2
- 238000004090 dissolution Methods 0.000 claims description 2
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims 1
- 159000000007 calcium salts Chemical class 0.000 claims 1
- 159000000003 magnesium salts Chemical class 0.000 claims 1
- XAEFZNCEHLXOMS-UHFFFAOYSA-M potassium benzoate Chemical compound [K+].[O-]C(=O)C1=CC=CC=C1 XAEFZNCEHLXOMS-UHFFFAOYSA-M 0.000 claims 1
- 159000000000 sodium salts Chemical class 0.000 claims 1
- 150000003751 zinc Chemical class 0.000 claims 1
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 abstract description 35
- 238000003756 stirring Methods 0.000 abstract description 28
- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid Substances OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 abstract description 13
- 230000021736 acetylation Effects 0.000 abstract description 8
- 238000006640 acetylation reaction Methods 0.000 abstract description 8
- 230000000694 effects Effects 0.000 abstract description 7
- 238000009776 industrial production Methods 0.000 abstract description 5
- 238000001914 filtration Methods 0.000 abstract description 4
- 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 2
- 239000002351 wastewater Substances 0.000 abstract description 2
- 239000003053 toxin Substances 0.000 abstract 1
- 231100000765 toxin Toxicity 0.000 abstract 1
- 229920002385 Sodium hyaluronate Polymers 0.000 description 83
- 229940010747 sodium hyaluronate Drugs 0.000 description 83
- 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 83
- 238000000967 suction filtration Methods 0.000 description 13
- 125000000218 acetic acid group Chemical group C(C)(=O)* 0.000 description 10
- 230000000052 comparative effect Effects 0.000 description 10
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 9
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 9
- 239000011777 magnesium Substances 0.000 description 9
- 229910052749 magnesium Inorganic materials 0.000 description 9
- 239000011591 potassium Substances 0.000 description 9
- 229910052700 potassium Inorganic materials 0.000 description 9
- 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
- 238000005886 esterification reaction Methods 0.000 description 5
- 230000003020 moisturizing effect Effects 0.000 description 5
- WETWJCDKMRHUPV-UHFFFAOYSA-N acetyl chloride Chemical compound CC(Cl)=O WETWJCDKMRHUPV-UHFFFAOYSA-N 0.000 description 4
- 238000006555 catalytic reaction Methods 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 238000001291 vacuum drying Methods 0.000 description 4
- -1 7-azabenzotriazol-1-yl Chemical group 0.000 description 3
- 241000282414 Homo sapiens Species 0.000 description 3
- 239000012346 acetyl chloride Substances 0.000 description 3
- 238000005917 acylation reaction Methods 0.000 description 3
- 239000003054 catalyst Substances 0.000 description 3
- 230000002349 favourable effect Effects 0.000 description 3
- 230000001050 lubricating effect Effects 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 239000002994 raw material Substances 0.000 description 3
- 230000001105 regulatory effect Effects 0.000 description 3
- 150000001242 acetic acid derivatives Chemical class 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 238000003912 environmental pollution Methods 0.000 description 2
- 230000032050 esterification Effects 0.000 description 2
- 230000014759 maintenance of location Effects 0.000 description 2
- 230000004060 metabolic process 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
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 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
- BDNKZNFMNDZQMI-UHFFFAOYSA-N 1,3-diisopropylcarbodiimide Chemical compound CC(C)N=C=NC(C)C BDNKZNFMNDZQMI-UHFFFAOYSA-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
- 241000283690 Bos taurus Species 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-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
- 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 description 1
- OFOBLEOULBTSOW-UHFFFAOYSA-N Malonic acid Chemical compound OC(=O)CC(O)=O OFOBLEOULBTSOW-UHFFFAOYSA-N 0.000 description 1
- 108010040201 Polymyxins Proteins 0.000 description 1
