CN101735336B - Oligomeric fucosylated glycosaminoglycan and preparation method thereof - Google Patents
Oligomeric fucosylated glycosaminoglycan and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a method for preparing oligomeric fucosylated glycosaminoglycan which is prepared by depolymerizing fucosylated glycosaminoglycan by a depolymerization method of peroxide catalyzed by a 4th period transition metal ion in an aqueous medium, and the preparation method has mild reaction condition, good reproducibility and stability, high pyrolysis selectivity and uniform and controllable product quality. The polysaccharide molecule number of the obtained oligomeric fucosylated glycosaminoglycan using GalNAc as a reducing end is not less than 80 percent, the weight average molecular weight is about 6, 000-20, 000Da, and the protein disulfide isomerase (PDI) is 1.0-2.0.
Description
Technical field
The invention belongs to medical technical field, specifically, relate to a kind of oligomeric fucosylated glycosaminoglycan (dFG) and preparation method thereof, and the pharmaceutical composition that contains this oligomeric fucosylated glycosaminoglycan.
Background technology
Fucosylated glycosaminoglycan (Fucosylated Glycosaminoglycan, Fucose-branchedGlycosamino-glycan or fucose-containing glycosaminoglycan are called for short FG); Or (Fucosylated chondroitin sulfate FCS), is meant that one type extract to obtain from echinoderms body wall or internal organ to be referred to as fucosylated CHS; Has the CHS of being similar to backbone structure; But have the substituted TGSS C3 verivate of side chain sulfated fucose (J Biol Chem, 1988,263 (34): 18176-83 and J Biol Chem; 1991,266 (21): 13530-6).
Natural FG has anticoagulant active, and (Fan painted once and waited, Acta Pharmaceutica Sinica, 1980; 15:267), this activity makes it to have potential therapeutic action for some thrombotic diseases, yet FG has induced platelet aggregation activity (Jia-zeng Li etc. simultaneously; Thronbosis and Haemostasis; 1988,54 (3): 435-9), and the thrombocyte induced activity has had a strong impact on the conversion of the potential therapeutic action of FG to practical clinical.In recent years research data shows, lower molecular weight FG possibly eliminate or avoid the thrombocyte induced activity of prototype polysaccharide in the anticoagulant active that keeps certain intensity (Fan painted once and waits, journal of biological chemistry, 1993,9 (2): 146-151; Toshio Imanari etc., Thrombosis Research, 1997,129:27-31).
Lower molecular weight FG can be from the stage treatment of FG, also can be from the FG depolymerization.The low molecule FG method of stage treatment gained simple (Fan painted once and waited, journal of biological chemistry, 1993,9 (2): 146-151), but since among the natural FG contained oligomeric component less, the stage treatment method prepares the lower and serious waste of resources of yield of oligomeric FG.
The enzyme process depolymerization, the nitrous acid depolymerization method that are usually used in the TGSS C3 depolymerization all are not suitable for depolymerization FG, and its reason is: the existence of Fucose side chain makes that known TGSS C3 restriction endonuclease all can not hydrolysis FG; Hexosamine among the FG is acetylamino sugars, and no free amine group or sulfation are amino to be existed, thereby can not be by the nitrous acid depolymerization.
H
2O
2Method depolymerization FG be shown in report (Fan Hui-Zeng etc., WO 90/08784; Ken-ichiroYoshida etc., Tetrahedron Letters, 1992,33 (34) 4959-62).Owing to there is the glycosidic link of number of different types among the FG; For example be present in glucuronic acid (GlcUA) β (1 → 3) glycosidic link, acetylamino galactosamine (GalNAc) β (1 → 4) glycosidic link of main chain and be present in α-Fucose (Fuc) glycosidic link of side chain etc., conventional H
2O
2Depolymerization method is limited to the selectivity of these glycosidic links.Non-selective cracking main chain glycosidic link can form the oligomeric FG product with different reducing end under neutral, promptly exists the GlcUA of high level and GalNAc terminal simultaneously, thereby influences the homogeneity of product; And the cracking of side chain glycosidic link can cause Fucose to replace coming off of side chain.
In addition, conventional H
2O
2The required reaction conditions of method depolymerization FG is violent relatively, and its temperature of reaction is had relatively high expectations, the reaction times is longer, and its critical defect is, the controllability of this reaction is relatively poor with repeatability.
Therefore, the method for developing the oligomeric FG of preparation that a kind of reaction conditions is gentle, circulation ratio is high, the product homogeneity is good is significant to the suitability for industrialized production of oligomeric FC.
Summary of the invention
One object of the present invention is to overcome the deficiency of prior art; Provide a kind of preparation quality homogeneous controlled; The method of the oligomeric fucosylated glycosaminoglycan (Depolymerized fucose-containingglycosaminoglycan abbreviates dFG or oligomeric FG as) that preparation repeatability is high.
The method of the dFG of preparation according to the invention is in aqueous media, to adopt the catalyzer that contains the period 4 transition metal ion, and catalysis superoxide depolymerization depolymerization fucosylated glycosaminoglycan (FG) specifically may further comprise the steps:
1) in the presence of the catalyzer of period 4 transition metal ion, in aqueous media, adds the fucosylated glycosaminoglycan of superoxide with depolymerization sea cucumber source;
2) stopped reaction, the oligomeric fucosylated glycosaminoglycan of collection and purifying desired molecule weight range.
