A method for preparing Isaridin E cyclic ester peptide

Abstract

This invention belongs to the field of peptide synthesis technology, specifically relating to a method for preparing Isaridin E cyclic ester peptides. This invention successfully prepares Isaridin E cyclic ester peptides through ester bond closure, solid-phase peptide synthesis, and ring-closure reaction. Specifically, compound A is linked to Fmoc-β-alanine, and then reduced with hydrogen gas to close the ester bond precursor, providing a reaction basis for subsequent solid-phase peptide synthesis. Overall, this invention is the first to successfully prepare Isaridin E cyclic ester peptides using a chemical synthesis method. Compared to traditional methods of isolation, purification, and extraction from marine fungi, this method not only achieves controllable and stable yield but also provides sufficient material support for the widespread application of Isaridin E cyclic ester peptides.

Description

Technical Field

[0001] This invention belongs to the field of polypeptide synthesis technology. More specifically, it relates to a method for preparing Isaridin E cyclic ester peptide. Background Technology

[0002] Isaridin E is a cyclic ester peptide isolated from the marine fungus *Amphichorda felina* SYSU-MS7908. Studies have shown that this compound exhibits significant ameliorative effects in both arterial thrombosis and venous thrombosis models. It significantly inhibits platelet activation and aggregation, thereby suppressing platelet-mediated inflammatory responses and effectively preventing thrombosis. Compared to traditional antithrombotic drugs, Isaridin E cyclic ester peptide not only has similar antithrombotic effects but also significantly reduces bleeding side effects, resulting in higher safety. Therefore, Isaridin E cyclic ester peptide has broad application prospects in antithrombotic drug development and may be applied in other pharmaceutical and biotechnology fields. However, currently, the acquisition of Isaridin E cyclic ester peptide is limited to direct extraction from the ocean, a process that faces significant challenges such as low extraction efficiency, difficult separation and purification, and high costs. Summary of the Invention

[0003] The technical problem to be solved by the present invention is to overcome the defects and shortcomings of Isaridin E cyclic ester peptide, such as low extraction efficiency, high difficulty in separation and purification, and high cost, and to provide a method for preparing Isaridin E cyclic ester peptide.

[0004] The above-mentioned objective of this invention is achieved through the following technical solution:

[0005] This invention protects a method for preparing Isaridin E cyclic ester peptide, characterized by the following synthetic route:

[0006]

[0007] Specifically, the following steps are included:

[0008] S1. Compound A was reacted with Fmoc-β-alanine under the conditions of a first solvent, a first condensing agent, and an acylation catalyst to obtain compound B;

[0009] S2. The compound B obtained in step S1 is reduced to obtain compound C;

[0010] S3. Compound C obtained in step S2 was synthesized into a linear peptide NH-Pro-Phe-N(Me)Val-N(Me)Val-βAla-D-Oleu-OH by solid-phase peptide synthesis.

[0011] S4. The linear peptide obtained in step S3 is subjected to a ring-closing reaction to obtain Isaridin E cyclic ester peptide.

[0012] This invention innovatively proposes a method for preparing Isaridin E cyclic ester peptides. The method sequentially includes the closure of the ester bond precursor (steps S1-S2), the preparation of a linear peptide synthesized in a solid phase, and a ring-closing reaction step. Specifically, the closure of the ester bond precursor involves compound A reacting with Fmoc-β-alanine, followed by debenzylation using hydrogen gas, thus achieving the closure of the ester bond precursor. Overall, this invention is the first to successfully prepare Isaridin E cyclic ester peptides using a chemical synthesis method. Compared to traditional methods of isolation, purification, and extraction from marine fungi, this method not only achieves controllable and stable yield but also provides sufficient material support for the widespread application of Isaridin E cyclic ester peptides.

[0013] Further, in step S1, the preparation method of compound A includes the following steps:

[0014] (S)-2-hydroxy-4-methylvaleric acid was reacted with benzyl alcohol under the conditions of a first solvent and an esterification catalyst, and then post-treated to obtain compound A.

[0015] Furthermore, the first solvent includes one or more of toluene, dichloromethane, diethyl ether, tetrahydrofuran, and methanol.

[0016] Furthermore, the esterification catalyst includes one or more of p-toluenesulfonic acid, o-toluenesulfonic acid, and m-toluenesulfonic acid.

[0017] Further, the molar ratio of (S)-2-hydroxy-4-methylvaleric acid, esterification catalyst, and benzyl alcohol is 1:(0.1-0.2):(1.1-1.5).

