CN103694316A - Preparation method of argireline - Google Patents

Preparation method of argireline Download PDF

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Publication number
CN103694316A
CN103694316A CN201310755456.4A CN201310755456A CN103694316A CN 103694316 A CN103694316 A CN 103694316A CN 201310755456 A CN201310755456 A CN 201310755456A CN 103694316 A CN103694316 A CN 103694316A
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resin
preparation
dmf
resins
peptide
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CN201310755456.4A
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CN103694316B (en
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顾含华
俞保彬
周天琼
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Zhejiang Hua Jun Pharmaceutical Co., Ltd.
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HANGZHOU HUAJIN PHARMACEUTICAL CO Ltd
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Abstract

The invention belongs to the technical field of medicinal chemistry and discloses a preparation method of argireline. The preparation method provided by the invention comprises the steps: activating resin to obtain amino resin; gradually enabling the obtained amino resin, guanidyl-protected L-arginine, the other guanidyl-protected L-arginine and side chain amino protected L-glutamine to be subjected to the first coupling reaction to obtain tripeptide resin; gradually enabling the obtained tripeptide resin, L-methionine, side chain carboxyl protected L-glutamic acid and side chain carboxyl protected N-acetyl-L-glutamic acid to be subjected to second coupling reaction to obtain hexapeptide resin; cracking the obtained hexapeptide resin to obtain a first product; and purifying the obtained first product to obtain the argireline. The preparation method provided by the invention is used for preparing the argireline through solid-phase synthesis so as to be simple in operation, capable of remarkably increasing the yield of the argireline and greatly improving the purity of the argireline, suitable for large-scale industrial production and more beneficial to the popularization and application of the argireline.

Description

The preparation method of a kind of A Ji Rayleigh
Technical field
The invention belongs to pharmaceutical chemistry technical field, particularly the preparation method of a kind of A Ji Rayleigh.
Background technology
A Ji Rayleigh (Argireline), has another name called acetyl Argireline-3, class novain bacillin, derivative by the N-terminal district of SNAP-25 albumen (SNAP of 25kDa), six amino-acid residues, consists of, and molecular formula is C 34h 60n 14o 12s, molecular weight 888.4, it has structure shown in formula I,
A Ji Rayleigh participates in the competition SNAP-25 in the site of melting bubble complex body, thereby the formation of bubble complex body is melted in impact, cause effectively release neurotransmitters of vesica, Muscle contraction is weakened, and then can relax one's muscles, the microgroove of releiving presents clearly thoroughly skin to have no time, also can impel cell regeneration to make skin recover resilient flexible, therefore be widely used in preparing makeup and healthcare products.Along with the enhancing of people's health care consciousness, makeup and health-product market were more and more wide in recent years, and the market requirement of A Ji Rayleigh is also increasing.
The preparation method of A Ji Rayleigh is mainly segment condense method at present, for example, and the preparation method that the patent document that publication number is CN102603869A is recorded.In this patent document, first disclosed preparation method for to adopt different preparation methods to synthesize two tripeptide fragments: solid phase synthesis process has been prepared NH 2-Gln (Trt)-Arg (pbf)-Arg (pdf)-NH-resin peptide resin; liquid-phase synthesis process has been prepared Fmoc-Glu (tbu)-Gln (tbu)-Met-COOH; again both are carried out to coupling, deprotection, acylations, cracking, obtain.This preparation method's complex steps, yield is lower, and Yin Aji Rayleigh peptide chain is shorter, certainly will cause a large amount of wastes of fragment by segment condense method, and these all cause the cost of A Ji Rayleigh higher, are unfavorable for its popularization and use.
Summary of the invention
In view of this, goal of the invention of the present invention has been to provide a kind of A Ji preparation method of Rayleigh.Preparation method provided by the invention adopts solid-phase synthesis to prepare A Ji Rayleigh, simple to operate, has significantly improved yield and the purity of A Ji Rayleigh, thereby has reduced its production cost, is more conducive to popularization and the use of A Ji Rayleigh.
In order to realize goal of the invention of the present invention, the present invention adopts following technical scheme:
The preparation method who the invention provides a kind of A Ji Rayleigh, comprises the following steps:
Step 1: get resin, activation, obtains aminoresin;
Step 2: get step 1 gained aminoresin, the first L-arginine that progressively coupling guanidine radicals is protected, the L-arginine of guanidine radicals protection, the L-glutaminate of side chain amido protecting, obtain three peptide resins;
Step 3: get step 2 gained three peptide resins, the second Pidolidone that progressively coupling METHIONINE, side chain carboxyl group are protected is, the N-acetyl-Pidolidone of side chain carboxyl group protection obtains six peptide resins;
Step 4: cleavage step 3 gained six peptide resins, obtain the first product;
Step 5: get step 4 gained the first product, purifying, obtains.
Preferably, in preparation method provided by the invention, in step 1, resin is Rink Amide-MBHA Resin.
Preferably, preparation method provided by the invention, in step 1, the substitution degree of Rink Amide-MBHA Resin resin is 0.4mmol/g～0.6mmol/g.
Preparation method provided by the invention adopts solid-phase synthesis to prepare A Ji Rayleigh; selected resin is preferably Rink Amide-MBHA Resin resin; this resin can overcome the impact of steric effect better; easier coupling, containing the amino acid of Side chain protective group, is conducive to improve the yield of A Ji Rayleigh.
Preferably, in preparation method provided by the invention, in step 1, the reagent of activation is alkali reagent.
In some embodiments of the invention, in step 1, the alkali reagent of activation is hexahydropyridine.
In other embodiment of the present invention, in preparation method provided by the invention, in volume percent, the DMF solution of the hexahydropyridine that in step 1, the alkali reagent of activation is 20%～30%.
Preferably, in preparation method provided by the invention, in step 1, the temperature of activation is 15 ℃～30 ℃.
Preferably, in preparation method provided by the invention, in step 1, the time of activation is 20 minutes～25 minutes.
Preferably, in preparation method provided by the invention, in step 2 first progressively coupling be specially:
Get the L-arginine of step 1 gained aminoresin and the protection of N end, guanidine radicals protection under the first action of coupling agents, the first linked reaction occurs, through the first deprotection, obtain the first amino-acid resin;
Get the L-arginine of gained the first amino-acid resin and the protection of N end, guanidine radicals protection under the second action of coupling agents, the second linked reaction occurs, through the second deprotection, obtain two peptide resins;
Get the L-glutaminate of gained two peptide resins and the protection of N end, side chain amido protecting under the 3rd action of coupling agents, the 3rd linked reaction occurs, through the 3rd deprotection, obtain three peptide resins.
Preferably, preparation method provided by the invention, in step 3 second progressively coupling be specially:
Get the METHIONINE of step 2 gained three peptide resins and the protection of N end under the 4th action of coupling agents, the 4th linked reaction occurs, through the 4th deprotection, obtain tetrapeptide resin;
Get the Pidolidone of gained tetrapeptide resin and the protection of N end, side chain carboxyl group protection under the 5th action of coupling agents, the 5th linked reaction occurs, through the 5th deprotection, obtain pentapeptide resin;
Get the N-acetyl-Pidolidone of gained pentapeptide resin and side chain carboxyl group protection under the 6th action of coupling agents, the 6th linked reaction occurs, obtain six peptide resins.
Preferably, in preparation method provided by the invention, the first coupling agent is EDCI/HOBT/DIEA or HOBT/DIC.
Preferably, in preparation method provided by the invention, in the first linked reaction, the L-arginine of aminoresin, the protection of N end, guanidine radicals protection, EDCI, HOBT are 1:2～5:4～9:2～6:3～9 with the amount of substance ratio of DIEA.
Preferably, in preparation method provided by the invention, in the first linked reaction, the L-arginine of aminoresin, the protection of N end, guanidine radicals protection, HOBT are 1:2～5:2～6:3～6 with the amount of substance ratio of DIC.。
Preferably, in preparation method provided by the invention, the temperature of the first linked reaction is 15 ℃～30 ℃.
Preferably, in preparation method provided by the invention, the time of the first linked reaction is 0.5 hour～2 hours.
Preferably, in preparation method provided by the invention, the reaction soln of the first linked reaction is DMF, THF or NMP.
Preferably, in preparation method provided by the invention, the first deprotection agents useful for same is alkali reagent.
In some embodiments of the invention, in preparation method provided by the invention, the first deprotection alkali reagent used is in volume percent, the DMF solution of 20%～30% hexahydropyridine.
Preferably, in preparation method provided by the invention, the temperature of the first deprotection is 15 ℃～30 ℃.
Preferably, in preparation method provided by the invention, the time of the first deprotection is 20 minutes～25 minutes.
Preferably, in preparation method provided by the invention, in the first linked reaction, the N end protecting group of the L-arginine of the protection of N end, guanidine radicals protection is Fmoc.
Preferably, in preparation method provided by the invention, in the first linked reaction, the guanidine radicals protecting group of the L-arginine of the protection of N end, guanidine radicals protection is Pbf.
Preferably, in preparation method provided by the invention, the second coupling agent is EDCI/HOBT/DIEA or HOBT/DIC.
Preferably, in preparation method provided by the invention, in the second linked reaction, the L-arginine of the first amino-acid resin, the protection of N end, guanidine radicals protection, EDCI, HOBT are 1:2～5:4～9:2～6:3～9 with the amount of substance ratio of DIEA.
Preferably, in preparation method provided by the invention, in the second linked reaction, the L-arginine of the first amino-acid resin, the protection of N end, guanidine radicals protection, HOBT are 1:2～5:2～6:3～6 with the amount of substance ratio of DIC.
Preferably, in preparation method provided by the invention, the temperature of the second linked reaction is 15 ℃～30 ℃.
Preferably, in preparation method provided by the invention, the time of the second linked reaction is 0.5 hour～2 hours.
Preferably, in preparation method provided by the invention, the reaction soln of the second linked reaction is DMF, THF or NMP.
Preferably, in preparation method provided by the invention, the reagent of the second deprotection is alkali reagent.
More preferably, in preparation method provided by the invention, the alkali reagent of the second deprotection is in volume percent, the DMF solution of 20%～30% hexahydropyridine.
Preferably, in preparation method provided by the invention, the temperature of the second deprotection is 15 ℃～30 ℃.
Preferably, in preparation method provided by the invention, the time of the second deprotection is 20 minutes～25 minutes.
Preferably, in preparation method provided by the invention, in the second linked reaction, the N end protecting group of the L-arginine of the protection of N end, guanidine radicals protection is Fmoc.
Preferably, in preparation method provided by the invention, in the second linked reaction, the guanidine radicals protecting group of the L-arginine of the protection of N end, guanidine radicals protection is Pbf.
Preferably, in preparation method provided by the invention, the 3rd coupling agent is EDCI/HOBT/DIEA or HOBT/DIC.
Preferably, in preparation method provided by the invention, in the 3rd linked reaction, the L-glutaminate of two peptide resins, the protection of N end, side chain amido protecting, EDCI, HOBT are 1:2～5:4～9:2～6:3～9 with the amount of substance ratio of DIEA.