In the step 1), the fucosylated glycosaminoglycan (FG) in said sea cucumber source is meant the acidic mucopolysaccharide that contains the Fucose component that from the said animal of Echinodermata Holothuroidea, extracts preparation.Its constitutional features is: in its monose is formed; GlcUA (glucuronic acid) and GalNAc (2-deoxidation-2-amino-N-ethanoyl-semi-lactosi or its sulfuric ester) exist mol ratio (1: 1 ± 0.3) such as to be bordering on; And GlcUA and GalNAc interconnect the formation polysaccharide main chain with β 1 → 3 with β 1 → 4 glycosidic link respectively; Fucose (Fuc) or its sulfuric ester are connected in main chain with the side chain form, and with molar ratio computing, Fuc: the GalNAc ratio range can be about 0.5～2.5.FG backbone structure unit can be expressed as formula (I):
In the formula (I) :-OR is hydroxyl (OH), sulfate group (OSO
3 -) or be suc as formula the sulphating fucosido shown in (II):
In the formula (II) :-OR is hydroxyl (OH), sulfate group (OSO
3 -) define same following formula.
From the side chain fucosido proportion of composing of the FG of different sea cucumber kind/kinds and be connected in main chain position mode and can there are differences; The sea cucumber kind is identical but tissue-derived difference or process for extracting difference also possibly cause the difference of the aspects such as monose proportion of composing, side chain existence form and polysaccharide sulfated degree of FG; But these differences all do not relate to the essential change of FG constitutional features and glycosidic link type, do not influence the effective enforcement and the application of the method for the invention.Therefore, in the present invention, said FC can derive from the sea cucumber of different varieties all over the world, includes but not limited to stichopus japonicus, Stichopus chloronotus (Brandt)., hojothuria leucospilota, Black Sea ginseng, Holothuria nobilis Selenka, rough sea cucumber etc.
In the step 1), depolymerization is in aqueous media, to carry out, and adopts the depolymerization reaction of metals ion as the catalyst superoxide, to generate oligomeric FG.
Said superoxide can produce radical in reaction system, and through free chain reaction cracking FG glycosidic link, and then form said dFG product.These superoxide include but not limited to Peracetic Acid, hydrogen peroxide, 3-chloro-peroxybenzoic acid, cumene hydroperoxide, Sodium Persulfate, Lucidol and their salt or ester, are preferably hydrogen peroxide.
The massfraction of said FG in reaction system is about 0.05%-15%, and the massfraction of superoxide in reaction system is about 0.5% to about 30%.In the depolymerization reaction process of FG, peroxide reactants can be before reaction disposable all joining in the reaction system, also can adopt to continue or the intermittence mode progressively joins peroxide reactants in the reaction system.The present invention preferably continues the mode of peroxide reactants according to controlled rate to join in the reaction system.
Said metals ion as catalyzer is the period 4 transition metal ion, comprises Cu
+, Cu
2+, Fe
2+, Fe
3+, Cr
3+, Cr
2O
7 2-, Mn
2+, Zn
2+, Ni
2+Deng, these metals ions can use separately, also can make up each other as composite catalyst and use.Wherein, preferred catalyzer is Cu
+, Cu
2+, Fe
2+, Fe
3+, Zn
2+, most preferably be Cu
2+Because metals ion is not self-existent chemical reagent, what reality was used is the inorganic or organic salt of these metals ions.In reaction system, the concentration range of said metals ion can be about 1nmol/L～0.1mol/L, and preferred concentration range is 10 μ mol/L～10mmol/L.
The common process parameter of said depolymerization reaction process is: TR is 10 ℃～75 ℃; Reaction times is 20 minutes～8 hours; Reaction can be carried out under normal pressure or pressurized conditions; Reaction can be selected to carry out under nitrogen, the protection of inert gas, also can be under condition of normal pressure communicates with atmospheric environment to carry out.
When reaction finishes, can in reaction system, add sequestrant and make it to suppress catalyzed reaction speed, then through technique means termination reactions such as cooling, organic solvent deposits with the metal ion catalyst chelating.Sequestrant is meant the material that can form inner complex with metals ion, and it includes but not limited to YD 30 (EDTA), diethylene triamine pentacetic acid (DTPA) (DTPA), 3-trimethylenedinitrilo-tertraacetic acid (PDTA), nitrilotriacetic base ammonia (NTA) or their salt.The preferred EDTA Disodium of the inventive method or its hydrate.The reaction product precipitator method are directly or under the condition that further adds inorganic salt (like potassium acetate) to add organic solvent is separated out glycan class material from reaction system method; Said organic solvent comprises low-carbon alcohol/ketone such as methyl alcohol, ethanol, acetone, wherein is preferably ethanol and acetone.
Reaction product dFG can pass through the means known in the art purifying; For example remove small molecule salt through dialysis method or ultrafiltration process; Be further purified etc. through gel chromatography or DEAE ion exchange chromatography; Gained dFG product can also be through cationic exchange to be prepared into single salt form, like sodium salt, sylvite or calcium salt etc.In the said dialysis impurity elimination treating processes, can select the dialysis membrane of suitable molecular weight cut-off according to order ground dFG molecular weight size requirements, preferred molecular weight cut-off is 3000Da.Dialysis time needs to confirm according to the particular procedure condition, is no less than 6 hours usually.The preferred dynamic ion exchange salt forming method of the salification process of dFG product wherein can select to adopt strongly acidic cationic exchange resin.Appearance all can be undertaken by ordinary method with wash-out on resin column pre-treatment, the sample.In the inventive method on the preferred sample appearance massfraction be about 2%～5%.