[0018] Furthermore, the time for the complete reaction is 6 to 8 hours.

[0019] Furthermore, the post-processing includes chromatographic purification and concentration.

[0020] Furthermore, the chromatographic purification is performed by eluting with an eluent of ethyl acetate and petroleum ether at a volume ratio of 1:(8-9), and the eluent is collected.

[0021] Furthermore, the concentration is a vacuum concentration.

[0022] Specifically, the post-processing includes eluting with an eluent of ethyl acetate and petroleum ether in a volume ratio of 1:(8-9), collecting the eluent, and concentrating it under reduced pressure.

[0023] Furthermore, in step S1, compound A can be synthesized via the above-described route or obtained through commercial purchase.

[0024] Further, in step S1, the first condensing agent is one or more of N,N-diisopropylcarbodiimide (DIC), dicyclohexylcarbodiimide (DCC), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC).

[0025] Further, in step S1, the acylation catalyst includes one or more of 4-dimethylaminopyridine (DMAP), 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), and 1,5-diazabicyclo[4.3.0]non-5-ene (DBN).

[0026] Further, in step S1, the molar ratio of compound A, Fmoc-β-alanine, the first condensing agent, and the acylation catalyst is 1:(0.8-1.5):(0.8-1.5):(0.1-0.2).

[0027] Furthermore, in step S1, the reaction time is 6 to 8 hours.

[0028] Furthermore, in step S1, the reaction also includes post-processing.

[0029] Furthermore, the post-processing includes chromatographic purification and concentration.

[0030] Preferably, the chromatographic purification is performed by eluting with an eluent of ethyl acetate and petroleum ether in a volume ratio of 1:(3-4), and the eluent is collected.

[0031] Preferably, the concentration is a vacuum concentration.

[0032] Specifically, the post-processing includes eluting with an eluent of ethyl acetate and petroleum ether in a volume ratio of 1:(3-4), collecting the eluent, and concentrating it under reduced pressure.

[0033] Furthermore, in step S2, the reduction reaction is carried out under the conditions of a first solvent and a hydrogenation catalyst.

[0034] Furthermore, the hydrogenation catalyst is palladium on carbon.

[0035] Preferably, the palladium on carbon has a mass volume of 5% to 10%.

[0036] More preferably, the palladium on carbon has a mass volume of 8% to 10%.

[0037] Furthermore, in step S2, the reduction reaction takes 18 to 24 hours.

[0038] Furthermore, in step S2, the reduction reaction also includes post-processing.

[0039] Furthermore, the post-processing includes chromatographic purification and concentration.

[0040] Preferably, the chromatographic purification involves first using an eluent of ethyl acetate and petroleum ether at a volume ratio of 1:(2-3) to remove unreacted compound B and excess condensing agent, followed by elution with pure ethyl acetate, and then collecting the eluent.

[0041] Preferably, the concentration is a vacuum concentration.

[0042] Specifically, the post-processing includes first using an eluent of ethyl acetate and petroleum ether in a volume ratio of 1:(2-3) to remove unreacted compound B and excess condensing agent, then using pure ethyl acetate as the eluent for elution, collecting the eluent, and concentrating it under reduced pressure.

[0043] Furthermore, in step S3, the polypeptide solid-phase synthesis method specifically includes the following steps:

[0044] Si. Compound C was reacted with 2-chlorotriphenylmethyl chloride resin under the conditions of a second solvent and a second condensing agent to obtain an amino acid-resin complex;

[0045] Sii. The amino acid-resin complex obtained in step Si is subjected to an amino acid de-Fmoc group reaction under the conditions of a second solvent and a protecting group removal agent to obtain an amino acid-resin complex with de-Fmoc groups.

[0046] Siii. Repeat steps Si to Sii above, and sequentially react the amino acid-resin complex obtained in step Sii with Fmoc-N-methyl-L-Val, Fmoc-N-methyl-L-Val, Fmoc-L-Phe-OH, and Fmoc-L-Pro-OH to couple amino acids and remove Fmoc groups, thereby gradually extending the polypeptide chain and obtaining a polypeptide chain resin with the target amino acid sequence.

[0047] Siv. The polypeptide chain resin with the target amino acid sequence obtained in step Siii is cleaved to obtain crude linear peptide NH-Pro-Phe-N(Me)Val-N(Me)Val-βAla-D-Oleu-OH with the target amino acid sequence.

[0048] Further, in step Si, the second solvent includes one or more of N,N-dimethylformamide (DMF), tetrahydrofuran, and methanol.

