CN1240712C - Method of preparing tyrosine-serine-leucine tripeptide - Google Patents
Method of preparing tyrosine-serine-leucine tripeptide Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 27
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- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims abstract description 10
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- 238000006243 chemical reaction Methods 0.000 claims description 28
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- 239000000243 solution Substances 0.000 claims description 22
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Abstract
The present invention relates to a method for preparing tyrosine-serine-leucine tripeptide, which uses leucine, serine of which the hydroxy of two side chains is not protected, and tyrosine as raw materials to synthesize the tyrosine-serine-leucine tripeptide (CMS024). The method of the present invention has the advantages of safety and low cost, and is suitable for industrial production.
Description
Technical field
The present invention relates to the preparation method of polypeptide.More particularly, the present invention relates to the preparation method of junket-Si-bright tripeptides (CMS024).
Technical background
In polypeptide synthesis method; liquid-phase synthesis process commonly used is to adopt the maximum protection strategy; promptly to as monomeric amino acid whose relevant functional group, especially amino, carboxyl and hydroxyl are all made protection, thereby carry out the synthetic polypeptide of condensation reaction between amino acid again.Describing in detail about the polypeptide synthetic can be referring to document " polypeptide is synthetic ",, Huang Weide, Chen Changqing work in 1985; " polypeptide drugs chemistry ", nineteen ninety, Peng Shiqi.
For example; when synthetic junket-Si-bright tripeptides, adopt the maximum protection strategy process, can Boc-Leu-OH be the feedstock production cesium salt; then with the esterification of carboxylic end benzyl; slough Boc with HCl subsequently, expose the ammonia end, then the Serine of protecting with benzyl (Bzl) according to DCC method (HOBT is auxiliary condensing agent) and side chain and 2; the tyrosine of 6-dichloro benzyl (Dcb) protection progressively connects peptide; then adopt HCl to slough Boc, slough various protecting groups with HF at last, obtain junket-Si-bright tripeptides.Described employing maximum protection strategy process, the simple and clear route of synthetic junket-Si-bright tripeptides scheme is seen Fig. 1:
The maximum protection strategy adopts the amino acid price costliness of side chain protected, need pass through violent HF deprotection at last at synthetic.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 ofly to avoid adopting that strong toxicity and irritating HF carry out deprotection, cost is cheap and can be in the method for the synthetic CMS024 of industrial enforcement.
Summary of the invention
The invention provides a kind of method that adopts the synthetic described junket-Si of minimum protection strategy-bright tripeptides (CMS024), said method comprising the steps of:
(1) makes L-Leu-OH and SOCl
2In alcoholic solution, react, obtain the ester of L-Leu-OH;
(2) ester that makes the L-Leu-OH that makes in (1) carries out condensation reaction with the L-Ser-OH to the hydroxyl protection of side chain to the protection of ammonia end but not in the presence of condensing agent, make the Serine that the ammonia end protects and the ester of leucic dipeptides condenses;
(3) product that makes in (2) is carried out deprotection, remove blocking group;
(4) make the product that makes in (3) and L-Tyr-OH to the protection of ammonia end but not in the presence of condensing agent, carry out condensation reaction, make the ester of tyrosine, Serine and leucic tripeptides condenses that the ammonia end protects the hydroxyl protection of side chain;
(5) make the product that makes in (4) in alcoholic solvent, in the presence of alkali, be hydrolyzed the H-Tyr-Ser-Leu-OH that is protected;
(6) product that makes in (5) is carried out deprotection, remove blocking group, obtain H-Tyr-Ser-Leu-OH.
According to one embodiment of the invention, the product that makes in (6) in Sephadex G 15, is carried out purifying with the 5%HOAC aqueous solution, obtain H-Tyr-Ser-Leu-OH.
With respect to the deprotection condition of HF, the security of the inventive method increases, and simultaneously industrialization is not had obstacle.In addition,, therefore adopt the synthetic cost of protection route of the present invention also less, and do not need the device of HF deprotection of the costliness of maximum protection route because the amino acid whose price of side chain no protective is comparatively cheap.