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 1
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 230000032683 aging 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
- 210000004204 blood vessel Anatomy 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 239000012295 chemical reaction liquid Substances 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 238000002425 crystallisation Methods 0.000 description 1
- 230000008025 crystallization Effects 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
- 239000003792 electrolyte Substances 0.000 description 1
- 239000003623 enhancer Substances 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 239000000446 fuel Substances 0.000 description 1
- 229960002442 glucosamine Drugs 0.000 description 1
- 229940097043 glucuronic acid Drugs 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 239000005457 ice water Substances 0.000 description 1
- 239000011261 inert gas Substances 0.000 description 1
- WFKAJVHLWXSISD-UHFFFAOYSA-N isobutyramide Chemical compound CC(C)C(N)=O WFKAJVHLWXSISD-UHFFFAOYSA-N 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
- 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
- 239000002244 precipitate Substances 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000004537 pulping Methods 0.000 description 1
- 230000003252 repetitive effect Effects 0.000 description 1
- 230000009759 skin aging Effects 0.000 description 1
- 230000008591 skin barrier function Effects 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 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
- 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
- 230000029663 wound healing Effects 0.000 description 1
- 230000037303 wrinkles Effects 0.000 description 1
- 239000011701 zinc Substances 0.000 description 1
- 229910052725 zinc Inorganic materials 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
Landscapes
- Polysaccharides And Polysaccharide Derivatives (AREA)
Abstract
The invention relates to a preparation method of acetylated hyaluronate, belonging to the technical field of biological medicine, and the method 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 for 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 to obtain the acetylated hyaluronate. In the preparation process, reagents of acetic anhydride and concentrated sulfuric acid which are easy to prepare toxin are not used, only a small amount of acetic acid is used, so that the production cost is reduced to the maximum extent, the preparation reagents are easy to obtain, the preparation method is more suitable for industrial production, the discharge amount of wastewater is reduced, the pollution to the environment is reduced, and the yield and the quality of the acetylated hyaluronate are improved. 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 medicines, and particularly relates to a preparation method of acetylated hyaluronate.
Background
Hyaluronic acid, also known as hyaluronic acid, is an acidic linear polymyxin formed by the repetitive arrangement of disaccharides of glucuronic acid and glucosamine. In 1934 Meyer et al, university of Columbia, USA, first separated the material from the vitreous of bovine eyes. Hyaluronic acid exhibits various important physiological functions in the body with its unique molecular structure and physicochemical properties, such as lubricating joints, regulating permeability of blood vessel walls, regulating proteins, regulating diffusion and operation of aqueous electrolytes, promoting wound healing, and the like. More importantly, hyaluronic acid has a special water retention effect, is a substance which is found in nature and has better moisture retention property, and is called as an ideal natural moisture retention 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 significant amount of hyaluronic acid. The skin aging process of human beings also changes along with the content and metabolism of hyaluronic acid, which can improve the nutrition metabolism of the skin, make the skin tender and smooth, remove wrinkles, increase the elasticity and prevent aging, and is a good transdermal absorption enhancer while preserving moisture.
