A production process of melittin

Abstract

The present invention discloses a production process of melittin, belonging to the technical field of polypeptide synthesis, and specifically relates to a method for preparing melittin. The method comprises synthesizing a melittin-peptide resin by solid-phase synthesis in the order of the melittin sequence, and cutting and purifying the melittin. The solid-phase synthesis method adopts coupling amino acid reagents one by one or coupling using at least one melittin peptide segment and a single amino acid reagent, and the number of amino acids in the melittin peptide segment is 4-8. In the present invention, the synthesis of the melittin peptide segment adopts a liquid-phase synthesis carrier, which is synthesized from 2,4-dihydroxybenzaldehyde, 1,4-dibromocyclohexane and diethyl bromomalonate and obtained by reduction.

Description

Technical Field

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

[0002] Bee venom is a complex composition. As a naturally occurring mixture extracted from bees, its properties are difficult to directly study and apply clinically. Melittin, isolated and purified from bee venom, is its primary active ingredient. Melittin is highly effective at disrupting cell membranes, including the plasma membrane and the membranes of several intracellular organelles. Its mechanism of action involves its amphipathic structure embedding into phospholipid membranes, perforating them and causing rupture. Alterations in the membrane's internal and external environments ultimately lead to cell death. Melittin can also affect key proteins in multiple intracellular signaling pathways that mediate tumor growth, leading to apoptosis. Melittin also exhibits anti-inflammatory, antibacterial, anti-cancer, anti-radiation, and platelet aggregation inhibition properties. It is effective in treating various diseases, including periarthritis of the shoulder, rheumatoid arthritis, and rheumatoid arthritis, and has considerable medical applications. Melittin's high biological activity and environmental friendliness make it a promising candidate for development as an immune modulator, attracting considerable attention and promising market development. Summary of the Invention

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

[0004] The technical solutions adopted by the present invention to achieve the above-mentioned purpose are:

[0005] A method for preparing melittin comprises: synthesizing melittin-peptide resin using a solid-phase synthesis method according to the order of the melittin sequence, and cleaving and purifying the melittin; wherein the solid-phase synthesis method uses a coupling amino acid reagent one by one or uses at least one melittin peptide segment and a single amino acid reagent for coupling, wherein the number of amino acids in the melittin peptide segment is 4-8. The present invention can greatly improve the yield of melittin by synthesizing melittin using at least one melittin peptide segment with a specific number of amino acids. The number of amino acids in the melittin peptide segment cannot be too large, as too many amino acids 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.

[0006] Preferably, the melittin peptide fragment is at least one of the following:

[0007] The sequence of peptide 1 is Fmoc-Leu-Pro-Ala-Leu-Ile- Ser(tBu)-Trp(Boc);

[0008] The sequence of peptide 2 is Fmoc-Leu-Lys(Boc)-Val-Leu-Thr(tBu)-Thr(tBu)-Gly;

[0009] The sequence of peptide 3 is Fmoc-Gly-Ile-Gly-Ala-Val;

[0010] The sequence of peptide 4 is Fmoc-Ala-Leu-Ile-Ser(tBu)-Trp(Boc)-Ile;

[0011] The sequence of peptide 5 is Fmoc-Val-Leu-Thr(tBu)-Thr(tBu)-Gly-Leu-Pro;

[0012] The sequence of peptide 6 is Fmoc-Gly-Ile-Gly-Ala-Val-Leu-Lys(Boc).

[0013] 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, the use of peptide segments 1-3 and a single amino acid reagent in the synthesis of melittin can increase 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, resulting in a high yield of melittin.

[0014] Preferably, 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.

[0015] 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.

[0016] 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 obtained by reduction with sodium borohydride.

[0017] 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.

[0018] 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.

[0019] Preferably, the cutting is carried out using 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.