The inventive method by selecting proper reaction conditions, prevents the generation of side reaction effectively in preparation process, the product that makes is determined as single-point through thin-layer chromatography.
Description of drawings
Fig. 1 is the simple and clear route map that adopts the synthetic junket-Si of maximum protection strategy process-bright tripeptides scheme.
Fig. 2 is the simple and clear route map when adopting the synthetic junket-Si of minimum protection strategy process-bright tripeptides.
Detailed Description Of The Invention
Although do not wish to be subjected to the restriction of any theory, thinking that the present invention is based on the following fact: because 1) CMS024 is for only containing the little peptide of three amino-acid residues; 2) the nucleophilic reaction activity of the hydroxyl of the side chain of tyrosine and Serine is relative amino less; Therefore in building-up process, can the hydroxyl of two side chains not protected.
At first, the applicant has prepared leucic ester, progressively connects peptide with the Serine and the tyrosine of side chain no protective, and ester group is sloughed in saponification, sloughs the salt that the Boc protection obtains CMS024 at last.
Simple and clear route map when the present invention of being shown in Figure 2 adopts the synthetic junket-Si of minimum protection strategy process-bright tripeptides.
1.L-Leu-OH esterification
Adopt SOCl
2Alcoholic solution with the Leu-OH esterification, with the protection Leu the carboxylic end.Wherein Leu-OH elder generation and SOCl
2Form Leu-Cl, then be reacted into ester with alcohol.Used alcoholic solvent can be methyl alcohol, ethanol or benzylalcohol etc.The present invention preferably uses methyl alcohol.Reaction was carried out 4 hours at 0 ℃ earlier, progressively was warming up to room temperature subsequently, by the thin-layer chromatography monitoring, disappeared until raw material point.Be generally about 8 hours.The product that makes is the hydrochloride form of L-Leu-OH ester.
2.Boc the preparation of the ester of the leucine of protection and the dipeptides condenses of Serine
With the ester dissolution with solvents of above-mentioned product HClLeu-OH, regulate the pH value to certain value with organic bases.On the other hand; to only protect the Boc-L-Ser-OH dissolution with solvents of (alcoholic extract hydroxyl group is unbound state) to amino; add condensing agent, subsequently at a certain temperature with the ester solution hybrid reaction of HClLeu-OH, in reaction process, adopt organic bases to keep the pH value within the specific limits.Obtain the ester of the dipeptides condenses of the leucine of Boc protection and Serine.
Adopt Boc-as amino protecting agent in this step reaction, described in addition amino protecting agent also can be selected from and be usually used in any protective material of polypeptide in synthetic, as carbobenzoxy-(Cbz) (Z).
DMF, NMP etc. that used solvent can be selected from tetrahydrofuran (THF), heavily steam preferably use tetrahydrofuran (THF).
Used organic bases can be selected from N-methylmorpholine (NMM), triethylamine and DIEA (diisopropyl ethyl amine) etc., preferably uses NMM.
Select for use DCC/HOBt as condensing agent in this reaction, described in addition condensing agent also can be selected from DIC, HBTU, BOP etc. and be usually used in any condensing agent of polypeptide in synthetic in step.
In 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 8-10.
Temperature of reaction can be 0 ℃ to room temperature.When at room temperature reacting, the reaction times is shorter, is multiple spot but detect through thin-layer chromatography, and product purity descends, and productive rate descends.And, under room temperature, react again after 2 hours in reaction under 0 ℃ earlier, then obtaining detecting through thin-layer chromatography is pure product.
3. deprotection
The deprotection of the dipeptides condenses of Boc protection 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 HCl/ ethyl acetate as deprotecting regent.
4.Boc the preparation of the ester of the tripeptides condenses of leucine, Serine and the tyrosine of protection
The selection of the preparation condition of the ester of the leucine of the selection of the preparation condition of the ester of the tripeptides condenses of leucine, Serine and the tyrosine of Boc protection and Boc protection and the dipeptides condenses of Serine is similar.
