CN102060908B - Novel synthetic antithrombotic polypeptide, and preparation method and application thereof - Google Patents
Novel synthetic antithrombotic polypeptide, and preparation method and application thereof Download PDFInfo
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- CN102060908B CN102060908B CN 201010284849 CN201010284849A CN102060908B CN 102060908 B CN102060908 B CN 102060908B CN 201010284849 CN201010284849 CN 201010284849 CN 201010284849 A CN201010284849 A CN 201010284849A CN 102060908 B CN102060908 B CN 102060908B
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Abstract
The invention relates to the field of biomedicine, and discloses a synthetic polypeptide BNC (basal nucleus complex) with antithrombotic activity, a preparation method thereof and an application of the synthetic polypeptide BNC as an antithrombotic agent. The BNC is synthesized by using a Fomc solid-phase chemical process, and contains 3 amino acid residues; the sequence of the BNC is Pyr-Asn-Cys; the molecular weight of the BNC is 346; and the structural formula is shown in the specification. The animal experiment indicates that the polypeptide has inhibiting effects on the formation of thrombus after electrically stimulating the common carotid artery intima of a rat and the formation of ADP-induced (adenosine diphosphate-induced) acute lung thrombus of a mouse, and can prolong the coagulation time of the mouse. The structure formula of the BNC is shown in the specification.
Description
Technical field
The present invention relates to polypeptide, relate in particular to through the manually modified and synthetic polypeptide with antithrombotic acitivity.The invention still further relates to the preparation method of this polypeptide and as the application of antithrombotic agent, belong to biomedicine field.
Background technology
Along with the aging of population, the increase of cardiovascular disease incidence rate, existing antithrombotic standard drug can not adapt to and satisfy existing demand, and the market capacity of such medicine is still in continuous increase.Present antithrombotic reagent fail to satisfy active good, drug effect is measurable, can be oral, rapid-action, the requirement such as side effect is little.
Thereby, in view of larger and existing each defectiveness of medicine of the market opportunity, seek safety, novel medicine for treating thrombus thing has become the study hotspot of domestic and international the world of medicine easily, meanwhile, for the innovative research of this type of medicine, particularly polypeptide and small molecules class peptide that can be oral or the research of compound also just had more importantly meaning and market outlook.
Atherosclerosis is the main pathological basis of vascular system disease, and atherosclerotic plaque breaks and the thrombosis of secondary is the major cause that most of acute vascular events occur.Research in recent years shows, destruction and the reconstruct of breaking with extracellular matrix of atherosclerotic formation and patch have substantial connection, and matrix metalloproteinase (MMPs) is the Zn of gang
2+The dependent form proteolytic ferment mainly is responsible for the degradation of cell epimatrix, and increasing with active enhancing of its expression level can both be accelerated the formation of unstable spot and thrombus.Therefore, matrix metallo-proteinase inhibitor (MMPI) becomes a focus of pharmaceutical chemistry area research, and increasing research is attempted by suppressing the MMPs activity to delay atherosclerotic generation and to prevent plaque rupture, thrombosis.Wherein, matrix metalloprotease tissue depressant (TIMPs) belongs to the L-Cysteine HCL Anhydrous superfamily, be one group of multifunctional agents family among the MMPI, the activity of inhibition MMPs that can be special, its N end functional zone cysteine residues is combined with the zine ion active centre of MMPs, is combined with other positions of MMPs in C end functional zone, with 1: 1 ratio non covalent bond in conjunction with formation MMP-TIMP complex body, the combination of blocking-up MMPs and substrate suppresses the MMPs proteolytic activity.Therefore, the activity of regulating MMPs may be a new direction for the treatment of cardiovascular disease, particularly thrombus treatment, will promote clinical early application to the further research of its antagonist.
Summary of the invention
One of purpose of the present invention provides a kind of novel polypeptide BNC with good antithrombotic acitivity of synthetic, and its aminoacid sequence is Pyr-Asn-Cys.