The inventive method can significantly be improved the reaction conditions of superoxide depolymerization FG; Promptly; When adopting identical peroxide reactants to prepare the dFG of identical or approximately equal molecular weight, under the proximate situation of reaction conditionss such as temperature, with respect to direct superoxide depolymerization (promptly not having the superoxide depolymerization method under the metal ion catalyst existence) with prototype FG initiator; The inventive method can improve speed of response, shortens response time history; Similarly, under the condition of controlling reaction time, it is temperature required that the inventive method can significantly reduce reaction, so that can accomplish required reaction at ambient temperature.
Repeating in the preparation process of dFG, under the identical or proximate condition of maintenance reaction conditions, to compare with direct oxidation thing depolymerization, batch differences of the inventive method products therefrom significantly reduces.For the dFG preparation of different target molecular weight; The inventive method can realize through changing reaction conditions easily; And for direct superoxide depolymerization, then change reaction conditions afterreaction product and depart from the uncertainty comparatively serious (difference of gained dFG molecular weight and target molecular weight can reach more than 5%) of estimating the result.
The inventive method is significantly improved the repeatability and the controllability of dFG preparation; Possibly reduce with activation energy due to the catalyzer and then to form metastable speed of reaction relevant, also possibly reduce with temperature of reaction or the controllability and the improved stability of gentle reaction conditions such as response time history shortening and reaction conditions thereof relevant.
For the refining FG raw material of pure relatively, homogeneous, under the same reaction conditions, the inventive method products therefrom has the repeatability of height; But for containing the containing for the FG " Crude polysaccharides " of more impurity component, the stability of depolymerization reaction and controllability still might exist than big-difference.The inventor has now found that, exists the inorganic or organic salt of certain ionic strength can improve the stability of depolymerization reaction in the reaction system.
For this reason, the present invention also further provides the method that improves and improve FG depolymerization reaction controllability,, in the reaction system of the superoxide depolymerization fucosylated glycosaminoglycan of metal ion catalysis, adds certain density inorganic and/or organic salt that is.Said inorganic and/or organic salt comprises the salt that metallic element (like basic metal, alkali earth metal etc.) and halogen, organic acid etc. form; The salt that organic acid or mineral acid and organic bases form; And their composite salt of making up each other, wherein preferred sodium-chlor, Repone K, sodium acetate, sodium acetate trihydrate, potassium acetate.In the inventive method, the preferred salt ionic concentration that is used to improve the depolymerization reaction speed and the inorganic and/or organic salt of reaction controllability is about 0.1mmol/L about 1.0mol/L extremely.Salt ion improves the mechanism of reaction conditions can not illustrate fully, for the inventive method, and maybe be relevant with the conformation of its stopping reaction thing FG.
FG according to the invention is the fucosylated glycosaminoglycan that derives from the echinoderms Holothuroidea, and its constitutional features is to have the GlcUA of about 1: 1 ± 0.3 mol ratio and the Fucose (sulfuric ester) of GalNAc (sulfuric ester) and different mol ratio.The difference of sea cucumber kind and tissue-derived difference or process for extracting can cause the difference of the aspects such as monose proportion of composing, side chain existence form and polysaccharide sulfated degree of FG; But these differences all do not relate to the essential change of FG constitutional features and glycosidic link type, therefore do not influence the enforcement and the application of depolymerization method according to the invention.The source animal of FG according to the invention can be selected from but be not limited to stichopus japonicus, Stichopus chloronotus (Brandt)., hojothuria leucospilota, Black Sea ginseng, Holothuria nobilis Selenka, rough sea cucumber etc.Obviously; It will be appreciated by those skilled in the art that; For other kind sea cucumber that originates in all over the world; The fucosylated glycosaminoglycan that meets the said structure characteristic in its source all can adopt the method for the invention depolymerization to obtain required oligomeric FG product, and therefore, the inventive method does not receive the restriction of specific sea cucumber kind.
The reducing end under neutral of gained dFG product of the present invention can detect through the NMR method.NMR detects and can also be advantageously used in confirming Fuc/GalNAc proportion of composing in the product.
More direct superoxide depolymerization and catalysis superoxide depolymerization product; The present invention finds; Catalysis superoxide depolymerization has beat all glycosidic link cracking selectivity; Its outstanding behaviours at gained dFG in GalNAc be basically reducing end under neutral (with terminal quantity generally more than 80% and even 95%) and less be GlcUA; This exists high-load relatively reductibility GlcUA end that marked difference is arranged with direct superoxide depolymerization product, shows that catalysis superoxide depolymerization gained dFG has better terminal homogeneity, thereby has better quality homogeneity and controllability.
In addition, the variation of comparison FG depolymerization front and back Fuc/GalNAc proportion of composing can be judged the cracking degree of side chain Fucose.Several no cracking of FG side chain Fucose in the catalytic degradation process according to the invention, the Fuc/GalNAc proportion of composing does not have considerable change among FG and the dFG; And directly superoxide is separated in the collecting process, and can there be about reduction of about 5% in this ratio, and this proves that further the inventive method has superior glycosidic link cracked selectivity.
Thus, another purpose of the present invention provides a kind of oligomeric FG (dFG), and said dFG is through the inventive method preparation; It is characterized in that; The reducing end under neutral of said dFG is mainly GalNAc, can confirm that through nucleus magnetic resonance (NMR) technology for detection its proportion of composing is not less than 80%, correspondingly; Among the said dFG, be less than 20% as the glycan molecule number of reducing end under neutral with GlcUA.Among the preferred dFG of the present invention, be that the polysaccharide molecule number of reducing end under neutral is no less than 90% with GalNAc.
