CN103910886A - Cysteine functionalized hyaluronic acid conjugate, synthetic method and application in injectable in-situ hydrogel thereof - Google Patents

Cysteine functionalized hyaluronic acid conjugate, synthetic method and application in injectable in-situ hydrogel thereof Download PDF

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CN103910886A
CN103910886A CN201410110610.7A CN201410110610A CN103910886A CN 103910886 A CN103910886 A CN 103910886A CN 201410110610 A CN201410110610 A CN 201410110610A CN 103910886 A CN103910886 A CN 103910886A
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hyaluronic acid
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CN103910886B (en
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张歆
胡碧煌
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Hainan University
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Abstract

The invention discloses a cysteine functionalized hyaluronic acid conjugate, a synthetic method and an application in preparation of injectable in-situ hydrogel thereof. According to the method, a hydroxy of hyaluronic acid is modified to obtain the cysteine functionalized hyaluronic acid conjugate with a stable ether bond, and tetrabutyl ammonium hydroxide is introduced in the reaction process. The new method improves the solubility of hyaluronic acid and increases the reaction efficiency. The obtained cysteine functionalized hyaluronic acid conjugate has a sulfhydryl active group which not only improves the adhesion of hyaluronic acid, but also can be further functionalized. According to the invention, injectable in-situ hydrogel is generated by a native chemical ligation reaction or a Michael addition reaction of the prepared cysteine functionalized hyaluronic acid conjugate with a polyethylene glycol conjugate; rheological research shows that the generated hydrogel has good rheological properties, the gel performance is controllable, and the hydrogel has wide application prospects in the biological medicine field.

Description

The conjugates of hyaluronic acid of halfcystine functionalization and synthetic method thereof and the application in the hydrogel of Injectable in-situ formation
Technical field
The present invention relates to a kind of conjugates of hyaluronic acid and synthetic method thereof of halfcystine functionalization, with and prepare Injectable in-situ form hydrogel in application, belong to biomedicine field.
Background technology
Hydrogel (Hydrogel) is the reticulated structure of hydrophilic polymkeric substance, can absorb and retain a large amount of moisture, expands at aqueous solution camber, has good biocompatibility.They are widely used in surgery encapsulant and tackiness agent, drug release, tissue filling, tissue repair and organizational project in medicine bioengineering field.When hydrogel is used to wound dressing, directly contact with tissue, can prevent external infected by microbes, effectively prevent the loss of body fluid, promote wound healing, pharmaceutical pack can also be rolled in hydrogel, medicine can see through gel and slowly be discharged into injury.The Sorbalgon hydrogel that for example has literature research to make with Sorbsan, for wound dressing wound healing [Hajek M, Sedlarik K M, Advantages of alginate bandages for coverage of extensive and poorly healing wounds, RozhiChir, 1992,71 (324): 152].Also there is reported in literature hydrogel to be used to the carrier of drug release, be rolled in hydrogel by pharmaceutical pack, by injecting or be implanted into after organism, maintain the long-time slowly controlled medicine that discharges, long-time performance drug effect, such as Miyata etc. has prepared Ag-Ab sensitive aqueous gel, for drug release carrier, plays the effect of specific drugs slowly-releasing.【Schmedlen?R?H，Masters?K?S，West?J?L.?Photocrosslindable?polyvinyl?alcohol-jydrogels?that?call?be?modified?with?celladhision?peptides?for?use?in?tissue?engineering.Biomaterials，2002，23∶4325】。Due to the good physical properties of hydrogel and biological characteristics, solid, the abdominal postoperative that also can be applicable to improve looks, dermatoplasty is skin graft prevents the aspects such as the sealing of adhesion and cranial cavity operation simultaneously, and applied research in this respect is more and more subject to scholars' concern.
Although its application in medicine has obtained huge progress, develop the still Challenge of hydrogel of the covalency interlinkage of the Injectable in-situ formation that does not use toxic reagent under mild conditions meeting clinical needs.The major defect of most of application aspect organizational project needs surgical operation exactly.But the hydrogel that Injectable in-situ forms has significant advantage: inject by needle tubing without operation, minimally body tissue is invaded, can fill the damaged of arbitrary shape, and can mix with medicine, play the effect of medicament slow release.
At organic sphere, sulfur ester (Thioesters) is the intermediate extensively existing, such as acetyl-CoA.Under the catalysis of enzyme, sulfur ester participates in the synthetic of the interior important component of organism, such as polypeptide hormone and lipid.In vitro, the relatively torpescence of chemical reaction of sulfur ester and primary amine compounds.But, it is found that, the compound of sulfur ester and N-end cysteine (phosphate buffer soln under physiological condition, pH7-9) after mixing, the sulfydryl of cysteine and the mercaptan of sulfur ester exchange, and after the intermediate of five annulus of formation, spontaneously reset, and produce a new amido linkage, be called nature chemistry and connect (Native Chemical Ligation, NCL).This chemical reaction oneself for polypeptide, protein, the synthetic of enzyme, also produces the polypeptide of tumor-necrosis factor glycoproteins for self-assembling polypeptide.Having report is to use NCL reaction that the hydrogel of the autonomous dress of short polypeptide has been increased to the hardness of assembling in advance polypeptide hydrogel by extending polypeptide length, also has the hydrogel that with NCL reaction, the polyethylene glycol conjugate interlinkage of branch is formed to covalency interlinkage in document.As the method for hydrogel covalency interlinkage, NCL has several obvious advantages: 1. have chemo-selective, thioester only reacts with the compound of halfcystine or N-end cysteine and forms new amido linkage, the interference that not existed by other mercaptan and sulfydryl; 2. under gentle condition, do not use the poisonous compound of possibility such as catalyzer, initiator and highly effective reaction; 3. different from other hydrogel that uses the chemical ligation of sulfydryl to form, naturally chemical ligation, in forming new amido linkage, also produces a mercapto groups on skeleton.So, this sulfydryl can be used for further functionalization hydrogel on skeleton according to different objects.And the polymkeric substance of sulfhydrylation (mercaptan) has better bioadhesive.4. in addition, mercaptan in thioester structure is discharged in solution in the time that NCL reacts, so, in design thioester, likely this mercaptan is designed to the medicine of sulfhydrylation and the precursor of cell growth factor, thereby when hydrogel forms, discharge these medicines and somatomedin in hydrogel.Therefore, utilize the hydrogel of the covalency interlinkage of the feature of NCL reaction and the Injectable in-situ formation that advantage obtains to there is significant superiority.For example: have document take polyoxyethylene glycol as main body, end thioesters or halfcystine functionalization, obtain polyoxyethylene glycol hydrogel [Bi-Huang Hu by naturally chemical ligation, Jing Su, and Phillip B.Messersmith, Hydrogels cross-linked by native chemical ligation, Biomacromolecules2009,10,2194-2200].
Hyaluronic acid (Hyaluronic acid, HA) is distributed widely in partes corporis humani position, is the main component of joint cavity synovia, is one of composition of cartilage matrix, and its reduces the friction between tissue, performance lubrication closing internode.HA is a kind of macromolecule polysaccharide base polymer of straight chain, is made up of N-ACETYL-D-GLUCOSAMINE and D-Glucose aldehydic acid disaccharide unit.In extracellular matrix, hyaluronic acid by with cell and the special and non-specific combination of other compositions, bringing into play important function of organization, such as, hyaluronic acid is the part of cell surface receptor CD44, and CD44 acceptor participates in various kinds of cell process, such as cell adhesion, cell migration and increment.Due to its important physiological action, after body or surface of a wound direct injection hyaluronic acid, can obviously alleviate and eliminate the inflammatory reaction of tissue, promote the surface of a wound and wound healing.But because hyaluronic acid is eliminated in vivo rapidly, such mitigation is also of short duration, so the hyaluronic acid gel of the covalent cross-linking that Injectable in-situ forms has just demonstrated its superiority.
The hyaluronic hydrogel of covalency interlinkage is mainly that the derivative that interlinkage can occur the reaction preparation by its carboxyl obtains hyaluronic acid gel.For example: having document is to modify hyaluronic carboxyl, obtain having by amido linkage combination and end the conjugates of hyaluronic acid of free sulfhydryl groups, again by being cross-linked to form injectable hyaluronic acid gel [Xiao Zheng Shu with polyethyleneglycol derivative, Yanchun Liu, Fabio S.Palumbo, Yi Luo, Glenn D.Prestwich, In situ crosslinkable hyaluronan hydrogels for tissue engineering, Biomaterials2004,25,1339-1348].Also there is document to modify by adipic dihydrazide, and add EDC and HOBt as couplant and catalyzer, modify hyaluronic carboxyl, obtain conjugates of hyaluronic acid again by being cross-linked to form hyaluronic acid derivatives [Yoon Yeo, Christopher B.Highley, Evangelia Bellas, Taichi Ito, Robert Marmi, Robert Langer, Daniel S.Kohane, In situ cross-linkable hyaluronic acid hydrogels prevent post-operative abdominal adhesions in a rabbit model, Biomaterials2006, 27, 4698-4705].Also have by forming the interlinkage chemical reaction of hydrogel with hyaluronic hydroxyl reaction, interlinkage compound is mainly 1,4-butanediol diglycidyl ether, polyethyleneglycol diglycidylether and divinylsulfone, also have and first use glycidyl ether and hyaluronic hydroxyl reaction, then carry out photopolymerization reaction formation hydrogel.For example: Chinese patent application 201010250802.X " a kind of method of preparing hyaluronic acid gel by using macromolecule photocrosslinking agent ", it is main body with hyaluronic acid, using macromolecule photocrosslinking agent is linking agent, under the irradiation of UV-light, prepares hyaluronic acid gel.Again for example: Chinese patent application 03156633.2 " a kind of hyaluronic acid derivatives and preparation method thereof " is carried out crosslinking reaction with glycidyl ether and divinylsulfone to hyaluronic hydroxyl respectively with Chinese patent application 200610024700.X " a kind of cross-linked hyaluronic acid derivatives preparation and preparation technology thereof ", obtains hyaluronic acid derivatives.And also there is document to modify hyaluronic hydroxyl by illumination reaction, the hyaluronic acid decorated thing that obtains connecting by ester bond, obtain hyaluronic acid gel [JimmyMergy by crosslinking reaction again, Audrey Fournier, Emilie Hachet, Rachel Auz_ely-Velty, Modification of polysaccharides via thiol-ene chemistry: a versatile route to functional biomaterials.J.Polym.Sci.Part A: Polym.Chem.2012.50,4019-3028].There is obvious prolongation the time that these crosslinked hyaluronic acids retain in vivo.
