CN1298732C - polypeptide microwave solid phase synthesis method - Google Patents
polypeptide microwave solid phase synthesis method Download PDFInfo
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- CN1298732C CN1298732C CNB2005100400363A CN200510040036A CN1298732C CN 1298732 C CN1298732 C CN 1298732C CN B2005100400363 A CNB2005100400363 A CN B2005100400363A CN 200510040036 A CN200510040036 A CN 200510040036A CN 1298732 C CN1298732 C CN 1298732C
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- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 54
- 229920001184 polypeptide Polymers 0.000 title claims abstract description 45
- 102000004196 processed proteins & peptides Human genes 0.000 title claims abstract description 45
- 238000000034 method Methods 0.000 title claims abstract description 24
- 238000010532 solid phase synthesis reaction Methods 0.000 title abstract description 8
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims abstract description 81
- 239000011347 resin Substances 0.000 claims abstract description 77
- 229920005989 resin Polymers 0.000 claims abstract description 77
- 238000006243 chemical reaction Methods 0.000 claims abstract description 59
- 150000001413 amino acids Chemical class 0.000 claims abstract description 44
- 238000009833 condensation Methods 0.000 claims abstract description 37
- 230000005494 condensation Effects 0.000 claims abstract description 37
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 32
- 230000005855 radiation Effects 0.000 claims abstract description 27
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims abstract description 16
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims abstract description 15
- 239000000706 filtrate Substances 0.000 claims abstract description 12
- PSHKMPUSSFXUIA-UHFFFAOYSA-N n,n-dimethylpyridin-2-amine Chemical compound CN(C)C1=CC=CC=N1 PSHKMPUSSFXUIA-UHFFFAOYSA-N 0.000 claims abstract description 12
- 238000002360 preparation method Methods 0.000 claims abstract description 12
- 125000006239 protecting group Chemical group 0.000 claims abstract description 9
- 238000001914 filtration Methods 0.000 claims abstract description 7
- 239000007788 liquid Substances 0.000 claims abstract description 7
- 238000010511 deprotection reaction Methods 0.000 claims abstract description 6
- 239000002904 solvent Substances 0.000 claims abstract description 6
- 238000005520 cutting process Methods 0.000 claims abstract description 5
- 229920003002 synthetic resin Polymers 0.000 claims abstract description 4
- 239000000057 synthetic resin Substances 0.000 claims abstract description 4
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract 2
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 84
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 57
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims description 52
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 claims description 26
- 239000000243 solution Substances 0.000 claims description 24
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 22
- 238000005303 weighing Methods 0.000 claims description 18
- 239000007790 solid phase Substances 0.000 claims description 17
- 230000035484 reaction time Effects 0.000 claims description 16
- 230000015572 biosynthetic process Effects 0.000 claims description 14
- 230000008569 process Effects 0.000 claims description 14
- 238000003786 synthesis reaction Methods 0.000 claims description 13
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 claims description 13
- 238000005406 washing Methods 0.000 claims description 13
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 claims description 12
- 239000000126 substance Substances 0.000 claims description 12
- 239000012043 crude product Substances 0.000 claims description 10
- 239000002253 acid Substances 0.000 claims description 9
- ASOKPJOREAFHNY-UHFFFAOYSA-N 1-Hydroxybenzotriazole Chemical compound C1=CC=C2N(O)N=NC2=C1 ASOKPJOREAFHNY-UHFFFAOYSA-N 0.000 claims description 8
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 claims description 8
- HNKJADCVZUBCPG-UHFFFAOYSA-N thioanisole Chemical compound CSC1=CC=CC=C1 HNKJADCVZUBCPG-UHFFFAOYSA-N 0.000 claims description 8
- -1 1-hydroxyl phenylpropyl Chemical group 0.000 claims description 7
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 7
- ZGYICYBLPGRURT-UHFFFAOYSA-N tri(propan-2-yl)silicon Chemical compound CC(C)[Si](C(C)C)C(C)C ZGYICYBLPGRURT-UHFFFAOYSA-N 0.000 claims description 7
- VYMPLPIFKRHAAC-UHFFFAOYSA-N 1,2-ethanedithiol Chemical compound SCCS VYMPLPIFKRHAAC-UHFFFAOYSA-N 0.000 claims description 6
- BWGNESOTFCXPMA-UHFFFAOYSA-N Dihydrogen disulfide Chemical compound SS BWGNESOTFCXPMA-UHFFFAOYSA-N 0.000 claims description 6
- BLRPTPMANUNPDV-UHFFFAOYSA-N Silane Chemical compound [SiH4] BLRPTPMANUNPDV-UHFFFAOYSA-N 0.000 claims description 6
- 239000007864 aqueous solution Substances 0.000 claims description 6
- 239000012964 benzotriazole Substances 0.000 claims description 6
- 150000002148 esters Chemical class 0.000 claims description 6
- 239000012261 resinous substance Substances 0.000 claims description 6
- QOSSAOTZNIDXMA-UHFFFAOYSA-N Dicylcohexylcarbodiimide Chemical compound C1CCCCC1N=C=NC1CCCCC1 QOSSAOTZNIDXMA-UHFFFAOYSA-N 0.000 claims description 5
- SECXISVLQFMRJM-UHFFFAOYSA-N N-Methylpyrrolidone Chemical compound CN1CCCC1=O SECXISVLQFMRJM-UHFFFAOYSA-N 0.000 claims description 5
- 238000001556 precipitation Methods 0.000 claims description 5
- 238000002390 rotary evaporation Methods 0.000 claims description 5
- 230000008961 swelling Effects 0.000 claims description 5
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 claims description 4
