A method for synthesizing a mirror image cyclic peptide using an OaAEP1 enzyme with the assistance of a thioester

Abstract

The present application relates to the technical field of cyclic peptide drug synthesis, and particularly relates to a method for synthesizing mirror image cyclic peptide by using OaAEP1 enzyme with the assistance of thioester, which comprises the following steps: firstly, a linear thioester precursor of mirror image cyclic peptide is synthesized by using an N-fluorenylmethyloxy carbonyl (Fmoc) solid-phase polypeptide synthesis method; and then the thioester precursor can generate the mirror image cyclic peptide under the mediation of OaAEP1 enzyme. The present application first synthesizes mirror image melittin (D-cMelittin) by using OaAEP1 enzyme with the assistance of thioester, and the mirror image melittin has high protease stability and good antibacterial activity.

Description

Technical Field

[0001] This invention relates to the field of cyclic peptide drug synthesis technology, and specifically to a method for synthesizing mirror-image cyclic peptides using OaAEP1 enzyme with the assistance of thioesters. Background Technology

[0002] Cyclic peptides, which can regulate protein function by specifically binding to targets with high affinity, have become important therapeutic agents and diagnostic reagents. However, natural cyclic peptides are composed of L-amino acids, which can be recognized by antibodies, hydrolases, and isomerases in the body, thus posing problems such as immunogenicity and short half-life. In contrast, mirror-shaped cyclic peptides composed of D-amino acids have low immunogenicity and high stability, making them promising drug candidates, such as the inhibitor D-H101 of the cancer target protein MDM2 and the antimicrobial peptide P-15.

[0003] The ligation of mirror-image peptides is fundamental to the synthesis of mirror-image cyclic peptides. Enzyme-catalyzed peptide ligation reactions, such as Sortase A and OaAEP1, can occur in a mild aqueous phase and exhibit high chemoselectivity, making them ideal strategies for the synthesis of mirror-image cyclic peptides. However, since natural peptide ligases are composed of L-amino acids, they can only recognize L-peptides and cannot mediate the ligation of mirror-image peptides. Current research has demonstrated the enzymatic ligation of mirror-image peptides by chemically synthesizing Sortase, a mirror-image peptide ligase composed of D-amino acids, proving for the first time the feasibility of enzymatic synthesis of mirror-image cyclic peptides. Intein-assisted natural chemical ligation methods have also been used to achieve the ligation of mirror-image peptides. However, the efficiency of obtaining mirror-image enzymes through total synthesis is extremely low, making it difficult to widely apply to the synthesis of mirror-image cyclic peptides; intein-assisted mirror-image peptide ligation relies on stoichiometric amounts of intein protein, resulting in poor atom economy. Therefore, the synthesis of mirror-image cyclic peptides using natural enzymes obtained through recombinant expression under catalytic stoichiometry remains a major challenge.

[0004] In view of the above-mentioned defects, the inventors of this invention have finally obtained this invention after a long period of research and practice. Summary of the Invention

[0005] The purpose of this invention is to solve the problem that natural protein ligases cannot recognize mirror-image peptides and mediate their cyclization, and to provide a method for synthesizing mirror-image cyclic peptides using OaAEP1 enzyme with the assistance of thioesters.

[0006] To achieve the above objectives, this invention discloses a method for synthesizing mirror-shaped cyclic peptides using OaAEP1 enzyme with the assistance of thioesters, comprising the following steps:

[0007] S1, OaAEP1 was expressed in E. coli Shuffle T7 competent cells and purified by nickel column, activated by dialysis with activation buffer at room temperature and further purified by AKTA Pure;

[0008] S2, 2,2'-Dithiodiacetic acid was coupled with L-leucine tert-butyl ester. After the reaction was complete, the mixture was extracted, and the organic phase was washed with HCl and Na2CO3 salt solutions, respectively. Dithiothreitol and triethylamine were added to the organic phase, and the reaction was allowed to proceed for 1 hour. After the reaction was complete, the reaction solution was washed, dried, and concentrated under vacuum. The product was then loaded onto silica gel and purified to obtain (2-mercaptoacetyl)-L-leucine tert-butyl ester (hereinafter referred to as thiol S1). The reaction process is shown below:

[0009]

[0010] S3, following the linear amino acid sequence of D-Melittin, firstly, the carboxyl group of the Fmoc-Asp-Oall side chain is anchored onto the resin, then amino acids are sequentially coupled from the C-terminus. The Fmoc protecting group of the terminal amino acid is removed, and the terminal amino group is protected with Boc2O. The allyl protecting group at the end of the peptide chain is removed, and it is coupled with (2-mercaptoacetyl)-L-leucine tert-butyl ester. The thioester precursor is cleaved from the resin using a trifluoroacetic acid cleavage reagent. After separation and purification, the linear melitrix thioester precursor is obtained.

