CN114369139B - Preparation method of snake venom tripeptide - Google Patents
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- CN114369139B CN114369139B CN202011097855.2A CN202011097855A CN114369139B CN 114369139 B CN114369139 B CN 114369139B CN 202011097855 A CN202011097855 A CN 202011097855A CN 114369139 B CN114369139 B CN 114369139B
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- 239000003998 snake venom Substances 0.000 title claims abstract description 37
- 238000002360 preparation method Methods 0.000 title claims abstract description 13
- LIWKOFAHRLBNMG-FQEVSTJZSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-4-[(2-methylpropan-2-yl)oxycarbonylamino]butanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CCNC(=O)OC(C)(C)C)C(O)=O)C3=CC=CC=C3C2=C1 LIWKOFAHRLBNMG-FQEVSTJZSA-N 0.000 claims abstract description 36
- 238000000034 method Methods 0.000 claims abstract description 34
- WGQKYBSKWIADBV-UHFFFAOYSA-N benzylamine Chemical compound NCC1=CC=CC=C1 WGQKYBSKWIADBV-UHFFFAOYSA-N 0.000 claims abstract description 25
- 238000003786 synthesis reaction Methods 0.000 claims abstract description 22
- 230000015572 biosynthetic process Effects 0.000 claims abstract description 21
- TVWATMRQKCTKAU-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 3-[(2-methylpropan-2-yl)oxycarbonylamino]propanoate Chemical compound CC(C)(C)OC(=O)NCCC(=O)ON1C(=O)CCC1=O TVWATMRQKCTKAU-UHFFFAOYSA-N 0.000 claims abstract description 20
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 claims abstract description 14
- 238000004519 manufacturing process Methods 0.000 claims abstract description 7
- 238000000926 separation method Methods 0.000 claims abstract description 7
- -1 Boc groups Chemical group 0.000 claims abstract description 5
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 111
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 53
- 238000006243 chemical reaction Methods 0.000 claims description 43
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 claims description 36
- 239000002904 solvent Substances 0.000 claims description 32
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 claims description 26
- 239000007864 aqueous solution Substances 0.000 claims description 24
- FDKXTQMXEQVLRF-ZHACJKMWSA-N (E)-dacarbazine Chemical compound CN(C)\N=N\c1[nH]cnc1C(N)=O FDKXTQMXEQVLRF-ZHACJKMWSA-N 0.000 claims description 22
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 21
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 21
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 19
- 239000000243 solution Substances 0.000 claims description 19
- 239000003795 chemical substances by application Substances 0.000 claims description 17
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 17
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 15
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 15
- 239000003960 organic solvent Substances 0.000 claims description 15
- 239000007787 solid Substances 0.000 claims description 15
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 14
- 229910000029 sodium carbonate Inorganic materials 0.000 claims description 13
- YEDUAINPPJYDJZ-UHFFFAOYSA-N 2-hydroxybenzothiazole Chemical compound C1=CC=C2SC(O)=NC2=C1 YEDUAINPPJYDJZ-UHFFFAOYSA-N 0.000 claims description 11
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 claims description 11
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 claims description 10
- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide Substances CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 claims description 9
- QOSSAOTZNIDXMA-UHFFFAOYSA-N Dicylcohexylcarbodiimide Chemical compound C1CCCCC1N=C=NC1CCCCC1 QOSSAOTZNIDXMA-UHFFFAOYSA-N 0.000 claims description 9
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 9
- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 claims description 8
- 230000002378 acidificating effect Effects 0.000 claims description 8
- PFKFTWBEEFSNDU-UHFFFAOYSA-N carbonyldiimidazole Chemical compound C1=CN=CN1C(=O)N1C=CN=C1 PFKFTWBEEFSNDU-UHFFFAOYSA-N 0.000 claims description 8
- 230000018044 dehydration Effects 0.000 claims description 7
- 238000006297 dehydration reaction Methods 0.000 claims description 7
- 239000003208 petroleum Substances 0.000 claims description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 6
- 238000006482 condensation reaction Methods 0.000 claims description 6
- 239000012046 mixed solvent Substances 0.000 claims description 6
- 239000003513 alkali Substances 0.000 claims description 5
- 239000007788 liquid Substances 0.000 claims description 4
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 claims description 4
- 239000007821 HATU Substances 0.000 claims description 3
- 239000002585 base Substances 0.000 claims description 3
- MVEAAGBEUOMFRX-UHFFFAOYSA-N ethyl acetate;hydrochloride Chemical compound Cl.CCOC(C)=O MVEAAGBEUOMFRX-UHFFFAOYSA-N 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 3
- 238000005406 washing Methods 0.000 claims description 3
- GSNUFIFRDBKVIE-UHFFFAOYSA-N DMF Natural products CC1=CC=C(C)O1 GSNUFIFRDBKVIE-UHFFFAOYSA-N 0.000 claims description 2
- 239000012074 organic phase Substances 0.000 claims description 2
- 239000011736 potassium bicarbonate Substances 0.000 claims description 2
- 229910000028 potassium bicarbonate Inorganic materials 0.000 claims description 2
- 229910000027 potassium carbonate Inorganic materials 0.000 claims description 2
- TYJJADVDDVDEDZ-UHFFFAOYSA-M potassium hydrogencarbonate Chemical compound [K+].OC([O-])=O TYJJADVDDVDEDZ-UHFFFAOYSA-M 0.000 claims description 2
- 238000002156 mixing Methods 0.000 claims 2
- 239000012071 phase Substances 0.000 claims 1
- 238000000746 purification Methods 0.000 abstract description 10
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 abstract description 5