[0049] Further, in step Si, the second condensing agent includes one or more of 2-(7-azabenzotriazole)-N,N,N,N-tetramethylurea hexafluorophosphate (HATU), (2-oxime-cyanoethyl acetate)-N,N-dimethyl-morpholinourea hexafluorophosphate (COMU), and 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethylurea hexafluorophosphate (HBTU).

[0050] Furthermore, in step Si, the 2-chlorotriphenylmethyl chloride resin is a fully swollen 2-chlorotriphenylmethyl chloride resin.

[0051] Furthermore, the swelling solvent is a second solvent.

[0052] Further, in step Si, the molar ratio of compound C, the second condensing agent, and 2-chlorotriphenylmethyl chloro resin is 1:(6-10):(4-8).

[0053] Further, in step Si, the molar volume ratio of compound C to the second solvent is 1:(15-30) mmol / mL.

[0054] Furthermore, in step Si, the reaction also includes post-processing.

[0055] Furthermore, the post-processing includes washing, filtration, sealing, filtration, and washing.

[0056] Preferably, the washing involves adding a second solvent to wash the reacted material 4 to 6 times.

[0057] Preferably, the filtration is performed by filtration of the washed product and collection of the filter residue.

[0058] Preferably, the end cap is obtained by using a sealing liquid to seal unreacted resin sites.

[0059] More preferably, the sealing fluid is a mixed solution of methanol, N,N-diisopropylethylamine (DIEA), and DMF.

[0060] Preferably, the volume ratio of methanol, DIEA, and DMF is (1-2):(2-3):(15-17).

[0061] Preferably, the filtration is performed after the head reaction is completed, and the filter residue is collected.

[0062] Preferably, the washing involves washing the filter residue with a second solvent 4 to 6 times.

[0063] Specifically, the post-treatment includes washing the reaction product 4 to 6 times with a second solvent, filtering the washed product and collecting the filter residue, using a blocking solution with a volume ratio of methanol, DIEA, and DMF of (1 to 2): (2 to 3): (15 to 17) to seal the unreacted resin sites in the filter residue, filtering, collecting the filter residue, and washing the filter residue 4 to 6 times with the second solvent.

[0064] Furthermore, in step Si, the success of the reaction is determined by a ninhydrin colorimetric reaction.

[0065] Furthermore, as a preferred embodiment, the ninhydrin colorimetric reaction includes the following steps:

[0066] Dip a clean, thin iron wire into the sintered metal tube and place a small amount of resin into the test tube. Add Kaiser's reagent (6% ninhydrin ethanol solution: 80% phenol ethanol solution: pyridine = 2:1:1, volume ratio). Heat the test tube at 120°C and observe the color after the reaction. If the resin in the test tube shows no color change, the condensation reaction between compound C and the resin is successful. If blue resin is visible in the test tube, the reaction is incomplete. Weigh out more compound C and add it back into the sintered metal tube to continue the reaction until the resin shows no color change.

[0067] Furthermore, in step Sii, the protecting group removal agent includes one or more of piperidine, diethylamine, and morpholine.

[0068] Preferably, the protecting group remover is piperidine.

[0069] More preferably, the volume fraction of the piperidine is 15% to 25%.

[0070] Furthermore, in step Sii, the reaction time is 10–20 min.

[0071] Furthermore, in step Sii, the reaction needs to be repeated 2 to 3 times.

[0072] Furthermore, in step Sii, the reaction also includes post-processing.

[0073] Furthermore, the post-processing is washing.

[0074] Preferably, the washing involves washing the reaction product with a second solvent for 5–10 seconds, repeated 4–6 times.

[0075] Furthermore, in step Sii, the success of the Fmoc group removal reaction is determined by a tetrachlorobenzoquinone colorimetric reaction.

[0076] Furthermore, as a preferred embodiment, the tetrachlorobenzoquinone colorimetric reaction includes the following steps:

[0077] Dip a clean, thin iron wire into the sapphire crystal tube and place a small amount of resin into the test tube. Add a DMF (1:49) solution containing acetaldehyde and a tetrachlorobenzoquinone solution (0.02 g / mL) to the test tube and react at room temperature for 5 minutes. After the reaction is complete, if the resin in the test tube shows no color change, the Fmoc removal reaction is successful. If the resin in the test tube turns blue, the Fmoc removal reaction is incomplete, and the required amount of protecting group remover needs to be weighed again and added to the sapphire crystal tube for further reaction until the resin shows no color.

