CN102382178B - Separation method of sialoglycopeptide - Google Patents
Separation method of sialoglycopeptide Download PDFInfo
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- CN102382178B CN102382178B CN 201110322663 CN201110322663A CN102382178B CN 102382178 B CN102382178 B CN 102382178B CN 201110322663 CN201110322663 CN 201110322663 CN 201110322663 A CN201110322663 A CN 201110322663A CN 102382178 B CN102382178 B CN 102382178B
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- elutriant
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Abstract
The invention discloses a separation method of sialoglycopeptide, including the following steps: (1) taking a fresh egg yolk, treating the fresh egg yolk with phenol and then conducting rotation evaporation to obtain a sample containing the sialoglycopeptide; (2) separating the sample with sephadex G-50 and collecting eluent; (3) concentrating the eluent, absorbing the concentrated sample with active carbon and then eluting the sample in acetonitrile solution to obtain eluent containing the sialoglycopeptide; and (4) freezing and drying the eluent to obtain pure sialoglycopeptide. In the method, the steps of G-25 desalting, HPLC (High Performance Liquid Chromatography) purification, ion exchange chromatography purification and the like of the existing method are eliminated, therefore the purification period is shortened greatly, the efficiency is improved, the cost is saved and inspected by HPLC, and the purity of the prepared sialoglycopeptide can achieve 99%. The separation method is used for not only laboratories but also small-scale industrial production.
Description
Technical field
The present invention relates to a kind of separation purification method of glycopeptide compound, relate in particular to a kind of separation purification method with natural complexity N sugar chain glycopeptide such as sialoglycopeptide.
Background technology
Sialoglycopeptide (Sialylglycopeptides) structure is:
Sialoglycopeptide (Sialylglycopeptides) is a kind of glycopeptide with complete natural complexity N sugar chain, and relative molecular mass is 2866.Although can synthesize multiple oligosaccharides by chemical method now, present technology still can't be synthesized this complete sialylated N-glucosides.Therefore, can only be from natural product this sialoglycopeptide of separation and purification.
At present, in the world from egg yolk the method for separation and purification sialoglycopeptide mainly be that the purification process of the Japanese AkiraSeko report take 1997 is as main.After the method is processed yolk first, separate through twice sephadex G-50, again through the G-25 desalination, DEAE-Toyopearl 650M ion-exchange column purification, then after CM-Sephadex C-25 separated, freeze-drying obtained sample.But the aforesaid method step is many, and the cycle is long, and sample loss is serious in purge process.Further improvement had once been carried out to purification process in the contriver laboratory, proposed to use gac and high performance liquid chromatography (HPLC) to carry out purifying.Although the method has been simplified some steps, need still could realize through a plurality of steps that when particularly using the HPLC purifying, because applied sample amount is few, productive rate is low, cost is high, therefore generally is merely able to for a small amount of separation in laboratory.
Because the complicacy of glycoprotein candy chain so that the research of the structure of sugar chain and function is biological and the difficult point of field of medicaments research always, with the albumen of homogeneous sugar chain, is absolutely necessary for its research.And sialoglycopeptide is a kind of humanized glycopeptide, can be used as the humanization modified glucosides donor of glycoprotein.Therefore, it is low to invent a kind of cost, and the cycle is short, and step is few, and the separation method that efficient is high is very urgent and necessary.
Summary of the invention
For the deficiency that the separation method step is many, the cycle is long, cost is high that exists in the prior art, the purpose of this invention is to provide the separation purification method of the sialoglycopeptide that a kind of cycle is short, cost is low, efficient is high.