The molecular weight of dFG product can adopt the efficient gel chromatography to detect.Active and avoid the induced platelet aggregation activity to consider from keeping the FG hematology, in weight-average molecular weight, the molecular weight ranges of the dFG that the present invention selects is about 6,000～20,000Da, and preferred molecular weight range is about 10,000～15,000Da.
The polydispersity index of dFG according to the invention (PDI, the ratio of weight-average molecular weight and number-average molecular weight is Mw/Mn) generally between 1.0 to 2.0; For preferred oligomeric FG, its PDI is between 1.2 to 1.6.
DFG according to the invention can be the salt of its pharmaceutically acceptable basic metal, earth alkali metal etc., for example sodium salt, sylvite and calcium salt etc.; Similarly, oligomeric FG according to the invention also can be the ester of its pharmaceutically acceptable alkaline organic group.
DFG according to the invention has definite anticoagulant active, therefore has clear and definite pharmaceutical potential.DFG has good water-solubility, therefore is easy to be prepared into solution type preparation or its freeze-dried products.As the polyose composition, its oral bioavailability is limited, so preferred preparation becomes parenteral dosage forms, and its formulation preparation can be carried out according to the technological method of knowing in this area.
Another purpose of the present invention provides a kind of medicinal compsns, and said medicinal compsns comprises dFG according to the invention and acceptable accessories.DFG according to the invention has the anticoagulating active of certain intensity; Therefore can be used for the prevention and the treatment of thrombotic diseases in various degree; For example thrombotic cardiovascular disorder, thrombus property cerebro-vascular diseases, pulmonary vein thrombus, PeV thrombus, DVT, property arterial thrombus etc. on every side.In view of the above, the present invention can provide the application of said compsn in the medication preparation of treatment and preventing cardiovascular disease.
Description of drawings
Fig. 1 is the HPGPC collection of illustrative plates of stichopus japonicus FG and degraded product dFG thereof;
Fig. 2 is for showing Fuc/GalNA mol ratio (methyl signals integral area ratio) among FG, the dFG
1H NMR spectrogram;
Fig. 3 A is dFG-2's
1H-
1H COSY spectrogram;
Fig. 3 B is the COSY spectrogram cross section at Fig. 3 A 5.22ppm place, shows the NMR signal of α-H of reducing end under neutral GalNAc and GlcUA.
Embodiment
Following examples are used to further specify the present invention, do not constitute limitation of the scope of the invention.
The superoxide depolymerization of the FG of embodiment 1 metal ion catalysis
1.1 material:
FG: the fucosylated glycosaminoglycan in stichopus japonicus source, press literature method (J Biol Chem, 1991,266 (21): 13530-6) extract preparation.Purity 98% (HPGPC, area normalization method), molecular weight (Mw), 69800.
H
2O
2, CH
3COONa3H
2O, NaCl, NaOH, CuCl
2, FeCl
2, ZnCl
2Deng agents useful for same: be commercially available AR.
1.2 method:
Four parts of each 5.0g of stichopus japonicus FG are dissolved in the 180ml water in the round-bottomed flask, and 45 ℃ of water bath heat preservations also continue evenly to stir, and add the cupric chloride (Cu of 10ml pure water or 20mmol/L concentration respectively
2+) solution, iron protochloride (Fe
2+) solution or zinc chloride (Zn
2+) behind the solution, in 2 hours, drip 10% H with 15ml/h speed
2O
2, using the NaOH solution control pH value scope of 1N in the reaction process is 7.2～7.8, the continuously stirring reaction is 2～8 hours under the above-mentioned condition.Begin back 30min, 1h, 2h, 6h, 8h from each reaction flask sampling 5ml respectively at reaction; Add 15mg EDTA disodium salt and mixing, frozen water cools off, and adds 95% ethanol sedimentation polysaccharide of 3 times of volumes; Twice of 5ml 60% washing with alcohol; Warm air dries up ethanol, 10ml pure water constant volume, and efficient gel chromatogram (HPGPC) method detects molecular weight of product.
1.3 result:
Having or not in the presence of the period 4 transition metal ion catalyzer, hydrogen peroxide is seen table 1 and accompanying drawing 1 to the depolymerization effect of stichopus japonicus FG.
Table 1:FG peroxo-depolymerization product molecular weight detection
The result of table 1 shows, metals ion Cu
2+, Fe
2+, Zn
2+Superoxide depolymerization reaction to FG all has obvious catalysis, and depolymerization reaction speed significantly promotes.Catalytic efficiency (through comparing these three kinds of catalyzer can be found out metals ion Cu
2+Katalysis efficient higher, it can realize the preparation of dFG in the short period of time.
2.1 material:
FG: the fucosylated glycosaminoglycan in stichopus japonicus source, press literature method (J Biol Chem, 1991,266 (21): 13530-6) extract preparation.Purity 98% (HPGPC, area normalization method), molecular weight (Mw), 69800.
H
2O
2, CH
3COONa3H
2O, NaCl, NaOH, Cu (CH3COO)
2H
2Agents useful for same such as O: be commercially available AR.