[0078] Further, in step Siii, the amino acid-resin complex de-Fmoc-group described in step Si serves as the resin in step Si, acting as the 2-chlorotriphenylmethyl chloride resin. Fmoc-N-methyl-L-Val serves as the amino acid in step Si, acting as compound C. Under the conditions of the second condensing agent and the second solvent in step Si, an amino acid-resin coupling reaction is performed to obtain an amino acid-resin complex with extended peptide chains. After the reaction, a ninhydrin colorimetric reaction is used to ensure complete reaction. Then, the obtained amino acid-resin complex with extended peptide chains is subjected to a de-Fmoc-group reaction under the conditions of the second solvent and protecting group removal agent in step Sii, resulting in an amino acid-resin complex with extended peptide chains and de-Fmoc-group. After the reaction, a tetrachlorobenzoquinone colorimetric reaction is used to ensure complete reaction. Fmoc-N-methyl-L-Val, Fmoc-L-Phe-OH, and Fmoc-L-Pro-OH are reacted sequentially to finally obtain a polypeptide chain resin with the target amino acid sequence.

[0079] Further, in step Siv, the acidic lysis solution includes one or more of an aqueous solution of trifluoroacetic acid and a dichloromethane solution of trifluoroacetic acid.

[0080] Preferably, in step Siv, the acidic lysis solution is an aqueous solution of trifluoroacetic acid.

[0081] More preferably, the trifluoroacetic acid solution is an aqueous solution containing 90% to 95% trifluoroacetic acid.

[0082] Furthermore, in step Siv, the solvent for pH adjustment is DIEA and / or DBU.

[0083] Further, in step 4, the ring-closing reaction is to perform a ring-closing reaction on the linear peptide obtained in step S3 under a second solvent, a second condensing agent, and alkaline conditions, followed by post-treatment to obtain Isaridin E cyclic ester peptide.

[0084] Furthermore, in step 4, the alkaline conditions include one or more of DIEA, DBN, and DBU.

[0085] Furthermore, in step 4, the molar ratio of the second condensing agent to the linear peptide is (2.5-3):1.

[0086] Furthermore, in step 4, the time for the complete reaction is 12 to 16 hours.

[0087] Furthermore, in step 4, the closed-loop reaction also includes post-processing.

[0088] Furthermore, the post-processing includes concentration, dissolution, filtration, separation, and purification.

[0089] Furthermore, the concentration involves evaporating the reaction solution under reduced pressure to obtain a concentrated, pale yellow oily substance.

[0090] Furthermore, the dissolution involves dissolving a concentrated, pale yellow oily substance using acetonitrile and water.

[0091] Furthermore, the filtration involves filtering the dissolved solution and collecting the filtrate.

[0092] Furthermore, the separation and purification are performed using high-performance liquid chromatography.

[0093] Specifically, the post-treatment involves evaporating the reaction solution under reduced pressure to obtain a concentrated pale yellow oily substance, dissolving the concentrated pale yellow oily substance in acetonitrile and water, filtering, collecting the filtrate, and purifying the filter residue using high performance liquid chromatography.

[0094] Compared with the prior art, the present invention has the following beneficial effects:

[0095] This invention successfully prepared Isaridin E cyclic ester peptides via ester bond closure, solid-phase peptide synthesis, and ring-closure reaction. Specifically, compound A was linked to Fmoc-β-alanine, followed by hydrogen reduction to close the ester bond, providing a reaction basis for subsequent solid-phase peptide synthesis. Overall, this invention represents the first successful chemical synthesis of Isaridin E cyclic ester peptides. Compared to traditional methods of isolation, purification, and extraction from marine fungi, this method not only achieves controllable and stable yields but also provides sufficient material support for the widespread application of Isaridin E cyclic ester peptides. Attached Figure Description

[0096] Figure 1 The image shows the carbon NMR spectrum of compound B prepared in Example 1.

[0097] Figure 2 The image shows the carbon NMR spectrum of compound C prepared in Example 1.

[0098] Figure 3 The mass spectrum is that of the linear peptide NH-Pro-Phe-N(Me)Val-N(Me)Val-βAla-D-Oleu-OH prepared in Example 1.

[0099] Figure 4 The image shows the carbon NMR spectrum of the linear peptide NH-Pro-Phe-N(Me)Val-N(Me)Val-βAla-D-Oleu-OH prepared in Example 1.

[0100] Figure 5 This is the mass spectrum of the Isaridin E cyclic ester peptide prepared in Example 1.

