CN1308343C - Process for preparing thymopentin - Google Patents
Process for preparing thymopentin Download PDFInfo
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- CN1308343C CN1308343C CNB2004100227416A CN200410022741A CN1308343C CN 1308343 C CN1308343 C CN 1308343C CN B2004100227416 A CNB2004100227416 A CN B2004100227416A CN 200410022741 A CN200410022741 A CN 200410022741A CN 1308343 C CN1308343 C CN 1308343C
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Abstract
The present invention provides a method for synthesizing thymopentin Arg-Lys-Asp-Val-Tyr-OH by a minimum protection strategy. In the method, Arg and Tyr unprotected by lateral-chain functional groups are used as raw materials for synthesizing the thymopentin. The method of the present invention has the advantages of simplicity, rapidness, low cost, easy obtainment of raw materials and capability of forming a scale.
Description
Technical field
The present invention relates to the preparation method of polypeptide.More particularly, the present invention relates to the preparation method of essence-Lai-sky-figured silk fabrics-junket pentapeptide (Arg-Lys-Asp-Val-Tyr-OH).
Background technology
Thymopeptide-5 is the pentapeptide active fragments of synthetic, its sequence is corresponding to 32 to 36 amino acids residues (Arg-Lys-Asp-Val-Tyr-OH) of thymopoietin, have important drug effect (Goldstein G, Audhya TK.Thymopoietin to thymopentin:experimental studies.Surv Immunol Res.1985; 4 Suppl1:1-10.).Clinical various malignant tumours, hepatitis B, infection, autoimmune disorder, type ii diabetes, climacteric syndrome and worn with age, lower immune function etc. of being used for the treatment of.Best (the Gonser S of application prospect in malignant tumour and treating hepatitis B, Weber E, Folkers G.Peptides and polypeptides as modulators ofthe immune response:thymopentin-an example with unknown mode of action.Pharm ActaHelv.1999 Jun; 73 (6): 265-73.).According to Chinese antitumor drug market intelligence, the present annual sales amount of thymopeptide-5 is about 600,000,000 dollars.By 2006, the annual sales amount of thymopeptide-5 estimated to reach 1,700,000,000 dollars (progress of solid phase synthesis thymopeptide-5, Wang Ling, Wei Hao, chemical industry progress, 2003 the 2nd phases).
At present, there is pharmaceutical factory of tens of family to adopt solid phase synthesis process to produce thymopeptide-5 both at home and abroad for clinical application.Minority pharmaceutical factory adopts the strategy of orthogonally protect, and liquid phase is synthesized thymopeptide-5 (IT1245230, Procedure for thepreparation of thymopentin, GOFFREDO MAURIZIO; MONTI FRANCESCO; SANNICOLO ' FRANCESCO).These method cost height, feed ratio cause very big waste generally at 3～5 times.Solid-phase synthesis also needs the polypeptide solid phase synthetic instrument, and cost is higher.In addition, adopt the amino acid price costliness of side chain protected, need pass through violent HF deprotection reaction at last at synthetic as the tyrosine of side chain protected.Because the hypertoxicity of HF, the danger of reaction is bigger, must possess special HF deprotection device.And because the reaction vessel volume of this class deprotection device is limited, therefore deprotection products obtained therefrom limited amount has limited the application on industry each time.
Therefore, need a kind of simple, quick, cost is low, raw material is easy to get and can form the method for the synthetic thymopeptide-5 of scale.
