CN103724458B - Containing preparation and the purifying thereof of N-non-substituted glucosamine heparin six sugar - Google Patents

Containing preparation and the purifying thereof of N-non-substituted glucosamine heparin six sugar Download PDF

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CN103724458B
CN103724458B CN201410019945.8A CN201410019945A CN103724458B CN 103724458 B CN103724458 B CN 103724458B CN 201410019945 A CN201410019945 A CN 201410019945A CN 103724458 B CN103724458 B CN 103724458B
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heparin
sugar
glcnh
sodium
pyridine
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CN103724458A (en
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魏峥
梁群焘
林江慧
魏可镁
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Fuzhou University
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Fuzhou University
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Abstract

The present invention relates to a kind of containing the non-substituted glucosamine (GlcNH of <i>N</iGreatT.Gr eaT.GT- 3 +) preparation of heparin six sugar and purifying thereof.Described method is with enzymolysis low molecular weight heparin for raw material, by the method such as gel chromatography and Ion-exchange high-performance liquid chromatography separating-purifying heparin sodium six sugar; Zeo-karb and pyridine neutralisation is utilized to prepare heparin pyridine six sugar; Finally standby containing different GlcNH by de-<i>N</iGreatT.Gr eaT.GT-sulfation legal system 3 +heparin six sugar of number, and carry out separating-purifying by methods such as SAX-HPLC.Present method can specificity be prepared containing different GlcNH 3 +the serial Heparin Oligosaccharides of number and sequence is GlcNH in heparitin sulfate 3 +the structure and fuction of residue and provide important sugared storehouse with the research of disease relationship.

Description

Containing preparation and the purifying thereof of N-non-substituted glucosamine heparin six sugar
Technical field
The invention relates to preparation containing GlcNH 3 +the method of Heparin Oligosaccharides and purifying thereof, belong to natural product and prepare purification art.
Background technology
There are 12 kinds of representative disaccharides, wherein containing GlcNAc, GlcNSO in heparitin sulfate 3the disaccharides of residue is main existence form, and positively charged GlcNH 3 +the disaccharides content pettiness of residue, and its existence is also because cell is different and different from the source of tissue.Although containing GlcNH 3 +heparitin sulfate content pettiness, but it with virus intrusion, the generation of inflammation, the diseases such as brain tissue impairment have close relationship, play important biology and physiopathology role in vivo.To containing GlcNH 3 +the research of saccharide compound structure function, has important meaning in disease treatment and new Carbohydrate drugs exploitation.
Along with going deep into of research, the demand containing different structure heparin sugar chain sample is also significantly increased.Bibliographical information past according to this, adopts separation means from biomaterial, directly obtain homogeneous, that structure the is clear and definite sugar chain of component very difficult.The method of many employings chemically modified, by changing the polarity of solvent, optionally removes the sulfate group on different positions on heparin long-chain, thus obtains target product.InoueY etc., use 5% water (or methyl alcohol)/dimethyl sulfoxide solution optionally to remove sulfate group in heparin pyridinium salt on N position; JasejaM etc., by the Na0H aqueous solution of pH=12.5-12.8, in α-L-iduronic acid fragment, 2S can remove quantitatively; AyotteL etc., use the Me of 9:1 2sO-water, HannoB etc. use N-Methyl pyrrolidone aqueous solution alternative to remove 6S; DulcePG etc. use SO 3-Py, SO 3the sulfur acidizing reagents such as-DMF can carry out sulfating reaction by the different positions on sugar chain, obtain the polysaccharide that various difference replaces type.
Up to now, the method steps preparing Heparin Oligosaccharides is various, expensive.The report skeleton structure of the oligose fragment obtained after heparitin sulfate enzymolysis directly being carried out to chemically modified is little.Batch obtains containing GlcNH 3 +the preparation method of Heparin Oligosaccharides is rare especially.The present invention is raw material with the low molecular weight heparin easily obtained, and easy method, prepares containing different GlcNH 3 +the serial Heparin Oligosaccharides of number and sequence is GlcNH in heparitin sulfate 3 +the structure and fuction of residue and provide important sugared storehouse with the research of disease relationship.