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 5
- 238000004128 high performance liquid chromatography Methods 0.000 abstract description 4
- 238000011282 treatment Methods 0.000 abstract description 3
- 238000003756 stirring Methods 0.000 description 19
- NQRYJNQNLNOLGT-UHFFFAOYSA-N Piperidine Chemical compound C1CCNCC1 NQRYJNQNLNOLGT-UHFFFAOYSA-N 0.000 description 10
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 9
- 150000001413 amino acids Chemical class 0.000 description 9
- 238000012544 monitoring process Methods 0.000 description 9
- 150000003839 salts Chemical class 0.000 description 8
- 239000008346 aqueous phase Substances 0.000 description 6
- 238000001914 filtration Methods 0.000 description 6
- 239000011541 reaction mixture Substances 0.000 description 6
- 238000005160 1H NMR spectroscopy Methods 0.000 description 5
- 241000270295 Serpentes Species 0.000 description 5
- 150000001875 compounds Chemical class 0.000 description 5
- 108090000765 processed proteins & peptides Proteins 0.000 description 5
- IAZDPXIOMUYVGZ-WFGJKAKNSA-N Dimethyl sulfoxide Chemical compound [2H]C([2H])([2H])S(=O)C([2H])([2H])[2H] IAZDPXIOMUYVGZ-WFGJKAKNSA-N 0.000 description 4
- 102000004196 processed proteins & peptides Human genes 0.000 description 4
- 230000002194 synthesizing effect Effects 0.000 description 4
- 238000004090 dissolution Methods 0.000 description 3
- 239000012535 impurity Substances 0.000 description 3
- 239000011734 sodium Substances 0.000 description 3
- HPALAKNZSZLMCH-UHFFFAOYSA-M sodium;chloride;hydrate Chemical class O.[Na+].[Cl-] HPALAKNZSZLMCH-UHFFFAOYSA-M 0.000 description 3
- ASOKPJOREAFHNY-UHFFFAOYSA-N 1-Hydroxybenzotriazole Chemical compound C1=CC=C2N(O)N=NC2=C1 ASOKPJOREAFHNY-UHFFFAOYSA-N 0.000 description 2
- WMFOQBRAJBCJND-UHFFFAOYSA-M Lithium hydroxide Chemical compound [Li+].[OH-] WMFOQBRAJBCJND-UHFFFAOYSA-M 0.000 description 2
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 239000002537 cosmetic Substances 0.000 description 2
- 238000002425 crystallisation Methods 0.000 description 2
- 230000008025 crystallization Effects 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 239000007791 liquid phase Substances 0.000 description 2
- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 description 2
- 125000006239 protecting group Chemical group 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 231100000611 venom Toxicity 0.000 description 2
- BDNKZNFMNDZQMI-UHFFFAOYSA-N 1,3-diisopropylcarbodiimide Chemical compound CC(C)N=C=NC(C)C BDNKZNFMNDZQMI-UHFFFAOYSA-N 0.000 description 1
- WCFJUSRQHZPVKY-UHFFFAOYSA-N 3-[(2-methylpropan-2-yl)oxycarbonylamino]propanoic acid Chemical compound CC(C)(C)OC(=O)NCCC(O)=O WCFJUSRQHZPVKY-UHFFFAOYSA-N 0.000 description 1
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
- 101100149256 Mus musculus Sema6b gene Proteins 0.000 description 1
- 102000019315 Nicotinic acetylcholine receptors Human genes 0.000 description 1
- 108050006807 Nicotinic acetylcholine receptors Proteins 0.000 description 1
- 241000271897 Viperidae Species 0.000 description 1
- 101800002700 Waglerin-1 Proteins 0.000 description 1
- 238000010306 acid treatment Methods 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 239000005557 antagonist Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- DNOFIHGTRCZHPH-UHFFFAOYSA-N benzotriazol-4-one Chemical compound O=C1C=CC=C2N=NN=C12 DNOFIHGTRCZHPH-UHFFFAOYSA-N 0.000 description 1
- 238000009833 condensation Methods 0.000 description 1
- 230000005494 condensation Effects 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- BGRWYRAHAFMIBJ-UHFFFAOYSA-N diisopropylcarbodiimide Natural products CC(C)NC(=O)NC(C)C BGRWYRAHAFMIBJ-UHFFFAOYSA-N 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000003818 flash chromatography Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 238000009499 grossing Methods 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- XETISTBVDHNRBD-FJXQXJEOSA-N methyl (2s)-2-amino-4-[(2-methylpropan-2-yl)oxycarbonylamino]butanoate;hydrochloride Chemical compound Cl.COC(=O)[C@@H](N)CCNC(=O)OC(C)(C)C XETISTBVDHNRBD-FJXQXJEOSA-N 0.000 description 1
- HQEIPVHJHZTMDP-JEDNCBNOSA-N methyl (2s)-pyrrolidine-2-carboxylate;hydrochloride Chemical compound Cl.COC(=O)[C@@H]1CCCN1 HQEIPVHJHZTMDP-JEDNCBNOSA-N 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000001308 synthesis method Methods 0.000 description 1
- 238000007039 two-step reaction Methods 0.000 description 1
- 239000002435 venom Substances 0.000 description 1
- 210000001048 venom Anatomy 0.000 description 1
- 230000037303 wrinkles Effects 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/08—Tripeptides
- C07K5/0802—Tripeptides with the first amino acid being neutral
- C07K5/0804—Tripeptides with the first amino acid being neutral and aliphatic
- C07K5/0806—Tripeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atoms, i.e. Gly, Ala
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Life Sciences & Earth Sciences (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a preparation method of snake venom tripeptide, which comprises the steps of reacting Boc-beta-Ala-OSu with L-Pro-OH to obtain Boc-beta-Ala-Pro-OH, dehydrating and condensing Fmoc-Dab (Boc) -OH with benzylamine to remove Fmoc groups to obtain H-Dab (Boc) -NHBn, dehydrating and condensing Boc-beta-Ala-Pro-OH with H-Dab (Boc) -NHBn to obtain Boc-beta-Ala-Pro-Dab (Boc) -NHBn, and finally removing Boc groups from Boc-beta-Ala-Pro-Dab (Boc) -NHBn to obtain the snake venom tripeptide. The method has the advantages of high efficiency and convenience in synthesis process, high yield, simple post-treatment, less reagent consumption, capability of obtaining a high-purity product without HPLC separation and purification, capability of greatly reducing production cost and suitability for practical production and application.