- QWVGKYWNOKOFNN-UHFFFAOYSA-N o-cresol Chemical compound CC1=CC=CC=C1O QWVGKYWNOKOFNN-UHFFFAOYSA-N 0.000 claims description 4
- QRUDEWIWKLJBPS-UHFFFAOYSA-N benzotriazole Chemical compound C1=CC=C2N[N][N]C2=C1 QRUDEWIWKLJBPS-UHFFFAOYSA-N 0.000 claims description 3
- 230000008859 change Effects 0.000 claims description 3
- 229910052760 oxygen Inorganic materials 0.000 claims description 3
- 239000001301 oxygen Substances 0.000 claims description 3
- 229910052698 phosphorus Inorganic materials 0.000 claims description 3
- 239000011574 phosphorus Substances 0.000 claims description 3
- 238000001308 synthesis method Methods 0.000 claims description 3
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 claims description 2
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical class CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 2
- DYUQAZSOFZSPHD-UHFFFAOYSA-N Phenylpropyl alcohol Natural products CCC(O)C1=CC=CC=C1 DYUQAZSOFZSPHD-UHFFFAOYSA-N 0.000 claims description 2
- 229920003180 amino resin Polymers 0.000 claims description 2
- 239000003795 chemical substances by application Substances 0.000 claims description 2
- 239000000460 chlorine Substances 0.000 claims description 2
- 229910052801 chlorine Inorganic materials 0.000 claims description 2
- 238000004090 dissolution Methods 0.000 claims description 2
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims description 2
- 238000005304 joining Methods 0.000 claims description 2
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 claims description 2
- 239000006228 supernatant Substances 0.000 claims description 2
- 150000003852 triazoles Chemical class 0.000 claims description 2
- FDKXTQMXEQVLRF-ZHACJKMWSA-N (E)-dacarbazine Chemical compound CN(C)\N=N\c1[nH]cnc1C(N)=O FDKXTQMXEQVLRF-ZHACJKMWSA-N 0.000 claims 1
- 239000004202 carbamide Substances 0.000 claims 1
- 239000012071 phase Substances 0.000 abstract description 4
- 150000001875 compounds Chemical class 0.000 abstract description 3
- 239000000047 product Substances 0.000 abstract description 3
- 238000001704 evaporation Methods 0.000 abstract 1
- 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 10
- JAUKCFULLJFBFN-VWLOTQADSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-[4-[(2-methylpropan-2-yl)oxy]phenyl]propanoic acid Chemical compound C1=CC(OC(C)(C)C)=CC=C1C[C@@H](C(O)=O)NC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21 JAUKCFULLJFBFN-VWLOTQADSA-N 0.000 description 7
- 230000000694 effects Effects 0.000 description 7
- 239000007791 liquid phase Substances 0.000 description 7
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 6
- 238000005516 engineering process Methods 0.000 description 6
- KLBPUVPNPAJWHZ-UMSFTDKQSA-N (2r)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-tritylsulfanylpropanoic acid Chemical compound C([C@@H](C(=O)O)NC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21)SC(C=1C=CC=CC=1)(C=1C=CC=CC=1)C1=CC=CC=C1 KLBPUVPNPAJWHZ-UMSFTDKQSA-N 0.000 description 5
- 229910021529 ammonia Inorganic materials 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 description 3
- 230000004913 activation Effects 0.000 description 3
- 238000006482 condensation reaction Methods 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- 238000002330 electrospray ionisation mass spectrometry Methods 0.000 description 3
- 230000008030 elimination Effects 0.000 description 3
- 238000003379 elimination reaction Methods 0.000 description 3
- 238000004128 high performance liquid chromatography Methods 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 238000005374 membrane filtration Methods 0.000 description 3
- 239000003960 organic solvent Substances 0.000 description 3
- 150000003053 piperidines Chemical class 0.000 description 3
- 239000002244 precipitate Substances 0.000 description 3
- 239000013558 reference substance Substances 0.000 description 3
- 230000004044 response Effects 0.000 description 3
- 238000007039 two-step reaction Methods 0.000 description 3
- 238000001291 vacuum drying Methods 0.000 description 3
- FZTIWOBQQYPTCJ-UHFFFAOYSA-N 4-[4-(4-carboxyphenyl)phenyl]benzoic acid Chemical compound C1=CC(C(=O)O)=CC=C1C1=CC=C(C=2C=CC(=CC=2)C(O)=O)C=C1 FZTIWOBQQYPTCJ-UHFFFAOYSA-N 0.000 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 description 2
- DHKHKXVYLBGOIT-UHFFFAOYSA-N acetaldehyde Diethyl Acetal Natural products CCOC(C)OCC DHKHKXVYLBGOIT-UHFFFAOYSA-N 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 description 2
- 230000004060 metabolic process Effects 0.000 description 2
- 238000010647 peptide synthesis reaction Methods 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- JFLSOKIMYBSASW-UHFFFAOYSA-N 1-chloro-2-[chloro(diphenyl)methyl]benzene Chemical compound ClC1=CC=CC=C1C(Cl)(C=1C=CC=CC=1)C1=CC=CC=C1 JFLSOKIMYBSASW-UHFFFAOYSA-N 0.000 description 1
- 238000005160 1H NMR spectroscopy Methods 0.000 description 1
- GOJUJUVQIVIZAV-UHFFFAOYSA-N 2-amino-4,6-dichloropyrimidine-5-carbaldehyde Chemical group NC1=NC(Cl)=C(C=O)C(Cl)=N1 GOJUJUVQIVIZAV-UHFFFAOYSA-N 0.000 description 1
- DYNFCHNNOHNJFG-UHFFFAOYSA-N 2-formylbenzoic acid Chemical compound OC(=O)C1=CC=CC=C1C=O DYNFCHNNOHNJFG-UHFFFAOYSA-N 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 239000007821 HATU Substances 0.000 description 1
- 238000005481 NMR spectroscopy Methods 0.000 description 1
- 230000006399 behavior Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
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- 230000005669 field effect Effects 0.000 description 1
- 125000005519 fluorenylmethyloxycarbonyl group Chemical group 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 239000013067 intermediate product Substances 0.000 description 1
