CN111850066A - A kind of preparation method of precursor-directed Bacillamide halogenated analog - Google Patents

Abstract

The invention discloses a precursor-guided preparation method of a Bacillus amide halogenated analogue, which comprises the following steps: activating marine-source Bacillus atrophaeus C89 with strains, culturing seeds, then using the strains for shake flask fermentation, adding 6-chloro-tryptophan with the final concentration of 0.2-0.6mM in 5-7h of the strain shake flask fermentation, terminating the fermentation after 48h of the fermentation, and separating and purifying the fermentation culture solution to obtain 6-Cl-Bacillamide C. The method utilizes the substrate universality of the microbial intracellular enzyme, adds the derivative 6-chloro-tryptophan of the tryptophan into the culture medium, and introduces the chlorine element into the natural product by a biosynthesis method.

Description

A kind of preparation method of precursor-directed Bacillamide halogenated analog

technical field

The invention belongs to the field of microbial molecular biology and natural product chemistry, relates to a method for preparing a halogenated analog of Bacillamide guided by a precursor, and in particular relates to a method for synthesizing Bacillamide by using a method for biosynthesis guided by a precursor in marine-derived Bacillus atrophicus Chlorinated analogs.

Background technique

Bacillamide family compounds belong to non-ribosomal peptide compounds, and five members of Bacillamide A, B, C, D and E have been found [[Bloudoff K, Fage CD, Marahiel MA, Schmeing TM: Structural andmutational analysis of the nonribosomal peptide synthetase heterocyclizationdomain Provides insight into catalysis.Proc Natl Acad Sci U S A 2017, 114(1): 95-100.], the common feature in structure is that they all have an indolethiazole ring. Its structure is as follows:

It is known that this family of compounds can effectively inhibit the growth of harmful dinoflagellates, dinoflagellates and cyanobacteria, etc. In addition, Bacillamides analogs are effective against human colorectal cancer cells HCT-116, human breast cancer cells MDA-MB-231 and immune system A cancer cell line (JURKAT) showed comparable cytotoxicity to the commercially available drug doxorubicin. In the activity experiments of Bacillamide A and its derivatives against cyanobacteria and microalgae, it was also found that the halogenated derivatives of Bacillamide showed higher biological activities [Kumar S, Aggarwal R, Kumar V, Sadana R, Patel B, Kaushik P , Kaushik D: Solvent-free synthesis of bacillamide analogues as novel cytotoxic and anti-inflammatory agents. Eur J Med Chem 2016, 123:718-726.].

CN201010278110 and CN201310677108 all provide biosynthesis methods for Bacillamide compounds. The Bacillamide family compounds that have been discovered so far include Bacillamide A, B, C, D, E, five members. Among them, CN201010278110 synthesized Bacillamide C, and CN201310677108 synthesized Bacillamide A, Bacillamide B, Bacillamide C and Bacillamide precursor compounds. However, none of them involves the introduction of halogen elements into the backbone of Bacillamide to synthesize halogenated derivatives of the Bacillamide family.

The present invention is a method for precursor-directed biosynthesis, which utilizes the substrate breadth of tryptophan decarboxylase in the Bacillamide biosynthesis pathway of Bacillus atrophicus B. atrephaeus C89, and adds 6-chloro-tryptophan, an analog of tryptophan. As the substrate, halogen elements were successfully introduced into the skeleton of Bacillamide C to obtain 6-Cl-Bacillamide C. Due to the strong electronegativity of halogen atoms, the introduction of halogen elements into natural products usually affects the spatial and electronic properties of molecules. The addition of halogen in drug development also has the effect of increasing drug effectiveness, bioavailability, and prolonging half-life. . Therefore, halogenated Bacillamdie derivatives have important research and development value.

SUMMARY OF THE INVENTION

In view of the defects in the prior art, the present invention provides a method for preparing a precursor-directed halogenated analog of Bacillamide. In the method, chlorotryptophan precursor is added to the fermentation medium of Bacillus atrophicus B. atrephaeus C89 of marine origin, and the halogenated derivative 6-Cl-Bacillamide C is obtained by extracting and preparing from the extraction liquid of the fermentation medium. Its molecular formula is is C ₁₈ H ₁₉ ClN ₄ O ₂ S with the following structure:

The present invention is achieved through the following technical solutions:

The invention provides a preparation method of a precursor-directed Bacillamide halogenated analog, comprising the following steps:

S1. Bacillus atrophicus B.atrephaeus C89 of marine origin is activated by strain, and the seeds are cultivated for subsequent fermentation;

S2, prepare precursor compound: configure 6-chloro-tryptophan mother liquor with artificial seawater, filter and sterilize for use;

S3, adding the precursor compound prepared in step S2 at the initial stage of the logarithmic growth phase of B.atrephaeus C89 fermentation and then continuing the fermentation;

S4. After the fermentation of B. atrephaeus C89, the chlorinated analog of Bacillamide: 6-Cl-Bacillamide C can be obtained by separating and purifying the fermentation broth.

As an embodiment of the present invention, in step S1, the medium used for activation of bacterial species is LB solid medium, and the medium used for seed culture and fermentation is LB liquid medium.

As an embodiment of the present invention, the LB liquid medium is prepared by dissolving 10g tryptone, 5g yeast extract, and 10g sodium chloride in each 1L of artificial seawater; the LB solid medium is prepared by dissolving each 1L of artificial seawater. It is prepared by dissolving 10g tryptone, 5g yeast extract, 10g sodium chloride and 15-20g agar powder in artificial seawater.

As an embodiment of the present invention, the artificial seawater components are: sodium chloride 26-27g/L, magnesium chloride 2-3g/L, magnesium sulfate 3-4g/L, calcium chloride 1-2g/L, chloride Potassium 0.5-1g/L, sodium bicarbonate 0.1-0.2g/L, sodium bromide 0.05-0.1g/L, dissolve each component one by one with ultrapure water, and adjust pH to 7.0-7.2.

