CN117285592A

CN117285592A - Preparation method of cyclohexalipopeptides natural product Cladoamide B

Info

Publication number: CN117285592A
Application number: CN202210690428.8A
Authority: CN
Inventors: 贾雪雷; 游恒志; 沈桂富; 卿晶; 廖竞远
Original assignee: Shenzhen Zhonghe Headway Bio Sci & Tech Co ltd; Shenzhen Graduate School Harbin Institute of Technology
Current assignee: Shenzhen Zhonghe Headway Bio Sci & Tech Co ltd; Shenzhen Graduate School Harbin Institute of Technology
Priority date: 2022-06-17
Filing date: 2022-06-17
Publication date: 2023-12-26

Abstract

The invention relates to the technical field of organic synthesis, and particularly discloses a preparation method of a high-efficiency cyclohexalipopeptides natural product Cladoamide B, which comprises the steps of connecting Fmoc-beta-alanine to 2-CTC resin to obtain a resin intermediate; deprotection of the resin intermediate and grafting Fmoc-N-methyl-L-phenylalanine to the resin intermediate to obtain a primary resin body; deprotection of the primary resin body, and sequentially peptide grafting Fmoc-N-methyl-L-phenylalanine, fmoc-L-valine, fmoc-L-proline and L-alpha-hydroxyisocaproic acid to the primary resin body to obtain a chain hexapeptide crude product; the polypeptide chain in the crude product of chain hexapeptide acid is excised from the resin and cyclized through macrolide to obtain Cladoamide B.

Description

Preparation method of cyclohexalipopeptides natural product Cladoamide B

Technical Field

The invention relates to the technical field of organic synthesis, in particular to a preparation method of a cyclohexalipopeptides natural product Cladoamide B.

Background

The fungus life activities can generate secondary metabolites with various biological activities so as to avoid predation, kill competitors and single prey, and avoid host immune response so as to maintain infection, and the fungus life activities can be used as an important source of various biological active compounds and can be used as a clinically useful medicine resource.

The university of Fushan county, japan, university of Biotechnology research center Yasuhiro Igarashi, 2021 teaches that the group extracts and separates the natural product Cladoamide B from a culture broth of fungal pathogen Cladobotrym varium that causes mushroom arachnoid disease. Cladobotrym is a pathogen of spider web disease on mushroom crops, and up to now 32 secondary metabolites have been reported, and in the continued study of chemical diversity of underdeveloped microbial metabolites, a strain Cladobotrym varium, numbered TAMA 582, was isolated from a mildew-producing porous fungus, which was found to produce two novel cyclic peptides Cladoamides A and B, a novel cyclic peptide Cladoamide C, and three known compounds cladobotryal, ternatin and melinacidin IV. Wherein the chemical structure of the natural product Cladoamide B is prepared by applying HR-ESITOFMS, ¹ H NMR、 ¹³ The C NMR, HSQC, COSY, HMBC and Marfey methods are characterized, the structure of the C NMR, HSQC, COSY, HMBC and Marfey methods contains several unusual amino acid residue sequences, and the biological activity of the C NMR, HSQC, COSY, HMBC and Marfey methods is not reported in the related art.

Since the compound is reported from the separation, the compound has low abundance in the nature, little research on the biological activity of the compound is carried out, and no related synthesis report exists at present. Therefore, the Cladoamide B is obtained in a sufficient amount through total synthesis, and has great significance for verifying the chemical structure and researching the biological activity.

Disclosure of Invention

Based on the above, it is necessary to provide a method for preparing Cladoamide B, which is a natural product of cyclohexalipopeptides, aiming at the technical problems of low natural abundance and insufficient artificial synthesis of Cladoamide B.

A preparation method of a cyclohexalipopeptides natural product Cladoamide B comprises the following steps:

s1: fmoc-beta-alanine was attached to 2-CTC resin to give a resin intermediate;

s2: deprotection is carried out on the resin intermediate, fmoc-N-methyl-L-phenylalanine is connected to the resin intermediate, and a primary resin body is obtained;

s3: deprotection of a primary resin body, sequentially peptide grafting Fmoc-N-methyl-L-phenylalanine, fmoc-L-valine, fmoc-L-proline and L-alpha-hydroxyisohexanoic acid to the primary resin body, deprotection is carried out after each amino acid is connected to the primary resin body, and then peptide grafting of the next amino acid to the primary resin body is carried out, so that a chain hexapeptide crude product is obtained;

s4: the polypeptide chain in the crude product of chain hexapeptide acid is excised from the resin and cyclized through macrolide to obtain Cladoamide B.