Be present in that in human body, to have the hydrogel that the hyaluronic acid of important physiological function forms after interlinkage should be more satisfactory biomaterial, it has overcome natural short shortcoming of hyaluronic acid transformation period.But the hydrogel of the interlinkage of preparing by hyaluronic carboxyl, may produce some side effects.Cell CD44 acceptor it is believed that it is by hyaluronic negative charge and hyaluronic acid effect [Morra, M., Engineering of biomaterials surfaces by hyaluronan, Biomacromolecules2005,6: 1205-1223.].Reduce hyaluronic negative charge and can affect cell CD44 acceptor and hyaluronic effect, thereby can reduce ability [the M B Herrera of cytothesis damage, B.B., S Brumo, L Morandol, G Mauriello-Romanazzi, F Sanavio3, I Stamenkovic, L Biancone and G Camussi, Exogenous mesenchymal stem cells localize to the kidney by means of CD44following acute tubular injury, Kidney Int.2007,72: 430-441.].Often need, by illumination reaction or with preformed hydrogel of additive method, so just can not realize in-situ injection and obtain hyaluronic acid gel by its hydroxyl reaction.Therefore, the present invention is take hyaluronic acid as raw material, by its hydroxyl reaction (not changing its negative charge), preparation have good biocompatibility, can obtain by naturally chemical ligation can in-situ injection covalent cross-linking hyaluronic acid gel there is superiority significantly.
Summary of the invention
The object of the present invention is to provide a kind of conjugates of hyaluronic acid of halfcystine functionalization.
Another object of the present invention is to the synthetic method of the conjugates of hyaluronic acid that a kind of halfcystine functionalization is provided.
The conjugates of hyaluronic acid that a further object of the present invention is to provide halfcystine functionalization is in the application of preparing in the hydrogel that Injectable in-situ forms.
The technical solution used in the present invention is:
A conjugates of hyaluronic acid for halfcystine functionalization, its structural formula is:
n is that 0～30, x is 200～3000.
A preparation method for the conjugates of hyaluronic acid of halfcystine functionalization, comprises the following steps:
1) getting structural formula is compound 1 be raw material, under acetone reflux condition, occur cyclization, generating structure formula is compound 2;
2) by compound 2 and tert-Butyl dicarbonate reaction, the amino structural formula by tertbutyloxycarbonyl (Boc) protection of generation is compound 3;
3) by compound 3 and N-hydroxy-succinamide under dicyclohexylcarbodiimide condition, there is esterification, generating structure formula is compound 4;
4) by compound 4 and cystamine under alkaline condition, there is acylation reaction, generating structure formula is compound 5;
5), by the disulfide bond reduction of compound 5, generating structure formula is compound 6;
6) by compound 6 and polyoxyethylene glycol compounds under alkaline condition, the ring-opening reaction of initial ring oxidative ethane, generating structure formula is compound 7;
7) by compound 7 and hyaluronic acid under TBAH condition, initial ring oxidative ethane ring-opening reaction, generating structure formula is compound 8;
8) compound 8 is sloughed to tertbutyloxycarbonyl (Boc) protecting group, then under concentrating under reduced pressure condition, ring-opening reaction is occurred, generating structure formula is compound 9, i.e. the conjugates of hyaluronic acid of halfcystine functionalization; Wherein, n is that 0～30, x is 200～3000.
Further, a kind of preparation method of conjugates of hyaluronic acid of halfcystine functionalization, its preparation method specifically comprises the following steps:
1) preparation of compound 2: get compound 1 cysteine hydrochloride, add dry acetone backflow 2～10h, be evaporated to the volume that no more than total reaction liquid one of is accumulated score of three, put into refrigerator and be no less than 0.5h, crystallization, cross leaching filter residue, obtain compound 2, wherein acetone is not less than 20 with the volume mass ratio of compound 1;
2) preparation of compound 3: compound 2 use acetonitriles are dissolved, add tert-Butyl dicarbonate, after mixing, dropwise add N, N-diisopropylethylamine, 15 ℃～37 ℃ stirring reaction 24～72h, concentrating under reduced pressure solvent, add again ether or hexanaphthene to continue concentrating under reduced pressure, add ether or hexanaphthene, with diatomite filtration, filtrate is used 0.01～0.1mol/LHCl solution, H successively again 2o, saturated NaCl extraction, dry, concentrating under reduced pressure, obtains compound 3, and wherein the amount of substance of tert-Butyl dicarbonate, DIPEA is independently 1.1～3 times of compound 2;
3) preparation of compound 4: compound 3 and N-hydroxy-succinamide are dissolved with tetrahydrofuran (THF), 0～4 ℃ of ice bath, toward wherein dripping N, the tetrahydrofuran solution of N '-dicyclohexylcarbodiimide, mix rear reaction 3～12h, complete by thin-layer chromatography detection reaction to reaction solution, put into 0～4 ℃ of refrigerator and leave standstill 0.5～12h, cross leaching filtrate, through concentrating under reduced pressure solvent, Virahol recrystallization, obtains compound 4, wherein N-hydroxy-succinamide, N, the amount of substance of N '-dicyclohexylcarbodiimide is independently 1.05～1.5 times of compound 2;
4) preparation of compound 5: the 2-aminoethyl disulfide dihydrochloride that is equivalent to 0.525～0.6 times of compound 4 molar weight by water dissolution molar weight, be equivalent to the sodium carbonate of 4 times of 2-aminoethyl disulfide dihydrochloride molar weights by water dissolution molar weight, drip in the 2-aminoethyl disulfide dihydrochloride aqueous solution, ice bath, stirs; With tetrahydrofuran (THF) dissolved compound 4, be added dropwise in above-mentioned reaction solution, 15 ℃～37 ℃ reaction 6～24h, until thin-layer chromatography detection reaction is complete; Concentrating under reduced pressure solvent, regulating pH is 3～7, then with ethyl acetate or dichloromethane extraction, 0.01～0.1mol/L HCl solution, H for organic phase 2o, saturated NaCl extraction, dry, concentrating under reduced pressure, obtains compound 5;
5) preparation of compound 6: by the ethanolic soln of compound 5, add again the water of 0.5～1.0 times of volume, ice bath stirs, slowly add again sodium borohydride, bubble to be no longer included is emerged, be placed in 15～37 ℃ of stirring reaction 4～12h, complete to thin-layer chromatography detection reaction, then add relative response liquid to amass the water termination reaction of 2～4 times; Regulating pH is 3～5, dichloromethane extraction, and washing, logical argon gas, sealing is dry, filters, and pressurization is concentrated obtains compound 6, and wherein, the amount of substance of sodium borohydride is 5～10 times of compound 5;
6) preparation of compound 7: toward the aqueous solution that adds sodium bicarbonate in the acetonitrile solution of polyoxyethylene glycol compounds, 15～37 ℃ stir, logical argon gas, again toward the acetonitrile solution that wherein drips compound 6, drip rear logical argon gas, acetonitrile in reaction solution: the volume ratio of water is (2: 3)～(3: 2), sealing, 15～37 ℃ of stirring reaction 6～24h, until thin-layer chromatography detection reaction is complete, Ellamn reagent colour development is not till displaing yellow, concentrating under reduced pressure solvent, with methylene dichloride or ethyl acetate extraction, organic phase washes with water rear dry, cross leaching filtrate, concentrating under reduced pressure obtains compound 7, wherein, sodium bicarbonate, the amount of substance of compound 6 is respectively 1～2 times of polyoxyethylene glycol compounds, 1.1～1.5 times,
7) preparation of compound 8: take its molecular weight and be 100,000～1,000,000 hyaluronic acid, add the TBAH aqueous solution, obtain hyaluronic acid reaction solution, wherein hyaluronic acid reaction solution mass concentration is 5%～20%, divide the acetonitrile solution that adds compound 7 for 1～4 time, 15～37 ℃ of stirring reaction 6～72h, with 0.01～1mol/LHCl solution neutralization reaction liquid, dialysis, freeze-drying, the washing of saturated sodium-chloride ethanolic soln, dry, obtain compound 8, wherein, TBAH, the amount of substance of compound 7 is respectively 0.5～2 times of hyaluronic acid primary alconol base, 0.5～10 times, acetonitrile volume is 0.5～2.5 times of the TBAH aqueous solution,
8) preparation of compound 9: it is in 80～100% trifluoroacetic acid aqueous solution that compound 8 is added to percent by volume, the concentration that makes compound 8 is 5～20g/L, 15～37 ℃ of stirring reaction 3～6h, concentrating under reduced pressure, adding volumetric concentration is 45～55% ethanolic soln, 30～40 ℃ of concentrating under reduced pressure 3～10 times, with after Ellnan reagent detection displaing yellow, separate out product with saturated sodium-chloride ethanolic soln, washes clean, dry, obtain product compound 9, i.e. the conjugates of hyaluronic acid of halfcystine functionalization.