The acetylated hyaluronate has the effects of moisturizing, repairing skin barrier, increasing skin elasticity and the like more efficiently than the traditional sodium hyaluronate. The acetyl is added to the hyaluronic acid structure, so that the biological activity difference is obvious, the solution has good elasticity and moisturizing and lubricating functions, and the hyaluronic acid solution is widely applied to moisturizing and beautifying cosmetics, ophthalmic surgery fuel agents and bone joint lubricating lubricants, greatly enhances the moisturizing performance, and is easier to absorb by a human body. The acetylated hyaluronic acid can be absorbed by skin when being used externally, increases the content of subcutaneous tissues, can effectively increase the skin elasticity, achieves the effect of deep moisturizing, can supplement the deficiency in the body when being taken orally, has obvious effects on beautifying, preventing and treating osteoarthritis, and shows good application prospects 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 a sodium hyaluronate acetylated, which is prepared by performing acylation reaction on hyaluronic acid or its salt in a mixed solvent of acetic acid and acetic anhydride under the catalysis of concentrated sulfuric acid, adding a reaction liquid stream into water after the reaction is finished to separate out a precipitate, filtering, and washing with a large amount of water; CN114133419B discloses a low molecular weight acetylated hyaluronate and a preparation method and application thereof, under the protection of inert gas, adding low molecular weight hyaluronic acid or a salt thereof into a mixed solution of acetic acid and acetic anhydride, and adding a concentrated sulfuric acid catalyst for acylation reaction; after the reaction is finished, adding the reaction solution into an ether solvent for crystallization, filtering, washing, adjusting the pH value with alkali liquor, and drying to obtain the catalyst. The reaction process of the two uses the controlled acetic anhydride and the concentrated sulfuric acid, has complex operation, large pollution to the environment, spray drying, higher cost and is not beneficial to industrial production. CN 113045686B discloses a preparation method of acetylated hyaluronic acid, which adopts organic base to activate hyaluronic acid, and then adopts acetyl chloride to acetylate the activated hyaluronic acid, and the patent adopts organic base to activate hyaluronic acid and finally prepares acetylated hyaluronate, although reagents such as acetic anhydride, concentrated sulfuric acid and the like are not used, the yield of the prepared product is low; and the catalyst is used singly for catalysis, so the catalytic effect is poor.
At present, great difficulties are encountered in improving yield and 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 improves the yield and quality of products, reduces the cost, simultaneously does not need raw materials such as acetic anhydride and sulfuric acid, and solves the problems of low yield of products, high cost, large environmental pollution, product preparation raw materials and difficult obtainment in the prior art. In order 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:
the method comprises the following steps: 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 and the first mixed solution, and reacting at a certain temperature to obtain a reaction solution;
step four: mixing the reaction solution obtained in the third step with a second organic solvent to separate out a solid crude product;
step five: purifying the crude solid product by using a second organic solvent, and drying to obtain an acetylated hyaluronate solid pure product; or the operation sequence of the step one and the step two can be exchanged in sequence.
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 a second reaction solution generated by the acetic acid and the hyaluronate are subjected to esterification reaction, and the acetylated hyaluronate is produced. Only with pre-activated acetic acid, the acetylated hyaluronate was obtained with better quality.
As an 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) urea hexafluorophosphate ], HBTU (benzotriazole-N, N, N ', N ' -tetramethyluronium 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), 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 favorable for 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 hyaluronic acid salt is 0.1:1 to 0.5:1. the amount of acetic acid is too small, and the acetylation degree is insufficient; the acetic acid content is too high, the acetylation degree is basically unchanged, and the cost is increased. The mass ratio range is favorable for reducing the cost while ensuring the acetylation degree. The mass ratio of the activating agent to the hyaluronate is 0.1:1 to 2:1, preferably 0.5:1 to 1.5:1; the mass ratio of the organic base to the hyaluronate is 0.1:1 to 2:1, preferably 0.5:1 to 1.5:1; the mass ratio range is favorable for reducing the cost while ensuring the acetylation degree.
As an embodiment of the present invention, the first organic solvent comprises one or more of DCM (dioxymethane), DMF (N, N-dimethylformamide), toluene, N-methylpyrrolidone; the mass ratio of the hyaluronic acid salt to the first organic solvent for dissolution is 1:3 to 1:10. the dosage of the first organic solvent is too small, and the product is not easy to stir, so that the reaction yield is influenced under the heterogeneous condition; when the amount of the first organic solvent is too large, the cost is increased. The mass ratio is selected to ensure homogeneous reaction and reduce cost.
As an embodiment of the invention, the reaction temperature is 25-60 ℃, and the reaction time is 4-24 h; the reaction temperature is preferably 35 to 50 ℃ and the reaction time is preferably 5 to 7 hours. When the reaction temperature is 45 ℃, the temperature is the preferable reaction temperature, and the obtained acetylated hyaluronate has optimal quality and highest yield.
As an embodiment of the present invention, the second organic solvent includes one or more of ethanol, methyl tert-butyl ether, methanol, and diethyl ether, preferably ethanol.