[0020] The present invention discloses the use of melittin peptide segments in the preparation of melittin, and the melittin peptide segments include at least one of the following:

[0021] The sequence of peptide 1 is Fmoc-Leu-Pro-Ala-Leu-Ile- Ser(tBu)-Trp(Boc);

[0022] The sequence of peptide 2 is Fmoc-Leu-Lys(Boc)-Val-Leu-Thr(tBu)-Thr(tBu)-Gly;

[0023] The sequence of peptide 3 is Fmoc-Gly-Ile-Gly-Ala-Val;

[0024] The sequence of peptide 4 is Fmoc-Ala-Leu-Ile-Ser(tBu)-Trp(Boc)-Ile;

[0025] The sequence of peptide 5 is Fmoc-Val-Leu-Thr(tBu)-Thr(tBu)-Gly-Leu-Pro;

[0026] The sequence of peptide 6 is Fmoc-Gly-Ile-Gly-Ala-Val-Leu-Lys(Boc).

[0027] 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. Under an inert gas atmosphere, the reaction is carried out at a temperature of 60-80°C for 3-12 hours. Then, diethyl bromomalonate is added and the reaction is carried out at a temperature of 60-80°C for 3-12 hours. After the reaction is completed, the intermediate product is separated, and the intermediate product is added to a tetrahydrofuran solution. Then, sodium borohydride is added and the reaction is carried out at a temperature of 30-50°C for 2-5 hours. 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 peptides. In the preparation of the above-mentioned liquid-phase synthesis carrier, if 1,4-dibromocyclohexane is replaced with bromocyclohexane, the resulting product cannot be used as a liquid-phase synthesis carrier in the preparation of peptides.

[0028] More preferably, in the preparation of the liquid phase synthetic carrier, the amount of potassium carbonate used is 10-20 wt % of DMF.

[0029] More preferably, in the preparation of the liquid phase synthesis carrier, the amount of 2,4-dihydroxybenzaldehyde used is 10-20 wt % of DMF.

[0030] 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.

[0031] 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.

[0032] More preferably, in the preparation of the liquid-phase synthetic carrier and the isolation 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.

[0033] 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 10-30 wt% of the tetrahydrofuran solution.

[0034] More preferably, in the preparation of the liquid phase synthesis carrier, the amount of sodium borohydride used is 10-30 wt % of the intermediate.

[0035] 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. The amino acid reagent is replaced in the order of the peptide segment and the coupling treatment and deprotection treatment are performed in sequence. After the last amino acid is coupled, no deprotection treatment is performed. The liquid-phase synthesis carrier is removed by cutting treatment using a cutting fluid to obtain the peptide segment.

[0036] More preferably, in the preparation of the peptide segment, the amount of the liquid phase synthesis carrier used is 10-30 wt % of DCM.

[0037] 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.

[0038] 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.

[0039] More preferably, in the preparation of the peptide segment, the amount of DMAP used is 1-4 wt % of the liquid phase synthesis carrier.

[0040] More preferably, in the preparation of the peptide segment, the peptide segment includes the above-mentioned peptide segments 1-6.

[0041] More preferably, in the coupling treatment for peptide preparation, the reaction is stirred at 20-40° C. for 2-8 h, acetonitrile is added to precipitate crystals, and the intermediate - liquid phase synthesis carrier is obtained by filtration.

[0042] More preferably, in the deprotection treatment of the peptide preparation, the intermediate-liquid phase synthesis support is added to the deprotection solution, treated at 20-40°C for 3-10 minutes, 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 sufficient to immerse the intermediate-liquid phase synthesis support.

[0043] 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 20-60 minutes, filtered, and recrystallized from isopropyl ether. The cleavage liquid is a DCM solution containing 5-20 vol% TFE and 0.5-2% TFA, and the amount of cleavage liquid used is sufficient to immerse the peptide 1-liquid phase synthesis carrier.

[0044] Preferably, in the preparation of melittin, AM resin is used to synthesize melittin, firstly Fmoc-Linker-OH is coupled to the AM resin, and then the amino acid reagent is coupled in the order of the melittin sequence. After the coupling is completed, the melittin-peptide resin is separated and treated to obtain the melittin-peptide resin, the melittin-peptide resin is cleaved to obtain the crude melittin, and the crude melittin is purified to obtain melittin.