Further, above-mentioned steps 4) and 5) described in to regulate the conditioning agent of pH be that volumetric concentration is 0.1%～2%HCl solution.
Further, above-mentioned steps 7) described in the dialyzate used of dialysis be the aqueous solution of mass percent 0.1～1.5% sodium-chlor and percent by volume 30～80% ethanol, at least dialyse 48 hours, at least change dialyzate 4 times.
The application of a kind of conjugates of hyaluronic acid of halfcystine functionalization in the hydrogel of preparing Injectable in-situ formation.
Further, above-mentioned application be specially Injectable in-situ form hydrogel generated by Michael reaction or naturally chemical ligation by conjugates of hyaluronic acid and the polyethylene glycol conjugate of halfcystine functionalization.
Further, above-mentioned polyethylene glycol conjugate is selected from structural formula and is polyethylene glycol acrylate binding substances, structural formula be polyoxyethylene glycol thioesters binding substances in a kind of, wherein m is that 50～500, y is 2～8, the number of polyethylene glycol conjugate side chain is that 2～8, R group is selected from middle one.
Further, above-mentioned polyoxyethylene glycol thioesters binding substances is selected from the polyoxyethylene glycol thioesters binding substances of 3-mercaptopropionic acid ethyl ester and polyoxyethylene glycol formation, the polyoxyethylene glycol thioesters binding substances that N-acetyl-L-cysteine and polyoxyethylene glycol form, the polyoxyethylene glycol thioesters binding substances that 2-ethane thiol sodium sulfonate and polyoxyethylene glycol form, the polyoxyethylene glycol thioesters binding substances that 4-sulfydryl phenylacetic acid and polyoxyethylene glycol form, 2-dimethylamino sulfur alcohol is a kind of in being combined with the polyoxyethylene glycol thioesters that polyoxyethylene glycol forms, wherein the polymerization degree of polyoxyethylene glycol thioesters binding substances is 50～500, side chain number is 2～8.
The invention has the beneficial effects as follows:
The invention provides a kind of novel halfcystine conjugates of hyaluronic acid and preparation method thereof, this derivatives of hyaluronic acids has terminal cysteine, even still keeping free end sulfydryl after forming hydrogel by the method that chemistry connects naturally, so can be according to further functionalization on skeleton of different objects, and the polymkeric substance of sulfhydrylation (mercaptan) has better bioadhesive, expand hyaluronic application, for biomedicine field provides a kind of desirable derivatives of hyaluronic acids; And the covalent cross-linking hyaluronic acid gel that the Injectable in-situ being prepared by this halfcystine conjugates of hyaluronic acid forms can be realized injectable and under physiological condition original position form, be the important breakthrough of technical field of biological material, have a good application prospect.
Accompanying drawing explanation
Fig. 1 is step 1 in embodiment 1) the hydrogen nuclear magnetic resonance spectrogram of synthetic intermediate;
Fig. 2 is step 2 in embodiment 1) the hydrogen nuclear magnetic resonance spectrogram of synthetic intermediate;
Fig. 3 is step 3 in embodiment 1) the hydrogen nuclear magnetic resonance spectrogram of synthetic intermediate;
Fig. 4 is step 4 in embodiment 1) the hydrogen nuclear magnetic resonance spectrogram of synthetic intermediate;
Fig. 5 is step 6 in embodiment 1) the UPLC-MS spectrogram of synthetic intermediate; ;
Fig. 6 is step 7 in embodiment 1) the hydrogen nuclear magnetic resonance spectrogram of synthetic intermediate;
Fig. 7 is step 8 in embodiment 1) the hydrogen nuclear magnetic resonance spectrogram of the conjugates of hyaluronic acid of synthetic end product halfcystine functionalization;
Fig. 8 is step 1 in embodiment 4) the hydrogen nuclear magnetic resonance spectrogram of synthetic intermediate E MPSA;
Fig. 9 is step 1 in embodiment 4) the hydrogen nuclear magnetic resonance spectrogram of synthetic intermediate four branch's polyoxyethylene glycol thioesters binding substancess;
Figure 10 is the typical curve of halfcystine absorbancy;
Figure 11 is the rheology time scan figure of the hydrogel that forms of the Injectable in-situ prepared of embodiment 4, storage modulus G ' and out-of-phase modulus G ";
Figure 12 is the rheology time scan detail view of the hydrogel that forms of the Injectable in-situ prepared of embodiment 4;
Figure 13 is the rheology frequency sweeping figure of the hydrogel that forms of the Injectable in-situ prepared of embodiment 4;
Figure 14 is the rheology stress scans figure of the hydrogel that forms of the Injectable in-situ prepared of embodiment 4;
Figure 15 is the rheology time scan figure of the hydrogel that forms of the Injectable in-situ prepared of embodiment 5;
Figure 16 is the rheology frequency sweeping figure of the hydrogel that forms of the Injectable in-situ prepared of embodiment 5;
Figure 17 is the rheology stress scans figure of the hydrogel that forms of the Injectable in-situ prepared of embodiment 5;
Figure 18 is the rheology time scan figure of the hydrogel that forms of the Injectable in-situ prepared of embodiment 6;
Figure 19 is the rheology frequency sweeping figure of the hydrogel that forms of the Injectable in-situ prepared of embodiment 6;
Figure 20 is the rheology stress scans figure of the hydrogel that forms of the Injectable in-situ prepared of embodiment 6;
Figure 21 is the rheology time scan figure of the hydrogel that forms of the Injectable in-situ prepared of embodiment 6;
Figure 22 is the rheology frequency sweeping figure of the hydrogel that forms of the Injectable in-situ prepared of embodiment 6;
Figure 23 is the rheology stress scans figure of the hydrogel that forms of the Injectable in-situ prepared of embodiment 6.
Embodiment
Injectable original position forms the preparation method of the hyaluronic acid gel of covalent cross-linking:
(1) preparation of the conjugates of hyaluronic acid of halfcystine functionalization
Synthetic route is as follows:
In conjunction with said synthesis route, specifically describe the synthesis step of the hyaluronic acid of cysteine modified:
1. the preparation of compound 2: get compound 1 cysteine hydrochloride, at least add the dry acetone backflow 2～10h with respect to compound 20 times of volumes of 1 quality (volume/mass), be evaporated to the no more than total reaction liquid volume of one of accumulating score of three, put into refrigerator and be no less than 0.5h, crystallization, Büchner funnel filters, and obtains compound 2, and the compound 2 in synthetic route be shown in its chemical structural formula;
2. the preparation of compound 3: compound 2 use acetonitriles are dissolved, add molar weight to be equivalent to the tert-Butyl dicarbonate of 1.1～3 times of the molar weights of compound 2, after mixing, dropwise add molar weight to be equivalent to the N of 1.1～3 times of the molar weights of compound 2, N-diisopropylethylamine, 15 ℃～37 ℃ stirring reaction 24～72h, concentrating under reduced pressure solvent, add again ether or hexanaphthene to continue concentrating under reduced pressure, add ether or hexanaphthene, with diatomite filtration, filtrate is with 0.01～0.1mol/L HCl solution, H again 2o, saturated NaCl extraction, dry, concentrating under reduced pressure, obtains compound 3, and the compound 3 in synthetic route be shown in its chemical structural formula;
3. the preparation of compound 4: the N-hydroxy-succinamide tetrahydrofuran (THF) that compound 3 and molar weight is equivalent to 1.05～1.5 times of the molar weights of compound 3 dissolves, 0 ℃～4 ℃ ice baths, molar weight is equivalent to the N of 1.05～1.5 times of compound 3 molar weights, N '-dicyclohexylcarbodiimide is dissolved with tetrahydrofuran (THF), drip in reaction solution, reaction 3～12h, to no longer variation of thin-layer chromatography detection for reaction solution, put into 0～4 ℃ of refrigerator and leave standstill 0.5～12h, cross leaching filtrate, concentrating under reduced pressure solvent, Virahol recrystallization, obtain compound 4, the compound 4 in synthetic route be shown in its chemical structural formula,
4. the preparation of compound 5: the 2-aminoethyl disulfide dihydrochloride that is equivalent to 0.525～0.6 times of compound 4 molar weight by water dissolution molar weight, be equivalent to the sodium carbonate of 4 times of 2-aminoethyl disulfide dihydrochloride molar weights by water dissolution molar weight, drip in the 2-aminoethyl disulfide dihydrochloride aqueous solution, ice bath, stirs; With tetrahydrofuran (THF) dissolved compound 4, be added dropwise in above-mentioned reaction solution, 15 ℃～37 ℃ reaction 6～24h, until TLC detection reaction is complete; Concentrating under reduced pressure solvent, is that 0.1%～2%HCl solution is acidified to pH3～7 by volumetric concentration, then with ethyl acetate or dichloromethane extraction, 0.01～0.1mol/LHCl solution, H for organic phase 2o, saturated NaCl extraction, dry, concentrating under reduced pressure, obtains compound 5; The compound 5 in synthetic route be shown in its chemical structural formula;