In one embodiment, step S4 is performed using liquid phase condensation conditions to cyclize the macrolide to give Cladoamide B.

In one embodiment, step S4 uses a continuous flow technique to cyclize the macrolide to give Cladoamide B.

In one example, in step S1, after swelling and washing the 2-CTC resin, fmoc- β -alanine is added to react to obtain a resin intermediate.

In one example, in step S1, unreacted sites in the 2-CTC resin after reaction with Fmoc- β -alanine are blocked.

In one embodiment, in step S2, the resin intermediate is deprotected in a mixture of morpholine and dimethylformamide solution.

In one embodiment, in step S4, when the macrolide cyclization reaction is performed under the condition of liquid phase condensation, the crude product of chain hexapeptide acid is reacted under the condition of DMAP and MNBA, and then subjected to reduced pressure rotary evaporation and silica gel column chromatography to obtain Cladoamide B.

In one embodiment, in step S4, when the macrolide cyclization reaction is performed using continuous flow techniques, the crude chain hexapeptide acid is reacted with a coupling reagent solution in a continuous flow reactor to yield Cladoamide B. By implementing the preparation method of the cyclic hexapeptide natural product Cladoamide B, the full synthesis process of the natural product Cladoamide B is efficiently completed in a solid phase synthesis mode, the problems of low natural abundance and insufficient artificial synthesis of the Cladoamide B are solved, a large amount of raw materials are provided for verifying the chemical structure and the biological activity of the Cladoamide B, the research on the Cladoamide B is facilitated, and the application of the Cladoamide B is accelerated.

Drawings

FIG. 1 is a flow chart of a process for preparing a cyclic hexapeptide natural product Cladoamide B in accordance with one embodiment of the present invention;

FIG. 2 is a synthetic route for preparing Cladoamide B using conventional solid phase synthesis in example 1 of the present invention;

FIG. 3 is a schematic diagram showing the structure of a macrolide cyclization reaction system in example 2 of the present invention;

FIG. 4 is a synthetic route for preparing Cladoamide B using solid phase synthesis and continuous flow techniques in example 2 of the present invention;

FIG. 5 is a hydrogen spectrum of the naturally occurring product Cladoamide B of phenolic peptides in one embodiment of the invention;

FIG. 6 is a graph of the carbon spectrum of the phenolic acid peptide natural product Cladoamide B in one embodiment of the invention.

Detailed Description

In order that the above objects, features and advantages of the invention will be readily understood, a more particular description of the invention will be rendered by reference to the appended drawings. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. The present invention may be embodied in many other forms than described herein and similarly modified by those skilled in the art without departing from the spirit of the invention, whereby the invention is not limited to the specific embodiments disclosed below.

The invention is based on solid phase synthesis, and obtains cyclized raw materials of Cladoamide B by preparing chain hexapeptide acid crude products, and performs ring closure synthesis (cyclization reaction) on the chain hexapeptide acid crude products by adopting various methods to obtain Cladoamide B products so as to meet the requirement of Cladoamide B research. The solid phase synthesis (Solid Phase Peptide Synthesis, SPPS for short) has great advantages as a classical synthesis method of peptide because of easy control of the reaction flow and strong operational repeatability. The solid phase synthesis reaction of the peptide is carried out on the external hanging group of the solid phase carrier, and the reaction intermediates and the final products are purified by a separation technique of washing and filtering.

In the embodiment, 2-CTC resin is used as synthetic resin, a plurality of amino acids are sequentially connected into the 2-CTC resin under the deprotection condition, peptide grafting operation is carried out, so that a chain hexapeptide acid crude product is obtained, and then cyclization reaction is carried out on the chain hexapeptide acid crude product, so that a target product Cladoamide B is prepared, the problem that the natural abundance of Cladoamide B is low is solved, and a large amount of raw materials are provided for the research of Cladoamide B.

Specifically, referring to fig. 1, the preparation method of the cyclohexalipopeptides natural product Cladoamide B of the present embodiment includes the following steps:

s1: fmoc- β -alanine was attached to the 2-CTC resin to give a resin intermediate.

In step S1, after swelling and washing the 2-CTC resin, fmoc-beta-alanine is added to allow Fmoc-beta-alanine to react with the 2-CTC resin sufficiently to obtain a resin intermediate.

