Production process of melittin

Abstract

The invention discloses a production process of melittin, belongs to the technical field of polypeptide synthesis, and particularly relates to a preparation method of melittin, which comprises the following steps: synthesizing melittin-peptide resin by adopting a solid-phase synthesis method according to the sequence of melittin sequences, and cutting and purifying to obtain melittin; in the solid-phase synthesis method, amino acid reagents are coupled one by one or at least more than one melittin peptide fragment and a single amino acid reagent are adopted for coupling, and the number of amino acids in the melittin peptide fragment is 4-8. The melittin peptide fragment is synthesized by adopting a liquid-phase synthesis carrier, and the liquid-phase synthesis carrier is obtained by synthesizing 2, 4-dihydroxy benzaldehyde, 1, 4-dibromocyclohexane and diethyl bromomalonate and reducing the 2, 4-dihydroxy benzaldehyde, the 1, 4-dibromocyclohexane and the diethyl bromomalonate.

Description

Technical Field

[0001] The invention belongs to the technical field of polypeptide synthesis, and in particular relates to a production process of melittin. Background Art

[0002] Bee venom has complex components. As a mixture extracted from bees in a natural state, it is difficult to directly study and apply its characteristics in clinical practice. Melittin is separated and purified from bee venom and is the main active ingredient in bee venom. Melittin has a very efficient destructive effect on cell membrane structures, including plasma membranes and membrane structures of some intracellular organelles. Its mechanism of action mainly includes embedding into the phospholipid membrane structure through its amphipathic structure, and then punching holes in the membrane, causing the membrane structure to rupture. Changes in the internal and external environments of the membrane eventually lead to cell death; it can also affect the key proteins in multiple signal pathways that mediate tumor proliferation in cells, thereby causing cell apoptosis. At the same time, bee venom peptide also has anti-inflammatory, antibacterial, anti-cancer, anti-radiation, and platelet aggregation inhibition effects. It can effectively treat scapulohumeral periarthritis, rheumatoid arthritis, rheumatoid arthritis and other diseases, and has certain applications in the field of medicine. Because bee venom peptide has the characteristics of high biological activity and no environmental pollution, it can be developed into products such as body immune regulators, which has great market development prospects and has attracted widespread attention. Summary of the invention

[0003] The object of the present invention is to provide a preparation method of melittin with high yield and high purity by using solid phase synthesis, liquid phase synthesis of peptide segments, at least one peptide segment.

[0004] The technical solution adopted by the present invention to achieve the above-mentioned purpose is: A preparation method of melittin, comprising: synthesizing melittin-peptide resin by solid phase synthesis in the order of melittin sequence, cutting and purifying to obtain melittin; in the solid phase synthesis method, coupling amino acid reagents one by one or coupling by at least one melittin peptide segment and a single amino acid reagent, the number of amino acids in the melittin peptide segment is 4-8. The present invention can greatly improve the yield of melittin by using at least one melittin peptide segment with a specific number of amino acids to synthesize melittin. The number of amino acids in the melittin peptide segment cannot be too many, as too many will also affect the yield of melittin. After research, the present invention discloses the effect of using different melittin peptide segments to synthesize melittin to improve the yield of melittin.

[0005] Preferably, the melittin peptide segment is at least one of the following: The sequence of peptide 1 is Fmoc-Leu-Pro-Ala-Leu-Ile- Ser(tBu)-Trp(Boc); The sequence of peptide 2 is Fmoc-Leu-Lys(Boc)-Val-Leu-Thr(tBu)-Thr(tBu)-Gly; The sequence of peptide 3 is Fmoc-Gly-Ile-Gly-Ala-Val; The sequence of peptide 4 is Fmoc-Ala-Leu-Ile-Ser(tBu)-Trp(Boc)-Ile; The sequence of peptide 5 is Fmoc-Val-Leu-Thr(tBu)-Thr(tBu)-Gly-Leu-Pro; The sequence of peptide 6 is Fmoc-Gly-Ile-Gly-Ala-Val-Leu-Lys (Boc).

[0006] Preferably, the number of melittin peptide segments is 3, and peptide segments 1-3 are used in the synthesis of melittin-peptide resin; or, the number of melittin peptide segments is 3, and peptide segments 4-6 are used in the synthesis of melittin-peptide resin. In the present invention, peptide segments 1-3 and a single amino acid reagent are used in the synthesis of melittin, which can improve the yield of melittin and improve the preparation effect of melittin; peptide segments 4-6 and a single amino acid reagent can also be used in the synthesis of melittin, and the yield of melittin obtained is high.

[0007] Preferably, the synthesis of the bee venom peptide segment is performed using a liquid phase synthesis carrier, and the liquid phase synthesis carrier is synthesized from 2,4-dihydroxybenzaldehyde, 1,4-dibromocyclohexane and diethyl bromomalonate and reduced.

[0008] More preferably, the molar amount of 1,4-dibromocyclohexane used is 30-50% of the molar amount of 2,4-dihydroxybenzaldehyde used; or the molar amount of diethyl bromomalonate used is 80-120% of the molar amount of 2,4-dihydroxybenzaldehyde used.

[0009] More preferably, in the synthesis of the liquid phase synthetic carrier, 2,4-dihydroxybenzaldehyde and 1,4-dibromocyclohexane are first reacted, then diethyl bromomalonate is added for reaction, and finally the liquid phase synthetic carrier is reduced by sodium borohydride.