[0045] 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.

[0046] Preferably, in the preparation of the activated linker reagent, Fmoc-Linker-OH 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 linker reagent.

[0047] More preferably, in the preparation of the activated linker reagent, the relationship between the amount of Fmoc-Linker-OH used and the amount of DMF used is 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.

[0048] Preferably, in the preparation of Fmoc-Linker-AM resin, AM resin is added to the deprotection solution, treated at 30-35°C for 20-50 minutes, washed with DMF, and then the activated linker reagent is added, reacted at 30-35°C for 2-4 hours, and then DIEA is added to react for 20-50 minutes. After the reaction is completed, the Fmoc-Linker-AM resin is filtered to obtain.

[0049] Preferably, in the preparation of the activated amino acid reagent, the amino acid reagent and HOBt are added to DMF, stirred to dissolve, and then DIC is added at a temperature of 2-8°C. The mixture is treated at a temperature of 2-8°C for 10-20 minutes to obtain the activated amino acid reagent. The amount of the amino acid reagent used is 0.3-1.5 mol / L relative to the amount of DMF used, 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.

[0050] 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.

[0051] Preferably, in the preparation of melittin-peptide resin, the Fmoc-Linker-AM resin is deprotected, and then an activated amino acid reagent is added and reacted at 30-35° C. for 2-4 hours. After the reaction is completed, the deprotection treatment is performed, and the steps of coupling the amino acid reagent are repeated in the order of the melittin sequence. The first amino acid reagent is Fmoc-Gln(Trt)-OH, and 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.

[0052] More preferably, in the preparation of melittin-peptide resin, when coupling with amino acid reagents, the product of the previous coupling needs to be deprotected, and the deprotection treatment includes post-coupling washing, deprotection, and post-deprotection washing.

[0053] More preferably, in the preparation of melittin-peptide resin, in the post-coupling washing, the coupled product is washed 1-3 times with DMF, and then filtered after washing.

[0054] More preferably, during the deprotection process in the preparation of melittin-peptide resin, a deprotection solution is added to the washed product after coupling, treated at 30-35° C. for 20-40 minutes, and filtered after treatment. The deprotection solution is a DMF solution containing 15-25% Pip.

[0055] More preferably, in the preparation of melittin-peptide resin, the deprotected product is washed with DMF 2-6 times in the washing after deprotection, and filtered after washing.

[0056] Preferably, the crude melittin is obtained by cleaving melittin-peptide resin. In the cleavage treatment, the melittin-peptide resin is added to a cleavage solution and cleaved at 25-35° C. for 2-6 hours. The resin is separated by filtration, methyl tert-ether is added to the filtrate, a precipitate is precipitated, and the precipitate is filtered to obtain the crude melittin.

[0057] More preferably, the cutting fluid comprises TFA, TIS, EDT, PhOH, and water, wherein the 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 fluid is immersed in the melittin-peptide resin, and methyl tertiary ether is used in an appropriate amount to precipitate the crude melittin product.

[0058] Preferably, melittin is obtained by purifying crude melittin, and the purification is HPLC treatment.

[0059] The present invention utilizes a liquid-phase synthesis carrier to synthesize melittin peptide segments, which are then applied to solid-phase synthesis of melittin, followed by coupling with a single amino acid reagent of melittin. The method for synthesizing melittin of the present invention requires the design and use of melittin peptide segments with specific sequences, as well as the use of at least one melittin peptide segment. This method 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 method for preparing melittin using solid-phase synthesis, liquid-phase peptide synthesis, at least one peptide segment, with high yield and high purity. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 This is the mass spectrum of melittin.

[0061] Figure 2 This is a graph showing the yield of melittin. DETAILED DESCRIPTION

[0062] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. 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 making creative efforts are within the scope of protection of the present invention.

[0063] The following first describes the concepts involved in this application with reference to the accompanying drawings. It should be noted that the following description of each concept is intended only to make the content of this application easier to understand and does not limit the scope of protection of this application. At the same time, the embodiments and features in the embodiments of this application can be combined with each other unless there is a conflict. The following detailed description of this application will be made with reference to the accompanying drawings and in conjunction with the embodiments.