Further, in step S1, unreacted sites in the 2-CTC resin after reaction with Fmoc- β -alanine are blocked. That is, after the 2-CTC resin is reacted with Fmoc- β -alanine and a resin intermediate is obtained, unreacted active sites may exist on a part of the 2-CTC resin due to unequal or insufficient reaction of the reaction raw materials, and thus, it is necessary to perform a capping operation on these sites to prevent the resin intermediate from being substituted with unreacted sites of the 2-CTC resin mixed in the resin intermediate during substitution reaction with other amino acids, thereby generating byproducts.

In this embodiment, 2-CTC resin is used as the linear peptide precursor, and the 2-CTC resin can effectively inhibit side reactions such as racemization and diketopiperazine formation, and has milder reaction conditions and high product purity compared with other resins, and the amino acids protected by multiple Fomc sequentially undergo substitution reaction with the 2-CTC resin, and after each substitution reaction is finished, the site of the next substitution reaction is determined by removing Fomc protection, thereby avoiding the formation of byproducts and being beneficial to improving the product yield.

S2: deprotection of the resin intermediate and grafting Fmoc-N-methyl-L-phenylalanine to the resin intermediate gave a primary resin.

In step S2, the resin intermediate is deprotected in a mixture of morpholine and dimethylformamide to remove the Fomc protecting group on the resin intermediate, so that the amino function is exposed for peptide grafting reaction (also known as substitution reaction) with Fmoc-N-methyl-L-phenylalanine.

S3: deprotection of the primary resin body was performed, and Fmoc-N-methyl-L-phenylalanine, fmoc-L-valine, fmoc-L-proline and L-alpha-hydroxyisohexanoic acid were sequentially peptide-grafted to the primary resin body, each amino acid was deprotected after being attached to the primary resin body, and then the next amino acid was peptide-grafted to the primary resin body to obtain a crude chain hexapeptide acid product.

In this embodiment, after 2-CTC resin and Fmoc- β -alanine are reacted to form a resin intermediate, fmoc-N-methyl-L-phenylalanine is added to the resin intermediate twice in sequence, the first added Fmoc-N-methyl-L-phenylalanine and the resin intermediate form a primary resin body, the primary resin body is deprotected to remove Fmoc groups on the primary resin body, and the second added Fmoc-N-methyl-L-phenylalanine and the cleavage site are subjected to peptide grafting reaction to obtain the target intermediate product. In this example, the addition of Fmoc-N-methyl-L-phenylalanine was not repeated, but was not completed by adding an excessive amount of Fmoc-N-methyl-L-phenylalanine once, deprotection was required between the two reactions, and the reaction mechanism of the two reactions was different. After the 2-CTC resin reacts with Fmoc-beta-alanine to generate a resin intermediate, fmoc protection removal and peptide grafting operation are required to be circularly carried out, namely Fmoc protection groups are required to be removed before the resin intermediate reacts with amino acids each time, amino functional groups are exposed, sites for the reaction with the amino acids are provided, so that peptide grafting reaction is carried out on the amino acids sequentially, and the like until the reaction of the last amino acid with the resin is completed, and a chain hexapeptide crude product with a specified structure is obtained.

In this example, after obtaining the crude chain hexapeptide, the crude chain hexapeptide was subjected to ring closure synthesis by cyclization of macrolide. The conventional macrolide strategy is as follows:

1) Yamaguchi macrolide: 2,4, 6-trichlorobenzoyl chloride is firstly reacted with carboxylic acid in a substrate, the carboxyl end is activated by a mode of forming mixed anhydride, and then the lactone ring is built by closing the ring under alkaline conditions, so that the method is the most commonly used macrolide method.

2) Steglich esterification: firstly, carboxylic acid reacts with DCC to generate active ester, then exchanges with DMAP to generate active amide, and alcohol attacks the active amide to generate ester. Can be used for esterification of large-steric hindrance or acid sensitive substrates, and is suitable for preparing tert-butyl ester from tert-butyl alcohol.

3) Keck macrolide: DMAP-HCl salt is added into Steglich esterification (DCC, DMAP), so that proton transfer efficiency is improved, and the yield of esterification reaction is improved.