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- 210000005036 nerve Anatomy 0.000 description 1
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- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
Classifications
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- 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 polypeptide microwave solid phase synthesis method relates to a polypeptide solid phase synthesis method under the assistance of microwave. The polypeptide microwave solid phase synthesis method comprises the following steps: (1) preparation of amino acid-resin complex: adding dichloromethane to swell polypeptide synthetic resin; adding a solvent into the carboxyl-terminal amino acid and the condensation reagent, filtering after the reaction is finished, adding the filtrate into resin, and simultaneously adding dimethylaminopyridine for reaction. (2) Removing the amino protecting group by microwave radiation: adding a deprotection reagent into the amino acid-resin compound, and reacting under microwave radiation. (3) Microwave radiation condensation of amino acid: dissolving the next amino acid and condensation reagent, adding into the amino acid-resin compound, and reacting under microwave radiation. (4) And (3) repeating (2) and (3) to synthesize the desired polypeptide by replacing the amino acid. (5) Preparing a crude polypeptide product: adding a cutting reagent into the synthesized peptide chain, and performing microwave radiation. Filtering after the reaction is finished, rotationally evaporating the filtrate, and adding glacial ethyl ether into the residual liquid to separate out a polypeptide phase product.
Description
Technical field
Process for solid-phase microwave synthesis of polypeptide relates to is polypeptide solid phase synthesis process under a kind of microwave-assisted, the invention belongs to organic chemistry and pharmaceutical chemistry field.
Background technology
In general, polypeptide refers to that it has diversified biological activity by being lower than the compound that 50 amino acid are formed.It is found that the polypeptide that is present in organism has tens thousand of kinds, and find that all cells can both synthesize polypeptide.Simultaneously, nearly all cell also all is subjected to polypeptides for modulating, and it relates to every field such as hormone, nerve, cell growth and reproduction.They are the important chemical messengers of linking up information between iuntercellular and organ, exercise its delicate information transfer function by modes of action such as internal secretion, neuroendocrine and even neurosecretions, thereby make organism be combined into the tight Controlling System of a series of height, regulate vital processes such as growth, growth, breeding, metabolism and behavior, for growing and metabolic process has and comprises complicated adjusting and direct acting multi-biological functional.
The chemiluminescent polypeptide synthesis method can be divided into the synthetic and solid-phase synthesis of liquid phase.Being reflected in the liquid phase of liquid phase synthesizing method carried out, owing to all need the intermediate product separation and purification is come out after each step reaction, so technology is too numerous and diverse, relatively difficulty is implemented in operation.Solid-phase synthesis adopts the polymkeric substance of insoluble carrier bonding, as long as filter simply repeatedly and wash, the reagent and the separation of by-products of solubility can be come out.Technology is simple, is the prefered method of chemically synthesized polypeptide.At present, the subject matter one that exists during solid-phase polypeptide is synthetic is owing to the mass transfer problem between the solid-liquid makes speed of response slower, each amino acid whose tie-time two hours or more than, the peptide preparation technology time is long; The 2nd, increased the synthetic cost of peptide owing to the excessive multiple of amino acid is high.
Summary of the invention
The objective of the invention is at above-mentioned weak point, a kind of process for solid-phase microwave synthesis of polypeptide is provided, solid-phase peptide synthesis under microwave-assisted is compared with traditional method, shortened the reaction times greatly, reduced amino acid whose excessive multiple, improve degree of purity of production, finally reduced the production cost of polypeptide.
Microwave refers to wavelength 1 μ m~1mm, and frequency is from the uhf electromagnetic wave of 300MHz~300GHz.Microwave has been widely used in each ambit.Microwave has been opened up this chemical frontier of microwave chemical in Application in Chemistry, promptly microwave directly and chemical system have an effect, thereby promote the carrying out of all kinds of chemical reactions.The action principle of microwave is at present main to exist two kinds of theories: the one, and polarizable medium produces the motion that is equivalent to " molecule stirrings " with its high speed rotating under the microwave field effect, thereby is heated equably and mix the raising speed of reaction.The 2nd, effect is very complicated to microwave to chemical reaction, also existing a kind of except having heat effect is not the non-thermal effect that is caused by temperature, it can change the kinetic property of reaction, reduces the activation energy of reaction, exists the influence of some specific non-thermal effect.Microwave technology have cleaning, efficient, power consumption less, pollute characteristics such as low, in subjects such as inorganic chemistry, organic chemistry analytical chemistry and environmental chemistry, obtained using widely.
