CN114716580A - Preparation method of multiple modified hyaluronic acid derivative - Google Patents
Preparation method of multiple modified hyaluronic acid derivative Download PDFInfo
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- CN114716580A CN114716580A CN202210306731.3A CN202210306731A CN114716580A CN 114716580 A CN114716580 A CN 114716580A CN 202210306731 A CN202210306731 A CN 202210306731A CN 114716580 A CN114716580 A CN 114716580A
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- 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 title claims abstract description 59
- 238000002360 preparation method Methods 0.000 title claims abstract description 10
- 229920002674 hyaluronan Polymers 0.000 claims abstract description 33
- 229960003160 hyaluronic acid Drugs 0.000 claims abstract description 33
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims abstract description 11
- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide Substances CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 claims description 17
- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 claims description 17
- -1 diamine compound Chemical class 0.000 claims description 15
- 239000012190 activator Substances 0.000 claims description 14
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 14
- 238000000034 method Methods 0.000 claims description 13
- 125000003396 thiol group Chemical group [H]S* 0.000 claims description 10
- 150000001875 compounds Chemical class 0.000 claims description 9
- 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 5
- PIICEJLVQHRZGT-UHFFFAOYSA-N Ethylenediamine Chemical compound NCCN PIICEJLVQHRZGT-UHFFFAOYSA-N 0.000 claims description 5
- 239000003638 chemical reducing agent Substances 0.000 claims description 5
- BWGNESOTFCXPMA-UHFFFAOYSA-N Dihydrogen disulfide Chemical compound SS BWGNESOTFCXPMA-UHFFFAOYSA-N 0.000 claims description 3
- CCMKPCBRNXKTKV-UHFFFAOYSA-N 1-hydroxy-5-sulfanylidenepyrrolidin-2-one Chemical compound ON1C(=O)CCC1=S CCMKPCBRNXKTKV-UHFFFAOYSA-N 0.000 claims description 2
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 claims description 2
- AOHJOMMDDJHIJH-UHFFFAOYSA-N propylenediamine Chemical compound CC(N)CN AOHJOMMDDJHIJH-UHFFFAOYSA-N 0.000 claims description 2
- KIDHWZJUCRJVML-UHFFFAOYSA-N putrescine Chemical compound NCCCCN KIDHWZJUCRJVML-UHFFFAOYSA-N 0.000 claims description 2
- 229960002317 succinimide Drugs 0.000 claims description 2
- JMTMSDXUXJISAY-UHFFFAOYSA-N 2H-benzotriazol-4-ol Chemical compound OC1=CC=CC2=C1N=NN2 JMTMSDXUXJISAY-UHFFFAOYSA-N 0.000 claims 1
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 claims 1
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 3
- 238000004132 cross linking Methods 0.000 abstract description 3
- 238000009776 industrial production Methods 0.000 abstract description 2
- 238000006243 chemical reaction Methods 0.000 description 31
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 30
- 239000000243 solution Substances 0.000 description 29
- 238000005160 1H NMR spectroscopy Methods 0.000 description 9
- 125000003277 amino group Chemical group 0.000 description 8
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- 238000010521 absorption reaction Methods 0.000 description 6
- 125000002777 acetyl group Chemical group [H]C([H])([H])C(*)=O 0.000 description 6
- 238000000502 dialysis Methods 0.000 description 6
- 125000000524 functional group Chemical group 0.000 description 6
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- 239000002904 solvent Substances 0.000 description 6
- 230000009257 reactivity Effects 0.000 description 5
- ASOKPJOREAFHNY-UHFFFAOYSA-N 1-Hydroxybenzotriazole Chemical compound C1=CC=C2N(O)N=NC2=C1 ASOKPJOREAFHNY-UHFFFAOYSA-N 0.000 description 4
- 230000001105 regulatory effect Effects 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- KIUMMUBSPKGMOY-UHFFFAOYSA-N 3,3'-Dithiobis(6-nitrobenzoic acid) Chemical compound C1=C([N+]([O-])=O)C(C(=O)O)=CC(SSC=2C=C(C(=CC=2)[N+]([O-])=O)C(O)=O)=C1 KIUMMUBSPKGMOY-UHFFFAOYSA-N 0.000 description 3
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 3
- 238000005481 NMR spectroscopy Methods 0.000 description 3
- 229920002385 Sodium hyaluronate Polymers 0.000 description 3
- 230000003213 activating effect Effects 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 238000004737 colorimetric analysis Methods 0.000 description 3
