CN116178588A - Preparation method of sulfhydrylation natural polysaccharide derivative - Google Patents
Preparation method of sulfhydrylation natural polysaccharide derivative Download PDFInfo
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- CN116178588A CN116178588A CN202310443410.2A CN202310443410A CN116178588A CN 116178588 A CN116178588 A CN 116178588A CN 202310443410 A CN202310443410 A CN 202310443410A CN 116178588 A CN116178588 A CN 116178588A
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- natural polysaccharide
- thiolated
- sulfhydrylation
- polysaccharide derivative
- ratio
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- 150000004676 glycans Chemical class 0.000 title claims abstract description 104
- 229920001282 polysaccharide Polymers 0.000 title claims abstract description 104
- 239000005017 polysaccharide Substances 0.000 title claims abstract description 104
- 238000002360 preparation method Methods 0.000 title claims abstract description 23
- 238000006243 chemical reaction Methods 0.000 claims abstract description 76
- 239000003054 catalyst Substances 0.000 claims abstract description 66
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 60
- 239000000126 substance Substances 0.000 claims abstract description 58
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims abstract description 49
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims abstract description 42
- 230000003213 activating effect Effects 0.000 claims abstract description 39
- 238000000034 method Methods 0.000 claims abstract description 21
- 239000000463 material Substances 0.000 claims abstract description 10
- 238000011065 in-situ storage Methods 0.000 claims abstract description 9
- 239000003431 cross linking reagent Substances 0.000 claims abstract description 8
- 238000004132 cross linking Methods 0.000 claims abstract description 6
- 125000006239 protecting group Chemical group 0.000 claims abstract description 6
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- 230000001590 oxidative effect Effects 0.000 claims abstract 2
- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide Chemical group CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 claims description 102
- 125000003396 thiol group Chemical group [H]S* 0.000 claims description 53
- 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 group 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 36
- 229920002674 hyaluronan Polymers 0.000 claims description 26
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- 239000011243 crosslinked material Substances 0.000 claims description 9
- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical group Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 claims description 8
- 229940099500 cystamine Drugs 0.000 claims description 8
- 238000000746 purification Methods 0.000 claims description 8
- GVJXGCIPWAVXJP-UHFFFAOYSA-N 2,5-dioxo-1-oxoniopyrrolidine-3-sulfonate Chemical compound ON1C(=O)CC(S(O)(=O)=O)C1=O GVJXGCIPWAVXJP-UHFFFAOYSA-N 0.000 claims description 7
- 229920001287 Chondroitin sulfate Polymers 0.000 claims description 6
- 229940059329 chondroitin sulfate Drugs 0.000 claims description 6
- 239000003638 chemical reducing agent Substances 0.000 claims description 5
- 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 claims description 4
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 claims description 4
- 229940014041 hyaluronate Drugs 0.000 claims description 4
- PBVAJRFEEOIAGW-UHFFFAOYSA-N 3-[bis(2-carboxyethyl)phosphanyl]propanoic acid;hydrochloride Chemical compound Cl.OC(=O)CCP(CCC(O)=O)CCC(O)=O PBVAJRFEEOIAGW-UHFFFAOYSA-N 0.000 claims description 2
- 150000001252 acrylic acid derivatives Chemical class 0.000 claims description 2
- AFOSIXZFDONLBT-UHFFFAOYSA-N divinyl sulfone Chemical compound C=CS(=O)(=O)C=C AFOSIXZFDONLBT-UHFFFAOYSA-N 0.000 claims description 2
- 150000002734 metacrylic acid derivatives Chemical class 0.000 claims description 2
- PEEHTFAAVSWFBL-UHFFFAOYSA-N Maleimide Chemical compound O=C1NC(=O)C=C1 PEEHTFAAVSWFBL-UHFFFAOYSA-N 0.000 claims 1
- 238000005859 coupling reaction Methods 0.000 abstract description 19
- 239000012535 impurity Substances 0.000 abstract description 13
- 230000008878 coupling Effects 0.000 abstract description 8
- 238000010168 coupling process Methods 0.000 abstract description 8
- 125000003277 amino group Chemical group 0.000 abstract description 7
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- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 87
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- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 14
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- 239000012153 distilled water Substances 0.000 description 7
- 239000000203 mixture Substances 0.000 description 7
- 239000011780 sodium chloride Substances 0.000 description 7
- NGDIAZZSCVVCEW-UHFFFAOYSA-M sodium;butyl sulfate Chemical compound [Na+].CCCCOS([O-])(=O)=O NGDIAZZSCVVCEW-UHFFFAOYSA-M 0.000 description 7
- 229920002385 Sodium hyaluronate Polymers 0.000 description 6
- 229940010747 sodium hyaluronate Drugs 0.000 description 6
- 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 6
- 239000000047 product Substances 0.000 description 5
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 4
- PVFIZYYTRXHJPI-UHFFFAOYSA-N carbamimidoyl acetate Chemical compound CC(=O)OC(N)=N PVFIZYYTRXHJPI-UHFFFAOYSA-N 0.000 description 4
- 210000000845 cartilage Anatomy 0.000 description 4
- 230000007547 defect Effects 0.000 description 4
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- 238000006460 hydrolysis reaction Methods 0.000 description 4
- 239000001301 oxygen Substances 0.000 description 4
- 229910052760 oxygen Inorganic materials 0.000 description 4
- 150000003141 primary amines Chemical class 0.000 description 4
- 230000001105 regulatory effect Effects 0.000 description 4
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 239000002202 Polyethylene glycol Substances 0.000 description 3
- 230000002378 acidificating effect Effects 0.000 description 3
- 239000003513 alkali Substances 0.000 description 3
- 239000007864 aqueous solution Substances 0.000 description 3
- 239000006227 byproduct Substances 0.000 description 3
- 230000003197 catalytic effect Effects 0.000 description 3
- UFULAYFCSOUIOV-UHFFFAOYSA-N cysteamine Chemical class NCCS UFULAYFCSOUIOV-UHFFFAOYSA-N 0.000 description 3
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 3
- 235000018417 cysteine Nutrition 0.000 description 3
- 125000004386 diacrylate group Chemical group 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 239000000499 gel Substances 0.000 description 3
- 125000004435 hydrogen atom Chemical group [H]* 0.000 description 3
- -1 mercaptoamino Chemical group 0.000 description 3
- GKRZNOGGALENQJ-UHFFFAOYSA-N n-carbamoylacetamide Chemical compound CC(=O)NC(N)=O GKRZNOGGALENQJ-UHFFFAOYSA-N 0.000 description 3
- 239000003960 organic solvent Substances 0.000 description 3
- 229920001223 polyethylene glycol Polymers 0.000 description 3
- 230000009467 reduction Effects 0.000 description 3
- 238000006177 thiolation reaction Methods 0.000 description 3
- 238000005160 1H NMR spectroscopy Methods 0.000 description 2
- OISVCGZHLKNMSJ-UHFFFAOYSA-N 2,6-dimethylpyridine Chemical compound CC1=CC=CC(C)=N1 OISVCGZHLKNMSJ-UHFFFAOYSA-N 0.000 description 2
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 2
- XLYOFNOQVPJJNP-ZSJDYOACSA-N Heavy water Chemical compound [2H]O[2H] XLYOFNOQVPJJNP-ZSJDYOACSA-N 0.000 description 2
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
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- 125000002947 alkylene group Chemical group 0.000 description 2
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- 239000001257 hydrogen Substances 0.000 description 2
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- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 2
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- VDZOOKBUILJEDG-UHFFFAOYSA-M tetrabutylammonium hydroxide Chemical compound [OH-].CCCC[N+](CCCC)(CCCC)CCCC VDZOOKBUILJEDG-UHFFFAOYSA-M 0.000 description 2
- 230000017423 tissue regeneration Effects 0.000 description 2