Process for solid-phase microwave synthesis of polypeptide is to take following scheme to realize:
The present invention uses the solvent such as the dimethyl formamide (DMF) that can absorb microwave to be medium; microwave technology is applied in polypeptide to be removed in synthetic in the formation four-step reaction of the preparation of amino protecting group, amino acid condensation, polypeptide crude product and disulfide linkage; utilize the special effects of microwave; reduce the activation energy of reaction, improve speed of response.Used microwave frequency is 2450 hertz, and temperature and pressure can on-line Control, is control condition to keep steady temperature, and the power of microwave changes thereupon.
Process for solid-phase microwave synthesis of polypeptide is:
The preparation of amino acid-resin complexes: a polypeptide synthetic resins of weighing, can be a kind of in hydroxy resin such as king (wang) resin, chlorine resin such as 2-chloro-trityl chloride resin and aminoresin such as the Rink Amide resin, put into the reactor of band filtration unit, to wherein adding methylene dichloride (DCM), swelling resin 30~120min takes out dichloromethane solution then.Weighing carboxyl terminal amino acid and condensation reagent---dicyclohexylcarbodiimide (DCC) or DIC (DIC) and 1-hydroxyl phenylpropyl alcohol triazole (HOBt), put into the container that can seal, add dimethyl formamide or tetrahydrofuran (THF) (THF) or N-Methyl pyrrolidone (NMP), reaction 30~150min.Described amino acid is the protection amino acid that side chain functionalities and aminoterminal all are protected.The ratio of the amount of described amino acid and resinous substances is 7~3: 1; The ratio of amino acid and condensation reagent amount of substance is 1: 1~1.2.After reaction finishes, filter out precipitation, filtrate joining is equipped with in the reactor of band filtration unit of resin, add Dimethylamino pyridine (DMAP) simultaneously, reacted 4~20 hours.Wherein the ratio of the amount of Dimethylamino pyridine and resinous substances is 0.1~0.5: 1.Take out reaction solution then, with dimethyl formamide, methyl alcohol, methylene dichloride washing resin successively three times.
Microwave radiation removes amino protecting group: add deprotecting regent in amino acid-resin complexes that washing obtains in the above---piperidines/dimethyl formamide solution of 20~30%; to place microwave reactor to react then; microwave condition is: temperature is 20~70 ℃, and the reaction times is 3~10min.After reaction finishes, filter and take out solution, with dimethyl formamide, methyl alcohol (MeOH), washed with dichloromethane resin.
Microwave radiation condensation amino acid: weighing next protection amino acid and condensation reagent, put into can be airtight container, add dissolution with solvents.Wherein said condensation reagent comprises dicyclohexylcarbodiimide (DCC), 1-hydroxy benzo triazole (HOBt), benzotriazole-N, N, N ', N ' ,-tetramethyl-urea phosphofluoric acid ester (HBTU), 2-(7-azo benzotriazole)-N, N, N ', N '-tetramethyl-urea phosphofluoric acid ester (HATU), N-hydroxyl-7-azo benzotriazole (HOAT), block special condensing agent BOP reagent, phosphofluoric acid benzotriazole-1-base-oxygen base tripyrrole alkyl phosphorus (PyBOP), several in diisopropyl ethyl amine (DIEA) and their derivative.The ratio of the amount of described amino acid and resinous substances is 4~2: 1; The ratio of amino acid and condensation reagent amount of substance is 1: 1~1.2.Described solvent is: dimethyl formamide, pyridine, N-Methyl pyrrolidone, tetrahydrofuran (THF).Then solution is joined in the reactor that the amino acid-resin complexes that removes amino protecting group is housed, place microwave reactor to react, microwave condition is: temperature is 20~70 ℃, and the reaction times is 3~10min.After reaction finishes, take out reaction solution, with dimethyl formamide, methyl alcohol, washed with dichloromethane resin.
According to amino acid from carboxyl terminal to N-terminal order, change amino acid, repeat microwave radiation deprotection and the amino acid whose reaction of microwave radiation condensation, synthetic required peptide chain.
The preparation of polypeptide crude product: protection peptide chain-resin that will obtain above put into can be airtight container, add cutting reagent, react under the microwave radiation.Described cutting reagent comprises: trifluoroacetic acid (TFA), redistilled water, triethyl silicane (TES), tri isopropyl silane (TIS), phenol, thioanisole, methyl-phenoxide, dithioglycol (EDT) and methylene dichloride.Combination between them and mutually ratio can be 1. trifluoroacetic acid: tri isopropyl silane (or triethyl silicane): redistilled water=95%: 2.5%: 2.5% (v/v); 2. trifluoroacetic acid: phenol: redistilled water: methyl-phenoxide: thioanisole=80%: 5%: 5%: 2.5%: 2.5% (v/v); 3. trifluoroacetic acid: phenol: redistilled water: dithioglycol: thioanisole=80%: 5%: 5%: 2.5%: 2.5% (v/v); 4. trifluoroacetic acid: redistilled water=95%: 5% (v/v); 5. trifluoroacetic acid: methylene dichloride=50%: 50% (v/v).Microwave condition is: temperature is 30~60 ℃, and the reaction times is 10~20min.Filter out resin then, collect filtrate, rotary evaporation is removed trifluoroacetic acid.In remaining liquid, add the ice ether, be settled out polypeptide, centrifugal then, remove supernatant liquor, obtain required polypeptide.