2.2 method:
Preparation dFG: two parts of each 5.0g of stichopus japonicus FG are dissolved in the 180ml aqueous solution in the round-bottomed flask,, add the venus crystals (Cu of 10ml pure water or 60mmol/L concentration then respectively respectively with 70 ℃ and 35 ℃ of water bath heat preservations and lasting evenly stirring
2+) solution, in 2 hours, drip 10% H then with 10ml/h speed
2O
2, using the NaOH solution control pH value scope of 1N in the reaction process is 7.2～7.8.The continuously stirring reaction added 500mg EDTA disodium salt and mixing respectively after 4 hours under the above-mentioned condition in reaction solution, and frozen water cools off, and adds 95% ethanol sedimentation polysaccharide of 3 times of volumes, centrifugal must the deposition, 100ml 60% washing with alcohol twice, centrifugal must the deposition.Resolution of precipitate becomes sodium salt through 001 * 7 type resin cation exchange in 150ml water, dialysed 6 hours with the dialysis membrane of molecular weight cut-off 3500Da then, holds back product and concentrates, and lyophilize obtains 3.23g (dFG-1, directly H respectively
2O
2Depolymerization product) and 3.52g (dFG-2, Cu
2+Catalytic H
2O
2Depolymerization product) depolymerization sample.
Physics and chemistry detects: HPGPC detection molecules amount and distribution; Conductometric titration detection-OSO
3 -/-COO
-Mol ratio.
NMR detects:
Instrument, AVANCE AV 400 superconduction nuclear magnetic resonance spectrometers (400MHz of Switzerland Bruker company);
Solvent, D
2O 99.9Atom%D (Norell company);
Interior mark, trimethylsilyl-propionic acid (TSP-D4); Temperature, 45 ℃.
2.3 result
Detected result sees the following form 2.The NMR spectrogram is seen accompanying drawing 2.
The physics and chemistry of table 2:FG, dFG-1 and dFG-2 detects with NMR and detects
Mw: weight-average molecular weight; Mn: number-average molecular weight; PDI: molecular weight polydispersity index (PDI=Mw/Mn)
*Reducing end under neutral glycosyl mol ratio
Reaction conditions through more direct hydrogen peroxide depolymerization, catalyzing hydrogen peroxide depolymerization (70 ℃/4h, 35 ℃/4h) and the molecular weight of two kinds of reaction product (about 1.9kDa and about 1.5kDa) can find out, at Cu
2+Ion exists down, H
2O
2The temperature of reaction of depolymerization FG reduces, and efficient is higher.
Visible from the physics and chemistry and the Spectrum Analysis result of two kinds of products, the PDI value of dFG-1 and dFG-2 all raises than FG to some extent, and this conforms to the peroxide radical depolymerization characteristic of mucopolysaccharide, and dFG-2's is dispersed relatively low.
From-OSO
3 -/-COO
-Ratio sees, the value of dFG-1 compare with FG reduce about 4%, and the basic no change of dFG-2.
Visible according to the GalNAc/Fuc mol ratio (Fucose methyl peak-to-peak signal appears at about 1.3ppm, and the methyl peak position of acetylamino galactosamine is in about 2.1ppm) of NMR methyl signals integral and calculating, dFG-1 reduces about 9% than FG, and the basic no change of dFG-2.This result shows, at Cu
2+Ion exists down, H
2O
2The process of depolymerization FG does not almost have influence to the Fucose side chain.Obviously, can judge that according to the Fuc ratio variation of direct superoxide depolymerization gained dFG-1 directly the peroxo-depolymerization can cause Fucose sulfuric ester side chain cleavage.
Can judge the mol ratio of the GalNAc and the GlcUA of depolymerization product reducing end under neutral from accompanying drawing 3A; Wherein the end of the bridge hydrogen of GalNAc in non reducing end and the polysaccharide main chain and GlcUA is beta configuration; Its NMR signal occurs between about 4.4～4.6ppm, and the α-configuration end of the bridge hydrogen of the about half of each existence of reducing end under neutral GalNAc and GlcUA.
1The α of GalNAc and GlcUA in the H NMR spectrogram-configuration end of the bridge hydrogen all appears at about 5.2～5.3ppm; Both are difficult to distinguish; But about 4.2～4.5ppm and 3.6～3.9ppm appear respectively in the chemical shift at the H2 peak relevant with them; Therefore can from the relevant peaks spectrogram, distinguish (prototype FG is bigger because of molecular weight, and the sugar radix is less and be difficult to obtain enough strong differentiation signal).
Calculate and to know that GalNAc on the reducing end under neutral of dGAG-1 and dGAG-2 and the mol ratio of GlcUA were respectively about 1: 0.45 and about 1: 0.08, this shows Cu from accompanying drawing 3B
2+Catalytic hydrogen peroxide depolymerization method has more superior selectivity to GalNAc β (1 → 4) glycosidic link among the FG.
Result of study of the present invention shows that metal ion catalysis can significantly improve the selectivity of superoxide depolymerization to glycosidic link.Although report Cu is arranged
2+Ionic catalysis is separated at the superoxide of heparin and is also demonstrated certain selectivity in the collecting process, but its precise mechanism is not clear.Judge that according to depolymerization reaction mechanism the selectivity of the present invention's catalytic degradation reaction should difference be relevant to some extent to the influence of the activation energy of the dissimilar glycosidic links of radical cracking with catalyzer.
Result of study of the present invention is also found; Superoxide depolymerization for the metal ion catalysis of FG; The glycan molecule number that in the dFG product with GalNAc is reducing end under neutral can reach more than 80% usually, and in the present embodiment, is that the ratio of reducing end under neutral has reached about 95% with GalNAc.
