A kind of synthetic method of CARB OXYMETHYL-CHITOSAN sulfation product
Technical field
The present invention relates to medical polymer field, particularly relate to a kind of synthetic method of CARB OXYMETHYL-CHITOSAN sulfation product.
Background technology
Since expensive and supply falls short of demand heparin becomes anticoagulation medicine, investigator is finding the method for heparin alternative medicine that synthesis has highly blood coagulation resistant activity, low side effect always, at present the relevant report of existing many chemosynthesis heparin alternative medicine.The molecular chain structure schematic diagram of heparin is as shown below.
Research both domestic and external is drug target mainly with chitosan sulfated derivatives greatly, and chitosan is the functional biological polymer obtained through deacetylation by chitin, and chemically reactive is relatively high.G.Vikhoreva (G.Vikhoreva, G.Bannikova, etal.Preparationandanticoagulantactivityofalow-molecular-weightsulfatedchitosan [J] .CarbohydratePolymers, 2005,62:327-332.) in chitosan, add oleum and N, the mixed solution of dinethylformamide (DMF), be uniformly mixed at low temperature and be obtained by reacting sulfated chitosan, product replaces on hydroxyl and amino simultaneously.PreeyanatV. (PreeyanatV, WarayuthS, DumratS, eta1.Anficoagulantactivityofasulfatedchitosan [J] .CarbohydrateResearch, 2002, 337:1239-1242) and (RonghuaHuang such as Huang, YuminDu, LiamhuangZheng, etal.Anewapproachtochemicallymodifiedchitosansulfatesand studyoftheirinfluenoesontheinhibitionofEscherichiacolian dStaphyloooccusaureusgrowth [J] .Reactive & FunctionalPolymers, 2004, 59:41-51.) with sulfonated reagent (chlorsulfonic acid/DMF) temperature control at 5 DEG C with chitosan reaction, obtain C equally
2-N, C
3-O, C
6the chitosan sulfate ester products that-O replaces.The signal formula of these reactions is as shown below:
Introducing-SO on chitin/chitosan molecular chain
3h group is the common method preparing heparinoid polysaccharide, but heparin contains C
2-N position ethanoyl and C
6the groups such as-O position carboxyl, introducing-SO on chitosan molecule chain separately
3h group at utmost can not imitate heparin, and therefore some scholars consider at chitin or chitosan C
6-O introduces carboxyl in position simultaneously, and product structure is closer to heparin.YuedongYang (YuedongYang, YongguoZhou, WenlongHou, etal.PreparationandAnticoagulantActivityofSulfated6-Carb oxychitosan [J] .BioinformaticsandBiomedicalEngineering, 2008,2:982-985.) study in glacial acetic acid, pass through NO
2realize chitosan C
6-O position is oxidized, and the Carboxy Chitosan of preparation continues to react in chlorsulfonic acid/formamide soln, realizes C
2-N position sulphating.But because intermediate product preparation condition is harsh, be difficult to the shortcomings such as control, output is also uncontrollable, and implements chitosan C
6-O position oxygenizement, product carboxyl distribution irregularity, cannot realize controlled synthesis, the side reactions such as the oxidative degradation of molecular chain also easily occur as carboxymethyl chitin is prepared in Mono Chloro Acetic Acid substitution reaction, and this reaction signal formula is as shown below:
Chinese patent application 200910273257.3 proposes to be scattered in water through the acid-treated chitin of phosphorus, 2 are added, 2,6 under condition of ice bath, 6-tetramethyl piperidine-1-oxyradical and NaBr solid, add 3wt%-10wt%NaClO solution after being stirred to dissolving completely and carry out C
6-O position selective oxidation reaction, then heparinoid polysaccharide is obtained after deacetylation, sulfonation/sulfuric acid acetify reaction, its sulphating substitution value is 0.3-1.35.And C
6-O position oxygenizement, the phenomenon such as oxidative degradation and group distribution irregularity can be produced too, cannot carry out as Mono Chloro Acetic Acid substitution reaction gentleness, its sulphur trioxide/N used, dinethylformamide sulphonating agent in aqueous activity are comparatively strong, are difficult to control replacement degree.