[0011] S4, the linear melitoxin thioester precursor obtained in step S3 undergoes an enzyme-catalyzed intramolecular cyclization reaction in phosphate buffer solution in the presence of OaAEP1 enzyme to produce cyclic peptide molecules.

[0012] In step S1, E. coli cells are grown in YT medium at 37°C, induced with 0.4 mM IPTG at 16°C for 16 hours, collected by centrifugation at 4300 rpm, resuspended in lysis buffer, and then lysed by sonication in an ice bath.

[0013] In step S1, the specific steps for nickel column purification are as follows:

[0014] S11, the pyrolysis solution was centrifuged at 9500 rpm for 30 minutes at 4°C, and the supernatant was loaded into a nickel column;

[0015] S12 was washed with a washing buffer consisting of 25 mM HEPES, pH 7.7, and 150 mM NaCl.

[0016] S13, Elute impurities with a low-concentration elution buffer, wherein the low-concentration elution buffer is 25 mM HEPES, pH=7.7, 150 mM NaCl, and 30 mM imidazole.

[0017] S14, elute the target protein with a high-concentration elution buffer, wherein the high-concentration elution buffer is 25 mM HEPES, pH=7.7, 150 mM NaCl, and 250 mM imidazole.

[0018] In step S1, the activation buffer is 50 mM sodium acetate, 1 mM EDTA, pH=4.0, and activated by dialysis at room temperature for 4 hours.

[0019] In step S3, the side-chain carboxyl anchoring resin is obtained by condensing Fmoc-Asp-Oall onto an amino resin using a standard Fmoc solid-phase peptide synthesis method, followed by sequential washing with DMF, DCM, and DMF, and then drying after thorough washing.

[0020] In step S3, the amino acid coupling conditions are: reacting at 37°C for 20 min in a system with a molar ratio of HATU: amino acid: DIEA = 5:5:10;

[0021] The conditions for removing the Fmoc protecting group are: reacting with 20% piperidine at 37°C for 10 min or at 75°C for 5 min.

[0022] The allyl protecting group removal conditions were as follows: tetrakis(triphenylphosphine)palladium was dissolved in DCM, then benzylsilane was added, and the reaction solution was added to the synthesis tube and reacted at 37°C for 1.5 h.

[0023] The coupling conditions for (2-mercaptoacetyl)-L-leucine tert-butyl ester were as follows: 1-hydroxybenzotriazole (HOBT):(2-mercaptoacetyl)-L-leucine tert-butyl ester in a molar ratio of 4:4 was dissolved in an equal volume mixture of DCM and DMF, and then 5 eq DIC and 5 eq DIEA were added and mixed well. The mixture was then added to a synthesis tube and reacted at 37°C for 4 h.

[0024] The TFA cleavage reagent was used to cleave the thioester precursor from the resin under the following conditions: a cleavage solution with a volume ratio of TFA:water:Tips = 95:2.5:2.5 was used, and the reaction was carried out at 37°C for 2.5 hours. Then, the cleavage solution was concentrated by nitrogen bubbling. Finally, ice-cold ether was added, the supernatant was removed by centrifugation, ice-cold ether was added again, the supernatant was removed, and the solution was air-dried to obtain a solid crude peptide.

[0025] The crude peptide was purified by high performance liquid chromatography (HPLC) with 10.0 mg of crude peptide, and then freeze-dried to obtain the target linear melitoxin thioester precursor.

[0026] In step S4, the sequence of the linear melitoxin thioester precursor is GiGavikvlttGlpaliswikrkrqqn(S)GL.

[0027] In step S4, the concentration of the linear melitoxin thioester precursor is 200 μM, and the concentration of the OaAEP1 enzyme is 0.9 μM.

[0028] In step S4, the phosphate buffer solution is 100mM disodium hydrogen phosphate, 150mM sodium chloride, and pH=7.

[0029] In step S4, the enzyme-catalyzed intramolecular cyclization reaction takes 2 to 6 hours and the reaction temperature is 20 to 25°C.