The separation method of sialoglycopeptide of the present invention comprises 1) get Fresh Egg yolk, after processing through phenol, obtain containing the sialoglycopeptide sample behind the rotary evaporation; 2) separate with sephadex G-50 pair upper step sample, collect elutriant; 3) concentrated to elutriant, with gac concentrating sample is adsorbed, obtain to contain again the elutriant of sialoglycopeptide with the acetonitrile solution wash-out; 4) the elutriant lyophilize is obtained the sterling of sialoglycopeptide;
Wherein:
1) the described method that Fresh Egg yolk is processed is: get first fresh unfertilized egg yolk, add the dilution of isopyknic deionized water, add again its 1/5 volume phenol/water (phenol: water volume ratio 9: 1), 4 ℃ of stirrings; Add again the deionized water of two volumes in the mixed solution, centrifugal behind the mixing, supernatant liquor is concentrated with Rotary Evaporators, obtain containing the sample of sialoglycopeptide.
2) describedly carried out separation method with sample of upper step of sephadex G-50 pair and be: sephadex G-50 post is put into 23-27 ℃ thermostat container, take 1.6cm * 100cm sephadex G-50 post, when flow velocity carries out as 1ml/min, collect the elutriant of 110-140 milliliter;
3) described concentrated to elutriant, with gac concentrating sample to be adsorbed, the method that contains the elutriant of sialoglycopeptide with the acquisition of acetonitrile solution wash-out again is:
The elutriant of collecting is put into beaker, again beaker is placed-80 ℃ freezing, then freezing rear sample is put into vacuum freeze drier and carries out lyophilize, the dried frozen aquatic products that obtains containing sialoglycopeptide is dry powder;
The freeze-drying sample is made into the solution that concentration is the 50-150 mg/ml, the ratio that adds sample 400-500 microlitre in per 500 milligrams of gacs, sample is added in the activated carbon column, absorption kept 30-40 minute, then the 3-5 ml pure water is added in the activated carbon column, the flushing gac is removed salt and impurity, 3-5 milliliter 20-40% acetonitrile solution is added in the activated carbon column again, and wash-out obtains containing the elutriant of sialoglycopeptide.
4) describedly with the cryodesiccated method of elutriant be: elutriant is put into container, volume does not surpass 1/2 of this container, container is placed-80 ℃ freezing, more freezing rear sample is put into vacuum freeze drier and carries out lyophilize, obtain at last the sterling of sialoglycopeptide.
In the separation method of above-mentioned sialoglycopeptide, describedly carried out sephadex G in the separation method-50 post with sample of upper step of sephadex G-50 pair and preferably put into 25 ℃ thermostat container.
In the separation method of above-mentioned sialoglycopeptide, step 3) described concentrated sample is made into the solution that concentration is preferably the 100-120 mg/ml.
In the separation method of above-mentioned sialoglycopeptide, step 3) Supelclean of the preferred SUPELCO of described activated carbon column
TMENVI-Carb
TMSPE Tube.
In the separation method of above-mentioned sialoglycopeptide, step 3) described acetonitrile solution concentration is preferably 30% in volume ratio.
Sialoglycopeptide is carried out Structural Identification and purity testing:
Utilize mass spectrum and nucleus magnetic resonance sample to be detected the result: molecular weight analyte and theoretical value are coincide, and nuclear magnetic spectrum and reference result coincide.
Utilize HPLC test sample purity, the result: substantially free of impurities, purity reaches more than 99%.
Above-mentioned during with sephadex G-50 purifying, use temperature is 23 ℃-27 ℃, is because temperature variation can make experimental result unstable, and too high or too low temperature all can't realize best separating effect.Therefore our preferred temperature is 25 ℃, and under this temperature, separating effect is stable, efficient.
The present invention substitutes traditional Resorcinol salt acid system with UV-detector and detects sialoglycopeptide.By finding that with traditional chemical method contrast 214nm test sample effect is basically identical with traditional method.This step has been saved loaded down with trivial details detecting step, has reduced simultaneously the sample loss that causes because of the chemical method detection.
In the separation method of above-mentioned sialoglycopeptide, during described collection sephadex G-50 elutriant, preferably collect the 110-140 ml sample, if be because will contain the elutriant of sialic acid sample all collects, can cause sample purity to descend; If only collect the highest elutriant of samples contg, then cause the sample waste, experiment screening confirms: collect 110-140 milliliter elutriant, both guaranteed the purity of sample, at utmost reduce again the loss of sample.