For the polypeptide that contains disulfide linkage, adopting 5~30% methyl-sulphoxides (DMSO)/aqueous solution is oxygenant, synthetic disulfide linkage under microwave radiation, and microwave condition is: temperature is 20~60 ℃, the reaction times is 5~30min.
Described microwave reactor is made by U.S. CE M company, and model is Mars5.This microwave reactor temperature and pressure can on-line Control, makes reaction result have good repeatability and reliability.
The present invention has compared following characteristics with prior art:
1. improve the speed of reaction greatly.Conventional solid-phase peptide synthesis connects an amino acid and generally needs two hours time or longer time, and under microwave radiation, the time that connects an amino is no more than 10min, and speed of reaction has improved 11 times at least.Because microwave radiation has special effect, can reduce the activation energy of reaction, thereby add the carrying out of fast response.
2. under microwave radiation, carry out the synthetic of disulfide linkage, shortened the time of reaction, improved degree of purity of production and productive rate.
3. reduce the synthetic cost.In ordinary method, the ratio of amino acid and condensation reagent and resin is generally 5~3: 1, the excessive multiple that is to say amino acid and condensation reagent is 4~2 times, and amino acid and condensation reagent account for synthetic more than 80% of cost, is the major cause under the synthetic cost of solid-phase polypeptide is in not.And under microwave radiation, the excessive multiple of amino acid and condensation reagent can be reduced to 1 times, this just greatly reduces the synthetic cost of polypeptide.
4. shorten the synthesis cycle of polypeptide, improve synthetic efficient.
Embodiment
The present invention further illustrates by following example, and these examples should not be construed as limitation of the present invention.
The microwave method preparation of embodiment 1 thymopeptide-5
(1) weighing king resin (0.93mmol/g) 3 grams (2.79mmol) place reactor, add methylene dichloride 15mL swelling resin 120 minutes, take out dichloromethane solution.Weighing 19.53mmol Fmoc-Tyr (tBu)-OH (protection tyrosine) adds the dissolving of 10mL tetrahydrofuran (THF), and then adds 19.53mmol DIC and 19.53mmol 1-hydroxy benzo triazole, reaction 150min.After reaction finishes, cross the elimination precipitation, filtrate is joined in the resin, add the 1.395mmol Dimethylamino pyridine simultaneously, reacted 20 hours.The ratio of each material amount of substance is Fmoc-Tyr (tBu)-OH in the reaction: resin=7: 1; Fmoc-Tyr (tBu)-OH: condensation reagent=1: 1; Dimethylamino pyridine: resin=0.5: 1.Reaction filters out reaction solution after finishing, and adds methylene dichloride, each washing resin of methyl alcohol and dimethyl formamide 15mL three times.The vacuum-drying resin, the joint efficiency that records Fmoc-Tyr (tBu)-OH and resin is 85%.
(2) microwave-assisted removes fluorenylmethyloxycarbonyl (Fmoc) protecting group
Tyrosine-king's resin is placed reaction tubes, add 10mL 30% piperidines/dimethyl formamide solution then, be reflected in the microwave reactor and carry out.Microwave condition is: temperature is 70 ℃, and the reaction times is 10min.After reaction finishes, add methylene dichloride, each washing resin of methyl alcohol and dimethyl formamide 20mL three times.
(3) microwave-assisted condensation reaction
1) condensation Fmoc-Val-OH.Weighing Fmoc-Val-OH, phosphofluoric acid benzotriazole-1-base-oxygen base tripyrrole alkyl phosphorus and each 9.487mmol of 1-hydroxy benzo triazole are with the dissolving of 10mL N-Methyl pyrrolidone.Join then in the resin, add the diisopropyl ethyl amine (Fmoc-Val-OH: condensation reagent=1: 1 of 9.487mmol simultaneously; Fmoc-Val-OH: resin=4: 1), react under the microwave radiation.Microwave condition is: temperature is 70 ℃, and the reaction times is 10min.Take out reaction solution then, add methylene dichloride, each washing resin of methyl alcohol and dimethyl formamide 20mL three times.
2) prolongation of peptide chain.According to amino acid in the thymopeptide-5 from carboxyl terminal to N-terminal order (Tyr-Val-Asp-Lys-Arg), the consumption of amino acid and condensation reagent is identical with Fmoc-Val-OH, repeats condensation and deprotection two-step reaction, synthetic thymopeptide-5.
(4) preparation of thymopeptide-5 crude product
About weighing synthetic thymopeptide-5-resin 40mg, place the container that can seal, add 0.125mL redistilled water, 0.125mL triethyl silicane and 4.75mL trifluoroacetic acid (redistilled water: triethyl silicane: trifluoroacetic acid=2.5%: 2.5%: 95%, v/v), react 10min under the microwave radiation, temperature is 30 ℃.Reaction filters out resin after finishing, and uses 10mL washed with dichloromethane resin three times, collects filtrate, and rotary evaporation is removed trifluoroacetic acid and other organic solvents.The anhydrous diethyl ether that adds an amount of ice in remaining liquid is separated out thymopeptide-5, the centrifugal then thymopeptide-5 crude product that obtains.
