CN103103239A - Method for synthesizing thymopentin by enzyme - Google Patents
Method for synthesizing thymopentin by enzyme Download PDFInfo
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- CN103103239A CN103103239A CN2013100241605A CN201310024160A CN103103239A CN 103103239 A CN103103239 A CN 103103239A CN 2013100241605 A CN2013100241605 A CN 2013100241605A CN 201310024160 A CN201310024160 A CN 201310024160A CN 103103239 A CN103103239 A CN 103103239A
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Abstract
The invention discloses a method for synthesizing thymopentin by an enzyme. According to the method, papain is utilized as a catalyst to synthesize thymopentin in ethyl acetate-water, so that the process is simple, the synthetic efficiency of thymopentin is improved, the utilization rate of enzyme is high, and the production cost is low. The product is easy to recover, and no chemical pollution is caused, so that the method has important social meaning and huge market prospect. The thymopentin is synthesized in ethyl acetate-water, so that the reaction condition is mild. The stereospecificity is strong, so that product racemization in synthesis by the chemical method is avoided. No side chain protecting groups are used, and no side effects are available. The organic substrate is good in solubility in water rather than in an organic solvent, while the enzyme is insoluble to organic media, so that the enzyme is easy to recover. The enzyme is more stable in the organic solvent rather than in water, so that the problem that the enzyme is inactivated due to microbial pollution is avoided.
Description
Technical field
The invention belongs to the polypeptide hormone preparing technical field, particularly a kind of method of enzymic synthesis thymopeptide-5.
Background technology
Zadaxin is a kind of polypeptide hormone that animal thymus produces, and in China's clinical application history of existing more than 20 year, common Zadaxin belongs to biochemical product, is the thymic tissue extract of the animals such as healthy calf or pig.This medicine is a kind of biological respinse regulatory factor, can promote lymphocyte ripe, regulates and strengthens human immunity mechanism, have clinically anti-ageing, antivirally copy, the effect of antitumor cell differentiation.Indefinite due to Zadaxin effective constituent, content is low, contain the sensitization high molecular weight protein, does not meet WHO to five standard (specific chemical components of immunomodulator; Be easy to degraded; Stimulate moderate; Not carcinogenic, aberration inducing; Without cumulative toxicity, have no adverse reaction and follow-up effect), thereby curative effect is low, poor stability, untoward reaction especially severe allergic reaction frequently occurs, and has caused thus national drug adverse reaction monitoring center this similar drug is circulated a notice of.Use Thymopeptide anaphylactic shock, fash, heating, shiver with cold, chilly once to occur, the multinomial untoward reactions such as uncomfortable in chest, palpitaition, expiratory dyspnea, headache, cyanosis, need do the intracutaneous sensitization test before using, when skin test is negative, still need strengthen the clinical application monitoring.
thymopeptide-5 is the pentapeptide of synthetic, its aminoacid sequence is the Arg-Lys-Asp-Val-Tyr(Arg-Lys-Asp-Val-Tyr), take from the 32-36 amino acids of Zadaxin, main reactive site for Zadaxin, with Zadaxin, identical physiological function and drug effect are arranged, be characterized in that pharmaceutical purity is high, stable content, safe and reliable, and do not contain macro-molecular protein, in the clinical expansion process, thymopeptide-5 is because purity is high, steady quality, determined curative effect, safe and reliable and be subject to numerous doctors and patients' welcome, need not skin test before use.Thymopeptide-5 is the pentapeptide class medicine with good drug effect, and exploitation is ripe, lower-cost synthetic thymopeptide-5 method, has important social effect and huge market outlook.
The enterprise of the such high-end product of domestic production thymopeptide-5 only has several families, and is all that the applied chemistry method is synthetic.The synthetic shortcoming of chemical process shows that reaction conditions is violent, has the problem of product racemization in synthesizing, and need to protect side-chain radical, and side reaction is arranged, and product is difficult to reclaim, and easily causes chemical pollution, complex process, and cost is high, and production efficiency is low.
Summary of the invention
The shortcoming that the object of the invention is to overcome prior art provides a kind of method of enzymic synthesis thymopeptide-5 with not enough.