[0010] More preferably, 2,4-dihydroxybenzaldehyde and 1,4-dibromocyclohexane are reacted in a DMF solution containing potassium carbonate, and the reduction is carried out using sodium borohydride in a tetrahydrofuran solution.

[0011] Preferably, in the solid phase synthesis, a deprotection solution is used to remove the protecting group, and the deprotection solution is a DMF solution containing 15-25% Pip.

[0012] Preferably, the cutting is processed with a cutting fluid, which consists of TFA, TIS, EDT, PhOH and water, wherein TFA, TIS, EDT, PhOH and water are mixed in a volume ratio of 1:0.02-0.1:0.01-0.05:0.01-0.05:0.01-0.05.

[0013] The present invention discloses the use of a melittin peptide segment in the preparation of melittin, wherein the melittin peptide segment is at least one of the following: The sequence of peptide 1 is Fmoc-Leu-Pro-Ala-Leu-Ile- Ser(tBu)-Trp(Boc); The sequence of peptide 2 is Fmoc-Leu-Lys(Boc)-Val-Leu-Thr(tBu)-Thr(tBu)-Gly; The sequence of peptide 3 is Fmoc-Gly-Ile-Gly-Ala-Val; The sequence of peptide 4 is Fmoc-Ala-Leu-Ile-Ser(tBu)-Trp(Boc)-Ile; The sequence of peptide 5 is Fmoc-Val-Leu-Thr(tBu)-Thr(tBu)-Gly-Leu-Pro; The sequence of peptide 6 is Fmoc-Gly-Ile-Gly-Ala-Val-Leu-Lys (Boc).

[0014] Preferably, in the preparation of the liquid phase synthesis carrier, potassium carbonate is added to DMF, and then 2,4-dihydroxybenzaldehyde and 1,4-dibromocyclohexane are added, and the reaction is carried out at a temperature of 60-80°C for 3-12h under an inert gas atmosphere, and then diethyl bromomalonate is added, and the reaction is carried out at a temperature of 60-80°C for 3-12h. After the reaction is completed, the intermediate product is separated, and the intermediate product is added to a tetrahydrofuran solution, and then sodium borohydride is added to react at a temperature of 30-50°C for 2-5h. After the reaction is completed, pure water is added to quench the reaction, and the liquid phase synthesis carrier is separated. The present invention also discloses a liquid phase synthesis carrier that can be used for liquid synthesis of peptide segments. In the preparation of the above liquid phase synthesis carrier, 1,4-dibromocyclohexane is replaced with bromocyclohexane, and the obtained product cannot be used as a liquid phase synthesis carrier in the preparation of peptide segments.

[0015] More preferably, in the preparation of the liquid phase synthetic carrier, the amount of potassium carbonate used is 10-20 wt % of DMF.

[0016] More preferably, in the preparation of the liquid phase synthesis carrier, the amount of 2,4-dihydroxybenzaldehyde used is 10-20 wt % of DMF.

[0017] More preferably, in the preparation of the liquid phase synthesis carrier, the molar amount of 1,4-dibromocyclohexane used is 30-50% of the molar amount of 2,4-dihydroxybenzaldehyde used.

[0018] More preferably, in the preparation of the liquid phase synthesis carrier, the molar amount of diethyl bromomalonate used is 80-120% of the molar amount of 2,4-dihydroxybenzaldehyde used.

[0019] More preferably, in the preparation of the liquid phase synthesis carrier and the separation of the intermediate product, the reaction solution is cooled to 0-10°C, 0.1-1M hydrochloric acid and water are added, and the volume ratio of hydrochloric acid to water is 1:0.1-1. After the precipitate is precipitated, it is filtered, washed, and dried to obtain the intermediate.

[0020] More preferably, in the preparation of the liquid phase synthesis carrier, the tetrahydrofuran solution is prepared by mixing tetrahydrofuran and methanol, wherein tetrahydrofuran and methanol are mixed in a volume ratio of 1:0.01-0.2, and the amount of the intermediate used is 10-30wt% of the tetrahydrofuran solution.

[0021] More preferably, in the preparation of the liquid phase synthesis carrier, the amount of sodium borohydride used is 10-30wt% of the intermediate.

[0022] Preferably, in the preparation of the peptide segment, a liquid phase synthesis carrier is added to DCM, and then an amino acid reagent, DIC and DMAP are added for coupling treatment. After the coupling is completed, a deprotection treatment is performed, and the amino acid reagent is replaced in the order of the peptide segment to perform coupling treatment and deprotection treatment in sequence. After the last amino acid is coupled, no deprotection treatment is performed, and a cutting liquid is used to perform a cutting treatment to remove the liquid phase synthesis carrier to obtain a peptide segment.

[0023] More preferably, in the preparation of the peptide segment, the amount of the liquid phase synthesis carrier used is 10-30 wt % of DCM.

[0024] More preferably, in the preparation of the peptide segment, the amount of the amino acid reagent used is 40-80 wt % of the liquid phase synthesis carrier.

[0025] More preferably, in the preparation of the peptide segment, the amount of DIC used is 0.1-1 wt % of the liquid phase synthesis carrier.

[0026] More preferably, in the preparation of the peptide segment, the amount of DMAP used is 1-4 wt % of the liquid phase synthesis carrier.

[0027] More preferably, in the preparation of the peptide segment, the peptide segment includes the above-mentioned peptide segments 1-6.