(5) analysis of thymopeptide-5
The white precipitate that obtains after centrifugal is dissolved in the 5mL redistilled water,, adds 10 times of redistilled water dilutions then, make standard specimen, analyze the purity and the productive rate of thymopeptide-5 with high performance liquid chromatography (HPLC) with the thymopeptide-5 reference substance through the membrane filtration of 0.22 μ.Liquid-phase condition is: moving phase is 0.1% trifluoroacetic acid aqueous solution and methyl alcohol, and ratio is 85%: 15%; Flow velocity is 1mL/min; Wavelength is 275nm.Recording purity is 96.5%, calculates productive rate to be: 92.4%.
The microwave method of embodiment 2 amino acid and the excessive one times of following thymopeptide-5 of condensation reagent is synthetic
(1) weighing king resin (0.93mmol/g) 1 gram (0.93mmol) places reactor, adds methylene dichloride 10mL swelling resin 90 minutes, takes out dichloromethane solution.Weighing 3.4875mmol Fmoc-Tyr (tBu)-OH adds the dissolving of 10mL dimethyl formamide, and then adds 4.185mmol DIC and 4.185mmol 1-hydroxy benzo triazole, reaction 30min.After reaction finishes, cross the elimination precipitation, filtrate is joined in the resin, add the 0.465mmol Dimethylamino pyridine simultaneously, reacted 4 hours.The ratio of each material amount of substance is an amino acid in the reaction: resin=3: 1; Fmoc-Tyr (tBu)-OH: condensation reagent=1: 1.2; Dimethylamino pyridine: resin=0.1: 1.Reaction filters out reaction solution after finishing, and adds methylene dichloride, each washing resin of methyl alcohol and dimethyl formamide 10mL three times.The vacuum-drying resin, the joint efficiency that records Fmoc-Tyr (tBu)-OH and resin is 95%.
(2) microwave-assisted removes the Fmoc protecting group
Tyrosine-king's resin is placed reaction tubes, add 20mL 25% piperidines/dimethyl formamide solution then, be reflected in the microwave reactor and carry out.Microwave condition is: 40 ℃ of temperature, reaction times 6min.After reaction finishes, add methylene dichloride, each washing resin of methyl alcohol and dimethyl formamide 10mL three times.
(3) microwave-assisted condensation reaction
1) condensation Fmoc-Val-OH.Weighing Fmoc-Val-OH 1.767mmol, benzotriazole-N, N, N ', N ' ,-tetramethyl-urea phosphofluoric acid ester and each 1.945mmol of 1-hydroxy benzo triazole are with the dissolving of 10mL dimethyl formamide.Join then in the resin, add the diisopropyl ethyl amine (Fmoc-Val-OH: condensation reagent=1: 1.1 of 1.945mmol simultaneously; Fmoc-Val-OH: resin=2: 1), react under the microwave radiation.Microwave condition is: 30 ℃ of temperature, reaction times 5min.Take out reaction solution then, add methylene dichloride, each washing resin of methyl alcohol and dimethyl formamide 10mL three times.
2) prolongation of peptide chain.According to amino acid in the thymopeptide-5 from carboxyl terminal to N-terminal order, the consumption of amino acid and condensation reagent is identical with Fmoc-Val-OH, repeats condensation and deprotection two-step reaction, synthetic thymopeptide-5.
(4) preparation of thymopeptide-5 crude product
About weighing synthetic thymopeptide-5-resin 40mg, place the container that can seal, add 0.125mL redistilled water, 0.125mL tri isopropyl silane and 4.75mL trifluoroacetic acid (redistilled water: tri isopropyl silane: trifluoroacetic acid=2.5%: 2.5%: 95%, v/v), react 20min under the microwave radiation, temperature is 60 ℃.Reaction filters out resin after finishing, and uses 10mL washed with dichloromethane resin three times, collects filtrate, and rotary evaporation is removed trifluoroacetic acid and other organic solvents.The anhydrous diethyl ether that adds an amount of ice in remaining liquid is separated out thymopeptide-5, the centrifugal then thymopeptide-5 crude product that obtains.
(5) analysis of thymopeptide-5
The white precipitate that obtains after centrifugal is dissolved in the 5mL redistilled water,, adds 10 times of redistilled water dilutions then, make standard specimen, analyze the purity and the productive rate of thymopeptide-5 with high performance liquid chromatography (HPLC) with the thymopeptide-5 reference substance through the membrane filtration of 0.22 μ.Liquid-phase condition is: moving phase is 0.1% aqueous solution and methyl alcohol, and ratio is 85%: 15%; Flow velocity is 1mL/min; Wavelength is 275nm.Recording purity is 91%, calculates productive rate to be: 88.7%.
The evaluation of thymopeptide-5: the ESI-MS mass spectroscopy is target product M+1=680.4, and is consistent with theoretical molecular.