In the above-mentioned activated carbon purification method, sample concentration is made into the 50-150 mg/ml, if be because sample concentration is too high, then exceeds activated carbon adsorptive capacity, the waste sample; If sample concentration is excessively low, then cause separation efficiency to descend, lose time and reagent.The preferred 100-120 mg/ml of sample concentration among the present invention neither causes the sample waste to improve again separation efficiency, has saved cost.
In the above-mentioned activated carbon purification method, use 20-40% acetonitrile solution wash-out sialoglycopeptide, if be because acetonitrile concentration is too high, many impurity are together eluted, cause sample purity to descend; If acetonitrile concentration is excessively low, then can't the wash-out sialoglycopeptide.Experiment screening confirms: acetonitrile solution concentration preferred 30% is most economical.
Sialoglycopeptide purification process of the present invention is mainly through pre-treatment, sephadex G-50 separation, three steps of activated carbon purification, the steps such as the G-25 desalination in the previous methods, HPLC purifying, ion-exchange chromatogram purification have been omitted, simultaneously, condition to each purification process is groped, obtain the optimum condition of sialoglycopeptide separation and purification, make purification process more stable, efficient, and do not affect product purity, thereby greatly shortened the purifying cycle, improve purification efficiency, saved cost.The inventive method not only can be used for the laboratory preparation, also can be used for small-scale industrial production, for sialoglycopeptide purifying or exploitation provide again a kind of new technology.
Description of drawings
Fig. 1: fast protein liquid chromatography detects sephadex G-50 elutriant collecting region.
Fig. 2: mass spectrum (ESI-MS) detects sialoglycopeptide.
Wherein: positive ion mode detects sialoglycopeptide, and 956.7 is sample tricharged peak, and is identical with the molecular weight of sialoglycopeptide 2866.
Fig. 3: 300MHz
1H-NMR detects sialoglycopeptide.
Wherein: after NMR collection of illustrative plates and bibliographical information result compared, structure was coincide.
Fig. 4: HPLC detects the sialoglycopeptide sample.
Wherein: pillar: reverse C18 post; Moving phase: 10% acetonitrile; Flow velocity: 0.5ml/min; Detect wavelength: 214nm.
Embodiment
By the following examples content of the present invention is described in further detail.But this should be interpreted as that the scope of the above-mentioned theme of the present invention only limits to following example.Do not breaking away from the above-mentioned technological thought situation of the present invention, various replacements of making according to ordinary skill knowledge and customary means or more become all should comprise within the scope of the invention.
Embodiment 1
1, egg yolk pre-treatment
At first get 100 fresh unfertilized egg yolks (1.9L), add the dilution of isopyknic deionized water, (phenol: water volume ratio 9: 1), 4 ℃ continue to stir 3 hours to add the phenol/water of its 1/5 volume (380ml) again.Add the 4.8L deionized water in mixed solution, centrifugal behind the mixing (10000g, 25 minutes) are concentrated into 50 milliliters with supernatant liquor with Rotary Evaporators, obtain containing the sample of sialoglycopeptide.
2, sephadex G-50 separates
(1.6cm * 100cm) be connected with the fast liquid chromatography instrument uses 0.1M NaCl as elutriant, flow velocity: 1ml/min, ultraviolet detection wavelength: 214nm to sephadex G-50 post.Pillar is positioned in the incubator 25 ℃ of temperature.Add the described sample 2ml of step 1, collect elutriant 110-140 ml sample, as shown in Figure 1, collect the sample that is labeled as " collecting region ".
3, concentrated to elutriant, and use the activated carbon column desalting and purifying
50 milliliters of elutriants are put into 100 ml beakers, again beaker is placed-80 ℃ freezing 1 hour, then freezing rear sample is put into vacuum freeze drier and carries out lyophilize after 12 hours, the dried frozen aquatic products that obtains containing sialoglycopeptide is dry powder.
