CN101161672A - Method for preparing arginine-glycine-aspartic acid cell adhesion tripeptide - Google Patents
Method for preparing arginine-glycine-aspartic acid cell adhesion tripeptide Download PDFInfo
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- CN101161672A CN101161672A CNA2007100563603A CN200710056360A CN101161672A CN 101161672 A CN101161672 A CN 101161672A CN A2007100563603 A CNA2007100563603 A CN A2007100563603A CN 200710056360 A CN200710056360 A CN 200710056360A CN 101161672 A CN101161672 A CN 101161672A
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Abstract
The present invention belongs to biochemistry field, in particular to a preparation method of cellular adhesion tripeptide of arginine-glycin-aspartic acid, which is an active peptide substance. The method mainly includes the procedures of the synthesis of a dipeptide and a tripeptide, and needs not the elimination of the protective base. Aspartic acid is adopted as the raw material, and three steps are involved, including: chloroacetyl chloride reacts with aspartic acid to obtain chloroacetyl aspartic acid, which undergoes amonolysis to obtain a dipeptide of glycin-aspartic acid, then benzyloxycarbonyl aspartic acid (Z-Arg-OH) reacts with phosphoric tribromide to obtain N-carboxyl-acid-inner anhydride-aspartic acid (NCA-Arg), which reacts with the dipeptide of glycin-aspartic acid to obtain the tripeptide of arginine-glycin-aspartic acid. The present invention has the advantages of simplified techics, lowered cost, decreased man-hour, excellent effect of safe manipulation, and capability to prepare the product of the tripeptide of arginine-glycin-aspartic acid with high purity and high activity.
Description
Technical field
The invention belongs to biochemical field, be specifically related to a kind of preparation method of bioactive peptide material arginine-glycine-aspartic acid cell adhesion tripeptide.
Background technology
Fibronectin in extracellular matrix and the basilar membrane (Fn), vitreum connect aminoacid sequence-arginine-glycine-aspartic acid tripeptide sequence that albumen (VN), collagen protein (Collagen), von Wilebrand factor (vWF) etc. all contain a high conservative.The Pierschbacherherhe in american cancer research centre and Puoslahti found and had identified the cell adhesion effect of this tripeptides first in 1984.The arginine-glycine-aspartic acid tripeptide sequence is the main recognition site of integrin, and the cell adhesion factor that integrin is a class to play a significant role in cell signalling, so thereby this tripeptide sequence can be regulated by pair cell that integrin is discerned by analog cell epimatrix composition.
Arginine-glycine-aspartic acid tripeptide sequence function in vivo is a lot.Ln comes across in the early embryo, and for keeping the iuntercellular adhesion, the polarity of cell and the differentiation of cell are all significant.Ln is also relevant with the transfer of tumour cell.And ln combines with integrin on the cytolemma by this tripeptide sequence just, thereby plays a significant role in fetal development and tissue differentiation.
In the fibronectin in extracellular matrix, the arginine-glycine-aspartic acid tripeptide sequence is that it is by the minimal structure unit of cell recognition.Its acceptor also is one of integrin family member.The function of fibronectin mainly is the mediated cell adhesion.By adhesion, fibronectin can promote cell to sprawl by the tissue that cellular signal transduction pathways is regulated the shape of cell and cytoskeleton.In embryo development procedure, fibronectin is essential for the cell migration and the differentiation of many types.In trauma repair, fibronectin promotes scavenger cell and other immunocytes to move to damaged part.In the blood clot forming process, fibronectin promotes that thrombocyte is attached to the vascular injury position.The antibody of microinjection fibronectin receptor or the small peptide that contains this tripeptide sequence can be blocked the migration of cell.