Purpose of the present invention is achieved through the following technical solutions: a kind of method of enzymic synthesis thymopeptide-5 comprises the steps:
(1) tertbutyloxycarbonyl-α-amino-isovaleric acid-tyrosine-methyl esters (Boc-Val-Tyr-OMe) dipeptides is synthetic: 0.2～0.8mol/L pH5.0～8.0 Tutofusin triss-hydrochloric acid (Tris-HCl) damping fluid is added in ethyl acetate, stir, then 4～10:40～55:20～35 add papoid (papain successively in mass ratio, PA), tertbutyloxycarbonyl-α-amino-isovaleric acid (Boc-Val) and tyrosine-methyl esters (Tyr-OMe), in 30～60 ℃ of reaction 12～48h, obtain tertbutyloxycarbonyl-α-amino-isovaleric acid-tyrosine-methyl esters dipeptides;
(2) [tertbutyloxycarbonyl-arginine (2, 2, 4, 6, 7-pentamethyl-benzo furans-5-alkylsulfonyl)-Methionin-methyl esters] (synthesizing of Boc-Arg (pbf)-Lys-Ome) dipeptides: 0.6～1.2mol/L pH4.0～7.0 tris-HCI buffers are added in ethyl acetate, stir, then 4～10:45～55:40～55 add papoid successively in mass ratio, [tertbutyloxycarbonyl-arginine (2, 2, 4, 6, 7-pentamethyl-benzo furans-5-alkylsulfonyl)] (Boc-Arg (pbf)) and Methionin-methyl esters (Lys-OMe), in 30～60 ℃ of reaction 36～72h, obtain [tertbutyloxycarbonyl-arginine (2, 2, 4, 6, 7-pentamethyl-benzo furans-5-alkylsulfonyl)-Methionin-methyl esters] dipeptides,
(3) [tertbutyloxycarbonyl-arginine (2, 2, 4, 6, 7-pentamethyl-benzo furans-5-alkylsulfonyl)-LYS-ASP-methyl esters] (synthesizing of Boc-Arg (pbf)-Lys-Asp-OMe) tripeptides: 0.2～0.8mol/L pH5.0～8.0 tris-HCI buffers are added in ethyl acetate, stir, then 3～10:2～7:4～10 add papoid successively in mass ratio, [tertbutyloxycarbonyl-arginine (2, 2, 4, 6, 7-pentamethyl-benzo furans-5-alkylsulfonyl)-Methionin] (Boc-Arg (pbf)-Lys) and aspartic acid-methyl esters (Asp-OMe), in 30～60 ℃ of reaction 36～72h, obtain [tertbutyloxycarbonyl-arginine (2, 2, 4, 6, 7-pentamethyl-benzo furans-5-alkylsulfonyl)-LYS-ASP-methyl esters] tripeptides,
(4) Arg-Lys-Asp-Val-Tyr (Arg-Lys-Asp-Val-Tyr) pentapeptide is synthetic: 0.2～0.8mol/L pH5.0～8.0 tris-HCI buffers are added in ethyl acetate, stir, then 2～6:4～10:4～10 add papoid successively in mass ratio, [tertbutyloxycarbonyl-arginine (2, 2, 4, 6, 7-pentamethyl-benzo furans-5-alkylsulfonyl)-LYS-ASP] (Boc-Arg (pbf)-Lys-Asp) and α-amino-isovaleric acid-tyrosine-methyl esters (Val-Tyr-OMe), in 30～60 ℃ of reaction 36～72h, obtain mixture, after mixture process, obtain the Arg-Lys-Asp-Val-Tyr pentapeptide,
In step (1):
Described 0.2～0.8mol/L pH5.0～8.0 tris-HCI buffers and ethyl acetate volume ratio are 0.5～2:1, are preferably 1:1;
The synthetic preferred following methods that adopts of described tertbutyloxycarbonyl-α-amino-isovaleric acid-tyrosine-methyl esters dipeptides carries out: 0.2～0.6mol/L pH5.0～7.0 tris-HCI buffers are added in ethyl acetate, stir, then 8:44:23 adds papoid, tertbutyloxycarbonyl-α-amino-isovaleric acid and tyrosine-methyl esters successively in mass ratio, in 40～60 ℃ of reaction 12～48h, obtain tertbutyloxycarbonyl-α-amino-isovaleric acid-tyrosine-methyl esters dipeptides;
In step (2):
The volume ratio of described 0.6～1.2mol/L pH4.0～7.0 tris-HCI buffers and ethyl acetate is 0.05～0.2:1, is preferably 0.11:1;