Summary of the invention
The invention provides a kind of method that adopts the synthetic thymopeptide-5 Arg-Lys-Asp-Val-Tyr-OH of minimum protection strategy, it is raw material that described method adopts unprotected Arg of side chain functionalities and Tyr, carries out the reaction of following steps:
(1) under alkaline condition, Tyr-OR and B-Val-OH are reacted in the presence of condensing agent, obtain the B-Val-Tyr-OR dipeptides;
(2) adopt the ammonia end deprotection of deprotecting regent, obtain the Val-Tyr-OR dipeptides the dipeptides that makes in (1);
(3) under alkaline condition, make the product that makes in (2) and B-Asp (OR)-OH reacts in the presence of condensing agent, obtain B-Asp (OR)-the Val-Tyr-OR tripeptides;
(4) adopt the ammonia end deprotection of deprotecting regent with the tripeptides that makes in (3), obtain Asp (OR)-the Val-Tyr-OR tripeptides;
(5) under alkaline condition, make the product that makes in (4) and B-Lys (Z)-OH reacts in the presence of condensing agent, obtain B-Lys (Z)-Asp (OR)-the Val-Tyr-OR tetrapeptide;
(6) adopt the ammonia end deprotection of deprotecting regent with the tetrapeptide that makes in (5), obtain Lys (Z)-Asp (OR)-the Val-Tyr-OR tetrapeptide;
(7) under alkaline condition, the product and the Z-Arg-OH that make in (6) are reacted in the presence of condensing agent, the Z-Arg-Lys that is protected (Z)-and Asp (OR)-the Val-Tyr-OR pentapeptide;
(8) with the carboxyl-protecting group of aspartic acid and tyrosine on the pentapeptide chain that makes in (7) through the saponification deprotection, obtain Z-Arg-Lys (Z)-the Asp-Val-Tyr-OH pentapeptide;
(9) with arginine and lysine amino protecting group on the pentapeptide chain that makes in (8) through the hydrogenolysis deprotection, obtain the Arg-Lys-Asp-Val-Tyr-OH pentapeptide;
Wherein the protecting group B of the α amino of Xie Ansuan, aspartic acid, Methionin is a tertbutyloxycarbonyl, the different third oxygen carbonyl of biphenyl, the carbobenzoxy-(Cbz) of carbobenzoxy-(Cbz) and replacement or fluorenylmethyloxycarbonyl, the protecting group Z of the side chain amino of arginic α amino and Methionin is the different third oxygen carbonyl or the fluorenylmethyloxycarbonyl of carbobenzoxy-(Cbz), tertbutyloxycarbonyl, biphenyl of carbobenzoxy-(Cbz) and replacement, the protecting group R of the α carboxyl of tyrosine is methyl esters, ethyl ester, the tert-butyl ester, benzyl ester and replaces the benzyl ester, the protecting group R of the side chain carboxyl group of aspartic acid is the benzyl ester, methyl esters, ethyl ester;
In described step (1), (3), (5) and (7), described alkaline condition is meant the pH value for 7-11, and connects reactive polypeptide and carry out to the temperature condition of room temperature at 0 ℃.
According to one embodiment of the invention, the product that makes in (9) through the desalination of C18 reversed-phase column purifying, is obtained purified product.
With respect to art methods; unprotected arginine of side chain and tyrosine that the present invention adopts are raw material; because the amino acid whose price of side chain no protective is comparatively cheap; therefore adopt the synthetic cost of this protection route also less, and do not need the device of HF deprotection of the costliness of maximum protection route.
The inventive method passes through to select proper reaction conditions in preparation process; prevent that effectively side reaction from taking place, the yield that connects reactive polypeptide is higher than 95%, and the yield of final saponification and hydrogenolysis deprotection base is not less than 90%; the product that makes is determined as single-point through thin-layer chromatography, and can form 100g level scale.
Embodiment
Although do not wish to be subjected to the restriction of any theory, think that the present invention is with the following basis that is found to be: the side chain functionalities of Arg and Tyr can not docked reactive polypeptide yet and be caused negative impact under the situation that does not add protection.Therefore adopt the strategy of minimum protection; is raw material with side chain without the Arg and the Tyr that protect; Lys (side chain amino protecting group)-OH with the Val-OH of α amido protecting, the Asp of α amido protecting (side chain carboxyl group protecting group)-OH, α amido protecting; progressively connect peptide; the thymopeptide-5 intermediate Z-Arg-Lys of synthetic minimum protection (Z)-Asp (OR)-and Val-Tyr-OR, through saponification and the synthetic thymopeptide-5 Arg-Lys-Asp-Val-Tyr-OH of hydrogenolysis.
A synthetic schemes of the present invention is as follows:
1. amino acid starting material
The amino acid starting material that is used for synthetic thymopeptide-5 of the present invention is: Z-Arg-OH and Tyr-OR, B-Val-OH, B-Asp (OR)-and OH, B-Lys (Z)-OH.Wherein the implication of each protecting group Z, B, R is as described below, and has independently implication when occurring in each compound.