- 150000003923 2,5-pyrrolediones Chemical class 0.000 description 1
- 241000251730 Chondrichthyes Species 0.000 description 1
- BWGNESOTFCXPMA-UHFFFAOYSA-N Dihydrogen disulfide Chemical compound SS BWGNESOTFCXPMA-UHFFFAOYSA-N 0.000 description 1
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- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 206010023232 Joint swelling Diseases 0.000 description 1
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 1
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- HPYNZHMRTTWQTB-UHFFFAOYSA-N dimethylpyridine Natural products CC1=CC=CN=C1C HPYNZHMRTTWQTB-UHFFFAOYSA-N 0.000 description 1
- RTZKZFJDLAIYFH-UHFFFAOYSA-N ether Substances CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 1
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- 231100000053 low toxicity Toxicity 0.000 description 1
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- 239000011159 matrix material Substances 0.000 description 1
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- 230000003020 moisturizing effect Effects 0.000 description 1
- 238000007344 nucleophilic reaction Methods 0.000 description 1
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- 150000003839 salts Chemical class 0.000 description 1
- 238000007086 side reaction Methods 0.000 description 1
- RPENMORRBUTCPR-UHFFFAOYSA-M sodium;1-hydroxy-2,5-dioxopyrrolidine-3-sulfonate Chemical compound [Na+].ON1C(=O)CC(S([O-])(=O)=O)C1=O RPENMORRBUTCPR-UHFFFAOYSA-M 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
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Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
- C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
- C08B37/0072—Hyaluronic acid, i.e. HA or hyaluronan; Derivatives thereof, e.g. crosslinked hyaluronic acid (hylan) or hyaluronates
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
- C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
- C08B37/0069—Chondroitin-4-sulfate, i.e. chondroitin sulfate A; Dermatan sulfate, i.e. chondroitin sulfate B or beta-heparin; Chondroitin-6-sulfate, i.e. chondroitin sulfate C; Derivatives thereof
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F251/00—Macromolecular compounds obtained by polymerising monomers on to polysaccharides or derivatives thereof
-
- 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
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- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicinal Chemistry (AREA)
- Polymers & Plastics (AREA)
- Organic Chemistry (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Materials Engineering (AREA)
- Dermatology (AREA)
- Polysaccharides And Polysaccharide Derivatives (AREA)
Abstract
The invention discloses a preparation method of a sulfhydrylation natural polysaccharide derivative, which comprises two steps of reducing amide bonds and disulfide bonds generated by the reaction of side chain carboxyl of the natural polysaccharide with amino groups of a sulfhydrylation amino reagent under the action of a catalyst and an activating reagent, removing sulfhydrylation protecting groups and purifying or directly purifying. The method has the advantages of remarkably reducing the consumption of the activating reagent by controlling the ratio of the substances of the catalyst to the activating reagent, remarkably improving the coupling efficiency between the side chain carboxyl of the natural polysaccharide and the amino of the sulfhydrylation amino reagent, along with mild reaction conditions, simple process, less consumption of the reagent, high reaction efficiency, less residual impurities, good safety and the like; in addition, the sulfhydrylation natural polysaccharide derivative prepared by the invention can form disulfide bond crosslinking materials under the oxidation action of an oxidant without a crosslinking agent; the method can also realize rapid in-situ crosslinking through the biocompatible crosslinking agent, has no reactive impurities, and has good application prospect in the field of biological medicine.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a preparation method of a sulfhydrylation natural polysaccharide derivative.
Background
Hyaluronic Acid (HA) and chondroitin sulfate (Chondroitin Sulfate, CS) are two important natural glycosaminoglycans, with important physiological functions. Hyaluronic acid, also known as "hyaluronic acid", widely distributed in the extracellular matrix of animals and humans, is an important constituent of cellular matrix and various tissues, having various important physiological functions, such as: regulating cell proliferation, migration and differentiation, natural moisturizing effect, lubricating joint protecting cartilage, regulating protein synthesis, regulating inflammatory reaction, regulating immunity, and promoting wound healing. Hyaluronic acid has found wide application in the biomedical field due to its unique viscoelastic properties, biocompatibility and degradability. Chondroitin sulfate is also widely used in animal and human cartilage tissues, can relieve pain of osteoarthritis patients, improve joint functions, effectively reduce joint swelling, accelerate absorption of effusion and promote cartilage repair. However, these natural glycosaminoglycans are readily degraded and absorbed in vivo, and have a relatively short residence time, limiting their use in the biomedical field (Brown et al, exp Physiol 1991,76:125-134; johns et al, feril Steril 1997, 68:37-42).
The natural glycosaminoglycan is chemically modified and crosslinked to form the glycosaminoglycan crosslinked material, so that the glycosaminoglycan crosslinked material not only can endow the glycosaminoglycan with better mechanical strength, rheological property, enzymolysis resistance and the like, but also can effectively overcome the defects of easy degradation and absorption in vivo and short retention time, and can effectively expand the application of the glycosaminoglycan crosslinked material in the field of biological medicine. The sulfhydryl glycosaminoglycan derivative is an important starting material for preparing crosslinked glycosaminoglycans, and has many advantages. For example, thiolated hyaluronic acid derivatives can form in situ crosslinked hydrogels under the influence of oxygen without the need for a crosslinking agent, which is more biocompatible (Shu et al, biomacromolecules 2002, 3:1304-1311); for another example, the thiolated hyaluronic acid derivative and the thiolated chondroitin sulfate derivative can be rapidly crosslinked in situ with a biocompatible crosslinking agent such as polyethylene glycol diacrylate, and the like, has no reactive impurities, can be used for in-situ embedding of cells, slow release of growth factors, and the like, and has important prospects in the field of tissue regeneration and repair (Shu et al, biomaterials 2004, 25:1339-1348; cai et al, biomaterials 2005:6054-6067).
However, the preparation of thiolated glycosaminoglycan derivatives still has some technical problems to be solved. For example, the prior patent CN103910886a discloses a cysteine functionalized hyaluronic acid conjugate, a synthesis method thereof and application thereof in hydrogel formed in situ by injection, the synthesis method disclosed in the prior patent is to modify hydroxyl groups of hyaluronic acid to obtain the cysteine functionalized hyaluronic acid conjugate with stable ether bond, but the preparation method has the defects of complex reaction process, long reaction route, use of a large amount of organic solvents as auxiliary agents and the like; for another example, the prior patent CN112842929a also discloses a thiolated hyaluronic acid, a preparation method and an application thereof, wherein the thiolated hyaluronic acid is obtained by thiol modification of a hydroxyl site of hyaluronic acid or a salt thereof, and in the reaction process, assistants such as tetrabutylammonium hydroxide, lutidine, methanol, ethanol, isopropanol, acetone, dimethyl sulfoxide and the like are used, and the defects of complex reaction process, long reaction route, use of a large amount of organic solvents as the assistants and the like exist.