[0101] Figure 6 The image shows the carbon NMR spectrum of the Isaridin E cyclic ester peptide prepared in Example 1. Detailed Implementation

[0102] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0103] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0104] Example 1: A method for preparing Isardin-E cyclic ester peptide

[0105] (1) Preparation of compound A

[0106] 5 g of (S)-2-hydroxy-4-methylpentanoic acid was dissolved in 50 mL of dichloromethane. 0.8 g of p-toluenesulfonic acid and 5 mL of benzyl alcohol were added at room temperature. The mixture was reacted overnight at room temperature. The reaction solution was purified by silica gel chromatography, eluting with ethyl acetate and petroleum ether in a 1:9 volume ratio. After concentration under reduced pressure, 6.5 g of compound A was obtained, with a yield of 45.8%.

[0107] (2) Preparation of compound B

[0108] 8 g of Fmoc-β-alanine was dissolved in 300 mL of dichloromethane (DCM), and 3.4 mL of N,N-dicyclohexylcarbodiimide (DCC) was added at room temperature. After stirring for 0.5 h, 6 g of compound A and 0.4 g of 4-dimethylaminopyridine were added, and the reaction was carried out at room temperature for 16 h. The reaction solution was purified by silica gel chromatography, eluted with ethyl acetate and petroleum ether in a volume ratio of 1:4, and concentrated under reduced pressure to obtain 4.3 g of compound B, with a yield of 68.5%. The carbon NMR data of compound B are as follows: Figure 1 ):13 C NMR (100MHz, CDCl3) δ171.98,170.89,156.49,144.09,141.40,135.25,128.75,128.70,128.62,128.35, 127.76,127.13,125.27,120.05,71.39,67.31,66.92,47.33,39.65,36.96,34.77,24.76,23.06,21.67.

[0109] (3) Preparation of compound C

[0110] 4.3 g of compound B was dissolved in DCM, 0.43 g of palladium on carbon was added, hydrogen gas was introduced, and the mixture was stirred at atmospheric pressure for 24 h. The reaction solution was purified by silica gel chromatography. First, unreacted compound B was removed by elution with ethyl acetate and petroleum ether in a 1:4 volume ratio. Then, pure ethyl acetate was used for further elution. The ethyl acetate eluent was collected, concentrated under reduced pressure, and 3.2 g of compound C was obtained, with a yield of 90.5%. The carbon NMR data of compound C are as follows: Figure 2 ): 13 C NMR (100MHz, CDCl3) δ175.20,172.09,141.36,129.12,128.31,127.77,127.15, 120.04,71.13,67.12,53.52,47.21,39.61,36.86,34.58,24.78,23.04,21.57.

[0111] (4) Peptide solid-phase synthesis steps

[0112] (A) Coupling reaction of resin and amino acids

[0113] Weigh 5g of 2-chlorotriphenylmethyl chloride resin and add it to a sintered sand tube for peptide synthesis. Add DCM to soak the resin and purge for 10 minutes to allow it to fully swell, then filter. Weigh 1g of compound C and add it to the sintered sand tube. Add 60mL of dimethylformamide (DMF) and 3mL of DIEA. Purge at room temperature for 2 hours, then remove the liquid. Wash with DMF four times and filter. Add 40mL of DMF again, add 4mL of anhydrous methanol and 4mL of DIEA, and purge for 30 minutes. Cap any unreacted sites on the resin. After the reaction is complete, filter and wash with DMF four times to obtain the amino acid-resin complex.

[0114] Ninhydrin colorimetric reaction detection: Using a clean, fine iron wire, dip the obtained amino acid-resin complex from the sintered metal tube and place it into the detection tube. Add Kaiser's reagent (6% ninhydrin ethanol solution: 80% phenol ethanol solution: pyridine = 2:1:1, volume ratio). Place the detection tube in a 120℃ thermostat and heat for 1 minute, then observe the color. If the resin in the detection tube shows no color change, the condensation reaction is successful, and the removal of the Fmoc group can proceed. If the resin in the detection tube turns blue, the reaction is incomplete, and compound C required for the reaction needs to be weighed again and added to the sintered metal tube until the resin shows no color change.

[0115] (B) Linear chain peptide synthesis

[0116] A piperidine solution (piperidine:DMF = 1:4, volume ratio) was added to a sand core containing the amino acid-resin complex obtained in step (A). After 15 min of blowing reaction, the mixture was filtered to carry out the de-Fmoc group removal reaction of amino acids. After the reaction was completed, DMF was added for washing for 5-10 s, and this was repeated 5 times to obtain the amino acid-resin complex with the de-Fmoc protecting group.