[0030] Compared with the prior art, the beneficial effects of the present invention are as follows: the natural antimicrobial peptide L-cMelittin is composed of L amino acids, which are easily hydrolyzed by proteases and have a short half-life; the present invention is the first to synthesize mirror-ring meliothin (D-cMelittin) using OaAEP1 enzyme with the assistance of thioesters. Tests show that D-cMelittin has extremely high resistance to protease hydrolysis and good antimicrobial activity. Attached Figure Description

[0031] Figure 1 This is a schematic diagram of the synthesis of mirror-shaped cyclic peptides using OaAEP1 enzyme with the assistance of thioesters. Note: G represents glycine, n represents D-asparagine, x represents 19 D-amino acids and glycine, and L represents L-leucine.

[0032] Figure 2 This is a schematic diagram of the synthesis of the compound thiol S1;

[0033] Figure 3 SDS-PAGE gel image of OaAEP1 enzyme;

[0034] Figure 4 The image shows the 1H NMR spectrum of the compound thiol S1.

[0035] Figure 5 The image shows the carbon NMR spectrum of the compound thiol S1.

[0036] Figure 6 The image shows the high-resolution mass spectrum of compound thiol S1. In the image, the green shading represents the molecular weight of the sodium salt adduct of thiol S1, and 284.12833 and 285.13116 are the molecular weights corresponding to the two isotopic peaks of this compound.

[0037] Figure 7 A schematic diagram of the cyclization of mirror-image linear melitoxin 1 catalyzed by the OaAEP1 enzyme;

[0038] Figure 8 The images show the high-performance liquid chromatogram and mass spectrum of linear melitoxin 1, with * indicating sodium salt addition products.

[0039] Figure 9 High-performance liquid chromatography (HPLC) chromatogram of linear melitoxin cyclization catalyzed by OaAEP1 enzyme;

[0040] Figure 10 High-performance liquid chromatography and mass spectra of D-cMelittin;

[0041] Figure 11 Here are high-phase liquid chromatograms of D-cMelittin before and after incubation with trypsin;

[0042] Figure 12 High-performance liquid chromatography (HPLC) chromatogram of the cyclized linear precursor of amide bond (D-cMelittin-nGL). Detailed Implementation

[0043] The above-mentioned and other technical features and advantages of the present invention will be described in more detail below with reference to the accompanying drawings.

[0044] The following is a list of the abbreviations used in the text and their corresponding information:

[0045] OaAEP1: OaAEP1b-C247A;

[0046] IPTG: Isopropyl-β-D-thiogalactoside;

[0047] DTT: Dithiothreitol;

[0048] EDTA: Ethylenediaminetetraacetic acid;

[0049] HATU: 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate;

[0050] DMF: N,N-dimethylformamide;

[0051] DIEA: N,N-diisopropylethylamine;

[0052] DCM: Dichloromethane;

[0053] PE: Petroleum ether;

[0054] EA: Ethyl acetate;

[0055] Fmoc: N-fluorenemethyloxycarbonyl;

[0056] Asp: Aspartic acid;

[0057] Boc2O: ditert-butyl dicarbonate;

[0058] Na2CO3: Sodium carbonate;

[0059] TFA: Trifluoroacetic acid;

[0060] HOBT: 1-Hydroxybenzotriazole;

[0061] Tips: Triisopropylsilane;

[0062] D-cMelittin: Mirror-shaped melioidin;

[0063] Example 1

[0064] Expression of OaAEP1b-C247A enzyme:

[0065] The target plasmid OaAEP1b-C247A (hereinafter referred to as OaAEP1) was transformed into *E. coli* Shuffle T7 competent cells and sputtered onto LB agar plates containing kanamycin for overnight growth. Single colonies were selected and inoculated into 5 mL of 2×YT medium (containing kanamycin) and cultured overnight at 37°C. 1 L of 2×YT medium was taken, 500 μL of kanamycin stock solution (100.0 mg / mL) was added, followed by 3 mL of the overnight cultured strain. The cells were incubated at 37°C for 3 hours until the OD600 reached approximately 0.8. Then, 400.0 μL of IPTG stock solution (0.4 mmol / L) was added, and the cells were incubated at 16°C for 16 hours. The strains were collected by centrifugation at 4300 rpm and resuspended in lysis buffer (25.0 mM HEPES, pH 7.7, 150.0 mM NaCl), and then sonicated on ice. The lysis solution was centrifuged at 9500 rpm for 30 minutes at 4°C to obtain a supernatant containing the target OaAEP1[C247A] enzyme. The supernatant was loaded into a nickel column and washed with wash buffer (25 mM HEPES, pH 7.7, 150 mM NaCl). Impurities were eluted first with a low-concentration elution buffer (25 mM HEPES, 150 mM NaCl, 30 mM imidazole, pH 7.7), followed by elution with a high-concentration elution buffer (25 mM HEPES, 150 mM NaCl, 250 mM imidazole, pH 7.7) to obtain the target protein with approximately 90% purity. The high-concentration eluent was collected and dialyzed against dialysis buffer (25 mM HEPES, 2 mM DTT, pH 7.7) for 4 hours, followed by activation by dialyzing against activation buffer (50 mM sodium acetate, 1 mM EDTA, pH 4.0) at room temperature for 4 hours. OaAEP1 was further purified using an AKTA Pure HiTrap SP HP cation exchange chromatography column, followed by ultrafiltration to replace the buffer with dialysis buffer (100 mM Na2HPO4, 150 mM NaCl, pH 7.4, 10% glycerol). Finally, the purified product was aliquoted and stored at -80°C, 40 μL per vial containing 40 μg of OaAEP1 enzyme, for use in enzyme-catalyzed cyclization reactions (SDS-PAGE gel image of activated OaAEP1 enzyme is shown below). Figure 3 ).

[0066] The amino acid sequence of the OaAEP1b-C247A enzyme is shown below:

[0067] VGTRWAVLIAGSKGYANYRHQAGVCHAYQILKRGGLKDENIVVFMYDDIAYNESNPRPGVIINSPHGSDVYAGVPKDYTGEEVNAKNFLAAILGNKSAITGGSGKVVDSGPNDHIFIYYTDHGAAGVIGMPSKPYLYADELNDALKK KHASGTYKSLVFYLEACESGSMFEGILPEDLNIYALTSTNTTESSWAYYCPAQENPPPPEYNVCLGDLFSVAWLEDSDVQNSWYETLNQQYHHVDKRISHASHATQYGNLKLGEEEGLFVYMGSNPANDNYTSLDGNALTPSSIVVNQ. (SEQ ID NO.1)

[0068] Example 2

[0069] Synthesis of (2-mercaptoacetyl)-L-leucine tert-butyl ester (thiol S1):

[0070] Dissolve 3.33 mmol of 2,2'-dithiodiacetic acid (607 mg, 1 eq), 6.66 mmol of HATU (2.54 g, 2 eq), and 6.66 mmol of HOAT (907 mg, 2 eq) in a small amount of N,N-dimethylformamide (DMF), and activate with 6 mL of DIEA (12 eq). Immediately afterwards, add 6.66 mmol of L-leucine tert-butyl ester (1.5 g, 2 eq) and react at room temperature for 2 hours. After the reaction is complete, perform thin-layer chromatography analysis using a PE:EA = 1:1 developing solvent. The product shows a weak UV color at 254 nm. Dilute the reaction solution with 4 times its volume of water, and extract the product with a small amount of DCM, extracting 2-3 times. Wash the organic phase four times with 1M HCl to remove DIEA and unreacted L-leucine tert-butyl ester, then wash the organic phase four times with Na2CO3 salt solution to remove unreacted 2,2'-dithiodiacetic acid. After drying with anhydrous sodium sulfate, the product was concentrated under vacuum to obtain 2,2'-(2,2'-dithioalkylbis(acetyl))bis(azadiyl)bis(4-methylvalerate ditert-butyl ester).

[0071] 1.9 g of 2,2'-(2,2'-dithioalkylbis(acetyl))bis(azadiyl)bis(4-methylvalerate ditert-butyl ester) (1 eq) and 0.844 mg of DTT (1.5 eq) were dissolved in DCM, and 600 μL of triethylamine was added. The reaction was allowed to proceed for 1 h. After the reaction was complete, thin-layer chromatography was performed using a PE:EA ratio of 1:1 as the developing solvent. The reaction solution was washed four times with 1M HCl, followed by four washes with Na₂CO₃ salt solution. The solution was dried over anhydrous sodium sulfate, concentrated under vacuum, and then loaded onto silica gel. The product was purified by rapid column chromatography (PE:EA = 10:1-3:1) to obtain purified thiol S1 (the 1H NMR / C NMR / high-resolution mass spectra of thiol S1 are shown in [link to chromatogram]). Figure 4-6 ).