Carboxyl groups of glycosaminoglycans such as hyaluronic acid and chondroitin sulfate can be catalyzed by water-soluble carbodiimide such as 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride (EDCI) and the like under the aqueous condition to form an O-acetyl isourea intermediate, and then the O-acetyl isourea intermediate and a sulfhydryl amino reagent undergo an amide bond coupling reaction; the sulfhydrylation amino reagents contain free or protected sulfhydrylation or disulfide bond, and the sulfhydrylation hyaluronic acid derivatives with amide bond connection structures (amino modification) can be obtained by direct purification after coupling reaction or purification after removal of sulfhydrylation protecting groups and disulfide bond reduction; the preparation method has the advantages of short reaction route, simple reaction process, no need of using organic solvent and the like. However, the O-acetylisourea intermediate is susceptible to reacting with water to form a more stable N-acetylurea byproduct which cannot continue to undergo a coupling reaction with a mercaptoamino reagent, affecting the synthesis of the final product, and in order to avoid this rearrangement reaction, it is usually necessary to add a certain amount of an activating reagent to form a stable hydrolysis-resistant reactive intermediate, which is then subjected to a coupling reaction with an amino group to form an amide bond, to give a mercaptohyaluronic acid derivative having an amide bond-linked structure (amino-modified), N-succinimide (NHS) and N-hydroxysulfosuccinimide (Sulfo-NHS) being the most commonly used activating reagents. Ding et al (Acta Biomaterialia 2012, 8:3643-3651) disclose the use of cysteamine salts to modify synthetic thiolated hyaluronic acid derivatives, and prior patent CN101367884A, CN104892962A, CN109432496A, CN113185716A et al also disclose cysteamine, cysteine, etc. modified synthetic thiolated hyaluronic acid derivatives, however, these disclosed methods all use an amino reagent containing free thiol groups to modify the carboxyl groups of hyaluronic acid, no protection of the thiol groups during the whole reaction process, and the free thiol groups can also undergo EDCl catalytic coupling reaction with the carboxyl groups of hyaluronic acid, so the end products obtained by these methods are actually thiol-coupled and amino-coupled modified hyaluronic acid mixtures. Furthermore, the suitable pH for the catalytic activation of the carboxyl group reaction is weakly acidic, whereas the amino group has a higher pKa (typically 8-11), under which weakly acidic conditions the nucleophilicity of the protonated amino group is reduced, as is the coupling reactivity with the activated carboxyl group; although the use of an activating reagent such as N-succinimido (NHS) and N-hydroxysulfosuccinimido (Sulfo-NHS) and the optimization of the reaction conditions improves the coupling reactivity of glycosaminoglycans such as hyaluronic acid with a thiolated amino reagent to some extent (CN 115466411A), the reaction efficiency thereof has yet to be improved.
Therefore, there is a need in the art for a preparation method of a sulfhydrylation natural polysaccharide derivative, which reduces the amount of an activating reagent, can improve the coupling efficiency between the side chain carboxyl of the natural polysaccharide and the amino group of the sulfhydrylation amino reagent, and has the advantages of mild reaction conditions, simple process, less reagent amount, high reaction efficiency, less residual impurities, good safety and the like.
Disclosure of Invention
The invention aims to solve the technical problem of providing a preparation method of a sulfhydrylation natural polysaccharide derivative, which can obviously improve the coupling reaction efficiency of a sulfhydrylation amino reagent and the side chain carboxyl of the natural polysaccharide, thereby effectively improving the sulfhydrylation modification degree.
In order to solve the technical problems, the preparation method of the sulfhydrylation natural polysaccharide derivative provided by the invention comprises the following steps:
step 1: dissolving natural polysaccharide in water, adding a sulfhydrylation amino reagent under the action of a catalyst and an activating reagent, and reacting side chain carboxyl of the natural polysaccharide with amino of the sulfhydrylation amino reagent to generate an amide bond;
wherein the natural polysaccharide is hyaluronic acid, hyaluronate, derivatives obtained by modifying hyaluronate, chondroitin sulfate salt and/or derivatives obtained by modifying chondroitin sulfate;
The catalyst is water-soluble carbodiimide;
the activating reagent is one or more of N-hydroxysuccinimide (NHS), N-hydroxysulfosuccinimide (Sulfo-NHS) and N-hydroxysulfosuccinimide salt;
the sulfhydrylation amino reagent is diammine containing disulfide bond, primary amine containing free sulfhydryl or primary amine with sulfhydryl protecting group;
the ratio of the amounts of the substances of the catalyst and the activating reagent is 1:0.25-1:0.75;
step 2: collecting the product obtained in the step 1, treating, and purifying the treated product to obtain the sulfhydrylation natural polysaccharide derivative;
when the sulfhydrylation amino reagent is diammine containing disulfide bond, the product obtained in the step 1 is reacted with a reducing agent, wherein the disulfide bond is reduced into sulfhydryl, and the sulfhydryl natural polysaccharide derivative is obtained after purification;
when the sulfhydrylation amino reagent is primary amine containing free sulfhydrylation, purifying the product obtained in the step 1 to obtain a sulfhydrylation natural polysaccharide derivative;
when the sulfhydrylation amino reagent is primary amine with sulfhydrylation protecting group, the sulfhydrylation protecting group of the product obtained in the step 1 is removed, and the sulfhydrylation natural polysaccharide derivative is obtained by purification.
Specifically, in step 1, the concentration of the solution of the natural polysaccharide in water is not more than 2.0% (w/v).
Specifically, the water-soluble carbodiimide is 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide (EDCI) and/or 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide salt.
In particular, the disulfide bond containing diamino is cystamine and/or cystamine salt.
Specifically, the ratio of the amounts of the substances of the catalyst and the activating agent is 1:0.25-1:0.6.
Preferably, the ratio of the amounts of the substances of the catalyst and the activating agent is 1:0.35-1:0.5.
Specifically, the ratio of the catalyst to the amount of the substance of the side chain carboxyl of the natural polysaccharide is 0.01:1-1:1.
Preferably, the ratio of the amount of the catalyst to the amount of the substance of the side chain carboxyl group of the natural polysaccharide is 0.01:1 to 0.75:1.
Preferably, the ratio of the amount of the catalyst to the amount of the substance of the side chain carboxyl group of the natural polysaccharide is 0.05:1-0.5:1.
Specifically, the ratio of the sulfhydrylation amino reagent to the mass of the side chain carboxyl of the natural polysaccharide is 1:1-6:1.
Preferably, the ratio of the amount of the sulfhydrylation amino reagent to the amount of the substance of the side chain carboxyl group of the natural polysaccharide is 1:1-4:1.
Preferably, the ratio of the amount of the sulfhydrylation amino reagent to the amount of the substance of the side chain carboxyl group of the natural polysaccharide is 1:1-2:1.
Specifically, in the step 2, the reducing agent is dithiothreitol and/or tris (2-carboxyethyl) phosphine hydrochloride.
Specifically, in the step 1, the pH value of the reaction system is 4.5 to 6.5.
Preferably, in the step 1, the pH value of the reaction system is: firstly, reacting for 15-120min under the condition of pH value of 4.5-5.0, then adjusting the pH value to 5.5-6.5, and continuing reacting for 8-16 h.
More preferably, in the step 1, the pH value of the reaction system is: firstly, reacting for 30min under the condition of pH value of 4.75-5.0, then adjusting the pH value to 6.0, and continuing to react for 8-16 h.
Specifically, when the sulfhydrylation amino reagent is cystamine and the activating reagent is N-hydroxysulfosuccinimide (Sulfo-NHS), the chemical reaction process of the preparation method provided by the invention can be expressed as the following reaction formula:
wherein P is a natural polysaccharide (hyaluronic acid or chondroitin sulfate) residue.
In the reaction formula, the side chain carboxyl of the natural polysaccharide firstly reacts with an activating reagent (Sulfo-NHS) under the catalysis of a catalyst (EDCI) to generate a stable hydrolysis resistant activity intermediate (I); then the free amino group of cystamine and the hydrolysis resistant activity intermediate (I) are coupled to generate a cystamine modified natural polysaccharide intermediate (II); finally, the cystamine modified natural polysaccharide intermediate (II) is reduced to a cysteamine modified thiolated natural polysaccharide derivative (III) using a reducing agent.