Description
Technical Field
The invention relates to the field of chemical synthesis, in particular to a preparation method of snake venom tripeptide.
Background
A snake venom tripeptide (H-beta-Ala-Pro-DAB-NH-Bzl) is a small peptide that mimics the activity of snake venom WAGLERINI, WAGLERINI is found in the venom of TEMPLE VIPER snake (Tripidolaemus wagleri) and is a muscle nicotinic acetylcholine receptor (nmAChR) antagonist. The H-beta-Ala-Pro-DAB-NH-Bzl is synthesized by the complete company PENTAPHARM of the Royal Seman group of DSM Netherlands through high-tech biochemical technology at the earliest, imitates the fragment of the effective molecular protein (Waglerin 1), has the chemical structure similar to that of the venomous snake serum, but is safer and more effective than the botulinum, so that the H-beta-Ala-Pro-DAB-NH-Bzl can be safely applied to cosmetics and has excellent skin smoothing and wrinkle removing performances.
At present, most of the conventional methods for synthesizing snake venom tripeptide (H-beta-Ala-Pro-DAB-NH-Bzl) adopt a solid phase and liquid phase combined method, the synthetic process is relatively complicated and the cost is high, so that in order to simplify the process to a certain extent, researchers develop a method for synthesizing the snake venom tripeptide in an all-liquid phase manner, for example, a patent CN107936108A discloses the following synthetic route:
The method comprises the steps of reacting Boc-beta-Ala-OH, N-ethyl-5-phenylisoxazole-3' -sulfonate and H-Pro-OMe-HCl to obtain Boc-beta-Ala-Pro-OMe, and reacting Boc-beta-Ala-Pro-OMe with LiOH to obtain Boc-beta-Ala-Pro-OH; boc-beta-Ala-Pro DAB (Boc) -OH was then synthesized in the same manner using Boc-beta-Ala-Pro-OH and H-DAB (Boc) -OMe-HCl; and then Boc-beta-Ala-Pro-DAB (Boc) -OH, 1-hydroxybenzotriazole, N-diisopropylcarbodiimide and benzylamine are reacted to obtain Boc-beta-Ala-Pro-DAB (Boc) -NH-Bzl, finally trifluoroacetic acid is used for removing Boc protecting group, and HPLC separation and purification are carried out to obtain the snake venom tripeptide with the purity of about 98%, the total yield is only about 38%, the synthetic route is longer, the treatment steps are complicated, and the dosage of a plurality of reagents in the synthetic process is larger.
Patent CN107857797a discloses another method for synthesizing snake venom tripeptide, the synthesis route is as follows:
the total yield of the method is only about 30%, and the high-purity snake venom tripeptide product can be obtained after HPLC separation and purification treatment, so that the synthesis steps are long and the operation is complicated.
Therefore, the existing method for synthesizing the snake venom tripeptide has the problems of low yield, complex synthesis process and the like, and further directly leads to the problem of higher cost in actual production.
Therefore, the synthesis method of the snake venom tripeptide with simple and convenient development process, high yield and low cost has important significance for the production of the snake venom tripeptide and the application of the snake venom tripeptide in the field of cosmetics.
Disclosure of Invention
The invention mainly solves the technical problem of providing a preparation method of snake venom tripeptide, which can efficiently synthesize the snake venom tripeptide, and has simple process and high yield.
In order to solve the technical problems, the invention adopts a technical scheme that:
The invention provides a preparation method of Boc-beta-Ala-Pro-Dab (Boc) -NHBn, which comprises the following steps: the amino acid dehydration condensation reaction is carried out between Boc-beta-Ala-Pro-OH and H-Dab (Boc) -NHBn to obtain the amino acid:
further, boc-beta-Ala-Pro-OH: the molar usage ratio of H-Dab (Boc) -NHBn is 1.0-1.5: 1.0, preferably 1.1:1.0.
Further, the reaction conditions include a first condensing agent; further, the first condensing agent is selected from one or more of DIC, DCC, EDCI, preferably DIC;
further, the first condensing agent: the molar usage ratio of H-Dab (Boc) -NHBn is 1.0-1.5: 1.0, preferably 1.2:1.0.
Further, the solvent of the reaction is a first solvent selected from one or more of DCM, etOAc, THF, preferably DCM.
Further, H-Dab (Boc) -NHBn: the mass and the dosage ratio of the first solvent is 1: 15-30; further 1:20.
Further, after the reaction, the reaction solution was washed with an alkaline aqueous solution and an acidic aqueous solution, respectively, and after washing, the solid-liquid separation was performed, and the solid was ethyl acetate: petroleum ether mass ratio is 0.8-1.2: and (3) crystallizing and purifying the organic solvent of 0.8-1.2.
The crystallization and purification are common purification methods in the synthesis process, namely, solid matters are completely dissolved in a solvent through means such as temperature rising and the like, and then the solid matters are separated out through means such as temperature reduction and the like.
The alkaline aqueous solution means an aqueous solution which is alkaline, and the acidic aqueous solution means an aqueous solution which is acidic, and the solute is not limited, and any alkaline aqueous solution or acidic aqueous solution which does not react with the product Boc-beta-Ala-Pro-Dab (Boc) -NHBn is applicable.
Further, the alkaline aqueous solution is a sodium carbonate aqueous solution with a mass fraction of 2-10%, preferably a sodium carbonate aqueous solution with a mass fraction of 5%.