The dFG's of embodiment 3 direct superoxide depolymerizations and the preparation of metal ion catalysis superoxide depolymerization
Repeatability and controllability are relatively
3.1 material:
With 2.1
3.2 method:
Direct hydrogen peroxide method depolymerization FG: five parts of each 300g of stichopus japonicus FG are dissolved in the 9L water, and 60 ℃ of water bath heat preservations also continue evenly to stir, and in 2 hours, drip 15% H then with 0.6L/h speed
2O
2, using the NaOH solution control pH value scope of 1N in the reaction process is 7.2～7.8.The continuously stirring reaction added 3.0g EDTA disodium salt and mixing respectively, the frozen water cooling after 6.5 hours under this condition in reaction solution; The 95% ethanol sedimentation polysaccharide that adds 3 times of volumes, centrifugal must the deposition is with twice of 0.6L 60% washing with alcohol; Be dissolved in then in the 10L water with the modified cellulose membrane ultrafiltration of molecular weight cut-off 3000Da 6 hours; Hold back product and concentrate, lyophilize is calculated efficiency of pcr product and is detected molecular weight of product and distribution.
Cu
2+Catalytic hydrogen peroxide method depolymerization FG: five parts of each 300g of stichopus japonicus FG are dissolved in the 9L water, and 45 ℃ of water bath heat preservations also continue evenly to stir, and add the venus crystals (Cu of 0.6L pure water or 20mmol/L concentration then respectively
2+) solution, in 2 hours, drip 15% H then with 0.6L/h speed
2O
2, using the NaOH solution control pH value scope of 1N in the reaction process is 7.2～7.8.The continuously stirring reaction added 3.0g EDTA disodium salt and mixing respectively, the frozen water cooling after 4 hours under this condition in reaction solution; The 95% ethanol sedimentation polysaccharide that adds 3 times of volumes, centrifugal must the deposition is with twice of 0.6L 60% washing with alcohol; Be dissolved in then in the 10L water with the modified cellulose membrane ultrafiltration of molecular weight cut-off 3000Da 6 hours; Hold back product and concentrate, lyophilize is calculated efficiency of pcr product and is detected molecular weight of product and distribution.
3.3 result
The result sees table 3.
Table 3: direct hydrogen peroxide depolymerization FG and Cu
2+The product of catalyzing hydrogen peroxide depolymerization FG
Mw: weight-average molecular weight, Mn: number-average molecular weight, PDI: molecular weight polydispersity index (PDI=Mw/Mn)
RSD: RSD
The result of table 3 shows that directly hydrogen peroxide depolymerization gained dFG molecular weight of product differences between batches are bigger, and the RSD value of its weight-average molecular weight and number-average molecular weight is all greater than 5%; And catalyzing hydrogen peroxide depolymerization products therefrom molecular weight differences between batches are less, and the RSD value of its weight-average molecular weight and number-average molecular weight is all less than 5%.And directly the RSD of hydrogen peroxide depolymerization products therefrom Mw, Mn, three detected values of PDI all obviously greater than the product of gained according to the method for the invention, shows that catalyzing hydrogen peroxide depolymerization of the present invention has better stability and controllability.
Embodiment 4 transition metal ion catalysis superoxide method depolymerization hojothuria leucospilota Crude polysaccharides
4.1 material:
Hojothuria leucospilota: commercially available kind
Reagent: with 2.1
4.2 method:
The preparation of hojothuria leucospilota fucosylated glycosaminoglycan raw sugar (preparation of reference method): get dry hojothuria leucospilota 1000g, pulverize, in the water-bath device of packing into, add 10L water and stir immersion.Adding solid potassium hydroxide to concentration is 1N, 60 ℃ of constant temperature alkaline hydrolysis reaction 100min.After the cooling, 6N hydrochloric acid adjust pH is 8.5, adds 50g trypsinase, and 50 ℃ of following insulated and stirred were reacted 3 hours.After the cooling, the centrifugal deposition of going.Get supernatant, using the salt acid for adjusting pH value is 2.5, centrifugal removal acidic protein deposition.Get supernatant, add the neutralization of 2N NaOH solution, the centrifugal deposition of removing.Get supernatant, add 95% ethanol and make its concentration reach 60%, 4 ℃ of placement to spend the night.Centrifugal must the deposition adds the water dissolution of 10 times of weight, centrifugal removal insolubles.Get supernatant, add Potassium ethanoate and stir and to dissolve that to make its ultimate density be 2.0mol/L entirely, adding 95% ethanol to its final volume concentration is 30%, staticly settles.The centrifugal deposition that obtains is used 95% ethanol, absolute ethanol washing successively, and reduced vacuum is dry, promptly gets thick sea cucumber polysaccharide 8.1g.
Cu
2+Catalytic hydrogen peroxide method depolymerization FG: five parts of each 5.0g of hojothuria leucospilota Crude polysaccharides are dissolved in the 190ml water in the round-bottomed flask, and 35 ℃ of water bath heat preservations also continue evenly to stir, and add the venus crystals (Cu of 50mmol/L concentration then respectively
2+) solution 10ml, drip 10% H then with 10ml/h speed
2O
2, using the NaOH solution control pH value scope of 1N in the reaction process is 7.2～7.8.The continuously stirring reaction added 500mg EDTA disodium salt and mixing respectively, the frozen water cooling after 3 hours under the above-mentioned condition in reaction solution; The 95% ethanol sedimentation polysaccharide that adds 3 times of volumes, centrifugal must the deposition is with twice of 100ml 60% washing with alcohol; After 100ml is water-soluble, adds Potassium ethanoate and stir and to dissolve that to make its ultimate density be 2.0mol/L, hold over night entirely; Centrifugal collecting precipitation, 100ml is water-soluble, dialyses 8 hours with the dialysis membrane of molecular weight cut-off 3500Da then; Hold back the product lyophilize, calculate efficiency of pcr product and detect molecular weight of product and distribution.