5. the preparation of compound 6: the water of 0.5～1.0 times that adds volume after compound 5 use dissolve with ethanol, ice bath stirs, slowly add molar weight to be equivalent to the sodium borohydride of 5～10 times of compound 5 molar weights, bubble to be no longer included is emerged, be placed in 15～37 ℃ of stirring reaction 4～12h, treat that thin-layer chromatography detection reaction liquid no longer includes variation, add with respect to 2～4 times of volume water termination reactions of reaction solution volume; With 0.01-1mol/LHCl solution acidifying reaction solution be pH3～5, dichloromethane extraction, washing, logical argon gas, sealing is dry, filters, pressurization is concentrated obtains compound 6, the compound 6 in synthetic route be shown in its chemical structural formula;
6. the preparation of compound 7: polyoxyethylene glycol compounds is dissolved with acetonitrile, be equivalent to the sodium bicarbonate of 1～2 times of polyoxyethylene glycol compounds molar weight and add wherein by water dissolution molar weight, 15～37 ℃ stir, logical argon gas, the compound 6 use acetonitriles that molar weight are equivalent to 1.1～1.5 times of polyoxyethylene glycol compounds molar weights dissolve, be added dropwise in reaction solution, drip rear logical argon gas, acetonitrile in reaction solution: the volume ratio of water is (2: 3)～(3: 2), sealing, 15～37 ℃ of stirring reaction 6～24h, react to thin-layer chromatography and detect, Ellamn reagent colour development is not till displaing yellow, concentrating under reduced pressure solvent, with methylene dichloride or ethyl acetate extraction, organic phase washes with water rear dry, cross leaching filtrate, concentrating under reduced pressure obtains compound 7, the compound in synthetic route be shown in its chemical structural formula,
7. the preparation of compound 8: take its molecular weight and be 100,000～1,000,000 hyaluronic acid (HA), add molar weight to be equivalent to 0.5～2 times of TBAH aqueous solution of hyaluronic acid primary alconol base molar weight, obtain hyaluronic acid reaction solution, wherein hyaluronic acid reaction solution mass concentration is 5%～20%, be equivalent to the compound 7 of 0.5～10 times of hyaluronic acid primary alconol base molar weight by the acetonitrile dissolving molar weight that volume is 0.5～2.5 times of the TBAH aqueous solution, dividing 1～4 batch adds in above-mentioned reaction solution, 15～37 ℃ of stirring reaction 6～72h, with 0.01～1mol/L HCl solution neutralization reaction liquid, dialysis, freeze-drying, the washing of saturated sodium-chloride ethanolic soln, dry, obtain compound 8, the compound 8 in synthetic route be shown in its chemical structural formula,
When dialysis wherein, dialyzate used is 30%-80% ethanolic soln (containing mass percent is that 0.1%-1.5% sodium-chlor and percent by volume are the aqueous solution of the 30%-80% ethanol) dialysis at least 48 hours of 0.1%-1.5% sodium-chlor, changes dialyzate at least four times;
8. the preparation of compound 9: the trifluoroacetic acid aqueous solution that is 80～100% compound 8 use percent by volumes, the concentration that makes compound 8 is 5～20g/L, 15～37 ℃ of stirring reaction 3～6h, concentrating under reduced pressure, adding volumetric concentration is 45～55% ethanolic soln, 30～40 ℃ of concentrating under reduced pressure 3～10 times, with after Ellman reagent detection displaing yellow, separate out product with saturated sodium-chloride ethanolic soln, wash for several times, dry, obtain product compound 9, be halfcystine conjugates of hyaluronic acid, the compound 9 in synthetic route be shown in its chemical structural formula.
(2) preparation of polyethylene glycol conjugate
Polyethylene glycol conjugate is preferably structural formula polyethylene glycol acrylate binding substances, structural formula be polyoxyethylene glycol thioesters binding substances in a kind of, wherein m is that 50～500, y is 2～8, the number of polyethylene glycol conjugate side chain is that 2～8, R group is selected from middle one.
Wherein, polyoxyethylene glycol thioesters binding substances is preferably the polyoxyethylene glycol thioesters binding substances of 3-mercaptopropionic acid ethyl ester and polyoxyethylene glycol formation, the polyoxyethylene glycol thioesters binding substances that N-acetyl-L-cysteine and polyoxyethylene glycol form, the polyoxyethylene glycol thioesters binding substances that 2-ethane thiol sodium sulfonate and polyoxyethylene glycol form, the polyoxyethylene glycol thioesters binding substances that 4-sulfydryl phenylacetic acid and polyoxyethylene glycol form, 2-dimethylamino sulfur alcohol is a kind of in being combined with the polyoxyethylene glycol thioesters that polyoxyethylene glycol forms, wherein the polymerization degree of polyoxyethylene glycol thioesters binding substances is 50～500, the number of side chain is 2～8.
(3) preparation of the hydrogel that Injectable in-situ forms
Compound 9 and polyethylene glycol conjugate can be obtained to the hydrogel that Injectable in-situ forms by Michael reaction or naturally chemical ligation, and concrete operations are: make compound 9 and polyethylene glycol conjugate at 5% (m/v) NaHCO 3the aqueous solution or pH are in 7.0～8.0 phosphate buffered saline buffer, and 20 ℃～37 ℃ to reacting completely.
Below in conjunction with specific embodiment, the present invention is further illustrated, but be not limited to this.
Embodiment 1: the preparation of the conjugates of hyaluronic acid of halfcystine functionalization
1) preparation of Apc: get 10g cysteine hydrochloride and add 1000mL dry acetone backflow 6h, be evaporated to about 200mL, put into refrigerator overnight, crystallization, Büchner funnel filters, obtain intermediate white crystals Apc (compound 2) quality 11g, productive rate is 88%, and its proton nmr spectra as shown in Figure 1.
2) preparation of Boc-Apc: 10.9g (55mmol) compound 2 use 250mL acetonitriles are dissolved, add 16g (73mmol) tert-Butyl dicarbonate, after mixing, dropwise add 12.8g (99mmol) DIPEA, 28 ℃ of stirring reaction 48h.Concentrating under reduced pressure solvent, then add ether or hexanaphthene to continue concentrating under reduced pressure, then add ether or hexanaphthene, with diatomite filtration, filtrate is used 0.01mol/LHCl solution, H 2o respectively washes 3 times, and saturated NaCl washes 1 time, anhydrous magnesium sulfate drying 2 hours, and concentrating under reduced pressure, obtaining intermediate B oc-Apc (compound 3) quality is 6g, and productive rate is 40%, and its proton nmr spectra is as shown in Figure 2.
3) preparation of Boc-Apc-OSU: 3g (11.5mmol) compound 3 and 1.45g (12.6mmol) N-hydroxy-succinamide are dissolved with 30mL tetrahydrofuran (THF), 0 ℃ of ice bath, 2.6g (12.6mmol) N, N '-dicyclohexylcarbodiimide is dissolved with 30mL tetrahydrofuran (THF), drip in reaction solution, reaction 6h, thin-layer chromatography detection reaction liquid no longer changes, put into refrigerator and leave standstill 0.5h, filter, filtrate decompression concentrated solvent, Virahol recrystallization, obtaining intermediate white crystal Boc-Apc-OSU (compound 4) quality is 3.7g, productive rate is 90.42%.
4) preparation of Boc-Apc-cys: with 20mL ultrapure water dissolving 2.24g (9.94mmol) halfcystine dihydrochloride, then with 10mL ultrapure water dissolving 4.2g sodium carbonate, drip in the halfcystine dihydrochloride aqueous solution, ice bath, stirs.With 60mL tetrahydrofuran (THF) dissolved compound 4, be added dropwise in above-mentioned reaction solution, 28 ℃ of reaction 12h, thin-layer chromatography detection reaction is complete.Concentrating under reduced pressure solvent, is that 2%HCl solution is acidified to pH5 with mass percent, then is extracted with ethyl acetate 3 times, the ethyl acetate of merging is used 0.01mol/L HCl solution, H mutually 2o respectively washes three times, and saturated NaCl washes once, anhydrous magnesium sulfate drying 2 hours, and concentrating under reduced pressure, obtaining the transparent oily matter Boc-Apc-cys of intermediate (compound 5) quality is 5.7g, productive rate is 94.4%.
5) preparation of Boc-Apc-SH: by after 6.37g (10mmol) compound 5 use 30mL dissolve with ethanol, add 30ml ultrapure water, ice bath stirs, slowly add 3.8g (100mmol) sodium borohydride, bubble to be no longer included is emerged, 28 ℃ of stirring reaction 6h, thin-layer chromatography detection reaction liquid no longer includes variation, adds 150mL ultrapure water termination reaction.With 1mol/LHCl solution acidifying reaction solution pH=3, dichloromethane extraction 3 times, the methylene dichloride merging is washed 3 times mutually, logical argon gas, sealing anhydrous magnesium sulfate drying 2 hours, filter, it is 8.38g that concentrating under reduced pressure obtains the transparent oily matter Boc-Apc-SH of intermediate (compound 6) quality, and productive rate is 99.8%.