The arginine-glycine-aspartic acid tripeptide sequence combines with the integrin receptor on tumour cell and newborn interior cutaneous vessel surface thereof, participates in the impregnation process of tumour cell.Adhesion is the initiating step of cancer cells invasion and attack.Tumour cell adheres to basilar membrane and extracellular matrix components by the film surface receptor.The mutual adhesion of the multiple composition of tumour cell and extracellular matrix (as ln, fiber adhesion albumen etc.) is the important step of metastases process, and this type of peptide has limited adhesion and the transfer of tumour cell to extracellular matrix.The aminoacid sequence of this peptide class of synthetic combines with the integrin receptor of tumor cell surface competitively, suppresses the adhesion between cell and the matrix, reaches the purpose that suppresses tumor cell invasion, transfer.In recent years, find that also this tripeptides has good effect at aspects such as wound healing, neurotization, skin repair.
In sum, the arginine-glycine-aspartic acid tripeptide sequence is a common basal component in multiple biomass cells epimatrix and the plasma proteins structure, it also is the fundamental unit that extensively is present in the iuntercellular recognition system, can combine with cell surface integrin specificity, thereby mediate many important vital movements.Exogenous arginine-glycine-aspartic acid three Toplink and body include the material competition integrin binding of this tripeptide sequence, thereby have a series of biological functions.
There has been enzyme process method synthetic, that chemical method synthesizes, chemical method combines with enzyme process to synthesize respectively for the synthetic of arginine-glycine-aspartic acid tripeptides.
Summary of the invention
The preparation method who the purpose of this invention is to provide the arginine-glycine-aspartic acid cell adhesion tripeptide product of a kind of work simplification, cost reduction, minimizing in man-hour, operational safety.
We have selected liquid chemical method to synthesize the arginine-glycine-aspartic acid tripeptides.Used chemical method is not that the peptide that carries out of the classical amino acid with the band protecting group is synthetic, but utilizes the cheap amino acid that does not have protecting group or the amino acid only protected with carbobenzoxy-(Cbz) synthesizes the arginine-glycine-aspartic acid tripeptides.Why we select this method to compare with additive method because of this method, and expense is lower, reaction is simple, efficient also is very high.
The present invention is a kind of liquid phase chemical synthesis method, and process mainly contains steps such as dipeptides is synthetic, tripeptides is synthetic, does not need to remove protecting group in the production.Be to be raw material with the aspartic acid, the reaction by chloroacetyl chloride and aspartic acid obtains the chloroacetylation aspartic acid, and chloroacetylation aspartic acid ammonia is separated and obtained the Gly-Asp dipeptides; Carbobenzoxy-(Cbz) arginine (Z-Arg-OH) obtains nitrogen carboxyl inner-acid anhydride arginine (NCA-Arg) with the phosphorus tribromide reaction; Nitrogen carboxyl inner-acid anhydride arginine (NCA-Arg) obtains the arginine-glycine-aspartic acid tripeptides with Gly-Asp two reactive polypeptides.Use the described method of this patent, can also synthesize other dipeptides, tripeptides or polypeptide products.
Concrete processing step is as follows:
(1) getting 25 grams～35 gram L-aspartic acids, to be suspended in 40～60 milliliters, mass concentration be in 17%～27% concentration hydrogen aqueous solution of sodium oxide it to be dissolved fully, between this concentration hydrogen aqueous solution of sodium oxide adjust pH 8～12, add 30～60 milliliters of ethyl acetate then; Under 0 ℃～10 ℃ condition, drip 15～30 milliliters of 10～13mol/l chloroacetyl chlorides and 15～40 milliliter of 17%～27% aqueous sodium hydroxide solution respectively in above-mentioned solution with the speed of 0.10～0.20ml/min, stir while dripping, control aqueous sodium hydroxide solution rate of addition keeps the pH of solution in 8～12 scopes in the dropping process, dropwise, continue to stir 20～50 minutes; Transfer below the PH to 3 with concentrated hydrochloric acid when finishing reaction, with ethyl acetate extracting three times of this reaction solution, united extraction liquid adds anhydrous sodium sulfate drying then, and ethyl acetate is removed in decompression, promptly obtains chloroacetylation aspartic acid white powder;