4) Corey-Nicolaou esterification: the reaction and the modified version thereof have mild conditions, simple operation and good yield by forming thioester to activate the carboxyl end, and the yield of the reaction can be improved by adding Lewis acid, which has the defect that the thioester has a bad taste, and the excessive phosphorus-oxygen-triphenyl is generated after the reaction, which is unfavorable for purification and is adopted less at present.

The invention adopts two methods to cyclize the macrolide of the chain hexapeptide acid crude product to obtain Cladoamide B product, one is to cyclize the macrolide to obtain Cladoamide B by adopting the conventional liquid phase condensation condition in the step S4; secondly, in step S4, the continuous flow technique is adopted to cyclize the macrolide to obtain Cladoamide B, and the yield and other conditions of the Cladoamide B synthesized by two modes are specifically compared. Specifically, when the macrolide cyclization reaction is performed under the condition of liquid-phase condensation, in the step S4, the crude product of chain hexapeptide acid is reacted under the condition of DMAP and MNBA, and then subjected to reduced pressure rotary evaporation and silica gel column chromatography separation to obtain Cladoamide B. When the continuous flow technology is adopted for the cyclization reaction of macrolide, in the step S4, the chain hexapeptide acid crude product and the coupling reagent solution are put into a continuous flow reactor for reaction, thus obtaining Cladoamide B.

The preparation of Cladoamide B is described below in connection with specific examples.

Example 1

1.0g of 2-CTC resin with the concentration of 1.08mmol/g is filled into a solid-phase polypeptide synthesis tube with the capacity of 100mL, 20mL of dichloromethane is added, nitrogen is then introduced for bubbling, and the resin is swelled for 30min, so that the 2-CTC resin is fully swelled under the action of the nitrogen bubbling. The 2-CTC resin was then washed with 3X10mL dimethylformamide and 3X10mL dichloromethane to remove residual dichloromethane from the surface of the swollen 2-CTC resin. In this example, 3X10mL of dimethylformamide and 3X10mL of methylene chloride are used to wash 2-CTC resin, meaning that 10mL of dimethylformamide and 10mL of methylene chloride are used to wash 2-CTC resin each time, and the washing is repeated three times, reference being made to 3X10mL in the subsequent operations. 1.68g, 5.40mmol, 5equiv. (i.e., equivalent concentration) of Fmoc- β -Ala-OH (i.e., fmoc- β -alanine) and 1.9mL, 10.80mmol, 10equiv. Diisopropylethylamine were dissolved in 10mL dimethylformamide, added to a solid-phase polypeptide synthesis tube equipped with a swollen 2-CTC resin, and reacted under nitrogen bubbling for 4 hours to give a resin-attached Fmoc- β -Ala-OH, i.e., a resin intermediate. The resin intermediate was washed with 3x10mL dimethylformamide and 3x10mL dichloromethane, then a mixed solution of 8mL dichloromethane, 1.5mL diisopropylethylamine, 5mL methanol was added, the mixture was bubbled with nitrogen gas, the reaction was carried out for 30 minutes, the unreacted sites on the 2-CTC resin were blocked, and the blocked resin intermediate was washed with 3x10mL dimethylformamide and 3x10mL dioxymethane.

To 10mL of dimethylformamide solution was added 50% concentration of morpholine and the resin intermediate after the end-capping and washing was treated for 1 hour to remove Fmoc protecting groups on the resin intermediate, and the resin intermediate was washed with 3X10mL of dimethylformamide and 3X10mL of methylene chloride for use. 2.17g, 5.40mmol, 5equiv. Fmoc-N-Me-Phe-OH (i.e., fmoc-N-methyl-L-phenylalanine), 2.05g, 5.40mmol, 5equiv. HATU and 1.9mL, 10.80mmol, 10equiv. Diisopropylethylamine were dissolved in 10mL dimethylformamide and poured into a solid-phase polypeptide synthesis tube containing the deprotected resin intermediate, bubbled with nitrogen, after 2 hours of reaction, the solution was poured out, one peptide grafting reaction of the resin intermediate was completed, and the peptide grafted resin was washed with 3X10mL dimethylformamide and 3X10mL dioxymethane to obtain a primary resin, thereby completing the deprotection and peptide grafting operation of the resin intermediate.