- 239000012153 distilled water Substances 0.000 description 3
- 238000004108 freeze drying Methods 0.000 description 3
- 239000001257 hydrogen Substances 0.000 description 3
- 229910052739 hydrogen Inorganic materials 0.000 description 3
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 3
- 239000012535 impurity Substances 0.000 description 3
- 238000011065 in-situ storage Methods 0.000 description 3
- FEMOMIGRRWSMCU-UHFFFAOYSA-N ninhydrin Chemical compound C1=CC=C2C(=O)C(O)(O)C(=O)C2=C1 FEMOMIGRRWSMCU-UHFFFAOYSA-N 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 229940010747 sodium hyaluronate Drugs 0.000 description 3
- 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 3
- 239000007787 solid Substances 0.000 description 3
- 230000004913 activation Effects 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- QVYARBLCAHCSFJ-UHFFFAOYSA-N butane-1,1-diamine Chemical compound CCCC(N)N QVYARBLCAHCSFJ-UHFFFAOYSA-N 0.000 description 2
- 239000006227 byproduct Substances 0.000 description 2
- PVFIZYYTRXHJPI-UHFFFAOYSA-N carbamimidoyl acetate Chemical compound CC(=O)OC(N)=N PVFIZYYTRXHJPI-UHFFFAOYSA-N 0.000 description 2
- 239000003431 cross linking reagent Substances 0.000 description 2
- 239000000017 hydrogel Substances 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000000269 nucleophilic effect Effects 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 230000017423 tissue regeneration Effects 0.000 description 2
- 230000029663 wound healing Effects 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 206010072170 Skin wound Diseases 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 229940041181 antineoplastic drug Drugs 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 150000001718 carbodiimides Chemical class 0.000 description 1
- 210000000845 cartilage Anatomy 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 230000012292 cell migration Effects 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 238000010924 continuous production Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 125000004386 diacrylate group Chemical group 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 239000003937 drug carrier Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000028709 inflammatory response Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 230000001050 lubricating effect Effects 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 238000006011 modification reaction Methods 0.000 description 1
- 230000003020 moisturizing effect Effects 0.000 description 1
- GKRZNOGGALENQJ-UHFFFAOYSA-N n-carbamoylacetamide Chemical compound CC(=O)NC(N)=O GKRZNOGGALENQJ-UHFFFAOYSA-N 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 230000002980 postoperative effect Effects 0.000 description 1
- XAEFZNCEHLXOMS-UHFFFAOYSA-M potassium benzoate Chemical compound [K+].[O-]C(=O)C1=CC=CC=C1 XAEFZNCEHLXOMS-UHFFFAOYSA-M 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000001243 protein synthesis Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000008707 rearrangement Effects 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 159000000000 sodium salts Chemical class 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 125000000020 sulfo group Chemical group O=S(=O)([*])O[H] 0.000 description 1
- 238000002626 targeted therapy Methods 0.000 description 1
- 230000014616 translation Effects 0.000 description 1
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
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- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Materials Engineering (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicinal Chemistry (AREA)
- Polymers & Plastics (AREA)
- Organic Chemistry (AREA)
- Polysaccharides And Polysaccharide Derivatives (AREA)
Abstract
The invention discloses a preparation method of a multiple modified hyaluronic acid derivative, which can simultaneously introduce amino and sulfydryl into a side chain of hyaluronic acid, effectively avoids forming a cross-linking structure, and has the characteristics of simple preparation process, small reagent dosage, suitability for industrial production and the like.
Description
Technical Field
The invention relates to the field of biomedicine, and particularly relates to a preparation method of a multiple modified hyaluronic acid derivative.