[0028] More preferably, in the coupling treatment for peptide preparation, the reaction is stirred at 20-40° C. for 2-8 hours, acetonitrile is added to precipitate crystals, and the intermediate-liquid phase synthesis carrier is obtained by filtration.

[0029] More preferably, in the deprotection treatment of peptide preparation, the intermediate-liquid phase synthesis carrier is added to the deprotection solution, treated at 20-40°C for 3-10 min, the pH is adjusted to neutral, and the product is precipitated. The deprotection solution is a THF solution containing 5-20 vol% piperidine and 0.5-2 vol% DBU, and the amount of deprotection solution used is enough to immerse the intermediate-liquid phase synthesis carrier.

[0030] More preferably, in the cleavage treatment of peptide preparation, the cleavage liquid is added to the peptide 1-liquid phase synthesis carrier, stirred at 20-40°C for 20-60min, filtered, and recrystallized from isopropyl ether. The cleavage liquid is a DCM solution containing 5-20vol% TFE and 0.5-2% TFA, and the amount of cleavage liquid used is enough to immerse the peptide 1-liquid phase synthesis carrier.

[0031] Preferably, in the preparation of melittin, AM resin is used to synthesize melittin, firstly Fmoc-Linker-OH is coupled to AM resin, and then amino acid reagents are coupled in the order of melittin sequence, after coupling is completed, melittin-peptide resin is separated and treated, melittin-peptide resin is cut to obtain crude melittin, and crude melittin is purified to obtain melittin.

[0032] The sequence of melittin is: H-Gly-Ile-Gly-Ala-Val-Leu-Lys-Val-Leu-Thr-Thr-Gly-Leu-Pro-Ala-Leu-Ile-Ser-Trp-Ile-Lys-Arg-Lys-Arg-Gln-Gln-NH2.

[0033] Preferably, in the preparation of the activated linker reagent, Fmoc-Linker-OH and HOBt are added to DMF, stirred and dissolved, and DIC is added at a temperature of 2-8°C, and treated at a temperature of 2-8°C for 10-20 minutes to obtain the activated linker reagent.

[0034] More preferably, in the preparation of the activated Linker reagent, the amount of Fmoc-Linker-OH used is related to the amount of DMF used in a ratio of 0.3-1.5 mol / L, the molar amount of HOBt used is 60-120% of the molar amount of Fmoc-Linker-OH used, and the molar amount of DIC used is 60-120wt% of the molar amount of Fmoc-Linker-OH used.

[0035] Preferably, in the preparation of Fmoc-Linker-AM resin, AM resin is added to a deprotection solution, treated at 30-35° C. for 20-50 min, washed with DMF, and then an activated Linker reagent is added, reacted at 30-35° C. for 2-4 h, and then DIEA is added to react for 20-50 min. After the reaction is completed, the Fmoc-Linker-AM resin is obtained by suction filtration.

[0036] Preferably, in the preparation of the activated amino acid reagent, the amino acid reagent and HOBt are added to DMF, stirred and dissolved, and then DIC is added at a temperature of 2-8°C, and treated at a temperature of 2-8°C for 10-20 minutes to obtain the activated amino acid reagent. The relationship between the amount of the amino acid reagent used and the amount of DMF used is 0.3-1.5 mol / L, the molar amount of HOBt used is 80-120% of the molar amount of the amino acid reagent used, and the molar amount of DIC used is 80-120% of the molar amount of the amino acid reagent used. Amino acid reagents include: Fmoc-Gln(Trt)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ile-OH, Fmoc-Trp(Boc)-OH, Fmo c-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-Ala-OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Thr(tBu)-OH, Fmoc-Val-OH.

[0037] The sequence of solid phase synthesis Fmoc-Gln(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH, F moc-Lys(Boc)-OH, Fmoc-Ile-OH, Fmoc-Trp(Boc)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Ala -OH, Fmoc-Pro-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Thr(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Leu-OH, Fmoc-Va l-OH, Fmoc-Lys(Boc)-OH, Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ile-OH, Fmoc-Gly-OH.

[0038] Preferably, in the preparation of bee venom peptide-peptide resin, the Fmoc-Linker-AM resin is deprotected, and then an activated amino acid reagent is added, and the reaction is carried out at 30-35° C. for 2-4 hours. After the reaction is completed, the deprotection treatment is carried out, and the above-mentioned steps of coupling amino acid reagents are repeated in the order of the bee venom peptide sequence. The first amino acid reagent is Fmoc-Gln(Trt)-OH, and then the amino acid reagents coupled in sequence are as follows: Fmoc-Gln(Trt)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ile-OH, Fmoc -Trp(Boc)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Ala-OH, Fmoc-Pro-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Thr(tBu)-OH, Fmoc- Thr(tBu)-OH, Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Lys(Boc)-OH, Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ile-OH, Fmoc-Gly-OH.

[0039] More preferably, in the preparation of bee venom peptide-peptide resin, when coupling with amino acid reagent, the product of the previous coupling needs to be deprotected, and the deprotection treatment includes post-coupling washing, deprotection and post-deprotection washing.

[0040] More preferably, in the preparation of bee venom peptide-peptide resin, in the post-coupling washing, the coupled product is washed 1-3 times with DMF, and filtered after washing.

[0041] More preferably, in the preparation of bee venom peptide-peptide resin, during deprotection, a deprotection solution is added to the washed product after coupling, treated at a temperature of 30-35° C. for 20-40 min, and filtered after treatment. The deprotection solution is a DMF solution containing 15-25% Pip.

