CN1442428A - Technology of one kettle method for liquid phase synthesizing thymopentapeptide - Google Patents
Technology of one kettle method for liquid phase synthesizing thymopentapeptide Download PDFInfo
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- CN1442428A CN1442428A CN03111273A CN03111273A CN1442428A CN 1442428 A CN1442428 A CN 1442428A CN 03111273 A CN03111273 A CN 03111273A CN 03111273 A CN03111273 A CN 03111273A CN 1442428 A CN1442428 A CN 1442428A
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- protecting group
- fmoc
- obzl
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- 238000000034 method Methods 0.000 title claims abstract description 35
- 239000007791 liquid phase Substances 0.000 title claims abstract description 10
- 230000002194 synthesizing effect Effects 0.000 title claims abstract description 6
- 238000005516 engineering process Methods 0.000 title claims description 13
- 125000006239 protecting group Chemical group 0.000 claims abstract description 25
- 108010016626 Dipeptides Proteins 0.000 claims abstract description 8
- ITMCEJHCFYSIIV-UHFFFAOYSA-N triflic acid Chemical compound OS(=O)(=O)C(F)(F)F ITMCEJHCFYSIIV-UHFFFAOYSA-N 0.000 claims abstract description 8
- 150000003862 amino acid derivatives Chemical class 0.000 claims abstract description 4
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 19
- 238000006243 chemical reaction Methods 0.000 claims description 19
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims description 18
- 239000000243 solution Substances 0.000 claims description 16
- 239000012043 crude product Substances 0.000 claims description 15
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 15
- 229960001701 chloroform Drugs 0.000 claims description 14
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 claims description 13
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 claims description 13
- 238000010511 deprotection reaction Methods 0.000 claims description 13
- 239000002253 acid Substances 0.000 claims description 11
- 238000001035 drying Methods 0.000 claims description 10
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 9
- -1 fluorenylmethyloxycarbonyl aspartic acid Chemical compound 0.000 claims description 9
- 239000000047 product Substances 0.000 claims description 9
- 238000003756 stirring Methods 0.000 claims description 9
- 238000010168 coupling process Methods 0.000 claims description 8
- 239000002994 raw material Substances 0.000 claims description 8
- KSDTXRUIZMTBNV-INIZCTEOSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)butanedioic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CC(=O)O)C(O)=O)C3=CC=CC=C3C2=C1 KSDTXRUIZMTBNV-INIZCTEOSA-N 0.000 claims description 6
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 6
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 6
- 230000008878 coupling Effects 0.000 claims description 6
- 238000005859 coupling reaction Methods 0.000 claims description 6
- 150000003667 tyrosine derivatives Chemical class 0.000 claims description 6
- HNKJADCVZUBCPG-UHFFFAOYSA-N thioanisole Chemical compound CSC1=CC=CC=C1 HNKJADCVZUBCPG-UHFFFAOYSA-N 0.000 claims description 5
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 claims description 4
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 claims description 4
- 125000006847 BOC protecting group Chemical group 0.000 claims description 4
- 235000003704 aspartic acid Nutrition 0.000 claims description 4
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 claims description 4
- 238000004821 distillation Methods 0.000 claims description 4
- 239000003456 ion exchange resin Substances 0.000 claims description 4
- 229920003303 ion-exchange polymer Polymers 0.000 claims description 4
- 239000003960 organic solvent Substances 0.000 claims description 4
- 150000003053 piperidines Chemical class 0.000 claims description 4
- 238000000746 purification Methods 0.000 claims description 4
- 239000007787 solid Substances 0.000 claims description 4
- 238000001291 vacuum drying Methods 0.000 claims description 4
- 125000005519 fluorenylmethyloxycarbonyl group Chemical group 0.000 claims description 3
- 238000004062 sedimentation Methods 0.000 claims description 3
- 229910000029 sodium carbonate Inorganic materials 0.000 claims description 3
