CN100441225C - Amino-acid modified chitin nucleophic NO donor and its synthesis method - Google Patents
Amino-acid modified chitin nucleophic NO donor and its synthesis method Download PDFInfo
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- CN100441225C CN100441225C CNB2006100283167A CN200610028316A CN100441225C CN 100441225 C CN100441225 C CN 100441225C CN B2006100283167 A CNB2006100283167 A CN B2006100283167A CN 200610028316 A CN200610028316 A CN 200610028316A CN 100441225 C CN100441225 C CN 100441225C
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Abstract
The present invention relates to an amino-acid modified chitosan nucleophilic NO donor and a synthesis method thereof, which belongs to the technical field of medicine engineering. In the present invention, chitosan is used as base material, amino acid is used for modifying the chitosan so as to increase the nucleophilic sites (NH groups) of the chitosan, and the modified chitosan reacts with NO so as to generate the nucleophilic NO donor containing N(O)NO functional groups, wherein the molecular structure of the donor is disclosed in the specification. The new nucleophilic NO donor has different NO releasing speed and half life and can overcome difficulties existing in the clinical application of NO donors at present, namely the cytotoxicity of a nucleophilic reagent (polyamine) and the generation of a carcinogenic side product-ammonium nitrite. Therefore, the present invention has important clinical application value and application prospects.
Description
Technical field
The present invention relates to a kind of medicine and synthetic method thereof of pharmaceutical engineering technical field, specifically is a kind of amino-acid modified chitin nucleophic NO donor and synthetic method thereof.
Background technology
In nearest 10 years, abroad to based on containing [N (O) NO]
-The NO donor controlled-release material of functional group has carried out a large amount of research.The nucleophic NO donor NONOate that initial research concentrates on ion-type is scattered in the various hydrophobic polymers, strengthens the blood compatibility of these materials by the release of NO.Though these researchs have obtained considerable success, discover, hydrophilic NO donor can ooze out from polymer, forms the N-nitrosamine material of carcinogenecity in blood.This class material is applied to clinical, also must consider following some: as the biocompatibility and the biodegradability of polymeric matrix material.The cytotoxicity of nucleopilic reagent and biocompatibility.[N (O) NO] arranged
-Group carrier such as diethylenetriamine are found health harmful; Polymine (PEI) can be induced cell death widely, and biocompatibility is relatively poor.Therefore select for use the nontoxic NO carrier that good biocompatibility is arranged to cause people's attention.One of solution wherein be exactly to adopt the harmless nucleophilic carrier with secondary amine group of health such as polyamino acid, micromolecule polypeptide.
Aminoacid is the difunctional life micromolecule part that has amino and carboxyl in the organism a large amount of the existence time.Aminoacid is the raw material of synthesized human protein, hormone, enzyme and antibody, participates in normal metabolism and physiological activity in the human body.And aminoacid is living matter important in the organism, is constitutive enzyme and proteinic elementary cell.As micromolecule, aminoacid plays a part very important to the activity and the physiological function thereof of biomacromolecule; As part, it can with multiple metallic ion coordination, for research antitumor, cancer therapy drug provide information.Each seed amino acid has different biological functions in organism, as the homergy of tryptophan in the organism and brain confidential relation is arranged, and the L-cysteine can strengthen the resistance against diseases of organism etc.Therefore at present, polyamino acid class material has caused concern widely again in the application aspect the medicine sustained release.Aminoacid by the effect of enzyme, participates in metabolism in human body, nutrient substance is absorbed by the body, and metabolite is an inorganic molecules, is excreted by Excretory system, so have excellent biological compatibility.
Find through literature search prior art, Bohl etc. are at " Biomaterials " (biomaterial) (2000, delivered " Nitric oxide-generating polymers reduceplatelet adhesion and smooth muscle cell proliferation " (NO produces polymer and reduces platelet adhesion and smooth muscle cell proliferation) 21:2273-2278), this article has proposed employing aminoacid to having optically active Polyethylene Glycol (PEG) and polyvinyl alcohol (PVA) modification makes it have nucleophilic NH group.After the NO reaction, obtain the NO release gels material of a series of different half-life.Its weak point is that used matrix material biocompatibility is not good enough, because the water solublity of matrix material can not well behaved thin film of processing machinery and coating.