[0042] More preferably, in the preparation of bee venom peptide-peptide resin, in the washing after deprotection, the deprotected product is washed with DMF for 2-6 times, and filtered after washing.

[0043] Preferably, the crude melittin is obtained by cutting melittin-peptide resin, wherein the melittin-peptide resin is added to a cutting solution, cut at 25-35° C. for 2-6 hours, the resin is separated by filtration, tertiary methyl ether is added to the filtrate, a precipitate is precipitated, and the crude melittin is obtained by filtration.

[0044] More preferably, the cutting liquid is composed of TFA, TIS, EDT, PhOH and water, wherein TFA, TIS, EDT, PhOH and water are mixed in a volume ratio of 1: 0.02-0.1: 0.01-0.05: 0.01-0.05: 0.01-0.05. The cutting liquid is immersed in the melittin-peptide resin, and tertiary methyl ether is used in an appropriate amount to precipitate the crude melittin.

[0045] Preferably, the melittin is obtained by purifying a crude melittin product, and the purification process is HPLC treatment.

[0046] The present invention adopts a liquid phase synthesis carrier to synthesize the melittin peptide segment, then applies the melittin peptide segment to the solid phase synthesis of melittin, and then assists with the coupling of a single amino acid reagent of melittin. In the method for synthesizing melittin of the present invention, it is necessary to design and use a melittin peptide segment of a specific sequence, and the use of at least one melittin peptide segment, so it has the following beneficial effects: the yield of melittin prepared by the present invention is high, and the purity of the purified melittin is high. Therefore, the present invention is a preparation method of melittin using solid phase synthesis, liquid phase peptide synthesis, at least one peptide segment, high yield and high purity. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 This is the mass spectrum of bee venom peptide.

[0048] Figure 2 This is a graph showing the yield of melittin. DETAILED DESCRIPTION

[0049] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0050] The following first describes the concepts involved in the present application in conjunction with the accompanying drawings. It should be noted that the following description of each concept is only to make the content of the present application easier to understand, and does not limit the scope of protection of the present application; at the same time, the embodiments and features in the embodiments of the present application can be combined with each other in the absence of conflict. The present application will be described in detail with reference to the accompanying drawings and in conjunction with the embodiments.

[0051] Example 1: A method for preparing melittin Preparation of melittin: AM resin is used to synthesize melittin. First, Fmoc-Linker-OH is coupled to AM resin, and then amino acid reagents are coupled in the order of melittin sequence. After coupling, melittin-peptide resin is separated and treated. The melittin-peptide resin is cut to obtain crude melittin. The crude melittin is purified to obtain melittin.

[0052] The sequence of melittin is: H-Gly-Ile-Gly-Ala-Val-Leu-Lys-Val-Leu-Thr-Thr-Gly-Leu-Pro-Ala-Leu-Ile-Ser-Trp-Ile-Lys-Arg-Lys-Arg-Gln-Gln-NH2.

[0053] Preparation of activated linker reagent: Fmoc-Linker-OH and HOBt were added to DMF, stirred and dissolved, and then DIC was added at 5°C, and treated at 5°C for 20 minutes to obtain an activated linker reagent. The relationship between the amount of Fmoc-Linker-OH used and the amount of DMF used was 0.72 mol / L, the molar amount of HOBt used was 100% of the molar amount of Fmoc-Linker-OH used, and the molar amount of DIC used was 100% of the molar amount of Fmoc-Linker-OH used.

[0054] Preparation of Fmoc-Linker-AM resin: Add AM resin to the deprotection solution, treat at 35°C for 30 minutes, wash with DMF, then add the activated Linker reagent, react at 35°C for 3 hours, then add DIEA to react for 30 minutes, and after the reaction is completed, filter to obtain Fmoc-Linker-AM resin.

[0055] Preparation of activated amino acid reagent: Add amino acid reagent and HOBt into DMF, stir and dissolve, then add DIC at 5°C, treat at 5°C for 20 minutes to obtain activated amino acid reagent. The relationship between the amount of amino acid reagent and the amount of DMF is 0.72 mol / L, the molar amount of HOBt is 100% of the molar amount of amino acid reagent, and the molar amount of DIC is 100% of the molar amount of amino acid reagent. Amino acid reagents include: Fmoc-Gln(Trt)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ile-OH, Fmoc-Trp(Boc)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-Ala-OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Thr(tBu)-OH, Fmoc-Val-OH.

[0056] Preparation of bee venom peptide-peptide resin: Fmoc-Linker-AM resin was deprotected, and then an activated amino acid reagent was added and reacted at 35°C for 3 hours. After the reaction was completed, deprotection was performed, and the aforementioned steps of coupling amino acid reagents were repeated in the order of the bee venom peptide sequence. The first amino acid reagent was Fmoc-Gln(Trt)-OH, and then the amino acid reagents coupled in sequence were as follows: Fmoc-Gln(Trt)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ile-OH, Fmoc-Trp( Boc)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Ala-OH, Fmoc-Pro-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Thr(tBu)-OH, Fmoc-Thr (tBu)-OH, Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Lys(Boc)-OH, Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ile-OH, Fmoc-Gly-OH.

[0057] When coupling amino acid reagents, the product of the previous coupling needs to be deprotected. The deprotection treatment includes post-coupling washing, deprotection, and post-deprotection washing. In the post-coupling washing, the coupled product is washed 3 times with DMF, and filtered after washing. In the deprotection, the deprotection solution is added to the post-coupling washing product, treated at 35°C for 30 minutes, and filtered after treatment. The deprotection solution is a DMF solution containing 20% ​​Pip. In the post-deprotection washing, the deprotected product is washed 5 times with DMF, and filtered after washing.