Nuclear magnetic resonance result is: 1H NMR (500MHz, D2O) δ: 6.67~6.98 (m, 4H, ArH), 3.34~4.48 (m, 5H, CH), 2.46~3.05 (m, 8H, Arg δ-, Lys ε-, Asp β-, Tyr β-, CH
2), 1.30~1.58 (m, 11H, Arg β-, γ-, Lys α-, β-, γ-, Val β-, CH
2), 0.69~0.72 (m, 6H, CH
3).The result is consistent with bibliographical information.
Embodiment 3 microwave-assisted solid phase synthesis Sostatins
(1) weighing Rink Amide resin (0.4mmol/g) 1g (0.4mmol) places reactor, adds methylene dichloride 5mL swelling resin 30 minutes, takes out dichloromethane solution.Weighing 1.6mmol Fmoc-L-Soviet Union ammonia alcohol and the bisacetal that carboxyl benzaldehyde is formed add the dissolving of 8mLN-methyl-2-pyrrolidone, and then add 1.76mmol dicyclohexylcarbodiimide and 1.76mmol 1-hydroxy benzo triazole, reaction 30min.After reaction finishes, cross the elimination precipitation, filtrate is joined in the resin, add the 0.08mmol Dimethylamino pyridine simultaneously, reacted 4 hours.The ratio of each material amount of substance is an amino acid in the reaction: resin=4: 1; : Soviet Union's ammonia acetal: condensation reagent=1: 1.1; Dimethylamino pyridine: resin=0.2: 1.Reaction filters out reaction solution after finishing, and adds methylene dichloride, each washing resin of methyl alcohol and dimethyl formamide 10mL three times.The vacuum-drying resin, the joint efficiency that records Soviet Union's ammonia acetal and resin is 75%.
(2) microwave-assisted removes the Fmoc protecting group
Ammonia alcohol-DHP the resin of will reviving places the solid-phase peptide reaction tubes, adds 8mL 20% piperidines/dimethyl formamide solution then, is reflected in the microwave reactor and carries out.Microwave condition is: 20 ℃ of temperature, reaction times 3min.After reaction finishes, add methylene dichloride, each washing resin of methyl alcohol and dimethyl formamide 8mL three times.
(3) microwave-assisted condensation reaction
1) condensation Fmoc-Cys (trt)-OH.Weighing Fmoc-Cys (trt)-OH 0.9mmol, 2-(7-azo benzotriazole)-N, N, N ', N '-tetramethyl-urea phosphofluoric acid ester and N-hydroxyl-each 1.08mmol of 7-azo benzotriazole is with the dissolving of 4mL tetrahydrofuran (THF), add 1 then) in the resin that obtains, diisopropyl ethyl amine (the Fmoc-Cys (trt)-OH: condensation reagent=1: 1.2, Fmoc-Cys (trt)-OH: resin=3: 1), react under the microwave radiation that adds 1.08mmol simultaneously.Microwave condition is: 20 ℃ of temperature, reaction times 3min.Take out reaction solution then, add methylene dichloride, each washing resin of methyl alcohol and dimethyl formamide 20mL three times.
2) prolongation of peptide chain.According to amino acid in the Sostatin from carboxyl terminal to N-terminal order (Thr-ol-Cys-Thr-Lys-D-Trp-Phe-Cys-D-Phe); the consumption of amino acid and condensation reagent is with Fmoc-Cys (trt)-OH is identical; repeat to contract and the deprotection two-step reaction synthetic Sostatin.
(4) preparation of reduced form Sostatin crude product and analysis
About weighing synthetic Sostatin-resin 200mg, place the container that can seal, 8mL trifluoroacetic acid, 0.5mL redistilled water, 0.5mL phenol and 0.25mL dithioglycol, 0.25mL thioanisole (trifluoroacetic acid: phenol: redistilled water: dithioglycol: thioanisole=80%: 5%: 5%: 2.5%: 2.5%, v/v), react 15min under the microwave radiation, temperature is 40 ℃.Reaction filters out resin after finishing, and uses 10mL washed with dichloromethane resin three times, collects filtrate, and rotary evaporation is removed trifluoroacetic acid and other organic solvent.The anhydrous diethyl ether that adds an amount of ice in remaining liquid is separated out Sostatin, the centrifugal then Sostatin crude product that obtains.
The white precipitate that obtains after centrifugal is dissolved in the 5mL redistilled water,, adds 10 times of redistilled water dilutions then, make standard specimen, analyze the purity and the productive rate of Sostatin with high performance liquid chromatography (HPLC) with the Sostatin reference substance through the membrane filtration of 0.22 μ.Liquid-phase condition is: moving phase is 1 ‰ trifluoroacetic acid/aqueous solution and methyl alcohol, and ratio is 85%: 15%; Flow velocity is 1mL/min; Wavelength is 280nm.Recording purity is 91.6%, calculates productive rate to be: 85.5%.ESI-MS m/z:1021.1[M+], actual relative molecular mass is 1021.1.
(5) preparation of oxidized form Sostatin and analysis
Get the reduced form Sostatin 50mg after the freeze-drying, add the DMSO/ aqueous solution of 10mL 5%, react in microwave reactor, the microwave reaction condition is: 30 ℃ of temperature, reaction times 5min.With the result of efficient liquid phase chromatographic analysis reaction, the productive rate of oxidized form Sostatin reaches 90.8%.ESI-MS m/z:1019.5[M+], actual relative molecular mass is 1019.2.