Summary of the invention
The present invention tests the low molecular weight heparin that starting material are enzymolysis.Utilize heparin sodium six sugar mixture of gel chromatography separation different polymerization degree, remove the NH in moving phase 4hCO 3after, obtain various heparin sodium six sugar by SAX-HPLC method separated and collected; After desalination and lyophilize, utilize cationic exchange and the preparation of pyridine neutralisation to regulate the pH of elutriant to slightly acidic, obtain heparin pyridine six sugar; After use 95%DMSO:5%H 2o solvent, optionally removes the sulfate radical on N position, then obtains containing different GlcNH by SAX-HPLC method separated and collected 3 +heparin six sugar of number.
Concrete technical scheme of the present invention is:
One contains nthe preparation of-non-substituted glucosamine heparin six sugar and purification process thereof, the steps include: that enzymolysis low molecular weight heparin utilizes gel chromatography to be separated; After removing moving phase, lyophilize, the heparin sodium oligosaccharide mixture of different polymerization degree is obtained after separation; Highly Sulfated heparin sodium six sugar is obtained by SAX-HPLC separated and collected; Heparin sodium six sugar collected after desalination and lyophilize, by cationic exchange, then to use in pyridine and the pH of elutriant to slightly acidic, obtain heparin pyridine six sugar; By heparin pyridine six sugar DMSO:H 2o(v/v)=95:5 dissolution with solvents, optionally removes nsulfate radical on position, then obtain containing different GlcNH by SAX-HPLC method separated and collected 3 +number heparin six sugar, through desalination with obtain after lyophilize purifying contain different GlcNH 3 +heparin six sugar of number.
Concrete steps are as follows:
1) preparation of heparin sodium six sugar and purification: enzymolysis low molecular weight heparin through polyacrylamide gel chromatographic column, with 0.2MNH 4hCO 3for moving phase, and under UV232nm, measure the absorbancy of elutriant; Collect the elution peak of heparin sodium six sugar mixture, remove NH through 50-60 DEG C of water-bath 4hCO 3, after vacuum lyophilization, obtain heparin sodium six sugar mixture; Utilize SAX-HPLC, at different N aCl concentration lower linear gradient elution, the sugared crude product of the Sulfated heparin sodium of separated and collected height six; After the sugared crude product of heparin sodium six collected is dialysed 3 days in ultrapure water, lyophilize;
2) preparation of heparin pyridine six sugar: at 4 DEG C, heparin sodium six sugar obtained in the dialysis of step 1) process also lyophilize through H type Zeo-karb, remove the sodium ion on sugar chain, it is 6-6.5 that the elutriant of gained is at room temperature neutralized to pH with pyridine; The elutriant lyophilize after neutralization, obtain the sugared dry product of heparin pyridine six;
3) containing GlcNH 3 +the preparation of Heparin Oligosaccharides and purifying: step 2) in obtain the sugared dry product of heparin pyridine six be dissolved in DMSO:H 2o(v/v) in=95:5 solvent, through 30-60 DEG C of water-bath 15-20min, the sulfate radical on heparin chain N position is optionally removed; After dialysis lyophilize, with SAX-HPLC, under NaCl linear gradient elution, separated and collected is containing different GlcNH 3 +heparin six sugar of number; Different GlcNH is contained what collect 3 +heparin six sugar of number, dialysis also lyophilize respectively, what obtain purifying contains different GlcNH 3 +heparin six sugar of number.
When water bath condition is under 30 DEG C of water-bath 20min reaction conditionss in step 3), obtain three kinds of heparin six sugar containing one, two and three GlcNH3+ of purifying.
When water bath condition is under 60 DEG C of water-bath 20min reaction conditionss, what obtain purifying contains three GlcNH 3 +heparin six sugar.
concrete operation step is as follows:
1. the preparation of heparin sodium oligosaccharides and purification
Enzymolysis low molecular weight heparin through polyacrylamide gel chromatographic column (Bio-GelP-10), with 0.2MNH 4hCO 3for moving phase, and under UV232nm, measure the absorbancy of elutriant.Collect each component, remove NH through 50-60 DEG C of water-bath 24h × 3 4hCO 3, after vacuum lyophilization, obtain serial heparin sodium six sugar mixture of different polymerization degree.
Utilize SAX-HPLC, at different N aCl concentration lower linear gradient elution, separated and collected heparin sodium six sugar.Heparin sodium six sugar collected is loaded the dialysis membrane of dimension, after dialysing 3 days in ultrapure water, lyophilize.