- 125000005931 tert-butyloxycarbonyl group Chemical group [H]C([H])([H])C(OC(*)=O)(C([H])([H])[H])C([H])([H])[H] 0.000 claims description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 3
- 239000004475 Arginine Substances 0.000 claims description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 2
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 claims description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 2
- 230000002378 acidificating effect Effects 0.000 claims description 2
- 229910021529 ammonia Inorganic materials 0.000 claims description 2
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 claims description 2
- BVCTWRNVKLXEQC-HNNXBMFYSA-N benzyl (2s)-2-amino-3-(4-hydroxyphenyl)propanoate Chemical compound C([C@H](N)C(=O)OCC=1C=CC=CC=1)C1=CC=C(O)C=C1 BVCTWRNVKLXEQC-HNNXBMFYSA-N 0.000 claims description 2
- 238000000605 extraction Methods 0.000 claims description 2
- 229930182817 methionine Natural products 0.000 claims description 2
- 239000012074 organic phase Substances 0.000 claims description 2
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 claims description 2
- 238000005342 ion exchange Methods 0.000 abstract description 2
- 238000003786 synthesis reaction Methods 0.000 abstract description 2
- 230000015572 biosynthetic process Effects 0.000 abstract 1
- 238000005580 one pot reaction Methods 0.000 abstract 1
- 230000002992 thymic effect Effects 0.000 abstract 1
- SJRJJKPEHAURKC-UHFFFAOYSA-N N-Methylmorpholine Chemical compound CN1CCOCC1 SJRJJKPEHAURKC-UHFFFAOYSA-N 0.000 description 18
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 6
- KRHYYFGTRYWZRS-UHFFFAOYSA-N Fluorane Chemical compound F KRHYYFGTRYWZRS-UHFFFAOYSA-N 0.000 description 6
- 238000004809 thin layer chromatography Methods 0.000 description 5
- DVBUCBXGDWWXNY-SFHVURJKSA-N (2s)-5-(diaminomethylideneamino)-2-(9h-fluoren-9-ylmethoxycarbonylamino)pentanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CCCN=C(N)N)C(O)=O)C3=CC=CC=C3C2=C1 DVBUCBXGDWWXNY-SFHVURJKSA-N 0.000 description 4
- 230000000975 bioactive effect Effects 0.000 description 4
- OZECDDHOAMNMQI-UHFFFAOYSA-H cerium(3+);trisulfate Chemical compound [Ce+3].[Ce+3].[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O OZECDDHOAMNMQI-UHFFFAOYSA-H 0.000 description 4
- 229910000333 cerium(III) sulfate Inorganic materials 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 238000012544 monitoring process Methods 0.000 description 4
- QPJSUIGXIBEQAC-UHFFFAOYSA-N n-(2,4-dichloro-5-propan-2-yloxyphenyl)acetamide Chemical compound CC(C)OC1=CC(NC(C)=O)=C(Cl)C=C1Cl QPJSUIGXIBEQAC-UHFFFAOYSA-N 0.000 description 4
- 238000012545 processing Methods 0.000 description 4
- 238000001953 recrystallisation Methods 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 description 3
- 239000002904 solvent Substances 0.000 description 3
- RDOXTESZEPMUJZ-UHFFFAOYSA-N anisole Chemical compound COC1=CC=CC=C1 RDOXTESZEPMUJZ-UHFFFAOYSA-N 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000003921 oil Substances 0.000 description 2
- 239000012071 phase Substances 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 238000010926 purge Methods 0.000 description 2
- 239000011347 resin Substances 0.000 description 2
- 229920005989 resin Polymers 0.000 description 2
- PSWFFKRAVBDQEG-YGQNSOCVSA-N thymopentin Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 PSWFFKRAVBDQEG-YGQNSOCVSA-N 0.000 description 2
- 210000001541 thymus gland Anatomy 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- UGNIYGNGCNXHTR-SFHVURJKSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-methylbutanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](C(C)C)C(O)=O)C3=CC=CC=C3C2=C1 UGNIYGNGCNXHTR-SFHVURJKSA-N 0.000 description 1
- FODJWPHPWBKDON-IBGZPJMESA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-4-[(2-methylpropan-2-yl)oxy]-4-oxobutanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CC(=O)OC(C)(C)C)C(O)=O)C3=CC=CC=C3C2=C1 FODJWPHPWBKDON-IBGZPJMESA-N 0.000 description 1
- UMRUUWFGLGNQLI-QFIPXVFZSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-6-[(2-methylpropan-2-yl)oxycarbonylamino]hexanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CCCCNC(=O)OC(C)(C)C)C(O)=O)C3=CC=CC=C3C2=C1 UMRUUWFGLGNQLI-QFIPXVFZSA-N 0.000 description 1
- SZXBQTSZISFIAO-ZETCQYMHSA-N (2s)-3-methyl-2-[(2-methylpropan-2-yl)oxycarbonylamino]butanoic acid Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)OC(C)(C)C SZXBQTSZISFIAO-ZETCQYMHSA-N 0.000 description 1