[0117] Tetrachlorobenzoquinone colorimetric reaction detection: Dip a clean, thin iron wire into the resin from the sapphire crystal tube and place it into the detection tube. Add a DMF (1:49) solution containing acetaldehyde and a tetrachlorobenzoquinone solution (0.02 g / mL) to the detection tube and react at room temperature for 5 minutes. After the reaction, if the resin in the detection tube does not change color, the Fmoc group removal reaction is successful. If the resin in the detection tube turns blue, the Fmoc group removal reaction is incomplete, and the required amount of protecting group remover needs to be weighed again and added to the sapphire crystal tube for further reaction until the resin test shows no color.

[0118] Repeat steps (A) to (B) above, and sequentially react 3g Fmoc-N-methyl-L-Val, 3g Fmoc-N-methyl-L-Val, 3.4g Fmoc-L-Phe-OH, and 3.2g Fmoc-L-Pro-OH with the amino acid-resin complex after the Fmoc protecting group has been removed to perform amino acid coupling and Fmoc protecting group removal reactions, and use colorimetric reactions (ninhydrin or tetrachlorobenzoquinone colorimetric reactions) to detect after each reaction.

[0119] Specifically, the Fmoc-de-Fmoc-group-free amino acid-resin complex serves as the resin in step (A) as 2-chlorotriphenylmethyl chloride resin, and Fmoc-N-methyl-L-Val serves as the amino acid in step (A) as compound C. Under the conditions of the second condensing agent and the second solvent in step (A), an amino acid-resin coupling reaction is carried out to obtain an amino acid-resin complex with extended peptide chains. After the reaction, the ninhydrin colorimetric reaction is used to detect whether the reaction has been completed. If there is no color change in the resin in the detection tube, it proves that the reaction has been completed; if the resin in the detection tube is blue, it proves that the reaction has not been completed, and the required amount of Fmoc-N-methyl-L-Val needs to be weighed again and added to the sappan core tube for reaction until there is no color change in the resin detection. Then, the obtained amino acid-resin complex with extended peptide chains is subjected to a de-Fmoc-group-free reaction under the conditions of the second solvent and the protecting group removal agent in step (B) to obtain an amino acid-resin complex with extended peptide chains and de-Fmoc-group-free. After the reaction, the tetrachlorobenzoquinone colorimetric reaction was used to detect whether the deprotection reaction of the Fmoc group was complete. If there was no color change in the resin in the detection tube, the reaction was complete; if the resin in the detection tube turned blue, the reaction was incomplete, and the required deprotecting agent needed to be weighed again and added to the sand core tube for reaction until the resin showed no color. Fmoc-N-methyl-L-Val, Fmoc-L-Phe-OH, and Fmoc-L-Pro-OH were reacted in sequence to ensure successful amino acid coupling and complete deprotection of the Fmoc group, resulting in an extended polypeptide chain. In the final step of deprotection of the Fmoc group, the resin was washed 5 times with DMF and 5 times with DCM, and then the resin was dried under vacuum until it was in dry granular form, yielding a polypeptide chain resin with the target amino acid sequence.

[0120] (C) Acid pyrolysis

[0121] The lysis buffer was a 95% trifluoroacetic acid aqueous solution, and the usage of the lysis buffer was calculated at 10 mL of lysis buffer per 1 g of resin. The polypeptide chain resin with the target amino acid sequence obtained in step (B) was transferred to a flask, 50 mL of lysis buffer was added, and the reaction was allowed to proceed for 1.5 h. The lysis buffer was collected in a new flask, concentrated under reduced pressure, and then slowly adjusted to neutral pH by adding DIEA dropwise. This yielded the crude solution of the linear peptide NH-Pro-Phe-N(Me)Val-N(Me)Val-βAla-D-Oleu-OH with the target amino acid sequence.

[0122] (5) Synthesis of Isaridin E cyclic ester peptide

[0123] Take another clean round-bottom flask and add 300 mL of THF, 600 mg of COMU, and 3 mL of DIEA as the reaction system for cyclic peptide synthesis. Fix the round-bottom flask on a magnetic stirrer and add a ceramic rotor at 800 rpm. Pour the crude solution of the linear peptide NH-Pro-Phe-N(Me)Val-N(Me)Val-βAla-D-Oleu-OH with the amino acid sequence obtained in part (C) of step (4) above into a dropping funnel. Control the dropping rate and add the crude peptide solution drop by drop into the reaction system. Control the reaction time to 4 h and let it react overnight at room temperature.