Further, the acidic aqueous solution is an aqueous solution of citric acid with a mass fraction of 2 to 10%, preferably an aqueous solution of citric acid with a mass fraction of 5%.
Further, the mass amount of the organic solvent is 15 to 25 times, and further 20 times the mass amount of the H-Dab (Boc) -NHBn.
In the invention, the crystallization and purification are to mix the solid and the organic solvent, heat the mixture until the solid is completely dissolved, cool the mixture and separate out the solid. In general, the solid is completely dissolved when the temperature is raised to about 60 ℃.
In a specific embodiment of the invention, the solid and ethyl acetate are: petroleum ether mass ratio 1:1, heating until the solid is completely dissolved, cooling, and separating out the solid.
Further, the H-Dab (Boc) -NHBn is obtained by carrying out amino acid dehydration condensation reaction on Fmoc-Dab (Boc) -OH and benzylamine, and removing Fmoc groups:
The amino acid molecules are bound by binding between the carboxyl group (-COOH) of one amino acid molecule and the amino group (-NH 2) of another amino acid molecule, and simultaneously removing one molecule of water, and the binding mode is called amino acid dehydration condensation.
Further, fmoc-Dab (Boc) -OH: the molar dosage ratio of the benzylamine is 0.8-1.2: 0.8 to 1.2, preferably 1:1.
Further, the conditions for the amino acid dehydration condensation reaction of Fmoc-Dab (Boc) -OH with benzylamine include a second condensing agent; further, the second condensing agent is selected from one or more of DIC, DCC, EDCI, HOBT, CDI, HBTU, HATU, preferably DIC and/or HOBT, or EDCI; more preferred are DIC and HOBT, DIC: the molar usage ratio of HOBT is 1:1.
Further, the second condensing agent: the molar ratio of Fmoc-Dab (Boc) -OH is 1.0-3.0: 1.0, preferably 1.2 to 2.4:1.0.
Further, the solvent for the dehydration condensation reaction of Fmoc-Dab (Boc) -OH with the amino acid of benzylamine is a second solvent selected from one or more of DCM, THF, acetonitrile, preferably DCM and/or THF, more preferably DCM.
Further, fmoc-Dab (Boc) -OH: the mass and the dosage ratio of the second solvent is 1:5 to 15, preferably 1:10.
Removal of Fmoc groups is a common removal of protecting groups, and methods known in the art for removing Fmoc groups are applicable to the present invention.
In a specific embodiment of the present invention, fmoc-Dab (Boc) -NHBn, a third solvent, and piperidine are mixed and reacted, wherein the solvent is one or more selected from methanol, DCM and THF, preferably methanol.
Further, fmoc-Dab (Boc) -NHBn: the mass dosage ratio of the third solvent is 1.0:5 to 20, preferably 1.0:10.
Further, fmoc-Dab (Boc) -NHBn: the molar ratio of the piperidine is 1.0:2 to 10, preferably 1.0:5.
Further, after removing the solvent from the reacted system, the residue is mixed with dilute hydrochloric acid, extracted with DCM and/or ethyl acetate, the aqueous phase is adjusted to be alkaline, extracted with ethyl acetate, and the organic phase is removed from the solvent to obtain the Fmoc group-removed product H-Dab (Boc) -NHBn.
In a specific embodiment of the invention, the aqueous phase is adjusted to alkaline by adjusting the aqueous phase to a pH of 10 with sodium carbonate.
Further, the Boc-beta-Ala-Pro-OH is obtained by reacting Boc-beta-Ala-OSu with L-Pro-OH as follows:
Further, boc-beta-Ala-OSu: the molar usage ratio of L-Pro-OH is 1.0:1.0-1.5, preferably 1;1.2;
Further, the conditions under which Boc-beta-Ala-OSu reacts with L-Pro-OH include a base; further, the alkali is selected from one or more of NaHCO 3、KHCO3、Na2CO3、K2CO3, preferably NaHCO 3;
Further, boc-beta-Ala-OSu: the molar usage ratio of the alkali is 1.0:1.5 to 3, preferably 1.0:2.0.
Further, the solvent for the reaction of Boc-beta-Ala-OSu with L-Pro-OH is water: the molar use ratio of the organic solvent is 0.8-1.2: 0.8 to 1.2, preferably water: the molar usage ratio of the organic solvent is 1:1, wherein the organic solvent is selected from one or more of tetrahydrofuran, acetonitrile, acetone, methanol, ethanol and DMF, preferably one or more of tetrahydrofuran, acetonitrile and acetone, more preferably tetrahydrofuran;
Still further, boc-beta-Ala-OSu: the mass and the dosage ratio of the mixed solvent is 1:5 to 15, preferably 1:10.
The invention also provides a method for preparing the snake venom tripeptide dihydrochloride, which is obtained by removing Boc groups from Boc-beta-Ala-Pro-Dab (Boc) -NHBn; wherein Boc-beta-Ala-Pro-Dab (Boc) -NHBn is prepared by any of the methods described above.
Further, boc-beta-Ala-Pro-Dab (Boc) -NHBn is mixed with ethyl acetate, then HCl ethyl acetate solution is added to the solution in batches, and after the reaction is finished, solid-liquid separation is carried out to obtain solid-like snake venom tripeptide dihydrochloride.
The batch addition refers to non-disposable addition, and the raw materials can be added in a period of time at a certain or different feeding speed, or the raw materials to be added can be divided into a plurality of parts and added in a plurality of times in a certain period of time.
Further, boc-beta-Ala-Pro-Dab (Boc) -NHBn: the mass consumption of the ethyl acetate is 1:5 to 15, preferably 1:10; boc-beta-Ala-Pro-Dab (Boc) -NHBn: the mass and consumption ratio of the ethyl acetate solution of the HCl is 1:5 to 15, preferably 1:10.