SAX-HPLC method design parameter is as follows:
Chromatographic column: ProPacPA1(9 × 250mm) Semi-Prep
Flow velocity: 4ml/min
Applied sample amount: < 3mg
Moving phase: A:pH3.5H 2oB:pH3.52MNaCl
Column temperature: room temperature
Detector: UV detector
Determined wavelength: 232nm
the preparation of 2 heparin pyridine oligosaccharides
At 4 DEG C, heparin sodium six sugar obtained after step 1 lyophilize through H type Zeo-karb, remove the sodium ion on sugar chain, it is 6-6.5 that the elutriant of gained is at room temperature neutralized to pH with pyridine.The elutriant lyophilize after neutralization, obtain heparin pyridine oligosaccharides dry product.
containing GlcNH 3 + the preparation of Heparin Oligosaccharides and purification
The heparin pyridine oligosaccharides obtained in step 2 is dissolved in DMSO:H 2o(v/v), in=95:5 solvent, water-bath for some time under certain temperature, the sulfate radical on heparin chain N position is optionally removed.After dialysis lyophilize, with SAX-HPLC, under NaCl linear gradient elution, separated and collected is containing different GlcNH 3 +heparin six sugar of number.Different GlcNH is contained what collect 3 +heparin six sugar of number, dialysis also lyophilize, what obtain purifying contains different GlcNH 3 +heparin six sugar of number.
SAX-HPLC method design parameter is as follows:
Chromatographic column: ProPacPA1(4 × 250mm)
Flow velocity: 1ml/min
Applied sample amount: < 500ug
Moving phase: A:pH3.5H 2oB:pH3.52MNaCl
Column temperature: room temperature;
Detector: UV detector;
Determined wavelength: 232nm.
Embodiment
More being convenient to make content of the present invention understand, below in conjunction with embodiment, technical solutions according to the invention are described further, but the present invention being not limited only to this.
Embodiment 1
with preparation containing GlcNH 3 + heparin six sugar is example:
the preparation of 1 heparin sodium six sugar and purification
Heparinase I, II, III 37 DEG C of enzymolysis low molecular weight heparin (3-8kD) 48h(are commercial) enzymolysis low molecular weight heparin is through gel chromatographic columns (Bio-GelP-10), moving phase is 0.2MNH 4hCO 3, at ultraviolet 232nm place, measure the absorbancy of elutriant.Collect the elution peak of heparin sodium six sugar mixture, NH is removed in rear 55 DEG C of water-bath 24h × 3 4hCO 3,-80 DEG C of freezing 5h postlyophilizations.Use SAX-HPLC again, two-phase linear gradient elution under 0-0.6MNaCl, 0.6-1.3MNaCl condition, be separated and collect the sugared crude product of highly Sulfated heparin sodium six.Design parameter is as follows:
Chromatographic column: ProPacPA1(9 × 250mm) Semi-Prep
Flow velocity: 4ml/min
Moving phase: A:pH3.5H 2oB:pH3.52MNaCl
Column temperature: room temperature
Detector: UV detector
Determined wavelength: 232nm
Sulfated for the height collected heparin sodium six sugar is poured into dialysis membrane (MW:500), dialyses 3 days in ultrapure water, after removing NaCl ,-80 DEG C of freezing 5h postlyophilizations.
the preparation of heparin pyridine six sugar
At 4 DEG C, height Sulfated heparin sodium six sugar obtained in step 1 through H type Zeo-karb (AmberliteIR-120H +), remove the sodium ion on sugar chain, then exchange upper pyridine.Concrete steps are as follows:
(1) according to the exchange capacity E(of resin in specification sheets namely: 1 gram of AmberliteIR-120H +permutable sodium ion mole number) calculate amount of resin needed for exchange 1 gram of heparin sodium six sugar.Method of calculation are as follows:
The sugared molar mass number of heparin sodium six is about 1947g/mol, and 1moldp6 contains 12mol sodium ion.
1 gram of heparin sodium six sugar is containing sodium ion number (mol): (1g ÷ 1947g/mol) x12=0.0062mol
1 gram of required amount of resin (g): 0.0062/E=Yg of heparin sodium six sugar
(2) glass column that blade diameter length ratio is 1 to 20 is chosen.Y gram of resin is immersed in ultrapure water, repeatedly rinses until fill post after neutrality.