described [tertbutyloxycarbonyl-arginine (2, 2, 4, 6, 7-pentamethyl-benzo furans-5-alkylsulfonyl)-Methionin-methyl esters] dipeptides preferably adopts following methods to synthesize: 0.8～1.0mol/L pH4.0～7.0 tris-HCI buffers added in ethyl acetate, stir, then 4～8:53:46 adds papoid successively in mass ratio, [tertbutyloxycarbonyl-arginine (2, 2, 4, 6, and Methionin-methyl esters 7-pentamethyl-benzo furans-5-alkylsulfonyl)], in 50～60 ℃ of reaction 48～72h, obtain [tertbutyloxycarbonyl-arginine (2, 2, 4, 6, 7-pentamethyl-benzo furans-5-alkylsulfonyl)-Methionin-methyl esters] dipeptides,
In step (3):
The volume ratio of described 0.2～0.8mol/L pH5.0～8.0 tris-HCI buffers and ethyl acetate is 0.05～0.2:1, is preferably 0.11:1;
described [tertbutyloxycarbonyl-arginine (2, 2, 4, 6, 7-pentamethyl-benzo furans-5-alkylsulfonyl)-LYS-ASP-methyl esters] tripeptides preferably adopts following methods to synthesize: 0.3～0.6mol/L pH6.0～7.0 tris-HCI buffers added in ethyl acetate, stir, then 3～6:7:6 adds papoid successively in mass ratio, [tertbutyloxycarbonyl-arginine (2, 2, 4, 6, 7-pentamethyl-benzo furans-5-alkylsulfonyl)-Methionin] and aspartic acid-methyl esters, in 40～50 ℃ of reaction 36～48h, obtain [tertbutyloxycarbonyl-arginine (2, 2, 4, 6, 7-pentamethyl-benzo furans-5-alkylsulfonyl)-LYS-ASP-methyl esters] tripeptides,
In step (4):
The volume ratio of described 0.2～0.8mol/L pH5.0～8.0 tris-HCI buffers and ethyl acetate is 0.05～0.2:1, is preferably 0.11:1;
Described Arg-Lys-Asp-Val-Tyr pentapeptide preferably adopts following methods to synthesize: 0.2～0.6mol/L pH5.0～7.0 tris-HCI buffers are added in ethyl acetate, stir, then 2～6:8:8 adds papoid, [tertbutyloxycarbonyl-arginine (2 successively in mass ratio, 2,4,6,7-pentamethyl-benzo furans-5-alkylsulfonyl)-LYS-ASP] and α-amino-isovaleric acid-tyrosine-methyl esters, in 40～50 ℃ of reaction 36～72h, obtain mixture; After mixture process, obtain the Arg-Lys-Asp-Val-Tyr pentapeptide;
Described mixture process preferably adopts following methods to carry out: mixture is added in 95% trifluoroacetic acid, under the lucifuge condition, room temperature reaction 1～2h obtains reaction solution; Reaction solution is added in methyl alcohol, add LiOH, transfer pH to 13, room temperature reaction 1～2h obtains the Arg-Lys-Asp-Val-Tyr pentapeptide;
Described 95% trifluoroacetic acid refers to trifluoroacetic acid and the water mixed solution that is mixed to get of 95:5 by volume;
The present invention has following advantage and effect with respect to prior art:
The present invention is synthetic thymopeptide-5 take papoid as catalyzer, in ethyl acetate-water, has improved the combined coefficient of thymopeptide-5, and the utilization ratio of enzyme is high, and production cost is low; Product easily reclaims, and can not cause chemical pollution, and technique is simple, has important social effect and huge market outlook.Synthetic thymopeptide-5 in ethyl acetate-water, reaction conditions is gentle; Stereospecificity is strong, has avoided in chemical method is synthetic problem, the problem includes: product racemization problem; Without Side chain protective group, several without side reaction; Organic substrates is better than solvability in water in organic solvent, and enzyme is insoluble to organic medium, so enzyme is easy to reclaim; Reaction thermodynamic(al)equilibrium in water by the amino acid synthetic peptide is disadvantageous, if carry out in organic solvent, is conducive to balance and moves to the direction that forms peptide bond; Enzyme is more stable in organic solvent than in water, does not have the problem that makes enzyme deactivation due to microbial contamination.