In the above reaction scheme, it can be understood that, under the same reaction conditions, increasing the amount of the activating reagent (Sulfo-NHS) helps to avoid the rearrangement side reaction of the O-acetylisourea intermediate (formation of N-acetylurea by-product), and accelerates the reaction to generate more hydrolysis-resistant active intermediate (I) and natural polysaccharide intermediate (II), thereby increasing the degree of thiolation modification of the thiolated natural polysaccharide derivative (III). Therefore, in the prior art, the amount of the activating reagent is large, and the ratio of the amount of the catalyst to the amount of the substance of the activating reagent is usually not less than 1, which not only increases the cost, but also increases the burden of the subsequent purification process. In addition, the reaction is suitably weakly acidic in pH, and the amino group has a relatively high pKa (usually 8-11), which results in a decrease in the coupling reaction with the activated carboxyl group (nucleophilic reaction activity), so that in the prior art, an excessive amount of catalyst is usually used, and the ratio of the amount of the catalyst to the amount of the substance of the side chain carboxyl group of the natural polysaccharide is usually not less than 1, which increases not only the cost but also the burden of the subsequent purification process.
In order to improve the coupling reaction efficiency of the side chain carboxyl of the natural polysaccharide and the sulfhydrylation amino reagent, thereby improving the sulfhydrylation modification degree of the sulfhydrylation natural polysaccharide derivative, a great deal of researches are carried out. The invention discovers that increasing the dosage of the activating reagent can not improve the coupling efficiency, but can reduce the coupling efficiency, and the cause of the obvious reduction of the sulfhydrylation modification degree is not clear at present and needs to be further studied. For example, in example one, the ratio of the amounts of catalyst and activator substances was increased from 1.0:1.0 to 1.0:3.0 and 1.0:6.0, respectively, and the 3-fold and 6-fold incremental use of the activators resulted in a 51.7% and 67.6% reduction in the degree of thiol-modification, respectively (relative).
In step 1 of the method, when the mass ratio of the catalyst to the activating agent is 1.0:0.25-1.0:0.75, the coupling efficiency is higher, and the thiol modification degree is higher. For example, decreasing the ratio of the amounts of the substances of the catalyst and the activating agent from 1.0:1.0 to 1.0:0.5 and 1.0:0.25, decreasing the activating agent to 0.5 and 0.25 times, resulted in an increase in the degree of thiol modification of 36.5% and 20.6%, respectively (relative values).
In a second aspect of the present invention, there is provided a thiolated natural polysaccharide derivative prepared by the aforementioned method for preparing thiolated natural polysaccharide derivatives.
In a third aspect of the invention, there is provided the use of the above-described thiolated natural polysaccharide derivatives in the preparation of biomedical materials.
According to a fourth aspect of the present invention, there is provided a cross-linked material of a thiolated natural polysaccharide derivative, the cross-linked material of a thiolated natural polysaccharide derivative being prepared based on the thiolated natural polysaccharide derivative described above, the preparation being carried out by oxidation of the thiolated natural polysaccharide derivative using an oxidizing agent or rapid in situ cross-linking using a biocompatible cross-linking agent containing two or more activated unsaturated double bond groups, the unsaturated double bond groups being maleimides, vinyl sulfones, unsaturated acrylates or unsaturated methacrylates.
In a fifth aspect, the invention provides the use of the cross-linked material of a thiolated natural polysaccharide derivative as described above in the preparation of biomedical materials.
The preparation method of the sulfhydrylation natural polysaccharide derivative has the following beneficial effects:
1. according to the preparation method of the sulfhydrylation natural polysaccharide derivative, provided by the invention, a sulfhydrylation amino reagent with sulfhydrylation protection or disulfide bond is adopted, only amino coupling reaction is carried out in a reaction system, after the amino coupling reaction is finished, sulfhydrylation is deprotected or disulfide bond is reduced to free sulfhydryl, and the defect that catalytic coupling reaction occurs due to unprotected measure of sulfhydryl groups is effectively avoided; and Cystamine (Cystamine) which can be adopted is the most commonly used sulfhydrylation amino reagent containing disulfide bonds at present, and has the advantages of wide sources, low price, low toxicity, low cost, contribution to amplified production and strong practicability.
2. The preparation method of the sulfhydrylation natural polysaccharide derivative provided by the invention takes water-soluble carbodiimide (1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide (EDCI) and/or 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide salt) as catalysts, and by adjusting the mass ratio of the catalyst to the substance of the activating reagent, the use amount of the activating reagent is reduced, and the coupling efficiency of the side chain carboxyl of the natural polysaccharide and the amino sulfhydrylation reagent under the EDCI catalysis is unexpectedly improved, so that the sulfhydrylation modification degree is obviously improved, and meanwhile, the preparation method has the advantages of mild reaction condition, simple process, less reagent use amount, high reaction efficiency, less residual impurities, good safety and the like.
3. The natural polysaccharide derivative prepared by the invention has good biocompatibility, and can form disulfide bond crosslinking materials under the oxidation action of oxidizing agents such as oxygen and the like without a crosslinking agent; the method can also realize rapid in-situ crosslinking through mercapto and biocompatible crosslinking agents (such as polyethylene glycol diacrylate and the like) containing two or more activated unsaturated double bonds, has no reactive impurities, can be used for in-situ embedding of cells, slow release of growth factors and the like, and has good application prospect in the field of biological medicine.
Drawings
In order to more clearly illustrate the technical solutions of the present invention, the following brief description of the drawings is given for the purpose of the present invention, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and that other drawings can be obtained according to these drawings without the need for inventive work for a person skilled in the art.
FIG. 1 shows the results of the thiol-modification degree of the final product (the ratio of the amounts of the catalyst and the substance of the side chain carboxyl groups of the natural polysaccharide is 0.75:1.0) by adjusting the ratio of the amounts of the catalyst and the substance of the activating reagent (1.0:0 to 1.0) in the first embodiment of the present invention;
FIG. 2 shows the results of the thiol-modification of the final product by adjusting the ratio of the amounts of the catalyst and the activating reagent materials (1.0:0 to 1.0:2.0) (the ratio of the amounts of the catalyst and the material of the side chain carboxyl groups of the natural polysaccharide: 0.5:1.0) in example II of the present invention;
FIG. 3 is the result of the thiol-modification of the final product by adjusting the ratio of the amounts of catalyst and activator substances (1.0:0 to 1.0:2.0) (the ratio of the amounts of catalyst and substance of the side chain carboxyl groups of the natural polysaccharide: 0.25:1.0) in example III of the present invention;
FIG. 4 shows the results of the thiol-modification of the final product by adjusting the ratio of the amounts of the catalyst and the activator substances (1.0:0 to 1.0:2.0) (the ratio of the amounts of the catalyst and the substances of the side chain carboxyl groups of the natural polysaccharide: 0.15:1.0) in example IV of the present invention;
FIG. 5 is the result of the thiol-modification of the final product by adjusting the ratio of the amounts of catalyst and activator substances (1.0:0 to 1.0:2.0) in example five of the present invention (the ratio of the amounts of catalyst and substance of the side chain carboxyl groups of natural polysaccharide is 0.05:1.0);
FIG. 6 shows the hydrogen nuclear magnetic resonance detection of the thiol-based final product of the fourth embodiment of the invention 1 H-NMR), wherein: FIG. 6 (a) is a hyaluronic acid source material; FIG. 6 (b) ratio of amounts of catalyst and activator species 1.0:0; FIG. 6 (c) ratio of amounts of catalyst and activator species 1.0:0.25; FIG. 6 (d) catalyst and living The ratio of the amounts of the chemical reagent substances is 1.0:0.5; FIG. 6 (e) ratio of amounts of catalyst and activator species 1.0:0.75; FIG. 6 (f) ratio of amounts of catalyst and activator species 1.0:1.0; FIG. 6 (g) ratio of amounts of catalyst and activator species 1.0:1.5; FIG. 6 (h) ratio of amounts of catalyst and activator species 1.0:2.0.
Detailed Description
The following description of the present invention will be made clearly and fully, and it is apparent that the embodiments described are some, but not all, of the embodiments of the present invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
In the following examples, sulfo-NHS is N-hydroxysulfosuccinimide and EDCI is 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide, D 2 O is heavy water.