6) preparation of Boc-Apc-oxyethane: 1.84mL (10mmol) 1, 4-butanediol diglycidyl ether dissolves with 5ml acetonitrile, dissolving 840mg (10mmol) sodium bicarbonate with 30mL ultrapure water adds wherein, 28 ℃ of stirrings, logical argon gas, 3.52g (11mmol) compound 6 use 15mL acetonitriles are dissolved, be added dropwise in above-mentioned reaction solution, drip rear logical argon gas, sealing, 28 ℃ of stirring reaction 12h, thin-layer chromatography detects, not displaing yellow of Ellamn reagent colour development, stopped reaction, concentrating under reduced pressure solvent, with dichloromethane extraction water 3 times, the methylene dichloride phase being associated with, wash with water after 3 times, anhydrous magnesium sulfate drying 2 hours, filter, it is 5.15g that concentrating under reduced pressure obtains the transparent oily matter Boc-Apc-of intermediate oxyethane (compound 7) quality, productive rate 98.6%.
7) preparation of Boc-Apc-HA: take the hyaluronic acid that 500mg (1.3055mmol primary alconol base) molecular weight is 100,000, add the TBAH aqueous solution of 1.35mL25% (mass/volume), add again 1.15mL ultrapure water, obtain hyaluronic acid reaction solution, dissolving 1.37g compound 7 with 1.5mL acetonitrile adds in above-mentioned reaction solution, 28 ℃ of stirring reaction 12h, dissolving 1.37g compound 7 with 1.5mL acetonitrile again adds in above-mentioned reaction solution, 28 ℃ of stirring reaction 24h, reaction total time is 36h, reaction finishes rear with 0.1mol/LHCl solution neutralization reaction liquid, with the aqueous solution dialysis 48h of (percent by volume) 50% ethanol of (mass percent) 0.9% sodium-chlor, change dialyzate four times, dialyse with ultrapure water for the last time, freeze-drying, obtain intermediate B oc-Apc-HA (compound 8).
8) preparation of the conjugates of hyaluronic acid of halfcystine functionalization: 28 ℃ of stirring reaction 3h of trifluoroacetic acid-aqueous solution of compound 8 use 80% (percent by volume), the concentration that makes compound 8 is 5～15g/L, concentrating under reduced pressure, add ethanol: water=1: 35 ℃ of concentrating under reduced pressure of l (volume ratio) 10 times, with Ellman reagent detection displaing yellow, separate out product with saturated sodium-chloride ethanolic soln, wash 3 times, dry, obtain compound 9, i.e. the conjugates of hyaluronic acid of halfcystine functionalization.
Embodiment 2: the preparation of the conjugates of hyaluronic acid of halfcystine functionalization
The preparation method of embodiment 2 in the same manner as in Example 1, except by the step 6 in embodiment 1) replace with:
The preparation of Boc-Apc-PEG-500: the polyoxyethylene glycol (PEG-500) that is 500 5.26g (10mmol) molecular weight dissolves with 5ml acetonitrile, dissolving 840mg (10mmol) sodium bicarbonate with 30mL ultrapure water adds wherein, 28 ℃ of stirrings, logical argon gas, 3.52g (11mmol) compound 6 use 15mL acetonitriles are dissolved, be added dropwise in above-mentioned reaction solution, drip rear logical argon gas, sealing, 28 ℃ of stirring reaction 12h, thin-layer chromatography detects, not displaing yellow of Ellamn reagent colour development, stopped reaction, concentrating under reduced pressure solvent, with dichloromethane extraction water 3 times, the methylene dichloride being associated with washes with water after 3 times mutually, anhydrous magnesium sulfate drying 2 hours, filter, it is 6.03g that concentrating under reduced pressure obtains transparent oily compounds 7 quality, productive rate 71.3%, then by Boc-Apc-PEG-500 for embodiment 1 step 7) and 8) reaction, can obtain the conjugates of hyaluronic acid of halfcystine functionalization.
Embodiment 3: the preparation of the conjugates of hyaluronic acid of halfcystine functionalization
The preparation method of embodiment 3 in the same manner as in Example 1, except by the step 6 in embodiment 1) replace with:
The preparation of Boc-Apc-PEG-1000: the polyoxyethylene glycol (PEG-1000) that is 1000 10g (10mmol) molecular weight dissolves with 40ml acetonitrile, dissolving 840mg (10mmol) sodium bicarbonate with 30mL ultrapure water adds wherein, 28 ℃ of stirrings, logical argon gas, 3.52g (11mmol) compound 6 use 10mL acetonitriles are dissolved, be added dropwise in above-mentioned reaction solution, after dripping, be to adjust reaction solution to clear state, add again 40mL pure water, logical argon gas, sealing, 28 ℃ of stirring reaction 15h, thin-layer chromatography detects, not displaing yellow of Ellamn reagent colour development, stopped reaction, concentrating under reduced pressure solvent, with dichloromethane extraction water 3 times, the methylene dichloride phase being associated with, wash with water after 3 times, anhydrous magnesium sulfate drying 2 hours, filter, it is 10.86g that concentrating under reduced pressure obtains transparent oily compounds 7 quality, productive rate 82.3%, then by Boc-Apc-PEG-1000 for embodiment 1 step 7) and 8) reaction, can obtain the conjugates of hyaluronic acid of halfcystine functionalization.
Embodiment 4: the preparation of the hydrogel that Injectable in-situ forms
1) prepare the four polyoxyethylene glycol thioesters binding substances 4-arm-PEG-EMP of branch:
Preparation EMPSA: take succinyl oxide 2.0g (20mmol) and DMAP 122mg (DMAP, 1mmol) be dissolved in 25mL acetonitrile: acetone=9: in 1 solvent systems, add 3-mercaptopropionic acid ethyl ester 2.95g (EMP, 22mmol), and pass into argon gas in reaction solution, stirring reaction spends the night, after concentrating under reduced pressure, dissolve in 50mL ethyl acetate, extract 3 times with 30mL0.1mol/L hydrochloric acid soln and water respectively, after anhydrous magnesium sulfate drying, after 2h, evaporate to dryness obtains white solid, be EMPSA (EMP-succinyl oxide binding substances), its proton nmr spectra as shown in Figure 8.
Preparation 4-arm-PEG-EMP: take respectively molecular weight and be 10000 the four polyoxamide 1g (4-arm-PEG of branch, 0.4mnol), 0.234g EMPSA (1mmol) and 0.221g (1mmol) benzotriazole-1-base oxygen base three (dimethylamino) phosphorus hexafluorophosphate (BOP) are placed in a bottle, add 2mL methylene dichloride to dissolve, add again 0.348mL (2mmol) N, N '-dicyclohexylcarbodiimide concuss 5 minutes, mix completely, stirring reaction 2 hours, thin-layer chromatography detects with the colour developing of acid ninhydrine test solution, after reacting completely, except desolventizing, add after 50mL dissolve with methanol, being placed in-20 ℃ of refrigerators separates out after white solid, be placed in rapidly at-10 ℃ whizzer centrifugal, liquid inclines, repeat after this purification step 3 times, dry, obtain white solid, obtain the four polyoxyethylene glycol thioesters binding substances 4-arm-PEG-EMP of branch, its structural formula is m is about 220, and molecular-weight average is about 10000, and its proton nmr spectra as shown in Figure 9.
2) conjugates of hyaluronic acid 100 μ L5% (m/v) sodium hydrogen carbonate solutions (pH8.0) that take halfcystine functionalization prepared by 10mg embodiment 1 dissolve, taking 1mg4-arm-PEG-EMP 100 μ L5% (m/v) sodium hydrogen carbonate solutions (pH8.0) dissolves, two solution mix, mix rapidly, in 28 ℃ of 3min, form hyaluronic acid gel, the hydrogel that Injectable in-situ forms.
Embodiment 5: the preparation of the hydrogel that Injectable in-situ forms
Conjugates of hyaluronic acid 100 μ L5% (m/v) sodium hydrogen carbonate solutions that take halfcystine functionalization prepared by 10mg embodiment 1 dissolve, and (structural formula is to take 2mg straight chain polyoxyethylene glycol thioesters binding substances m is about 220, and molecular-weight average is about 10000) dissolve with 100 μ L5% (m/v) sodium hydrogen carbonate solutions, two solution mix, and mix rapidly, form hyaluronic acid gels, the hydrogel that Injectable in-situ forms in 28 ℃ of 3min.
Embodiment 6: the preparation of the hydrogel that Injectable in-situ forms
The preparation method of embodiment 6 in the same manner as in Example 4, except by the step 2 in embodiment 4) replace with:
Take the 100 μ L pH7.2 phosphate buffered saline buffers for conjugates of hyaluronic acid of halfcystine functionalization prepared by 10mg embodiment 1, taking 2mg4-arm-PEG-EMP 100pL pH7.2 phosphate buffered saline buffer dissolves, two solution mix, mix rapidly, in 28 ℃ of 3min, form hyaluronic acid gel, the hydrogel that Injectable in-situ forms.
Embodiment 7: the preparation of the hydrogel that Injectable in-situ forms
Conjugates of hyaluronic acid 100 μ L5% (m/v) sodium hydrogen carbonate solutions that take halfcystine functionalization prepared by 10mg embodiment 1 dissolve, and (structural formula is to take 1mg molecular weight and be four branch's polyethylene glycol acrylate binding substancess of 10k m is about 220, molecular-weight average is about 10000) with 100 μ L5% (m/v) sodium hydrogen carbonate solutions (pH8.0) dissolve, two solution mix, and form hyaluronic acid gel, the hydrogel that Injectable in-situ forms in 28 ℃ of 3min.
Further effect detection is made in the preparation of the hydrogel that the conjugates of hyaluronic acid to the halfcystine functionalization of preparing in embodiment, Injectable in-situ form below.