Adopt Boc as amino protecting agent in this step reaction, described in addition amino protecting agent also can be selected from and be usually used in any protective material of polypeptide in synthetic, as carbobenzoxy-(Cbz) (Z).In one embodiment of the invention, employing only protects the Boc-L-Tyr-OH of (phenolic hydroxyl group is unbound state) to react as raw material to amino.
DMF, NMP etc. that used solvent can be selected from tetrahydrofuran (THF), heavily steam preferably use tetrahydrofuran (THF).Used organic bases can be selected from N-methylmorpholine, triethylamine and DIEA etc., preferably uses NMM.Condensing agent can be selected from DCC/HOBt, DIC, HBTU, BOP etc., preferably uses DCC/HOBt.The pH of reaction process is controlled at 7-11, more preferably in the scope of 8-10.Temperature of reaction is controlled to be earlier reacted 2 hours down at 0 ℃, reacted under room temperature again.
5.Boc-Tyr-Ser-Leu-OH preparation
The previous step reaction product is dissolved with alcoholic solvent, slowly be added dropwise to alkali at a certain temperature and carry out saponification reaction, slough the ester group of carboxylic acid end.
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 ℃.
6.Boc-Tyr-Ser-Leu-OH deprotection
Similar with the deprotection condition in 3, can adopt deprotecting regents such as HCl/ ethyl acetate or TFA/ methylene dichloride to implement.But more preferably use the HCl/ ethyl acetate as deprotecting regent.
7. refining
With the HClTyr-Ser-Leu-OH deionized water dissolving that makes, adopt Sephadex G15 gel column to carry out desalination, finished product is tyroserleutide H-Tyr-Ser-Leu-OH.
Description of drawings
Fig. 1 is the simple and clear route map when adopting the synthetic junket-Si of maximum protection strategy process-bright tripeptides.
Fig. 2 is the simple and clear route map of the present invention when adopting the Minimal Protective strategy process to synthesize junket-Si-bright tripeptides.
Preferred embodiment of the present invention
Below in conjunction with Fig. 2, by non-limiting embodiment the present invention is further specified.
In this article, being abbreviated as of agents useful for same:
DCC: dicyclohexylcarbodiimide
The HOBt:1-hydroxy benzo triazole
The NMM:N-methyl morpholine
THF: oxolane
Embodiment 1
1.HClLeu-OMe preparation
L-LeuOH (Sigma) and the SOCl for preparing in advance with 20g (0.152mol)2(chemical pure, Beijing Chemical Plant)/CH3OH (chemical pure, Beijing Chemical Plant) solution (is chilled to-10 ℃, by the CH of 260ml3OH and 70ml newly steam SOCl2Consist of) progressively to mix, outer temperature is kept-10 ℃, and L-Leu-OH is progressively dissolved. Then the reactant liquor that obtains progressively is warming up to room temperature in 0 ℃ of reaction 4 hours, reacts 8 hours again, disappears through TLC (chloroform/methanol, 20/1) detection display raw material point. Reactant liquor is evaporated to dried, the Ex-all hydrogen chloride that dissociates. Residue dissolves with a small amount of methyl alcohol, adds absolute ether to muddy, places, and crystallization filters and obtains 22.02g (74.8%) target compound. In chloroform/methanol (5/1) solvent, it is single-point. Fusing point: 151～153 ℃ [α]D 20＝20°(C＝2 MeOH)
2.Boc-Ser-Leu-OMe preparation (1)
With 100ml anhydrous tetrahydro furan (analyzes pure, Beijing Chemical Plant) dissolving, the solution that obtains is with N-methylmorpholine accent pH to 7 with the HClLeu-OMe of 22.02g (0.121mol), be cooled to 0 ℃ for subsequent use.