In order to overcome C
6the defect of-O position oxidation, in chitin or chitosan molecule chain, introduce carboxymethyl provide new thinking, product structure is also close with heparin.Muzzarelli (MuzzarelliRAA, GiaeomeljiG.ThebloodanticoagulantactivityofN-earboxymeth ylchitosantrisulfate [J] .CarbohydratePolymers, 1987,7:87.) in chitosan molecule, introduce-COOH and-SO simultaneously
3h, by C
2the carboxymethylated cm-chitosan of-N bit position prepares C
2-N, C
3-O, C
6-O position replaces the cm-chitosan sulphating derivative of rich sulphur, with Trombin inhibiting and Xa factor associativity good.Shin-ichiroNishimura (Shin-ichiroNishimuraa, SeiichiTokuraa.Preparationandantithrombogenicactivitieso fheparinoidfrom6-O-(carboxymethyl) chitin [J] .InternationalJournalofBiologicalMacromolecules, 1987,9 (4): 225-232.) research finds at C
2-the SO of-N position distribution
3h group may be vital for selecting in conjunction with Antithrombin III anticoagulant enzymic activity, and kinetic assay shows C
6-O position carboxymethyl group has synergy.Baumann.H (BaumannH; FaustV.ConceptsforimprovedregioseleetiveplacementofO-sul fo; N-acetyl; andN-carboxylmethylgroupsinchitosanderivatives [J] .CarbohvdrateResearch; 2001; 331:43.) utilize in chitosan molecule structure containing the very strong amino of reactive behavior easily and metal copper ion chelating protect amino group, and utilize C
3-O, C
6the reactive behavior difference of-O makes-SO
3h is only at C
6-O position selectivity replaces, and needs after the reaction to remove metal ion with ion exchange column, product purification operational difficulty.TokuraSehchi (TOKURASehchi.Selectivesulfationofchitinderivativesforbio medicalfunctions [J] .JMS-PureApplChem, 1994, A31 (11): 1710.) once report, N-COOH, O-SO
3the possibility causing vascular hypertension is there is in H chitosan while having anticoagulation function, and O-COOH, N-SO
3h chitosan, then without this side effect, shows that the modification on molecular chain should select corresponding substituted radical.YuquanZou (YuquanZou, EugeneKhor.Preparationofsulfated-chitinsunderhomogeneous conditions [J] .CarbohydratePolymers, 2009,77:516-525.) experiment finds that sulfonation chitin sulphur content is higher, activity is higher, uses HMQC-NMR determination part separation structure to think N position-SO
3h is the major site of anticoagulant active.These reaction signal formulas are as shown below.
Above carboxymethylation and sulphating modification are all raw material with chitosan, and chitosan is that raw material is through repeatedly deacetylation is obtained with chitin, deacetylation is higher causes active higher amino to exist in a large number, causes the replacement of carboxymethyl selectivity and control substitution value very difficult.In addition, select sulphur trioxide/N, dinethylformamide as sulphonating agent, and the synthetic method such as the chelating protection of amino is all undesirable.
Summary of the invention
In order to make up above-mentioned the deficiencies in the prior art, the present invention proposes a kind of synthetic method of CARB OXYMETHYL-CHITOSAN sulfation product.