(3) 100mg heparin sodium six sugar to be dissolved in 1ml ultrapure water 4 DEG C to spend the night, the ultrapure water of the resin filled in and 3 times of resin volumes to be also placed in 4 DEG C simultaneously and to spend the night.
(4) in 4 DEG C, heparin sodium six sugar in (3) is slow transitted through H type Zeo-karb, 4 DEG C of ultrapure water wash-outs of rear use 3 times of resin volumes;
(5) at room temperature, with pyridine by the elutriant of gained stir state under, being neutralized to pH is 6-6.5.Elutriant after neutralization is put-80 DEG C of freezing 5h, uses vacuum freeze drier lyophilize, obtain the sugared sterling of heparin pyridine six.
containing GlcNH 3 + the preparation of heparin six sugar and purification
The sugared sterling of heparin pyridine six obtained in step 2 is dissolved in 3ml95% (v/v) DMSO:5% (v/v) H 2o solvent, 30 DEG C of water-bath 20min, optionally remove the sulfate radical on N site, obtain containing different GlcNH 3 +heparin six sugar mixture of number, after pour dialysis membrane (MW:500) into, dialyse 3 days in ultrapure water,-80 DEG C of freezing 5h afterwards, use vacuum freeze drier lyophilize, then use SAX-HPLC, at 0-0.6MNaCl (2.1-7.1min), 0.6-1.3MNaCl (7.1-47.1min) condition lower linear is gradient elution separation, collects containing a GlcNH respectively 3 +the sugared crude product of heparin six, containing two GlcNH 3 +the sugared crude product of heparin six and containing three GlcNH 3 +the sugared crude product of heparin six.Design parameter is as follows:
Chromatographic column: ProPacPA1(4 × 250mm)
Flow velocity: 1ml/min
Applied sample amount: 500ug
Moving phase: A:pH3.5H 2oB:pH3.52MNaCl
Column temperature: room temperature
Detector: UV detector
Determined wavelength: 232nm
These collecting are contained different GlcNH 3 +heparin six sugar of number does not pour dialysis membrane (MW:500) into, dialyses 3 days in ultrapure water, and postlyophilization obtains containing three GlcNH 3 +the sugared sterling of heparin six, containing two GlcNH 3 +the sugared sterling of heparin six and containing a GlcNH 3 +the sugared sterling of heparin six.Its molar percentage is respectively 14%, and 42%, 34%.
containing GlcNH 3 + the sign of heparin six sugar
One, two, three GlcNH are contained what obtain in step 3 3 +heparin six sugar, analyze under high performance liquid phase ion hydrazine flight time mass spectrum (LCMS-IT-TOF) negative ion mode of electron spray(ES) (ESI) source respectively, design parameter is as follows: chromatographic column: 3.5umEclipsePlusC18column(2.1 × 100mm); Flow velocity: 0.2mL/min; Applied sample amount: 10ul; Moving phase: A phase pH8.8 is containing the H of 15mM amylamine 2o, B phase pH8.875%/25% (v/v) acetonitrile/water is containing 15mM amylamine; Column temperature: 35 DEG C; Detector: photodiode array, determined wavelength: 190-800nm.Mass spectrum full scan scope: 200-1800m/z; Nitrogen (N2) flow velocity 1.5L/min; Detect voltage+1.7Kv; IT vacuum ranges: 1.8e-002Pa; TOF vacuum ranges: 1.6e-004Pa; CDL temperature 200 DEG C, heating block temperature 200 DEG C.
Under the LCMS-IT-TOF negative ion mode in ESI source, all have found containing one, two, three GlcNH 3 +the characteristic ion peak of heparin six sugar, they are with two electric charges, and have occurred that sulfate radical is lost and the phenomenon (as following table) of amylamine (PTA) adduction: M3: containing three GlcNH 3 +heparin six sugar; M2: containing two GlcNH 3 +heparin six sugar; M1: containing a GlcNH 3 +heparin six sugar.