The invention also provides application of the snake venom tripeptide dihydrochloride in preparing a product related to the snake venom tripeptide, wherein the snake venom tripeptide dihydrochloride is prepared by the preparation method.
The snake venom tripeptide related products generally refer to products containing compounds in different forms of the snake venom tripeptide, such as salts in different forms of the snake venom tripeptide, and the like, wherein the salts can be hydrochloride, acetate and the like.
After the snake venom tripeptide dihydrochloride is prepared by the method, the compound in various forms can be obtained after the compound is treated by the conventional method, for example, different acid treatments are adopted to convert the compound into other salts (such as snake venom tripeptide diacetate) and the salt is alkalized to obtain the snake venom tripeptide which is used as a product; or further processing different forms of salt and/or tripeptide of snake venom to obtain product such as skin care product and medicine. As long as the snake venom tripeptide dihydrochloride is prepared by adopting the method of the invention in the preparation process of the product, the snake venom tripeptide dihydrochloride belongs to the protection scope of the invention.
The beneficial effects of the invention are as follows:
(1) The method adopts a new synthetic route, can obtain the product with the purity of more than 98 percent without HPLC separation and purification, and has simple process, convenient operation, less reagent consumption and high yield.
(2) The synthesis process of the method is efficient and convenient, can greatly reduce the production cost, is more suitable for actual production and application, is suitable for large-scale industrial production, and is worthy of popularization.
The following abbreviations have the meanings indicated below:
DCC: dicyclohexylcarbodiimide;
DIC: diisopropylcarbodiimide;
EDCI:1- (3-dimethylaminopropyl) -3-ethylcarbodiimide;
HATU:2- (7-oxo-benzotriazol) -N, N' -tetramethyl urea hexafluorophosphate;
HBTU: o-benzotriazol-tetramethylurea hexafluorophosphate;
HOBT: 1-hydroxybenzotriazole;
CDI: n, N' -carbonyldiimidazole;
DCM: dichloromethane;
DMF: n, N-dimethylformamide;
THF: tetrahydrofuran.
Detailed Description
The following description of the present invention will be made clearly and fully, and it is apparent that the embodiments described are some, but not all, of the embodiments of the present invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The yields in the examples of the present invention are all molar yields.
In the examples, "eq" means equivalent weight, and is used to indicate the multiple of the molar amount of one reagent (i.e., 1 eq) and the "n-fold weight part" means the n-fold mass amount of the other reagent based on the mass of 1 eq.
For example, "Fmoc-Dab (Boc) -OH (1 eq) was added to a reaction flask, dichloromethane (10-fold parts by weight), and DIC (1.2 eq) was added in sequence after stirring, meaning that the molar amount of DIC was 1.2 times that of Fmoc-Dab (Boc) -OH, and if the amount of Fmoc-Dab (Boc) -OH was 1mol, the amount of DIC was 1.2mol; the mass usage amount of dichloromethane is 10 times of that of Fmoc-Dab (Boc) -OH, and if the mass usage amount of Fmoc-Dab (Boc) -OH is 1kg, the mass usage amount of dichloromethane is 10kg; the rest of the situation is the same.
The synthesis scale of the scheme recorded in the embodiment of the invention is 10 kg/batch, namely the mass of the snake venom tripeptide dihydrochloride prepared in each batch is about 10 kg.
The synthetic route is as follows:
Step 1: boc-beta-Ala-Pro-OH Synthesis:
Step 2: synthesis of H-Dab (Boc) -NHBn:
step 3: synthesis of snake venom-like peptides:
the specific effects of the preparation method of the present invention will be described below by means of different examples:
example 1
(1) Synthesis of Boc-beta-Ala-Pro-OH
L-Pro-OH (1.2 eq), water (5-fold weight parts), naHCO 3 (2 eq) and tetrahydrofuran (5-fold weight parts) were added to the flask, and after stirring to dissolve, boc-beta-Ala-OSu (1 eq) was added at room temperature, followed by stirring at room temperature until the TLC monitored reaction was complete. The reaction mixture was concentrated under reduced pressure, diluted hydrochloric acid was used to adjust the pH to 3-4, extracted with ethyl acetate (3X 5 parts by weight), dried over anhydrous Na 2SO4, and concentrated by filtration to give Boc-beta-Ala-Pro-OH as a reddish brown oil (yield 80%).
Boc-beta-Ala-Pro-OH 1H NMR(d6-DMSO)：1.38(9H,s,3xCH3);1.80-1.95(2H,m,CH2);2.08-2.20(1H,m,CHH);2.33-2.48(3H,m,CH2,CHH);3.10-3.15(2H,m,CH2);3.43-3.55(2H,m,CH2);4.21(1H,dd,J 8.0,4.0Hz,CH);6.64(1H,m,NH);12.5(1H,br,OH).
(2) Synthesis of H-Dab (Boc) -NHBn
Fmoc-Dab (Boc) -OH (1 eq) and dichloromethane (10 times weight parts based on Fmoc-Dab (Boc) -OH) were added to the reaction flask, DIC (1.2 eq) was added in sequence after stirring, HOBT (1.2 eq) was added after stirring, and benzylamine (1 eq) was added after stirring for 30 minutes at room temperature; the reaction was allowed to proceed to room temperature until completion of the TLC monitoring. The reaction mixture was washed with dilute hydrochloric acid, water, saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated to give Fmoc-Dab (Boc) -NHBn, which was taken directly to the next reaction without purification (a small amount of analytically pure sample was obtained by flash column chromatography for structural identification).