- HNICLNKVURBTKV-NDEPHWFRSA-N (2s)-5-[[amino-[(2,2,4,6,7-pentamethyl-3h-1-benzofuran-5-yl)sulfonylamino]methylidene]amino]-2-(9h-fluoren-9-ylmethoxycarbonylamino)pentanoic acid Chemical compound C12=CC=CC=C2C2=CC=CC=C2C1COC(=O)N[C@H](C(O)=O)CCCN=C(N)NS(=O)(=O)C1=C(C)C(C)=C2OC(C)(C)CC2=C1C HNICLNKVURBTKV-NDEPHWFRSA-N 0.000 description 1
- 208000023275 Autoimmune disease Diseases 0.000 description 1
- 101000753683 Bos taurus Thymopoietin-2 Proteins 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 239000000898 Thymopoietin Substances 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 230000000274 adsorptive effect Effects 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 235000011114 ammonium hydroxide Nutrition 0.000 description 1
- DYLKTBYVTWFYGQ-FTBISJDPSA-N benzyl (2s)-2-amino-3-(4-phenylmethoxyphenyl)propanoate;hydrochloride Chemical compound Cl.C([C@H](N)C(=O)OCC=1C=CC=CC=1)C(C=C1)=CC=C1OCC1=CC=CC=C1 DYLKTBYVTWFYGQ-FTBISJDPSA-N 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 238000009104 chemotherapy regimen Methods 0.000 description 1
- 239000000287 crude extract Substances 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 208000002672 hepatitis B Diseases 0.000 description 1
- 230000036737 immune function Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 239000013067 intermediate product Substances 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000012264 purified product Substances 0.000 description 1
- 238000001959 radiotherapy Methods 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000004366 reverse phase liquid chromatography Methods 0.000 description 1
- 238000004007 reversed phase HPLC Methods 0.000 description 1
- YCRCICOIWZWOTQ-UHFFFAOYSA-N silicon;trifluoromethanesulfonic acid Chemical compound [Si].OS(=O)(=O)C(F)(F)F YCRCICOIWZWOTQ-UHFFFAOYSA-N 0.000 description 1
- 238000010532 solid phase synthesis reaction Methods 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 238000010189 synthetic method Methods 0.000 description 1
- WROMPOXWARCANT-UHFFFAOYSA-N tfa trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F.OC(=O)C(F)(F)F WROMPOXWARCANT-UHFFFAOYSA-N 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Landscapes
- Peptides Or Proteins (AREA)
Abstract
A process for liquid-phase synthesis of thymic pentapeptide from amino acid derivatives by one-pot method includes such steps as synthesizing dipeptide, sythesizing tripeptide, synthesizing tetrapeptide, synthesizing pentapeptide, removing protecting group by TFMSA system to generate coarse product, and purifying by ion exchange. Its advantages are simple process, low cost, and high quality and purity (more than 95%).
Description
Technical field
The invention belongs to biochemical field, particularly a kind of preparation technology's method of bioactive peptide material.
Background technology
Contain many bioactive peptide materials relevant in the thymus gland, contain the bioactive peptide more than 15 kinds in its crude extract with immunocompetence.Thymopoietin II (ThymopoietinII) is wherein a kind of important active ingredient.This bioactive peptide is made of 49 amino acid, and molecular weight is 5562 dalton, and the 32-36 position residue segment of this molecule is the active fragments that thymus gland generates II, has the various activity of original molecule, is called as thymopeptide-5 (TP-5).The thymopeptide-5 molecular structure is simple, and (TP-5 Arg-Lys-Asp-Val-Tyr), is easy to the chemical synthesis of peptide synthetic.TP-5 went on the market in Italy in 1985, and commodity are called Timunox, also had the man pharmaceutical factory of number to research and develop in the U.S..Injection " thymopeptide-5 " lyophilized injectable powder of producing with pharmaceutcal corporation, Ltd in domestic Hainan Province is sold at home.This medicine recovers to have obvious curative effects to the cancer patient immunologic function; But assisted surgery, radiotherapy, chemotherapy regimen are able to fine finishing, and can prevent and treat complication such as infection.The treatment of hepatitis B, great surgical operation and severe infections, autoimmune disorder also there is good effect.At present the synthetic method of TP-5 is divided into two kinds of liquid phase synthesizing method and solid-phase synthesis substantially.