Background
Hyaluronic acid is an important constituent of cell matrices and of various tissues, having a variety of important physiological functions, such as: regulating cell proliferation, migration and differentiation, natural moisturizing effect, lubricating joint to protect cartilage, regulating protein synthesis, regulating inflammatory response, regulating immunity, and promoting wound healing. The unique viscoelasticity, biocompatibility and degradability of hyaluronic acid enables the hyaluronic acid to be widely applied to the field of biomedicine, and the hyaluronic acid can be used as an ophthalmic operation aid, a postoperative anti-adhesion agent, a skin wound healing regeneration aid, a drug carrier, a tissue engineering scaffold and the like.
However, hyaluronic acid is easily degraded and absorbed in vivo, and the retention time is short, which greatly limits the application of hyaluronic acid in the biomedical field. For example, the half-life is no more than 24h after injection into the skin or joint (Brown et al, Exp Physiol 1991,76: 125-. Therefore, it is necessary to chemically modify hyaluronic acid to impart more excellent mechanical strength, rheological properties, and anti-enzymatic hydrolysis capability, and to expand its application range in the biomedical field.
The side chain of hyaluronic acid has carboxyl and hydroxyl functional groups for coupling reaction, but the reaction efficiency of the two functional groups is low, and the reaction conditions are harsh. For example, hydroxyl groups of hyaluronic acid have certain nucleophilic reactivity only when being strongly alkaline, and the reaction conditions are harsh (Liu et al, CN106589424A), which can cause significant backbone hydrolytic cleavage of hyaluronic acid; the carboxyl group of hyaluronic acid is usually coupled with a specific group (amino group) only by activation of carbodiimide and the like, and the reaction is limited (Nakajima et al, Bioconjugate Chem 1995, 6: 123-.
The carboxyl and/or hydroxyl of the hyaluronic acid are chemically modified to prepare the derivative with higher reactive functional groups, so that the application of the derivative in the field of biomedicine can be effectively expanded, and the derivative has many advantages. For example, thiol-modified derivatives of hyaluronic acid can form in-situ cross-linked hydrogel under the action of oxygen, and has many advantages such as no need of cross-linking agent, no impurities, good biocompatibility, etc. (Shu et al, Biomacromolecules 2002,3: 1304-1311); meanwhile, the thiolated derivative of hyaluronic acid can also realize rapid in-situ crosslinking with biocompatible crosslinking agents such as polyethylene glycol diacrylate and the like, has no reaction impurities, can be used for in-situ embedding of cells, and has important prospects in the field of tissue regeneration and repair (Shu and the like, Biomaterials 2004,25: 1339-.
However, the current hyaluronic acid derivatives usually have only one highly reactive functional group, which is not favorable for expanding the application range in the biomedical field. The hyaluronic acid derivative with a plurality of high-reactivity functions has better flexibility and advantages in the aspects of preparing multiple cross-linked hyaluronic acid hydrogel, preparing hyaluronic acid-drug conjugates with various small-molecule anticancer drugs for tumor targeted therapy and the like. However, different modification of the side chain of hyaluronic acid to add different active functional groups has certain difficulties, because the addition of the high-reactivity functional group introduced first may interfere with the subsequent modification reaction, and thus the introduction of the subsequent active group may be affected.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a preparation method of a multiple modified hyaluronic acid derivative, which can simultaneously introduce sulfydryl and amino into a side chain of hyaluronic acid to obtain the hyaluronic acid derivative with two high-reactivity functional groups.
In order to solve the above technical problems, the present invention provides a method for preparing a multiple modified hyaluronic acid derivative, comprising the steps of:
s1, reacting the side chain carboxyl of hyaluronic acid with diamine compound in the presence of a first activator to obtain a product containing hyaluronic acid derivative with side chain containing amino;
s2, step S1, reacting the side chain carboxyl group of the product with a dithiodihydrazide compound in the presence of a second activator to obtain a product including a hyaluronic acid derivative having an amino-containing side chain and a side chain having a disulfide dihydrazide structure;
s3, reacting the product obtained in step S2 with a reducing agent to reduce the disulfide bond to a thiol group, and purifying to obtain the hyaluronic acid derivative having both an amino-containing side chain and a thiol-containing side chain.