Cu under certain ionic strength
2+Catalytic hydrogen peroxide method depolymerization FG: five parts of each 5.0g of hojothuria leucospilota Crude polysaccharides are dissolved in round-bottomed flask and contain in the 190ml water of 12.21g sodium acetate trihydrate and 6.02g sodium-chlor; 35 ℃ of water bath heat preservations also continue evenly to stir, and add the venus crystals (Cu of 50mmol/L concentration then respectively
2+) solution 10ml, drip 10% H then with 10ml/h speed
2O
2, using the NaOH solution control pH value scope of 1N in the reaction process is 7.2～7.8.The continuously stirring reaction added 500mg EDTA disodium salt and mixing respectively, the frozen water cooling after 4 hours under the above-mentioned condition in reaction solution; The 95% ethanol sedimentation polysaccharide that adds 3 times of volumes, centrifugal must the deposition, twice of 100ml 60% washing with alcohol; After 100ml is water-soluble, adds Potassium ethanoate and stir and to dissolve that to make its ultimate density be 2.0mol/L, hold over night entirely; Centrifugal collecting precipitation, 100ml is water-soluble, dialyses 8 hours with the dialysis membrane of molecular weight cut-off 3500Da then; Hold back the product lyophilize, calculate efficiency of pcr product and detect molecular weight of product and distribution.
4.3 result
The result sees table 4.
Table 4: the hydrogen peroxide method depolymerization of hojothuria leucospilota Crude polysaccharides
Mw: weight-average molecular weight, Mn: number-average molecular weight, PDI: molecular weight polydispersity index (PDI=Mw/Mn)
RSD: RSD
The result of table 4 shows; For Crude polysaccharides; Because its foreign matter content is higher relatively, adopt the hydrogen peroxide depolymerization of metal ion catalysis this moment, and still possibly there are bigger differences between batches in its products therefrom; It shows that mainly there is notable difference (RSD＞10%) in molecular-weight average, and dispersion coefficient does not have significant difference.
In reaction medium, add the inorganic and/or organic ion of certain intensity,, possibly make speed of reaction that reduction to a certain degree takes place although add these ions can significantly improve the repeatability of depolymerization reaction.The mechanism that inorganic and/or organic ion improves reaction repeatability is unclear fully as yet, and it is generally acknowledged maybe be relevant with the conformation of keeping polysaccharide stabilizes.
Correlative study of the present invention shows that in the peroxo-depolymerization reaction of the non-pure FG in different genera sea cucumber source, for said inorganic and/or organic ion, suitable positively charged ion intensity is about 0.1mol/L to 1.0mol/L.
The anticoagulant active of embodiment 5 oligomeric fucosylated glycosaminoglycans (dFG)
5.1 material
Series hojothuria leucospilota dFG:Mw is respectively 25,380,18,050,14,350,11,450,8,800.
Press Cu among the embodiment 4
2+Catalytic depolymerization preparation (depolymerization asynchronism(-nization));
Reagent: rabbit platelet poor plasma, the special bio tech ltd of Guangzhou stamen;
Activated partial thromboplastin time (APTT) is measured test kit (gallogen): Shanghai Sun Bio-Tech Co., Ltd.
Instrument: BICO-double-channel coagulo meter, Minivolt company (Italy).
5.2 method
Accurately take by weighing each sample 13.0mg, dissolving is fixed dissolves to 100ml, dilutes ten times then.Press the APTT time of each sample of method survey of test kit specification sheets, the clotting time of deduction blank plasma promptly is the time Δ APTT (Sec) that sample prolongs blood coagulation.
5.3 result
Experimental result is as shown in table 5.
Table 5: the dFG of the different molecular weight in hojothuria leucospilota source is to the influence of plasma in rabbit APTT time
Result in the table 5 shows, the dFG in hojothuria leucospilota source can the significant prolongation rabbit plasma APTT time, shows that it can suppress intrinsic coagulation.
The freeze-dried products of embodiment 6 oligomeric fucosylated glycosaminoglycans (dFG)
6.1 material
Stichopus japonicus dFG:Mw 14,350.Press Cu among the embodiment 3
2+Catalytic depolymerization preparation.
20mmol/L pH 7.0 phosphoric acid buffers (PBS)
6.2 prescription
| The supplementary material title | Consumption |
| dFG | 50g |
| Seminose | 50g |
| 20mmol/L pH 7.0 phosphoric acid buffers (PBS) | 500ml |
| Process altogether | 1000 |
6.3 preparation technology
The stichopus japonicus dFG and the seminose that take by weighing recipe quantity add 20mmol/L pH 7.0PBS to full dose, stir to make dissolving fully.The medicinal carbon of adding 0.6% stirs 20min; Use B and 3.0 μ m millipore filtration decarbonization filterings.Survey midbody content.Qualified back is with the filtering with microporous membrane of 0.22 μ m; Can in the control cillin bottle, every bottle of 0.5ml, pouring process monitoring loading amount, half tamponade is put in the freeze drying box, carries out freeze-drying by the freeze-drying curve of setting, tamponade, outlet rolls lid, visual inspection is qualified, finished product.