With the most close prior art of the present invention is the synthetic thymopeptide-5 of liquid phase method.This method is a raw material with the amino acid derivative of tertbutyloxycarbonyl (tBoc) protection; the synthetic------building-up process that crude product generates that tetrapeptide is synthetic---pentapeptide is synthetic---that tripeptides is synthetic through dipeptides; building-up process will be passed through deprotection, and---working cycle of coupling---purifying---recrystallization, the intermediate product of promptly synthetic peptide all need purifying and recrystallization again.Said deprotection is to remove protecting group with hydrofluoric acid (HF), and said purifying is with C
18RPLC (C
18RP-HPLC) purified product.
Existing liquid phase method synthesizes thymopeptide-5, owing to use acetonitrile to make solvent in purge process, consumption is big, the cost height; Because repeatedly many purifying and recrystallization process consuming time are carried out in circulation, make that the process time of synthetic thymopeptide-5 is oversize; Because the high risk of HF, the HF of final step cuts peptide and resin key and removes protecting group and needs finish in special device, makes technological process and equipment used numerous and diverse.
Summary of the invention
The technical problem to be solved in the present invention be simplify technology, reduce work hours, reduce cost, safety operation, preparation high reactivity, highly purified thymopeptide-5 product.
The technical problem to be solved in the present invention realizes by changing technological process.Promptly having developed a kind of novel process---one kettle way synthesizes thymopeptide-5 (TP-5).This technology is a kind of liquid phase method between tBoc and Fmoc from principle, and system removes protecting group with trifluoromethanesulfonic acid (TFMSA), and finishes purifying products with ion exchange method.
Concrete technology content is, is raw material with the amino acid derivative, and process has dipeptides synthetic, and---tripeptides is synthetic, and---tetrapeptide is synthetic---pentapeptide is synthetic---, and crude product generates; It is characterized in that the added coupling raw material of peptide building-up process is synthetic by each peptide to be tyrosine derivative and tertbutyloxycarbonyl Xie Ansuan (BOC-Val-OH) or tyrosine derivative and fluorenylmethyloxycarbonyl Xie Ansuan (Fmoc-Val-OH), fluorenylmethyloxycarbonyl aspartic acid or tertbutyloxycarbonyl aspartic acid, (Fmoc-Asp-OH or BOC-Asp-OH), fluorenylmethyloxycarbonyl Methionin (Fmoc-Lys-OH), fluorenylmethyloxycarbonyl arginine (Fmoc-Arg-OH) in proper order; Each peptide building-up process comprises the process of the coupling-soda acid wash-out-drying that dewaters-deprotection base; Said coupling is to carry out linked reaction at the coupling raw material that adds in the small peptide of deprotection base that trichloromethane dissolved tyrosine or building-up process obtain; Said soda acid wash-out is that the reaction solution that will obtain through coupling process is washed with sodium carbonate solution, water, citric acid solution extraction, and every kind of solution is washed the collection organic phase 2～5 times; The said drying that dewaters is that organic addition anhydrous magnesium sulfate is dewatered, and organic solvent is removed in underpressure distillation, gets solid vacuum-drying again; Said deprotection base is that the sample that will obtain after the drying that dewater is dissolved in trichloromethane, adds trifluoroacetic acid (TFA) solution or piperidines/chloroform soln, stirs and removes BOC protecting group or Fmoc protecting group; After said crude product generates and is synthetic pentapeptide; take off BOC protecting group or Fmoc protecting group; adding trifluoromethanesulfonic acid/trifluoroacetic acid (TMFSA/TFA), thioanisole or adding trifluoromethanesulfonic acid silicon ester/trifluoroacetic acid, thioanisole remove the residue protecting group, and ether sedimentation makes crude product.
Aforesaid tyrosine derivative can be tyrosine benzyl ester hydrochloride (H-Tyr-ObzlHCl), and synthetic dipeptides or synthetic tripeptides both can adopt tertbutyloxycarbonyl (Boc) system, also can adopt fluorenylmethyloxycarbonyl (Fmoc) system.Be that the synthetic dipeptides is Boc-Val-Tyr (Bzl) Obzl or Fmoc-Val-Tyr (Bzl)-Obzl; The synthetic tripeptides is Fmoc-Asp (Obut)-Val-Tyr (Bzl)-Obzl or Boc-Asp (OBzl)-Val-Tyr (Bzl)-OBzl.Do like this and help improving productive rate.