U.S. Pat P6261594 (Chitosan-based nitric oxide donor compositions) and USP6451337 (Chitosan-based nitric oxide donor compositions) have proposed modification of chitosan to synthesize serial chitosan-NO addition product as nucleophilic NO carrier.The concrete modification of chitosan is divided into two classes: (1) hydrophilic group modification: N-carboxylic butyl chitosan, N-carboxyetbyl chitosan, N-carboxymethyl chitosan.(2) hydrophobic group modification: N-propyl group chitosan, N-carboxyetbyl chitosan methyl ester.Its weak point is that the biocompatibility of modification of chitosan is poor, and the loading level of NO is lower.
Summary of the invention
The objective of the invention is at the deficiencies in the prior art, a kind of amino-acid modified chitin nucleophic NO donor and synthetic method thereof are provided.The present invention adopts aminoacid that chitosan is carried out modification, thereby provide more nucleophilic site, improve the load capacity of NO, by chitosan and these two kinds of combinations that all have the material of good biocompatibility of aminoacid, help to solve the problem that this type of NO donor medicine is applied to clinical existence simultaneously.
The present invention is achieved by the following technical solutions:
Amino-acid modified chitin nucleophic NO donor of the present invention, chitosan adopt its nucleophilic site of amino-acid modified increase (NH group) as matrix material, produce [N (O) NO] with the NO reaction
-Functional group, molecular structure is as follows:
Wherein [N (O) NO]
-Functional group is that the NH group forms with the NO reaction, and the NH group comes from amino acid whose amino N H
2Or the amino N H in the chitosan molecule structure
2Modified-reaction; R is the alkyl of straight or branched.
When reacting on the C6 position of aminoacid at chitosan, the amino N H on the amino acid molecular
2COOH reaction with on the using carboxyl chitosan molecule of C6 position forms the NH site, forms [N (O) NO] with the NO reaction
-Group.
When aminoacid reacts in the C2 position of chitosan, carboxyl on the amino acid molecular and the amino N H in the chitosan molecule
2Reaction forms the NH site, forms [N (O) NO] with the NO reaction
-Group.
Amino-acid modified chitin nucleophic NO donor synthetic method of the present invention, can adopt in following two kinds any one:
Method (1): introduce aminoacid in the C6 site.With C
6The carboxy CO OH of-O using carboxyl chitosan activates with N-hydroxy-succinamide (NHS) and dicyclohexylcarbodiimide (DCC) or EDC earlier, again with amino acid whose amino (NH
2) reaction, can make C6 site amino-acid modified chitin.Modified product is carried out reaction under high pressure (Na with the NO gas molecule in the methanol solution of Feldalat NM
+/ NH 〉=3), pressure is 5-10atm, room temperature reaction 3-7 days, produces [N (O) NO]
-Group.
Method (2): introduce aminoacid in the C2 site.Amino acid whose carboxyl is activated again with the amino (NH on the chitosan molecule with N-hydroxy-succinamide (NHS) and dicyclohexylcarbodiimide (DCC) or EDC earlier
2) reaction, can make C2 site amino-acid modified chitin.Modified product is carried out reaction under high pressure (Na with the NO gas molecule in the methanol solution of Feldalat NM
+/ NH 〉=3), pressure is 5-10atm, room temperature reaction 3-7 days, produces [N (O) NO]
-Group.
The viscosity-average molecular weight of used chitosan is 1-100 ten thousand, deacetylation 50-100%.
The carboxylation substitution value of used using carboxyl chitosan molecule is 1.1, with C
6-O carboxymethyl chitosan is main.
Used aminoacid comprises common acidity, alkalescence and neutral amino acids such as glycine, lysine, alanine, cysteine, arginine, aspartic acid, glutamic acid.