Preferably, in preparation method provided by the invention, in the 3rd linked reaction, the L-glutaminate of two peptide resins, the protection of N end, side chain amido protecting, HOBT are 1:2～5:2～6:3～6 with the amount of substance ratio of DIC.
Preferably, in preparation method provided by the invention, the temperature of the 3rd linked reaction is 15 ℃～30 ℃.
Preferably, in preparation method provided by the invention, the time of the 3rd linked reaction is 0.5 hour～2 hours.
Preferably, in preparation method provided by the invention, the reaction soln of the 3rd linked reaction is DMF, THF or NMP.
Preferably, in preparation method provided by the invention, the reagent of the 3rd deprotection is alkali reagent.
More preferably, in preparation method provided by the invention, the alkali reagent of the 3rd deprotection is, in volume percent, and the DMF solution of 20%～30% hexahydropyridine.
Preferably, in preparation method provided by the invention, the temperature of the 3rd deprotection is 15 ℃～30 ℃.
Preferably, in preparation method provided by the invention, the time of the 3rd deprotection is 20 minutes～25 minutes.
Preferably, in preparation method provided by the invention, in the 3rd linked reaction, the N end amino protecting group of the L-glutaminate of the protection of N end, side chain amido protecting is Fmoc.
Preferably, in preparation method provided by the invention, in the 3rd linked reaction, the side chain amino protecting group of the L-glutaminate of the protection of N end, side chain amido protecting is Trt, tBu or StBu.
In the preparation process of A Ji Rayleigh, first three amino acid whose coupling, i.e. the coupling of the L-arginine of guanidine radicals protection, the L-arginine of guanidine radicals protection and the L-glutaminate of side chain amido protecting, is the committed step that affects A Ji Rayleigh yield and purity.Because; in the process of three peptide resins; first three amino acid whose Side chain protective group and side-chain radical are larger; cause need to overcoming during coupling one by one at amino acid sterically hindered and make the failure of coupling one by one compared with large; produce a large amount of by products such as polypeptide fragment, had a strong impact on yield and the purity of A Ji Rayleigh.In preparation method provided by the invention, step 2 is progressively during coupling, adopt EDCI/HOBT/DIEA composite coupler or the HOBT/DIC composite coupler as coupling agent, successfully overcome the difficulty that first three amino acid steric hindrance is difficult for greatly coupling, improved yield and the purity of A Ji Rayleigh.Experimental result invention, preparation method provided by the invention has significantly improved yield and the purity of A Ji Rayleigh, is more conducive to the industrialized production of A Ji Rayleigh.
Preferably, in preparation method provided by the invention, the 4th coupling agent is HOBT/DIC.
Preferably, in preparation method provided by the invention, in the 4th linked reaction, the METHIONINE of three peptide resins, the protection of N end, HOBT are 1:2～5:2～6:3～6 with the amount of substance ratio of DIC.
Preferably, in preparation method provided by the invention, the temperature of the 4th linked reaction is 15 ℃～30 ℃.
Preferably, in preparation method provided by the invention, the time of the 4th linked reaction is 2 hours～4 hours.
Preferably, in preparation method provided by the invention, the reaction soln of the 4th linked reaction is DMF, THF or NMP.
Preferably, in preparation method provided by the invention, the 4th deprotection reagent used is alkali reagent.
In some embodiments of the invention, in preparation method provided by the invention, the 4th deprotection alkali reagent used is, in volume percent, and the DMF solution of 20%～30% hexahydropyridine.
Preferably, in preparation method provided by the invention, the temperature of the 4th deprotection is 15 ℃～30 ℃.
Preferably, in preparation method provided by the invention, the time of the 4th deprotection is 20 minutes～25 minutes.
Preferably, in preparation method provided by the invention, the amino protecting group of the METHIONINE of the N of the 4th linked reaction end protection is Fmoc.
Preferably, in preparation method provided by the invention, the 5th coupling agent is HOBT/DIC.
Preferably, in preparation method provided by the invention, in the 5th linked reaction, the Pidolidone of tetrapeptide resin, the protection of N end, side chain carboxyl group protection, HOBT are 1:2～5:2～6:3～6 with the amount of substance ratio of DIC.
Preferably, in preparation method provided by the invention, the temperature of the 5th linked reaction is 15 ℃～30 ℃.
Preferably, in preparation method provided by the invention, the time of the 5th linked reaction is 2 hours～4 hours.
Preferably, in preparation method provided by the invention, the reaction reagent of the 5th linked reaction is DMF, THF or NMP.
Preferably, in preparation method provided by the invention, the 5th deprotection reagent used is alkali reagent.
In some embodiments of the invention, in preparation method provided by the invention, the 5th deprotection alkali reagent used is, in volume percent, and the DMF solution of 20%～30% hexahydropyridine.
Preferably, in preparation method provided by the invention, in the 5th linked reaction, the amino protecting group of the Pidolidone of the protection of N end, side chain carboxyl group protection is Fmoc.
Preferably, in preparation method provided by the invention, in the 5th linked reaction, the side chain carboxyl group protecting group of the Pidolidone of the protection of N end, side chain carboxyl group protection is tBu.
Preferably, in preparation method provided by the invention, the 6th coupling agent is HOBT/DIC.
Preferably, in preparation method provided by the invention, in the 6th linked reaction, the N-acetyl-Pidolidone of pentapeptide resin, side chain carboxyl group protection, HOBT are 1:2～5:2～6:3～6 with the amount of substance ratio of DIC.
Preferably, in preparation method provided by the invention, the temperature of the 6th linked reaction is 15 ℃～30 ℃.
Preferably, in preparation method provided by the invention, the time of the 6th linked reaction is 2 hours～4 hours.
Preferably, in preparation method provided by the invention, the reaction reagent of the 6th linked reaction is DMF, THF or NMP.
Preferably, in preparation method provided by the invention, in the 6th linked reaction, the side chain carboxyl group protecting group of the N-acetyl-Pidolidone of side chain carboxyl group protection is tBu.
In preparation method provided by the invention, first by using High Efficient Bonding Agents successfully to overcome larger steric effect, sterically hindered, obtained three peptide resins, afterwards, adopt progressively three amino acid after coupling of HOBT/DIC composite coupler.Because HOBT, DIC low price, so this composite coupler has further reduced the production cost of A Ji Rayleigh, more have the popularization and the use that utilize A Ji Rayleigh.
Preferably, in preparation method provided by the invention, in step 4, the lytic reagent of cracking comprises trifluoroacetic acid, thioanisole and water.
In some embodiments of the invention, in preparation method provided by the invention, in step 4, the lytic reagent of cracking also comprises phenol and 1，2-ethandithiol.
In other embodiment of the present invention, in preparation method provided by the invention, the lytic reagent of step 4 is specially the mixing solutions of trifluoroacetic acid, thioanisole and water, and wherein the volume ratio of trifluoroacetic acid, thioanisole and water is 95:2.5:2.5.
In other embodiment of the present invention, in preparation method provided by the invention, the lytic reagent of step 4 is specially trifluoroacetic acid, phenol, 1, the mixing solutions of 2-dithioglycol, thioanisole and water, wherein the volume ratio of trifluoroacetic acid, phenol, 1，2-ethandithiol, thioanisole and water is 83.0:6.3:2.1:4.3:4.3.
Preferably, in preparation method provided by the invention, in g/mL, the mass volume ratio of step 3 gained six peptide resins and lytic reagent is 1:8～12.
Preferably, in preparation method provided by the invention, in step 4, the temperature of cracking is 15 ℃～30 ℃.
Preferably, in preparation method provided by the invention, in step 4, the time of cracking is 2 hours～3 hours.
Preferably, in preparation method provided by the invention, in step 5, purifying is specially:
Get step 4 gained the first product, through first reverse-phase chromatographic column the first separation, the SDS aqueous solution-acetonitrile system gradient elution of 10mmol/L, collects elution fraction corresponding to characteristic peak, obtains purified for the first time; In volume percent, gradient elution is acetonitrile by 15% to 21%, the SDS aqueous solution of 10mmol/L is by 85% to 79%;
Get gained purified for the first time, through second reverse-phase chromatographic column the second separation, with volume percent, count after acetonitrile solution the first wash-out of 5%, with volume percent, count acetonitrile solution the second wash-out of 40%, collect the second wash-out obtained component, obtain.
In some embodiments of the invention, in purification process provided by the invention, the first reverse-phase chromatographic column is C18 reverse-phase chromatographic column.
In other embodiment of the present invention, in purification process provided by the invention, the second reverse-phase chromatographic column is C18 reverse-phase chromatographic column.
In some embodiments of the invention, in purification process provided by the invention, separated for the first time, in volume percent, the elutriant of the elution fraction that characteristic peak is corresponding is the SDS aqueous solution 81% to 79% of acetonitrile 19% to 21%, 10mmol/L.
The invention provides the preparation method of a kind of A Ji Rayleigh.This preparation method comprises: get resin, activation, obtains aminoresin; Get gained aminoresin, the first L-arginine that progressively coupling guanidine radicals is protected, the L-arginine of guanidine radicals protection, the L-glutaminate of side chain amido protecting, obtain three peptide resins; Get gained three peptide resins, the second Pidolidone that progressively coupling METHIONINE, side chain carboxyl group are protected is, the N-acetyl-Pidolidone of side chain carboxyl group protection obtains six peptide resins; Cracking gained six peptide resins, obtain the first product; Get gained the first product, purifying, obtains.Preparation method provided by the invention adopts solid phase synthesis process, using EDCI/HOBT/DIEA composite coupler or HOBT/DIC composite coupler as coupling agent, successfully overcome the difficulty that first three amino acid steric hindrance is difficult for greatly coupling, improved yield and the purity of A Ji Rayleigh.Experimental result confirmation, preparation method provided by the invention has significantly improved yield and the purity of A Ji Rayleigh, and the coupling agent low price of the present invention's employing, has further reduced the production cost of A Ji Rayleigh, is more conducive to popularization and the use of A Ji Rayleigh.
Accompanying drawing explanation
Fig. 1 shows the high performance liquid chromatography spectrogram of A Ji Rayleigh standard substance; Retention time is 9.33min;
Fig. 2 shows the high performance liquid chromatography spectrogram of the product that embodiment 9 makes; Retention time is that the component that 9.33min is corresponding is A Ji Rayleigh component;
Fig. 3 shows the mass spectrogram of the product that embodiment 9 makes.
Embodiment
The invention discloses the preparation method of a kind of A Ji Rayleigh.Those skilled in the art can be with reference to this paper content, obtains A Ji Rayleigh, special needs to be pointed out is, and all similar replacements and change apparent to those skilled in the artly, they are all deemed to be included in the present invention.Preparation method of the present invention and application are described by preferred embodiment, related personnel obviously can change this paper preparation method and application or suitably change and combination within not departing from content of the present invention, spirit and scope, realizes and apply the technology of the present invention.
In the preparation method of a kind of A Ji provided by the invention Rayleigh, reagent used and raw material all can be buied by market.
The bilingual of the english abbreviation relating in the present invention is in Table 1.