In the above reaction step, for step S1, the first activator is preferably a carbodiimide compound, and more preferably, the first activator is water-soluble 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC); the reaction principle of step S1 is: activating side chain carboxyl of hyaluronic acid by EDC to form an O-acetyl isourea intermediate, and performing nucleophilic attack on amino to form an amido bond; however, since the O-acetylisourea intermediate is also liable to react with water to undergo rapid rearrangement to form a stable N-acetylurea by-product, and the reactivity is affected, in order to avoid the by-product, N-succinimide (NHS), N-hydroxythiosuccinimide (Sulfo NHS) or hydroxybenzotriazole (HOBt) may be added to the solution of step S1 to form a stable hydrolysis-resistant intermediate; it should be noted that, because the most suitable pH for the reaction of activating carboxyl group by EDC is weak acidity (pH is about 4.5), and the amino group has a high pKa (usually greater than 8), the protonized nucleophilicity of the amino group is reduced under the weak acidic pH condition, and the reactivity with the activated carboxyl group is also reduced correspondingly, so that the formation of a cross-linked structure by both terminal amino groups and side chain carboxyl group can be effectively avoided, and at the same time, in order to further avoid the formation of a cross-linked structure by both terminal amino groups and side chain carboxyl group, the molar ratio of the usage amount of diamine compound to the usage amount of EDC is not less than 2, that is, the usage amount of diamine compound is at least twice as much as that of EDC; preferably, the molar ratio of the amount of the diamine compound to the amount of EDC is more than or equal to 4; the diamine compound used herein does not contain a disulfide bond, and is preferably one of ethylenediamine, propylenediamine, or butylenediamine; more preferably, the diamine compound is ethylenediamine; in the reaction step, the dihydrazide compound has a lower pKa (about 3) and a higher reactivity, and although the dihydrazide compound can also be used in the reaction step, the reaction activity is too high, double-end reaction is easily caused, and a cross-linked structure is produced.
In the above reaction step, for step S2, the second activator is preferably a carbodiimide compound, and more preferably, the second activator is water-soluble 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC); activating the side chain carboxyl of the hyaluronic acid derivative in the product of the step S1 by using EDC, and reacting with a dithiodihydrazide compound after activation to generate the hyaluronic acid derivative with a side chain containing a disulfide dihydrazide structure; in the reaction step, although the dithiodihydrazide compound has higher reactivity with the side chain carboxyl of hyaluronic acid and a cross-linked structure exists after the reaction, the disulfide bond of the cross-linked structure is reduced to the sulfhydryl group in the step S3 to be dissociated, and the preparation result is not influenced, so that the excessive dithiodihydrazide compound is not required to be used, and the reduction of the using amount of a reagent and the reduction of the workload of subsequent purification and impurity removal are facilitated; preferably, the dithiodihydrazide compound is selected from dithiodipropylhydrazide or dithiodibutylhydrazide.
In the above reaction step, as for step S3, the reducing agent is selected from dithiothreitol or tris (2-carboxyethyl) phosphine hydrochloride, and the disulfide bond of the side chain in the product of step S2 is reduced to a thiol group by the reducing agent, to obtain a multiple-modified hyaluronic acid derivative having both an amino-containing side chain and a thiol-containing side chain. It is emphasized that the molecular weight of the hyaluronic acid starting material employed in the present invention is typically between 1 million and 1000 million daltons, preferably between 10 million and 300 million daltons, and particularly preferably between 20 million and 100 million daltons. In the present invention, the hyaluronic acid raw material includes the form of salt (such as sodium salt, potassium salt, etc.), and also includes modified derivatives such as carboxymethyl hyaluronic acid, acetylated hyaluronic acid, etc.
The preparation method is a continuous process, can simultaneously introduce amino and sulfydryl into the side chain of the hyaluronic acid, effectively avoids forming a cross-linking structure, and has the characteristics of simple preparation process, small reagent dosage, suitability for industrial production and the like.