The amino-acid modified chitin nucleophic NO donor of the present invention's preparation, can be prepared into pharmaceutical film, microsphere, the nanosphere that can discharge the NO molecule, can be widely used in the treatment cardiovascular system diseases, pulmonary's high pressure, promote wound healing, effectively prevent the molding postoperative restenosis, improve the antithrombotic property of medical treatment device.
The specific embodiment
The synthetic route of synthetic method of the present invention is as follows:
Method (1): introduce aminoacid (is example with glycine (neutrality) GLY and lysine (alkalescence) LYS) in the C6 site
Activated carboxylic
The glycine modification
The lysine modification
React with NO
Method (2): introduce aminoacid in the C2 site
Because of also there being carboxyl in the aminoacid, earlier with the activated carboxylic in the aminoacid, again with chitosan in No. 2 carbon potentials-NH
2Reaction.
Provide embodiment below in conjunction with the technology of the present invention content:
Embodiment 1: introduce glycine in the C6 site
Preparation of Carboxymethylchitosan
Chitosan 15g and isopropyl alcohol 200ml are added in the three-necked bottle, and stirring is dispersed in the isopropyl alcohol chitosan, and 50ml is divided into 5 parts with 10mol/L NaOH solution, adds portion every 30min, and stirring at room 1 hour gets the reactant slurry.24g solid monoxone is dissolved in the 70ml isopropyl alcohol, divides 4 times and add in the reactant slurry.After adding reactant is heated to 70 ℃ with water-bath, temperature control is opened reflux condensate device down, stirs 2 hours.Stop the back and regulate pH value to 7-8 with glacial acetic acid, separate out a large amount of light yellow flocculent deposits, vacuum filtration must yellow solid, and with methanol wash 3 times, and with gained yellow solid vacuum drying 1 day, temperature was 60 ℃.
The activation of carboxymethyl chitosan
Carboxymethyl chitosan (CMCS) is added in the reactor, add solvent (water, second eyeball, alcohol) then, stirring is fully dissolved carboxymethyl chitosan.Dissolved N-hydroxy-succinamide (N-Hydroxysuccinimide NHS) aqueous solution and dicyclohexylcarbodiimide (Dicuclohexylcarbodiimide DCC) alcoholic solution add above-mentioned solution respectively, in above-mentioned reaction system, the mol ratio of CMCS: NHS: DCC is 1: 1.5: 1.Stirring at room 24h gets the muddy liquid of milk yellow.After the filtration, white solid with organic solvent thorough washing final vacuum drying, is precipitated out after scouring with the reactant in the filtrate, vacuum drying is standby then once more.
Glycine modified carboxy methyl chitosan
Taking by weighing mol ratio is the NaHCO that 1: 1 glycine and activatory carboxymethyl chitosan add 0.05mol/L
3In the buffer solution, stirring reaction gets yellow solution after 1 day.After the gained solution concentration, dialysed three days, get white fiber shape material after the lyophilization.
Glycine modification of chitosan FTIR:1650-1655cm
-1(amide I), 1560cm
-1(amide II), 1600cm
-1(-COONa),
13C NMR:176.0ppm ,-COOH; 43.0ppm ,-CH
2-,
1H NMR:4.22 (1H), 2.52 (2H), 3.91 (3H), 3.0-3.3 (4H, 5H, 6H), 1.42 (7H), 4.33 (8H), 3.62 (9H)
Embodiment 2: introduce lysine in the C6 site
Take by weighing mol ratio and be activatory carboxymethyl chitosan among 1: 1 lysine and the embodiment 1 and add the NaHCO of 0.05mol/L
3In the buffer solution, stirring reaction gets yellow solution after 1 day.After the gained solution concentration, dialysed three days, get white fiber shape material after the lyophilization.
Lysine modification of chitosan FTIR:1730cm
-1(-COOH), 1630cm
-1And 1514cm
-1(NH
3 +), 1415cm
-1(CH
2With-CH
3), 2928cm
-1(CH
2).
1H?NMR：4.33(8H)，3.41(9H)，1.05(10H)，0.88(11H)，2.35(12H)，3.60(13H).