One, mass spectrometric detection
Conjugates of hyaluronic acid and the each intermediate of getting the target product halfcystine functionalization in embodiment 1,4 carry out proton nmr spectra detection or UPLC-MS detection, detect collection of illustrative plates as shown in Fig. 1～10, learn that by atlas analysis the each intermediate and the end product that prepare are the synthetic target product of expectation.
Fig. 1 is step 1 in embodiment 1) the hydrogen nuclear magnetic resonance spectrogram of synthetic intermediate A pc, signal in figure is analyzed to ownership: 1h NMR (600MHz, MeOD), δ h4.78 (1H, t, J=8.4Hz ,-C h-COOH), δ h3.46 (1H, dd, J=12.0Hz, 8.4Hz ,-C h 2-SH), δ h3.32 (1H, dd, J=12.0Hz, 7.8Hz ,-C h 2-SH), δ h1.61 (3H, s ,-C-C h 3), δ h1.60 (3H, s ,-C-C h 3), illustrate that this intermediate is target intermediate product Apc.
Fig. 2 is step 2 in embodiment 1) the hydrogen nuclear magnetic resonance spectrogram of synthetic intermediate, signal in figure is analyzed to ownership: 1h NMR (600MHz, DMSO), δ h12.74 (1H, br s ,-COO h), δ h4.74 (1H, dd, J=8.4Hz, 5.4Hz ,-C h-COOH), δ h3.36 (1H, d, J=12.0Hz ,-C h 2-SH), δ h3.04 (1H, d, J=12.0Hz ,-C h 2-SH), δ h1.75 (3H, s ,-C-C h 3), δ h1.73 (3H, s ,-C-C h 3), 1.42 (3H, s, ter-butyl), δ H1.35 (6H, s, ter-butyl), illustrates that this intermediate is target intermediate product Boc-Apc.
Fig. 3 is step 3 in embodiment 1) the hydrogen nuclear magnetic resonance spectrogram of synthetic intermediate, signal in figure is analyzed to ownership: 1h NMR (600MHz, CDCl 3), δ h5.11 (1H, d, J=6.0 ,-C h-CH 2-), δ h3.43 (1H, d, J=12.0Hz ,-C h 2-S-), δ h3.30 (1H, d, J=12.0Hz ,-C h 2-S-), δ h2.82 (4H, t, J=6.6Hz ,-CO-C h 2-C h 2-CO-), δ h1.87 (3H, s ,-C-C h 3), δ h1.79 (3H, s ,-C-C h 3), 1.48 (3H, s, ter-buty1), δ h1.45 (6H, s, ter-butyl), illustrate that this intermediate is target intermediate product Boc-Apc-OSU.
Step 4 in Fig. 4 embodiment 1) the hydrogen nuclear magnetic resonance spectrogram of synthetic intermediate; , signal in figure is analyzed to ownership: 1h NMR (600MHz, DMSO), δ h7.97 (2H, br s ,-CON h-), δ h4.57 (2H, br s ,-C h-CONH-), δ h3.42 (2H, m ,-CONH-C h 2-), δ h3.34 (2H, d, J=10.8Hz ,-C h 2-SH), δ h3.28 (2H, m ,-CONH-C h 2-), δ h2.94 (2H, d, J=10.8Hz ,-C h 2-SH), δ h2.79 (4H, br s ,-C h 2-S-S-), δ h1.77 (6H, s ,-C-C h 3), δ h1.70 (6H, s ,-C-C h 3), δ h1.34 (18H, br s, ter-butyl), illustrate that this intermediate is target intermediate product Boc-Apc-cys.
Step 6 in Fig. 5 embodiment 1) the UPLC-MS figure (Ultra Performance Liquid Chromatography tandem mass spectrometry) of synthetic intermediate, signal in figure is analyzed to ownership: have three peaks through detecting in ultra-high efficiency liquid phase uv-spectrogram, their peaks while being time=5.5min respectively of connexus spectrum analysis are the peak of product compound 7 again, account for 50%; The peak going out when time=7.5min is that BDDE two ends are all reacted the by product (by product 1) obtaining and accounted for 30% with compound 6; The peak occurring when time=9.5min is that the oxidation products (by product 2) that compound 6 autoxidations obtain having disulfide linkage accounts for 20%.In mass spectrum, specific charge is 545.24 for product compound 7[M+Na] peak, specific charge is the 865.37 [M that are by product 1 1+ Na] peak, specific charge is 661.22 to be the [M of by product 2 2-2H+Na] peak, illustrate that this intermediate is target intermediate product Boc-Apc-oxyethane (compound 7).
Fig. 6 is step 7 in embodiment 1) the hydrogen nuclear magnetic resonance spectrogram of synthetic intermediate; , signal in figure is analyzed to ownership, at δ hthere is tertbutyloxycarbonyl signal in 1.34ppm place, so can judge that this this intermediate is target intermediate product Boc-Apc-HA, but Boc-Apc-HA molecular weight is larger, when magnetic resonance detection, have a small amount of error, so it is Boc-Apc-HA that this proton nmr spectra can only judge this intermediate, but its modification rate still needs ultraviolet spectrophotometry further to detect.
Fig. 7 is the hydrogen nuclear magnetic resonance spectrogram of the conjugates of hyaluronic acid of halfcystine functionalization, and signal in figure is analyzed to ownership: on the basis of proton nmr spectra that can mesosome Boc-Apc-HA at δ hthe tertbutyloxycarbonyl blackout at 1.34ppm place, can judge that this compound is the conjugates of hyaluronic acid of target product halfcystine functionalization.
Fig. 8 is step 1 in embodiment 4) the hydrogen nuclear magnetic resonance spectrogram of synthetic intermediate E MPSA (EMP-succinyl oxide binding substances), signal in figure is analyzed to ownership: 1hNMR (600MHz, CDCl3), δ H9.91 (s, br), 4.13 (2H, q, J=7.2Hz ,-C h 2 -CH 3), 3.12 (2H, t, J=7.2Hz ,-S-C h 2-), 2.87 (2H, t, J=7.2Hz ,-S-CO-C h 2-), 2.68 (2H, t, J=7.2Hz ,-S-CH 2-C h 2-), 2.60 (2H, t, J=7.2Hz ,-C h 2-COOH), 1.24 (3H, t, J=7.2Hz ,-CH 24C h 3), illustrate that this intermediate is target intermediate product EMPSA (EMP-succinyl oxide binding substances).
Fig. 9 is step 1 in embodiment 4) the hydrogen nuclear magnetic resonance spectrogram of synthetic intermediate 4-arn-PEG-EMP, signal in figure is analyzed to ownership: 1h NMR (600MHz, CDCl 3), δ h4.15 (2H, q, J=7.0Hz ,-C h 2-CH3-), δ h3.79-3.41 (m, ,-O-C h 2 -CH 2-O-), δ h3.12 (2H, t, J=7.0Hz ,-S-C h 2 -), δ h2.92 (2H, t, J=7.0Hz ,-S-CO-C h 2 -), δ h2.61 (2H, t, J=7.0Hz ,-S-C h 2 -C h 2 -), δ h2.52 (2H, t, J=7.0Hz ,-C h 2-COOH), δ h1.26 (3H, t, J=7.0HZ ,-C h 2-CH 3-), illustrate that this intermediate is target intermediate product 4-arm-PEG-EMP.
Two, the modification rate of the conjugates of hyaluronic acid of halfcystine functionalization detects
The measuring method of the modification rate of the conjugates of hyaluronic acid of halfcystine functionalization is ultraviolet spectrophotometry, detects the hyaluronic sulfhydryl content of terminal cysteine calculate modification rate by Ellman reagent.First make halfcystine typical curve, then use determined by ultraviolet spectrophotometry sample absorbancy, calculate sulfhydryl content, finally draw modification rate.
1) production standard curve:
The configuration of damping fluid: take 0.254gNaH 2pO 4, with 10.162g Na 2hPO 4the EDTA (ethylenediamine tetraacetic acid (EDTA)) that adds 148.896mg after mixing, is formulated as 500mL.
Ellman reagent solution: add 4mg Ellman reagent in 1ml damping fluid.
The preparation of table 1 cysteine standard solution
Standard reagent Volume of buffer solution Halfcystine amount Ultimate density
A 100mL 26.34mg 1.5mmol／L
B 5mL 25mL standard reagent solution A 1.25mmol／L
C 10mL 20mL standard reagent solution A 1.0mmol／L
D 15mL 15mL standard reagent solution A 0.75mmol／L
E 20mL 10mL standard reagent solution A 0.5mmol／L
F 25mL 5mL standard reagent solution A 0.25mmol／L
G 30mL 0mL standard reagent solution A 0mmol/L (blank)
Measure the absorbancy (as shown in table 2) of the cysteine standard solution prepared by table 1, and production standard curve is as shown in Figure 10, obtaining typical curve equation is y=0.1692x-0.0001, R 2=0.9994.
Table 2 cysteine standard solution absorbancy
2) measure the modification rate that embodiment 1 prepares the conjugates of hyaluronic acid of halfcystine functionalization
Preparation sample solution: the conjugates of hyaluronic acid (containing 0.0274mmol primary hydroxyl group) that takes halfcystine functionalization prepared by 10.5 milligrams of embodiment 1 dissolves with damping fluid and is settled to 5mL;
Preparation blank solution: take 10.5mg hyaluronic acid (containing 0.0274mmol primary hydroxyl group) and dissolve with damping fluid and be settled to 5mL;
Get test tube, in vitro add 50 μ L Ellman reagent and 2.5mL buffer solution of sodium phosphate at every respectively, respectively in vitro, add 50 μ L standardized solution or testing samples, mix, place 15 minutes in room temperature, take solution G as blank, when test sample, do blank with hyaluronic acid solution, measure the absorbance (A) of halfcystine reference liquid and testing sample in wavelength 412nm place, as shown in table 3.