Description of drawings
Fig. 1 is total ion current figure and the mass spectrum of the standard specimen Boc-Val-Tyr-OMe of embodiment 1; Wherein a is the total ion current figure of standard specimen Boc-Val-Tyr-OMe, and b is the mass spectrum of standard specimen Boc-Val-Tyr-OMe.
Fig. 2 is total ion current figure and the mass spectrum of the synthetic Boc-Val-Tyr-OMe of embodiment 1; Wherein a is the total ion current figure of Boc-Val-Tyr-OMe, and b is the mass spectrum of Boc-Val-Tyr-OMe.
Fig. 3 is total ion current figure and the mass spectrum of the standard specimen Boc-Arg (pbf)-Lys-OMe of embodiment 1; Wherein a is the total ion current figure of Boc-Arg (pbf)-Lys-OMe, and b is the mass spectrum of Boc-Arg (pbf)-Lys-OMe.
Fig. 4 is total ion current figure and the mass spectrum of the synthetic Boc-Arg (pbf) of embodiment 1-Lys-OMe; Wherein a is the total ion current figure of Boc-Arg (pbf)-Lys-OMe, and b is the mass spectrum of Boc-Arg (pbf)-Lys-OMe.
Fig. 5 is total ion current figure and the mass spectrum of the standard specimen Boc-Arg (pbf)-Lys-Asp-OMe of embodiment 1; Wherein a is the total ion current figure of Boc-Arg (pbf)-Lys-Asp-OMe, and b is the mass spectrum of Boc-Arg (pbf)-Lys-Asp-OMe.
Total ion current figure and the mass spectrum of the Boc-Arg (pbf) that Fig. 6 embodiment 1 synthesizes-Lys-Asp-OMe; Wherein a is the total ion current figure of Boc-Arg (pbf)-Lys-Asp-OMe, and b is the mass spectrum of Boc-Arg (pbf)-Lys-Asp-OMe.
Fig. 7 is total ion current figure and the mass spectrum of the standard specimen Arg-Lys-Asp-Val-Tyr of embodiment 1; Wherein a is the total ion current figure of Arg-Lys-Asp-Val-Tyr, and b is the mass spectrum of Arg-Lys-Asp-Val-Tyr.
Total ion current figure and the mass spectrum of the Arg-Lys-Asp-Val-Tyr that Fig. 8 embodiment 1 is synthetic; Wherein a is the total ion current figure of Arg-Lys-Asp-Val-Tyr, and b is the mass spectrum of Arg-Lys-Asp-Val-Tyr.
Embodiment
The present invention is described in further detail below in conjunction with embodiment and accompanying drawing, but embodiments of the present invention are not limited to this.