According to the mentality of designing of matrix metallo-proteinase inhibitor (MMPI), based on the design theory of the MMPI of substrate, utilize the solid-phase polypeptide synthetic technology, novel antithrombotic polypeptide BNC has been finished in design, and its aminoacid sequence is Pyr-Asn-Cys, and molecular weight is 346.
Two of purpose of the present invention provides a kind of preparation method with novel polypeptide BNC of good antithrombotic acitivity.
According to designed BNC aminoacid sequence, utilize Fmoc solid-phase polypeptide synthetic technology to synthesize.First the carboxyl that will synthesize the C-terminal halfcystine of peptide chain is linked to each other with 2-chlorine trityl chloride resin with the ester bond form; then be combined in halfcystine on the solid phase carrier as amino component with this, carry out condensation and deprotection through the deaminize protecting group and with excessive activated carboxyl component.Repeat (condensation → wash → go the condensation of protection → washing → next round) and operate, reach the peptide chain length that will synthesize, utilize at last cutting liquid that peptide chain cracking from the resin is got off, namely get crude product.After carrying out purifying, the HPLC reversed-phase column proves conclusively by mass spectroscopy.
Above-described Fmoc solid-phase peptide synthesis is the conventional and known technology of this area.
The chmice acute lung thrombosis that electricity irritation rat carotid artery thrombosis, ADP bring out and the experimentation on animalies such as coagulation time test of mouse prove, polypeptide BNC of the present invention has stronger antithrombotic acitivity.
Three of purpose of the present invention is that novel polypeptide BNC is applied in the preparation of antithrombotic reagent.
Beneficial effect of the present invention is:
Mentality of designing according to matrix metallo-proteinase inhibitor (MMPI), design theory based on the MMPI of substrate, utilize the solid-phase polypeptide synthetic technology, that the novel antithrombotic polypeptide BNC that finishes of design has is simple in structure, synthetic convenient and the good beneficial features such as antithrombotic acitivity, is expected to become a kind of have application potential and antithrombotic polypeptide drugs safely and effectively.
Embodiment
The below further specifies essentiality content of the present invention with embodiment.Should be pointed out that these embodiment only are used for the present invention is specifically described, not should be understood to limitation of the present invention.
The explanation of the shortenings that occurs among the present invention:
Preparation and the separation and purification of embodiment 1 novel antithrombotic polypeptide BNC
Prepare BNC by following sequence.
The aminoacid sequence of BNC is: Pyrrolidonecarboxylic acid-l-asparagine-halfcystine.
It is synthetic that the present embodiment adopts the solid-phase polypeptide synthetic technology to carry out craft, and synthetic polypeptide obtains crude product after the TFA of high density cutting.After the HPLC reversed-phase column carries out purifying, prove conclusively by mass spectroscopy.Concrete experimental procedure is as follows:
1) preparation of BNC (method of solid-phase synthetic peptide is described as example take the BNC of preparation 1.323mmol amount)
Below resin, Fmoc protected amino acid and condensation reagent, the cutting reagent of preparation antithrombotic polypeptide BNC are all bought in the biochemical company limited of Shanghai gill.