[0072] Example 3

[0073] Synthesis of mirror-image linear melitin peptide 1:

[0074] 0.2 mmol of Rink amide resin (360.0 mg, 0.56 mmol / g) was added to a 5.0 mL solid-phase synthesis reactor, and 3.0 mL of DMF was added. The resin was allowed to swell at room temperature for 15 minutes. Using a diaphragm pump as the power source, the swollen product was dried to obtain the swollen resin. The Fmoc protecting group was removed with a DMF solution containing 20% ​​piperidine, and the Fmoc deprotection process was repeated once. After washing the resin with DMF, 2.0 mL of DMF solution (containing 4.5 equivalents of Fmoc-Asp-Oall, 4.5 equivalents of HATU, and 9 equivalents of DIEA) was added, and the mixture was reacted at 37 °C with shaking for 20 minutes.

[0075] After the reaction was complete, the resin was washed six times sequentially with DMF, DCM, and DMF, and dried to obtain Fmoc-Asp-Oall side chain carboxyl groups anchored to the resin. Following the amino acid sequence, the resin was subjected to amino acid condensation using the standard Fmoc solid-phase peptide synthesis method, with amino acids sequentially coupled from the C-terminus. The Fmoc protecting group was removed with a 20% piperidine DMF solution, and the deprotection process was repeated once. The mixture was then reacted for 10 minutes with 4 mL of a mixed solvent of Boc2O, DIEA, and DMF (volume ratio 1:1:8). One eq of tetrakis(triphenylphosphine) palladium was dissolved in 3 mL of DCM, and 10 eq of phenylsilane was added dropwise to the synthesis tube, reacting for 1.5 hours. Sodium diethyldithiocarbamate (trihydrate) was dissolved in DMF and added to the synthesis tube, reacting for 10 minutes, repeated 2-3 times. Dissolve 5 eq HOBT and 5 eq thiol S1 in 4 mL of a 1:1 mixture of DCM and DMF, then add 10 eq DIC and 10 eq DIEA, mix well, and add to a synthesis tube to react for 4 hours.

[0076] After the reaction was complete, the resin was washed six times with DCM. Using a diaphragm pump as the power source, the resin was dried. Freshly prepared cleavage reagent (95% TFA, 2.5% Tips, 2.5% water) was added to the resin, and the reaction was carried out at room temperature for 2.5 hours. The cleavage solution was then transferred to a centrifuge tube and concentrated using nitrogen bubbling. Finally, ice-cold ether was added, and after centrifugation to remove the supernatant, ice-cold ether was added again. After removing the supernatant, the solution was air-dried to obtain a solid crude peptide. 10.0 mg of the crude peptide was purified by high-performance liquid chromatography and freeze-dried to obtain the target image-image linear meliosteum peptide 1 (…). Figure 8 (4mg, sequence is GiGavikvlttGlpaliswikrkrqqn(S)GL).

[0077] Example 4

[0078] OaAEP1 enzyme-mediated cyclization of mirror-image linear melitoxin 1:

[0079] Prepare a buffer solution (100 mM Na₂HPO₄, pH=7, 150 mM NaCl). Take 5.0 mL of the buffer solution, add 333.3 μL of LoaAEP1 (30 μM, final concentration 1 μM), add 200 μL of mirror-image linear melittin stock solution (10 mM, final concentration 200 μM), and then add 4.5 mL of pure water. After mixing the reaction solution, incubate at room temperature for 4-5 hours. Note that the mirror-image peptide is pre-dissolved in phosphate buffer. After the reaction is complete, add 5.0 mL of guanidine hydrochloride solution (6 M, pH=3) to terminate the reaction. Purify by high performance liquid chromatography, freeze-dry to obtain D-cMelittin. Figure 9-10 (1.1 mg, isolated yield 18%).

[0080] Example 5

[0081] D-cMelittin anti-trypsin enzymatic hydrolysis experiment:

[0082] D-cMelittin (final concentration 100 μM) was dissolved in buffer (100 mM Na2HPO4, pH=7.8, 150 mM NaCl, 10 mM CaCl2) and incubated with trypsin at a final concentration of 100 nM at 37 °C for 2 hours. The reaction was then terminated by adding 1% of the reaction solution volume of trifluoroacetic acid. High-performance liquid chromatography (HPLC) analysis showed that D-cMelittin was completely resistant to trypsin digestion. Figure 11 ).