[0064] Example 1: A method for preparing melittin

[0065] Preparation of melittin: AM resin is used to synthesize melittin. First, Fmoc-Linker-OH is coupled to the AM resin, and then amino acid reagents are coupled in the order of the melittin sequence. After coupling, the melittin-peptide resin is separated and treated. The melittin-peptide resin is cleaved to obtain crude melittin. The crude melittin is purified to obtain melittin.

[0066] 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.

[0067] Preparation of activated linker reagent: Add Fmoc-Linker-OH and HOBt to DMF, stir to dissolve, then add DIC at 5°C. Incubate at 5°C for 20 minutes to obtain the activated linker reagent. The amount of Fmoc-Linker-OH used is 0.72 mol / L relative to the amount of DMF used. The molar amount of HOBt used is 100% of the molar amount of Fmoc-Linker-OH used, and the molar amount of DIC used is 100% of the molar amount of Fmoc-Linker-OH used.

[0068] Preparation of Fmoc-Linker-AM resin: AM resin was added to the deprotection solution, treated at 35°C for 30 minutes, washed with DMF, and then the activated linker reagent was added and reacted at 35°C for 3 hours. Then DIEA was added and reacted for 30 minutes. After the reaction was completed, the Fmoc-Linker-AM resin was filtered to obtain the resin.

[0069] Preparation of activated amino acid reagent: Add the amino acid reagent and HOBt to DMF, stir to dissolve, then add DIC at 5°C. Incubate at 5°C for 20 min to obtain the activated amino acid reagent. The amount of amino acid reagent used is 0.72 mol / L relative to the amount of DMF used. The molar amount of HOBt and DIC should be 100% of the molar amount of the amino acid reagent. Examples of 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, and Fmoc-Val-OH.

[0070] Preparation of melittin-peptide resin: Fmoc-Linker-AM resin was deprotected, and then 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 melittin 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.

[0071] When coupling amino acid reagents, the product from the previous coupling needs to be deprotected. This deprotection process includes post-coupling washing, deprotection, and post-deprotection washing. During the post-coupling wash, the coupled product is washed three times with DMF and filtered. During the deprotection process, the deprotection solution is added to the post-coupling washed product, treated at 35°C for 30 minutes, and filtered. The deprotection solution is a DMF solution containing 20% ​​Pip. During the post-deprotection wash, the deprotected product is washed five times with DMF and filtered.

[0072] Crude melittin is obtained by cleaving melittin-peptide resin. The resin is added to a cleavage solution and cleaved at 35°C for 4 hours. The resin is then filtered and separated. Methyl tertiary ether is added to the filtrate to precipitate the precipitate, which is then filtered to obtain crude melittin. The cleavage solution consists of TFA, TIS, EDT, PhOH, and water in a volume ratio of 1:0.05:0.03:0.03:0.03. The cleavage solution is used to submerge the melittin-peptide resin, and an appropriate amount of methyl tertiary ether is used to precipitate the crude melittin.

[0073] Melittin is obtained by purifying crude melittin, and the purification process is HPLC treatment.

[0074] Example 2: A method for preparing melittin

[0075] Preparation of a liquid-phase synthetic support: Potassium carbonate is added to DMF, followed by 2,4-dihydroxybenzaldehyde and 1,4-dibromocyclohexane. The mixture is reacted at 70°C for 6 hours under an inert gas atmosphere. Diethyl bromomalonate is then added and the reaction is continued at 70°C for 6 hours. After the reaction is complete, the intermediate product is separated and added to a tetrahydrofuran solution. Sodium borohydride is then added and the reaction is continued at 40°C for 3 hours. After the reaction is complete, pure water is added to quench the reaction and the liquid-phase synthetic support is separated. The amount of potassium carbonate used is 15wt% of the DMF, the amount of 2,4-dihydroxybenzaldehyde used is 15wt% of the 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. To isolate the intermediate product, the reaction solution is cooled to 5°C, 0.5M hydrochloric acid and water are added in a volume ratio of 1:0.5. The precipitate is filtered, washed, and dried to obtain the intermediate. The tetrahydrofuran solution is prepared by mixing tetrahydrofuran and methanol in a volume ratio of 1:0.1. The intermediate is used in an amount of 20% by weight of the tetrahydrofuran solution, and the sodium borohydride is used in an amount of 20% by weight of the intermediate.