Table 1 bilingual table
English abbreviation Chinese
EDCI 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride
HOBT 1-hydroxy benzo triazole
HBTU Benzotriazole-N, N, N ', N '-tetramethyl-urea hexafluorophosphate
DIEA DIPEA
DIC N, N '-DIC
DMF DMF
THF Tetrahydrofuran (THF)
NMP N-Methyl pyrrolidone
DCM Methylene dichloride
Fmoc 9-fluorenylmethyloxycarbonyl
Pbf 2,2,4,6,7-pentamethyl-Dihydrobenzofuranes-5-alkylsulfonyl
Trt Trityl
tBu The tertiary butyl
StBu Tertiary fourth mercapto ether
Arg Arginine
Gln Glutamine
Met Methionine(Met)
Glu L-glutamic acid
Ac Ethanoyl
In order to make those skilled in the art can understand better technical scheme of the present invention, below in conjunction with embodiment, further set forth the present invention:
The preparation of embodiment 1 aminoresin
Take Rink Amide-MBHA Resin (buying in Shangyu pul resin company limited) 10g, substitution degree is for O.5mmol/g, the 100mL DMF agitator treating 10 minutes that adds resin, No. three core decompress filter is removed DMF, again adds the abundant swelling of DCM 1 hour of 80mL.
The DMF solution of the hexahydropyridine that is 25% by 80mL volume percent adds in the sufficient Rink Amide-MBHA of above-mentioned swelling Resin, reacts 20 minutes No. three core decompress filters at 25 ℃, resin 80mL DMF, 80mL DCM is washing alternately, totally six times, obtains aminoresin.
The preparation of embodiment 2 first amino-acid resins
Get Fmoc-L-ArgfPbf)-OH15mmol, HOBT20mmol, DIEA30mmol, after dissolving with 50mLDMF, join in the aminoresin that embodiment 1 makes and stir, to be cooled to-15 ℃ time, with moving liquid funnel, slowly drip EDCI30mmol, dropwise half an hour, it returns to 20 ℃ of reactions one hour naturally-15 ℃ of insulated and stirred reliefs half an hour, get several grainy resins and do the negative detection of triketohydrindene hydrate, resin water white transparency, reacts completely.No. three core decompress filter is removed reaction solution, resin 80mL DMF, and each washing of 80mLDCM is once.The DMF solution of the hexahydropyridine that is 25% by 80mL volume percent adds in gained resin, reacts 20 minutes at 25 ℃, and No. three core decompress filters, gained is alternately washing of 80mLDMF, 80mL DCM for resin, totally six times, obtains the first amino-acid resin.
The preparation of embodiment 3 two peptide resins
Get Fmoc-L-ArgfPbf)-OH15mmol, HOBT20mmol, DIEA30mmol, after dissolving with 50mLDMF, join in the first amino-acid resin that embodiment 2 makes and stir, to be cooled to-15 ℃ time, with moving liquid funnel, slowly drip EDCI30mmol, dropwise half an hour, it returns to 20 ℃ of reactions one hour naturally-15 ℃ of insulated and stirred reliefs half an hour, get several grainy resins and do the negative detection of triketohydrindene hydrate, resin water white transparency, reacts completely.No. three core decompress filter is removed reaction solution, peptide resin 80mLDMF, and each washing of 80mL DCM is once.The DMF solution of the hexahydropyridine that is 25% by 80mL volume percent adds in gained resin, reacts 20 minutes at 25 ℃, and No. three core decompress filters, for gained resin, alternately washing of 80mL DMF, 80mL DCM, totally six times, obtains two peptide resins.
The preparation of embodiment 4 three peptide resins
Get Fmoc-L-Gln (Trt)-OH15mmol, HOBT20mmol, DIEA30mmol, after dissolving with 50mLDMF, join in two peptide resins that embodiment 3 makes and stir, to be cooled to-15 ℃ time, with moving liquid funnel, slowly drip EDCI30mmol, dropwise half an hour, it returns to 20 ℃ of reactions one hour naturally-15 ℃ of insulated and stirred reliefs half an hour, get several grainy resins and do the negative detection of triketohydrindene hydrate, resin water white transparency, reacts completely.No. three core decompress filter is removed reaction solution, peptide resin 80mL DMF, and each washing of 80mL DCM is once.The DMF solution of the hexahydropyridine that is 25% by 80mL volume percent adds in gained resin, reacts 20 minutes at 25 ℃, and No. three core decompress filters, for gained resin, alternately washing of 80mL DMF, 80mL DCM, totally six times, obtains three peptide resins.
The preparation of embodiment 5 tetrapeptide resins
Get Fmoc-L-Met-OH15mmol, HOBT20mmol with after 80ml DMF dissolving, add DIC20mmol activation, at 20 ℃, activate 15 minutes, Bush's funnel filters out undissolved activation by product, filtrate is added in three peptide resins that embodiment 4 makes, and stirring reaction is 2 hours at 25 ℃, and triketohydrindene hydrate is done negative detection, resin water white transparency, reacts completely.No. three core decompress filter is removed reaction solution, peptide resin 80mL DMF, and each washing of 80mL DCM is once.The DMF solution of the hexahydropyridine that is 25% by 80mL volume percent adds in gained resin, reacts 20 minutes at 25 ℃, and No. three core decompress filters, for gained resin, alternately washing of 80mL DMF, 80mL DCM, totally six times, obtains tetrapeptide resin.
The preparation of embodiment 6 pentapeptide resins
Get after Fmoc-L-Glu (tBu)-OH15mmol, HOBT20mmol dissolves with 80mL DMF and add DIC20mmol activation, at 20 ℃, activate 15 minutes, Bush's funnel filters out undissolved activation by product, filtrate is added in the tetrapeptide resin that embodiment 5 makes, at 25 ℃, stirring reaction is 2 hours, triketohydrindene hydrate is done negative detection, and resin water white transparency, reacts completely.No. three core decompress filter is removed reaction solution, peptide resin 80mL DMF, and each washing of 80mL DCM is once.The DMF solution of the hexahydropyridine that is 25% by 80mL volume percent adds in gained resin, reacts 20 minutes at 25 ℃, and No. three core decompress filters, for gained resin, alternately washing of 80mL DMF, 80mL DCM, totally six times, obtains pentapeptide resin.
The preparation of embodiment 7 six peptide resins
Get Ac-L-Glu (tBu)-OH15mmol, HOBT20mmol, add DIC20mmol activation after dissolving with 80mL DMF, at 20 ℃, activate 15 minutes, Bush's funnel filters out undissolved activation by product, filtrate is added in the pentapeptide resin that embodiment 6 makes, at 25 ℃, stirring reaction is 2 hours, triketohydrindene hydrate is done negative detection, and resin water white transparency, reacts completely.Alternately washing of 80mL DMF, 80mLDCM for gained resin, is dried for six times totally afterwards, obtains six peptide resin 18.8g.
The preparation of embodiment 8 first products
Configuration lytic reagent: be that 83.0:6.3:2.1:4.3:4.3 mixes according to trifluoroacetic acid, phenol, 1，2-ethandithiol, thioanisole with water volume ratio, obtain lytic reagent.According to lytic reagent recipe configuration 188mL lytic reagent, join in Glass Containers and place 2 hours under 4 ℃ of conditions.
Excision resin and other amino acid side chain blocking group: take out the lysate 188mL of precooling, 18.8g six peptide resins that add embodiment 7 to make under stirring, 20 ℃ are reacted 2 hours; then filter, filtrate is added in the ether 1880mL of precooling under 4 ℃ of conditions, stirs; precipitation; precipitated liquid is centrifugal, removes supernatant liquor, ether washing four times for precipitation; each 50mL; vacuum-drying afterwards, obtains dry powder 6.2g, i.e. the first product.Gained the first product is carried out to efficient analysis liquid chromatographic detection, and the chromatographic purity that obtains A Ji Rayleigh in the first product is 92.7%.
The preparation of embodiment 9 A Ji Rayleighs
Get the first product 6.2g that embodiment 8 makes, by 124mL purified water, dissolve, with the filtering with microporous membrane of 0.22 micron.Gained solution is carried out to further separation and purification.
With C18 reversed-phase column (Beijing Chuangxin Tongheng Science and Technology Co., Ltd.) the first separation: chromatographic column internal diameter 100mm, detect wavelength 230nm, 5～30g/ pin applied sample amount, flow velocity loading with 200ml/ minute, after loading, use eluent gradient wash-out, A moving phase is the SDS solution of 10mmol/L, B is acetonitrile mutually, in volume percent, gradient is that B is by 15% to 21%, A is mutually by 85% to 79%, time is 40 minutes, when sharply rising suddenly, detector level starts Fractional Collections product, storing elution fraction is acetonitrile 19% to 21%, the elution fraction of the SDS aqueous solution 81% to 79% of 10mmol/L, it is the elution fraction that characteristic peak is corresponding, obtain purified for the first time.
Desalination: the present embodiment is obtained for the first time to purified sample introduction to C18 reversed-phase column (buying in Beijing innovation), A moving phase purified water, B phase acetonitrile, flow velocity 250ml/ minute, the detection wavelength of 230nm; Volume percent meter, 95%A phase and 5%B phase mixing solutions, rinse 40 minutes, then switches to volume percent meter, and 60%A phase and 40%B phase mixing solutions wash-out, collect this elution fraction.By gained elution fraction, concentrate, freeze-drying obtains product 3.8g, total recovery approximately 85.5%.
With mass spectrum and high performance liquid chromatography, identify products obtained therefrom.
The present embodiment obtains the high-efficient liquid phase chromatogram of product and sees Fig. 2, and with the high performance liquid chromatography spectrogram of A Ji Rayleigh standard substance, Fig. 1 compares, and both appearance times are basically identical, therefore the present embodiment obtains product, is A Ji Rayleigh, and purity is greater than 99%.The mass spectrogram of the product that the present embodiment obtains is simultaneously shown in Fig. 3, and the molecular weight of the molecular weight of this product and A Ji Rayleigh is basically identical as we know from the figure.Above qualification result has illustrated that the product that the present embodiment obtains is A Ji Rayleigh.
The preparation of embodiment 10 aminoresin
Take Rink Amide-MBHA Resin(Shangyu pul resin company limited) 12.5g, substitution degree is 0.4mmol/g, the 100mL DMF agitator treating 10 minutes that adds resin, No. three core decompress filter is removed DMF, again adds the abundant swelling of DCM 1 hour of 80mL.
The DMF solution of the hexahydropyridine that is 30% by 80mL volume percent adds in the sufficient Rink Amide-MBHA of above-mentioned swelling Resin, reacts 25 minutes No. three core decompress filters at 30 ℃, resin 80mL DMF, 80mL DCM is washing alternately, totally six times, obtains aminoresin.