[0058] The crude product of melittin is obtained by cutting melittin-peptide resin. In the cutting process, melittin-peptide resin is added to the cutting solution, cut at 35°C for 4 hours, the resin is separated by filtration, methyl tert-ether is added to the filtrate, the precipitate is precipitated, and the crude product of melittin is obtained by filtration. The cutting solution is composed of TFA, TIS, EDT, PhOH and water. TFA, TIS, EDT, PhOH and water are mixed in the cutting solution in a volume ratio of 1:0.05:0.03:0.03:0.03. The cutting solution is immersed in the melittin-peptide resin, and methyl tert-ether is used in an appropriate amount to precipitate the crude product of melittin.

[0059] Melittin is obtained by purifying a crude product of melittin, and the purification process is HPLC treatment.

[0060] Example 2: A method for preparing melittin Preparation of liquid phase synthetic carrier: Potassium carbonate is added to DMF, then 2,4-dihydroxybenzaldehyde and 1,4-dibromocyclohexane are added, reacted at 70°C for 6h under an inert gas atmosphere, then diethyl bromomalonate is added, reacted at 70°C for 6h, the intermediate product is separated after the reaction is completed, the intermediate product is added to tetrahydrofuran solution, then sodium borohydride is added and reacted at 40°C for 3h, pure water is added after the reaction is completed to quench the reaction, and the liquid phase synthetic carrier is separated. The amount of potassium carbonate used is 15wt% of DMF, the amount of 2,4-dihydroxybenzaldehyde used is 15wt% of DMF, the molar amount of 1,4-dibromocyclohexane used is 40% of the molar amount of 2,4-dihydroxybenzaldehyde used, and the molar amount of diethyl bromomalonate used is 100% of the molar amount of 2,4-dihydroxybenzaldehyde used. In the separation of the intermediate product, the reaction solution is cooled to 5°C, 0.5M hydrochloric acid and water are added, the volume ratio of hydrochloric acid to water is 1:0.5, and the precipitate is filtered, washed, and dried to obtain the intermediate. The tetrahydrofuran solution is prepared by mixing tetrahydrofuran and methanol, and the tetrahydrofuran and methanol in the tetrahydrofuran solution are mixed in a volume ratio of 1:0.1. The amount of the intermediate used is 20wt% of the tetrahydrofuran solution, and the amount of sodium borohydride used is 20wt% of the intermediate.

[0061] Preparation of peptide 1: Add the liquid phase synthesis carrier to DCM, then add Fmoc-Trp(Boc)-OH, DIC and DMAP for coupling treatment, perform deprotection treatment after coupling, replace Fmoc-Trp(Boc)-OH in the order of peptide 1, perform coupling treatment and deprotection treatment in sequence, do not perform deprotection treatment after coupling the last amino acid, use cutting liquid to perform cutting treatment to remove the liquid phase synthesis carrier, and obtain peptide 1. The amount of liquid phase synthesis carrier used is 20wt% of DCM, the amount of Fmoc-Trp(Boc)-OH used is 60wt% of the liquid phase synthesis carrier, the amount of DIC used is 0.5wt% of the liquid phase synthesis carrier, and the amount of DMAP used is 2wt% of the liquid phase synthesis carrier; the sequence of peptide 1 is Fmoc-Leu-Pro-Ala-Leu-Ile- Ser(tBu)-Trp(Boc); in the coupling treatment, the reaction was stirred at 30°C for 4h, acetonitrile was added to precipitate crystals, and the intermediate-liquid phase synthesis carrier was filtered; in the deprotection treatment, the intermediate-liquid phase synthesis carrier was added to the deprotection solution, treated at 35°C for 5min, the pH was adjusted to neutral, and the product was precipitated. The deprotection solution was a THF solution containing 10vol% piperidine and 1vol% DBU, and the amount of the deprotection solution used was to immerse the intermediate-liquid phase synthesis carrier; in the cleavage treatment, the cleavage solution was added to the peptide segment 1-liquid phase synthesis carrier, stirred at 35°C for 30min, filtered, and recrystallized from isopropyl ether. The cleavage solution was a DCM solution containing 10vol% TFE and 1vol% TFA, and the amount of the cleavage solution used was to immerse the peptide segment 1-liquid phase synthesis carrier.

[0062] Peptide 2 was synthesized according to the preparation steps of peptide 1. The sequence of peptide 2 is Fmoc-Leu-Lys(Boc)-Val-Leu-Thr(tBu)-Thr(tBu)-Gly.

[0063] Peptide 3 was synthesized according to the preparation steps of peptide 1. The sequence of peptide 3 is Fmoc-Gly-Ile-Gly-Ala-Val.

[0064] Preparation of melittin: AM resin is used to synthesize melittin. First, Fmoc-Linker-OH is coupled to AM resin, and then amino acid reagents are coupled in the order of melittin sequence, coupled to the previous amino acid reagent of melittin peptide segment, and melittin peptide segment is coupled according to the sequence of melittin. After completion, separation treatment is performed to obtain melittin-peptide resin, and melittin-peptide resin is cut to obtain crude melittin, and the crude melittin is purified to obtain melittin.