Fmoc-N-Me-Phe-OH (i.e. Fmoc-N-methyl-L-phenylalanine), fmoc-L-Val (i.e. Fmoc-L-valine), fmoc-L-Pro (i.e. Fmoc-L-proline) and L-Leuic Acid (i.e. L-alpha-hydroxyisohexanoic Acid) are sequentially connected by the deprotection and peptide grafting operations, and the deprotection operation is repeated each time before the peptide grafting product is obtained and the peptide grafting reaction is carried out with the next amino Acid under the conditions of HATU and diisopropylethylamine until the reaction of the last amino Acid L-Leuic Acid with the previous peptide grafting product is completed, thereby obtaining a primary product. After the end of each peptide reaction, 50% of morpholine is added into 10mL of dimethylformamide solution to treat the primary product for 1h so as to remove Fmoc protecting groups, 3x10mL of dimethylformamide and 3x10mL of dichloromethane are used for washing, the primary product is transferred from a solid-phase polypeptide synthesis tube into a round bottom flask, 9.5mL of trifluoroacetic acid, 0.25mL of water and 0.25mL of triisopropylsilane are added for reaction for 2h, resin is removed by filtration, and the solution is dried by nitrogen to obtain a chain hexapeptide crude product.

65mg, 0.09mmol of crude chain hexapeptide acid was taken and dissolved in 10mL of methylene chloride, the solution was cooled to 0℃and 6mg, 0.045mmol, 0.5equiv. DMAP (4-dimethylaminopyridine) and 46mg, 0.135mmol, 1.5equiv. MNBA (2-methyl-6-nitrobenzoic anhydride) were added and reacted at room temperature (20 ℃) for 24 hours. The solvent was removed by rotary evaporation under reduced pressure and separated by silica gel column chromatography (methanol/dichloromethane=1/25) to give Cladoamide B as a white solid in a yield of 37%.

As shown in FIG. 2, the synthetic route of this example is that Fmoc- β -Ala-OH is first connected to a resin by using 2-CTC resin as the synthetic resin, fmoc protecting group is removed under the basic condition of morpholine, amino functional group is exposed, primary peptide grafting reaction is performed with Fmoc-N-Me-Phe-OH, condensation connection is performed, after the primary resin body after primary peptide grafting is deprotected, fmoc-N-Me-Phe-OH is added again for secondary peptide grafting reaction, fmoc-L-Val, fmoc-L-Pro and L-Leuic Acid are connected by the same cyclic operation, chain polypeptide compounds 1-6 are obtained, fmoc protection is removed, polypeptide chain can be cut off from the resin by trifluoroacetic Acid treatment, and finally natural product Cladoamid B is obtained by adopting conventional liquid phase condensation condition Guan Huange, thereby realizing the breakthrough of artificial synthesis of Cladoamid B.

Example 2

This example uses a combination of solid phase synthesis and continuous flow to synthesize Cladoamide B, and differs from example 1 in that after obtaining crude chain hexapeptide, 2mmol/L of chain polypeptide solution is prepared by anhydrous DCM, 6mmol/L of MNBA solution and 12mmol/L of DMAP solution are respectively prepared by anhydrous DCM, and the volume ratio of MNBA solution to DMAP solution is 1:1 is configured as a coupling reagent solution.

The method comprises the steps of taking a tubular microreactor shown in fig. 3 and arranging a macrolide cyclization reaction system, wherein the macrolide cyclization reaction system comprises a microinjection pump 1 for containing a chain polypeptide solution and a coupling reagent solution, a T-shaped mixer 2 communicated with the output end of the microinjection pump 1, a micro-pipeline 3 communicated with the output end of the T-shaped mixer 2 and a sample outlet 4 arranged at the output end of the micro-pipeline, and the sample outlet 4 is communicated with a product collecting bottle. In this embodiment, the T-shaped mixer 2 includes two input ends, each of which is connected with a microinjection pump 1, the chain-shaped polypeptide solution is injected into the T-shaped mixer 2 through one microinjection pump 1, the coupling reagent solution is injected into the T-shaped mixer 2 through the other T microinjection pump 1, and the micro-pipe 3 is a pipe made of PP plastic and wound and stacked round by round, and has an inner diameter of 0.8mm and a length of 1m. The micro-pipeline 3 can also be made of PTFE, PEEK, PE or PP plastic, the inner diameter of which is between 10 mu m and 1mm, and the length of which is between 0.1m and 2m, and is specifically selected according to the synthesis requirement. Argon is introduced into the tubular microreactor to purge, so that oxygen and other gases possibly interfering with the reaction in the tubular microreactor are removed, and the prepared coupling reagent solution and chain polypeptide solution are pumped into the micro-pipeline 3 through the microinjection pump 1 at a flow rate of 50 mu L/min for reaction. The temperature was controlled at room temperature and the reaction solution was allowed to stand in the microchannel 3 for 10min. The exit material was collected and tested by HPLC (high Performance liquid chromatography) for a yield of 51% of the lactamized cyclic polypeptide product (i.e., cladoamide B).