(1) the Boc-Val-Tyr-OMe dipeptides is synthetic: the Tris-HCl damping fluid 0.5ml of 0.2mol/L pH6.0 is added in the 0.5ml ethyl acetate, stir, then add successively 8mg papoid (PA), 44mg Boc-Val and 23mg Tyr-OMe, in 40 ℃ of reaction 48h, obtain the Boc-Val-Tyr-OMe dipeptides; Productive rate is 63.7%;
Total ion current figure and the mass spectrum of standard specimen Boc-Val-Tyr-OMe are seen Fig. 1, and total ion current figure and the mass spectrum of the material that PA catalyzes and synthesizes are seen Fig. 2; Can find out that from total ion current figure and mass spectrum PA successfully synthesizes the Boc-Val-Tyr-OMe dipeptides, its molecular weight is 411.5, and is consistent with the molecular weight 411.5 of standard specimen;
(2) Boc-Arg (pbf)-Lys-OMe dipeptides is synthetic: the Tris-HCl damping fluid 0.1ml of 0.8mol/L pH7.0 is added in the 0.9ml ethyl acetate, stir, then add successively 4mg papoid (PA), 53mg Boc-Arg (pbf) and 46mg Lys-OMe, in 50 ℃ of reaction 72h, obtain Boc-Arg (pbf)-Lys-OMe dipeptides; Productive rate is 66.93%;
Total ion current figure and the mass spectrum of the Boc-Arg of standard specimen (pbf)-Lys-OMe are seen Fig. 3, and total ion current figure and the mass spectrum of the Boc-Arg that PA catalyzes and synthesizes (pbf)-Lys-OMe are seen Fig. 4; Can find out that from total ion current figure and mass spectrum PA successfully synthesizes Boc-Arg (pbf)-Lys-OMe dipeptides, its molecular weight is 669.2, and is almost consistent with the molecular weight 669.3 of standard specimen;
(3) Boc-Arg (pbf)-Lys-Asp-OMe tripeptides is synthetic: the Tris-HCl damping fluid 0.1ml of 0.3mol/L pH7.0 is added in the 0.9ml ethyl acetate, stir, then add successively 3mg papoid (PA), 7mg Boc-Arg (pbf)-Lys and 6mg Asp-OMe, in 40 ℃ of reaction 48h, obtain Boc-Arg (pbf)-Lys-Asp-OMe tripeptides, productive rate is 26.9%;
The Boc-Arg of standard specimen (pbf)-Lys-Asp-OMe total ion current figure and mass spectrum are seen Fig. 5, and total ion current figure and the mass spectrum of the Boc-Arg that PA catalyzes and synthesizes (pbf)-Lys-Asp-OMe are seen Fig. 6.Can find out that from total ion current figure and mass spectrum PA successfully synthesizes Boc-Arg (pbf)-Lys-Asp-OMe tripeptides, its molecular weight is 784.3, and is almost consistent with the molecular weight 784.2 of standard specimen;
(4) the Arg-Lys-Asp-Val-Tyr pentapeptide is synthetic: the Tris-HCl damping fluid 0.1ml of 0.3mol/L pH7.0 is added in the 0.9ml ethyl acetate, stir, then add successively 2mg papoid (PA), 8mg Boc-Arg (pbf)-Lys-Asp and 8mg Val-Tyr-OMe, in 40 ℃ of reaction 72h, obtain mixture; Mixture is added in 95% trifluoroacetic acid 0.2ml, under the lucifuge condition, room temperature reaction 1h obtains reaction solution; Reaction solution is added in methyl alcohol 0.3ml, add LiOH, transfer pH to 13, room temperature reaction 1h obtains the Arg-Lys-Asp-Val-Tyr pentapeptide; Productive rate is 17.8%;
Arg-Lys-Asp-Val-Tyr total ion current figure and the mass spectrum of standard specimen are seen Fig. 7, and Arg-Lys-Asp-Val-Tyr total ion current figure and mass spectrum that PA catalyzes and synthesizes are seen Fig. 8; Can find out that from total ion current figure and mass spectrum PA successfully synthesizes the Arg-Lys-Asp-Val-Tyr pentapeptide, its molecular weight is 679.8, and is almost consistent with the molecular weight 680.4 of standard specimen.