Example 1
Sodium hyaluronate (MW 200 kDa) 1 g (2.5 mmol) and cystamine dihydrochloride 1.69 g (7.5 mmol) were dissolved in 100ml distilled water, and 0 g (0 mmol), 0.102 g (0.47 mmol), 0.204 g (0.94 mmol), 0.305 g (1.41 mmol), 0.407 g (1.88 mmol), 0.611 g (2.81 mmol), 0.814 g (3.75 mmol), 1.221 g (5.63 mmol), 1.832 g (8.44 mmol) and 2.442 g (11.25 mmol) were added, respectively, and dissolved with stirring. An appropriate amount of acid or base (0.1. 0.1N hydrochloric acid or sodium hydroxide) was added to adjust the pH of the solution to 4.75.
EDCI solid (0.36 g, 1.88 mmol) was added, stirred and dissolved, and an appropriate amount of acid or base (0.1N hydrochloric acid or sodium hydroxide) was added to keep the pH of the reaction solution stable at 4.75, and the reaction was stirred overnight. The reaction was terminated by adding 1.0. 1.0N sodium hydroxide to adjust the pH of the reaction solution to 8.5. Then 6 g of dithiothreitol was added thereto, and the mixture was dissolved by stirring and reacted for 4 hours.
1.0. 1.0N hydrochloric acid was added to adjust the pH of the reaction solution to 3.0. The above solution was placed in a dialysis tube (molecular weight cut-off 3500 Da) (spectra Labs, U.S.A.), and dialyzed against a mixed solution of 0.001N hydrochloric acid and 0.2N sodium chloride, followed by Gel Permeation Chromatography (GPC) until no impurity absorption peak was visible.
Finally, the solution in the dialysis tube is collected and freeze-dried to obtain white flocculent solid.
The thiol content was measured and the degree of thiol modification calculated using the modified Ellman method reported by Shu et al (Biomacromolecules 2002, 3:1304-1311). The degree of thiol modification refers to the percentage of the total number of thiol groups introduced to the total number of carboxyl groups in the side chain of the hyaluronic acid.
In this example, the ratio of the amounts of the catalyst and the substance of the side chain carboxyl group of the natural polysaccharide was 0.75:1.0, and the ratio of the amounts of the catalyst and the substance of the activating agent was 1.0:0, 1.0:0.25, 1.0:0.5, 1.0:0.75, 1.0:1.0, 1.0:1.5, 1.0:2.0, 1.0:3.0, 1.0:4.5, and 1.0:6.0 in this order.
As shown in FIG. 1, the results showed that the mass ratios (Sulfo-NHS/EDCI) of 0.25, 0.5 and 0.75 had higher thiol-modification levels, which were 121%, 137% and 101% of the thiol-modification levels when the mass ratios (Sulfo-NHS/EDCI) were 1, respectively. The ratio of the amount of substances (Sulfo-NHS/EDCI) is more than 1, and the sulfhydrylation modification degree is obviously reduced. The thiol-modified degree at the mass ratios (Sulfo-NHS/EDCI) of 1.5, 2, 3, 4.5 and 6 were 74%, 63%, 48%, 40% and 32% at the mass ratios (Sulfo-NHS/EDCI) of 1, respectively.
Example two
Sodium hyaluronate (MW 200 kDa) 1 g (2.5 mmol) and cystamine dihydrochloride 1.126 g (5 mmol) were dissolved in 100ml distilled water, and 0 g (0 mmol), 0.068 g (0.31 mmol), 0.136 g (0.63 mmol), 0.205 g (0.94 mmol), 0.271 g (1.25 mmol), 0.407 g (1.875 mmol) and 0.543 g (2.5 mmol) of Sulfo-NHS were added, respectively, and dissolved with stirring. An appropriate amount of acid or base (0.1. 0.1N hydrochloric acid or sodium hydroxide) was added to adjust the pH of the solution to 4.75.
EDCI solid (0.24 g, 1.25 mmol) was added, stirred and dissolved, and an appropriate amount of acid or base (0.1N hydrochloric acid or sodium hydroxide) was added to keep the pH of the reaction solution stable at 4.75, and reacted for 30 minutes. Proper amount of 0.1. 0.1N sodium hydroxide was added to raise the pH of the reaction solution to 6.0, and the reaction was stirred overnight. The reaction was terminated by adding 1.0. 1.0N sodium hydroxide to adjust the pH of the reaction solution to 8.5. Then, 4 g of dithiothreitol was added thereto, and the mixture was dissolved by stirring and reacted for 4 hours.
1.0. 1.0N hydrochloric acid was added to adjust the pH of the reaction solution to 3.0. The above solution was placed in a dialysis tube (molecular weight cut-off 3500 Da) (spectra Labs, U.S.A.), and dialyzed against a mixed solution of 0.001N hydrochloric acid and 0.2N sodium chloride, followed by Gel Permeation Chromatography (GPC) until no impurity absorption peak was visible.
Finally, the solution in the dialysis tube is collected and freeze-dried to obtain white flocculent solid.
The thiol content was measured and the degree of thiol modification calculated using the modified Ellman method reported by Shu et al (Biomacromolecules 2002, 3:1304-1311). The degree of thiol modification refers to the percentage of the total number of thiol groups introduced to the total number of carboxyl groups in the side chain of the hyaluronic acid.
In this example, the ratio of the amounts of the catalyst and the substance of the side chain carboxyl group of the natural polysaccharide was 0.5:1.0, and the ratio of the amounts of the catalyst and the substance of the activating agent was 1.0:0, 1.0:0.25, 1.0:0.5, 1.0:0.75, 1.0:1.0, 1.0:1.5 and 1.0:2.0 in this order.
As shown in FIG. 2, the results revealed that the mass ratios (Sulfo-NHS/EDCI) of 0.25, 0.5 and 0.75 had higher thiol-modification levels, which were 141%, 149% and 108% of the thiol-modification levels when the mass ratios (Sulfo-NHS/EDCI) were 1, respectively. The ratio of the amount of substances (Sulfo-NHS/EDCI) is more than 1, and the sulfhydrylation modification degree is obviously reduced. The thiol modification degree at the mass ratio (Sulfo-NHS/EDCI) of 1.5 and 2 was 81% and 71% at the mass ratio (Sulfo-NHS/EDCI) of 1, respectively.
Example III
Sodium hyaluronate (MW 200 kDa) 1 gram (2.5 mmol) and cystamine dihydrochloride 1.126 gram (5 mmol) were dissolved in 100ml distilled water, and 0 gram (0 mmol), 0.034 gram (0.156 mmol), 0.068 gram (0.31 mmol), 0.102 gram (0.47 mmol), 0.136 gram (0.625 mmol), 0.204 gram (0.94 mmol) and 0.271 gram (1.25 mmol) were added respectively, and dissolved with stirring. An appropriate amount of acid or base (0.1. 0.1N hydrochloric acid or sodium hydroxide) was added to adjust the pH of the solution to 4.75.
EDCI solid 0.12 g (0.625 mmol) is added, stirred and dissolved, and proper acid or alkali (0.1N hydrochloric acid or sodium hydroxide) is added to keep the pH value of the reaction solution stable to 4.5-5.0, and the reaction is carried out for 120 minutes. And adding a proper amount of 0.1-N sodium hydroxide, adjusting the pH of the reaction solution to 5.5-6.5, and stirring for reaction overnight. The reaction was terminated by adding 1.0. 1.0N sodium hydroxide to adjust the pH of the reaction solution to 8.5. Dithiothreitol (4 g) was added thereto, and the mixture was dissolved by stirring and reacted for 4 hours. 1.0. 1.0N hydrochloric acid was added to adjust the pH of the reaction solution to 3.0.