With the HOBt (Fluka) of Boc-Ser-OH (available from Yangzhou Bao Sheng Corp.), the 17.2g (0.127mol) of 24.87g (0.121mol) with the dissolving of 100ml anhydrous tetrahydro furan, be chilled to 0 ℃, slowly add the DCC (Fluka) of 26.24g (0.127mol), stirred 5 minutes in 0 ℃. Then mix with top solution, stirred 2 hours in 0 ℃, (Fluka) keeps PH=7 with N-methylmorpholine, in stirring at room 24 hours, disappears through TLC (chloroform/methanol/acetic acid, (20/1/0.4)) detection display raw material point. Filter reactant mixture, reduced pressure concentration filtrate is ground residue repeatedly with benzinum, with the thick material that the 400ml acetic acid ethyl dissolution obtains, uses respectively saturated NaHCO3The aqueous solution (75ml * 5), the saturated NaCl aqueous solution (75ml * 1), 5%KHSO4The aqueous solution (75ml * 5) and the saturated NaCl aqueous solution (15ml * 3) washing, organic layer filters through anhydrous sodium sulfate drying, and filtrate decompression is spin-dried for and obtains the thick target compound of 40.0g (100.8%). Detect through TLC, adopt (20/1/0.4) two kind of system of ethyl acetate/petroleum ether (1/1) and chloroform/methanol/acetic acid to launch, obtain 3 points.
3.Boc-Ser-Leu-OMe preparation (2)
With 100ml anhydrous tetrahydro furan (analyzes pure, Beijing Chemical Plant) dissolving, the solution that obtains is with N-methylmorpholine accent pH to 8 with the HClLeu-OMe of 22.02g (0.121mol), be cooled to 0 ℃ for subsequent use.
Boc-Ser-OH (available from Yangzhou Bao Sheng Corp.) with 24.87g (0.121mol), 17.2g HOBt (0.127mol) (Fluka) dissolves with the 100ml anhydrous tetrahydro furan, be chilled to 0 ℃, the DCC (Fluka) that slowly adds 26.24g (0.127mol), 0 ℃ was stirred 5 minutes. Then mix with top solution, 0 ℃ was stirred 2 hours, and (Fluka) keeps PH=8 with N-methylmorpholine, stirring at room 10 hours, and chloroform/methanol/acetic acid, (20/1/0.4) shows the disappearance of raw material point. Filter reactant mixture, reduced pressure concentration filtrate, residue grinds repeatedly with benzinum, and the thick material that obtains is used the 400ml acetic acid ethyl dissolution, uses respectively saturated NaHCO3The aqueous solution (75ml * 5), the saturated NaCl aqueous solution (75ml * 1), 5%KHSO4The aqueous solution (75ml * 5) and the saturated NaCl aqueous solution (15ml * 3) washing, organic layer filters through anhydrous sodium sulfate drying, and filtrate decompression is spin-dried for and obtains the thick target compound of 41.2g (103.8%). Detect through TLC, adopt ethyl acetate/petroleum ether (1/1) and the good for both sides development system of chloroform/methanol/acetic acid (20/1/0.4), be single-point, this product can be directly used in next step reaction.
4.Boc-Ser-Leu-OMe preparation (3)
With 100ml anhydrous tetrahydro furan (analyzes pure, Beijing Chemical Plant) dissolving, the solution that obtains is with N-methylmorpholine accent pH to 10 with the HClLeu-OMe of 22.02g (0.121mol), be cooled to 0 ℃ for subsequent use.
Boc-Ser-OH (available from Yangzhou Bao Sheng Corp.) with 24.87g (0.121mol), 17.2g HOBt (0.127mol) (Fluka) dissolves with the 100ml anhydrous tetrahydro furan, be chilled to 0 ℃, slowly add the DCC (Fluka) of 26.24g (0.127mol), stirred 5 minutes in 0 ℃. Then mix with top solution, stirred 2 hours in 0 ℃, (Fluka) keeps PH10 with N-methylmorpholine, in stirring at room 10 hours, detects through TLC, adopts chloroform/methanol/acetic acid (20/1/0.4) to launch, and shows the disappearance of raw material point. Filter reactant mixture, reduced pressure concentration filtrate is ground residue repeatedly with benzinum, and the thick material 400ml acetic acid ethyl dissolution that obtains is used respectively saturated NaHCO3The aqueous solution (75ml * 5), the saturated NaCl aqueous solution (75ml * 1), 5%KHSO4The aqueous solution (75ml * 5) and the saturated NaCl aqueous solution (15ml * 3) washing, organic layer filters through anhydrous sodium sulfate drying, and filtrate decompression is spin-dried for and obtains the thick target compound of 41.2g (103.8%). Detect through TLC, adopt ethyl acetate/petroleum ether (1/1) and (20/1/0.4) two kind of development system of chloroform/methanol/acetic acid, be single-point, this product can be directly used in next step reaction.