(1) the Boc-Val-Tyr-OMe dipeptides is synthetic: the Tris-HCl damping fluid 0.5ml of 0.6mol/L pH5.0 is added in the 0.5ml ethyl acetate, stir, then add successively 8mg papoid, 44mgBoc-Val and 23mg Tyr-OMe, in 50 ℃ of reaction 36h, obtain the Boc-Val-Tyr-OMe dipeptides; Productive rate is 52.8%;
(2) Boc-Arg (pbf)-Lys-OMe dipeptides is synthetic: the Tris-HCl damping fluid 0.1ml of 1.0mol/L pH4.0 is added in the 0.9ml ethyl acetate, stir, then add successively 8mg papoid, 53mg Boc-Arg (pbf) and 46mg Lys-OMe, in 50 ℃ of reaction 60h, obtain Boc-Arg (pbf)-Lys-OMe dipeptides; Productive rate is 65.01%;
(3) Boc-Arg (pbf)-Lys-Asp-OMe tripeptides is synthetic: the Tris-HCl damping fluid 0.1ml of 0.4mol/L pH6.0 is added in the 0.9ml ethyl acetate, stir, then add successively 6mg papoid, 7mg Boc-Arg (pbf)-Lys and 6mg Asp-OMe, in 50 ℃ of reaction 36h, Boc-Arg (pbf)-Lys-Asp-OMe tripeptides, productive rate are 21.1%;
(4) the Arg-Lys-Asp-Val-Tyr pentapeptide is synthetic: the Tris-HCl damping fluid 0.1ml of 0.2mol/L pH6.0 is added in the 0.9ml ethyl acetate, stir, then add successively 6mg papoid, 8mgBoc-Arg (pbf)-Lys-Asp and 8mg Val-Tyr-OMe, in 50 ℃ of reaction 36h, obtain mixture; Mixture is added in 95% trifluoroacetic acid 0.2ml, under the lucifuge condition, room temperature reaction 1.5h obtains reaction solution; Reaction solution is added in methyl alcohol 0.3ml, add LiOH, transfer pH to 13, room temperature reaction 1.5h obtains the Arg-Lys-Asp-Val-Tyr pentapeptide; Productive rate is 15.1%.
(1) the Boc-Val-Tyr-OMe dipeptides is synthetic: the Tris-HCl damping fluid 0.5ml of 0.4mol/L pH7.0 is added in the 0.5ml ethyl acetate, stir, then add successively 8mg papoid, 44mgBoc-Val and 23mg Tyr-OMe, in 60 ℃ of reaction 12h, obtain the Boc-Val-Tyr-OMe dipeptides; Productive rate is 48.4%;
(2) Boc-Arg (pbf)-Lys-OMe dipeptides is synthetic: the Tris-HCl damping fluid 0.1ml of 1.0mol/L pH4.0 is added in the 0.9ml ethyl acetate, stir, then add successively 4mg papoid, 53mg Boc-Arg (pbf) and 46mg Lys-OMe, in 60 ℃ of reaction 48h, obtain Boc-Arg (pbf)-Lys-OMe dipeptides; Productive rate is 52.6%;
(3) Boc-Arg (pbf)-Lys-Asp-OMe tripeptides is synthetic: the Tris-HCl damping fluid 0.1ml of 0.6mol/L pH7.0 is added in the 0.9ml ethyl acetate, stir, then add successively 4mg papoid, 7mg Boc-Arg (pbf)-Lys and 6mg Asp-OMe, in 40 ℃ of reaction 48h, Boc-Arg (pbf)-Lys-Asp-OMe tripeptides, productive rate are 17.3%;
(4) the Arg-Lys-Asp-Val-Tyr pentapeptide is synthetic: the Tris-HCl damping fluid 0.1ml of 0.6mol/L pH5.0 is added in the 0.9ml ethyl acetate, stir, then add successively 4mg papoid (papain, PA), 8mg Boc-Arg (pbf)-Lys-Asp and 8mg Val-Tyr-OMe, in 40 ℃ of reaction 48h, obtain mixture; Mixture is added in 95% trifluoroacetic acid 0.2ml, under the lucifuge condition, room temperature reaction 2h obtains reaction solution; Reaction solution is added in methyl alcohol 0.3ml, add LiOH, transfer pH to 13, room temperature reaction 2h obtains the Arg-Lys-Asp-Val-Tyr pentapeptide; Productive rate is 11.9%.
Above-described embodiment is the better embodiment of the present invention; but embodiments of the present invention are not restricted to the described embodiments; other any do not deviate from change, the modification done under spirit of the present invention and principle, substitutes, combination, simplify; all should be the substitute mode of equivalence, within being included in protection scope of the present invention.