Embodiment 2
with preparation containing GlcNH 3 + heparin six sugar is example:
the preparation of 1 heparin sodium six sugar and purification
Heparinase I, II, III 37 DEG C of enzymolysis low molecular weight heparin (3-8kD) 48h(are commercial) enzymolysis low molecular weight heparin is through gel chromatographic columns (Bio-GelP-10), moving phase is 0.2MNH 4hCO 3, at ultraviolet 232nm place, measure the absorbancy of elutriant.Collect the elution peak of heparin sodium six sugar mixture, NH is removed in rear 55 DEG C of water-bath 24h × 3 4hCO 3,-80 DEG C of freezing 5h postlyophilizations.Use SAX-HPLC again, two-phase linear gradient elution under 0-0.6MNaCl, 0.6-1.3MNaCl condition, be separated and collect the sugared crude product of highly Sulfated heparin sodium six.Design parameter is as follows:
Chromatographic column: ProPacPA1(9 × 250mm) Semi-Prep
Flow velocity: 4ml/min
Moving phase: A:pH3.5H2OB:pH3.52MNaCl
Column temperature: room temperature
Detector: UV detector
Determined wavelength: 232nm
Sulfated for the height collected heparin sodium six sugar is poured into dialysis membrane (MW:500), dialyses 3 days in ultrapure water, after removing NaCl ,-80 DEG C of freezing 5h postlyophilizations.
the preparation of heparin pyridine six sugar
At 4 DEG C, height Sulfated heparin sodium six sugar obtained in step 1 through H type Zeo-karb (AmberliteIR-120H +), remove the sodium ion on sugar chain, then exchange upper pyridine.Concrete steps are as follows:
(1) according to the exchange capacity E(of resin in specification sheets namely: 1 gram of AmberliteIR-120H +permutable sodium ion mole number) calculate amount of resin needed for exchange 1 gram of heparin sodium six sugar.Method of calculation are as follows:
The sugared molar mass number of heparin sodium six is about 1947g/mol, and 1moldp6 contains 12mol sodium ion.
1 gram of heparin sodium six sugar is containing sodium ion number (mol): (1g ÷ 1947g/mol) x12=0.0062mol
1 gram of required amount of resin (g): 0.0062/E=Yg of heparin sodium six sugar
(2) glass column that blade diameter length ratio is 1 to 20 is chosen.Y gram of resin is immersed in ultrapure water, repeatedly rinses until fill post after neutrality.
(3) 100mg heparin sodium six sugar to be dissolved in 1ml ultrapure water 4 DEG C to spend the night, the ultrapure water of the resin filled in and 3 times of resin volumes to be also placed in 4 DEG C simultaneously and to spend the night.
(4) in 4 DEG C, heparin sodium six sugar in (3) is slow transitted through H type Zeo-karb, 4 DEG C of ultrapure water wash-outs of rear use 3 times of resin volumes;
(5) at room temperature, with pyridine by the elutriant of gained stir state under, being neutralized to pH is 6-6.5.Elutriant after neutralization is put-80 DEG C of freezing 5h, uses vacuum freeze drier lyophilize, obtain the sugared sterling of heparin pyridine six.
containing GlcNH 3 + the preparation of heparin six sugar and purification
The sugared sterling of heparin pyridine six obtained in step 2 is dissolved in 3ml95% (v/v) DMSO:5% (v/v) H 2o solvent, 60 DEG C of water-bath 20min, remove the sulfate radical on N site, obtain containing three GlcNH 3 +heparin six sugar, after pour dialysis membrane (MW:500) into, dialyse 3 days in ultrapure water,-80 DEG C of freezing 5h afterwards, use vacuum freeze drier lyophilize, then use SAX-HPLC, at 0-0.6MNaCl (2.1-7.1min), 0.6-1.3MNaCl (7.1-47.1min) condition lower linear is gradient elution separation, collects containing three GlcNH 3 +the sugared crude product of heparin six.Design parameter is as follows:
Chromatographic column: ProPacPA1(4 × 250mm)
Flow velocity: 1ml/min
Applied sample amount: 500ug
Moving phase: A:pH3.5H 2oB:pH3.52MNaCl
Column temperature: room temperature
Detector: UV detector
Determined wavelength: 232nm
Heparin six sugar collected is poured into dialysis membrane (MW:500), dialyses 3 days in ultrapure water, postlyophilization, obtain containing three GlcNH 3 +the sugared sterling of heparin six.Its molar percentage is 45%.
Characterizing method, also have found containing three GlcNH with embodiment 1. under the LCMS-IT-TOF negative ion mode in ESI source 3 +the characteristic ion peak of heparin six sugar.