Preparation holds the N end to carry out one by one from C.Take by weighing 1.01g (1.323mmol) 2-chlorine trityl chloride resin and put into small beaker, add DMF (not having resin) swelling 30min, after pour out half volume DMF add again swelling 30min of isopyknic DCM, pour afterwards the sand core funnel suction filtration into and do.Connect device, and pass into nitrogen, regulate making air-flow steady.Taking by weighing Fmoc-Cys (Trt)-OH 1.93g (3.31mmol) dissolves with DMF, afterwards in the adding apparatus, and add DIEA 1.2ml (7.69mmol), and then add cumulative volume that DMF makes reaction solution and be column volume 2～3 times, room temperature reaction 1h.After utilizing the ninhydrin detection reaction fully, the piperidines/DMF that adds part 20% reacts first 5min, adds afterwards remainder and reacts 15min (altogether 30ml) again, sloughs the Fmoc protecting group.Detect with ninhydrin, the result shows that deprotection reaction has carried out fully.The elimination reaction solution with DMF washing 4 times, adds water without muddiness to filtrate with resin, is filtered dry.Take by weighing the 2nd amino acid Fmoc-Asn (Trt)-OH1.97g (3.31mmol), other takes by weighing condensing agent HBTU 1.57g (3.80mmol), dissolve with DMF after mixing, consoluet mixed solution is joined in the sand core funnel that contains polypeptide resin afterwards, the flow velocity of regulating nitrogen makes it to be in the speed stabilizing state, add afterwards DIEA 1.2ml (7.69mmol), it is 2～3 times of column volumes that rear adding DMF makes the total amount of reaction solution, room temperature reaction 1h again.Detect with ninhydrin, the result shows that condensation reaction carried out fully.Reacted resin is filtered dry, washes 4 times with DMF, suction filtration is done, and the piperidines of adding 20%/DMF repeats the deprotection operation.The elimination reaction solution with DMF washing 6 times, adds water without muddiness to filtrate with resin, is filtered dry.Then take by weighing the 3rd amino acid Pyr 0.55g (4.26mmol), condensing agent HBTU1.57g, mix afterwards dissolve complete in DMF, add afterwards in the reaction unit, regulating nitrogen makes flow velocity steady, add afterwards DIEA 1.2ml (7.69mmol), it is 2～3 times of column volumes that rear adding DMF makes the total amount of reaction solution, room temperature reaction 1h again.Detect with ninhydrin, the result shows that condensation reaction carried out fully.Reacted resin is filtered dry, washes 4 times with DMF, anhydrous MeOH washes 1 time, and DCM washes 2 times, and anhydrous MeOH washes 2 times, and each washing is no less than 1min, and suction filtration is done afterwards.
After resin transfer, add cutting liquid TFA: TIS: EDT: H
2O (9.4: 0.1: 0.25: 0.25) stir 30min.Suction filtration, the ice ether of 10 times of volumes of adding fully leaves standstill 30min after the concussion in filtrate.Drain, vacuum-drying namely gets the thick peptide of BNC.
2) purifying of BNC
The above-mentioned thick peptide of 200mg is dissolved in the 50ml pure water, with preparation type reversed-phase HPLC purifying.Chromatographic column is Delta-PakC1825 * 200mm; Moving phase is A liquid: 5% methyl alcohol+aqueous phase 0.05% trifluoroacetic acid, B liquid: 95% methyl alcohol+aqueous phase 0.05% trifluoroacetic acid, B liquid take 70% is the moving phase isocratic elution, flow velocity is 5ml/min, detecting wavelength is 280nm and 214nm, and the main peak retention time is about 17min, and is proper, separating effect is better, is fit to a large amount of preparations of sample.After collecting main peak peak nose part, minute detect being gathered into the analysis mode reversed-phase HPLC, chromatographic column is the global post in Suzhou, moving phase is the same, and elution program is 49%～54%B liquid 20min, and flow velocity is lml/min, detect wavelength 214nm, the composition of collection reaches purity requirement.Collect liquid concentrated through rotary evaporation, then lyophilize obtains the pure peptide of BNC.The mass spectrum of pure peptide shows, its molecular weight is 346, conforms to calculated value.
Embodiment 2BNC on electricity irritation rat carotid artery inner membrance after thrombotic impact
1) preparation of sample solution
Positive controls becomes clopidogrel the liquid (2mg/ml) of desired concn before use with normal saline dilution; The BNC group is dissolved with the 0.2%L-arginine before use, and becomes the liquid (2mg/ml) of desired concn with normal saline dilution.Each organizes equal tail intravenously administrable, and volume is 10ml/kg.