Claims (9)
1. the method for an enzymic synthesis thymopeptide-5, is characterized in that comprising the steps:
(1) tertbutyloxycarbonyl-α-amino-isovaleric acid-tyrosine-methyl esters dipeptides is synthetic: 0.2～0.8mol/L pH5.0～8.0 tris-HCI buffers are added in ethyl acetate, stir, then 4～10:40～55:20～35 add papoid, tertbutyloxycarbonyl-α-amino-isovaleric acid and tyrosine-methyl esters successively in mass ratio, in 30～60 ℃ of reaction 12～48h, obtain tertbutyloxycarbonyl-α-amino-isovaleric acid-tyrosine-methyl esters dipeptides;
(2) [tertbutyloxycarbonyl-arginine (2, 2, 4, 6, 7-pentamethyl-benzo furans-5-alkylsulfonyl)-Methionin-methyl esters] dipeptides synthetic: 0.6～1.2mol/L pH4.0～7.0 tris-HCI buffers are added in ethyl acetate, stir, then 4～10:45～55:40～55 add papoid successively in mass ratio, [tertbutyloxycarbonyl-arginine (2, 2, 4, 6, and Methionin-methyl esters 7-pentamethyl-benzo furans-5-alkylsulfonyl)], in 30～60 ℃ of reaction 36～72h, obtain [tertbutyloxycarbonyl-arginine (2, 2, 4, 6, 7-pentamethyl-benzo furans-5-alkylsulfonyl)-Methionin-methyl esters] dipeptides,
(3) [tertbutyloxycarbonyl-arginine (2, 2, 4, 6, 7-pentamethyl-benzo furans-5-alkylsulfonyl)-LYS-ASP-methyl esters] tripeptides synthetic: 0.2～0.8mol/L pH5.0～8.0 tris-HCI buffers are added in ethyl acetate, stir, then 3～10:2～7:4～10 add papoid successively in mass ratio, [tertbutyloxycarbonyl-arginine (2, 2, 4, 6, 7-pentamethyl-benzo furans-5-alkylsulfonyl)-Methionin] and aspartic acid-methyl esters, in 30～60 ℃ of reaction 36～72h, obtain [tertbutyloxycarbonyl-arginine (2, 2, 4, 6, 7-pentamethyl-benzo furans-5-alkylsulfonyl)-LYS-ASP-methyl esters] tripeptides,
(4) the Arg-Lys-Asp-Val-Tyr pentapeptide is synthetic: 0.2～0.8mol/LpH5.0～8.0 tris-HCI buffers are added in ethyl acetate, stir, then 2～6:4～10:4～10 add papoid, [tertbutyloxycarbonyl-arginine (2 successively in mass ratio, 2,4,6,7-pentamethyl-benzo furans-5-alkylsulfonyl)-LYS-ASP] and α-amino-isovaleric acid-tyrosine-methyl esters, in 30～60 ℃ of reaction 36～72h, obtain mixture; After mixture process, obtain the Arg-Lys-Asp-Val-Tyr pentapeptide.
2. the method for enzymic synthesis thymopeptide-5 according to claim 1, it is characterized in that: the 0.2～0.8mol/L pH5.0 described in step (1)～8.0 tris-HCI buffers and ethyl acetate volume ratio are 0.5～2:1.
3. the method for enzymic synthesis thymopeptide-5 according to claim 1, it is characterized in that: the volume ratio of the 0.6～1.2mol/L pH4.0 described in step (2)～7.0 tris-HCI buffers and ethyl acetate is 0.05～0.2:1.
4. the method for enzymic synthesis thymopeptide-5 according to claim 1, it is characterized in that: the volume ratio of the 0.2～0.8mol/L pH5.0 described in step (3)～8.0 tris-HCI buffers and ethyl acetate is 0.05～0.2:1.
5. the method for enzymic synthesis thymopeptide-5 according to claim 1, it is characterized in that: the volume ratio of the 0.2～0.8mol/L pH5.0 described in step (4)～8.0 tris-HCI buffers and ethyl acetate is 0.05～0.2:1.
6. the method for enzymic synthesis thymopeptide-5 according to claim 1, it is characterized in that: the synthetic employing following methods of the tertbutyloxycarbonyl-α-amino-isovaleric acid described in step (1)-tyrosine-methyl esters dipeptides carries out: 0.2～0.6mol/L pH5.0～7.0 tris-HCI buffers are added in ethyl acetate, stir, then 8:44:23 adds papoid, tertbutyloxycarbonyl-α-amino-isovaleric acid and tyrosine-methyl esters successively in mass ratio, in 40～60 ℃ of reaction 12～48h, obtain tertbutyloxycarbonyl-α-amino-isovaleric acid-tyrosine-methyl esters dipeptides.