The preparation of embodiment 11 first amino-acid resins
Get Fmoc-L-Arg (Pbf)-OH25mmol, HOBT30mmol, DIEA45mmol, after dissolving with 60mLTHF, join in the aminoresin that embodiment 10 makes and stir, to be cooled to-15 ℃ time, with moving liquid funnel, slowly drip EDCI45mmol, dropwise half an hour, it returns to 30 ℃ of reactions 2 hours naturally-15 ℃ of insulated and stirred reliefs half an hour, get several grainy resins and do the negative detection of triketohydrindene hydrate, resin water white transparency, reacts completely.No. three core decompress filter is removed reaction solution, peptide resin 80mL DMF, each washing of 80mL DCM once, the DMF solution of the hexahydropyridine that is 30% by 80mL volume percent adds in gained resin, at 30 ℃, react No. three core decompress filters, alternately washing of 80mL DMF, 80mL DCM for gained resin 25 minutes, totally six times, obtain the first amino-acid resin.
The preparation of embodiment 12 2 peptide resins
Get Fmoc-L-Arg (Pbf)-OH25mmol, HOBT30mmol, DIEA45mmol, after dissolving with 60mLTHF, join in the first amino-acid resin that embodiment 11 makes and stir, to be cooled to-15 ℃ time, with moving liquid funnel, slowly drip EDCI45mmol, dropwise half an hour, it returns to 30 ℃ of reactions 2 hours naturally-15 ℃ of insulated and stirred reliefs half an hour, get several grainy resins and do the negative detection of triketohydrindene hydrate, resin water white transparency, reacts completely.No. three core decompress filter is removed reaction solution, peptide resin 80mLDMF, each washing of 80mL DCM once, the DMF solution of the hexahydropyridine that is 30% by 80mL volume percent adds in gained resin, at 30 ℃, react No. three core decompress filters, alternately washing of 80mL DMF, 80mL DCM for gained resin 25 minutes, totally six times, obtain two peptide resins.
The preparation of embodiment 13 3 peptide resins
Get Fmoc-L-Gln (tBu)-OH25mmol, HOBT30mmol, DIEA45mmol, after dissolving with 60mLTHF, join in two peptide resins that embodiment 12 makes and stir, to be cooled to-15 ℃ time, with moving liquid funnel, slowly drip EDCI45mmol, dropwise half an hour, it returns to 30 ℃ of reactions 2 hours naturally-15 ℃ of insulated and stirred reliefs half an hour, get several grainy resins and do the negative detection of triketohydrindene hydrate, resin water white transparency, reacts completely.No. three core decompress filter is removed reaction solution, peptide resin 80mL DMF, each washing of 80mL DCM once, the DMF solution of the hexahydropyridine that is 30% by 80mL volume percent adds in gained resin, at 30 ℃, react No. three core decompress filters, alternately washing of 80mL DMF, 80mL DCM for gained resin 25 minutes, totally six times, obtain three peptide resins.
The preparation of embodiment 14 tetrapeptide resins
Get Fmoc-L-Met-OH25mmol, HOBT30mmol with after 60mL THF dissolving, add DIC30mmol activation, at 30 ℃, activate 20 minutes, Bush's funnel filters out undissolved activation by product, filtrate is added in three peptide resins that embodiment 13 makes, and stirring reaction is 4 hours at 30 ℃, and triketohydrindene hydrate is done negative detection, resin water white transparency, reacts completely.No. three core decompress filter is removed reaction solution, peptide resin 80mL DMF, each washing of 80mL DCM once, the DMF solution of the hexahydropyridine that is 30% by 80mL volume percent adds in gained resin, at 30 ℃, react No. three core decompress filters, alternately washing of 80mL DMF, 80mL DCM for gained resin 25 minutes, totally six times, obtain tetrapeptide resin.
The preparation of embodiment 15 pentapeptide resins
Get after Fmoc-L-Glu (tBu)-OH25mmol, HOBT30mmol dissolves with 60mL THF and add DIC30mmol activation, at 30 ℃, activate 20 minutes, Bush's funnel filters out undissolved activation by product, filtrate is added in the tetrapeptide resin that embodiment 14 makes, at 30 ℃, stirring reaction is 4 hours, triketohydrindene hydrate is done negative detection, and resin water white transparency, reacts completely.No. three core decompress filter is removed reaction solution, peptide resin 80mL DMF, each washing of 80mL DCM once, the DMF solution of the hexahydropyridine that is 30% by 80mL volume percent adds in gained resin, at 30 ℃, react No. three core decompress filters, alternately washing of 80mL DMF, 80mL DCM for gained resin 25 minutes, totally six times, obtain pentapeptide resin.
The preparation of embodiment 16 6 peptide resins
Get Ac-L-Glu (tBu)-OH25mmol, HOBT30mmol, add DIC30mmol activation after dissolving with 60mL THF, at 30 ℃, activate 20 minutes, Bush's funnel filters out undissolved activation by product, filtrate is added in the pentapeptide resin that embodiment 15 makes, at 30 ℃, stirring reaction is 4 hours, triketohydrindene hydrate is done negative detection, and resin water white transparency, reacts completely.No. three core decompress filter is removed reaction solution, peptide resin 80mL DMF, and 80mL DCM is washing alternately, and totally six times, dry, obtain six peptide resin 20.4g.
The preparation of embodiment 17 first products
Configuration lytic reagent: be that 83.0:6.3:2.1:4.3:4.3 mixes according to trifluoroacetic acid, phenol, 1，2-ethandithiol, thioanisole with water volume ratio, obtain lytic reagent.According to lytic reagent recipe configuration 244.8mL lytic reagent, join in Glass Containers and place 2 hours under 4 ℃ of conditions.
Excision resin and other amino acid side chain blocking group: take out the lysate 244.8mL of precooling, 20.4g six peptide resins that add embodiment 16 to make under stirring, 30 ℃ are reacted 3 hours; then filter, filtrate is added in the ether 2448mL of precooling under 4 ℃ of conditions, stirs; precipitation; precipitated liquid is centrifugal, removes supernatant liquor, ether washing four times for precipitation; each 50mL; vacuum-drying afterwards, obtains dry powder 6.0g, i.e. the first product.Gained the first product is carried out to efficient analysis liquid chromatographic detection, and with standard substance comparison, both appearance times are basically identical, and the chromatographic purity that obtains A Ji Rayleigh in the first product is 89%.
The preparation of embodiment 18 A Ji Rayleighs
Get the first product 6.0g that embodiment 17 makes, by 75mL purified water, dissolve, with the filtering with microporous membrane of 0.45 micron.Gained solution is carried out to further separation and purification.
With C18 reversed-phase column (Beijing Chuangxin Tongheng Science and Technology Co., Ltd.) the first separation: chromatographic column internal diameter 100mm, detect wavelength 230nm, 5～30g/ pin applied sample amount, flow velocity loading with 200ml/ minute, after loading, use eluent gradient wash-out, A moving phase is the SDS solution of 10mmol/L, B is acetonitrile mutually, in volume percent, gradient is that B is by 15% to 21%, A is by 85% to 79%, time is 40 minutes, when sharply rising suddenly, detector level starts Fractional Collections product, storing elution fraction is acetonitrile 19% to 21%, the elution fraction of the SDS aqueous solution 81% to 79% of 10mmol/L, it is the elution fraction that characteristic peak is corresponding, obtain purified for the first time.
Desalination: the present embodiment is obtained for the first time to purified sample introduction to C18 reversed-phase column (Beijing Chuangxin Tongheng Science and Technology Co., Ltd.), A moving phase purified water, B phase acetonitrile, flow velocity 250ml/ minute, the detection wavelength of 230nm; Volume percent meter, 95%A phase and 5%B phase mixing solutions, rinse 40 minutes, then switches to volume percent meter, and 60%A phase and 40%B phase mixing solutions wash-out, collect this elution fraction.By gained elution fraction, concentrate, freeze-drying obtains product 3.6g, total recovery approximately 81.0%.Products obtained therefrom is carried out to high performance liquid chromatography detection, and basically identical with the appearance time of A Ji Rayleigh standard substance, obtaining the present embodiment products obtained therefrom is A Ji Rayleigh, and purity is greater than 99%.
The preparation of embodiment 19 aminoresin
Take Rink Amide-MBHA Resin(Shangyu pul resin company limited) 8.33g, substitution degree is 0.6mmol/g, the 100mL DMF agitator treating 10 minutes that adds resin, No. three core decompress filter is removed DMF, again adds the abundant swelling of DCM 1 hour of 80mL.
The DMF solution of the hexahydropyridine that is 20% by 80mL volume percent adds in the sufficient Rink Amide-MBHA of above-mentioned swelling Resin, reacts 22 minutes No. three core decompress filters at 15 ℃, resin 80mL DMF, 80mL DCM is washing alternately, totally six times, obtains aminoresin.
The preparation of embodiment 20 first amino-acid resins
Get Fmoc-L-Arg (Pbf)-OH10mmol, HOBT10mmol, DIEA15mmol, after dissolving with 40mLNMP, join in the aminoresin that embodiment 19 makes and stir, to be cooled to-15 ℃ time, with moving liquid funnel, slowly drip EDCI20mmol, dropwise half an hour, it returns to 15 ℃ of reactions 0.5 hour naturally-15 ℃ of insulated and stirred reliefs half an hour, get several grainy resins and do the negative detection of triketohydrindene hydrate, resin water white transparency, reacts completely.No. three core decompress filter is removed reaction solution, peptide resin 80mL DMF, and each washing of 80mL DCM is once.The DMF solution of the hexahydropyridine that is 20% by 80mL volume percent adds in gained resin, reacts 22 minutes at 15 ℃, and No. three core decompress filters, for gained resin, alternately washing of 80mL DMF, 80mL DCM, totally six times, obtains the first amino-acid resin.
The preparation of embodiment 21 2 peptide resins
Get Fmoc-L-Arg (Pbf)-OH10mmol, HOBT10mmol, DIEA15mmol, after dissolving with 60mLNMP, join in the first amino-acid resin that embodiment 20 makes and stir, to be cooled to-15 ℃ time, with moving liquid funnel, slowly drip EDCI20mmol, dropwise half an hour, it returns to 15 ℃ of reactions 0.5 hour naturally-15 ℃ of insulated and stirred reliefs half an hour, get several grainy resins and do the negative detection of triketohydrindene hydrate, resin water white transparency, reacts completely.No. three core decompress filter is removed reaction solution, peptide resin 80mLDMF, and each washing of 80mL DCM is once.The DMF solution of the hexahydropyridine that is 20% by 80mL volume percent adds in gained resin, reacts 22 minutes at 15 ℃, and No. three core decompress filters, for gained resin, alternately washing of 80mL DMF, 80mL DCM, totally six times, obtains two peptide resins.
The preparation of embodiment 22 3 peptide resins
Get Fmoc-L-Gln (StBu)-OH10mmol, HOBT10mmol, DIEA15mmol, after dissolving with 70mL NMP, join in two peptide resins that embodiment 21 makes and stir, to be cooled to-15 ℃ time, with moving liquid funnel, slowly drip EDCI20mmol, dropwise half an hour, it returns to 15 ℃ of reactions 0.5 hour naturally-15 ℃ of insulated and stirred reliefs half an hour, get several grainy resins and do the negative detection of triketohydrindene hydrate, resin water white transparency, reacts completely.No. three core decompress filter is removed reaction solution, peptide resin 80mLDMF, and each washing of 80mL DCM is once.The DMF solution of the hexahydropyridine that is 20% by 80mL volume percent adds in gained resin, reacts 22 minutes at 15 ℃, and No. three core decompress filters, for gained resin, alternately washing of 80mL DMF, 80mL DCM, totally six times, obtains three peptide resins.