5.Boc-Ser-Leu-OMe preparation (4)
With 100ml anhydrous tetrahydro furan (analyzes pure, Beijing Chemical Plant) dissolving, the solution that obtains is with N-methylmorpholine accent pH to 11 with the HClLeu-OMe of 22.02g (0.121mol), be cooled to 0 ℃ for subsequent use.
Boc-Ser-OH (available from Yangzhou Bao Sheng Corp.) with 24.87g (0.121mol), 17.2g HOBt (0.127mol) (Fluka) dissolves with the 100ml anhydrous tetrahydro furan, be chilled to 0 ℃, the DCC (Fluka) that slowly adds 26.24g (0.127mol), 0 ℃ was stirred 5 minutes. Then mix with top solution, stirred 2 hours in 0 ℃, (Fluka) keeps PH 11 with N-methylmorpholine, in stirring at room 10 hours, detects through TLC, adopts chloroform/methanol/acetic acid (20/1/0.4) to launch, and shows the disappearance of raw material point. Filter reactant mixture, reduced pressure concentration filtrate is ground residue repeatedly with benzinum, and the thick material 400ml acetic acid ethyl dissolution that obtains is used respectively saturated NaHCO3The aqueous solution (75ml * 5), the saturated NaCl aqueous solution (75ml * 1), 5%KHSO4The aqueous solution (75ml * 5) and the saturated NaCl aqueous solution (15ml * 3) washing, organic layer filters through anhydrous sodium sulfate drying, and filtrate decompression is spin-dried for and obtains the thick target compound of 32.9g (83%).
6.HClSer-Leu-OMe preparation
At room temperature stirred 1 hour 30 minutes after the Boc-Ser-Leu-OMe of 41.2g (0.124mol) mixed with 200ml 4N HCL/ ethyl acetate solution, a large amount of white precipitates appear in solution, detect through TLC, adopt TLC (chloroform/methanol, 20/1) launches, show that raw material point disappears. Filter, with ethyl acetate cyclic washing filter cake, with the free HCl of Ex-all. Repeatedly grind residue with absolute ether, obtain target compound 26.23g (78.7%). Detect through TLC, adopt chloroform/methanol (10/1) development system, be single-point. Fusing point: 128～129 ℃, [α]D 20＝-18°(C＝2
MeOH)
7.Boc-Tyr-Ser-Leu-OMe preparation (1)
With 125ml anhydrous tetrahydro furan (analytical pure, Beijing Chemical Plant) dissolving, the solution that obtains is transferred pH to 8 with N-methylmorpholine (Fluka) with the HClSer-Leu-OMe of 26.23g (0.097mol), be cooled to 0 ℃ standby.
Boc-Tyr-OH (available from Sichuan three high biochemical company limiteds) with 27.45g (0.097mol), 13.85g HOBt (0.102mol) (Fluka) dissolves with the 125ml anhydrous tetrahydro furan, be chilled to 0 ℃, slowly add the DCC (Fluka) of 21.13g (0.102mol), stirred 5 minutes in 0 ℃.Mix with top solution then, stirred 2 hours in 0 ℃ again, keep PH=8,, detect, adopt chloroform/methanol/acetate (20/1/0.4) to launch, show raw material point disappearance through TLC in stirring at room 28 hours with N-methylmorpholine.Filter reaction mixture, concentrating under reduced pressure filtrate is ground residue repeatedly with sherwood oil, and the solid that obtains 400ml acetic acid ethyl dissolution is used saturated NaHCO respectively
3The aqueous solution (75ml * 3), the saturated NaCl aqueous solution (75ml * 1), 5%KHSO
4The anhydrous sodium sulfate drying organic layer is used in the aqueous solution (75ml * 3) and the saturated NaCl aqueous solution (15ml * 3) washing, filters, and filtrate decompression is spin-dried for and obtains 49.12g (101.6%) target compound.Detect through TLC, adopt ethyl acetate/petroleum ether (1/1) and (20/1/0.4) two kind of development system of chloroform/methanol/acetate, be single-point.Fusing point: 147.4～147.8 ℃ [α]
D 20=-22 ° (C=2 MeOH)
8.Boc-Tyr-Ser-Leu-OMe preparation (2)
With 125ml anhydrous tetrahydro furan (analytical pure, Beijing Chemical Plant) dissolving, the solution that obtains is transferred pH to 10 with N-methylmorpholine (Fluka) with the HClSer-Leu-OMe of 26.23g (0.097mol), be cooled to 0 ℃ standby.