Detailed Description
The technical solutions in the present invention will be described clearly and completely below, and it is obvious that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example one
1g (2.5mmol) of sodium hyaluronate (molecular weight 100 ten thousand daltons) was dissolved in 500ml of distilled water at room temperature, 0.667g (5.0mmol) of 1-hydroxybenzotriazole was added, 0.6g (10mmol) of ethylenediamine was added, and then the pH of the solution was adjusted to 4.75 with 0.5mol/L hydrochloric acid, 0.96g (5mmol) of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride was added, and 0.5mol/L hydrochloric acid was added to maintain the pH of the solution at 4.75, and the reaction was allowed to proceed at room temperature overnight. Then, 0.3g (1.25mmol) of dithiodipropylhydrazide was added to the above reaction solution, 0.24g (1.25mmol) of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride was added thereto, and 0.5mol/L hydrochloric acid was added to maintain the pH of the solution at 4.75, followed by reaction at room temperature for 4 hours. After the completion of the reaction, 1.43g (5mmol) of tris (2-carboxyethyl) phosphine hydrochloride was added to the solution after the reaction, and the reaction was stirred for 4 hours. The above solution was put into a dialysis tube (molecular weight cut off 3500), dialyzed with a large amount of 0.001 mol/l hydrochloric acid and 0.2 mol/l sodium chloride solution for 2 days, and then dialyzed with a large amount of 0.001 mol/l hydrochloric acid solution for 1 day. And finally, collecting the solution in the dialysis tube, and freeze-drying to obtain flocculent solid, namely the multiple modified hyaluronic acid derivative.
The side chain sulfhydryl content of the multiple modified hyaluronic acid derivative of the invention can be detected by an improved Ellman reagent method (Shu et al, Biomacromolecules 2002,3:1304-1311) or hydrogen nuclear magnetic resonance (H-NMR)1H-NMR) (D2O is solvent), and the content is 747 mu mol/g (taking the characteristic methyl absorption peak of acetyl of hyaluronic acid as an internal standard); the content of the side chain amino group can be detected by conventional ninhydrin colorimetry (Chen et al, J. Pharma analysis 2005, 25: 526-1H-NMR)(D2O as a solvent), and the like (with the characteristic methyl absorption peak of acetyl group of hyaluronic acid as an internal standard), and the content thereof was 76 μmol/g.
Example 2
1g (2.5mmol) of sodium hyaluronate (molecular weight 20 ten thousand daltons) was dissolved in 500ml of distilled water at room temperature, 0.667g (5.0mmol) of 1-hydroxybenzotriazole was added, 1.76g (20mmol) of butanediamine was added, and then the pH of the solution was adjusted to 4.75 with 0.5mol/L hydrochloric acid, 0.96g (5mmol) of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride was added, and 0.5mol/L hydrochloric acid was added to maintain the pH of the solution at 4.75 and reacted at room temperature for 4 hours. Then, 0.3g (1.25mmol) of dithiodipropylhydrazide was added to the above reaction solution, 0.12g (0.625mmol) of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride was added thereto, and 0.5mol/L hydrochloric acid was added to maintain the pH of the solution at 4.75, followed by reaction at room temperature for 4 hours. After the reaction, 1.43g (5mmol) of tris (2-carboxyethyl) phosphine hydrochloride was added to the reacted solution, and the reaction was stirred for 4 hours. The above solution was put into a dialysis tube (molecular weight cut off 3500), dialyzed with a large amount of 0.001 mol/l hydrochloric acid and 0.2 mol/l sodium chloride solution for 2 days, and then dialyzed with a large amount of 0.001 mol/l hydrochloric acid solution for 1 day. And finally, collecting the solution in the dialysis tube, and freeze-drying to obtain flocculent solid, namely the multiple modified hyaluronic acid derivative.
The side chain sulfhydryl content of the multiple modified hyaluronic acid derivative of the invention can be detected by an improved Ellman reagent method (Shu et al, Biomacromolecules 2002,3:1304-1311) or hydrogen nuclear magnetic resonance (H-NMR)1H-NMR) (D2O is used as a solvent), and the content of the hyaluronic acid is 366 mu mol/g (taking a characteristic methyl absorption peak of acetyl of the hyaluronic acid as an internal standard); the content of amino groups in the side chains can be detected by conventional ninhydrin colorimetry (Chen et al, J. Pharma. Anal., 2005, 25: 526-1H-NMR)(D2O is a solvent), and the content thereof is 81. mu. mol/g (with the characteristic methyl absorption peak of acetyl group of hyaluronic acid as an internal standard).