The preparation of embodiment 23 tetrapeptide resins
Get Fmoc-L-Met-OH10mmol, HOBT10mmol with after 60mL NMP dissolving, add DIC15mmol activation, at 20 ℃, activate 15 minutes, Bush's funnel filters out undissolved activation by product, filtrate is added in three peptide resins that embodiment 22 makes, and stirring reaction is 2 hours at 15 ℃, and triketohydrindene hydrate is done negative detection, resin water white transparency, reacts completely.No. three core decompress filter is removed reaction solution, peptide resin 80mL DMF, and each washing of 80mL DCM is once.The DMF solution of the hexahydropyridine that is 20% by 80mL volume percent adds in gained resin, reacts 22 minutes at 15 ℃, and No. three core decompress filters, for gained resin, alternately washing of 80mL DMF, 80mL DCM, totally six times, obtains tetrapeptide resin.
The preparation of embodiment 24 pentapeptide resins
Get after Fmoc-L-Glu (tBu)-OH10mmol, HOBT10mmol dissolves with 60mL NMP and add DIC15mmol activation, at 20 ℃, activate 15 minutes, Bush's funnel filters out undissolved activation by product, filtrate is added in the tetrapeptide resin that embodiment 23 makes, at 15 ℃, stirring reaction is 2 hours, triketohydrindene hydrate is done negative detection, and resin water white transparency, reacts completely.No. three core decompress filter is removed reaction solution, peptide resin 80mL DMF, and each washing of 80mL DCM is once.The DMF solution of the hexahydropyridine that is 20% by 80mL volume percent adds in gained resin, reacts 22 minutes at 15 ℃, and No. three core decompress filters, for gained resin, alternately washing of 80mL DMF, 80mL DCM, totally six times, obtains pentapeptide resin.
The preparation of embodiment 25 6 peptide resins
Get Ac-L-Glu (tBu)-OH10mmol, HOBT10mmol, add DIC15mmol activation after dissolving with 60mL NMP, at 20 ℃, activate 15 minutes, Bush's funnel filters out undissolved activation by product, filtrate is added in the pentapeptide resin that embodiment 24 makes, at 15 ℃, stirring reaction is 2 hours, triketohydrindene hydrate is done negative detection, and resin water white transparency, reacts completely.No. three core decompress filters, alternately washing of 80mL DMF, 80mL DCM for gained resin, totally six times, dry, obtain six peptide resin 15.6g.
The preparation of embodiment 26 first products
Configuration lytic reagent: be that 95:2.5:2.5 mixes according to trifluoroacetic acid, thioanisole with water volume ratio, obtain lytic reagent.According to lytic reagent recipe configuration 124.8mL lytic reagent, join in Glass Containers and place 2 hours under 0 ℃ of condition.
Excision resin and other amino acid side chain blocking group: take out the lysate 124.8mL of precooling, 15.6g six peptide resins that add embodiment 25 to make under stirring, 15 ℃ are reacted 2 hours; then filter, filtrate is added in the ether 1248mL of precooling under 4 ℃ of conditions, stirs; precipitation; precipitated liquid is centrifugal, removes supernatant liquor, ether washing four times for precipitation; each 50mL; vacuum-drying afterwards, obtains dry powder 6.1g, i.e. the first product.Gained the first product is carried out to efficient analysis liquid chromatographic detection, efficient analysis liquid chromatogram retention time under its high-efficient liquid phase chromatogram and A Ji Rayleigh standard substance the same terms is basically identical, in gained the first product, contain A Ji Rayleigh, in the first product, the chromatographic purity of A Ji Rayleigh is 83%.
The preparation of embodiment 27 A Ji Rayleighs
Get the first product 6.1g that embodiment 26 makes, by 100mL purified water, dissolve, with the filtering with microporous membrane of 0.45 micron.Gained solution is carried out to further separation and purification.
With C18 reversed-phase column (Beijing Chuangxin Tongheng Science and Technology Co., Ltd.) the first separation: chromatographic column internal diameter 100mm, detect wavelength 230nm, 5～30g/ pin applied sample amount, flow velocity loading with 200ml/ minute, after loading, use eluent gradient wash-out, A moving phase is the SDS solution of 10mmol/L, B is acetonitrile mutually, in volume percent, gradient is that B is by 15% to 21%, time is 40 minutes, when sharply rising suddenly, detector level starts Fractional Collections product, storing elution fraction is acetonitrile 19% to 21%, the elution fraction of the SDS aqueous solution 81% to 79% of 10mmol/L, it is the elution fraction that characteristic peak is corresponding, obtain purified for the first time.
Desalination: the present embodiment is obtained for the first time to purified sample introduction to C18 reversed-phase column (Beijing Chuangxin Tongheng Science and Technology Co., Ltd.), A moving phase purified water, B phase acetonitrile, flow velocity 250ml/ minute, the detection wavelength of 230nm; Volume percent meter, 95%A phase and 5%B phase mixing solutions, rinse 40 minutes, then switches to volume percent meter, and 60%A phase and 40%B phase mixing solutions wash-out, collect this elution fraction.By gained elution fraction, concentrate, freeze-drying obtains product 3.2g, total recovery approximately 72.0%.Products obtained therefrom is carried out to high performance liquid chromatography detection, and basically identical with the appearance time of A Ji Rayleigh standard substance, obtaining the present embodiment products obtained therefrom is A Ji Rayleigh, and purity is greater than 99%.
The preparation of embodiment 28 aminoresin
Take Rink Amide-MBHA Resin(Shangyu pul resin company limited) 10g, substitution degree is 0.5mmol/g, the 100mL DMF agitator treating 10 minutes that adds resin, No. three core decompress filter is removed DMF, again adds the abundant swelling of DCM 1 hour of 80mL.
The DMF solution of the hexahydropyridine that is 25% by 80mL volume percent adds in the sufficient Rink Amide-MBHA of above-mentioned swelling Resin, reacts 22 minutes No. three core decompress filters at 25 ℃, resin 80mL DMF, 80mL DCM is washing alternately, totally six times, obtains aminoresin.
The preparation of embodiment 29 first amino-acid resins
Get Fmoc-L-Arg (Pbf)-OH10mmol, HOBT10mmol, after dissolving with 40mL DMF, add DIC15mmol activation, at 25 ℃, activate 15 minutes, Bush's funnel filters out undissolved activation by product, filtrate is added in the aminoresin that embodiment 28 makes, and stirring reaction is 2 hours at 25 ℃.Get several grainy resins and do the negative detection of triketohydrindene hydrate, resin water white transparency, reacts completely.No. three core decompress filter is removed reaction solution, peptide resin 80mL DMF, and each washing of 80mL DCM is once.The DMF solution of the hexahydropyridine that is 25% by 80mL volume percent adds in gained resin, reacts 22 minutes at 25 ℃, and No. three core decompress filters, for gained resin, alternately washing of 80mL DMF, 80mL DCM, totally six times, obtains the first amino-acid resin.
The preparation of embodiment 30 2 peptide resins
Get Fmoc-L-Arg (Pbf)-OH10mmol, HOBT10mmol, after dissolving with 40mL DMF, add DIC15mmol activation, at 25 ℃, activate 15 minutes, Bush's funnel filters out undissolved activation by product, filtrate is added in the first amino-acid resin that embodiment 29 makes, and stirring reaction is 2 hours at 25 ℃.Get several grainy resins and do the negative detection of triketohydrindene hydrate, resin water white transparency, reacts completely.No. three core decompress filter is removed reaction solution, peptide resin 80mL DMF, and each washing of 80mL DCM is once.The DMF solution of the hexahydropyridine that is 25% by 80mL volume percent adds in gained resin, reacts 22 minutes No. three core decompress filters, alternately washing of 80mL DMF, 80mL DCM for gained resin, totally six times at 25 ℃.Obtain two peptide resins.
The preparation of embodiment 31 3 peptide resins
Get Fmoc-L-Gln (Trt)-OH10mmol, HOBT10mmol, after dissolving with 70mL DMF, add DIC15mmol activation, at 25 ℃, activate 15 minutes, Bush's funnel filters out undissolved activation by product, and filtrate is added in two peptide resins that embodiment 30 makes, at 25 ℃, stirring reaction is 2 hours, get several grainy resins and do the negative detection of triketohydrindene hydrate, resin water white transparency, reacts completely.No. three core decompress filter is removed reaction solution, peptide resin 80mL DMF, and each washing of 80mL DCM is once.The DMF solution of the hexahydropyridine that is 25% by 80mL volume percent adds in gained resin, reacts 22 minutes at 25 ℃, and No. three core decompress filters, for gained resin, alternately washing of 80mL DMF, 80mL DCM, totally six times, obtains three peptide resins.
The preparation of embodiment 32 tetrapeptide resins
Get Fmoc-L-Met-OH10mmol, HOBT10mmol with after 60mL NMP dissolving, add DIC15mmol activation, at 25 ℃, activate 15 minutes, Bush's funnel filters out undissolved activation by product, filtrate is added in three peptide resins that embodiment 31 makes, and stirring reaction is 2 hours at 25 ℃, and triketohydrindene hydrate is done negative detection, resin water white transparency, reacts completely.No. three core decompress filter is removed reaction solution, peptide resin 80mL DMF, and each washing of 80mL DCM is once.The DMF solution of the hexahydropyridine that is 25% by 80mL volume percent adds in gained resin, reacts 22 minutes at 25 ℃, and No. three core decompress filters, for gained resin, alternately washing of 80mL DMF, 80mL DCM, totally six times, obtains tetrapeptide resin.
The preparation of embodiment 33 pentapeptide resins
Get after Fmoc-L-Glu (tBu)-OH10mmol, HOBT10mmol dissolves with 60mL THF and add DIC15mmol activation, at 25 ℃, activate 15 minutes, Bush's funnel filters out undissolved activation by product, filtrate is added in the tetrapeptide resin that embodiment 32 makes, at 25 ℃, stirring reaction is 2 hours, triketohydrindene hydrate is done negative detection, and resin water white transparency, reacts completely.No. three core decompress filter is removed reaction solution, peptide resin 80mL DMF, and each washing of 80mL DCM is once.The DMF solution of the hexahydropyridine that is 25% by 80mL volume percent adds in gained resin, reacts 22 minutes at 25 ℃, and No. three core decompress filters, for gained resin, alternately washing of 80mL DMF, 80mL DCM, totally six times, obtains pentapeptide resin.