Boc-Tyr-OH (available from Sichuan three high biochemical company limiteds) with 27.45g (0.097mol), 13.85g HOBt (0.102mol) (Fluka) dissolves with the 125ml anhydrous tetrahydro furan, be chilled to 0 ℃, slowly add the DCC (Fluka) of 21.13g (0.102mol), stirred 5 minutes in 0 ℃.Mix with top solution then, stirred 2 hours in 0 ℃ again, keep PH=8,, detect, adopt chloroform/methanol/acetate (20/1/0.4) to launch, show raw material point disappearance through TLC in stirring at room 28 hours with N-methylmorpholine.Filter reaction mixture, concentrating under reduced pressure filtrate is ground residue repeatedly with sherwood oil, and the solid that obtains 400ml acetic acid ethyl dissolution is used saturated NaHCO respectively
3The aqueous solution (75ml * 3), the saturated NaCl aqueous solution (75ml * 1), 5%KHSO
4The aqueous solution (75ml * 3) and the saturated NaCl aqueous solution (15ml * 3) washing, the organic layer anhydrous sodium sulfate drying filters, and filtrate decompression is spin-dried for and obtains 48.30g (100.0%) target compound.Detect through TLC, adopt ethyl acetate/petroleum ether (1/1) and (20/1/0.4) two kind of development system of chloroform/methanol/acetate, be single-point.Fusing point: 147.4～147.8 ℃ [α]
D 20=-22 ° (C=2 MeOH)
9.Boc-Tyr-Ser-Leu-OH preparation (1)
With the Boc-Tyr-Ser-Leu-OMe of 49.12g (0.099mol), use 400ml CH
3OH (chemical pure, Beijing Chemical Plant) dissolving slowly splashes into NaOH (chemical pure, Beijing Chemical Plant)/H of 120ml 2N under 0 ℃
2O in 0 ℃ of stirring 5 hours, detects through TLC, adopts chloroform/methanol (10/1) to launch, and shows the disappearance of raw material point.Reaction solution KHSO
4/ H
2O transfers to neutrality, evaporated under reduced pressure methyl alcohol, KHSO
4/ H
2O continues to be acidified to pH value 1～2, with ethyl acetate (150ml * 3) extraction, merges organic layer, and uses H
2O (20ml * 3) repetitive scrubbing, filters through anhydrous sodium sulfate drying to neutral, and filtrate decompression is spin-dried for, and gets target compound 48.32g (100.8%).Detect through TLC, adopt chloroform/methanol/acetate (5/1/0.4) development system, be single-point, R
f=0.31, can be directly used in next step reaction.
Fusing point: 103.2～105.1 ℃.[α]
D 20＝-7°(C＝2 MeOH)
10.Boc-Tyr-Ser-Leu-OH preparation (2)
With the Boc-Tyr-Ser-Leu-OMe of 49.12g (0.099mol), use 400ml CH
3OH (chemical pure, Beijing Chemical Plant) dissolves, and slowly splashes into NaOH (chemical pure, Beijing Chemical Plant)/H of 120ml 2N under room temperature
2O at room temperature stirred 2 hours, detected through TLC, adopted chloroform/methanol (10/1) to launch, and showed that raw material point disappears.Reaction solution KHSO
4/ H
2O transfers to neutrality, evaporated under reduced pressure methyl alcohol, KHSO
4/ H
2O continues to be acidified to pH value 1～2, with ethyl acetate (150ml * 3) extraction, merges organic layer, and uses H
2O (20ml * 3) repetitive scrubbing, filters through anhydrous sodium sulfate drying to neutral, and filtrate decompression is spin-dried for, and gets target compound 47.50g (99.1%).Detect through TLC, adopt chloroform/methanol/acetate (5/1/0.4) development system, be multiple spot, show serious racemization has taken place.