Technical problem of the present invention is solved by following technical scheme:
A synthetic method for CARB OXYMETHYL-CHITOSAN sulfation product, comprises following steps:
(1) chitin alkalinisation treatment
Chitin is mixed in the ratio that chitin and the mol ratio of NaOH are 1:5 ~ 1:10 with the NaOH aqueous solution of 20wt%-60wt%, 20-25 DEG C of dispersed with stirring, within negative pressure of vacuum 1-5 hour, treats the infiltration of the NaOH aqueous solution completely, at-20 ~ 0 DEG C of freezing 12-48 hour;
(2) chitin C
6-O position carboxymethylation
Chitin after step (1) alkalinisation treatment is added in dispersion agent to be stirred to and disperses completely, the volume of dispersion agent and the mass ratio of chitin are 10 ~ 20ml:1g, slowly add Mono Chloro Acetic Acid, add in 10min ~ 1 hour, the mass ratio of Mono Chloro Acetic Acid and chitin is 0.47 ~ 2.33g:1g, at 25-65 DEG C, react 2-24 hour; Reaction terminates rear mistake and filters dispersion agent, and filter residue being dissolved in adjust ph in deionized water is 5 ~ 9, is the ultra-filtration membrane ultrafiltration purification of 10,000, obtains 6-O-carboxymethyl chitin (6-O-CMCH) finally by drying with molecular weight cut-off value;
(3) deacetylation of 6-O-CMCH
6-O-CMCH is scattered in dispersion agent, the volume of dispersion agent and the mass ratio of 6-O-CMCH are 10 ~ 40ml:1g, add the NaOH aqueous solution of 20wt%-60wt%, a repeating unit mol ratio of NaOH and 6-O-CMCH is 1 ~ 15:1 (a repeating unit molecular mass of 6-O-CMCH is 203-42 × DD+58 × DSC), then stirring reaction 2-8 hour at 50-100 DEG C, reaction terminates rear mistake and filters dispersion agent, it is 5 ~ 9 that filter residue is dissolved in regulation system pH value after in deionized water, be 10 with molecular weight cut-off value, the ultra-filtration membrane ultrafiltration purification of 000, drying obtains CARB OXYMETHYL-CHITOSAN (6-O-CMCS),
(4) sulphating of 6-O-CMCS
6-O-CMCS is scattered in dispersion agent, the volume of dispersion agent and 6-O-CMCS mass ratio are 10 ~ 20ml:1g, 0.5-2 hour is stirred at 0-10 DEG C, then the mix acid liquor of chlorsulfonic acid and the vitriol oil is added, the volume ratio of chlorsulfonic acid and the vitriol oil is 2:1, the volume of mix acid liquor and 6-O-CMCS mass ratio are 5 ~ 15ml:1g, constant temperature stirring reaction 2-16 hour at 0-65 DEG C, reaction terminates the NaOH aqueous solution neutralization of rear 2-10wt%, add ether, at 0-4 DEG C, refrigerate 12-48h, the supernatant liquid that inclines is to remove free SO
4 2-ion, then add organic solvent deposit and go out solid, cross and filter organic solvent, it is 10 that filter residue is dissolved in rear molecular weight cut-off value in deionized water, the ultra-filtration membrane ultrafiltration purification of 000, obtains CARB OXYMETHYL-CHITOSAN sulfation product (sulfated6-O-CMCS) finally by drying.
Preferably, the dispersion agent in described step (2) is at least one in n-propyl alcohol, Virahol and ethanol; Dispersion agent in described step (3) is at least one in n-propyl alcohol, Virahol and ethanol.
Preferably, dispersion agent in described step (4) is at least one in dimethyl formamide (DMF), dimethyl sulfoxide (DMSO) (DMSO) and methane amide, the DMF solution of CARB OXYMETHYL-CHITOSAN carries out heterogeneous sulfuric acid esterification and is easy to control, sulphur content is higher, reacts in DMF so preferred further.
Preferably, the organic solvent in described step (4) is methyl alcohol or ethanol or acetone or ether or at least both mixing.
Preferably, the chitin in described step (1) is 50 ~ 200 orders, and deacetylation (DD) is greater than 0 and is less than or equal to 15%.
Preferably, 6-O-CMCH degree of substitution by carboxymethyl (DSC) for 20-80%, DD be 15 ~ 50%.
Preferably, the DSC of 6-O-CMCS and 6-O-CMCH is identical, be 20 ~ 80%, DD is 50 ~ 90%.
Preferably, the sulphating degree (DSS) of sulfated6-O-CMCS is 50 ~ 150%.
The CARB OXYMETHYL-CHITOSAN sulfation product that synthetic method described in a kind of above-mentioned any one prepares.
Synthetic method of the present invention is a kind of completely new approach using chitin selectivity to replace, control Replacement rate, and the synthetic product (macromolecule polysaccharide) of the method not only provides the N position-SO of main anticoagulant active
3h, and introduce-COOH and to make on molecular structure electronegative-COOH and-SO in a large number
3h distributes regularly, and synergy produces the anticoagulation effect of heparitin.This macromolecule polysaccharide is the heparinoid drug being selected modification by chitin through safe agent, decrease bulk drug reagent contamination, more avoid the virus contamination risk that heparin organism is extracted, in theory, comparatively heparin sodium is more excellent for the safety of medicine performance of its clinical experiment, be hopeful the direct thrombin inhibitor as cheapness, substitute heparin sodium anticoagulation medicine.