Dried frozen aquatic products is made into the solution that concentration is 100 mg/ml, for subsequent use;
Activated carbon column (Supelclean
TMENVI-Carb
TMSPE Tube, SUPELCO) use before with 5 ml methanol cleaning active charcoal posts, then use pure water balance pillar.
The ratio that adds sample 500 microlitres in per 500 milligrams of gacs, the sample for subsequent use of above-mentioned preparation is added in the activated carbon column, absorption kept 40 minutes, then 5 ml pure waters are added in the activated carbon column, the flushing gac is removed salt and impurity, 5 milliliter of 30% acetonitrile solution added in the activated carbon column, wash-out obtains containing the elutriant of sialoglycopeptide again.
4, lyophilize obtains the gac sterling.
50 milliliters of elutriants are put into 100 ml beakers, beaker is placed-80 ℃ freezing 1 hour, more freezing rear sample is put into vacuum freeze drier and carries out lyophilize, after dry 12 hours, obtain the sterling of sialoglycopeptide.
5, Structural Identification and purity testing.
Utilize mass spectrum (result as shown in Figure 2) and nucleus magnetic resonance (result as shown in Figure 3) that sample is detected, molecular weight analyte and structure and theoretical value are coincide.Utilize HPLC test sample purity (result is as shown in Figure 4), substantially free of impurities, purity is more than 99%.
Use aforesaid method, the applicant is from 100 egg yolks, and separation and purification has gone out the sialoglycopeptide of 640mg purity 99%, only one month consuming time.
Embodiment 2
1, egg yolk pre-treatment
At first get 100 fresh unfertilized egg yolks (1.9L), add isopyknic deionized water dilution, add phenol/water (volume ratio 9: 1) of 1/5 volume (380ml) again, 4 ℃ continue to stir 3 hours.Add the 4.8L deionized water in mixed solution, centrifugal behind the mixing (10000g, 25 minutes) are concentrated into 50 milliliters with supernatant liquor with Rotary Evaporators, obtain containing the sample of sialoglycopeptide.
2, sephadex G-50 separates
(1.6cm * 100cm) be connected with the fast liquid chromatography instrument uses 0.1M NaCl as elutriant, flow velocity: 1ml/min, ultraviolet detection wavelength: 214nm to sephadex G-50 post.Pillar is positioned in the incubator 23 ℃ of temperature.Add above-mentioned 1 described sample 2.5ml, collect elutriant 110-140 ml sample.
3, concentrated to elutriant, and use the activated carbon column desalting and purifying
50 milliliters of elutriants are put into 100 ml beakers, again beaker is placed-80 ℃ freezing 1 hour, then freezing rear sample is put into vacuum freeze drier and carries out lyophilize after 12 hours, the dried frozen aquatic products that obtains containing sialoglycopeptide is dry powder.
Dried frozen aquatic products is made into the solution that concentration is 120 mg/ml, for subsequent use;
Activated carbon column (Supelclean
TMENVI-Carb
TMSPE Tube, SUPELCO) use before with 5 ml methanol cleaning active charcoal posts, then use pure water balance pillar.
The ratio that adds sample 400 microlitres in per 500 milligrams of gacs, above-mentioned sample for subsequent use is added in the activated carbon column, absorption kept 30 minutes, then 4 ml pure waters are added in the activated carbon column, the flushing gac is removed salt and impurity, 4 milliliter of 25% acetonitrile solution added in the activated carbon column, wash-out obtains containing the elutriant of sialoglycopeptide again.
4, lyophilize obtains the gac sterling.
50 milliliters of elutriants are put into 100 ml beakers, again beaker is placed-80 ℃ freezing 1 hour, freezing rear sample is put into vacuum freeze drier carries out lyophilize, after dry 12 hours, obtain the sterling of sialoglycopeptide.
5, Structural Identification and purity testing.