The present invention selects for use side chain functionalities not add the arginine (Arg) and the tyrosine (Tyr) of protection, promptly is for Arg:
Guanidine radicals on the side chain does not add protection and to Tyr:
Phenolic hydroxyl group on the side chain does not add protection.
The α amino of Xie Ansuan (Val), aspartic acid (Asp), Methionin (Lys) can be according to method well known in the art; adopt blocking group well known in the art to protect; the protecting group B that is α amino in each raw material can independently be selected from tertbutyloxycarbonyl (Boc) separately; the different third oxygen carbonyl (Bpoc) of biphenyl, the carbalkoxy that carbobenzoxy-(Cbz) (Cbz) and the carbobenzoxy-(Cbz) that replaces or fluorenylmethyloxycarbonyl (Fmoc) wait other.Preferably all adopt the Boc-group to protect.
Amino on Methionin (Lys) side chain:
According to method well known in the art; adopt blocking group well known in the art to protect, promptly the protecting group Z of side chain amino can be selected from the carbalkoxy that carbobenzoxy-(Cbz) (Cbz) and the different third oxygen carbonyl (Bpoc) of carbobenzoxy-(Cbz), tertbutyloxycarbonyl (Boc), biphenyl that replaces or fluorenylmethyloxycarbonyl (Fmoc) wait other among the Lys.
The α amino of arginine (Arg) can be according to method well known in the art; adopt blocking group well known in the art to protect; the protecting group Z that is α amino among the Arg can independently be selected from tertbutyloxycarbonyl (Boc) separately; the different third oxygen carbonyl (Bpoc) of biphenyl, the carbalkoxy that carbobenzoxy-(Cbz) (Cbz) and the carbobenzoxy-(Cbz) that replaces or fluorenylmethyloxycarbonyl (Fmoc) wait other.The identical protecting group protection of the amino employing of the amino side chain with Methionin of preferred arginic α preferably all adopts carbobenzoxy-(Cbz) (Cbz) to protect.
The α carboxyl of tyrosine can be according to method well known in the art; adopt blocking group well known in the art to protect, promptly the protecting group R of the α carboxyl of tyrosine can be selected from the form of other esters such as methyl esters (OMe), ethyl ester (OEt), the tert-butyl ester, benzyl ester (OBzl) and replacement benzyl ester.Be preferably methyl esters.
The side chain carboxyl group of aspartic acid:
Can adopt blocking group well known in the art to protect according to method well known in the art, promptly the protecting group R of the side chain carboxyl group of aspartic acid can be selected from benzyl ester (Obzl), methyl esters (OMe), and ethyl ester (OEt) waits the form of other ester.Be preferably the benzyl ester.
As mentioned above, being used for amino acid starting material of the present invention can adopt blocking group well known in the art that each amino acid protection is made according to method well known in the art, maybe can buy by the commercial channel.
2. connect reactive polypeptide
The present invention adopts liquid phase method, and each amino acid as raw material is progressively connect peptide, synthetic final product.
Connect reactive polypeptide and carry out in solvent usually, DMF, NMP etc. that used solvent can be selected from tetrahydrofuran (THF), heavily steam preferably use tetrahydrofuran (THF).
The control of pH value is comparatively crucial in the reaction process.When pH is lower than 7, the purity of reaction product will reduce, and pH is higher than 11, and productive rate will be reduced.Therefore preferably pH is controlled at 7-11, more preferably in the scope of 8-9.Can adopt the pH value in the organic bases conditioned reaction system.Organic bases can be selected from N-methylmorpholine (NMM), triethylamine and DIEA (diisopropyl ethyl amine) etc., preferably uses NMM.
The condensing agent of selecting for use in reaction is well known to those skilled in the art, and the available condensing agent is selected from DCC/HOBt, DIC, HBTU, BOP etc., preferably adopts DCC/HOBt as condensing agent.
Temperature of reaction can be 0 ℃ to room temperature.When at room temperature reacting, the reaction times is short, is multiple spot but detect through thin-layer chromatography, and product purity descends, and productive rate descends.And react for some time down at 0 ℃ earlier, and after preferred 2 hours, under room temperature, react again, then obtaining detecting through thin-layer chromatography is pure product.