[0083] Example 6

[0084] D-cMelittin antibacterial activity test:

[0085] Two bacterial strains, one Gram-positive bacterium (Staphylococcus aureus) and one Gram-negative bacterium (Escherichia coli), were cultured on LB medium at 37°C for 10 weeks. 8 CFU. Add 100 μL of cultured Staphylococcus aureus to wells A1-A12, B1-B12, C1-C12, and D1-D12 of a 96-well plate, and add 100 μL of cultured Escherichia coli to wells E1-E12, F1-F12, G1-G12, and H1-H12. Prepare a 1 mM MD-cMelittin stock solution using distilled water. Add 100 μL of the D-cMelittin stock solution to wells B1-G1 and mix well. Add 100 μL of distilled water to wells H and H1 of A1 and mix well. Then, aspirate 100 μL from wells A1-H1 and add it to wells A2-H2 and mix well. Then, aspirate 100 μL from wells A2-H2 and add it to wells A3-H3 and mix well. Continue this process until wells A11-H11. The 96-well plates were incubated at 37°C for 16-20 hours. The minimum inhibitory concentration (MIC) of D-cMelittin was determined using the standard two-fold dilution method. The results showed that the MIC of D-cMelittin against Staphylococcus aureus was 3.1 μM and the MIC against Escherichia coli was 6.3 μM.

[0086] Example 7

[0087] Cyclolation control of the linear amide precursor (D-cMelittin-nGL):

[0088] The amide-linked linear precursor GiGavikvlttGlpaliswikrkrqqnGL (D-cMelittin-nGL) was purchased from Genscript Biotech Inc. D-cMelittin-nGL was dissolved in a buffer solution containing 1 μM OaAEP1 (100 mM Na2HPO4, pH=7, 150 mM NaCl), resulting in a final concentration of 200 μM. The reaction solution was incubated at room temperature for 4–5 hours. Note that the mirror peptide was pre-dissolved in phosphate buffer. After the reaction was complete, 5.0 mL of guanidine hydrochloride solution (6 M, pH=3) was added to terminate the reaction, and the analysis was performed using high-performance liquid chromatography (HPLC). The results showed that the amide-linked linear precursor D-cMelittin-nGL could not cyclize under the same reaction conditions. Figure 12 ).

[0089] The above description is merely a preferred embodiment of the present invention and is illustrative rather than restrictive. Those skilled in the art will understand that many changes, modifications, and even equivalents can be made within the spirit and scope defined by the claims of the present invention, all of which will fall within the protection scope of the present invention.

Claims

1. A method for synthesizing a mirror image cyclic peptide using OaAEP1 enzyme with the aid of a thioester, characterized in that, Comprise the following steps: S1, expressing OaAEP1 in E. coli Shuffle T7 competent cells and purifying by nickel column, activating by dialysis in activation buffer at room temperature, and further purifying by AKTA Pure; S2, coupling 2,2'-dithiodiglycolic acid with L-leucine tert-butyl ester, extracting after the reaction is completed, washing the organic phase with HCl and Na2CO3 salt solution respectively, adding dithiothreitol and triethylamine to the organic phase, reacting for 1 hour, washing and drying the reaction solution after the reaction is completed, vacuum concentrating, loading the product onto silica gel, and obtaining (2-mercaptoacetyl)-L-leucine tert-butyl ester after purification; S3, according to the linear amino acid sequence of D-Melittin, first anchoring the side chain carboxyl of Fmoc-Asp-Oall to the resin, then coupling amino acids from the C-terminal in turn, removing the last amino acid Fmoc protection group, protecting the terminal amino group with Boc2O, removing the allyl protection group at the end of the peptide chain, coupling with (2-mercaptoacetyl)-L-leucine tert-butyl ester, cutting the thioester precursor from the resin with trifluoroacetic acid as the cutting reagent, and obtaining the linear melittin thioester precursor after separation and purification; S4, using the linear melittin thioester precursor obtained in step S3 to generate an intramolecular cyclization reaction catalyzed by OaAEP1 enzyme in a phosphate buffer solution to produce a cyclic peptide molecule; In step S4, the amino acid sequence of the OaAEP1 enzyme is shown in SEQ ID NO. 1, and the sequence of the linear melittin thioester precursor is GiGavikvlttGlpaliswikrkrqqn(S)GL.