Utilize mass spectrum and nucleus magnetic resonance that sample is detected, results sample molecular weight and structure and theoretical value are coincide.
Utilize HPLC test sample purity, substantially free of impurities as a result, purity is more than 99%.
Claims (4)
1. the separation method of a sialoglycopeptide comprises 1) get Fresh Egg yolk, after processing through phenol, obtain containing the sialoglycopeptide sample behind the rotary evaporation; 2) separate with sephadex G-50 pair upper step sample, collect elutriant; 3) concentrated to elutriant, with gac concentrating sample is adsorbed, obtain to contain again the elutriant of sialoglycopeptide with the acetonitrile solution wash-out; 4) the elutriant lyophilize that step 3) is obtained obtains the sterling of sialoglycopeptide; It is characterized in that, 2) describedly carried out separation method with sample of upper step of sephadex G-50 pair and be: sephadex G-50 post is put into 23-27 ℃ thermostat container, take 1.6 ㎝ * 100 ㎝ sephadex G-50 posts, when flow velocity carries out as 1ml/min, collect the elutriant of 110-140 milliliter; 3) describedly to the concentrated method of elutriant be: the elutriant of collecting is put into beaker, again beaker is placed-80 ℃ freezing, then freezing rear sample is put into vacuum freeze drier and carries out lyophilize, the dried frozen aquatic products that obtains containing sialoglycopeptide is dry powder; Describedly with gac concentrating sample is adsorbed, the method that obtains to contain the elutriant of sialoglycopeptide with the acetonitrile solution wash-out again is: the freeze-drying sample that will contain first sialoglycopeptide is made into the solution that concentration is the 50-150 mg/ml, the ratio that adds sample 400-500 microlitre in per 500 milligrams of gacs, sample is added in the activated carbon column, absorption kept 30-40 minute, then the 3-5 ml pure water is added in the activated carbon column, the flushing gac is removed salt and impurity, again 3-5 milliliter 20-40% acetonitrile solution is added wash-out gac in the activated carbon column, obtain containing the elutriant of sialoglycopeptide;
Wherein, the described activated carbon column of step 3) selects the Supelclean of SUPELCO
TMENVI-Carb
TMSPE Tube.
2. the separation method of sialoglycopeptide as claimed in claim 1 is characterized in that, describedly carries out sephadex G in the separation method-50 post with sephadex G-50 pair upper step sample and puts into 25 ℃ thermostat container.
3. the separation method of sialoglycopeptide as claimed in claim 1 is characterized in that the described freeze-drying sample that contains sialoglycopeptide of step 3) is made into the solution that concentration is the 100-120 mg/ml.
4. the separation method of sialoglycopeptide as claimed in claim 1 is characterized in that the described acetonitrile solution concentration of step 3) counts 30% with volume ratio.
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| CN108107141B (en) * | 2017-12-19 | 2020-07-14 | 浙江丰安生物制药有限公司 | Method for extracting polypeptide in spleen aminopeptide |
| CN110373443A (en) * | 2018-04-13 | 2019-10-25 | 中国科学院大连化学物理研究所 | A kind of pilose antler glycopeptide and preparation and application |
| CN109280081B (en) * | 2018-10-26 | 2021-06-25 | 浙江海洋大学 | Method for preparing sialoglycopeptide from tuna eggs |
| CN109824762B (en) * | 2019-01-23 | 2022-11-22 | 西北大学 | Method for preparing Sialoglycopeptide (SGP) through large-scale separation and purification |
| CN117003827B (en) * | 2023-09-27 | 2023-12-08 | 山东睿鹰制药集团有限公司 | Separation and purification method of sialic acid glycopeptide |
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| CN1543471A (en) * | 2001-06-19 | 2004-11-03 | ��V��ѧ��ʽ���� | Process for producing sugar chain asparagine derivative |
| WO2011027868A1 (en) * | 2009-09-03 | 2011-03-10 | 公益財団法人野口研究所 | Method for producing 11-sugar sialyloligosaccharide-peptide |
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