11.HClTyr-Ser-Leu-OH preparation
Under 0 ℃, in the Boc-Tyr-Ser-Leu-OH of 48.32g, slowly add the 250ml4NHCl/ ethyl acetate, stirred 1 hour 30 minutes in 0 ℃, a large amount of precipitations appear in reaction solution, detect through TLC, adopt chloroform/methanol (10/1) to launch, show that raw material point disappears, filter, precipitation to there not being HCl, is used the 100ml dissolve with ethanol with the ethyl acetate repetitive scrubbing, places crystallization, filter collecting precipitation.In mother liquor, add sherwood oil again and carry out recrystallization, filter, collect crystallization.Merge target compound 35.5g (85.1%).[α]
D 20＝-6°(C＝2 H
2O)
12. the conversion of acid ion and desalination
35.5g HClTyr-Ser-Leu-OH is dissolved with small amount of deionized water, adopt SephadexG15 to change salt, eluent is the 5%HOAc aqueous solution, online detection of Ultraviolet Detector 215nm and triketohydrindene hydrate coloration method detect, collect principal constituent, freeze-drying gets 31.0gHOACTyr-Ser-Leu-OH[α]
D 20=+19 ° (C=2 Glacial acetic acid)
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.
Claims (10)
1. method for preparing tripeptide compound with following structure:
(H-Tyr-Ser-Leu-OH)
Said method comprising the steps of:
(1) makes L-Leu-OH and SOCl
2In alcoholic solution, react, obtain the ester of L-Leu-OH;
(2) ester that makes the L-Leu-OH that makes in (1) carries out condensation reaction with the L-Ser-OH to the hydroxyl protection of side chain to the protection of ammonia end but not in the presence of condensing agent, make the Serine that the ammonia end protects and the ester of leucic dipeptides condenses;
(3) product that makes in (2) is carried out deprotection, remove blocking group;
(4) make the product that makes in (3) and L-Tyr-OH to the protection of ammonia end but not in the presence of condensing agent, carry out condensation reaction, make the ester of tyrosine, Serine and leucic tripeptides condenses that the ammonia end protects the hydroxyl protection of side chain;
(5) make the product that makes in (4) in alcoholic solvent, in the presence of alkali, be hydrolyzed the H-Tyr-Ser-Leu-OH that is protected;
(6) product that makes in (5) is carried out deprotection, remove blocking group, obtain H-Tyr-Ser-Leu-OH.
2. the method for claim 1 also is included in after the step (6) product that will make through Sephadex G 15, carry out purifying with 5%HOAC aqueous solution wash-out.
3. the process of claim 1 wherein that the L-Ser-OH of protection is Boc-Ser-OH, and the L-Tyr-OH of protection is Boc-Tyr-OH.
4. the process of claim 1 wherein that used alcohol is methyl alcohol in step (1).
5. the process of claim 1 wherein that used condensing agent is DCC/HOBt in step (2) and step (4).
6. the process of claim 1 wherein that the pH value is 7-11 in the reaction of step (2) and step (4).
7. the method for claim 6, wherein the pH value is 8-10 in the reaction of step (2) and step (4).
8. the process of claim 1 wherein and all under 0 ℃, carry out earlier, under room temperature, carry out again in the reaction of step (2) and step (4).
9. the process of claim 1 wherein that the deprotection in step (3) adopts the HCl/ ethyl acetate to carry out.
10. the process of claim 1 wherein and implement down at 0 ℃ at the hydrolysis reaction of step (5).
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