Advantage of the present invention is: 1. preparation condition easy handling controls, reproducible, has and introduces-COOH and-SO simultaneously
3h group produces collaborative anticoagulant active effect.2. CARB OXYMETHYL-CHITOSAN sulphating prepares C
6-O position carboxymethylation, C
2-N position Sulfated chitosans anticoagulation alternative medicine, product location replaces structure-controllable.3. adopt chlorsulfonic acid/vitriol oil as sulphating reagent, its reagent properties gentleness, toxicity are little.4. the controlling sulfate polyose (i.e. CARB OXYMETHYL-CHITOSAN sulfation product sulfated6-O-CMCS) utilizing present method to prepare, in reaction process, polysaccharide degraded is little, and side reaction is few, and product purity is high.
Accompanying drawing explanation
Fig. 1 is the FT-IR spectrogram of the chitin prepared of a preferred embodiment of the invention and derivative thereof.
Embodiment
Below contrast accompanying drawing and combine preferred embodiment the invention will be further described.
The invention provides a kind of synthetic method of CARB OXYMETHYL-CHITOSAN sulfation product, in one embodiment, comprise following steps:
(1) chitin alkalinisation treatment
Chitin is mixed in the ratio that chitin and the mol ratio of NaOH are 1:5 ~ 1:10 with the NaOH aqueous solution of 20wt%-60wt%, 20-25 DEG C of dispersed with stirring, within negative pressure of vacuum 1-5 hour, treats the infiltration of the NaOH aqueous solution completely, at-20 ~ 0 DEG C of freezing 12-48 hour;
(2) chitin C
6-O position carboxymethylation
Chitin after step (1) alkalinisation treatment is added in dispersion agent to be stirred to and disperses completely, the volume of dispersion agent and the mass ratio of chitin are 10 ~ 20ml:1g, slowly add Mono Chloro Acetic Acid, add in 10min ~ 1 hour, the mass ratio of Mono Chloro Acetic Acid and chitin is 0.47 ~ 2.33g:1g, at 25-65 DEG C, react 2-24 hour; Reaction terminates rear mistake and filters dispersion agent, and filter residue being dissolved in adjust ph in deionized water is 5 ~ 9, is the ultra-filtration membrane ultrafiltration purification of 10,000, obtains 6-O-CMCH finally by drying with molecular weight cut-off value;
(3) deacetylation of 6-O-CMCH
6-O-CMCH is scattered in dispersion agent, the volume of dispersion agent and the mass ratio of 6-O-CMCH are 10 ~ 40ml:1g, add the NaOH aqueous solution of 20wt%-60wt%, a repeating unit mol ratio of NaOH and 6-O-CMCH is 1 ~ 15:1 (a repeating unit molecular mass of 6-O-CMCH is 203-42 × DD+58 × DSC), then stirring reaction 2-8 hour at 50-100 DEG C, reaction terminates rear mistake and filters dispersion agent, it is 5 ~ 9 that filter residue is dissolved in regulation system pH value after in deionized water, be 10 with molecular weight cut-off value, the ultra-filtration membrane ultrafiltration purification of 000, drying obtains 6-O-CMCS,
(4) sulphating of 6-O-CMCS
6-O-CMCS is scattered in dispersion agent, the volume of dispersion agent and 6-O-CMCS mass ratio are 10 ~ 20ml:1g, 0.5-2 hour is stirred at 0-10 DEG C, then the mix acid liquor of chlorsulfonic acid and the vitriol oil is added, the volume ratio of chlorsulfonic acid and the vitriol oil is 2:1, the volume of mix acid liquor and 6-O-CMCS mass ratio are 5 ~ 15ml:1g, constant temperature stirring reaction 2-16 hour at 0-65 DEG C, reaction terminates the NaOH aqueous solution neutralization of rear 2-10wt%, add ether, at 0-4 DEG C, refrigerate 12-48h, the supernatant liquid that inclines is to remove free SO
4 2-ion, then adds organic solvent deposit and goes out solid, and cross and filter organic solvent, filter residue is dissolved in the ultra-filtration membrane ultrafiltration purification that rear molecular weight cut-off value in deionized water is 10,000, obtains sulfated6-O-CMCS finally by drying.