[0065] The sequence of melittin is: H-Gly-Ile-Gly-Ala-Val-Leu-Lys-Val-Leu-Thr-Thr-Gly-Leu-Pro-Ala-Leu-Ile-Ser-Trp-Ile-Lys-Arg-Lys-Arg-Gln-Gln-NH2.

[0066] Preparation of activated linker reagent: Fmoc-Linker-OH and HOBt were added to DMF, stirred and dissolved, and then DIC was added at 5°C, and treated at 5°C for 20 minutes to obtain an activated linker reagent. The relationship between the amount of Fmoc-Linker-OH used and the amount of DMF used was 0.72 mol / L, the molar amount of HOBt used was 100% of the molar amount of Fmoc-Linker-OH used, and the molar amount of DIC used was 100% of the molar amount of Fmoc-Linker-OH used.

[0067] Preparation of Fmoc-Linker-AM resin: Add AM resin to the deprotection solution, treat at 35°C for 30 minutes, wash with DMF, then add the activated Linker reagent, react at 35°C for 3 hours, then add DIEA to react for 30 minutes, and after the reaction is completed, filter to obtain Fmoc-Linker-AM resin.

[0068] Preparation of activated amino acid reagent: Add amino acid reagent and HOBt into DMF, stir and dissolve, then add DIC at 5°C, treat at 5°C for 20 minutes to obtain activated amino acid reagent. The relationship between the amount of amino acid reagent and the amount of DMF is 0.72 mol / L, the molar amount of HOBt is 100% of the molar amount of amino acid reagent, and the molar amount of DIC is 100% of the molar amount of amino acid reagent. Amino acid reagents include: Fmoc-Gln(Trt)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ile-OH, Fmoc-Trp(Boc)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-Ala-OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Thr(tBu)-OH, Fmoc-Val-OH.

[0069] Preparation of bee venom peptide-peptide resin: Fmoc-Linker-AM resin was deprotected, and then an activated amino acid reagent was added and reacted at 35°C for 3 hours. After the reaction was completed, deprotection was performed and the aforementioned steps of coupling amino acid reagents were repeated in the order of the bee venom peptide sequence. The first amino acid reagent was Fmoc-Gln(Trt)-OH, and then the amino acid reagents coupled in sequence were as follows: Fmoc-Gln(Trt)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ile-OH, peptide 1, peptide 2 and peptide 3. The sequence of peptide 1 is Fmoc-Leu-Pro-Ala-Leu-Ile-Ser(tBu)-Trp(Boc), the sequence of peptide 2 is Fmoc-Leu-Lys(Boc)-Val-Leu-Thr(tBu)-Thr(tBu)-Gly, and the sequence of peptide 3 is Fmoc-Gly-Ile-Gly-Ala-Val.

[0070] When coupling amino acid reagents, the product of the previous coupling needs to be deprotected. The deprotection treatment includes post-coupling washing, deprotection, and post-deprotection washing. In the post-coupling washing, the coupled product is washed 3 times with DMF, and filtered after washing. In the deprotection, the deprotection solution is added to the product of the post-coupling washing, treated at 35°C for 30 minutes, and filtered after treatment. The deprotection solution is a DMF solution containing 20% ​​Pip. In the post-deprotection washing, the deprotected product is washed 5 times with DMF, and filtered after washing. When coupling peptides, the treatment is consistent with that of coupling amino acid reagents.

[0071] The crude product of melittin is obtained by cutting melittin-peptide resin. In the cutting process, melittin-peptide resin is added to the cutting solution, cut at 35°C for 4 hours, the resin is separated by filtration, methyl tert-ether is added to the filtrate, the precipitate is precipitated, and the crude product of melittin is obtained by filtration. The cutting solution is composed of TFA, TIS, EDT, PhOH and water. TFA, TIS, EDT, PhOH and water are mixed in the cutting solution in a volume ratio of 1:0.05:0.03:0.03:0.03. The cutting solution is immersed in the melittin-peptide resin, and methyl tert-ether is used in an appropriate amount to precipitate the crude product of melittin.

[0072] Melittin is obtained by purifying a crude product of melittin, and the purification process is HPLC treatment.

[0073] Example 3: A method for preparing melittin Compared with Example 2, this example is different in that a liquid phase synthesis carrier is used to synthesize peptide segment 4, peptide segment 5 and peptide segment 6.

[0074] The sequence of peptide 4 is Fmoc-Ala-Leu-Ile-Ser(tBu)-Trp(Boc)-Ile.

[0075] The sequence of peptide 5 is Fmoc-Val-Leu-Thr(tBu)-Thr(tBu)-Gly-Leu-Pro.

[0076] The sequence of peptide 6 is Fmoc-Gly-Ile-Gly-Ala-Val-Leu-Lys (Boc).

[0077] When synthesizing melittin, the preparation method of Example 2 was used for processing, and the preparation method was adaptively adjusted according to peptide segments 4, 5, and 6 in this example.

[0078] Comparative Example 1: A method for preparing bee venom peptide The difference between this comparative example and Example 2 lies in the preparation of the liquid phase synthetic carrier.