The preparation of embodiment 34 6 peptide resins
Get Ac-L-Glu (tBu)-OH10mmol, HOBT10mmol, add DIC15mmol activation after dissolving with 60mL THF, at 25 ℃, activate 15 minutes, Bush's funnel filters out undissolved activation by product, filtrate is added in the pentapeptide resin that embodiment 33 makes, at 25 ℃, stirring reaction is 2 hours, triketohydrindene hydrate is done negative detection, and resin water white transparency, reacts completely.No. three core decompress filters, alternately washing of 80mL DMF, 80mL DCM for gained resin, totally six times, dry, obtain six peptide resin 16.0g.
The preparation of embodiment 35 first products
Configuration lytic reagent: be that 83.0:6.3:2.1:4.3:4.3 mixes according to trifluoroacetic acid, phenol, 1，2-ethandithiol, thioanisole with water volume ratio, obtain lytic reagent.According to lytic reagent recipe configuration 160mL lytic reagent, join in Glass Containers and place 2 hours under 4 ℃ of conditions.
Excision resin and other amino acid side chain blocking group: take out the lysate 160mL of precooling, 16.0g six peptide resins that add embodiment 34 to make under stirring, 25 ℃ are reacted 2.2 hours; then filter, filtrate is added in the ether 1600mL of precooling under 4 ℃ of conditions, stirs; precipitation; precipitated liquid is centrifugal, removes supernatant liquor, ether washing four times for precipitation; each 50mL; vacuum-drying afterwards, obtains dry powder 4.2g, i.e. the first product.Gained the first product is carried out to efficient analysis liquid chromatographic detection, and the chromatographic purity that obtains A Ji Rayleigh in the first product is 71%.
The preparation of embodiment 36 A Ji Rayleighs
Get the first product 4.2g that embodiment 35 makes, by 100mL purified water, dissolve, with the filtering with microporous membrane of 0.45 micron.Gained solution is carried out to further separation and purification.
With C18 reversed-phase column (Beijing Chuangxin Tongheng Science and Technology Co., Ltd.) the first separation: chromatographic column internal diameter 100mm, detect wavelength 230nm, 5～30g/ pin applied sample amount, flow velocity loading with 200ml/ minute, after loading, use eluent gradient wash-out, A moving phase is the SDS solution of 10mmol/L, B is acetonitrile mutually, in volume percent, gradient is that B is by 15% to 21%, B is 85% to 79% mutually, time is 40 minutes, when sharply rising suddenly, detector level starts Fractional Collections product, storing elution fraction is acetonitrile 19% to 21%, the elution fraction of the SDS aqueous solution 81% to 79% of 10mmol/L, it is the elution fraction that characteristic peak is corresponding, obtain purified for the first time.
Desalination: the present embodiment is obtained for the first time to purified sample introduction to C18 reversed-phase column (Beijing Chuangxin Tongheng Science and Technology Co., Ltd.), A moving phase purified water, B phase acetonitrile, flow velocity 250ml/ minute, the detection wavelength of 230nm; Volume percent meter, 95%A phase and 5%B phase mixing solutions, rinse 40 minutes, then switches to volume percent meter, and 60%A phase and 40%B phase mixing solutions wash-out, collect this elution fraction.By gained elution fraction, concentrate, freeze-drying obtains product 2.3g, total recovery approximately 51.8%.Products obtained therefrom is carried out to high performance liquid chromatography detection, and basically identical with the appearance time of A Ji Rayleigh standard substance, obtaining the present embodiment products obtained therefrom is A Ji Rayleigh, and purity is greater than 99%.
The preparation of embodiment 37 first amino-acid resins
Get Fmoc-L-Arg (Pbf)-OH25mmol, HOBT30mmol, after dissolving with 40mL DMF, add DIC30mmol activation, at 25 ℃, activate 15 minutes, Bush's funnel filters out undissolved activation by product, filtrate is added in the aminoresin that embodiment 28 makes, and stirring reaction is 2 hours at 25 ℃.Get several grainy resins and do the negative detection of triketohydrindene hydrate, resin water white transparency, reacts completely.No. three core decompress filter is removed reaction solution, peptide resin 80mL DMF, and each washing of 80mL DCM is once.The DMF solution of the hexahydropyridine that is 25% by 80mL volume percent adds in gained resin, reacts 22 minutes at 25 ℃, and No. three core decompress filters, for gained resin, alternately washing of 80mL DMF, 80mL DCM, totally six times, obtains the first amino-acid resin.
The preparation of embodiment 38 2 peptide resins
Get Fmoc-L-Arg (Pbf)-OH25mmol, HOBT30mmol, after dissolving with 40mL DMF, add DIC30mmol activation, at 25 ℃, activate 15 minutes, Bush's funnel filters out undissolved activation by product, filtrate is added in the first amino-acid resin that embodiment 37 makes, and stirring reaction is 2 hours at 25 ℃.Get several grainy resins and do the negative detection of triketohydrindene hydrate, resin water white transparency, reacts completely.No. three core decompress filter is removed reaction solution, peptide resin 80mL DMF, and each washing of 80mL DCM is once.The DMF solution of the hexahydropyridine that is 25% by 80mL volume percent adds in gained resin, reacts 22 minutes at 25 ℃, and No. three core decompress filters, for gained resin, alternately washing of 80mL DMF, 80mL DCM, totally six times, obtains two peptide resins.
The preparation of embodiment 39 3 peptide resins
Get Fmoc-L-Gln (Trt)-OH25mmol, HOBT30mmol, after dissolving with 70mL DMF, add DIC30mmol activation, at 25 ℃, activate 15 minutes, Bush's funnel filters out undissolved activation by product, and filtrate is added in two peptide resins that embodiment 38 makes, at 25 ℃, stirring reaction is 2 hours, get several grainy resins and do the negative detection of triketohydrindene hydrate, resin water white transparency, reacts completely.No. three core decompress filter is removed reaction solution, peptide resin 80mL DMF, and each washing of 80mL DCM is once.The DMF solution of the hexahydropyridine that is 25% by 80mL volume percent adds in gained resin, reacts 22 minutes at 25 ℃, and No. three core decompress filters, for gained resin, alternately washing of 80mL DMF, 80mL DCM, totally six times, obtains three peptide resins.
The preparation of embodiment 40 tetrapeptide resins
Get Fmoc-L-Met-OH25mmol, HOBT30mmol with after 60mL DMF dissolving, add DIC30mmol activation, at 25 ℃, activate 15 minutes, Bush's funnel filters out undissolved activation by product, filtrate is added in three peptide resins that embodiment 39 makes, and stirring reaction is 2 hours at 25 ℃, and triketohydrindene hydrate is done negative detection, resin water white transparency, reacts completely.No. three core decompress filter is removed reaction solution, peptide resin 80mL DMF, and each washing of 80mL DCM is once.The DMF solution of the hexahydropyridine that is 25% by 80mL volume percent adds in gained resin, reacts 22 minutes at 25 ℃, and No. three core decompress filters, for gained resin, alternately washing of 80mL DMF, 80mL DCM, totally six times, obtains tetrapeptide resin.
The preparation of embodiment 41 pentapeptide resins
Get after Fmoc-L-Glu (tBu)-OH25mmol, HOBT30mmol dissolves with 60mL DMF and add DIC30mmol activation, at 25 ℃, activate 15 minutes, Bush's funnel filters out undissolved activation by product, filtrate is added in the tetrapeptide resin that embodiment 40 makes, at 25 ℃, stirring reaction is 2 hours, triketohydrindene hydrate is done negative detection, and resin water white transparency, reacts completely.No. three core decompress filter is removed reaction solution, peptide resin 80mL DMF, and each washing of 80mL DCM is once.The DMF solution of the hexahydropyridine that is 25% by 80mL volume percent adds in gained resin, reacts 22 minutes at 25 ℃, and No. three core decompress filters, for gained resin, alternately washing of 80mL DMF, 80mL DCM, totally six times, obtains pentapeptide resin.
The preparation of embodiment 42 6 peptide resins
Get Ac-L-Glu (tBu)-OH25mmol, HOBT30mmol, add DIC30mmol activation after dissolving with 60mL DMF, at 25 ℃, activate 15 minutes, Bush's funnel filters out undissolved activation by product, filtrate is added in the pentapeptide resin that embodiment 41 makes, at 25 ℃, stirring reaction is 2 hours, triketohydrindene hydrate is done negative detection, and resin water white transparency, reacts completely.No. three core decompress filters, alternately washing of 80mL DMF, 80mL DCM for gained resin, totally six times, dry, obtain six peptide resin 15.5g.
The preparation of embodiment 43 first products
Configuration lytic reagent: be that 83.0:6.3:2.1:4.3:4.3 mixes according to trifluoroacetic acid, phenol, 1，2-ethandithiol, thioanisole with water volume ratio, obtain lytic reagent.According to lytic reagent recipe configuration 186mL lytic reagent, join in Glass Containers and place 2 hours under 4 ℃ of conditions.
Excision resin and other amino acid side chain blocking group: take out the lysate 186mL of precooling, 15.5g six peptide resins that add embodiment 42 to make under stirring, 25 ℃ are reacted 2.2 hours; then filter, filtrate is added in the ether 1860mL of precooling under 4 ℃ of conditions, stirs; precipitation; precipitated liquid is centrifugal, removes supernatant liquor, ether washing four times for precipitation; each 50mL; vacuum-drying afterwards, obtains dry powder 4.0g, i.e. the first product.Gained the first product is carried out to efficient analysis liquid chromatographic detection, and the chromatographic purity that obtains A Ji Rayleigh in the first product is 69%.
The preparation of embodiment 44 A Ji Rayleighs
Get the first product 4.0g that embodiment 43 makes, by 100mL purified water, dissolve, with the filtering with microporous membrane of 0.45 micron.Gained solution is carried out to further separation and purification.
With C18 reversed-phase column (Beijing Chuangxin Tongheng Science and Technology Co., Ltd.) the first separation: chromatographic column internal diameter 100mm, detect wavelength 230nm, 5～30g/ pin applied sample amount, flow velocity loading with 200ml/ minute, after loading, use eluent gradient wash-out, A moving phase is the SDS solution of 10mmol/L, B is acetonitrile mutually, in volume percent, gradient is that B is by 15% to 21%, A phase 85% to 79%, when sharply rising suddenly, detector level starts Fractional Collections product, storing elution fraction is acetonitrile 19% to 21%, the elution fraction of the SDS aqueous solution 81% to 79% of 10mmol/L, it is the elution fraction that characteristic peak is corresponding, obtain purified for the first time.
Desalination: the present embodiment is obtained for the first time to purified sample introduction to C18 reversed-phase column (Beijing Chuangxin Tongheng Science and Technology Co., Ltd.), A moving phase purified water, B phase acetonitrile, flow velocity 250ml/ minute, the detection wavelength of 230nm; Volume percent meter, 95%A phase and 5%B phase mixing solutions, rinse 40 minutes, then switches to volume percent meter, and 60%A phase and 40%B phase mixing solutions wash-out, collect this elution fraction.By gained elution fraction, concentrate, freeze-drying obtains product 2.2g, total recovery approximately 49.5%.Products obtained therefrom is carried out to high performance liquid chromatography detection, and basically identical with the appearance time of A Ji Rayleigh standard substance, obtaining the present embodiment products obtained therefrom is A Ji Rayleigh, and purity is greater than 99%.