[0076] Preparation of peptide 1: The liquid phase synthesis carrier was added to DCM, and then Fmoc-Trp(Boc)-OH, DIC and DMAP were added for coupling treatment. After the coupling was completed, deprotection treatment was performed. Fmoc-Trp(Boc)-OH was replaced in the order of peptide 1 and coupling treatment and deprotection treatment were performed in sequence. No deprotection treatment was performed after coupling the last amino acid. The liquid phase synthesis carrier was removed by cutting with a cutting liquid to obtain peptide 1. The amount of liquid phase synthesis carrier used was 20wt% of DCM, the amount of Fmoc-Trp(Boc)-OH used was 60wt% of the liquid phase synthesis carrier used, the amount of DIC used was 0.5wt% of the liquid phase synthesis carrier used, and the amount of DMAP used was 2wt% of the liquid phase synthesis carrier used; the sequence of peptide 1 was Fmoc-Leu-Pro-Ala-Leu-Ile- Ser(tBu)-Trp(Boc); in the coupling treatment, the reaction was stirred at 30°C for 4 hours, acetonitrile was added to precipitate crystals, and the intermediate-liquid phase synthesis carrier was obtained by filtration; in the deprotection treatment, the intermediate-liquid phase synthesis carrier was added to the deprotection solution, treated at 35°C for 5 minutes, the pH was adjusted to neutral, and the product was precipitated. The deprotection solution was a THF solution containing 10 vol% piperidine and 1 vol% 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 30 minutes, filtered, and recrystallized from isopropyl ether. The cleavage solution was a DCM solution containing 10 vol% TFE and 1 vol% TFA, and the amount of the cleavage solution used was to immerse the peptide segment 1-liquid phase synthesis carrier.

[0077] Peptide 2 was synthesized according to the preparation steps of peptide 1 above. The sequence of peptide 2 is Fmoc-Leu-Lys(Boc)-Val-Leu-Thr(tBu)-Thr(tBu)-Gly.

[0078] Peptide 3 was synthesized according to the preparation steps of peptide 1. The sequence of peptide 3 is Fmoc-Gly-Ile-Gly-Ala-Val.

[0079] Preparation of melittin: AM resin is used to synthesize melittin. First, Fmoc-Linker-OH is coupled to the AM resin. Then, amino acid reagents are coupled in the order of the melittin sequence. The amino acid reagent is coupled to the preceding amino acid reagent of the melittin peptide segment. The melittin peptide segment is coupled according to the sequence of the melittin. After completion, separation is performed to obtain melittin-peptide resin. The melittin-peptide resin is cleaved to obtain crude melittin. The crude melittin is purified to obtain melittin.

[0080] 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.

[0081] Preparation of activated linker reagent: Add Fmoc-Linker-OH and HOBt to DMF, stir to dissolve, then add DIC at 5°C. Incubate at 5°C for 20 minutes to obtain the activated linker reagent. The amount of Fmoc-Linker-OH used is 0.72 mol / L relative to the amount of DMF used. The molar amount of HOBt used is 100% of the molar amount of Fmoc-Linker-OH used, and the molar amount of DIC used is 100% of the molar amount of Fmoc-Linker-OH used.

[0082] Preparation of Fmoc-Linker-AM resin: AM resin was added to the deprotection solution, treated at 35°C for 30 minutes, washed with DMF, and then the activated linker reagent was added and reacted at 35°C for 3 hours. Then DIEA was added and reacted for 30 minutes. After the reaction was completed, the Fmoc-Linker-AM resin was filtered to obtain the resin.