2) preparation of animal model
Rats by intraperitoneal injection 10mg/L Sodital sodium solution (30mg/kg), dorsal position is fixed after the anesthesia.Neck median line otch, free right carotid, proximal part is placed the stimulating electrode of BT87-3 type instrument for detecting internal thrombosis, distal end laying temperature probe.After the stimulating electrode making current, with 2mA galvanic current stimulation artery 7min, make the blood vessel endothelium of stimulation location impaired, activate thrombocyte and blood coagulation system, the tube chamber mixed thrombus forms gradually, and blood flow is blocked gradually, when blood flow is blocked fully, far-end temperature bust, instrument is reported to the police, and shows duration of congestion (t
Stop up), i.e. thrombus formation time.
3) experimental procedure
Get 32 of Wistar rats, body weight 200～250g, male and female half and half are divided into 4 groups at random by body weight, and 8 every group, i.e. blank group, model control group, positive controls (dosage is 20mg/kg), BNC organizes (dosage is 20mg/kg).Adopt the tail vein injection administration, the administration volume is 10ml/kg, and 15min begins to stimulate after the administration.The blank group gives the 0.2%L-arginine, and model control group gives physiological saline, and positive controls gives clopidogrel.
4) statistical procedures
Adopt the Excel software data processing.All data all are expressed as mean ± standard deviation
Expression relatively with the t check, the results are shown in Table 1 between two groups.
Each group of table 1 is on thrombotic impact behind the electricity irritation rat carotid artery inner membrance
Statistical method: t check; 1. compare with model control group P＜0.01; 2. compare with positive controls P＞0.05.
Can find out from the data of table 1, the positive controls of Isodose (20mg/kg) has been compared significant differences with the BNC group with model group, and BNC group and positive controls there was no significant difference, therefore, judge that BNC can effectively suppress the formation of thrombus behind the electricity irritation rat carotid artery inner membrance.
The thrombotic impact of chmice acute lung that embodiment 3BNC brings out ADP
1) preparation of sample solution
Positive controls becomes clopidogrel the liquid (dosage is as 4mg/kg) of desired concn before use take normal saline dilution; The BNC group becomes the liquid (dosage is as 4mg/kg) of desired concn before use take normal saline dilution; Inductor ADP solution is diluted to desired concn (200mg/kg) with pure water before use.Each organizes equal tail intravenously administrable, and volume is 10ml/kg.
2) experimental procedure
Get 24 of healthy ICR mouse, body weight 20～25g, male and female half and half are divided into 3 groups at random by body weight, and 8 every group, i.e. model control group, positive controls, BNC group.Each organizes equal tail intravenously administrable, and volume is 10ml/kg, and model control group gives isometric physiological saline, and positive controls gives clopidogrel.15min after the administration (behind the positive controls administration 2h), press 200mg/kg tail vein injection ADP solution, form acute lung thrombus (mouse breathing syndrome characterized by dyspnea, can not autonomic activities), behind the record injection ADP to time (min) that mouse recovers autonomic activities.
3) statistical procedures
Adopt the Excel software data processing.All data all are expressed as mean ± standard deviation
Expression relatively with the t check, the results are shown in Table 2 between two groups.
Each group of table 2 is brought out the thrombotic impact of chmice acute lung to ADP
Statistical method: t check; 1. compare with model control group P＜0.01; 2. compare with positive controls P＞0.05.
Can find out from the data of table 2, the positive controls of Isodose (4mg/kg) has been compared significant differences with the BNC group with model group, and BNC group and positive controls there was no significant difference, therefore, judge that BNC can effectively accelerate mouse and recover the autonomic activities time, thus the formation of the chmice acute lung thrombus that alleviation ADP brings out.
Embodiment 4BNC is on the impact of Mice Body intravascular coagulation time
1) preparation of sample solution
Positive controls becomes clopidogrel the liquid (dosage is as 4mg/kg) of desired concn before use take normal saline dilution; The BNC group becomes the liquid (dosage is as 4mg/kg) of desired concn before use take normal saline dilution; Each organizes equal tail intravenously administrable, and volume is 10ml/kg.