In one embodiment of the present invention, the molecular weight of the hyaluronate is 1 to 2000kDa. The preparation method is suitable for the hyaluronate with various sizes and molecular weights, and has wide application range.
As an embodiment of the present invention, the hyaluronic acid salt includes a combination of 1 or at least 2 of sodium, potassium, magnesium, calcium, zinc, or ammonium salts of hyaluronic acid. The preparation method is suitable for various hyaluronate salts and has wide application range.
Compared with the prior art, the invention has the following beneficial effects:
1. according to the invention, acetic acid is dissolved in a second organic solvent, an activating reagent/organic base is added for pre-activation, and then the acetic acid and the hyaluronate are subjected to esterification reaction, so that acetylated hyaluronate is obtained, acetic anhydride and concentrated sulfuric acid are not used in the preparation process, the amount of the used acetic acid is small, the production cost is reduced to the maximum extent, the preparation reagent is easy to obtain, and the method is more suitable for industrial production, reduces the wastewater discharge amount, and reduces the pollution to the environment. The yield and acetylation degree of the acetylated hyaluronate are improved by using the pre-activated acetic acid, and the obtained product has better quality. 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 base has the advantages of low cost and high esterification efficiency.
3. The preparation method is suitable for various hyaluronate salts 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 the embodiments of the present invention are not limited thereto.
A method for preparing acetylated hyaluronate, used in the examples, includes providing hyaluronate materials with different molecular weights from Nanjing Letao Biotech Limited.
The equipment or other reagents/materials used in the examples are commercially available.
The yield of the acetylated hyaluronate salt can be simply converted by the mass ratio of the obtained acetylated hyaluronate salt to the raw material hyaluronate salt.
The acetyl content of the acetylated hyaluronate is determined by an ultraviolet spectrophotometry.
Example 1
In this embodiment, a preparation method of acetylated sodium hyaluronate is provided, which includes the following steps:
the method comprises the following steps: 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: mixing acetic acid and sodium hyaluronate according to a mass ratio of 0.2:1 and DMF, adding an activating reagent DIC/organic base DMAP for preactivation, wherein the mass ratio of the added mass of the activating reagent DIC to the mass of sodium hyaluronate is 0.6:1, wherein the mass ratio of the added mass of the organic base DMAP to the mass of sodium hyaluronate is 0.6:1, obtaining a second mixed solution;
step three: uniformly mixing the second mixed solution and 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 obtained in the third step into 20 times of ethanol, stirring, and performing suction filtration to separate out a solid crude product;
step five: and purifying the crude solid product by using ethanol, and drying the crude solid product for 8 hours at 45 ℃ in vacuum to obtain a pure acetylated sodium hyaluronate solid product.
The relevant indexes of the obtained acetylated sodium hyaluronate are detailed in table 1.
Example 2
In this embodiment, a preparation method of acetylated sodium hyaluronate is provided, which includes the following steps:
the method comprises the following steps: 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: mixing acetic acid and sodium hyaluronate according to a mass ratio of 0.2:1 and DMF, adding an activating reagent DIC/organic base DMAP for preactivation, wherein the mass ratio of the added mass of the activating reagent DIC to the mass of sodium hyaluronate is 0.6:1, the mass ratio of the added mass of the organic base DMAP to the mass of the sodium hyaluronate is 0.6:1, obtaining a second mixed solution;
step three: uniformly mixing the second mixed solution and 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 obtained in the third step into 20 times of ethanol, stirring, and performing suction filtration to separate out a solid crude product;
step five: and purifying the crude solid product by using ethanol, and drying the crude solid product for 8 hours at 45 ℃ in vacuum to obtain a pure acetylated sodium hyaluronate solid product.
The relevant indexes of the obtained acetylated sodium hyaluronate are detailed in table 1.