The above solution was placed in a dialysis tube (molecular weight cut-off 3500 Da) (spectra Labs, U.S.A.), and dialyzed against a mixed solution of 0.001N hydrochloric acid and 0.2N sodium chloride, followed by Gel Permeation Chromatography (GPC) until no impurity absorption peak was visible. Finally, the solution in the dialysis tube is collected and freeze-dried to obtain white flocculent solid.
The thiol content was measured and the degree of thiol modification calculated using the modified Ellman method reported by Shu et al (Biomacromolecules 2002, 3:1304-1311). The degree of thiol modification refers to the percentage of the total number of thiol groups introduced to the total number of carboxyl groups in the side chain of the hyaluronic acid.
In this example, the ratio of the amounts of the catalyst and the substance of the side chain carboxyl group of the natural polysaccharide was 0.25:1.0, and the ratio of the amounts of the catalyst and the substance of the activating agent was 1.0:0, 1.0:0.25, 1.0:0.5, 1.0:0.75, 1.0:1.0, 1.0:1.5 and 1.0:2.0 in this order.
As shown in FIG. 3, the results revealed that the mass ratios (Sulfo-NHS/EDCI) of 0.25, 0.5 and 0.75 had higher thiol-modification levels, which were 137%, 144% and 108% of the thiol-modification levels when the mass ratios (Sulfo-NHS/EDCI) were 1, respectively. The ratio of the amount of substances (Sulfo-NHS/EDCI) is more than 1, and the sulfhydrylation modification degree is obviously reduced. The thiol modification degree at the mass ratio (Sulfo-NHS/EDCI) of 1.5 and 2 was 92% and 70% at the mass ratio (Sulfo-NHS/EDCI) of 1, respectively.
Example IV
Sodium hyaluronate (MW 200 kDa) 1 gram (2.5 mmol) and cystamine dihydrochloride 1.126 gram (5 mmol) were dissolved in 100ml distilled water, and 0 gram (0 mmol), 0.020 gram (0.094 mmol), 0.041 gram (0.19 mmol), 0.061 gram (0.28 mmol), 0.081 gram (0.375 mmol), 0.122 gram (0.56 mmol) and 0.163 gram (0.75 mmol) were added, respectively, and dissolved with stirring. An appropriate amount of acid or base (0.1. 0.1N hydrochloric acid or sodium hydroxide) was added to adjust the pH of the solution to 4.75.
EDCI solid 0.072 g (0.375 mmol) is added, stirred and dissolved, and proper acid or alkali (0.1N hydrochloric acid or sodium hydroxide) is added to keep the pH value of the reaction solution stable at 4.75-5.0, and the reaction is carried out for 60 minutes. Proper amount of 0.1. 0.1N sodium hydroxide was added to raise the pH of the reaction solution to 6.0, and the reaction was stirred overnight. The reaction was terminated by adding 1.0. 1.0N sodium hydroxide to adjust the pH of the reaction solution to 8.5. Then, 4 g of dithiothreitol was added thereto, and the mixture was dissolved by stirring and reacted for 4 hours.
1.0. 1.0N hydrochloric acid was added to adjust the pH of the reaction solution to 3.0. The above solution was placed in a dialysis tube (molecular weight cut-off 3500 Da) (spectra Labs, U.S.A.), and dialyzed against a mixed solution of 0.001N hydrochloric acid and 0.2N sodium chloride, followed by Gel Permeation Chromatography (GPC) until no impurity absorption peak was visible.
Finally, the solution in the dialysis tube is collected and freeze-dried to obtain white flocculent solid.
The thiol content was measured and the degree of thiol modification calculated using the modified Ellman method reported by Shu et al (Biomacromolecules 2002, 3:1304-1311). The degree of thiol modification refers to the percentage of the total number of thiol groups introduced to the total number of carboxyl groups in the side chain of the hyaluronic acid.
In this example, the ratio of the amounts of the catalyst and the substance of the side chain carboxyl group of the natural polysaccharide was 0.15:1.0, and the ratio of the amounts of the catalyst and the substance of the activating agent was 1.0:0, 1.0:0.25, 1.0:0.5, 1.0:0.75, 1.0:1.0, 1.0:1.5 and 1.0:2.0 in this order.
As shown in FIG. 4, the results revealed that the mass ratios (Sulfo-NHS/EDCI) of 0.25, 0.5 and 0.75 had higher thiol-modification levels, which were 117%, 126% and 103% of the thiol-modification levels when the mass ratios (Sulfo-NHS/EDCI) were 1, respectively. The ratio of the amount of substances (Sulfo-NHS/EDCI) is more than 1, and the sulfhydrylation modification degree is obviously reduced. The thiol modification degree at the mass ratio (Sulfo-NHS/EDCI) of 1.5 and 2 was 84% and 76% at the mass ratio (Sulfo-NHS/EDCI) of 1, respectively.
Example five
Sodium hyaluronate (MW 200 kDa) 1 gram (2.5 mmol) and cystamine dihydrochloride 1.126 gram (5 mmol) were dissolved in 100ml distilled water, and 0 gram (0 mmol), 0.068 gram (0.031 mmol), 0.014 gram (0.063 mmol), 0.020 gram (0.094 mmol), 0.027 gram (0.125 mmol), 0.041 gram (0.188 mmol) and 0.054 gram (0.25 mmol) were added, respectively, and dissolved with stirring. An appropriate amount of acid or base (0.1. 0.1N hydrochloric acid or sodium hydroxide) was added to adjust the pH of the solution to 4.75.
EDCI solid 0.024 g (0.125 mmol) was added, stirred and dissolved, and an appropriate amount of acid or base (0.1N hydrochloric acid or sodium hydroxide) was added to keep the pH of the reaction solution stable at 4.75, and reacted for 30 minutes. Proper amount of 0.1. 0.1N sodium hydroxide was added to raise the pH of the reaction solution to 6.0, and the reaction was stirred overnight. The reaction was terminated by adding 1.0. 1.0N sodium hydroxide to adjust the pH of the reaction solution to 8.5. Dithiothreitol (4 g) was added thereto, and the mixture was dissolved by stirring and reacted for 4 hours.
1.0. 1.0N hydrochloric acid was added to adjust the pH of the reaction solution to 3.0. The above solution was placed in a dialysis tube (molecular weight cut-off 3500 Da) (spectra Labs, U.S.A.), and dialyzed against a mixed solution of 0.001N hydrochloric acid and 0.2N sodium chloride, followed by Gel Permeation Chromatography (GPC) until no impurity absorption peak was visible.
Finally, the solution in the dialysis tube is collected and freeze-dried to obtain white flocculent solid.
The thiol content was measured and the degree of thiol modification calculated using the modified Ellman method reported by Shu et al (Biomacromolecules 2002, 3:1304-1311). The degree of thiol modification refers to the percentage of the total number of thiol groups introduced to the total number of carboxyl groups in the side chain of the hyaluronic acid.
In this example, the ratio of the amounts of the catalyst and the substance of the side chain carboxyl group of the natural polysaccharide was 0.05:1.0, and the ratio of the amounts of the catalyst and the substance of the activating agent was 1.0:0, 1.0:0.25, 1.0:0.5, 1.0:0.75, 1.0:1.0, 1.0:1.5 and 1.0:2.0 in this order.
As shown in FIG. 5, the results revealed that the mass ratios (Sulfo-NHS/EDCI) of the substances were 0.25, 0.5 and 0.75, with higher thiol-modification degrees, respectively, of 108%, 121% and 115% for the mass ratios (Sulfo-NHS/EDCI) of 1. The ratio of the amount of substances (Sulfo-NHS/EDCI) is more than 1, and the sulfhydrylation modification degree is obviously reduced. The thiol modification degree at the mass ratio (Sulfo-NHS/EDCI) of 1.5 and 2 was 92% and 89% at the mass ratio (Sulfo-NHS/EDCI) of 1, respectively.