Embodiment 3: introduce aminoacid in the C2 site
Because of also there being carboxyl in the aminoacid, earlier with the activated carboxylic in the aminoacid, again with chitosan in No. 2 carbon potentials-NH
2Reaction.Aminoacid is dissolved in the 100ml water alcohol (2: 1), adds N-hydroxy-succinamide (NHS), stirring reaction 1h, the alcoholic solution of adding dicyclohexylcarbodiimide (DCC), room temperature reaction 24h.In above-mentioned reaction system, the mol ratio of aminoacid: NHS: DCC is 1: 1.5: 1.(aminoacid: chitosan mol ratio 1: 1) be dissolved in the 0.1M acetum and under agitation be added dropwise in the above-mentioned solution, room temperature reaction 24h after the gained solution concentration, dialysed three days, got white fiber shape material after the lyophilization to get quantitative chitosan.
FTIR:1650cm
-1(amide I), 1560cm
-1(amide II), 1310cm
-1(amide III);
1H NMR:3.53 (8H)
Embodiment 4: amino-acid modified chitin load NO reaction
With CH
3(1.5g 13.9mmol) is dissolved in the 50ml methanol and prepares sodium methoxide solution ONa, and (1g 4.5mmol) is suspended in this solution, need satisfy Na with the chitosan after amino-acid modified then
+/ NH 〉=3.Be placed in the autoclave, react under the NO of 5atm pressure, lead to NO 60min every day, react after 3 days, filter and use the ether washed product, the room temperature vacuum drying is stored in the close drying device, and temperature is-20 ℃.
For containing [NONO]
-Functional group, the most effective characterizing method is the ultraviolet characteristic absorption: the product that takes a morsel is dissolved in the NaOH solution of 0.1M, records [NONO]
-The characteristic absorption 256nm of functional group.
The research of NO release performance
The amino-acid modified chitin product is because introduced carboxyl, so water solublity is better, and experimental study shows that it all has dissolubility preferably in acid and alkaline solution.Put it into the dispose procedure of measuring its NO in the bag filter.Release profiles is carried out the function match, obtain it and discharge the half-life.Discharging total amount for C6GLYCS/NO is 423nmol/mg, and the half-life is 0.143h; C6LYSCS/NO, the release total amount is 327nmol/mg, the half-life is 0.223h; C2GLYCS/NO, the release total amount is 310nmol/mg, the half-life is for O.311h.
From above-mentioned data as can be seen: the burst size of C6 site modification of chitosan NO nucleophilic donor is greater than C2 site modification of chitosan NO nucleophilic donor, but the half-life is significantly less than C2 site modification of chitosan NO nucleophilic donor.The burst size of C6 site glycine modification of chitosan NO is greater than the burst size of lysine modification of chitosan NO, and the half-life then is that the lysine modification of chitosan is greater than the glycine modification of chitosan.
Claims (8)
1, a kind of amino-acid modified chitin nucleophic NO donor is characterized in that: chitosan adopts glycine or lysine modification to increase its NH nucleophilic site as matrix material, produces [N (O) NO] with the NO reaction
-Functional group, molecular structure is as follows:
Wherein [N (O) NO]
-Functional group is that the NH group forms with the NO reaction, and the NH group comes from glycine or lysine amino NH
2Or the amino N H in the chitosan molecule structure
2Modified-reaction; R is CH
2Or (CH
2)
4NH
2The CH group.
2, amino-acid modified chitin nucleophic NO donor according to claim 1 is characterized in that: when reacting on the C6 position at chitosan when glycine or lysine, and the amino N H on glycine or the lysine molecule
2COOH reaction with on the using carboxyl chitosan molecule of C6 position forms the NH site, forms [N (O) NO] with the NO reaction
-Group.
3, amino-acid modified chitin nucleophic NO donor according to claim 1 is characterized in that: when glycine or lysine react in the C2 site of chitosan, and carboxyl on glycine or the lysine molecule and the amino N H in the chitosan molecule
2Reaction forms the NH site, forms [N (O) NO] with the NO reaction
-Group.