[0124] The reacted liquid was poured into a round-bottom flask and the solvent was removed by rotary evaporation under reduced pressure at 50°C, yielding a concentrated pale yellow oil. After dissolving in acetonitrile and pure water, the solution was filtered and purified by high-performance liquid chromatography (HPLC). The target product was collected and freeze-dried to obtain Isaridin E cyclic ester peptide. In the preparation of the final product Isaridin E cyclic ester peptide from (S)-2-hydroxy-4-methylvaleric acid, the overall yield from (S)-2-hydroxy-4-methylvaleric acid to Isaridin E cyclic ester peptide was 31%.

[0125] Example 2: Preparation of an Isaridin E cyclic ester peptide

[0126] The difference from Example 1 is that in step (1), the solvent is changed from dichloromethane to toluene; in step (2), the condensing agent is changed from DCC to N,N-diisopropylcarbodiimide (DIC). In Example 2, the total yield from (S)-2-hydroxy-4-methylvaleric acid to Isaridin E cyclic ester peptide was 14% during the preparation of the final product Isaridin E cyclic ester peptide from (S)-2-hydroxy-4-methylvaleric acid.

[0127] The other steps and conditions are the same as in Example 1.

[0128] Example 3: Preparation of an Isaridin E cyclic ester peptide

[0129] The difference from Example 1 is that in step (4) part (A), the original condensing agent DIEA is replaced with HBTU; in step (5), DIEA used to adjust the solution to neutrality is replaced with DBU. In Example 3, during the preparation of the final product Isaridin E cyclic ester peptide from (S)-2-hydroxy-4-methylvaleric acid, the total yield from (S)-2-hydroxy-4-methylvaleric acid to Isaridin E cyclic ester peptide was 16%.

[0130] The other steps and conditions are the same as in Example 1.

[0131] Experimental Example: Structural Characterization of Peptides

[0132] 1. Spectral Analysis

[0133] (1) Mass spectrometry and carbon NMR analysis of the linear peptide NH-Pro-Phe-N(Me)Val-N(Me)Val-βAla-D-Oleu-OH

[0134] like Figure 3 As shown, the main peak of the mass spectrum of the linear peptide NH-Pro-Phe-N(Me)Val-N(Me)Val-βAla-D-Oleu-OH prepared in Example 1 is [M+H]+674.4124, which is consistent with the theoretical molecular weight of the linear peptide, 673.4051. To further verify the structural accuracy of the linear peptide, carbon NMR spectroscopy was performed, and the results are as follows. Figure 4 As shown, 13 CNMR (150MHz, CDCl3) δ177.59172.47,172.10,171.30,171.03,169.22,136.71,129.86,129.47,127.89,73.55,59.96,59.04,51.6 5,47.22,41.16,38.22,35.95,35.61,31.50,31.06,30.77,28.24,27.27,25.54,24.85,23.61,21.66,20.40,19.62,19.59,17.92. Based on the combined analysis results of mass spectrometry and carbon spectroscopy, the structure of the linear peptide prepared in this application has been effectively verified, ensuring its correctness and purity.

[0135] (2) Mass spectrometry and carbon NMR analysis of Isaridin E cyclic ester peptide

[0136] like Figure 5 As shown, after the ring-closure reaction, the main peak of the Isaridin E cyclic ester peptide obtained in Example 1 was [M+H]+654.4029 in its mass spectrum, which is highly consistent with the theoretical molecular weight of 654.4023 for the Isaridin E cyclic ester peptide. To further verify the structural characteristics of the Isaridin E cyclic ester peptide, carbon NMR spectroscopy was performed, and the results are as follows... Figure 6 As shown, 13CNMR (100MHz, CDCl3) δ174.19,173.85,172.20,170.10,169.95,168.84,136.53,128.91,128.87,127.39,73.54,66.66,61.13,57.75, 53.97,47.32,38.92,35.74,35.49,35.21,32.43,29.80,29.19,27.85,27.80,24.91,23.50,22.14,20.67,20.41,19.86,19.64,19.00. Based on the above mass spectrometry and carbon spectroscopy analysis results, it is confirmed that this application has successfully prepared the Isaridin E cyclic ester peptide.

[0137] Examples 2 and 3, by changing only the solvent, condensing agent, and pH adjuster, experimentally verified that the same product, Isaridin E cyclic ester peptide, as in Example 1 could also be prepared, so they will not be described again here.