(2) under 0 ℃～10 ℃ cold condition, it is 25%～28% strong aqua that resulting chloroacetylation aspartic acid is added mass concentration according to the ratio of 1.0g: 5～10ml, stirred 24～48 hours, unnecessary ammonia is removed in decompression then, rotary evaporation obtains the thick material of little Huang, adding ethanol jolting is also outwelled, and adds ethanol again and is stirred to powdered, and rotary evaporation promptly gets Gly-Asp dipeptides white powder;
(3) in 20ml～200ml tetrahydrofuran solution, the suspend carbobenzoxy-(Cbz) arginine (Z-Arg-OH) of 1g～10g, add the tetrahydrofuran solution (V phosphorus tribromide/V tetrahydrofuran (THF)=1: 4) that 5ml～50ml contains phosphorus tribromide in room temperature, after the stirring at room 3～5 hours, the tetrahydrofuran (THF) on reaction solution upper strata is poured into down, lower floor is the bigger oily matter of proportion that precipitates, this oily matter is washed with the tetrahydrofuran (THF) of 200～400ml repeatedly, the vacuum drying gets flaxen tarry materials, promptly gets nitrogen carboxyl inner-acid anhydride arginine;
(4) in the container that has thermometer, pH electrode, outer refrigerating unit and whipping appts, the Gly-Asp dipeptides is dissolved in boric acid-potassium borate buffer system of 0.1～0.3mol/L of pH 8～12, the content of Gly-Asp dipeptides is 0.5mol/l～0.8mol/l in the system.Transfer pH 8～12 with KOH, add 0.01～0.03 milliliter of octanol, under high degree of agitation in nitrogen carboxyl inner-acid anhydride arginine amount of substance: Gly-Asp dipeptides amount of substance is 1～1.5: 1 ratio, once add nitrogen carboxyl inner-acid anhydride arginine (NCA-Arg), and the KOH solution maintenance pH that constantly adds 1～2mol/l is 8～12, alkali consumption finishes in 2～8 fens kinds, adds the vitriol oil and transfers the following termination reaction of pH to 3; With above-mentioned reaction solution freeze-drying, with the less water dissolving, with the alcohol precipitation, suction filtration must precipitate, and with SephadexG-10 column chromatography for separation product, obtains the described arginine-glycine-aspartic acid kyrine product of this patent.
Chloracetyl has three effects in synthetic: 1. be that amino is protected; 2. be that carboxyl to aspartic acid has activation; 3. be that chloracetyl can be converted into the amino glycine residue that becomes peptide bond of required and aspartic acid after ammonia is separated, saved one and gone on foot and connect amino acid whose step.
Description of drawings
Fig. 1: the mass spectrum of the arginine-glycine-aspartic acid tripeptides of this patent preparation.
By collection of illustrative plates as can be known, peak value is 347.2272 corresponding with the molecular weight Mw346 of arginine-glycine-aspartic acid tripeptides, confirms as its mass spectra peak, proves that product is the arginine-glycine-aspartic acid tripeptides.
Concrete embodiment
Embodiment 1: chloroacetylation aspartic acid synthetic
Get 25 gram L-aspartic acids and be suspended in 40 milliliters, the aqueous solution of 27% concentration hydrogen sodium oxide it is dissolved fully, with this concentration hydrogen aqueous solution of sodium oxide accent pH=10, add 30 milliliters of ethyl acetate then then; Under 0 ℃ condition, drip 15 milliliters of 12.54mol/l chloroacetyl chlorides and 15 milliliter of 27% sodium hydroxide respectively in above-mentioned solution with the speed of 0.16ml/min, stir while dripping, control aqueous sodium hydroxide solution rate of addition keeps the pH of solution 9.5 in the dropping process, dropwise, continue to stir 30 minutes; Transfer PH to 2.5 with concentrated hydrochloric acid (concentration is 37%) when finishing reaction; then this reaction solution is used 200ml ethyl acetate extracting three times, united extraction liquid adds 2 gram anhydrous sodium sulfate dryings; ethyl acetate is removed in decompression, promptly obtains about 30 grams of chloroacetylation aspartic acid white powder.