3. the deprotection of amido protecting group
The deprotection of amido protecting group is known in the art.In a preferred embodiment of the invention, it is protecting group that the α amino of Xie Ansuan (Val), aspartic acid (Asp), Methionin (Lys) all adopts Boc, and its deprotection can adopt deprotecting regents such as HCl/ ethyl acetate or TFA/ methylene dichloride to implement.
When adopting the TFA/ methylene dichloride as deprotecting regent, though its solvability to reaction raw materials is better, the product that obtains is thick material, and TFA needs underpressure distillation just can remove, so aftertreatment difficulty comparatively.
And in the HCl/ ethyl acetate because HCl is a volatile acid, the easy crystallization of the product that obtains get final product by filtering aftertreatment, so more preferably uses the deprotecting regent of HCl/ ethyl acetate as the amido protecting group.
In a preferred embodiment of the invention; arginic α side chain amino amino and Methionin all adopts carbobenzoxy-(Cbz) (Cbz) to protect, and its deprotection can adopt the hydrogenolysis method, by in solution; in the presence of palladium on carbon, at room temperature react with hydrogen and to carry out.
4. the deprotection of carboxyl-protecting group
The carboxylic end of the side chain carboxyl group of aspartic acid and synthetic pentapeptide intermediate adopts the formation ester to form and protects.This protecting group can slowly be added dropwise to alkali at a certain temperature and carry out saponification reaction and slough.
The solvent that uses in the reaction can be selected from reaction solvents commonly used such as methyl alcohol, ethanol.
Reaction can be carried out to room temperature at 0 ℃.At room temperature carry out to shorten the reaction times (contracting to 2 hours), but serious racemization takes place, produce the more by product that comprises racemic mixture by about 5 hours under 0 ℃.Then obtaining detecting through thin-layer chromatography when carrying out under 0 ℃ is pure compound.Therefore preferably implement saponification reaction down at 0 ℃.
5. refining
Adopt the C18 reversed-phase column to the desalination of crude product purifying.Behind the purifying, detect purity through HPLC.
The invention will be further described below by indefiniteness embodiment.
In this article, being abbreviated as of agents useful for same:
DCC: dicyclohexylcarbodiimide
The HOBt:1-hydroxy benzo triazole
The NMM:N-methylmorpholine
THF: tetrahydrofuran (THF)
Embodiment
1.Boc-Val-Tyr-OMe preparation
3.52g (15.2mmol) HClTyr-OMe is with the dissolving of 20ml anhydrous tetrahydro furan, the solution that obtains is regulated pH to 7～8 with N-methylmorpholine, be cooled to 0 ℃ standby.
3.29g (15.2mmol) Boc-L-Val-OH, 2.15g (16.0mmol) HOBt feeds dissolving with anhydrous tetrahydrochysene furan, is chilled to 0 ℃, slowly adds 3.29g (16.0mmol) DCC, and 0 ℃ was stirred 5 minutes.Mix with top solution then, 0 ℃ was stirred 2 hours, kept PH 8～9 with N-methylmorpholine, stirring at room 10 hours, and chloroform/methanol/acetate (20/1/0.4) shows raw material point disappearance.Reaction mixture filters, and filtrate decompression concentrates, and residue grinds repeatedly with sherwood oil, and the thick material 250ml acetic acid ethyl dissolution that obtains is used saturated NaHCO respectively
3The aqueous solution (30ml * 5), the saturated NaCl aqueous solution (30ml * 1), 5%KHSO
4The aqueous solution (30ml * 5), the saturated NaCl aqueous solution (30ml * 3) washing, the organic layer anhydrous sodium sulfate drying filters, and filtrate decompression is spin-dried for and obtains faint yellow solid powder 5.64g (productive rate 99.0%).In ethyl acetate/petroleum ether (1/3) and (50/1) two kind of development system of chloroform/methanol, be single-point, R
fValue is respectively 0.40 and 0.25, and this product can be directly used in next step reaction.The crude product that takes a morsel adopts ethyl acetate/petroleum ether to carry out recrystallization, obtains crystalline powder and is used to measure fusing point and specific optical rotation, records fusing point: 152.9～154.2 ℃, [α]
D 20=-17 ° (C=2MeOH).