[0079] Preparation of liquid phase synthetic carrier: Potassium carbonate is added to DMF, then 2,4-dihydroxybenzaldehyde and bromocyclohexane are added, reacted at 70°C for 6h under an inert gas atmosphere, then diethyl bromomalonate is added, reacted at 70°C for 6h, the intermediate product is separated after the reaction is completed, the intermediate product is added to tetrahydrofuran solution, then sodium borohydride is added and reacted at 40°C for 3h, pure water is added after the reaction is completed to quench the reaction, and the liquid phase synthetic carrier is separated. The amount of potassium carbonate used is 15wt% of DMF, the amount of 2,4-dihydroxybenzaldehyde used is 15wt% of DMF, the molar amount of bromocyclohexane used is 40% of the molar amount of 2,4-dihydroxybenzaldehyde used, and the molar amount of diethyl bromomalonate used is 100% of the molar amount of 2,4-dihydroxybenzaldehyde used. In the separation of the intermediate product, the reaction solution is cooled to 5°C, 0.5M hydrochloric acid and water are added, the volume ratio of hydrochloric acid to water is 1:0.5, and the precipitate is filtered, washed, and dried to obtain the intermediate. The tetrahydrofuran solution is prepared by mixing tetrahydrofuran and methanol, and the tetrahydrofuran and methanol in the tetrahydrofuran solution are mixed in a volume ratio of 1:0.1. The amount of the intermediate used is 20wt% of the tetrahydrofuran solution, and the amount of sodium borohydride used is 20wt% of the intermediate.

[0080] When the present invention adopts the method of the above comparative example to prepare the synthetic liquid phase carrier, the product of coupling the amino reagent cannot be separated from the reaction solution, so the liquid phase synthetic carrier prepared in comparative example 1 cannot be used in the synthesis of peptides.

[0081] Comparative Example 2: A method for preparing bee venom peptide The difference between this comparative example and Example 2 is that a liquid phase synthesis carrier is used to synthesize peptide segment 7.

[0082] The sequence of peptide 7 is Fmoc-Gly-Ile-Gly-Ala-Val-Leu-Lys(Boc)-Val-Leu-Thr(tBu).

[0083] The synthesis of melittin in this comparative example was processed according to the preparation method of Example 2, and the preparation method was adaptively adjusted according to peptide segment 7 in this comparative example.

[0084] Comparative Example 3: A method for preparing bee venom peptide The difference between this comparative example and Example 2 is that a liquid phase synthesis carrier is used to synthesize peptide segment 8, and the peptide segment 7 synthesized in comparative example 2.

[0085] The sequence of peptide 8 is Fmoc-Thr(tBu)-Gly-Leu-Pro-Ala-Leu-Ile-Ser(tBu)-Trp(Boc)-Ile.

[0086] The synthesis of melittin in this comparative example was processed according to the preparation method of Example 2, and the preparation method was adaptively adjusted according to peptide segments 7 and 8 in this comparative example.

[0087] Test example: The present invention uses mass spectrometry to identify the melittin prepared in Example 1. The mass spectrometry results are as follows: Figure 1 The mass spectrometry results of melittin prepared in Example 2 and Example 3 are basically consistent with the mass spectrometry results of Example 1, indicating that melittin was purified and prepared in Example 2 and Example 3.

[0088] The yields of melittin prepared in the embodiments and comparative examples of the present invention are as follows: Figure 2As shown, S1 is Example 1, S2 is Example 2, S3 is Example 3, D1 is Comparative Example 1, D2 is Comparative Example 2, and D3 is Comparative Example 3. In the present invention, the amino acid reagents are coupled one by one in the order of melittin by using AM resin in a solid phase synthesis method, and the yield of melittin obtained is relatively low. The present invention finds that when melittin peptide segments are used for synthesis, the yield of melittin can be improved. In the synthesis method of melittin of the present invention, a peptide resin having seven amino acids can be synthesized by solid phase synthesis, and then Fmoc-Leu-Pro-Ala-Leu-Ile-Ser(tBu)-Trp(Boc), Fmoc-Leu-Lys(Boc)-Val-Leu- The peptide segments of Thr(tBu)-Thr(tBu)-Gly and Fmoc-Gly-Ile-Gly-Ala-Val can be used to synthesize melittin, which can greatly improve the yield of melittin. In the synthesis method of melittin of the present invention, a peptide resin with six amino acids can also be synthesized, and then the peptide segments of Fmoc-Ala-Leu-Ile-Ser(tBu)-Trp(Boc)-Ile, Fmoc-Val-Leu-Thr(tBu)-Thr(tBu)-Gly-Leu-Pro and Fmoc-Gly-Ile-Gly-Ala-Val-Leu-Lys(Boc) can be synthesized, which can also greatly improve the yield of melittin. In the present invention, a peptide segment of Fmoc-Gly-Ile-Gly-Ala-Val-Leu-Lys(Boc)-Val-Leu-Thr(tBu) and a peptide segment of Fmoc-Thr(tBu)-Gly-Leu-Pro-Ala-Leu-Ile-Ser(tBu)-Trp(Boc)-Ile were prepared. However, when the above peptide segments were used for synthesis, the yield of the obtained melittin was relatively poor, indicating that the longer the peptide segment is in the present invention, the better. When the peptide segment meets a certain length and has a specific number of peptide segments, a higher yield of melittin can be obtained. The present invention can use a liquid phase synthesis carrier to synthesize the peptide segment. The liquid phase synthesis carrier is prepared by reacting 2,4-dihydroxybenzaldehyde, 1,4-dibromocyclohexane and diethyl bromomalonate and reducing them. The liquid phase synthesis carrier prepared and synthesized can be successfully applied to the synthesis of the peptide segment, and the amino acid protection group is retained, so that the synthesized peptide segment can be used in the synthesis of melittin and the yield of melittin is improved. However, if the product prepared by reacting 2,4-dihydroxybenzaldehyde, bromocyclohexane and diethyl bromomalonate and reducing them is used for the liquid phase synthesis carrier, the amino acid coupling product cannot be separated from the reaction solution and cannot be used to synthesize the melittin peptide segment.