Example six
Sodium hyaluronate (MW 200 kDa) 1 g (2.5 mmol) and Sulfo-NHS 0.020 g (0.094 mmol) were dissolved in 100ml distilled water, and cystamine dihydrochloride 0.563 g (2.5 mmol), 1.1263 g (5 mmol), 1.69 g (7.5 mmol) and 2.252 g (10 mmol) were added respectively and dissolved with stirring. An appropriate amount of acid or base (0.1. 0.1N hydrochloric acid or sodium hydroxide) was added to adjust the pH of the solution to 4.75. Then 0.072 g (0.375 mmol) of EDCI solid was added, stirred and dissolved, and an appropriate amount of acid or alkali (0.1N hydrochloric acid or sodium hydroxide) was added to keep the pH value of the reaction solution stable at 4.75, and the reaction was carried out for 30 minutes. Then, an appropriate amount of 0.1. 0.1N sodium hydroxide was added to raise the pH of the reaction solution to 6.0, and the reaction was stirred overnight. The reaction was terminated by adding 1.0. 1.0N sodium hydroxide to adjust the pH of the reaction solution to 8.5. Then adding 3-8 g of dithiothreitol, stirring and dissolving, and reacting for 4 hours. Then, 1.0. 1.0N hydrochloric acid was added to adjust the pH of the reaction solution to 3.0. The solution was placed in a dialysis tube (molecular weight cut-off 3500 Da) (Spectrum Labs, U.S.A.); then, the final product was purified by dialysis against a mixed solution of 0.001. 0.001N hydrochloric acid and 0.2. 0.2N sodium chloride, followed by Gel Permeation Chromatography (GPC) until the impurity absorption peak was not visible. Finally, the solution in the dialysis tube is collected and freeze-dried to obtain white flocculent solid.
The thiol content was measured and the degree of thiol modification calculated using the modified Ellman method reported by Shu et al (Biomacromolecules 2002, 3:1304-1311). The degree of thiol modification refers to the percentage of the total number of thiol groups introduced to the total number of carboxyl groups in the side chain of the hyaluronic acid.
In this example, the ratio of the amounts of the catalyst and the substance of the side chain carboxyl group of the natural polysaccharide was 0.15:1.0, the ratio of the amounts of the catalyst and the substance of the activating agent was 1.0:0.25, and the ratio of the amounts of the mercapto amino agent and the substance of the side chain carboxyl group of the natural polysaccharide was 1.0:1.0 to 4.0:1.0.
Experimental results show that the ratio of the amounts of the substances (sulfhydrylation amino reagent/carboxyl group of side chain of natural polysaccharide) is about 4% for all of sulfhydrylation modifications of 1, 2, 3 and 4, without obvious differences.
Example seven
1.2 g (2.5 mmol) of sodium chondroitin sulfate (derived from shark cartilage) and 1.126 g (5 mmol) of cystamine dihydrochloride were dissolved in 100ml of distilled water, and 0 g (0 mmol), 0.068 g (0.31 mmol), 0.136 g (0.63 mmol), 0.205 g (0.94 mmol), 0.271 g (1.25 mmol), 0.407 g (1.875 mmol) and 0.543 g (2.5 mmol) of Sulfo-NHS were added, respectively, and dissolved by stirring. An appropriate amount of acid or base (0.1. 0.1N hydrochloric acid or sodium hydroxide) was added to adjust the pH of the solution to 4.75.
EDCI solid (0.24 g, 1.25 mmol) was added, stirred and dissolved, and an appropriate amount of acid or base (0.1N hydrochloric acid or sodium hydroxide) was added to keep the pH of the reaction solution stable at 4.75, and reacted for 30 minutes. Proper amount of 0.1. 0.1N sodium hydroxide was added to raise the pH of the reaction solution to 6.0, and the reaction was stirred overnight. The reaction was terminated by adding 1.0. 1.0N sodium hydroxide to adjust the pH of the reaction solution to 8.5. Dithiothreitol (4 g) was added thereto, and the mixture was dissolved by stirring and reacted for 4 hours.
1.0. 1.0N hydrochloric acid was added to adjust the pH of the reaction solution to 3.0. The above solution was placed in a dialysis tube (molecular weight cut-off 3500 Da) (spectra Labs, U.S.A.), and dialyzed against a mixed solution of 0.001N hydrochloric acid and 0.2N sodium chloride, followed by Gel Permeation Chromatography (GPC) until no impurity absorption peak was visible.
Finally, the solution in the dialysis tube is collected and freeze-dried to obtain white flocculent solid.
The thiol content was measured and the degree of thiol modification calculated using the modified Ellman method reported by Shu et al (Biomacromolecules 2002, 3:1304-1311). The degree of sulfhydrylation modification refers to the percentage of the total number of sulfhydryl groups introduced to the total number of carboxyl groups of the chondroitin sulfate side chain.
In this example, the ratio of the amounts of the catalyst and the substance of the side chain carboxyl group of the natural polysaccharide was 0.5:1.0, and the ratio of the amounts of the catalyst and the substance of the activating agent was 1.0:0 to 1.0:2.0.
Experimental results show that the ratio of the amounts of the substances (Sulfo-NHS/EDCI) was 0.25, 0.5 and 0.75 with higher thiol-modification, respectively 128%, 141% and 112% of the thiol-modification when the ratio of the amounts of the substances (Sulfo-NHS/EDCI) was 1. The ratio of the amount of substances (Sulfo-NHS/EDCI) is more than 1, and the sulfhydrylation modification degree is obviously reduced. The thiol modification degree at the mass ratio (Sulfo-NHS/EDCI) of 1.5 and 2 was 85% and 73% at the mass ratio (Sulfo-NHS/EDCI) of 1, respectively.
Example eight
In D 2 O is used as solvent, hydrogen spectrum nuclear magnetic resonance detection is adopted 1 H-NMR) characterization of the thiolated natural polysaccharide derivatives prepared in each example, as shown in fig. 6, is a characterization result of the thiolated natural polysaccharide derivatives prepared in example four.
FIG. 6 (a) is a hyaluronic acid source material; FIG. 6 (b) ratio of amounts of catalyst and activator species 1.0:0; (c) The ratio of the amounts of catalyst and activator species was 1.0:0.25; (d) The ratio of the amounts of catalyst and activator species was 1.0:0.5; (e) The ratio of the amounts of catalyst and activator species was 1.0:0.75; (f) The ratio of the amounts of catalyst and activator species was 1.0:1.0; (g) The ratio of the amounts of catalyst and activator species was 1.0:1.5; (h) The ratio of the amounts of catalyst and activator species was 1.0:2.0. As shown in FIGS. 6 (a) -6 (h), the spectra of each thiolated natural polysaccharide derivative demonstrate thiolation modification Structure, new absorption peak (triplet peak) appears around δ2.53, and 2 hydrogens (P-C (O) -NH-CH, which are assigned to alkylene group near free mercapto group 2 -CH 2 -SH）；
As shown in fig. 6 (c) -6 (e), the thiolated natural polysaccharide derivatives prepared when the ratio of the amounts of the substances of the catalyst and the activating agent is 1.0:0.25 to 1.0:0.75 have a relatively larger new absorption peak area (corresponding to a higher thiolation degree of modification);
2 hydrogens of alkylene close to the amide bond (P-C (O) -NH-CH 2 -CH 2 The absorption peak of-SH) overlaps with the absorption peak of the hydrogen atom on the sugar ring (region of δ3.2-3.8) at δ3.5.
As shown in FIG. 6 (b), the thiol-modified degree of the thiol-modified natural polysaccharide derivative prepared without the addition of the activating agent was very low, the new absorption peak of δ2.53 was hardly visible, and a hetero peak ascribed to the N-acetylurea by-product appeared at δ2.74, which was not found in FIGS. 6 (c) -6 (h).