Claims (6)
1, a kind of process for solid-phase microwave synthesis of polypeptide is characterized in that process for solid-phase microwave synthesis is:
(1) preparation of amino acid-resin complexes: a polypeptide synthetic resins of weighing, put into the reactor of band filtration unit, to wherein adding methylene dichloride, swelling resin 30~120min, take out dichloromethane solution then, weighing carboxyl terminal amino acid and condensation reagent---dicyclohexylcarbodiimide or DIC and 1-hydroxyl phenylpropyl alcohol triazole, put into the container that can seal, add dimethyl formamide or tetrahydrofuran (THF), reaction 30~150min, the ratio of the amount of described amino acid and resinous substances is 7~3: 1; The ratio of amino acid and condensation reagent amount of substance is 1: 1~1.2, after reaction finishes, filter out precipitation, filtrate joining is equipped with in the reactor of band filtration unit of resin, add Dimethylamino pyridine simultaneously, reacted 3~20 hours, wherein the ratio of the amount of Dimethylamino pyridine and resinous substances is 0.5~0.1: 1, take out reaction solution then, with dimethyl formamide, methyl alcohol, methylene dichloride washing resin successively three times;
(2) microwave radiation removes amino protecting group: add deprotecting regent in amino acid-resin complexes that washing obtains in the above---piperidines/dimethyl formamide solution of 20~30%, to place microwave reactor to react then, microwave condition is: temperature is 20~70 ℃, reaction times is 3~10min, after reaction finishes, solution is taken out in filtration, with dimethyl formamide, methyl alcohol, washed with dichloromethane resin;
(3) microwave radiation condensation amino acid: weighing next protection amino acid and condensation reagent, put into can be airtight container, add dissolution with solvents, the ratio of the amount of described amino acid and resinous substances is 4~2: 1; The ratio of amino acid and condensation reagent amount of substance is 1: 1~1.2, described solvent is: dimethyl formamide, pyridine, N-Methyl pyrrolidone, tetrahydrofuran (THF), solution is joined in the reactor that the amino acid-resin complexes that removes amino protecting group is housed then, place microwave reactor to react, microwave condition is: temperature is 20~70 ℃, and the reaction times is 3~10min, after reaction finishes, take out reaction solution, with dimethyl formamide, methyl alcohol, washed with dichloromethane resin;
(4) according to amino acid from carboxyl terminal to N-terminal order, change amino acid, repeat microwave radiation deprotection and the amino acid whose reaction of microwave radiation condensation, synthetic required polypeptide;
(5) preparation of polypeptide crude product: protection peptide-resin that will obtain above put into can be airtight container, add cutting reagent, react under the microwave radiation; microwave condition is: 30~60 ℃ of temperature; reaction times is 10~20min, filters out resin then, collects filtrate; rotary evaporation is removed trifluoroacetic acid; in remaining liquid, add the ice ether, be settled out polypeptide, centrifugal then; remove supernatant liquor, obtain required polypeptide crude product.
2, process for solid-phase microwave synthesis of polypeptide according to claim 1, it is characterized in that for the polypeptide that contains disulfide linkage adopting 5~30% methyl-sulphoxides/aqueous solution is oxygenant, synthetic disulfide linkage under microwave radiation, microwave condition is: temperature is 20~60 ℃, and the reaction times is 5~30min.
3, process for solid-phase microwave synthesis of polypeptide according to claim 1 is characterized in that polypeptide synthetic resins adopts a kind of in hydroxy resin, chlorine resin and the aminoresin.
4, process for solid-phase microwave synthesis of polypeptide according to claim 1 is characterized in that described amino acid is the protection amino acid that side chain functionalities and aminoterminal all are protected.
5, process for solid-phase microwave synthesis of polypeptide according to claim 1, it is characterized in that the condensation reagent described in the synthesis method (3) comprises dicyclohexylcarbodiimide, DIC, the 1-hydroxy benzo triazole, benzotriazole-N, N, N ', N ',-tetramethyl-urea phosphofluoric acid ester, 2-(7-azo benzotriazole)-N, N, N ', N '-tetramethyl-urea phosphofluoric acid ester, N-hydroxyl-7-azo benzotriazole blocks special condensing agent BOPreagent, phosphofluoric acid benzotriazole-1-base-oxygen base tripyrrole alkyl phosphorus, diisopropyl ethyl amine.
6, process for solid-phase microwave synthesis of polypeptide according to claim 1, it is characterized in that the described cutting reagent described in the synthesis method (5) comprises: trifluoroacetic acid, redistilled water, triethyl silicane, tri isopropyl silane, phenol, thioanisole, methyl-phenoxide, dithioglycol and methylene dichloride, the combination between them and mutually ratio can be 1. trifluoroacetic acid: tri isopropyl silane or triethyl silicane: redistilled water=95%: 2.5%: 2.5% (v/v); 2. trifluoroacetic acid: phenol: redistilled water: methyl-phenoxide: thioanisole=80%: 5%: 5%: 2.5%: 2.5% (v/v); 3. trifluoroacetic acid: phenol: redistilled water: dithioglycol: thioanisole=80%: 5%: 5%: 2.5%: 2.5% (v/v); 4. trifluoroacetic acid: redistilled water=95%: 5% (v/v); 5. trifluoroacetic acid: methylene dichloride=50%: 50% (v/v).
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