Claims (2)

1. one kind contains nthe preparation of heparin six sugar of-non-substituted glucosamine and purification process thereof, is characterized in that: concrete steps are as follows:
1) preparation of heparin sodium six sugar and purification: adopt heparinase I, II, III in 37 DEG C of enzymolysis molecular weight be the low molecular weight heparin of 3-8kD, then by gained enzymolysis low molecular weight heparin through polyacrylamide gel chromatographic column, with 0.2MNH 4hCO 3for moving phase, and under UV232nm, measure the absorbancy of elutriant; Collect the elution peak of heparin sodium six sugar mixture, remove NH through 50-60 DEG C of water-bath 4hCO 3, after vacuum lyophilization, obtain heparin sodium six sugar mixture; Utilize SAX-HPLC, under 0-0.6MNaCl, 0.6-1.3MNaCl condition, carry out two-phase linear gradient elution, the sugared crude product of the Sulfated heparin sodium of separated and collected height six; After the sugared crude product of heparin sodium six collected is dialysed 3 days in ultrapure water, lyophilize;
2) preparation of heparin pyridine six sugar: at 4 DEG C, heparin sodium six sugar obtained in the dialysis of step 1) process also lyophilize through H type Zeo-karb, remove the sodium ion on sugar chain, it is 6-6.5 that the elutriant of gained is at room temperature neutralized to pH with pyridine; The elutriant lyophilize after neutralization, obtain the sugared dry product of heparin pyridine six;
3) containing GlcNH 3 +the preparation of Heparin Oligosaccharides and purifying: step 2) in obtain the sugared dry product of heparin pyridine six be dissolved in DMSO:H 2o(v/v) in=95:5 solvent, through 30 DEG C of water-bath 20min, the sulfate radical on heparin chain N position is optionally removed; After dialysis lyophilize, with SAX-HPLC, under 0-0.6MNaCl, 0.6-1.3MNaCl condition, carry out linear gradient elution, separated and collected is containing different GlcNH 3 +heparin six sugar of number; Different GlcNH is contained what collect 3 +heparin six sugar of number, dialysis also lyophilize respectively, what obtain purifying contains one, two and three GlcNH 3 +three kinds of heparin six sugar.
2. one kind contains nthe preparation of heparin six sugar of-non-substituted glucosamine and purification process thereof, is characterized in that: concrete steps are as follows:
1) preparation of heparin sodium six sugar and purification: adopt heparinase I, II, III in 37 DEG C of enzymolysis molecular weight be the low molecular weight heparin of 3-8kD, then by gained enzymolysis low molecular weight heparin through polyacrylamide gel chromatographic column, with 0.2MNH 4hCO 3for moving phase, and under UV232nm, measure the absorbancy of elutriant; Collect the elution peak of heparin sodium six sugar mixture, remove NH through 50-60 DEG C of water-bath 4hCO 3, after vacuum lyophilization, obtain heparin sodium six sugar mixture; Utilize SAX-HPLC, under 0-0.6MNaCl, 0.6-1.3MNaCl condition, carry out two-phase linear gradient elution, the sugared crude product of the Sulfated heparin sodium of separated and collected height six; After the sugared crude product of heparin sodium six collected is dialysed 3 days in ultrapure water, lyophilize;
2) preparation of heparin pyridine six sugar: at 4 DEG C, heparin sodium six sugar obtained in the dialysis of step 1) process also lyophilize through H type Zeo-karb, remove the sodium ion on sugar chain, it is 6-6.5 that the elutriant of gained is at room temperature neutralized to pH with pyridine; The elutriant lyophilize after neutralization, obtain the sugared dry product of heparin pyridine six;
3) containing GlcNH 3 +the preparation of Heparin Oligosaccharides and purifying: step 2) in obtain the sugared dry product of heparin pyridine six be dissolved in DMSO:H 2o(v/v) in=95:5 solvent, through 60 DEG C of water-bath 20min, the sulfate radical on heparin chain N position is optionally removed; After dialysis lyophilize, with SAX-HPLC, under 0-0.6MNaCl, 0.6-1.3MNaCl condition, carry out linear gradient elution, separated and collected is containing different GlcNH 3 +heparin six sugar of number; Different GlcNH is contained what collect 3 +heparin six sugar of number, dialysis also lyophilize respectively, what obtain purifying contains three GlcNH 3 +heparin six sugar.
CN201410019945.8A 2014-01-17 2014-01-17 Containing preparation and the purifying thereof of N-non-substituted glucosamine heparin six sugar Expired - Fee Related CN103724458B (en)

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