Example 3
The present embodiment provides a method for preparing acetylated sodium hyaluronate, including the following steps:
the method comprises the following steps: 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: mixing acetic acid and sodium hyaluronate according to a mass ratio of 0.2:1 and DMF, adding an activating reagent DIC/organic base DMAP for preactivation, wherein the mass ratio of the added mass of the activating reagent DIC to the mass of sodium hyaluronate is 0.6:1, wherein the mass ratio of the added mass of the organic base DMAP to the mass of sodium hyaluronate is 0.6:1, obtaining a second mixed solution;
step three: uniformly mixing the second mixed solution and 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 obtained in the third step into 20 times of ethanol, stirring, and performing suction filtration to separate out a solid crude product;
step five: and purifying the crude solid product by using ethanol, and drying the crude solid product for 8 hours at 45 ℃ in vacuum to obtain a pure acetylated sodium hyaluronate solid product.
The related indexes of the obtained acetylated sodium hyaluronate are detailed in table 1.
Example 4
The present embodiment provides a method for preparing acetylated sodium hyaluronate, including the following steps:
the method comprises the following steps: 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: mixing acetic acid and sodium hyaluronate according to a mass ratio of 0.2:1 and DMF, adding an activating reagent DIC/organic base DMAP for preactivation, wherein the mass ratio of the added mass of the activating reagent DIC to the mass of sodium hyaluronate is 0.6:1, wherein the mass ratio of the added mass of the organic base DMAP to the mass of sodium hyaluronate is 0.6:1, obtaining a second mixed solution;
step three: uniformly mixing the second mixed solution and 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 obtained in the third step into 20 times of ethanol, stirring, and performing suction filtration to separate out a solid crude product;
step five: and purifying the crude solid product by using ethanol, and drying the crude solid product for 8 hours at 45 ℃ in vacuum to obtain a pure acetylated sodium hyaluronate solid product.
The relevant indexes of the obtained acetylated sodium hyaluronate are detailed in table 1.
Comparative example 1
Comparative example acetic anhydride is acylated with sodium hyaluronate under the catalysis of concentrated sulfuric acid according to the method disclosed in CN110981991, and the related indexes of the obtained acetylated sodium hyaluronate are detailed in table 1.
The method comprises the following steps: mixing sodium hyaluronate and acetic anhydride according to a mass ratio of 1:13.5, mixing, and carrying out acylation reaction for 6 hours at 45 ℃ in concentrated sulfuric acid; wherein the molecular weight of the sodium hyaluronate is 500kDa; the addition 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 filtering to separate out a solid crude product;
step three: purifying the solid crude product by using ethanol, and drying the solid crude product for 8 hours in vacuum at the temperature of 45 ℃ to obtain the acetylated sodium hyaluronate.
The related indexes of the obtained acetylated sodium hyaluronate are detailed in Table 1
TABLE 1 indices of acetylated sodium hyaluronate obtained from 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 |
Table 1 shows that, in the preparation method provided by the present invention, acetic acid is pre-activated and then undergoes an esterification reaction with sodium hyaluronate, acetic anhydride and sulfuric acid are not used in the preparation process, the yield can reach more than 96%, the environmental protection pressure is reduced, and the material cost is saved; the acetic acid is only a small amount, compared with the acetic anhydride used in a large amount in the comparative example 1, the method is more suitable for industrialization, has less pollution to the environment and lower cost, and simultaneously, the acetyl content of the prepared acetylated sodium hyaluronate is over 27 percent and meets the requirement that the acetyl content is in the range of 23-29 percent, so the product quality is better.
Example 5
The present embodiment provides a method for preparing acetylated sodium hyaluronate, including the following steps:
the method comprises the following steps: 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: mixing acetic acid and sodium hyaluronate according to a mass ratio of 0.2:1 and DMF, adding an activating reagent EDCI/organic base TEA for preactivation, wherein the mass ratio of the added activating reagent DIC to the mass of the sodium hyaluronate is 0.6:1, the mass ratio of the added mass of the organic base DMAP to the mass of the sodium hyaluronate is 0.6:1, obtaining a second mixed solution;
step three: uniformly mixing the second mixed solution and 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 obtained in the third step into 20 times of ethanol, stirring, and performing suction filtration to separate out a solid crude product;
step five: and purifying the crude solid product by using ethanol, and performing vacuum drying at 45 ℃ for 8 hours to obtain an acetylated sodium hyaluronate solid pure product.