2.HClVal-Tyr-OMe preparation
After 5.64g (15mmol) Boc-Val-Tyr-OMe mixes with 20ml 4N HCL/ ethyl acetate solution stirring at room 2.5 hours, the yellow clarification of solution, TLC (chloroform/methanol, 10/1) shows raw material point disappearance.Decompression is spin-dried for solvent to there not being HCl, grinds repeatedly with sherwood oil at last, obtains yellow solid powder 4.49g (productive rate 90.7%).In chloroform/methanol/triethylamine (20/1/0.2) development system is single-point, R
f=0.28, [α]
D 20=+53 ° (C=2MeOH).Fusing point: 95.1～97.5 ℃.
3.Boc-Asp (Bzl)-preparation of Val-Tyr-OMe
4.49g (13.6mmol) HClVal-Tyr-OMe is with the dissolving of 15ml anhydrous tetrahydro furan, the solution that obtains is transferred pH to 7～8 with N-methylmorpholine, be cooled to 0 ℃ standby.
14.3g (14.3mmol) Boc-Asp (Obzl)-OH, 1.93g (14.3mmol) HOBt is chilled to 0 ℃ with the dissolving of 30ml anhydrous tetrahydro furan, slowly adds 2.94g (14.3mmol) DCC, and 0 ℃ was stirred 5 minutes.Mix with top solution then, 0 ℃ was stirred 2 hours, kept pH8～9 with N-methylmorpholine, stirring at room 12 hours, and chloroform/methanol/acetate (20/1/0.4) shows raw material point disappearance.Reaction mixture filters, and filtrate decompression concentrates, and residue grinds repeatedly with sherwood oil, and the solid that obtains adds the 300ml acetic acid ethyl dissolution, uses saturated NaHCO respectively
3The aqueous solution (30ml * 3), the saturated NaCl aqueous solution (30ml * 1), 5%KHSO
4The aqueous solution (30ml * 3), the saturated NaCl aqueous solution (30ml * 3) washing, the organic layer anhydrous sodium sulfate drying filters, and filtrate decompression is spin-dried for and obtains 7.83g crude product (productive rate 96.4%).In ethyl acetate/petroleum ether (1/3) and (50/1) two kind of development system of chloroform/methanol, be single-point, R
fValue is respectively 0.4 and 0.12, can be directly used in next step reaction.The crude product that takes a morsel adopts ethyl acetate/petroleum ether to carry out recrystallization, obtains crystalline powder and is used to measure fusing point and specific optical rotation, records fusing point: 148.3～149.3 ℃, [α]
D 20=-30 ° (C=2MeOH).
4.HClAsp (Bzl)-preparation of Val-Tyr-OMe
7.83g stirring at room 3 hours, TLC (chloroform/methanol, 20/1) showed raw material point disappearance after (13.1mmol) Boc-Asp (Obzl)-Val-Tyr-OMe mixed with 4N HCL/ ethyl acetate solution.Decompression is spin-dried for solvent to there not being HCl, grinds repeatedly with sherwood oil at last, obtains faint yellow solid powder 6.82g (productive rate 97.8%).In chloroform/methanol/triethylamine (20/1/0.2) development system is single-point, R
f=0.33, fusing point: 102.4～105.6 ℃, [α]
D 20=+35 ° (C=2MeOH).
5.Boc-Lys (Z)-preparation of Asp (Bzl)-Val-Tyr-OMe
6.82g (12.8mmol) HClAsp (Obzl)-Val-Tyr-OMe is with the dissolving of 35ml anhydrous tetrahydro furan, the solution that obtains is transferred pH to 7～8 with N-methylmorpholine, be cooled to 0 ℃ standby.