[0089] The embodiments and / or implementation methods described above are only used to illustrate the preferred embodiments and / or implementation methods for realizing the technology of the present invention, and are not intended to limit the implementation methods of the technology of the present invention in any form. Any technical personnel in this field may make slight changes or modifications to other equivalent embodiments without departing from the scope of the technical means disclosed in the content of the present invention, but they should still be regarded as technologies or embodiments that are essentially the same as the present invention.

[0090] This article uses specific examples to illustrate the principles and implementation methods of this application. The description of the above embodiments is only used to help understand the method and its core ideas of this application. The above is only the preferred implementation method of this application. It should be pointed out that due to the limitations of textual expression and the objective existence of infinite specific structures, ordinary technicians in this technical field can make several improvements, modifications or changes without departing from the principles of this application, and can also combine the above technical features in an appropriate manner; these improvements, modifications, changes or combinations, or the direct application of the concept and technical solution of the invention to other occasions without improvement, should be regarded as the scope of protection of this application.

Claims

1. A method for preparing melittin, comprising: Melittin-peptide resin is synthesized by solid phase synthesis in the order of the melittin sequence, and then cut and purified to obtain melittin; in the solid phase synthesis method, amino acid reagents are coupled one by one or at least one melittin peptide segment and a single amino acid reagent are used for coupling, and the number of amino acids in the melittin peptide segment is 4-8.

2. The method for preparing melittin according to claim 1, characterized in that: The bee venom peptide segment is at least one of the following: The sequence of peptide 1 is Fmoc-Leu-Pro-Ala-Leu-Ile- Ser(tBu)-Trp(Boc); The sequence of peptide 2 is Fmoc-Leu-Lys(Boc)-Val-Leu-Thr(tBu)-Thr(tBu)-Gly; The sequence of peptide 3 is Fmoc-Gly-Ile-Gly-Ala-Val; The sequence of peptide 4 is Fmoc-Ala-Leu-Ile-Ser(tBu)-Trp(Boc)-Ile; The sequence of peptide 5 is Fmoc-Val-Leu-Thr(tBu)-Thr(tBu)-Gly-Leu-Pro; The sequence of peptide 6 is Fmoc-Gly-Ile-Gly-Ala-Val-Leu-Lys (Boc).

3. The method for preparing melittin according to claim 2, characterized in that: The number of the melittin peptide segments is 3, and the peptide segments 1-3 are used in the synthesis of the melittin-peptide resin; or, the number of the melittin peptide segments is 3, and the peptide segments 4-6 are used in the synthesis of the melittin-peptide resin.

4. The method for preparing melittin according to claim 1, characterized in that: The synthesis of the bee venom peptide segment adopts a liquid phase synthesis carrier, and the liquid phase synthesis carrier is synthesized from 2,4-dihydroxybenzaldehyde, 1,4-dibromocyclohexane and diethyl bromomalonate and reduced.

5. The method for preparing melittin according to claim 4, characterized in that: The molar amount of the 1,4-dibromocyclohexane used is 30-50% of the molar amount of the 2,4-dihydroxybenzaldehyde used; or the molar amount of diethyl bromomalonate used is 80-120% of the molar amount of the 2,4-dihydroxybenzaldehyde used.

6. The method for preparing melittin according to claim 4, characterized in that: In the synthesis of the liquid phase synthetic carrier, 2,4-dihydroxybenzaldehyde and 1,4-dibromocyclohexane are first reacted, then diethyl bromomalonate is added for reaction, and finally the liquid phase synthetic carrier is reduced by sodium borohydride.

7. The method for preparing melittin according to claim 4, characterized in that: The 2,4-dihydroxybenzaldehyde and 1,4-dibromocyclohexane are reacted in a DMF solution containing potassium carbonate, and the reduction is carried out in a tetrahydrofuran solution using sodium borohydride.

8. The method for preparing melittin according to claim 1, characterized in that: In the solid phase synthesis, a deprotection solution is used to remove the protecting group, and the deprotection solution is a DMF solution containing 15-25% Pip.

9. The method for preparing melittin according to claim 1, characterized in that: The cutting is processed by cutting liquid, which is composed of TFA, TIS, EDT, PhOH and water. The TFA, TIS, EDT, PhOH and water in the cutting liquid are mixed in a volume ratio of 1:0.02-0.1:0.01-0.05:0.01-0.05:0.01-0.

05.

10. Use of a melittin peptide segment in the preparation of melittin, wherein the melittin peptide segment is at least one of the following: The sequence of peptide 1 is Fmoc-Leu-Pro-Ala-Leu-Ile- Ser(tBu)-Trp(Boc); The sequence of peptide 2 is Fmoc-Leu-Lys(Boc)-Val-Leu-Thr(tBu)-Thr(tBu)-Gly; The sequence of peptide 3 is Fmoc-Gly-Ile-Gly-Ala-Val; The sequence of peptide 4 is Fmoc-Ala-Leu-Ile-Ser(tBu)-Trp(Boc)-Ile; The sequence of peptide 5 is Fmoc-Val-Leu-Thr(tBu)-Thr(tBu)-Gly-Leu-Pro; The sequence of peptide 6 is Fmoc-Gly-Ile-Gly-Ala-Val-Leu-Lys (Boc).

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