The relevant indexes of the obtained acetylated sodium hyaluronate are detailed in table 2.
Example 6
In this embodiment, a preparation method of acetylated sodium hyaluronate is provided, which includes the following steps:
the method comprises the following steps: 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: mixing acetic acid and sodium hyaluronate according to a mass ratio of 0.2:1 and DMF, adding an activating reagent HATU/organic base DIEA for preactivation, wherein the mass ratio of the added mass of the activating reagent DIC to the mass of sodium hyaluronate is 0.6:1, the mass ratio of the added mass of the organic base DMAP to the mass of the sodium hyaluronate is 0.6:1, obtaining a second mixed solution;
step three: uniformly mixing the second mixed solution and 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 obtained in the third step into 20 times of ethanol, stirring, and performing suction filtration to separate out a solid crude product;
step five: and purifying the crude solid product by using ethanol, and drying the crude solid product for 8 hours at 45 ℃ in vacuum to obtain a pure acetylated sodium hyaluronate solid product.
The relevant indexes of the obtained acetylated sodium hyaluronate are detailed in table 2.
Example 7
The present embodiment provides a method for preparing acetylated sodium hyaluronate, including the following steps:
the method comprises the following steps: 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: mixing acetic acid and sodium hyaluronate according to a mass ratio of 0.2:1 and DMF, adding an activating reagent HBTU/organic base NMM for preactivation, wherein the mass ratio of the added mass of the activating reagent DIC to the mass of sodium hyaluronate is 0.6:1, the mass ratio of the added mass of the organic base DMAP to the mass of the sodium hyaluronate is 0.6:1, obtaining a second mixed solution;
step three: uniformly mixing the second mixed solution and 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 obtained in the third step into 20 times of ethanol, stirring, and performing suction filtration to separate out a solid crude product;
step five: and purifying the crude solid product by using ethanol, and drying the crude solid product for 8 hours at 45 ℃ in vacuum to obtain a pure acetylated sodium hyaluronate solid product.
The relevant indexes of the obtained acetylated sodium hyaluronate are detailed in table 2.
Example 8
In this embodiment, a preparation method of acetylated sodium hyaluronate is provided, which includes the following steps:
the method comprises the following steps: 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: mixing acetic acid and sodium hyaluronate according to a mass ratio of 0.2:1 and DMF, adding an activating reagent EEDQ/organic base DMAP for preactivation, wherein the mass ratio of the added mass of the activating reagent DIC to the mass of sodium hyaluronate is 0.6:1, the mass ratio of the added mass of the organic base DMAP to the mass of the sodium hyaluronate is 0.6:1, obtaining a second mixed solution;
step three: uniformly mixing the second mixed solution and 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 obtained in the third step into 20 times of ethanol, stirring, and performing suction filtration to separate out a solid crude product;
step five: and purifying the crude solid product by using ethanol, and drying the crude solid product for 8 hours at 45 ℃ in vacuum to obtain a pure acetylated sodium hyaluronate solid product.
The relevant indexes of the obtained acetylated sodium hyaluronate are detailed in table 2.
TABLE 2 indices of acetylated sodium hyaluronate obtained in example 1 and examples 5 to 8
| # | Molecular weight/kDa | Activating reagent | Organic base | Reaction temperature of | 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 found from Table 2, in the preparation method provided by the invention, the activating reagents DIC, EDCI, HATU, HBTU, EEDQ and organic bases DIEA, TEA, DMAP and NMM are used in combination with acetic acid, and the activating reagents and the organic bases have the advantages of high acetylation efficiency and less side reactions; during the reaction, the carboxyl of the acetic acid is pre-activated by an activating reagent to form an active intermediate, the active intermediate and the hydroxyl in the sodium hyaluronate are easier to esterify under the catalysis of organic alkali, the side reactions are fewer, the yield and the quality of the acetylated hyaluronate are finally improved, and the use effect of DIC (DiCarboxylic acid) in cooperation with DMAP (Dimethylacetylamide) is optimal, the yield is highest, and the acetyl content of the obtained product is highest.