5.11g (13.4mmol) Boc-Lys (Cbz)-OH, 1.81g (13.4mmol) HOBt is chilled to 0 ℃ with the dissolving of ml anhydrous tetrahydro furan, slowly adds 2.76g (13.4mmol) DCC, and 0 ℃ was stirred 5 minutes.Mix with top solution then, 0 ℃ was stirred 2 hours, kept pH8～9 with N-methylmorpholine, stirring at room 12 hours, and chloroform/methanol/acetate (20/1/0.4) shows raw material point disappearance.Reaction mixture filters, and filtrate decompression concentrates, and residue grinds repeatedly with sherwood oil, and the solid that obtains adds the 500ml acetic acid ethyl dissolution, uses saturated NaHCO respectively
3The aqueous solution (50ml * 3), the saturated NaCl aqueous solution (50ml * 1), 5%KHSO
4The aqueous solution (50ml * 3), the saturated NaCl aqueous solution (50ml * 3) washing, the organic layer anhydrous sodium sulfate drying filters, and filtrate decompression is spin-dried for and obtains 10.55g crude product (productive rate 96.0%).In ethyl acetate/petroleum ether (3/1) and (30/1) two kind of development system of chloroform/methanol, be single-point, R
fValue is respectively 0.38 and 0.27, can be directly used in next step reaction.The crude product that takes a morsel adopts ethyl acetate/petroleum ether to carry out recrystallization, obtains crystalline powder and is used to measure fusing point and specific optical rotation, records fusing point: 121.8～122.9 ℃, [α]
D 20=-22 ° (C=2MeOH).
6.HClLys (Cbz)-preparation of Asp (Obzl)-Val-Tyr-OMe
10.55g stirring at room 3 hours, TLC (chloroform/methanol, 20/1) showed raw material point disappearance after (12.3mmol) Boc-Lys (Cbz)-Asp (Obzl)-Val-Tyr-OMe mixed with 50ml 4N HCL/ ethyl acetate solution.Decompression is spin-dried for solvent to there not being HCl, grinds repeatedly with anhydrous diethyl ether at last, obtains faint yellow solid powder 9.55g (productive rate 97.7%).In chloroform/methanol/triethylamine (5/1/0.2) development system is single-point, R
f=0.29, fusing point: 144.8～150.1 ℃, [α]
D 20=-15 ° (C=2MeOH).
7.Z-Arg-Lys (Z)-preparation of Asp (Bzl)-Val-Tyr-OMe
9.55g (12.02mmol) HClLys (Cbz)-Asp (Obzl)-Val-Tyr-Ome dissolves with the anhydrous THF of 200ml.Splash into 1.54ml NMM under 0 ℃, regulate pH value 8～9.4.45g (14.42mmol) Z-Arg-OH and 6.84g (18.03mmol) HBTU adds reaction flask, NMM regulates PH to 8～9, and the variation of reaction monitoring pH value is to keep PH8～9, every 6 hours TLC (chloroform/methanol 10: 1) monitoring
The disappearance situation of HClLys (Cbz)-Asp (Obzl)-Val-Tyr-OMe.About 48 hours
HClLys (Cbz)-Asp (Obzl)-Val-Tyr-OMe primitive reaction is complete.Reaction solution decompression is spin-dried for, and uses sherwood oil (20ml * 3) successively, anhydrous diethyl ether (20ml * 3), and ethyl acetate (20ml * 5), unsaturated carbonate hydrogen is received the aqueous solution (50ml * 5), and pure water (50ml * 3) grinds bubble and washes.Get faint yellow solid powder 11.08g (productive rate 87.9%) behind the sample drying.In chloroform/methanol (9/1) development system is single-point, R
f=0.31, fusing point: 115.5～118.7 ℃, [α]
D 20=-11 ° (C=0.75MeOH).
8.Z-Arg-Lys (Z)-preparation of Asp-Val-Tyr-OH
11.08g (10.57mmol) Z-Arg-Lys (Cbz)-Asp (Obzl)-Val-Tyr-OMe uses 100ml CH
3OH dissolving slowly splashes into 20ml 2NNaOH/H under 0 ℃
2O, 0 ℃ was stirred 3～4 hours, and chloroform/methanol (10/1) shows that raw material point disappears.Reaction solution KHSO
4/ H
2O continues to be acidified to pH value about 7, a large amount of white precipitates occur, filters, and filtrate is spin-dried for methyl alcohol, occurs a large amount of the precipitation again, filters, and merges precipitation, pure water (20ml * 3) washing, the dry faint yellow solid powder 8.54g (productive rate 85.2%) that gets.At chloroform/methanol/H
2R in O (1/1/0.1) development system
f=0.5.Fusing point: 148.8～153.2 ℃.[α]
D 20＝-10°(C＝2MeOH)。
9.H-Arg-Lys-Asp-Val-Tyr-OH preparation
8.54g (9.00mmol) Z-Arg-Lys (Cbz)-Asp-Val-Tyr-OH, ethanol/water (1: 1) 200ml dissolving adds 5%Pd/C 1g, stirs, and feeds hydrogen, and reaction is about 5 hours under the room temperature, chloroform/methanol/H
2O (1/1/0.1) shows that raw material point disappears, and filters, and filtrate decompression is spin-dried for ethanol, and water is removed in lyophilize then, gets crude product 8.20g.