The absorbancy of the conjugates of hyaluronic acid of table 3 halfcystine functionalization
The sample solution optical density of utilizing typical curve and recording, the content of sulfydryl in calculation sample.
Bringing typical curve into calculates: 0.0385=0.1692x-0.0001, show that modification rate is 3.5% (sulfydryl number is with respect to disaccharide units in HA), can form the good gel of rheologic behavio(u)r with thioester compound in theory.
Three, rheology test
The hydrogel that the Injectable in-situ of embodiment 4～7 preparations is formed carries out respectively rheology test, and rheological parameters is set to: scan pattern: vibration; Rotor: PP25; Get dot frequency: 1/20 second; Probe temperature: 20 ℃.Time scan: frequency (f) is 1 hertz, stress (Strain) is 1%, the constant time gets a little.Frequency sweeping: frequency (f) is 10～0.01 hertz, stress (Strain) is 1%, 6 point/a be order of magnitude, gets altogether 19 points, No time Setting.Stress scans: frequency (f) is 1 hertz, stress (Strain) is 0.1%～100%, 6 points/a be order of magnitude, gets altogether 19 points, No timeSetting.
The rheology time scan figure of the hydrogel that Injectable in-situ prepared by embodiment 4 forms as shown in figure 11, time scan detail view as shown in figure 12, frequency sweeping figure as shown in figure 13, stress scans figure as shown in figure 14.Can find out that from Figure 11 and Figure 12 the time is 150s left and right, G ' (storage modulus, storage modulus) and G " there is intersection point in (loss modulus, loss modulus); this point is gelation point; be the time point that hydrogel forms, G ' >G after gelation point ", hydrogel formation in the time of 150s left and right be described, along with the increase of time, G ', in ascendant trend, finally reaches balance gradually, illustrates that gel reaction reacts completely; Figure 13 is frequency sweeping figure, and frequency sweeping figure is that time scan figure shows that G ' is not till changing, and gel reaction is complete, and along with the variation of frequency, G ', always in equilibrium state, does not have huge fluctuation, illustrates that this hydrogel has good rheological property; Figure 14 is stress scans figure, and stress scans figure detects after frequency sweeping figure detects, and along with the variation of stress, G ', always in equilibrium state, does not have huge fluctuation, illustrates that this hydrogel has good rheological property.
The rheology time scan figure of the hydrogel that Injectable in-situ prepared by embodiment 5 forms as shown in figure 15, frequency sweeping figure as shown in figure 16, stress scans figure as shown in figure 17.Time is 2000s left and right as can be seen from Figure 15, G ' and G " there is intersection point; this point is gelation point; be the time point that hydrogel forms, G ' >G after gelation point ", hydrogel formation in the time of 2000s left and right be described, along with the increase of time, G ', in ascendant trend, finally reaches balance gradually, illustrates that gel reaction reacts completely; Figure 16 is frequency sweeping figure, and frequency sweeping figure is that time scan figure shows that G ' is not till changing, and gel reaction is complete, and along with the variation of frequency, G ', always in equilibrium state, does not have huge fluctuation, illustrates that this hydrogel has good rheological property; Figure 17 is stress scans figure, and stress scans figure detects after frequency sweeping figure detects, and along with the variation of stress, G ', always in equilibrium state, does not have huge fluctuation, illustrates that this hydrogel has good rheological property.
The rheology time scan figure of the hydrogel that Injectable in-situ prepared by embodiment 6 forms as shown in figure 18, frequency sweeping figure as shown in figure 19, stress scans figure as shown in figure 20.Time is 3000s left and right as can be seen from Figure 18, G ' and G " there is intersection point; this point is gelation point; be the time point that hydrogel forms, G ' >G after gelation point ", hydrogel formation in the time of 3000s left and right be described, along with the increase of time, G ', in ascendant trend, finally reaches balance gradually, illustrates that gel reaction reacts completely; Figure 19 is frequency sweeping figure, and frequency sweeping figure is that time scan figure shows that G ' is not till changing, and gel reaction is complete, and along with the variation of frequency, G ', always in equilibrium state, does not have huge fluctuation, illustrates that this hydrogel has good rheological property; Figure 20 is stress scans figure, and stress scans figure detects after frequency sweeping figure detects, and along with the variation of stress, G ', always in equilibrium state, does not have huge fluctuation, illustrates that this hydrogel has good rheological property.
The rheology time scan figure of the hydrogel that Injectable in-situ prepared by embodiment 7 forms as shown in figure 21, frequency sweeping figure as shown in figure 22, stress scans figure as shown in figure 23.As can be seen from Figure 21, while starting to test, there is G ' >G "; and G ' balance; illustrate that this reaction occurs very fast; after the conjugates of hyaluronic acid solution of halfcystine functionalization is mixed with polyethylene glycol acrylate binding substances solution, form immediately hydrogel, and react completely.Figure 22 is frequency sweeping figure, and frequency sweeping figure is that time scan figure shows that G ' is not till changing, and gel reaction is complete, and along with the variation of frequency, G ', always in equilibrium state, does not have huge fluctuation, illustrates that this hydrogel has good rheological property; Figure 23 is stress scans figure, and stress scans figure detects after frequency sweeping figure detects, and along with the variation of stress, G ', always in equilibrium state, does not have huge fluctuation, illustrates that this hydrogel has good rheological property.
Rheology collection of illustrative plates contrast by embodiment 4, embodiment 5, embodiment 6, embodiment 7 draws to draw a conclusion:
1) embodiment 4 is conjugates of hyaluronic acid and four branch's polyoxyethylene glycol thioesters binding substancess formation hydrogels of halfcystine functionalization, the conjugates of hyaluronic acid of embodiment 7 halfcystine functionalization and four branch's polyethylene glycol acrylate binding substancess form hydrogel, these two embodiment gelatin forming conditions are identical, unique different be the reactive group difference of polyethylene glycol conjugate, embodiment 4 reaction mechanisms are the ligations of nature chemistry, embodiment 7 reaction mechanisms are Michael addition, find out that from their time scan figure contrast the gelation point of embodiment 4 is 150s, embodiment 7 starts to detect gel and reacts completely, illustrate that embodiment 7 Michael additions form gel reaction faster compared with the naturally chemical ligation formation of embodiment 4 gel.
2) embodiment 4 is conjugates of hyaluronic acid and four branch's polyoxyethylene glycol thioesters binding substancess formation hydrogels of halfcystine functionalization, embodiment 5 is that conjugates of hyaluronic acid and the straight chain polyoxyethylene glycol thioesters binding substances of halfcystine functionalization forms hydrogel, these two embodiment gelatin forming conditions are identical, unique different be the side chain number difference of polyoxyethylene glycol thioesters binding substances, find out that from their time scan figure contrast the gelation point of embodiment 4 is 150s, embodiment 5 gelation points are 2000s, illustrate that the conjugates of hyaluronic acid of halfcystine functionalization is in the time reacting formation hydrogel with polyoxyethylene glycol thioesters binding substances, the more reaction of side chain chain is very fast, can find out that from their frequency sweeping figure and stress scans figure the modulus (G ') of the hydrogel that embodiment 4 obtains is higher, illustrate that the gel modulus that the polyoxyethylene glycol thioesters binding substances of four branches forms compared with straight chain polyoxyethylene glycol thioesters binding substances is higher.
3) embodiment 4 is all conjugates of hyaluronic acid and four branch's polyoxyethylene glycol thioesters binding substancess formation hydrogels of halfcystine functionalization with embodiment 6, these two embodiment gelatin forming conditions are identical, unique different be the pH difference of damping fluid used in reaction process, embodiment 4 pH of buffer 8.0 used is higher than embodiment 6 pH of buffer 7.2, find out that from their time scan figure contrast the gelation point of embodiment 4 is 150s, embodiment 6 gelation points are 3000s, illustrate that in gel reaction process, damping fluid used pH within the scope of claim is higher, gel reaction is faster.
4) the hydrogel r frequency sweeping figure that the Injectable in-situ that prepared by embodiment 4, embodiment 5, embodiment 6, embodiment 7 forms and stress scans figure all reflect the prepared hydrogel of the present invention and have good rheological property.
So can pass through the control to pH of buffer, and select different polyethylene glycol conjugates, the polyoxyethylene glycol thioesters binding substances of different branches, obtains the hydrogel of different gelation points and different modulus, can meet the different needs.
In sum, the hydrogel that prepared by embodiment 4～7 explanation the present invention is at 5%NaHCO 3in the aqueous solution or phosphate buffered saline buffer, form, formation condition gentleness, can think that this condition is physiological condition, can find out by corresponding rheology test result, gel is to form within a certain period of time hydrogel after the conjugates of hyaluronic acid of halfcystine functionalization and the aqueous solution of polyethylene glycol conjugate, so the aqueous solution of the conjugates of hyaluronic acid of halfcystine functionalization and polyethylene glycol conjugate can be injected to rejected region by needle tubing, within the regular hour, original position forms hydrogel, therefore the present invention can realize injectable and original position formation covalent cross-linking hyaluronic acid gel under physiological condition, it is the important breakthrough of technical field of biological material, have a good application prospect.