2) experimental procedure
Get 24 of healthy ICR mouse, body weight 20～25g, male and female half and half are divided into 3 groups at random by body weight, and 8 every group, i.e. model control group, positive controls, BNC group.Each organizes equal tail intravenously administrable, volume is 10ml/kg, 1 time/d, continuous 4d behind the last administration 15min (administration of positive controls last is after 2 hours), inserts glass capillary blood is flowed out voluntarily, wiping the 1st with dry cotton ball bleeds, respectively drop of blood is dripped in the two ends of cleaning slide glass, drop of blood diameter 5～10mm begins timing immediately again.After this every 30s, provoke blood 1 time with dry syringe needle, can provoke fiber protein yarn to syringe needle till, be the clotting time, 1 bleed for last retrial in addition.Model control group gives isometric physiological saline, and positive controls gives clopidogrel.
3) statistical procedures
Adopt the Excel software data processing.All data all are expressed as mean ± standard deviation
Expression relatively with the t check, the results are shown in Table 3 between two groups.
Each group of table 3 is on the impact of Mice Body intravascular coagulation time
Statistical method: t check; 1. compare with model control group P＜0.05; 2. compare with positive controls P＞0.05.
The result shows: 2 groups of positive control clopidogrel group, 1 group of compound and compounds can prolong the Mice Body intravascular coagulation time significantly under same concentration, tentatively judge to have anti thrombotic action.And the positive with dosage has been compared significant difference with sample sets with the blank group, and sample sets and positive group there was no significant difference.
Can find out from the data of table 3, the positive controls of Isodose (4mg/kg) has been compared significant difference with the BNC group with the blank group, and BNC group and positive controls there was no significant difference, therefore, judge that BNC can effectively prolong clotting time of mice.
Claims (3)
1. polypeptide BNC with antithrombotic acitivity, its aminoacid sequence is Pyr-Asn-Cys, molecular weight is 346.
2. a method for preparing claim 1 polypeptide is characterized in that, described preparation method is the Fmoc solid-phase synthesis, comprising:
(1) take by weighing 2-chlorine trityl chloride resin and put into small beaker, add DMF swelling 30min, after pour out half volume DMF add again swelling 30min of isopyknic DCM, pour afterwards the sand core funnel suction filtration into and do;
(2) on resin, hold the N end to carry out one by one coupling and deprotection by design sequence in advance from C through the amino acid of protection, utilize nitrogen gas stirring in the reaction process;
(3) synthetic polypeptide is through cutting liquid TFA: TIS: EDT: H
2O=9.4: behind 0.1: 0.25: 0.25 shearing 30min, obtain the polypeptide crude product.
3. the polypeptide of claim 1 is in the purposes of preparation in the antithrombotic reagent.
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| CN103113456B (en) * | 2013-03-05 | 2014-07-16 | 中国药科大学 | Stiff silkworm polypeptide with antiplatelet aggregation activity as well as preparation method and application of stiff silkworm polypeptide |
| CN105254710A (en) * | 2015-11-26 | 2016-01-20 | 句容苏南生物科技有限公司 | Liquid phase synthesis method of antithrombotic compound BNW |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101058572A (en) * | 2007-03-13 | 2007-10-24 | 中国药科大学 | Peptides compound with antineoplastic activity |
| CN101239178A (en) * | 2008-03-13 | 2008-08-13 | 中国药科大学 | Antithrombotic use of peptide compounds |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101058572A (en) * | 2007-03-13 | 2007-10-24 | 中国药科大学 | Peptides compound with antineoplastic activity |
| CN101239178A (en) * | 2008-03-13 | 2008-08-13 | 中国药科大学 | Antithrombotic use of peptide compounds |
Non-Patent Citations (1)
| Title |
|---|
| 田垒等.新的抗凝血三肽及其改造物的合成和生物活性.《西北药学杂志》.2009,第24卷(第03期), * |
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