10.H-Arg-Lys-Asp-Val-Tyr-OH refining
Adopt the desalination of C18 reversed-phase column purifying.
The crude product pre-treatment: the 8.20gH-Arg-Lys-Asp-Val-Tyr-OH crude product, dissolved in distilled water filters.
Filler: C18,1 meter of column length, 0.1 meter of internal diameter,
Elution requirement: gradient elution, flow velocity 1ml/min with 1%HOAC aqueous solution wash-out, is changed to the 5% acetonitrile 1%HOAC aqueous solution earlier after 1 hour, be converted to the 15% acetonitrile 1%HOAC aqueous solution after 1 hour again.
Collect sample: the outflow situation that adopts TLC triketohydrindene hydrate coloration method test sample, and according to HPLC detection data gathering principal constituent, merge elutriant, after the lyophilize 24 hours, add the continuous freeze-drying of dissolved in distilled water once more and got 4.63g white lyophilized powder (productive rate 75.6%) finished product thymopeptide-5 (H-Arg-Lys-Asp-Val-Tyr-OH) in 36 hours.
[α]
D 20=° (C=1 Glacial acetic acid), HPLC detect purity greater than 99%.
The present invention has been made and having illustrated by indefiniteness embodiment.But person of skill in the art will appreciate that, do not departing under aim of the present invention and the scope, can make various modifications, replacement and change the present invention.For example, also can take " 2+3 " segment to contract and scheme synthesized thymopeptide-5.Synthetic at first respectively Z-Arg-Lys (Z)-OH (I) and H-Asp (OR)-Val-Tyr-OR (II), then with HBTU for contracting and agent synthesizes
Z-Arg-Lys (Z)-Asp (OR)-Val-Tyr-OR obtains thymopeptide-5 through saponification, hydrogenolysis again.Simple and clear route is shown in scheme 2.
Claims (11)
1. the method for a synthetic thymopeptide-5 Arg-Lys-Asp-Val-Tyr-OH, it is raw material that described method adopts unprotected Arg of side chain functionalities and Tyr, carries out the reaction of following steps:
(1) under alkaline condition, Tyr-OR and B-Val-OH are reacted in the presence of condensing agent, obtain the B-Val-Tyr-OR dipeptides;
(2) adopt the ammonia end deprotection of deprotecting regent, obtain the Val-Tyr-OR dipeptides the dipeptides that makes in (1);
(3) under alkaline condition, make the product that makes in (2) and B-Asp (OR)-OH reacts in the presence of condensing agent, obtain B-Asp (OR)-the Val-Tyr-OR tripeptides;
(4) adopt the ammonia end deprotection of deprotecting regent with the tripeptides that makes in (3), obtain Asp (OR)-the Val-Tyr-OR tripeptides;
(5) under alkaline condition, make the product that makes in (4) and B-Lys (Z)-OH reacts in the presence of condensing agent, obtain B-Lys (Z)-Asp (OR)-the Val-Tyr-OR tetrapeptide;
(6) adopt the ammonia end deprotection of deprotecting regent with the tetrapeptide that makes in (5), obtain Lys (Z)-Asp (OR)-the Val-Tyr-OR tetrapeptide;
(7) under alkaline condition, the product and the Z-Arg-OH that make in (6) are reacted in the presence of condensing agent, the Z-Arg-Lys that is protected (Z)-and Asp (OR)-the Val-Tyr-OR pentapeptide;
(8) with the carboxyl-protecting group of aspartic acid and tyrosine on the pentapeptide chain that makes in (7) through the saponification deprotection, obtain Z-Arg-Lys (Z)-the Asp-Val-Tyr-OH pentapeptide;
(9) with arginine and lysine amino protecting group on the pentapeptide chain that makes in (8) through the hydrogenolysis deprotection, obtain the Arg-Lys-Asp-Val-Tyr-OH pentapeptide;