Fmoc-Dab (Boc) -NHBn 1H NMR(d6-DMSO)：1.38(9H,s,3xCH3);1.66-1.87(2H,m,CH2);2.93-3.02(2H,m,CH2);3.35(2H,s,CH2);4.03-4.08(1H,m,CH);4.21-4.31(5H,m,CH,2xCH2);6.75(1H,br,NH);7.19-7.35(7H,m,CH);7.40-7.44(2H,m,2xCH);7.60(1H,br,NH);7.74-7.75(2H,m,2xCH);7.81-7.91(2H,m,2xCH);8.37-8.39(1H,br,NH).
Fmoc-Dab (Boc) -NHBn obtained in the above step was dissolved in methanol (10 times weight part), then piperidine (5 eq) was added, after the reaction was completed at room temperature, the residue was concentrated, added to dilute hydrochloric acid, then the impurity was extracted with dichloromethane, the aqueous phase was adjusted to pH 10 with sodium carbonate, extracted with ethyl acetate, dried over anhydrous sodium sulfate, and concentrated to obtain the product H-Dab (Boc) -NHBoc (purity more than 95%, yield of two steps 70%).
H-L-Dab (Boc) -NHBn 1H NMR(d6-DMSO)：1.38-1.46(10H,m,3xCH3,CHH);1.70-1.79(1H,m,CHH);1.92(2H,br,NH2);3.00-3.05(2H,m,CH2);3.15-3.19(1H,m,CH);4.28-4.30(2H,m,CH2);6.78(1H,br,NH);7.24-7.34(5H,m,5xCH);8.37-8.39(1H,br,NH).
(3) Synthesis of snake venom-like peptides
Dichloromethane (20 times weight part), boc-beta-Ala-Pro-OH (1.1 eq) and DIC (1.2 eq) were added after stirring and dissolution, H-Dab (Boc) -NHBn (1.0 eq) was added after stirring for 30 minutes, the reaction was stirred at room temperature, after TLC monitoring the reaction was completed, 5% sodium carbonate and 5% aqueous citric acid solution were used, washed with water, dried over anhydrous sodium sulfate, and the residue after filtration and concentration was crystallized with a mixed solvent (20 times weight part) of ethyl acetate/petroleum ether in a mass ratio of 1:1 to obtain Boc-beta-Ala-Pro-Dab (Boc) -NHBn (yield 88%, purity more than 98%).
Boc-beta-Ala-Pro-Dab (Boc) -NHBn 1H NMR(d6-DMSO)：1.30-1.35(18H,m,6xCH3);1.60-2.20(6H,m,3xCH2);2.45-2.50(2H,m,CH2);2.85-2.95(2H,m,CH2);3.10-3.20(2H,m,CH2);3.40-3.60(2H,m,CH2),4.20-4.40(4H,m,2xCH,CH2);6.50-6.75(2H,m,2x NH),7.24-7.34(5H,m,5xCH);7.8(1m,br,NH),8.20(1H,br,NH).
The Boc-beta-Ala-Pro-Dab (Boc) -NHBn (1 eq) obtained in the above step was dissolved in ethyl acetate (10 times weight portion), HCl/ethyl acetate solution (4 mol/L,10 times weight portion) was added dropwise, after TLC monitoring the reaction was completed, the precipitated solid in the reaction solution was filtered, rinsed (5 times weight portion of ethyl acetate) and dried to obtain the dihydrochloride salt of the snake venom tripeptide (yield 95%, purity more than 98%).
1H NMR(D2O):1.70-1.80(1H,m,CHH);1.81-1.98(2H,m,CH2);2.01-2.30(3H,m,CHH,CH2);2.73-2.78(2H,m,CH2);2.90-3.35(4H,m,2xCH2);3.48-3.6(2H,m,,CH2);4.30-4.40(4H,m,2xCH2)7.20-7.45(5H,m,5x CH).
Example 2
(1) Synthesis of Boc-beta-Ala-Pro-OH
L-Pro-OH (1.2 eq), water (5 parts by weight), naHCO 3 (2 eq), acetonitrile (5 parts by weight) and, after stirring to dissolve, boc-beta-Ala-OSu (1 eq, a known compound) were added at room temperature and then stirred at room temperature until the TLC monitored reaction was complete. The reaction mixture was concentrated under reduced pressure, diluted hydrochloric acid was used to adjust the pH to 3-4, extracted with ethyl acetate (3X 5 parts by weight), dried over anhydrous Na 2SO4, and concentrated by filtration to give Boc-beta-Ala-Pro-OH as a reddish brown oil (yield: 75%).
(2) Synthesis of H-Dab (Boc) -NHBn
Fmoc-Dab (Boc) -OH (1 eq) and tetrahydrofuran (10 times weight parts based on Fmoc-Dab (Boc) -OH) were added to the reaction flask, DIC (1.2 eq) was added in sequence after stirring, HOBT (1.2 eq) was added after stirring for 30 minutes at room temperature, and benzylamine (1 eq) was added; the reaction was allowed to proceed to room temperature until completion of the TLC monitoring. The reaction mixture was washed with dilute hydrochloric acid, water, saturated brine, extracted with ethyl acetate, dried over anhydrous sodium sulfate, filtered and concentrated to give Fmoc-Dab (Boc) -NHBn, which was taken directly to the next reaction without purification.
Fmoc-Dab (Boc) -NHBn obtained in the above step was dissolved in methylene chloride (10 eq), then piperidine (5 eq) was added, after the reaction was completed at room temperature, the residue was concentrated, added to dilute hydrochloric acid, then the impurity was extracted with methylene chloride, the aqueous phase was adjusted to pH 10 with sodium carbonate, extracted with ethyl acetate, dried over anhydrous sodium sulfate, and concentrated to obtain the product H-Dab (Boc) -NHBoc (purity more than 95%, two-step reaction yield 65%).