7. the method for enzymic synthesis thymopeptide-5 according to claim 1, it is characterized in that: [tertbutyloxycarbonyl-the arginine (2 described in step (2), 2, 4, 6, 7-pentamethyl-benzo furans-5-alkylsulfonyl)-Methionin-methyl esters] dipeptides adopts following methods to synthesize: 0.8～1.0mol/L pH4.0～7.0 tris-HCI buffers added in ethyl acetate, stir, then 4～8:53:46 adds papoid successively in mass ratio, [tertbutyloxycarbonyl-arginine (2, 2, 4, 6, and Methionin-methyl esters 7-pentamethyl-benzo furans-5-alkylsulfonyl)], in 50～60 ℃ of reaction 48～72h, obtain [tertbutyloxycarbonyl-arginine (2, 2, 4, 6, 7-pentamethyl-benzo furans-5-alkylsulfonyl)-Methionin-methyl esters] dipeptides.
8. the method for enzymic synthesis thymopeptide-5 according to claim 1, it is characterized in that: [tertbutyloxycarbonyl-the arginine (2 described in step (3), 2, 4, 6, 7-pentamethyl-benzo furans-5-alkylsulfonyl)-LYS-ASP-methyl esters] tripeptides adopts following methods to synthesize: 0.3～0.6mol/L pH6.0～7.0 tris-HCI buffers added in ethyl acetate, stir, then 3～6:7:6 adds papoid successively in mass ratio, [tertbutyloxycarbonyl-arginine (2, 2, 4, 6, 7-pentamethyl-benzo furans-5-alkylsulfonyl)-Methionin] and aspartic acid-methyl esters, in 40～50 ℃ of reaction 36～48h, obtain [tertbutyloxycarbonyl-arginine (2, 2, 4, 6, 7-pentamethyl-benzo furans-5-alkylsulfonyl)-LYS-ASP-methyl esters] tripeptides.
9. the method for enzymic synthesis thymopeptide-5 according to claim 1, it is characterized in that: the Arg-Lys-Asp-Val-Tyr pentapeptide described in step (4) adopts following methods to synthesize: 0.2～0.6mol/L pH5.0～7.0 tris-HCI buffers are added in ethyl acetate, stir, then 2～6:8:8 adds papoid successively in mass ratio, [tertbutyloxycarbonyl-arginine (2, 2, 4, 6, 7-pentamethyl-benzo furans-5-alkylsulfonyl)-LYS-ASP] and α-amino-isovaleric acid-tyrosine-methyl esters, in 40～50 ℃ of reaction 36～72h, obtain mixture, after mixture process, obtain the Arg-Lys-Asp-Val-Tyr pentapeptide.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106366177A (en) * | 2016-09-07 | 2017-02-01 | 烟台海安药物研发有限公司 | Thymopentin methyl ester conjugate and preparation method thereof |
| CN113244119A (en) * | 2021-05-27 | 2021-08-13 | 天津大学 | Method for dyeing hair in color based on enzymatic oxidation of tyrosine derivative |
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2013
- 2013-01-22 CN CN2013100241605A patent/CN103103239A/en active Pending
Non-Patent Citations (1)
| Title |
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| 邹倩儒: "有机相中木瓜蛋白酶催化性质及胸腺五肽合成", 《万方学位论文》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106366177A (en) * | 2016-09-07 | 2017-02-01 | 烟台海安药物研发有限公司 | Thymopentin methyl ester conjugate and preparation method thereof |
| CN113244119A (en) * | 2021-05-27 | 2021-08-13 | 天津大学 | Method for dyeing hair in color based on enzymatic oxidation of tyrosine derivative |
| CN113244119B (en) * | 2021-05-27 | 2022-10-04 | 天津大学 | Method for dyeing hair in color based on enzymatic oxidation of tyrosine derivative |
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