Freeze-drying process: with the sample inlet, fall the dividing plate temperature, keep 3h to-40 ℃; Cold-trap is reduced to-50 ℃, begins to be evacuated to 300 μ bar.Begin distillation: 1h at the uniform velocity is warming up to-30 ℃, keeps 2h; 2h at the uniform velocity is warming up to-20 ℃, keeps 8h, and vacuum keeps 200～300 μ bar; Carry out drying: 2h again and be warming up to-5 ℃, keep 2h, vacuum keeps 150～200 μ bar; 0.5h be warming up to 10 ℃, keep 2h, vacuum keeps 80～100 μ bar; 0.5h be warming up to 40 ℃, keep 4h, vacuum is evacuated to minimum.
Claims (12)
1. method for preparing oligomeric fucosylated glycosaminoglycan is included in that catalyzer exists down in the aqueous media, and catalysis superoxide depolymerization fucosylated glycosaminoglycan obtains oligomeric fucosylated glycosaminoglycan, wherein:
Said fucosylated glycosaminoglycan is meant that derives from an echinoderms TGSS C3 or derivatives thereof; Its constitutional features does; Contain molar ratio range in the said fucosylated glycosaminoglycan and be 0.7～1.3 glucal acidic group and acetylamino galactosamine base or its sulfuric ester; And contain Fucose or its sulfate group, the molar ratio range of Fucose or its sulfate group and acetylamino galactosamine or its sulfuric ester is 0.5～2.5;
Said catalyzer is to contain to be selected from the catalyzer that periodic table of elements period 4 transition metal ion forms;
Said oligomeric fucosylated glycosaminoglycan is meant that its weight-average molecular weight is the fucosylated glycosaminoglycan in 6000～20000Da scope;
Wherein the quality with reaction system is the basis; The massfraction of said fucosylated glycosaminoglycan is 0.05%～15%; The massfraction of superoxide is 0.5%～30%, and said catalyst concentration is 0.001mmol/L～100mmol/L, and the TR of reaction is 10 ℃～75 ℃.
2. the described method of claim 1, the wherein said catalyzer that is selected from periodic table of elements period 4 transition metal ion that contains is Cu
+, Cu
2+, Fe
2+, Fe
3+, Cr
3+, Cr
2O
7 2-, Mn
2+, Zn
2+, Ni
2+The inorganic salt or the organic salt that form, or its combination.
3. the described method of claim 2, wherein said metals ion is Cu
2+
4. the described method of claim 1, wherein said superoxide is selected from Peracetic Acid, hydrogen peroxide, 3-chloro-peroxybenzoic acid, cumene hydroperoxide, Sodium Persulfate, Lucidol and salt or ester.
5. the described method of claim 4, wherein said superoxide is a hydrogen peroxide.
6. each described method of claim 1-5; Inorganic and/or the organic salt that wherein in reaction system, further comprises 1mmol/L～1.0mol/L, said inorganic and/or organic salt be selected from sodium-chlor, Repone K, sodium acetate, potassium acetate and their hydrate, with and combination.
7. oligomeric fucosylated glycosaminoglycan or its pharmacy acceptable salt and/or ester according to claim 1 a method preparation; It is 1 that wherein said oligomeric fucosylated glycosaminoglycan contains mol ratio: the glucal acidic group of (1 ± 0.3) and acetylamino galactosamine or its sulfuric ester, and contain Fucose or its sulfate group; In the said oligomeric fucosylated glycosaminoglycan, be not less than 80% with acetylamino galactosamine or its sulfuric ester as the glycan molecule number of reducing end under neutral, polydispersity index is 1.0～2.0.
8. the described oligomeric fucosylated glycosaminoglycan of claim 7 or its pharmacy acceptable salt and/or ester; The weight-average molecular weight of wherein said oligomeric fucosylated glycosaminoglycan is 10; 000Da～15; 000Da, polydispersity index are 1.2～1.6, and it is not less than 90% with acetylamino galactosamine or its sulfuric ester as the glycan molecule number of reducing end under neutral.
9. each described oligomeric fucosylated glycosaminoglycan of claim 7-8 or its pharmacy acceptable salt and/or ester, wherein said pharmacy acceptable salt is its basic metal and/or alkaline earth salt.
10. the described oligomeric fucosylated glycosaminoglycan of claim 9 or its pharmacy acceptable salt and/or ester, wherein said pharmacy acceptable salt is sodium salt, sylvite and calcium salt.
11. a pharmaceutical composition is characterized in that, comprises the described oligomeric fucosylated glycosaminoglycan of claim 7 or its pharmacy acceptable salt and/or the ester of effective dose, and acceptable accessories.
12. the described oligomeric fucosylated glycosaminoglycan of claim 7 or its pharmacy acceptable salt and/or ester are in the purposes in the medicine that treats and/or prevents of preparation thrombotic diseases.
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| WO2014166282A1 (en) | 2013-04-12 | 2014-10-16 | 中国科学院昆明植物研究所 | Low-molecular-weight glycosaminoglycan derivative containing terminal 2, 5-anhydrated talose or derivative thereof |
| WO2015103921A1 (en) | 2014-01-08 | 2015-07-16 | 中国科学院昆明植物研究所 | Fuc3s4s substituted oligoglycosaminoglycan and preparation method thereof |
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| CN102329397B (en) * | 2011-10-19 | 2014-04-09 | 中国科学院昆明植物研究所 | Fucosylated glycosaminoglycan derivative and preparation method thereof |
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| WO2015103921A1 (en) | 2014-01-08 | 2015-07-16 | 中国科学院昆明植物研究所 | Fuc3s4s substituted oligoglycosaminoglycan and preparation method thereof |
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