As can be seen from the synthetic route of this example shown in FIG. 4, in this example, 2-CTC resin is used as the synthetic resin, fmoc- β -Ala-OH is first connected to the resin, fmoc-N-Me-Phe-OH, fmoc-L-Val, fmoc-L-Pro and L-Leuic Acid are then sequentially connected to obtain chain polypeptide compound 2-1, then the polypeptide chain can be excised from the resin by trifluoroacetic Acid treatment, and finally the natural product Cladoamid B is obtained by loop synthesis by adopting a continuous flow method, compared with the solid phase synthesis method, the synthetic efficiency of Cladoamid B is improved by adopting the continuous flow technique in this example.

The macrolide cyclization reactions and results in examples 1 and 2 are compared as follows:

among them, tests 1 to 4 are for preparing Cladoamide B by performing a macrolide cyclization reaction by using the conventional liquid phase condensation method of example 1, and tests 5 to 6 are for preparing Cladoamide B by performing a macrolide cyclization reaction by using the continuous flow technique of example 2, and it can be seen that the yield of Cladoamide B prepared by performing a macrolide cyclization reaction by using the continuous flow technique is greatly improved and the reaction time is remarkably shortened under the condition that the types, the temperatures and the concentrations of the reaction reagent and the solvent are the same.

Referring to fig. 5 and 6, the reaction product was analyzed by hydrogen and carbon spectra, and the separation data of the product from the compound was compared, and the synthesized sample was consistent with the data of the naturally separated spectrum, confirming that the product was Cladoamide B.

By implementing the preparation method of the cyclic hexapeptide natural product Cladoamide B, the full synthesis process of the natural product Cladoamide B is efficiently completed in a solid phase synthesis mode, the problems of low natural abundance and insufficient artificial synthesis of the Cladoamide B are solved, a large amount of raw materials are provided for verifying the chemical structure and the biological activity of the Cladoamide B, the research on the Cladoamide B is facilitated, and the application of the Cladoamide B is accelerated.

It should be noted that, in the above embodiments, the preparation process of Cladoamide B is only exemplified by specific dosage of materials, and in actual production, an expansion test may be performed according to the ratio of the reagent and the solvent in the above embodiments, so as to meet the needs of industrial production, which is not described herein.

The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.

The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.

Claims

1. A method for preparing a cyclohexalipopeptides natural product Cladoamide B, which is characterized by comprising the following steps:

s1: fmoc-beta-alanine was attached to 2-CTC resin to give a resin intermediate;

2. The method for producing Cladoamide B as defined in claim 1, wherein the step S4 is performed by cyclizing the macrolide under liquid phase condensation conditions to obtain Cladoamide B.

3. The method for preparing Cladoamide B, which is a natural product of cyclohexalipopeptides according to claim 1, wherein the step S4 is performed by continuous flow technique for cyclizing macrolides to obtain Cladoamide B.

4. A process for the preparation of Cladoamide B, a natural product of the cyclohexalipopeptides according to claim 2 or 3, characterized in that in step S1, after swelling and washing the 2-CTC resin, fmoc- β -alanine is added for reaction to obtain a resin intermediate.

5. The method according to claim 4, wherein in step S1, the unreacted sites in the 2-CTC resin after the Fmoc- β -alanine reaction are blocked.

6. The method according to claim 5, wherein in step S2, the resin intermediate is deprotected in a mixture of a solution of morpholine and dimethylformamide.

7. The method for producing Cladoamide B as defined in claim 6, wherein in step S4, when the macrolide cyclization reaction is performed under the condition of liquid phase condensation, the crude product of chain hexapeptide acid is reacted with DMAP and MNBA, and then subjected to rotary evaporation under reduced pressure and silica gel column chromatography to obtain Cladoamide B.

8. The method for preparing Cladoamide B as claimed in claim 6, wherein in step S4, when the macrolide cyclization reaction is performed by continuous flow technique, the crude product of chain hexapeptide acid and the coupling reagent solution are placed into a continuous flow reactor to react, thereby obtaining Cladoamide B.