Example 3
1g (2.5mmol) of sodium hyaluronate (molecular weight 50 ten thousand daltons) was dissolved in 500ml of distilled water at room temperature, 0.667g (5.0mmol) of 1-hydroxybenzotriazole was added, 1.48g (20mmol) of butanediamine was added, and then the pH of the solution was adjusted to 4.75 with 0.5mol/L hydrochloric acid, 0.96g (5mmol) of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride was added, and 0.5mol/L hydrochloric acid was added to maintain the pH of the solution at 4.75, followed by reaction at room temperature overnight. Then, 0.33g (1.25mmol) of dithiodibutylhydrazine was added to the above reaction solution, 0.12g (0.625mmol) of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride was added, and 0.5mol/L hydrochloric acid was added to maintain the pH of the solution at 4.75, followed by reaction at room temperature for 4 hours. After the reaction, 1.43g (5mmol) of tris (2-carboxyethyl) phosphine hydrochloride was added to the reacted solution, and the reaction was stirred for 4 hours. The above solution was put into a dialysis tube (molecular weight was cut off 3500), dialyzed with a large amount of 0.001 mol/l hydrochloric acid and 0.2 mol/l sodium chloride solution for 2 days, and then dialyzed with a large amount of 0.001 mol/l hydrochloric acid solution for 1 day. And finally, collecting the solution in the dialysis tube, and freeze-drying to obtain flocculent solid, namely the multiple modified hyaluronic acid derivative.
The side chain sulfhydryl content of the multiple modified hyaluronic acid derivative of the invention can be detected by an improved Ellman reagent method (Shu et al, Biomacromolecules 2002,3:1304-1311) or hydrogen nuclear magnetic resonance (H-NMR)1H-NMR) (D2O is used as a solvent), and the like (taking a characteristic methyl absorption peak of acetyl of the hyaluronic acid as an internal standard), the content of the hyaluronic acid is 298 mu mol/g; the content of the side chain amino group can be detected by conventional ninhydrin colorimetry (Chen et al, J. Pharma analysis 2005, 25: 526-1H-NMR)(D2O is a solvent), and the content thereof is 94. mu. mol/g (with the characteristic methyl absorption peak of acetyl group of hyaluronic acid as an internal standard).
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and should not be taken as limiting the scope of the present invention, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (10)
1. A preparation method of a multiple modified hyaluronic acid derivative is characterized by comprising the following steps:
s1, reacting the side chain carboxyl of hyaluronic acid with diamine compound in the presence of a first activator to obtain a product containing hyaluronic acid derivative with side chain containing amino;
s2, step S1, reacting the side chain carboxyl group of the product with a dithiodihydrazide compound in the presence of a second activator to obtain a product including a hyaluronic acid derivative having an amino-containing side chain and a side chain having a disulfide dihydrazide structure;
s3, reacting the product obtained in the step S2 with a reducing agent to reduce the disulfide bond into a sulfhydryl group, and purifying to obtain the hyaluronic acid derivative simultaneously having the amino-containing side chain and the sulfhydryl-containing side chain.
2. The method of claim 1, wherein the first activator and the second activator are both carbodiimide compounds.
3. The method of claim 2, wherein the first activator and the second activator are each 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride.
4. The method of claim 1, wherein in step S1, the diamine compound is one selected from ethylenediamine, propylenediamine, and butylenediamine.
5. The method of claim 4, wherein the diamine compound is ethylene diamine.
6. The method of claim 1, wherein step S1 further comprises adding N-succinimide, N-hydroxythiosuccinimide, or hydroxybenzotriazole.
7. The method according to claim 1, wherein in step S2, the dithiodihydrazide compound is selected from one of dithiodipropylhydrazide or dithiodibutylhydrazide.
8. The method of claim 1, wherein in step S3, the reducing agent is selected from one of dithiothreitol and tris (2-carboxyethyl) phosphine hydrochloride.
9. The method of claim 1, wherein in step S1, the molar ratio of the diamine compound to the first activator is greater than or equal to 2.
10. The method of claim 1, wherein in step S1, the molar ratio of the diamine compound to the first activator is greater than or equal to 4.
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| CN103613686A (en) * | 2013-11-12 | 2014-03-05 | 广州市一杰医药科技有限公司 | Preparation method and application of sulfhydryl hyaluronic acid |
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