[0083] Preparation of activated amino acid reagent: Add the amino acid reagent and HOBt to DMF, stir to dissolve, then add DIC at 5°C. Incubate at 5°C for 20 min to obtain the activated amino acid reagent. The amount of amino acid reagent used is 0.72 mol / L relative to the amount of DMF used. The molar amount of HOBt and DIC should be 100% of the molar amount of the amino acid reagent. Examples of 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, and Fmoc-Val-OH.

[0084] Preparation of melittin-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 melittin sequence. The first amino acid reagent was Fmoc-Gln(Trt)-OH, and 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.

[0085] When coupling amino acid reagents, the product from the previous coupling needs to be deprotected. This deprotection process includes post-coupling washes, deprotection, and post-deprotection washes. During the post-coupling washes, the coupled product is washed three times with DMF and filtered. During the deprotection process, the deprotection solution is added to the post-coupling washed product, treated at 35°C for 30 minutes, and filtered. The deprotection solution is a DMF solution containing 20% ​​Pip. During the post-deprotection washes, the deprotected product is washed five times with DMF and filtered. The same procedures are used for coupling peptides as for coupling amino acid reagents.

[0086] Crude melittin is obtained by cleaving melittin-peptide resin. The resin is added to a cleavage solution and cleaved at 35°C for 4 hours. The resin is then filtered and separated. Methyl tertiary ether is added to the filtrate to precipitate the precipitate, which is then filtered to obtain crude melittin. The cleavage solution consists of TFA, TIS, EDT, PhOH, and water in a volume ratio of 1:0.05:0.03:0.03:0.03. The cleavage solution is used to submerge the melittin-peptide resin, and an appropriate amount of methyl tertiary ether is used to precipitate the crude melittin.

[0087] Melittin is obtained by purifying crude melittin, and the purification process is HPLC treatment.

[0088] Example 3: A method for preparing melittin

[0089] Compared with Example 2, this example is different in that a liquid phase synthesis carrier is used to synthesize peptide segments 4, 5 and 6.

[0090] The sequence of peptide 4 is Fmoc-Ala-Leu-Ile-Ser(tBu)-Trp(Boc)-Ile.

[0091] The sequence of peptide 5 is Fmoc-Val-Leu-Thr(tBu)-Thr(tBu)-Gly-Leu-Pro.

[0092] The sequence of peptide 6 is Fmoc-Gly-Ile-Gly-Ala-Val-Leu-Lys(Boc).

[0093] 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.

[0094] Comparative Example 1: A method for preparing melittin

[0095] The difference between this comparative example and Example 2 lies in the preparation of the liquid phase synthetic carrier.

[0096] Preparation of a liquid-phase synthetic support: Potassium carbonate is added to DMF, followed by 2,4-dihydroxybenzaldehyde and bromocyclohexane. The mixture is reacted at 70°C for 6 hours under an inert gas atmosphere. Diethyl bromomalonate is then added and the mixture is reacted at 70°C for 6 hours. After the reaction is complete, the intermediate product is separated and added to a tetrahydrofuran solution. Sodium borohydride is then added and the mixture is reacted at 40°C for 3 hours. After the reaction is complete, pure water is added to quench the reaction and the liquid-phase synthetic support is separated. The amount of potassium carbonate used is 15wt% of the DMF, the amount of 2,4-dihydroxybenzaldehyde used is 15wt% of the 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. To isolate the intermediate product, the reaction solution is cooled to 5°C, 0.5M hydrochloric acid and water are added in a volume ratio of 1:0.5. The precipitate is filtered, washed, and dried to obtain the intermediate. The tetrahydrofuran solution is prepared by mixing tetrahydrofuran and methanol in a volume ratio of 1:0.1. The intermediate is used in an amount of 20% by weight of the tetrahydrofuran solution, and the sodium borohydride is used in an amount of 20% by weight of the intermediate.

[0097] 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. Therefore, the liquid phase synthetic carrier prepared in comparative example 1 cannot be used in peptide synthesis.