In the aforesaid soda acid wash-out, being used to extract sodium carbonate solution, the citric acid solution concentration of washing can be 3% to 10%.
In the deprotection of crude product generative process, be to remove protecting group, or remove protecting group with trifluoromethanesulfonic acid silicon ester-sulfydryl phenylmethylether (TMSOTf-thioanisole) system with trifluoromethanesulfonic acid-sulfydryl phenylmethylether (TFMSA-thioanisole) system.Compare with background technology, this is the method for an innovation.
Different with background technology is only final product to be carried out purification process.Purge process is with slightly acidic and strong-acid ion exchange resin purifying TP-5 crude product, with the weak ammonia eluted product; Or, carry out wash-out with acetic acid with weakly alkaline and strong basic ion exchange resin purifying TP-5.After the purified processing, the purity of product is higher.
The present invention makes technology simply quick because pilot process does not need long purifying and recrystallization process consuming time; Owing to do not use acetonitrile solvent, production cost reduces greatly; Owing to not using HF to make operational safety; The quality of product also reaches domestic medicine inspection standard, and purity reaches more than 95%.
Embodiment
(Boc-Val-Tyr's (Bzl)-Obzl) is synthetic for embodiment 1 dipeptides
Take by weighing H-Tyr (Bzl)-OBzlHCL (M.W 397.9,1.99 gram, 5 mmoles), add about 15 milliliters of trichloromethanes under 25-30 ℃, stir, dissolving.Transfer pH to 7 to 8 with N-methylmorpholine (NMM).To wherein add 1.3 times of excessive Boc-Val-OH (M.W217.27,1.41 the gram, 6.5 mmoles), bop reagent (M.W442.5,2.87 the gram, 6.5 mmoles), HOBt (M.W153.15,0.99 the gram, 6.5 mmoles), stirred, carry out linked reaction 2 hours.With thin-layer chromatography (TLC), cerous sulfate colour developing, monitoring reaction performance.
Reaction solution gets Boc-Val-Tyr (Bzl)-OBzl2.51 gram, yield 89.66% after soda acid wash-out, the drying treatment that dewaters.
(Fmoc-Asp (Obut)-Val-Tyr's (Bzl)-Obzl) is synthetic for embodiment 2 tripeptides
Get Boc-Val-Tyr (Bzl)-OBzl2.51 gram,, slough the Boc protecting group through the processing of deprotection base with 4 milliliters of trichloromethane dissolvings.Product dissolves with trichloromethane, and NMM transfers pH to 7-8.To wherein adding 1.3 times of excessive Fmoc-Asp (OBut)-OH, 2.4 grams, bop reagent 2.57 grams, the HOBt0.89 gram, NMM 640 microlitres are in stirring at room, carried out linked reaction 2 hours.With TLC, cerous sulfate colour developing, monitoring reaction performance.
Reaction solution gets Fmoc-Asp (Obut)-Val-Tyr (Bzl)-OBzl, 85% yield after soda acid wash-out, the drying treatment that dewaters.
(Fmoc-Lys (Boc)-Asp (Obut)-Val-Tyr's (Bzl)-Obzl) is synthetic for embodiment 3 tetrapeptides
Get Fmoc-Asp (Obut)-Val-Tyr (Bzl)-OBzl and dissolve, handle removing the Fmoc protecting group through the deprotection base with 10 to 15 milliliters of trichloromethanes.Product dissolves with trichloromethane, adds 1.3 times of excessive Fmoc-Lys-(Boc)-OH, BOP, and HOBt, NMM is in stirring at room, carried out linked reaction 2 hours.With TLC, cerous sulfate colour developing, monitoring reaction performance.
Reaction solution gets Fmoc-Lys (Boc)-Asp (Obut)-Val-Tyr (Bzl)-OBzl, 90% yield after soda acid wash-out, the drying treatment that dewaters.