The present invention utilizes the protection of chitin acetamido, directly with chitin (Chitin) for starting raw material, first the crystallizing field of chitin is destroyed by alkaline purification, reduce degree of crystallinity, obtain close to unbodied chitin, then, the water-soluble 6-O-CMCH of suitable DSC is synthesized with Mono Chloro Acetic Acid (MCA) inhomogeneous reaction and by controlling reaction conditions; Then, 6-O-CMCH utilizes concentrated base to slough part ethanoyl further in heterogeneous reaction system, and controls the 6-O-CMCS that reaction conditions prepares certain DD; In chlorsulfonic acid/sulphuric acid soln, carry out sulfuric acid esterification under last temperature control heterogeneous conditions obtain target product sulfated6-O-CMCS, control product C through above reactions steps
6-O position-COOH, C
2-N position ethanoyl and-SO
3h ratio, make product molecule chain structure and heparin similar.Reaction signal formula is as follows:
Wherein, step (2), (3) and (4) are heterogeneous reaction system.In step (2) and step (3), adjust ph can preferably regulate with the concentrated hydrochloric acid of massfraction >=10%, is adjusted to neutrality, and pH is 5 ~ 9 neutrality that can be called in the present invention.
Drying in step (2), (3), (4) can preferably be carried out under vacuo, can be faster more easily dry.
The vitriol oil in step (4) refers to the vitriol oil of massfraction >=98%.In step (4), the mix acid liquor of chlorsulfonic acid and the vitriol oil is first pre-chilled to 0 ~ 10 DEG C, then under ice bath, slowly can add mix acid liquor.
In other preferred embodiments, the dispersion agent in described step (2) can be at least one in n-propyl alcohol, Virahol and ethanol.Dispersion agent in described step (3) can be at least one in n-propyl alcohol, Virahol and ethanol.Dispersion agent in described step (4) can be at least one in DMF, DMSO and methane amide.Organic solvent in described step (4) can be methyl alcohol or ethanol or acetone or ether or at least both mixing.Chitin in described step (1) is 50 ~ 200 orders, and DD is greater than 0 and is less than or equal to 15%.The DSC of 6-O-CMCH is 20-80%, DD is 15 ~ 50%.The DSC of 6-O-CMCS and 6-O-CMCH is identical, be 20 ~ 80%, DD is 50 ~ 90%.The DSS of sulfated6-O-CMCS is 50 ~ 150%.
The CARB OXYMETHYL-CHITOSAN sulfation product that the present invention also provides the synthetic method described in a kind of above-mentioned any one to prepare.Product C is controlled through above reactions steps
6-O position-COOH, C
2-N position ethanoyl and-SO
3h ratio, CARB OXYMETHYL-CHITOSAN sulfation product main chain is the dextran ring of heparitin molecule, make product molecule chain structure and heparin similar.
Below by way of a preferred embodiment, the present invention is described in detail.
A synthetic method for CARB OXYMETHYL-CHITOSAN sulfation product, comprises the steps:
(1) chitin alkalinisation treatment
Take 150 object chitin 2g, add the NaOH aqueous solution that 6.23g concentration is 45wt%, dispersed with stirring is even, and in vacuum drying oven, room temperature vacuumizes 2 hours, in 24h freezing at-20 DEG C after alkali lye infiltration completely.
(2) chitin C
6-O position carboxymethylation
Aforementioned alkalization chitin is proceeded in reactor, adds 40ml Virahol (dispersion agent) in stirred at ambient temperature, slowly add (adding in 1 hour) 1.42g Mono Chloro Acetic Acid, constant temperature 40 DEG C reaction 4 hours.Reaction terminates rear mistake and filters dispersion agent, filter residue is dissolved in after in deionized water with commercially available concentrated hydrochloric acid regulation system pH value extremely neutral (pH is 5 ~ 9), be 10 with molecular weight cut-off value, the ultra-filtration membrane ultrafiltration purification of 000, weigh finally by vacuum-drying, obtain 2.13g6-O-CMCH.