The preparation of embodiment 45 first amino-acid resins
Get Fmoc-L-Arg (Pbf)-OH 15mmol, HOBT 20mmol, after dissolving with 40mL DMF, add DIC 20mmol activation, at 25 ℃, activate 15 minutes, Bush's funnel filters out undissolved activation by product, filtrate is added in the aminoresin that embodiment 28 makes, and stirring reaction is 2 hours at 25 ℃.Get several grainy resins and do the negative detection of triketohydrindene hydrate, resin water white transparency, reacts completely.No. three core decompress filter is removed reaction solution, peptide resin 80mL DMF, and each washing of 80mL DCM is once.The DMF solution of the hexahydropyridine that is 25% by 80mL volume percent adds in gained resin, reacts 22 minutes at 25 ℃, and No. three core decompress filters, for gained resin, alternately washing of 80mL DMF, 80mL DCM, totally six times, obtains the first amino-acid resin.
The preparation of embodiment 46 2 peptide resins
Get Fmoc-L-Arg (Pbf)-OH 15mmol, HOBT 20mmol, after dissolving with 40mL DMF, add DIC 20mmol activation, at 25 ℃, activate 15 minutes, Bush's funnel filters out undissolved activation by product, filtrate is added in the first amino-acid resin that embodiment 45 makes, and stirring reaction is 2 hours at 25 ℃.Get several grainy resins and do the negative detection of triketohydrindene hydrate, resin water white transparency, reacts completely.No. three core decompress filter is removed reaction solution, peptide resin 80mL DMF, and each washing of 80mL DCM is once.The DMF solution of the hexahydropyridine that is 25% by 80mL volume percent adds in gained resin, reacts 22 minutes at 25 ℃, and No. three core decompress filters, for gained resin, alternately washing of 80mL DMF, 80mL DCM, totally six times, obtains two peptide resins.
The preparation of embodiment 47 3 peptide resins
Get Fmoc-L-Gln (Trt)-OH 15mmol, HOBT 20mmol, after dissolving with 70mL DMF, add DIC 20mmol activation, at 25 ℃, activate 15 minutes, Bush's funnel filters out undissolved activation by product, and filtrate is added in two peptide resins that embodiment 46 makes, at 25 ℃, stirring reaction is 2 hours, get several grainy resins and do the negative detection of triketohydrindene hydrate, resin water white transparency, reacts completely.No. three core decompress filter is removed reaction solution, peptide resin 80mL DMF, and each washing of 80mL DCM is once.The DMF solution of the hexahydropyridine that is 25% by 80mL volume percent adds in gained resin, reacts 22 minutes at 25 ℃, and No. three core decompress filters, for gained resin, alternately washing of 80mL DMF, 80mL DCM, totally six times, obtains three peptide resins.
The preparation of embodiment 48 tetrapeptide resins
Get Fmoc-L-Met-OH15mmol, HOBT20mmol with after 60mL DMF dissolving, add DIC20mmol activation, at 25 ℃, activate 15 minutes, Bush's funnel filters out undissolved activation by product, filtrate is added in three peptide resins that embodiment 47 makes, and stirring reaction is 2 hours at 25 ℃, and triketohydrindene hydrate is done negative detection, resin water white transparency, reacts completely.No. three core decompress filter is removed reaction solution, peptide resin 80mL DMF, and each washing of 80mL DCM is once.The DMF solution of the hexahydropyridine that is 25% by 80mL volume percent adds in gained resin, reacts 22 minutes at 25 ℃, and No. three core decompress filters, for gained resin, alternately washing of 80mL DMF, 80mL DCM, totally six times, obtains tetrapeptide resin.
The preparation of embodiment 49 pentapeptide resins
Get after Fmoc-L-Glu (tBu)-OH15mmol, HOBT20mmol dissolves with 60mL DMF and add DIC20mmol activation, at 25 ℃, activate 15 minutes, Bush's funnel filters out undissolved activation by product, filtrate is added in the tetrapeptide resin that embodiment 48 makes, at 25 ℃, stirring reaction is 2 hours, triketohydrindene hydrate is done negative detection, and resin water white transparency, reacts completely.No. three core decompress filter is removed reaction solution, peptide resin 80mL DMF, and each washing of 80mL DCM is once.The DMF solution of the hexahydropyridine that is 25% by 80mL volume percent adds in gained resin, reacts 22 minutes at 25 ℃, and No. three core decompress filters, for gained resin, alternately washing of 80mL DMF, 80mL DCM, totally six times, obtains pentapeptide resin.
The preparation of embodiment 50 6 peptide resins
Get Ac-L-Glu (tBu)-OH15mmol, HOBT20mmol, add DIC20mmol activation after dissolving with 60mL DMF, at 25 ℃, activate 15 minutes, Bush's funnel filters out undissolved activation by product, filtrate is added in the pentapeptide resin that embodiment 49 makes, at 25 ℃, stirring reaction is 2 hours, triketohydrindene hydrate is done negative detection, and resin water white transparency, reacts completely.No. three core decompress filters, alternately washing of 80mL DMF, 80mL DCM for gained resin, totally six times, dry, obtain six peptide resin 16.4g.
The preparation of embodiment 51 first products
Configuration lytic reagent: be that 83.0:6.3:2.1:4.3:4.3 mixes according to trifluoroacetic acid, phenol, 1，2-ethandithiol, thioanisole with water volume ratio, obtain lytic reagent.According to lytic reagent recipe configuration 164mL lytic reagent, join in Glass Containers and place 2 hours under 4 ℃ of conditions.
Excision resin and other amino acid side chain blocking group: take out the lysate 164mL of precooling, 16.4g six peptide resins that add embodiment 50 to make under stirring, 25 ℃ are reacted 2.2 hours; then filter, filtrate is added in the ether 1640mL of precooling under 4 ℃ of conditions, stirs; precipitation; precipitated liquid is centrifugal, removes supernatant liquor, ether washing four times for precipitation; each 50mL; vacuum-drying afterwards, obtains dry powder 4.4g, i.e. the first product.Gained the first product is carried out to efficient analysis liquid chromatographic detection, and the chromatographic purity that obtains A Ji Rayleigh in the first product is 73%.
The preparation of embodiment 52 A Ji Rayleighs
Get the first product 4.4g that embodiment 51 makes, by 100mL purified water, dissolve, with the filtering with microporous membrane of 0.45 micron.Gained solution is carried out to further separation and purification.
With C18 reversed-phase column (Beijing Chuangxin Tongheng Science and Technology Co., Ltd.) the first separation: chromatographic column internal diameter 100mm, detect wavelength 230nm, 5～30g/ pin applied sample amount, flow velocity loading with 200ml/ minute, after loading, use eluent gradient wash-out, A moving phase is the SDS solution of 10mmol/L, B is acetonitrile mutually, in volume percent, gradient is that B is by 15% to 21%, B is by 85% to 79%, time is 40 minutes, when sharply rising suddenly, detector level starts Fractional Collections product, storing elution fraction is acetonitrile 19% to 21%, the elution fraction of the SDS aqueous solution 81% to 79% of 10mmol/L, it is the elution fraction that characteristic peak is corresponding, obtain purified for the first time.
Desalination: the present embodiment is obtained for the first time to purified sample introduction to C18 reversed-phase column (Beijing Chuangxin Tongheng Science and Technology Co., Ltd.), A moving phase purified water, B phase acetonitrile, flow velocity 250ml/ minute, the detection wavelength of 230nm; Volume percent meter, 95%A phase and 5%B phase mixing solutions, rinse 40 minutes, then switches to volume percent meter, and 60%A phase and 40%B phase mixing solutions wash-out, collect this elution fraction.By gained elution fraction, concentrate, freeze-drying obtains product 2.4g, total recovery approximately 54%.Products obtained therefrom is carried out to high performance liquid chromatography detection, and basically identical with the appearance time of A Ji Rayleigh standard substance, obtaining the present embodiment products obtained therefrom is A Ji Rayleigh, and purity is greater than 99%.
A Ji Rayleigh yield and the purity tool of preparation method's output that the patent document that the A Ji Rayleigh of above-described embodiment output is CN102603869A compared with publication number is recorded improve a lot; production cost has obvious reduction, has more the potentiality of industrialization and large-scale production.
Comparative example
Coupling agent is the important factor that affects polypeptide synthesis yield and purity, and the present invention, when conditional filtering, has investigated the application of other coupling agents in the preparation of A Ji Rayleigh simultaneously.
Taking Rink Amide-MBHA Resin(buys in Shangyu pul resin company limited) 10g, substitution degree is 0.5mmol/g, the 100mL DMF agitator treating 10 minutes that adds resin, No. three core decompress filter is removed DMF, again adds the abundant swelling of DCM 1 hour of 80mL.
The DMF solution of the hexahydropyridine that is 25% by 80mL volume percent adds in the sufficient Rink Amide-MBHA of above-mentioned swelling Resin, reacts 20 minutes No. three core decompress filters at 25 ℃, resin 80mL DMF, 80mL DCM is washing alternately, totally six times, obtains aminoresin.
Get Fmoc-L-Arg (Pbf)-OH15mmol, HOBT20mmol, after HBTU20mmol dissolves with 40mLDMF, add DIEA30mmol activation, activate 15 minutes at 25 ℃, activation solution is added in above-mentioned aminoresin, and stirring reaction is 4 hours at 25 ℃.Get several grainy resins and do the negative detection of triketohydrindene hydrate, the micro-indigo plant of resin, illustrates reaction not exclusively, and reactive behavior is inadequate.No. three core decompress filters, resin 80mLDMF, 80mL DCM is washing alternately, according to above-mentioned feeding intake, member is mended and is thrown once, and stirring reaction is 1 hour at 25 ℃, and get several grainy resins and do that triketohydrindene hydrate is negative to be detected, resin transparent, explanation reacts completely.No. three core decompress filters, resin 80mL DMF, 80mL DCM alternately washs each once.The DMF solution of the hexahydropyridine that is 25% by 80mL volume percent adds in gained resin, react 20 minutes at 25 ℃, No. three core decompress filters, alternately washing of 80mL DMF, 80mL DCM for gained resin, totally six times the first amino-acid resin.
Get Fmoc-L-Arg (Pbf)-OH15mmol, HOBT20mmol, after HBTU20mmol dissolves with 40mLDMF, add DIEA30mmol activation, activate 15 minutes at 25 ℃, activation solution is added in above-mentioned the first amino-acid resin, and stirring reaction is 4 hours at 25 ℃.Get several grainy resins and do the negative detection of triketohydrindene hydrate, the micro-indigo plant of resin, illustrates reaction not exclusively, and reactive behavior is inadequate.No. three core decompress filters, resin 80mL DMF, 80mL DCM is washing alternately, according to above-mentioned feeding intake, member is mended and is thrown once, and stirring reaction is 1 hour at 25 ℃, and get several grainy resins and do that triketohydrindene hydrate is negative to be detected, resin transparent, explanation reacts completely.No. three core decompress filters, resin 80mL DMF, 80mL DCM alternately washs each once.The DMF solution of the hexahydropyridine that is 25% by 80mL volume percent adds in gained resin, react 20 minutes at 25 ℃, No. three core decompress filters, alternately washing of 80mL DMF, 80mL DCM for gained resin, totally six times two peptide resins.