(Fmoc-Arg (Pbf)-Lys (Boc)-Asp (Obut)-Val-Tyr's (Bzl)-Obzl) is synthetic for embodiment 4 pentapeptides
Get Fmoc-Lys (Boc)-Asp (Obut)-Val-Tyr (Bzl)-OBzl with 15 milliliters of left and right sides trichloromethane dissolvings, remove the Fmoc protecting group through the processing of deprotection base.Product dissolves with trichloromethane, adds 1.3 times of excessive Fmoc-Arg (Pbf)-OH, bop reagent, and HOBt, NMM is in stirring at room, carried out linked reaction 2 hours.With TLC, cerous sulfate colour developing, monitoring reaction performance.
Reaction solution gets Fmoc-Arg (Pbf)-Lys (Boc)-Asp (Obut)-Val-Tyr (Bzl)-OBzl, 80% yield through after soda acid wash-out, the drying treatment that dewaters.
The generation of embodiment 5 crude products (H-Arg-Lys-Asp-Val-Tyr-OH)
1. take off the Fmoc protecting group
Fmoc-Arg (Pbf)-Lys (Boc)-Asp (Obut)-Val-Tyr (Bzl)-OBzl is sloughed the Fmoc protecting group through the processing of deprotection base.
2. take off remaining protecting group
In the pentapeptide that takes off the Fmoc protecting group, add an amount of 1M TFMSA/TFA, thioanisole, in ice bath, stir, reacted 2 hours, remove the residue protecting group.The reaction solution ether sedimentation of precooling, centrifugal, vacuum-drying gets the H-Arg-Lys-Asp-Val-Tyr-OH crude product, 95% yield.
Substitute trifluoromethanesulfonic acid (TFMSA) with the trifluoromethanesulfonic acid silicon ester and take off remaining protecting group, method is the same with effect.
Embodiment 6 purification process
Adopt the method for ion exchange chromatography that crude product is carried out purifying.Purifies and separates be according to TP-5 under condition of different pH, electrically charged difference, TP-5 is different with the adsorptive power of resin, adopts the pH gradient to carry out wash-out, collects the sample peak.
Concrete experiment condition:
Purifying instrument: the BioCAD 700E of P.E company type
Purification column: POROS CM 4.6mm * 100mm
Solvent systems: A phase water, pH=5.5
B phase ammoniacal liquor, pH=11.0
Gradient elution, B is by 0% to 100%.
Embodiment 7 takes off the Boc protecting group
Sample with an amount of trichloromethane dissolving, is added TFA solution, under room temperature, stir, reacted 1 hour, remove the Boc protecting group.The reaction solution underpressure distillation is removed excessive TFA, organic solvent, get yellow solid, vacuum-drying.
Embodiment 8 takes off the Fmoc protecting group
Sample with an amount of trichloromethane dissolving, is added 20% piperidines/chloroform soln, under room temperature, stir, reacted 1 hour, remove the Fmoc protecting group.The reaction solution underpressure distillation is removed excessive piperidines, organic solvent get yellow oil.With sherwood oil wash 2 times (2 * 25ml), solid.
Claims (3)
1, the technology of the synthetic thymopeptide-5 of a kind of one kettle way liquid phase is raw material with the amino acid derivative, and process has dipeptides synthetic, and---tripeptides is synthetic, and---tetrapeptide is synthetic---pentapeptide is synthetic---, and crude product generates; It is characterized in that the added coupling raw material of peptide building-up process is synthetic by each peptide to be tyrosine derivative and tertbutyloxycarbonyl Xie Ansuan or tyrosine derivative and fluorenylmethyloxycarbonyl Xie Ansuan, fluorenylmethyloxycarbonyl aspartic acid or tertbutyloxycarbonyl aspartic acid, fluorenylmethyloxycarbonyl Methionin, fluorenylmethyloxycarbonyl arginine in proper order; Each peptide building-up process comprises the process of the coupling-soda acid wash-out-drying that dewaters-deprotection base; Said coupling is to carry out linked reaction at the coupling raw material that adds in the small peptide of deprotection base that trichloromethane dissolved tyrosine or building-up process obtain; Said soda acid wash-out is that the reaction solution that will obtain through coupling process is washed with sodium carbonate solution, water, citric acid solution extraction, and every kind of solution is washed the collection organic phase 2～5 times; The said drying that dewaters is that organic addition anhydrous magnesium sulfate is dewatered, and organic solvent is removed in underpressure distillation, gets solid vacuum-drying again; Said deprotection base is that the sample that will obtain after the drying that dewater is dissolved in trichloromethane, adds trifluoroacetic acid solution or piperidines/chloroform soln, stirs and removes BOC protecting group or Fmoc protecting group; Said crude product takes off BOC protecting group or Fmoc protecting group after generating and being synthetic pentapeptide, and adding trifluoromethanesulfonic acid/trifluoroacetic acid, thioanisole or trifluoromethanesulfonic acid silicon ester/trifluoroacetic acid, thioanisole remove the residue protecting group, and ether sedimentation makes crude product.