(3) deacetylation of 6-O-CMCH
Above-mentioned product 6-O-CMCH is added in 40ml Virahol, and add the NaOH aqueous solution of 60wt%, the mol ratio of a repeating unit of NaOH and 6-O-CMCH is 5:1,80 DEG C of constant temperature deacetylations 4 hours, reaction terminates rear mistake and filters Virahol, filter residue is dissolved in after in deionized water with commercially available concentrated hydrochloric acid regulation system pH value extremely neutral (pH is 5 ~ 9), be 10 with molecular weight cut-off value, the ultra-filtration membrane ultrafiltration purification of 000, weigh finally by vacuum-drying, obtain 1.91g6-O-CMCS.
(4) sulphating of 6-O-CMCS
6-O-CMCS is joined in the DMF solvent of 40ml, stir 1 hour at 0 DEG C, chlorsulfonic acid/the vitriol oil mix acid liquor of slow dropping 30ml through being chilled to 0 DEG C in advance, the volume ratio of chlorsulfonic acid and the vitriol oil is 2:1, the massfraction of the vitriol oil is 98%, dropwise rear constant temperature 50 DEG C reaction and obtain yellow viscous liquid product in 4 hours, neutralize with the NaOH aqueous solution of 5wt%, add appropriate ether (those skilled in the art can select appropriate ether according to the amount of product), refrigerate 24h at 4 DEG C, the supernatant liquid that inclines is to remove free SO
4 2-ion.Add dehydrated alcohol and be settled out solid, cross and filter ethanol, filter residue is dissolved in the ultra-filtration membrane ultrafiltration purification that rear molecular weight cut-off value in deionized water is 10,000, obtains 2.56g end product sulfated6-O-CMCS finally by vacuum-drying.
Adopt FT-IR, potentiometric titration and elemental analysis method to characterize product structure at different levels, the DD being recorded its Raw chitin by potentiometric titration and elemental analysis method is 6.25%; The DSC of 6-O-CMCH is 53.47%, DD is 21.74%; The DSC of DSC and the 6-O-CMCH of 6-O-CMCS is identical, and DD is 73.34%; The DSS (the DSS sum of C2 and C6) of sulfated6-O-CMCS is 86.33%.Fig. 1 is the FT-IR spectrogram of chitin and derivative thereof, and wherein, curve a is chitin chitin, and curve b is 6-O-CMCH, and curve c is 6-O-CMCS, and curve d is sulfated6-O-CMCS.In the spectrogram of chitin, 2878cm
-1place is C-H stretching vibration peak; 3448cm
-1there is strong and wide absorption peak at place, indicates association-OH stretching vibration; 3271cm
-1near have weak N-H stretching vibration peak; 1660cm
-1there is stronger absorption peak at place, is the C=O stretching vibration of acid amides; 1315cm
-1and 1379cm
-1place is respectively CH
3and CH
2flexural vibration peak; 950 ~ 1100cm
-1there is a series of stronger absorption peak at place, is the stretching vibration of C-O-C.In the spectrogram of 6-O-CMCH, 1415cm
-1place is-COO-stretching vibration peak, and 1028cm
-1place C
6position C-O stretching vibration peak dies down, and the C of chitin is described
6-O position there occurs carboxymethylation.In the spectrogram of 6-O-CMCS, 1598cm
-1place-CH
2cOON
amiddle carbonylic stretching vibration characteristic peak and acid amides I bands of a spectrum and acid amides II bands of a spectrum overlap the strong absorption peak formed, and 1646cm
-1place's acid amides I bands of a spectrum weaken, 3294cm
-1place weakens at the N-H peak that stretches, and illustrates and there occurs deacetylation.In the spectrogram of sulfated6-O-CMCS, there is the characteristic peak of sulfonation cm-chitosan, wherein 1253cm
-1place is that C-O-S stretching vibration is bimodal, 1080 ~ 1000cm
-1place is for S-O symmetry is flexible and antisymmetric stretching vibration is bimodal, 802cm
-1place is S=O stretching vibration peak.The characterization result of FT-IR confirms the structure of target product.
Above content is in conjunction with concrete preferred implementation further description made for the present invention, can not assert that specific embodiment of the invention is confined to these explanations.For those skilled in the art, without departing from the inventive concept of the premise, some equivalent to substitute or obvious modification can also be made, and performance or purposes identical, all should be considered as belonging to protection scope of the present invention.