[0138] In summary, this invention successfully prepared Isaridin E cyclic ester peptides via ester bond closure, solid-phase peptide synthesis, and ring-closure reaction. The specific steps are as follows: First, (S)-2-hydroxy-4-methylvaleric acid was esterified with benzyl alcohol. The resulting ester was then linked to Fmoc-β-alanine, and finally, hydrogen reduction and debenzylation were performed to close the ester bond, yielding compound C. This compound provided a crucial reaction basis for subsequent solid-phase peptide synthesis. Next, compound C was coupled to a resin, and amino acids were sequentially linked to obtain a peptide resin chain with the target sequence. Subsequently, the linear peptide with the target sequence was released from the resin using acid cleavage technology. Finally, through a cyclic peptide synthesis step, the target product—Isaridin E cyclic ester peptide—was successfully obtained. In summary, this invention is the first to successfully prepare Isaridin E cyclic ester peptides using a chemical synthesis method. Compared with traditional methods of isolation, purification, and extraction from marine fungi, this method not only significantly improves the controllability and stability of the yield, but also provides sufficient material support for the widespread application of Isaridin E cyclic ester peptides.

[0139] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for preparing Isaridin E cyclic ester peptide, characterized in that, The synthesis route is as follows: Specifically, the following steps are included: S1. Compound A was reacted with Fmoc-β-alanine under the conditions of dichloromethane, N,N-dicyclohexylcarbodiimide, and 4-dimethylaminopyridine to give compound B; S2. The compound B obtained in step S1 is reduced to obtain compound C; S3. Compound C obtained in step S2 was synthesized into a linear peptide NH-Pro-Phe-N(Me)Val-N(Me)Val-βAla-D-Oleu-OH by solid-phase peptide synthesis. S4. The linear peptide obtained in step S3 is subjected to a ring-closing reaction to obtain Isaridin E cyclic ester peptide; In step S4, the ring-closing reaction involves subjecting the linear peptide obtained in step S3 to a ring-closing reaction under the conditions of tetrahydrofuran, (2-oxime-cyanoethyl acetate)-N,N-dimethyl-morpholinourea hexafluorophosphate, and N,N-diisopropylethylamine, followed by post-treatment to obtain Isaridin E cyclic ester peptide.

2. The preparation method according to claim 1, characterized in that, In step S3, the solid-phase synthesis method of the polypeptide includes the following steps in sequence: resin-amino acid coupling reaction, linear peptide synthesis, and acid cleavage.

3. The preparation method according to claim 2, characterized in that, The linear peptide synthesis includes a deFmoc group removal reaction and a coupling reaction.

4. The preparation method according to claim 1, characterized in that, In step S1, the preparation method of compound A includes the following steps: (S)-2-hydroxy-4-methylvaleric acid was reacted with benzyl alcohol under the conditions of a first solvent and an esterification catalyst, and then post-treated to obtain compound A.

5. The preparation method according to any one of claims 1 to 3, characterized in that, In step S3, the polypeptide solid-phase synthesis method specifically includes the following steps: Si. Compound C was reacted with 2-chlorotriphenylmethyl chloride resin under the conditions of dimethylformamide and N,N-diisopropylethylamine to obtain an amino acid-resin complex; Sii. The amino acid-resin complex obtained in step Si is subjected to an amino acid de-Fmoc group reaction under the conditions of a second solvent and a protecting group removal agent to obtain an amino acid-resin complex with de-Fmoc groups. Siii. Repeat steps Si to Sii above, and sequentially couple amino acids and remove Fmoc groups from the amino acid-resin complex obtained in step Siii with Fmoc-N-methyl-L-Val, Fmoc-N-methyl-L-Val, Fmoc-L-Phe-OH, and Fmoc-L-Pro-OH to gradually extend the polypeptide chain and obtain a polypeptide chain resin with the target amino acid sequence. Siv. The polypeptide chain resin with the target amino acid sequence obtained in step Siii is subjected to acid cleavage to obtain the linear peptide NH-Pro-Phe-N(Me)Val-N(Me)Val-βAla-D-Oleu-OH.

6. The preparation method according to claim 5, characterized in that, In step Siv, the acid lysis includes the following steps: adding the acidic lysis buffer to the obtained polypeptide chain resin with the target amino acid sequence, reacting fully, collecting the lysis buffer, adjusting the pH to neutral, and then obtaining the crude linear peptide NH-Pro-Phe-N(Me)Val-N(Me)Val-βAla-D-Oleu-OH with the target amino acid sequence.

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