Example 9
In this embodiment, a preparation method of acetylated potassium hyaluronate is provided, including the following steps:
the method comprises the following steps: mixing acetic acid and potassium hyaluronate according to a mass ratio of 0.1:1 and DCM are mixed, an activating reagent DIC/organic base DMAP is added for preactivation, and the mass ratio of the addition amount of the activating reagent DIC to the mass ratio of potassium hyaluronate is 0.1:1, wherein the mass ratio of the addition amount of the organic base DMAP to the potassium hyaluronate is 0.1:1, obtaining a second mixed solution;
step two: mixing potassium hyaluronate and DCM according to a mass-volume ratio of 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 and the first mixed solution, and stirring and reacting for 4 hours at 60 ℃ to obtain a reaction solution;
step four: slowly adding the reaction solution obtained in the third step into 20 times of ethanol, stirring, and performing suction filtration to separate out a solid crude product;
step five: and purifying the crude solid product by using ethanol, and performing vacuum drying at 45 ℃ for 8 hours to obtain an acetylated potassium hyaluronate solid pure product.
The related indexes of the obtained acetylated potassium hyaluronate are detailed in table 3.
Example 10
In this embodiment, a method for preparing acetylated magnesium hyaluronate is provided, which includes the following steps:
the method comprises the following steps: and (2) mixing magnesium hyaluronate and toluene according to a mass-volume ratio of 1:8, mixing to obtain a first mixed solution; the molecular weight of the magnesium hyaluronate is 1kDa;
step two: mixing acetic acid and magnesium hyaluronate according to a mass ratio of 0.5:1 and toluene, adding an activating reagent DIC/organic base DMAP for preactivation, wherein the mass ratio of the addition amount of the activating reagent DIC to the magnesium hyaluronate is 0.5:1, wherein the mass ratio of the addition 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 and 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 obtained in the third step into 20 times of ethanol, stirring, and performing suction filtration to separate out a solid crude product;
step five: and respectively purifying the crude solid products by using ethanol, and performing vacuum drying at 45 ℃ for 8h to obtain the acetylated magnesium hyaluronate solid pure product.
The related indexes of the obtained acetylated magnesium hyaluronate are detailed in table 3.
Example 11
The present embodiment provides a preparation method of acetylated zinc hyaluronate, including the following steps:
the method comprises the following steps: 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: mixing acetic acid and zinc hyaluronate according to a mass ratio of 0.4:1 and N-methylpyrrolidone, adding an activating reagent DIC/organic base DMAP for preactivation, wherein the mass ratio of the addition amount of the activating reagent DIC to zinc hyaluronate is 1.5:1, the mass ratio of the addition 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 and 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 obtained in the third step into 20 times of ethanol, stirring, and performing suction filtration to separate out a solid crude product;
step five: and respectively purifying the crude solid products by using ethanol, and performing vacuum drying at 45 ℃ for 8h to obtain pure acetylated zinc hyaluronate solid products.
The related indexes of the obtained zinc hyaluronate are shown in the table 3 in detail.
Comparative example 2
This example provides a process for the preparation of acetylated hyaluronate, referred to in patent CN113045686A, comprising the following steps:
the method comprises the following steps: sodium hyaluronate with the molecular weight of 1kDa and DMF are mixed according to the mass-volume ratio of 1:6, mixing, activating sodium hyaluronate by adopting 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: adding acetyl chloride into the mixture in an ice water bath, wherein the mass ratio of the acetic chloride to the sodium hyaluronate is 0.26: stirring and reacting for 6 hours at the temperature of 1,45 ℃;
step three: slowly adding the reacted liquid into 20 times of ethanol, stirring, and performing suction filtration to separate out a solid crude product;
step four: pulping the crude solid product with ethanol for 2 times respectively, and performing suction filtration to obtain a pure solid product;
step five: and (3) drying the solid pure product in vacuum at 45 ℃ for 8h to obtain the acetylated sodium hyaluronate.