Wherein the protecting group B of the α amino of Xie Ansuan, aspartic acid, Methionin is a tertbutyloxycarbonyl, the different third oxygen carbonyl of biphenyl, the carbobenzoxy-(Cbz) of carbobenzoxy-(Cbz) and replacement or fluorenylmethyloxycarbonyl, the protecting group Z of the side chain amino of arginic α amino and Methionin is the different third oxygen carbonyl or the fluorenylmethyloxycarbonyl of carbobenzoxy-(Cbz), tertbutyloxycarbonyl, biphenyl of carbobenzoxy-(Cbz) and replacement, the protecting group R of the α carboxyl of tyrosine is methyl esters, ethyl ester, the tert-butyl ester, benzyl ester and replaces the benzyl ester, the protecting group R of the side chain carboxyl group of aspartic acid is the benzyl ester, methyl esters, ethyl ester;
In described step (1), (3), (5) and (7), described alkaline condition is meant the pH value for 7-11, and connects reactive polypeptide and carry out to the temperature condition of room temperature at 0 ℃.
2. the method for claim 1, described method comprises that also the product that will make in (9) through the desalination of C18 reversed-phase column purifying, obtains purified product.
3. the method for claim 1; wherein the protecting group B of the α amino of Xie Ansuan, aspartic acid, Methionin is the Boc-group; the protecting group Z of the side chain amino of arginic α amino and Methionin is a carbobenzoxy-(Cbz); and the protecting group R of the α carboxyl of tyrosine is a methyl esters, and the protecting group R of the side chain carboxyl group of aspartic acid is the benzyl ester.
4. the process of claim 1 wherein that condensation step employing DCC/HOBt, HBTU, DIC, BOP in step (1), (3), (5) and (7) are condensing agent.
5. the method for claim 4, wherein to adopt HBTU be condensing agent to the condensation step in the step (7).
6. the process of claim 1 wherein in step (1), (3), (5) and (7) the pH value to 8-9.
7. the process of claim 1 wherein that the reaction elder generation of step (1), (3) and (5) carries out, and carries out again under room temperature under 0 ℃.
8. the process of claim 1 wherein that being reflected under 0 ℃ of step (7) carry out.
9. the process of claim 1 wherein that step (2), (4) and (6) middle HCl/ of employing ethyl acetate are as deprotecting regent.
10. the process of claim 1 wherein in the step (8) adopts aqueous sodium hydroxide solution to carry out the saponification deprotection under 0 ℃.
11. the step of the process of claim 1 wherein (9) is finished with hydrogen reaction in room temperature and in the presence of palladium on carbon.
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| CNB2004100227416A CN1308343C (en) | 2004-06-01 | 2004-06-01 | Process for preparing thymopentin |
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| CN1308343C true CN1308343C (en) | 2007-04-04 |
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| CN100455593C (en) * | 2005-04-08 | 2009-01-28 | 重庆华邦制药股份有限公司 | Process for synthesis of thymopentin |
| CN103450338B (en) * | 2012-05-29 | 2016-08-24 | 首都医科大学 | The Thymopentin that heterocyclic carboxylic acid is modified, it prepares, antitumor action and application |
| CN103421087A (en) * | 2013-04-12 | 2013-12-04 | 上海捌加壹医药科技有限公司 | Liquid-phase synthesizing method for polypeptide |
| CN109836455B (en) * | 2019-01-30 | 2021-11-16 | 西北工业大学 | Liquid phase synthesis method of thymopentin based on phosphorus or phosphorus acyloxy diphenyl methanol and derivatives thereof and assistance |
| CN117088944B (en) * | 2023-10-20 | 2023-12-19 | 深圳市维琪科技股份有限公司 | Pentapeptide, composition and application thereof |
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| Title |
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| 固相合成胸腺五肽的研究进展 王玲等，化工进展，第22卷第2期 2003 * |
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