[0098] Comparative Example 2: A method for preparing melittin

[0099] The difference between this comparative example and Example 2 is that a liquid phase synthesis carrier is used to synthesize peptide segment 7.

[0100] The sequence of peptide 7 is Fmoc-Gly-Ile-Gly-Ala-Val-Leu-Lys(Boc)-Val-Leu-Thr(tBu).

[0101] The synthesis of melittin in this comparative example was carried out according to the preparation method of Example 2, and the preparation method was adjusted adaptively according to peptide segment 7 in this comparative example.

[0102] Comparative Example 3: A method for preparing melittin

[0103] 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 synthesized in comparative example 2 is 7.

[0104] The sequence of peptide 8 is Fmoc-Thr(tBu)-Gly-Leu-Pro-Ala-Leu-Ile-Ser(tBu)-Trp(Boc)-Ile.

[0105] The synthesis of melittin in this comparative example was carried out according to the preparation method of Example 2, and the preparation method was adjusted adaptively according to peptide segments 7 and 8 in this comparative example.

[0106] Test example:

[0107] 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.

[0108] The yields of melittin prepared in the examples and comparative examples of the present invention are as follows: Figure 2As shown, wherein 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 first, and then Fmoc-Leu-Pro-Ala-Leu-Ile-Ser(tBu)-Trp(Boc), Fmoc-Leu-Lys(Boc)-Val-Leu- The peptide segments Thr(tBu)-Thr(tBu)-Gly and Fmoc-Gly-Ile-Gly-Ala-Val are 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 having six amino acids can also be synthesized, and then the peptide segments 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, the better it is. A higher yield of melittin can be obtained when the peptide segment meets a certain length and a specific number of peptide segments. The present invention can use a liquid-phase synthesis carrier to synthesize peptide segments. The liquid-phase synthesis carrier is prepared by reacting 2,4-dihydroxybenzaldehyde, 1,4-dibromocyclohexane, and diethyl bromomalonate and then reducing them. The liquid-phase synthesis carrier prepared above can be successfully applied to the synthesis of peptide segments, and retains the amino acid protecting groups, so that the synthesized peptide segments can be used in the synthesis of melittin and improve the yield of melittin. However, if the product obtained by reacting 2,4-dihydroxybenzaldehyde, bromocyclohexane, and diethyl bromomalonate and then reducing them is used as a liquid-phase synthesis carrier, the amino acid coupling product cannot be separated from the reaction solution and cannot be used to synthesize melittin peptide segments.

[0109] 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 do not impose any form of limitation on the implementation methods of the technology of the present invention. Any person skilled in the art 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.

[0110] 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 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, there are objectively infinite specific structures. For ordinary technicians in this technical field, without departing from the principles of this application, they can also make several improvements, modifications or changes, and can also combine the above technical features in an appropriate manner; these improvements, modifications, changes or combinations, or the direct application of the inventive concept and technical solution 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; the solid-phase synthesis method uses three melittin peptide segments and a single amino acid reagent for coupling, and the number of amino acids in the melittin peptide segment is 4-8; the synthesis of the melittin peptide segment is synthesized by 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; The molar amount of 1,4-dibromocyclohexane used is 30-50% of the molar amount of 2,4-dihydroxybenzaldehyde used; the molar amount of diethyl bromomalonate used is 80-120% of the molar amount of 2,4-dihydroxybenzaldehyde used; 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 reduced with sodium borohydride to obtain the liquid-phase synthetic carrier; 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; The synthesis of the melittin-peptide resin uses peptide segments 1-3; or, the synthesis of the melittin-peptide resin uses peptide segments 4-6; 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).

2. The method for preparing melittin according to claim 1, wherein: 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.

3. The method for preparing melittin according to claim 1, wherein: The cutting is processed by a cutting fluid, which consists of TFA, TIS, EDT, PhOH and water. The TFA, TIS, EDT, PhOH and water in the cutting fluid are mixed in a volume ratio of 1:0.02-0.1:0.01-0.05:0.01-0.05:0.01-0.05.

Images