2. The method for synthesizing a mirror image cyclic peptide with the aid of OaAEP1 enzyme in the presence of a thioester according to claim 1, characterized in that, In step S1, the E. coli cells are grown in YT medium at 37℃, and are collected by centrifugation at 4300 rpm after being induced with 0.4 mM IPTG for 16 hours at 16℃, and are resuspended in lysis buffer, and are then lysed by ultrasonic wave in an ice bath.

3. The method for synthesizing a mirror image cyclic peptide using OaAEP1 enzyme with the assistance of a thioester according to claim 1, wherein, In step S1, the specific steps of nickel column purification are as follows: S11, centrifuging the lysis solution at 9500 rpm for 30 minutes at 4℃, and loading the supernatant into a nickel column; S12, washing with a washing buffer, wherein the washing buffer is 25 mM HEPES, pH=7.7, 150 mM NaCl; S13, eluting impurities with a low-concentration elution buffer, wherein the low-concentration elution buffer is 25 mM HEPES, pH=7.7, 150 mM NaCl, 30 mM imidazole; S14, eluting the target protein with a high-concentration elution buffer, wherein the high-concentration elution buffer is 25 mM HEPES, pH=7.7, 150 mM NaCl, 250 mM imidazole.

4. The method for synthesizing a mirror image cyclic peptide with the aid of OaAEP1 enzyme in the presence of a thioester according to claim 1, characterized in that, In step S1, the activation buffer is 50 mM sodium acetate, 1 mM EDTA, pH=4.0, and the activation is performed by dialysis at room temperature for 4 hours.

5. The method for synthesizing a mirror image cyclic peptide using OaAEP1 enzyme with the aid of a thioester according to claim 1, characterized in that, In the step S3, the side chain carboxyl group anchors the resin by condensing Fmoc-Asp-Oall to the amino resin in a standard Fmoc solid-phase polypeptide synthesis method, and then washing with DMF, DCM and DMF in sequence, and drying after sufficient washing.

6. The method for synthesizing mirror-shaped cyclic peptides using OaAEP1 enzyme with thioester assistance as described in claim 1, characterized in that, In the step S3, the amino acid coupling conditions are: 37°C for 20 min in a system with a molar ratio of HATU:amino acid:DIEA=5:5:10; The Fmoc protecting group removal conditions are: 20% piperidine at 37°C for 10 min or 75°C for 5 min; The allyl protecting group removal conditions are: dissolving tetraphenylphosphonium palladium in DCM, adding phenylsilane, and then adding the reaction solution to a synthesis tube for 37°C reaction for 1.5 h; The coupling conditions of (2-mercaptoacetyl)-L-leucine tert-butyl ester are: dissolving 1-hydroxybenzotriazole (HOBT):(2-mercaptoacetyl)-L-leucine tert-butyl ester with a molar ratio of 4:4 in a mixed solvent of DCM and DMF with equal volume, then adding 5 eq DIC and 5 eq DIEA, mixing, adding to a synthesis tube, and reacting at 37°C for 4 h; The TFA cleavage reagent cuts the thioester precursor from the resin under the following conditions: using a cleavage solution with a volume ratio of TFA:water:Tips=95:2.5:2.5, 37°C for 2.5 h, then concentrating the cleavage solution by nitrogen bubbling, finally adding ice ethanol, centrifuging to remove the supernatant, adding ice ethanol again, removing the supernatant, and air-drying to obtain a solid crude peptide; The purification is high-performance liquid chromatography preparation of 10.0 mg of the crude peptide, and the target linear melittin thioester precursor is obtained after freeze-drying.

7. The method for synthesizing mirror-shaped cyclic peptides using OaAEP1 enzyme with thioester assistance as described in claim 1, characterized in that, In the step S4, the concentration of the linear melittin thioester precursor is 200 μM, and the concentration of the OaAEP1 enzyme is 0.9 μM.

8. The method for synthesizing mirror-shaped cyclic peptides using OaAEP1 enzyme with thioester assistance as described in claim 1, characterized in that, In the step S4, the phosphate buffer solution is 100 mM disodium hydrogen phosphate, 150 mM sodium chloride, and pH=7.

9. The method for synthesizing mirror-shaped cyclic peptides using OaAEP1 enzyme with thioester assistance as described in claim 1, characterized in that, In the step S4, the time for the enzyme-catalyzed intramolecular cyclization reaction is 2-6 h, and the reaction temperature is 20-25°C.

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