(3) Synthesis of snake venom-like peptides
Dichloromethane (20 times weight part), boc-beta-Ala-Pro-OH (1.1 eq) and DIC (1.2 eq) were added after stirring and dissolution, H-Dab (Boc) -NHBn (1.0 eq) was added after stirring for 30 minutes, the reaction was stirred at room temperature, after TLC monitoring the reaction was completed, 5% sodium carbonate and 5% aqueous citric acid solution were used, washed with water, dried over anhydrous sodium sulfate, and the residue after filtration and concentration was crystallized with a mixed solvent (20 times weight part) of ethyl acetate/petroleum ether in a mass ratio of 1:1 to obtain Boc-beta-Ala-Pro-Dab (Boc) -NHBn (yield 89%, purity more than 98%).
The Boc-beta-Ala-Pro-Dab (Boc) -NHBn (1 eq) obtained in the above step was dissolved in ethyl acetate (10 times by weight), HCl/ethyl acetate solution (4 mol/L,10 times by weight) was added dropwise, after TLC monitoring the reaction was completed, the precipitated solid in the reaction solution was filtered, rinsed (5 times by weight of ethyl acetate) and dried to obtain the dihydrochloride salt of the snake venom tripeptide (yield 93%, purity more than 98%).
Example 3
(1) Synthesis of Boc-beta-Ala-Pro-OH
L-Pro-OH (1.2 eq), water (5-fold weight), naHCO 3 (2 eq), acetone (5-fold weight) and, after stirring to dissolve, boc-beta-Ala-OSu (1 eq) were added at room temperature and then stirred at room temperature until the TLC monitored reaction was complete. The reaction mixture was concentrated under reduced pressure, diluted hydrochloric acid was used to adjust the pH to 3-4, extracted with ethyl acetate (3X 5 parts by weight), dried over anhydrous Na 2SO4, and concentrated by filtration to give Boc-beta-Ala-Pro-OH as a reddish brown oil (yield 78%).
(2) Synthesis of H-Dab (Boc) -NHBn
Fmoc-Dab (Boc) -OH (1 eq) and dichloromethane (10 times weight parts based on Fmoc-Dab (Boc) -OH) were added to the reaction flask, EDCI (1.2 eq) was added in sequence after stirring, and benzylamine (1 eq) was added after stirring at room temperature for 30 minutes; the reaction was allowed to proceed to room temperature until completion of the TLC monitoring. The reaction mixture was washed with dilute hydrochloric acid, water, saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated to give Fmoc-Dab (Boc) -NHBn, which was taken directly to the next reaction without purification.
Fmoc-Dab (Boc) -NHBn obtained in the above step was dissolved in tetrahydrofuran (10 times weight part), then piperidine (5 eq) was added, after the reaction was completed at room temperature, concentrated, the residue was added to dilute hydrochloric acid, then the impurity was extracted with ethyl acetate, the aqueous phase was adjusted to pH 10 with sodium carbonate, extracted with ethyl acetate, dried over anhydrous sodium sulfate, and concentrated to obtain the product H-Dab (Boc) -NHBoc (purity more than 95%, yield 68% in two steps).
(3) Synthesis of snake venom-like peptides
Dichloromethane (20 times weight part) and Boc-beta-Ala-Pro-OH (1.1 eq) are added into a reaction bottle, DIC (1.2 eq) is added after stirring and dissolution, H-Dab (Boc) -NHBn (1.0 eq) is added after stirring for 30 minutes, stirring and reaction is carried out at room temperature, after TLC monitoring reaction is finished, 5% sodium carbonate and 5% citric acid aqueous solution are respectively used, water washing and drying are carried out by anhydrous sodium sulfate, and the residue after filtration and concentration is crystallized by a mixed solvent (20 times weight part) with the mass ratio of ethyl acetate to petroleum ether being 1:1 to obtain Boc-beta-Ala-Pro-Dab (Boc) -NHBn (yield 88%, purity is more than 98%).
The Boc-beta-Ala-Pro-Dab (Boc) -NHBn (1 eq) obtained in the above step was dissolved in ethyl acetate (10 times weight portion), HCl/ethyl acetate solution (4 mol/L,10 times weight portion) was added dropwise, after TLC monitoring the reaction was completed, the precipitated solid in the reaction solution was filtered, rinsed (5 times weight portion of ethyl acetate) and dried to obtain the dihydrochloride salt of the snake venom tripeptide (yield 95%, purity more than 98%).
Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made therein without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (12)
- The preparation method of Boc-beta-Ala-Pro-Dab (Boc) -NHBn is characterized by comprising the following steps:Step 1: boc-beta-Ala-Pro-OH Synthesis:the conditions for the reaction of Boc-beta-Ala-OSu with L-Pro-OH include a base, and the solvent for the reaction of Boc-beta-Ala-OSu with L-Pro-OH is water: the mass ratio of the organic solvent is 0.8-1.2: 0.8-1.2, wherein the organic solvent is selected from one or more of tetrahydrofuran, acetonitrile, acetone, methanol, ethanol and DMF;Step 2: synthesis of H-Dab (Boc) -NHBn:the conditions for the dehydration condensation reaction of Fmoc-dab (Boc) -OH with benzylamine amino acid include a second condensing agent selected from one or more of DIC, DCC, EDCI, HOBT, CDI, HBTU, HATU; the solvent of the dehydration condensation reaction of Fmoc-Dab (Boc) -OH and benzylamine is a second solvent, wherein the second solvent is selected from one or more of DCM, THF and acetonitrile;Removing the solvent from the reacted system, mixing the residue with dilute hydrochloric acid, extracting with DCM and/or ethyl acetate, adjusting the water phase to be alkaline, extracting with ethyl acetate, and removing the solvent from the organic phase to obtain a Fmoc-group-removed product H-Dab (Boc) -NHBn;step 3: preparation of Boc-beta-Ala-Pro-Dab (Boc) -NHBn:The reaction conditions include a first condensing agent; the first condensing agent is selected from one or more of DIC, DCC, EDCI; the solvent for the reaction is a first solvent, and the first solvent is one or more selected from DCM, etOAc, THF;After the reaction, the reaction solution is respectively washed with an alkaline aqueous solution, an acidic aqueous solution and water, and after the washing, solid and liquid are separated, and the solid is ethyl acetate: petroleum ether mass ratio is 0.8-1.2: and (3) crystallizing and purifying the organic solvent of 0.8-1.2.