Example nine
The 3 thiol-modified hyaluronic acid derivatives (white flocculent solid) prepared in example four (the mass ratios of the substances of Sulfo-NHS/EDCI were 0.25, 0.5 and 0.75, respectively) were dissolved in physiological saline (pH 7.0) containing a phosphate buffer of 0.01. 0.01N to give clear and transparent aqueous solutions, the concentrations of the thiol-modified hyaluronic acid derivatives were 5, 7.5, 10, 15 and 20 mg/ml, respectively, and the pH values of the solutions were adjusted to 6.8-8.0 with an appropriate amount of 0.1N sodium hydroxide or hydrochloric acid, respectively. The solution 1 ml was placed in a 5 ml glass bottle and stored at room temperature under sealed conditions. After two weeks, the solution sealed in the glass bottle lost fluidity and had formed a gel. The gel was insoluble in water, but soluble in dithiothreitol solution, confirming that disulfide cross-links formed under oxidation of oxygen in the vial air and dissolved oxygen in the solution.
Examples ten
The 3 thiol-modified hyaluronic acid derivatives (white flocculent solid) prepared in example one (the mass ratios of the substances of Sulfo-NHS/EDCI were 0.25, 0.5 and 0.75, respectively) were dissolved in physiological saline (pH 7.0) containing a phosphate buffer of 0.01. 0.01N to give clear and transparent aqueous solutions, the concentrations of the thiol-modified hyaluronic acid derivatives were 20 mg/ml, respectively, and the pH of the solutions was adjusted to 7.4 with an appropriate amount of 0.1. 0.1N sodium hydroxide or hydrochloric acid, respectively. The above solution 4 ml and 1 ml polyethylene glycol diacrylate aqueous solution (40 mg/ml) were taken and mixed thoroughly, and then left to stand, and after two hours, it was observed that the mixed solution lost fluidity and had formed a gel.
In summary, the above embodiments are only preferred embodiments of the present invention, and are not intended to limit the scope of the present invention, but any modifications, equivalent substitutions, improvements, etc. within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (19)
1. A process for the preparation of a thiolated natural polysaccharide derivative, comprising the steps of:
step 1: dissolving natural polysaccharide in water, adding a sulfhydrylation amino reagent under the action of a catalyst and an activating reagent, and reacting side chain carboxyl of the natural polysaccharide with amino of the sulfhydrylation amino reagent to generate an amide bond;
Wherein the natural polysaccharide is hyaluronic acid, hyaluronate, derivatives obtained by modifying hyaluronate, chondroitin sulfate salt and/or derivatives obtained by modifying chondroitin sulfate;
the catalyst is water-soluble carbodiimide;
the activating reagent is one or a combination of more of N-hydroxysuccinimide, N-hydroxysulfosuccinimide and N-hydroxysulfosuccinimide salt;
the sulfhydrylation amino reagent is diammine containing disulfide bond, primary amine containing free sulfhydryl or primary amine with sulfhydryl protecting group;
the ratio of the amounts of the substances of the catalyst and the activating reagent is 1:0.25-1:0.75;
step 2: collecting the product obtained in the step 1, treating, and purifying the treated product to obtain the sulfhydrylation natural polysaccharide derivative;
when the sulfhydrylation amino reagent is diammine containing disulfide bond, the product obtained in the step 1 is reacted with a reducing agent, wherein the disulfide bond is reduced into sulfhydryl, and the sulfhydryl natural polysaccharide derivative is obtained after purification;
when the sulfhydrylation amino reagent is primary amine containing free sulfhydrylation, purifying the product obtained in the step 1 to obtain a sulfhydrylation natural polysaccharide derivative;
When the sulfhydrylation amino reagent is primary amine with sulfhydrylation protecting group, the sulfhydrylation protecting group of the product obtained in the step 1 is removed, and the sulfhydrylation natural polysaccharide derivative is obtained by purification.
2. The method for producing a thiolated natural polysaccharide derivative according to claim 1, wherein the water-soluble carbodiimide is 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide and/or 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide salt.
3. The method for producing a thiolated natural polysaccharide derivative according to claim 1, wherein the disulfide bond-containing diamino is cystamine and/or cystamine salt.
4. The method for producing a thiolated natural polysaccharide derivative according to claim 1, wherein the ratio of the amounts of the catalyst and the activating agent is 1:0.25 to 1:0.6.
5. The method for producing a thiolated natural polysaccharide derivative according to claim 1, wherein the ratio of the amounts of the catalyst and the activating agent is 1:0.35 to 1:0.5.
6. The method for producing a thiolated natural polysaccharide derivative according to claim 1, wherein the ratio of the amounts of the catalyst and the substance of the side chain carboxyl group of the natural polysaccharide is 0.01:1 to 1:1.
7. The method for producing a thiolated natural polysaccharide derivative according to claim 6, wherein the ratio of the amount of the catalyst to the amount of the substance of the side chain carboxyl group of the natural polysaccharide is 0.01:1 to 0.75:1.
8. The method for producing a thiolated natural polysaccharide derivative according to claim 7, wherein the ratio of the amount of the catalyst to the amount of the substance of the side chain carboxyl group of the natural polysaccharide is 0.05:1 to 0.5:1.
9. The method for producing a thiolated natural polysaccharide derivative according to claim 1, wherein the ratio of the thiolated amino reagent to the substance of the side chain carboxyl group of the natural polysaccharide is 1:1 to 6:1.
10. The method for producing a thiolated natural polysaccharide derivative according to claim 9, wherein the ratio of the thiolated amino reagent to the substance of the side chain carboxyl group of the natural polysaccharide is 1:1 to 4:1.
11. The method for producing a thiolated natural polysaccharide derivative according to claim 10, wherein the ratio of the thiolated amino reagent to the substance of the side chain carboxyl group of the natural polysaccharide is 1:1 to 2:1.
12. The method for producing a thiolated natural polysaccharide derivative according to claim 1, wherein the reducing agent in step 2 is dithiothreitol and/or tris (2-carboxyethyl) phosphine hydrochloride.
13. The method for preparing a thiolated natural polysaccharide derivative according to claim 1, wherein in the step 1, the pH value of the reaction system is 4.5-6.5.
14. The method for preparing a thiolated natural polysaccharide derivative according to claim 13, wherein in the step 1, the pH value of the reaction system is: firstly, reacting for 15-120min under the condition of pH value of 4.5-5.0, then adjusting the pH value to 5.5-6.5, and continuing reacting for 8-16 h.
15. The method for preparing a thiolated natural polysaccharide derivative according to claim 14, wherein in the step 1, the pH value of the reaction system is: firstly, reacting for 30min under the condition of pH value of 4.75-5.0, then adjusting the pH value to 6.0, and continuing to react for 8-16 h.
16. A thiolated natural polysaccharide derivative prepared by the process for preparing thiolated natural polysaccharide derivatives according to any one of claims 1-15.
17. Use of the thiolated natural polysaccharide derivatives of claim 16 for the preparation of biomedical materials.
18. A thiolated natural polysaccharide derivative cross-linked material, wherein the thiolated natural polysaccharide derivative cross-linked material is prepared based on the thiolated natural polysaccharide derivative according to claim 16, wherein the preparation is performed by oxidizing the thiolated natural polysaccharide derivative with an oxidizing agent or by rapid in situ cross-linking with a biocompatible cross-linking agent containing two or more activated unsaturated double bond groups, the unsaturated double bond groups being maleimide, vinyl sulfone, unsaturated acrylate or unsaturated methacrylate.
19. Use of the cross-linked material of thiolated natural polysaccharide derivatives according to claim 18 for the preparation of biomedical materials.
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