Claims (9)

1. a conjugates of hyaluronic acid for halfcystine functionalization, its structural formula is:
n is that 0～30, x is 200～3000.
2. a kind of preparation method of conjugates of hyaluronic acid of halfcystine functionalization described in claim 1, is characterized in that: comprise the following steps:
1) getting structural formula is compound 1 be raw material, under acetone reflux condition, there is cyclization l generating structure formula and be compound 2;
2) by compound 2 and tert-Butyl dicarbonate reaction, the amino structural formula by tertbutyloxycarbonyl (Boc) protection of generation is compound 3;
3) by compound 3 and N-hydroxy-succinamide under dicyclohexylcarbodiimide condition, there is esterification, generating structure formula is compound 4;
4) by compound 4 and cystamine under alkaline condition, there is acylation reaction, generating structure formula is compound 5;
5), by the disulfide bond reduction of compound 5, generating structure formula is compound 6;
6) by compound 6 and polyoxyethylene glycol compounds under alkaline condition, there is epoxy second
The ring-opening reaction of alkane, generating structure formula is compound 7;
7) by compound 7 and hyaluronic acid under TBAH condition, initial ring oxidative ethane ring-opening reaction, generating structure formula is compound 8;
8) compound 8 is sloughed to tertbutyloxycarbonyl (Boc) protecting group, then under concentrating under reduced pressure condition, ring-opening reaction is occurred, generating structure formula is compound 9, i.e. the conjugates of hyaluronic acid of halfcystine functionalization; Wherein, n is that 0～30, x is 200～3000.
3. the preparation method of the conjugates of hyaluronic acid of a kind of halfcystine functionalization according to claim 1, is characterized in that:
Its preparation method specifically comprises the following steps:
1) preparation of compound 2: get compound 1 cysteine hydrochloride, add dry acetone backflow 2～10h, be evaporated to the volume that no more than total reaction liquid one of is accumulated score of three, put into refrigerator and be no less than 0.5h, crystallization, cross leaching filter residue, obtain compound 2, wherein acetone is not less than 20 with the volume mass ratio of compound 1;
2) preparation of compound 3: compound 2 use acetonitriles are dissolved, add tert-Butyl dicarbonate, after mixing, dropwise add N, N-diisopropylethylamine, 15 ℃～37 ℃ stirring reaction 24～72h, concentrating under reduced pressure solvent, add again ether or hexanaphthene to continue concentrating under reduced pressure, add ether or hexanaphthene, with diatomite filtration, filtrate is used 0.01～0.1mol/L HCl solution, H successively again 2o, saturated NaCl extraction, dry, concentrating under reduced pressure, obtains compound 3, and wherein the amount of substance of tert-Butyl dicarbonate, DIPEA is independently 1.1～3 times of compound 2;
3) preparation of compound 4: compound 3 and N-hydroxy-succinamide are dissolved with tetrahydrofuran (THF), 0～4 ℃ of ice bath, toward wherein dripping N, the tetrahydrofuran solution of N '-dicyclohexylcarbodiimide, mix rear reaction 3～12h, complete by thin-layer chromatography detection reaction to reaction solution, put into 0～4 ℃ of refrigerator and leave standstill 0.5～12h, cross leaching filtrate, through concentrating under reduced pressure solvent, Virahol recrystallization, obtains compound 4, wherein N-hydroxy-succinamide, N, the amount of substance of N '-dicyclohexylcarbodiimide is independently 1.05～1.5 times of compound 2;
4) preparation of compound 5: the 2-aminoethyl disulfide dihydrochloride that is equivalent to 0.525～0.6 times of compound 4 molar weight by water dissolution molar weight, be equivalent to the sodium carbonate of 4 times of 2-aminoethyl disulfide dihydrochloride molar weights by water dissolution molar weight, drip in the 2-aminoethyl disulfide dihydrochloride aqueous solution, ice bath, stirs; With tetrahydrofuran (THF) dissolved compound 4, be added dropwise in above-mentioned reaction solution, 15 ℃～37 ℃ reaction 6～24h, until thin-layer chromatography detection reaction is complete; Concentrating under reduced pressure solvent, regulating pH is 3～7, then with ethyl acetate or dichloromethane extraction, 0.01～0.1mol/LHCl solution, H for organic phase 2o, saturated NaCl extraction, dry, concentrating under reduced pressure, obtains compound 5;
5) preparation of compound 6: by the ethanolic soln of compound 5, add again the water of 0.5～1.0 times of volume, ice bath stirs, slowly add again sodium borohydride, bubble to be no longer included is emerged, be placed in 15～37 ℃ of stirring reaction 4～12h, complete to thin-layer chromatography detection reaction, then add relative response liquid to amass the water termination reaction of 2～4 times; Regulating pH is 3～5, dichloromethane extraction, and washing, logical argon gas, sealing is dry, filters, and pressurization is concentrated obtains compound 6, and wherein, the amount of substance of sodium borohydride is 5～10 times of compound 5;
6) preparation of compound 7: toward the aqueous solution that adds sodium bicarbonate in the acetonitrile solution of polyoxyethylene glycol compounds, 15～37 ℃ stir, logical argon gas, again toward the acetonitrile solution that wherein drips compound 6, drip rear logical argon gas, acetonitrile in reaction solution: the volume ratio of water is (2: 3)～(3: 2), sealing, 15～37 ℃ of stirring reaction 6～24h, until thin-layer chromatography detection reaction is complete, Ellamn reagent colour development is not till displaing yellow, concentrating under reduced pressure solvent, with methylene dichloride or ethyl acetate extraction, organic phase washes with water rear dry, cross leaching filtrate, concentrating under reduced pressure obtains compound 7, wherein, sodium bicarbonate, the amount of substance of compound 6 is respectively 1～2 times of polyoxyethylene glycol compounds, 1.1～1.5 times,
7) preparation of compound 8: take its molecular weight and be 100,000～1,000,000 hyaluronic acid, add the TBAH aqueous solution, obtain hyaluronic acid reaction solution, wherein hyaluronic acid reaction solution mass concentration is 5%～20%, divide the acetonitrile solution that adds compound 7 for 1～4 time, 15～37 ℃ of stirring reaction 6～72h, with 0.01～1mol/L HCl solution neutralization reaction liquid, dialysis, freeze-drying, the washing of saturated sodium-chloride ethanolic soln, dry, obtain compound 8, wherein, TBAH, the amount of substance of compound 7 is respectively 0.5～2 times of hyaluronic acid primary alconol base, 0.5～10 times, acetonitrile volume is 0.5～2.5 times of the TBAH aqueous solution,
8) preparation of compound 9: it is in 80～100% trifluoroacetic acid aqueous solution that compound 8 is added to percent by volume, the concentration that makes compound 8 is 5～20g/L, 15～37 ℃ of stirring reaction 3～6h, concentrating under reduced pressure, adding volumetric concentration is 45～55% ethanolic soln, 30～40 ℃ of concentrating under reduced pressure 3～10 times, with after Ellman reagent detection displaing yellow, separate out product with saturated sodium-chloride ethanolic soln, washes clean, dry, obtain product compound 9, i.e. the conjugates of hyaluronic acid of halfcystine functionalization.
4. the preparation method of the conjugates of hyaluronic acid of a kind of halfcystine functionalization according to claim 3, is characterized in that: step 4) and 5) described in to regulate the conditioning agent of pH be that volumetric concentration is 0.1%～2%HCl solution.
5. the preparation method of the conjugates of hyaluronic acid of a kind of halfcystine functionalization according to claim 3, it is characterized in that: step 7) described in the dialyzate used of dialysis be the aqueous solution of mass percent 0.1～1.5% sodium-chlor and percent by volume 30～80% ethanol, at least dialyse 48 hours, at least change dialyzate 4 times.
6. the application of the conjugates of hyaluronic acid of a kind of halfcystine functionalization claimed in claim 1 in the hydrogel of preparing Injectable in-situ formation.
7. application according to claim 6, is characterized in that: what it was concrete be applied as, and hydrogel that Injectable in-situ forms is generated by Michael reaction or naturally chemical ligation by conjugates of hyaluronic acid and the polyethylene glycol conjugate of the halfcystine functionalization described in claim 1 right.
8. application according to claim 7, is characterized in that: described polyethylene glycol conjugate is selected from structural formula and is polyethylene glycol acrylate binding substances, structural formula be polyoxyethylene glycol thioesters binding substances in a kind of, wherein m is that 50～500, y is 2～8, the number of polyethylene glycol conjugate side chain is that 2～8, R group is selected from middle one.
9. application according to claim 8, it is characterized in that: described polyoxyethylene glycol thioesters binding substances is selected from the polyoxyethylene glycol thioesters binding substances of 3-mercaptopropionic acid ethyl ester and polyoxyethylene glycol formation, the polyoxyethylene glycol thioesters binding substances that N-acetyl-L-cysteine and polyoxyethylene glycol form, the polyoxyethylene glycol thioesters binding substances that 2-ethane thiol sodium sulfonate and polyoxyethylene glycol form, the polyoxyethylene glycol thioesters binding substances that 4-sulfydryl phenylacetic acid and polyoxyethylene glycol form, 2-dimethylamino sulfur alcohol is a kind of in being combined with the polyoxyethylene glycol thioesters that polyoxyethylene glycol forms, wherein the polymerization degree of polyoxyethylene glycol thioesters binding substances is 50～500, side chain number is 2～8.
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