Table 3 indices of acetylated hyaluronate salts obtained in examples 1, 9 to 11 and comparative example 2
As can be seen from Table 3, when comparing the data of the preparation methods provided by the invention, namely example 1, example 9, example 10, example 11 and comparative example 2, the yield of the method is more than 95%, the acetyl content is higher than comparative example 2, the heat release is severe when acetyl chloride is added in comparative example 2, the low temperature needs to be controlled during feeding, and the industrial production is not easy to realize.
In conclusion, the preparation method provided by the invention is suitable for various hyaluronate salts, has the yield of more than 95 percent and higher acetyl content, is suitable for industrial production, and has high use value.
Claims (10)
1. A method for preparing acetylated hyaluronate, which is characterized by comprising the following steps: the preparation method comprises the following steps:
the method comprises the following steps: 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 and the first mixed solution, and reacting at a certain temperature to obtain a reaction solution;
step four: mixing the reaction solution obtained in the third step with a second organic solvent to separate out a solid crude product;
step five: purifying the crude solid 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 changed in sequence.
2. The method of claim 1, wherein: the activating reagent is one or more of DIC, EDCI, HATU, HBTU and EEDQ, and the organic base is one or more of DIEA, TEA, DMAP and NMM.
3. The method of claim 2, wherein: the activating reagent/organic base is DIC/DMAP.
4. The method of claim 1, wherein: the mass ratio of the activating agent to the hyaluronate is 0.1:1 to 2:1, preferably 0.5:1 to 1.5:1; the mass ratio of the organic alkali to the hyaluronate is 0.1:1 to 2:1, preferably 0.5:1 to 1.5:1; the mass ratio of the acetic acid to the hyaluronate is 0.1:1 to 0.5:1.
5. the method according to any one of claims 1 to 4, wherein: the first organic solvent comprises one or more of DCM, DMF, toluene, N-methyl pyrrolidone; the mass ratio of the hyaluronic acid salt to the first organic solvent for dissolution is 1:3 to 1:10.
6. the method of claim 1, wherein: the reaction temperature is 25-60 ℃, and the reaction time is 4-24 h; the reaction temperature is preferably 35-50 ℃ and the reaction time is preferably 5-7 h.
7. The method of claim 6, wherein: the reaction temperature is 45 ℃, and the reaction time is 6h.
8. The method according to any one of claims 1 to 7, wherein: the second organic solvent comprises one or more of ethanol, methyl tert-butyl ether, methanol and diethyl ether, preferably ethanol.
9. The method of claim 1, wherein: the molecular weight of the hyaluronate is 1-2000 kDa.
10. The method according to any one of claims 1 to 9, wherein: the hyaluronic acid salt comprises 1 or a combination of at least 2 of sodium salt, potassium salt, magnesium salt, calcium salt, zinc salt or ammonium salt of hyaluronic acid.
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| JPH069707A (en) * | 1992-04-21 | 1994-01-18 | Shiseido Co Ltd | Highly acetylated hyaluronic acid and production thereof |
| US20060166930A1 (en) * | 2003-02-27 | 2006-07-27 | Norio Ueno | Medicinal composition for ophthal use containing acetylated hyaluronic acid |
| 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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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH069707A (en) * | 1992-04-21 | 1994-01-18 | Shiseido Co Ltd | Highly acetylated hyaluronic acid and production thereof |
| US20060166930A1 (en) * | 2003-02-27 | 2006-07-27 | Norio Ueno | Medicinal composition for ophthal use containing acetylated hyaluronic acid |
| 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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