The technology of 2, synthesizing thymopeptide-5 according to the described one kettle way liquid phase of claim 1, it is characterized in that, tyrosine derivative in the raw material is the tyrosine benzyl ester hydrochloride, and the synthetic dipeptides is Boc-Val-Tyr (Bzl) Obzl or Fmoc-Val-Tyr (Bzl)-Obzl; The synthetic tripeptides is Fmoc-Asp (Obut)-Val-Tyr (Bzl)-Obzl or Boc-Asp (OBzl)-Val-Tyr (Bzl)-OBzl.
3, according to the technology of claim 1 or the synthetic thymopeptide-5 of 2 described one kettle way liquid phases, it is characterized in that, after crude product generates, carry out purification process, with slightly acidic and strong-acid ion exchange resin purifying crude product, with the weak ammonia eluted product; Or, carry out wash-out with acetic acid with weakly alkaline and strong basic ion exchange resin purifying crude product.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1305895C (en) * | 2004-01-13 | 2007-03-21 | 兰州凯博生物化学技术有限公司 | Method for synthesizing thymic-pentapeptide by mixing acid anhydride method |
| 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 |
| CN1865279B (en) * | 2005-05-18 | 2010-07-14 | 周达明 | Solid phase polypeptide synthesis preparation method for thymopoietin pentapeptide |
| CN101724019B (en) * | 2008-10-13 | 2013-02-20 | 中国人民解放军军事医学科学院毒物药物研究所 | Thymopeptide-5 active esters, medicinal composition containing same and application thereof |
| CN101525369B (en) * | 2008-03-04 | 2014-04-09 | 赢创德固赛有限责任公司 | Method used for producing peptide |
| CN103819539A (en) * | 2013-05-22 | 2014-05-28 | 兰州大学第一医院 | Liquid phase synthesis method for thymopentin |
| CN106916219A (en) * | 2017-04-10 | 2017-07-04 | 上海化工研究院有限公司 | A kind of synthetic method of cold labeling alpha-lactalbumin feature peptide fragment |
| CN109836455A (en) * | 2019-01-30 | 2019-06-04 | 西北工业大学 | Thymopeptide-5 liquid-phase synthesis process based on phosphorus or phosphorous acyloxy benzhydrol and its derivative and auxiliary |
-
2003
- 2003-03-24 CN CNB031112730A patent/CN1202131C/en not_active Expired - Lifetime
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1305895C (en) * | 2004-01-13 | 2007-03-21 | 兰州凯博生物化学技术有限公司 | Method for synthesizing thymic-pentapeptide by mixing acid anhydride method |
| 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 |
| CN1865279B (en) * | 2005-05-18 | 2010-07-14 | 周达明 | Solid phase polypeptide synthesis preparation method for thymopoietin pentapeptide |
| CN101525369B (en) * | 2008-03-04 | 2014-04-09 | 赢创德固赛有限责任公司 | Method used for producing peptide |
| CN101724019B (en) * | 2008-10-13 | 2013-02-20 | 中国人民解放军军事医学科学院毒物药物研究所 | Thymopeptide-5 active esters, medicinal composition containing same and application thereof |
| CN103819539A (en) * | 2013-05-22 | 2014-05-28 | 兰州大学第一医院 | Liquid phase synthesis method for thymopentin |
| CN106916219A (en) * | 2017-04-10 | 2017-07-04 | 上海化工研究院有限公司 | A kind of synthetic method of cold labeling alpha-lactalbumin feature peptide fragment |
| CN109836455A (en) * | 2019-01-30 | 2019-06-04 | 西北工业大学 | Thymopeptide-5 liquid-phase synthesis process based on phosphorus or phosphorous acyloxy benzhydrol and its derivative and auxiliary |
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|---|---|
| CN1202131C (en) | 2005-05-18 |
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