Embodiment 2: Gly-Asp dipeptides synthetic
Under 2 ℃; the chloroacetylation aspartic acid of front gained is added strong aqua (27%) according to the ratio of 1.0g: 6ml; this solution stirring 24 hours; unnecessary ammonia is removed in decompression then; rotary evaporation obtains the thick material of little Huang; add 10ml ethanol jolting and outwell (removing alcohol soluble substance matter), add 10ml ethanol again and be stirred to powdered, rotary evaporation promptly gets about 0.9 gram of Gly-Asp dipeptides white powder.
Embodiment 3: nitrogen carboxyl inner-acid anhydride arginine (NCA-Arg) synthetic
The carbobenzoxy-(Cbz) arginine (Z-Arg-OH) of suspension 1g in the 20ml tetrahydrofuran solution, add the tetrahydrofuran solution (V phosphorus tribromide/V tetrahydrofuran (THF)=1: 4) that 5ml contains phosphorus tribromide in room temperature, after the stirring at room 3 hours, the tetrahydrofuran (THF) on reaction solution upper strata is poured into down, lower floor is the bigger oily matter of proportion that precipitates, this oily matter is washed 6～8 times with the tetrahydrofuran (THF) of 200ml repeatedly, the vacuum drying gets about flaxen tarry materials 0.5 gram, i.e. nitrogen carboxyl inner-acid anhydride arginine.
Embodiment 4: arginine-glycine-aspartic acid tripeptides synthetic
1 g Gly-Asp dipeptides is dissolved in 0.2mol/L boric acid-potassium borate buffer system of pH 9, the content of Gly-Asp dipeptides is 0.6mol/l in the system.In the container that has thermometer, pH electrode, outer refrigerating unit and whipping appts, transfer pH=10 with KOH, add an octanol (0.01 milliliter), once add nitrogen carboxyl inner-acid anhydride arginine (NCA-Arg) (nitrogen carboxyl inner-acid anhydride arginine amount of substance: Gly-Asp dipeptides amount of substance=1.05), and the KOH solution maintenance pH that constantly adds 1mol/l is that alkali consumption finishes in 9,5 minutes.Adding the vitriol oil subsequently transfers pH to 3 termination reactions.The 5ml water dissolution is used in the reaction solution freeze-drying, and with this liquid of 10ml ethanol sedimentation, suction filtration must precipitate, and with SephadexG-10 column chromatography for separation product, obtains about 0.2 gram of the described arginine-glycine-aspartic acid kyrine product of this patent.
Claims (5)
1. the preparation method of arginine-glycine-aspartic acid cell adhesion tripeptide, it comprises the steps:
(1) reaction of chloroacetyl chloride and aspartic acid obtains the chloroacetylation aspartic acid;
(2) chloroacetylation aspartic acid ammonia is separated and is obtained the Gly-Asp dipeptides;
(3) reaction of carbobenzoxy-(Cbz) arginine and phosphorus tribromide obtains nitrogen carboxyl inner-acid anhydride arginine;
(4) nitrogen carboxyl inner-acid anhydride arginine and Gly-Asp two reactive polypeptides obtain the arginine-glycine-aspartic acid tripeptides.
2. the preparation method of arginine-glycine-aspartic acid cell adhesion tripeptide as claimed in claim 1, it is characterized in that: getting 25 grams～35 gram L-aspartic acids, to be suspended in 40～60 milliliters, mass concentration be in 17%～27% concentration hydrogen aqueous solution of sodium oxide it to be dissolved fully, between the adjust pH 8～12, add 30～60 milliliters of ethyl acetate then; Under 0 ℃～10 ℃ condition, drip 15～30 milliliters of 10～13mol/L chloroacetyl chlorides and 15～40 milliliter of 17%～27% sodium hydroxide respectively in above-mentioned solution with the speed of 0.10～0.20ml/min, stir while dripping, the pH that keeps solution is in 8～12 scopes, dropwise, continue to stir 20～50 minutes; Transfer below the PH to 3 with concentrated hydrochloric acid when finishing reaction, with ethyl acetate extracting three times of this reaction solution, united extraction liquid adds anhydrous sodium sulfate drying then, and ethyl acetate is removed in decompression, promptly obtains chloroacetylation aspartic acid white powder.