Get Fmoc-L-Gln (Trt)-OH15mmol, HOBT20mmol, after HBTU20mmol dissolves with 40mLDMF, add DIEA30mmol activation, activate 15 minutes at 25 ℃, activation solution is added in above-mentioned two peptide resins, and stirring reaction is 4 hours at 25 ℃.Get several grainy resins and do the negative detection of triketohydrindene hydrate, the micro-indigo plant of resin, illustrates reaction not exclusively, and reactive behavior is inadequate.No. three core decompress filters, resin 80mLDMF, 80mL DCM is washing alternately, according to above-mentioned feeding intake, member is mended and is thrown once, and stirring reaction is 1 hour at 25 ℃, and get several grainy resins and do that triketohydrindene hydrate is negative to be detected, resin transparent, explanation reacts completely.No. three core decompress filters, resin 80mL DMF, 80mL DCM alternately washs each once.The DMF solution of the hexahydropyridine that is 25% by 80mL volume percent adds in gained resin, react 20 minutes at 25 ℃, No. three core decompress filters, alternately washing of 80mL DMF, 80mL DCM for gained resin, totally six times three peptide resins.
Get Fmoc-L-Met-OH15mmol, HOBT20mmol, after HBTU20mmol dissolves with 40mLDMF, add DIEA30mmol activation, activate 15 minutes at 25 ℃, activation solution is added in above-mentioned three peptide resins, and stirring reaction is 4 hours at 25 ℃.Get several grainy resins and do the negative detection of triketohydrindene hydrate, resin transparent, explanation reacts completely.No. three core decompress filters, resin 80mL DMF, 80mL DCM alternately washs each once.The DMF solution of the hexahydropyridine that is 25% by 80mL volume percent adds in gained resin, react 20 minutes at 25 ℃, No. three core decompress filters, gained is alternately washing of 80mLDMF, 80mL DCM for resin, totally six times tetrapeptide resin.
Get Fmoc-L-Glu (tBu)-OH15mmol, HOBT20mmol, after HBTU20mmol dissolves with 40mLDMF, add DIEA30mmol activation, activate 15 minutes at 25 ℃, activation solution is added in above-mentioned tetrapeptide resin, and stirring reaction is 4 hours at 25 ℃.Get several grainy resins and do the negative detection of triketohydrindene hydrate, resin transparent, explanation reacts completely.No. three core decompress filters, resin 80mL DMF, 80mL DCM alternately washs each once.The DMF solution of the hexahydropyridine that is 25% by 80mL volume percent adds in gained resin, react 20 minutes at 25 ℃, No. three core decompress filters, gained is alternately washing of 80mLDMF, 80mL DCM for resin, totally six times pentapeptide resin.
Get Ac-L-Glu (tBu)-OH15mmol, HOBT20mmol, after HBTU20mmol dissolves with 40mLDMF, add DIEA30mmol activation, activate 15 minutes at 25 ℃, activation solution is added in above-mentioned pentapeptide resin, and stirring reaction is 4 hours at 25 ℃.Get several grainy resins and do the negative detection of triketohydrindene hydrate, resin transparent, explanation reacts completely.No. three core decompress filters, resin 80mL DMF, 80mL DCM is washing alternately, totally six times, is dried to obtain six peptide resin 14.2g.
Configuration lytic reagent: be that 83.0:6.3:2.1:4.3:4.3 mixes according to trifluoroacetic acid, phenol, 1，2-ethandithiol, thioanisole with water volume ratio, obtain lytic reagent.According to lytic reagent recipe configuration 142mL lytic reagent, join in Glass Containers and place 2 hours under 4 ℃ of conditions.
Excision resin and other amino acid side chain blocking group: take out the lysate 142mL of precooling, add 14.2g six peptide resins of gained under stirring, 25 ℃ are reacted 2.2 hours; then filter, filtrate is added in the ether 1420mL of precooling under 4 ℃ of conditions, stirs; precipitation; precipitated liquid is centrifugal, removes supernatant liquor, ether washing four times for precipitation; each 50mL; vacuum-drying afterwards, obtains dry powder 2.9g, i.e. the first product.Gained the first product is carried out to efficient analysis liquid chromatographic detection, obtain the chromatographic purity 42% of A Ji Rayleigh in the first product, the necessity not being further purified.Gained the first product, it is A Ji Rayleigh crude product, hence one can see that, and A Ji Rayleigh crude product quality that preparation method that comparative example provides obtains is few, purity is low, compares with the first product that the corresponding embodiment of preparation method provided by the invention makes, yield and purity all significantly reduce, and whole preparation process time is long, especially first three amino acid whose coupling, length expends time in, and need a large amount of coupling reagents, cost significantly increases.
Below be only the preferred embodiment of the present invention, it should be pointed out that above-mentioned preferred implementation should not be considered as limitation of the present invention, protection scope of the present invention should be as the criterion with claim limited range.For those skilled in the art, without departing from the spirit and scope of the present invention, can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.

Claims (13)

1. a preparation method for A Ji Rayleigh, is characterized in that, comprises the following steps:
Step 1: get resin, activation, obtains aminoresin;
Step 2: get described aminoresin, the first L-arginine that progressively coupling guanidine radicals is protected, the L-arginine of guanidine radicals protection, the L-glutaminate of side chain amido protecting, obtain three peptide resins;
Step 3: get described three peptide resins, the second Pidolidone that progressively coupling METHIONINE, side chain carboxyl group are protected is, the N-acetyl-Pidolidone of side chain carboxyl group protection obtains six peptide resins;
Step 4: six peptide resins described in cracking, obtain the first product;
Step 5: get described the first product, purifying, obtains.
2. preparation method according to claim 1, is characterized in that, resin is Rink Amide-MBHA Resin described in step 1.
3. preparation method according to claim 1, is characterized in that, described in step 2 first progressively coupling be specially:
Get the L-arginine of described aminoresin and the protection of N end, guanidine radicals protection under the first action of coupling agents, the first linked reaction occurs, through the first deprotection, obtain the first amino-acid resin;
Get the L-arginine of described the first amino-acid resin and the protection of N end, guanidine radicals protection under the second action of coupling agents, the second linked reaction occurs, through the second deprotection, obtain two peptide resins;
Get the L-glutaminate of described two peptide resins and the protection of N end, side chain amido protecting under the 3rd action of coupling agents, the 3rd linked reaction occurs, through the 3rd deprotection, obtain three peptide resins.
4. preparation method according to claim 1, is characterized in that, described in step 3 second progressively coupling be specially:
Get the METHIONINE of described three peptide resins and the protection of N end under the 4th action of coupling agents, the 4th linked reaction occurs, through the 4th deprotection, obtain tetrapeptide resin;
Get the Pidolidone of described tetrapeptide resin and the protection of N end, side chain carboxyl group protection under the 5th action of coupling agents, the 5th linked reaction occurs, through the 5th deprotection, obtain pentapeptide resin;
Get the N-acetyl-Pidolidone of described pentapeptide resin and side chain carboxyl group protection under the 6th action of coupling agents, the 6th linked reaction occurs, obtain six peptide resins.
5. preparation method according to claim 3, is characterized in that, described the first coupling agent is EDCI/HOBT/DIEA or HOBT/DIC.
6. preparation method according to claim 3, is characterized in that, described the second coupling agent is EDCI/HOBT/DIEA or HOBT/DIC.
7. preparation method according to claim 3, is characterized in that, described the 3rd coupling agent is EDCI/HOBT/DIEA or HOBT/DIC.
8. preparation method according to claim 4, is characterized in that, described the 4th coupling agent is HOBT/DIC.
9. preparation method according to claim 4, is characterized in that, described the 5th coupling agent is HOBT/DIC.
10. preparation method according to claim 4, is characterized in that, described the 6th coupling agent is HOBT/DIC.
11. preparation methods according to claim 1, is characterized in that, the lytic reagent of cracking comprises trifluoroacetic acid, thioanisole and water described in step 4.
12. preparation methods according to claim 11, is characterized in that, described lytic reagent also comprises phenol and 1，2-ethandithiol.
13. preparation methods according to claim 1, is characterized in that, purifying is specially described in step 5:
Get described the first product, through first reverse-phase chromatographic column the first separation, the SDS aqueous solution-acetonitrile system gradient elution of 10mmol/L, collects elution fraction corresponding to characteristic peak, obtains purified for the first time; In volume percent, described gradient elution is acetonitrile by 15% to 21%, the SDS aqueous solution of 10mmol/L is by 85% to 79%;
Purified for the first time described in getting, through second reverse-phase chromatographic column the second separation, counts with volume percent after acetonitrile solution the first wash-out of 5%, counts acetonitrile solution the second wash-out of 40% with volume percent, collects the second wash-out obtained component, obtains.
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CN106632608A (en) * 2017-01-04 2017-05-10 陕西慧康生物科技有限责任公司 Purifying method for arigireline
CN106699847B (en) * 2017-01-04 2020-08-14 陕西慧康生物科技有限责任公司 Method for purifying hexapeptide at low cost
CN106699847A (en) * 2017-01-04 2017-05-24 陕西慧康生物科技有限责任公司 Method for purifying argireline at low cost
CN106749529A (en) * 2017-01-05 2017-05-31 陕西慧康生物科技有限责任公司 A kind of preparation method of Argireline and products thereof
WO2018126525A1 (en) * 2017-01-05 2018-07-12 陕西慧康生物科技有限责任公司 Method for preparing hexapeptide, and product thereof
CN106632609B (en) * 2017-01-05 2020-05-19 陕西慧康生物科技有限责任公司 Preparation method of hexapeptide and product thereof
CN106632609A (en) * 2017-01-05 2017-05-10 陕西慧康生物科技有限责任公司 Method for preparing hexapeptide and product thereof
WO2020085778A1 (en) * 2018-10-23 2020-04-30 웰펩 주식회사 Process for preparing acetyl hexapeptide-3
CN110426476A (en) * 2019-08-15 2019-11-08 珠海伊斯佳科技股份有限公司 The measuring method of Argireline content in a kind of cosmetics
CN110684077A (en) * 2019-10-18 2020-01-14 陕西慧康生物科技有限责任公司 Large-scale synthesis method of achirelin
CN112851746A (en) * 2019-11-26 2021-05-28 深圳翰宇药业股份有限公司 Desalination method utilizing freeze-drying principle
WO2021103454A1 (en) * 2019-11-26 2021-06-03 深圳翰宇药业股份有限公司 Desalination method using lyophilization principle
CN114057838A (en) * 2022-01-11 2022-02-18 浙江湃肽生物有限公司南京分公司 Solid-phase synthesis method of acetyl hexapeptide-8
CN114057838B (en) * 2022-01-11 2022-04-12 浙江湃肽生物有限公司南京分公司 Solid-phase synthesis method of acetyl hexapeptide-8

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