- 2. The method of claim 1, wherein in step 1, boc-beta-Ala-OSu: the molar dosage ratio of the L-Pro-OH is 1.0:1.0-1.5;the alkali is selected from one or more of NaHCO 3、KHCO3、Na2CO3、K2CO3;Boc-beta-Ala-OSu: the molar usage ratio of the alkali is 1.0:1.5 to 3;the solvent for the reaction of Boc-beta-Ala-OSu with L-Pro-OH is water: the mass ratio of the organic solvent is 1:1, wherein the organic solvent is selected from one or more of tetrahydrofuran, acetonitrile and acetone;Boc-beta-Ala-OSu: the mass and the dosage ratio of the mixed solvent is 1:5 to 15.
- 3. The method of claim 1, wherein in step 1, boc-beta-Ala-OSu: the molar usage ratio of L-Pro-OH is 1:1.2;The base is selected from NaHCO 3;Boc-beta-Ala-OSu: the molar usage ratio of the alkali is 1.0:2.0;The solvent for the reaction of Boc-beta-Ala-OSu with L-Pro-OH is water: the mass ratio of the organic solvent is 1:1, wherein the organic solvent is selected from tetrahydrofuran;Boc-beta-Ala-OSu: the mass and the dosage ratio of the mixed solvent is 1:10.
- 4. The method according to claim 1, wherein in step 2, fmoc-Dab (Boc) -OH: the molar dosage ratio of the benzylamine is 0.8-1.2: 0.8 to 1.2;The second condensing agent is selected from DIC and/or HOBT, or from EDCI; the second condensing agent: the molar usage ratio of Fmoc-Dab (Boc) -OH is 1.0-3.0: 1.0;the second solvent is selected from DCM and/or THF;Fmoc-Dab (Boc) -OH: the mass and the dosage ratio of the second solvent is 1:5 to 15.
- 5. The method according to claim 1, wherein in step 2, fmoc-Dab (Boc) -OH: the molar dosage ratio of the benzylamine is 1:1, a step of;The second condensing agent is selected from the group consisting of DIC and HOBT, DIC: the molar usage ratio of HOBT is 1:1, a step of;The second condensing agent: the molar usage ratio of Fmoc-Dab (Boc) -OH is 1.2-2.4: 1.0;the second solvent is selected from DCM;Fmoc-Dab (Boc) -OH: the mass and the dosage ratio of the second solvent is 1:10.
- 6. The method of claim 1, wherein in step 3, boc-beta-Ala-Pro-OH:The molar usage ratio of H-Dab (Boc) -NHBn is 1.0-1.5: 1.0; the first condensing agent is selected from DIC;The first condensing agent: the molar usage ratio of H-Dab (Boc) -NHBn is 1.0-1.5: 1.0;the first solvent is selected from DCM;H-Dab (Boc) -NHBn: the mass and the dosage ratio of the first solvent is 1: 15-30.
- 7. The method of claim 1, wherein in step 3, boc-beta-Ala-Pro-OH:The molar ratio of H-Dab (Boc) -NHBn was 1.1:1.0; the first condensing agent is selected from DIC; the first condensing agent:the molar ratio of H-Dab (Boc) -NHBn was 1.2:1.0;The first solvent is selected from DCM; H-Dab (Boc) -NHBn: the mass and the dosage ratio of the first solvent is 1:20.
- 8. The preparation method according to claim 1, wherein in the step 3, the alkaline aqueous solution is an aqueous sodium carbonate solution with a mass fraction of 2-10%; the acidic aqueous solution is 2-10% of citric acid aqueous solution by mass fraction; the mass dosage of the organic solvent is 15-25 times of the mass of the H-Dab (Boc) -NHBn.
- 9. The preparation method according to claim 1, wherein in the step 3, the alkaline aqueous solution is 5% by mass of sodium carbonate aqueous solution; the acidic aqueous solution is 5% citric acid aqueous solution by mass fraction; the mass dosage of the organic solvent is 20 times of the mass of the H-Dab (Boc) -NHBn; ethyl acetate: the mass ratio of petroleum ether is 1:1.
- 10. A process for preparing snake venom tripeptide dihydrochloride includes such steps as mixing Boc-beta-Ala-Pro-Dab (Boc) -NHBn with ethyl acetate, adding HCl ethyl acetate solution to the mixture, and solid-liquid separationBoc-beta-Ala-Pro-Dab (Boc) -NHBn prepared by the method of any one of claims 1 to 9.
- 11. The method of claim 10, wherein Boc-beta-Ala-Pro-Dab (Boc) -NHBn: the mass and dosage ratio of the ethyl acetate is 1:5 to 15; the concentration of the HCl ethyl acetate solution is 3-5 mol/L, boc-beta-Ala-Pro-Dab (Boc) -NHBn: the mass and consumption ratio of the ethyl acetate solution of the HCl is 1:5 to 15.
- 12. The method of claim 11, wherein Boc-beta-Ala-Pro-Dab (Boc) -NHBn: the mass and dosage ratio of the ethyl acetate is 1:10; the concentration of the HCl in ethyl acetate solution was 4mol/L, boc-beta-Ala-Pro-Dab (Boc) -NHBn: the mass and consumption ratio of the ethyl acetate solution of the HCl is 1:10.
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