3. the preparation method of arginine-glycine-aspartic acid cell adhesion tripeptide as claimed in claim 1; it is characterized in that: under 0 ℃～10 ℃ cold condition; it is 25%～28% strong aqua that resulting chloroacetylation aspartic acid is added mass concentration according to the ratio of 1.0g: 5～10ml; stirred 24～48 hours; unnecessary ammonia is removed in decompression then; rotary evaporation obtains the thick material of little Huang; adding ethanol jolting is also outwelled; add ethanol again and be stirred to powdered, rotary evaporation promptly gets Gly-Asp dipeptides white powder.
4. the preparation method of arginine-glycine-aspartic acid cell adhesion tripeptide as claimed in claim 1, it is characterized in that: the carbobenzoxy-(Cbz) arginine of the 1g～10g that in 20ml～200ml tetrahydrofuran solution, suspends, add the tetrahydrofuran solution that 5ml～50ml contains phosphorus tribromide in room temperature, both volume ratios are 1: 4, after the stirring at room 3～5 hours, the tetrahydrofuran (THF) on reaction solution upper strata is poured into down, lower floor is the bigger oily matter of proportion that precipitates, this oily matter is washed with the tetrahydrofuran (THF) of 200～400ml repeatedly, the vacuum drying gets flaxen tarry materials, promptly gets nitrogen carboxyl inner-acid anhydride arginine.
5. the preparation method of arginine-glycine-aspartic acid cell adhesion tripeptide as claimed in claim 1, it is characterized in that: the Gly-Asp dipeptides is dissolved in boric acid-potassium borate buffer system of 0.1～0.3mol/L of pH8～12, the content of Gly-Asp dipeptides is 0.5mol/l～0.8mol/l in the system, transfer pH 8～12 with KOH, add 0.01～0.03 milliliter of octanol, under high degree of agitation, disposable adding and Gly-Asp dipeptides mol ratio are 1～1.5: 1 nitrogen carboxyl inner-acid anhydride arginine, keeping pH is 8～12, alkali consumption finishes in 2～8 fens kinds, adds the vitriol oil and transfers the following termination reaction of pH to 3; With above-mentioned reaction solution freeze-drying, the less water dissolving adds the alcohol precipitation, and suction filtration must precipitate, and with SephadexG-10 column chromatography for separation product, obtains the arginine-glycine-aspartic acid kyrine product.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102105485A (en) * | 2008-05-21 | 2011-06-22 | 新世界实验室公司 | Selective caspase inhibitors and uses thereof |
| US9045524B2 (en) | 2009-05-21 | 2015-06-02 | Novagenesis Foundation | Selective caspase inhibitors and uses thereof |
| US9944674B2 (en) | 2011-04-15 | 2018-04-17 | Genesis Technologies Limited | Selective cysteine protease inhibitors and uses thereof |
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2007
- 2007-11-28 CN CNA2007100563603A patent/CN101161672A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102105485A (en) * | 2008-05-21 | 2011-06-22 | 新世界实验室公司 | Selective caspase inhibitors and uses thereof |
| CN102105485B (en) * | 2008-05-21 | 2016-01-13 | 新世界实验室公司 | Selectivity caspase inhibitors and uses thereof |
| US10167313B2 (en) | 2008-05-21 | 2019-01-01 | Genesis Technologies Limited | Selective caspase inhibitors and uses thereof |
| US9045524B2 (en) | 2009-05-21 | 2015-06-02 | Novagenesis Foundation | Selective caspase inhibitors and uses thereof |
| US9944674B2 (en) | 2011-04-15 | 2018-04-17 | Genesis Technologies Limited | Selective cysteine protease inhibitors and uses thereof |
| US10975119B2 (en) | 2011-04-15 | 2021-04-13 | Genesis Technologies Limited | Selective cysteine protease inhibitors and uses thereof |
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Open date: 20080416 |