4, a kind of amino-acid modified chitin nucleophic NO donor synthetic method as claimed in claim 1, it is characterized in that: the NH group on C6 site or C2 site glycine or the lysine modification of chitosan molecule reacts in the methanol solution of Feldalat NM with the NO gas molecule, wherein Na
+/ NH 〉=3, pressure are 5atm, and room temperature reaction 3 days produces [N (O) NO]
-Group is contained [N (O) NO]
-The nucleophic NO donor of group.
5, amino-acid modified chitin nucleophic NO donor synthetic method according to claim 4 is characterized in that, the NH group comes from the amino of chitosan and the carboxyl of using carboxyl chitosan and the carboxyl or the amino reaction back of glycine or lysine and forms.
6, amino-acid modified chitin nucleophic NO donor synthetic method according to claim 4, it is characterized in that the preparation of C6 site glycine or lysine modification of chitosan is that the COOH of C6 position using carboxyl chitosan is activated again with glycine or lysine amino NH with N-hydroxy-succinamide NHS and dicyclohexylcarbodiimide DCC or 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimide hydrochloride EDC earlier
2Reaction makes.
7, amino-acid modified chitin nucleophic NO donor synthetic method according to claim 4, it is characterized in that the preparation of C2 site glycine or lysine modification of chitosan is that the carboxy CO OH of glycine or lysine is activated amino N H with chitosan with N-hydroxy-succinamide NHS and dicyclohexylcarbodiimide DCC or 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimide hydrochloride EDC earlier again
2Reaction makes.
8, amino-acid modified chitin nucleophic NO donor synthetic method according to claim 6 is characterized in that, carries out the carboxymethyl reaction with monoxone and chitosan, and product is with C
6-O carboxymethyl chitosan is main.
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| CN106995502B (en) * | 2016-01-25 | 2021-12-14 | 中国科学院理化技术研究所 | Bifunctional group modified chitosan derivative and preparation method thereof |
| CN105832656B (en) * | 2016-05-25 | 2018-10-09 | 暨南大学 | It is a kind of to carry nitric oxide production carboxyl chitosan-polyethyleneimine hydrogel and its preparation method and application |
| EP3565848A4 (en) * | 2017-01-03 | 2020-09-02 | The University of North Carolina at Chapel Hill | Nitric oxide-releasing alginates as biodegradable antibacterial scaffolds and methods pertaining thereto |
| CN110483662A (en) * | 2019-07-05 | 2019-11-22 | 常州大学 | A kind of carboxymethyl chitosan cross-linked gel |
| CN112320914B (en) * | 2020-10-13 | 2022-09-27 | 神美科技有限公司 | Phosphorus removal agent with composite function and phosphorus removal method |
| CN112457358B (en) * | 2020-12-11 | 2022-05-31 | 上海交通大学 | Compound, nitric oxide donor prodrug compound, preparation method and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5691423A (en) * | 1992-08-24 | 1997-11-25 | The United States Of America As Represented By The Department Of Health And Human Services | Polysaccharide-bound nitric oxide-nucleophile adducts |
| US6261594B1 (en) * | 1998-11-25 | 2001-07-17 | The University Of Akron | Chitosan-based nitric oxide donor compositions |
| US6703046B2 (en) * | 2001-10-04 | 2004-03-09 | Medtronic Ave Inc. | Highly cross-linked, extremely hydrophobic nitric oxide-releasing polymers and methods for their manufacture and use |
-
2006
- 2006-06-29 CN CNB2006100283167A patent/CN100441225C/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5691423A (en) * | 1992-08-24 | 1997-11-25 | The United States Of America As Represented By The Department Of Health And Human Services | Polysaccharide-bound nitric oxide-nucleophile adducts |
| US6261594B1 (en) * | 1998-11-25 | 2001-07-17 | The University Of Akron | Chitosan-based nitric oxide donor compositions |
| US6451337B1 (en) * | 1998-11-25 | 2002-09-17 | The University Of Akron | Chitosan-based nitric oxide donor compositions |
| US6703046B2 (en) * | 2001-10-04 | 2004-03-09 | Medtronic Ave Inc. | Highly cross-linked, extremely hydrophobic nitric oxide-releasing polymers and methods for their manufacture and use |
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