A further example is: the artificial seawater components are: sodium chloride 26.518g/L, magnesium chloride 2.447g/L, magnesium sulfate 3.305g/L, calcium chloride 1.141g/L, potassium chloride 0.725g/L, hydrogen carbonate Sodium 0.202g/L, sodium bromide 0.083g/L, dissolve each component one by one with ultrapure water, and adjust the pH to 7.0-7.2.

As an embodiment of the present invention, in step S2, the 6-chloro-tryptophan mother liquor is prepared with artificial seawater, specifically: 6-chloro-tryptophan is first dissolved with formic acid, and then the artificial seawater is used to make a volume of 250 -300mM stock solution.

As an embodiment of the present invention, in step S1, the fermentation is shake flask fermentation, and the culture conditions are 28° C. and 150 rpm.

As an embodiment of the present invention, in step S2, the filtration sterilization adopts a 0.22 μm filter membrane filtration sterilization.

As an embodiment of the present invention, in step S3, the time for adding the precursor compound is 5-7 hours after the start of fermentation, and the time for ending the fermentation is 48 hours.

As an embodiment of the present invention, the precursor compound is added at a working concentration of 0.2-0.6 mM.

As an embodiment of the present invention, in step S4, the separation and purification steps are as follows: immediately after the fermentation is completed, a certain volume of ethyl acetate is added for extraction overnight, and the supernatant filtered under reduced pressure is continuously extracted with ethyl acetate. The crude extract containing 6-Cl-Bacillamide C was obtained after rotary evaporation and drying of the organic phase solution.

As an embodiment of the present invention, the separation and purification step further comprises: dissolving the crude extract with methanol, and separating the crude extract by gel filtration chromatography.

As an embodiment of the present invention, the packing material used in gel filtration chromatography is SephadexLH-20, the mobile phase is methanol, and the eluate is analyzed by high performance liquid chromatography (HPLC) to determine 6-Cl-BacillamideC The composition of the difference peaks.

As an embodiment of the present invention, the conditions used for HPLC detection of the eluent of gel filtration chromatography are: ultraviolet detection wavelength: 220 nm; mobile phase is A phase: ultrapure water, B phase: HPLC grade acetonitrile; column temperature : The elution conditions at 25°C are: 0-30min, B phase 10-100%; 30-35min, B phase 100-10%. The chromatographic column was ZORBAX Eclipse XDB-C18 (4.6×150 mm, 5 μm). The flow rate was 1 mL/min, the injection volume was 20 μL, and the sample concentration was 4 mg/mL.

As an embodiment of the present invention, 6-Cl-Bacillamide C can be obtained by further separating the composition containing the difference peak of 6-Cl-Bacillamide C by semi-preparative high performance liquid chromatography.

Semi-preparative high performance liquid chromatography was used to crudely prepare the composition containing the difference peak of 6-Cl-Bacillamide C. The semi-preparative high performance liquid chromatography conditions were: UV detection wavelength: 220 nm; mobile phase was A phase: ultrapure water, Phase B: HPLC grade acetonitrile; column temperature: 25°C; elution conditions are 0-30min, phase B is 10-100%; 30-35min, phase B is 100-10%; the column is Durashell C18-AM, (4.6× 250mm, 5μm).

The above crude product is subjected to secondary preparation by semi-preparative high performance liquid chromatography to obtain a single product of 6-Cl-Bacillamide C. The chromatographic conditions are: UV detection wavelength: 220 nm; mobile phase is A phase: ultrapure water, B phase: HPLC grade Acetonitrile; column temperature: 25°C; elution conditions: 0-11min, B phase 10-44%; 11-16min, B phase 44-60%; 16.01-20min, 100-100%; 20.01-25min, 100-10 %; the chromatographic column is Durashell C18-AM, (4.6×250 mm, 5 μm).

The present invention utilizes the method of biosynthesis guided by the precursor, adds 6-chloro-tryptophan precursor to the medium of Bacillus atrophicus B.atrephaeusC89 from marine sources, and successfully separates and identifies the halogenated derivative 6 of Bacillamide in the fermentation product. -Cl-Bacillamide C.

The present invention introduces halogen elements on the original skeleton of Bacillamide family compounds through the method of precursor-directed biosynthesis, and finally obtains a halogenated derivative of Bacillamide family, namely the Bacillamide chloro derivative 6-Cl-Bacillamide C of the present invention. Mainly using the substrate breadth of tryptophan decarboxylase in Bacillus atrophicus B. atrephaeus C89, by adding tryptophan derivative 6-chloro-tryptophan in the medium, and finally introducing the chlorine atom into Bacillamide C On the skeleton of , 6-Cl-Bacillamide C was synthesized.

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

1) Using the method of precursor-directed biosynthesis, the halogenated derivative 6-Cl-Bacillamide C of Bacillamide was isolated and identified in the fermentation medium of Bacillus atrophicus B. atrephaeus C89 of marine origin;

2) The first use of microorganisms to synthesize the chlorinated derivatives of Bacillamide, which has guiding significance for the biosynthesis of the halogenated derivatives of Bacillamide, and provides a basis for the research on the anti-algal and anti-tumor activities of Bacillamide analogs;

3) The elution reagents used in the separation and purification method of 6-Cl-Bacillamide C of the present invention are methanol and acetonitrile, the cost is low, the preparation method is simple, and has industrial research and development value;

4) The present invention broadens the types of Bacillamide family compounds, and provides more choices for subsequent activity screening.

Description of drawings

Other features, objects and advantages of the present invention will become more apparent by reading the detailed description of non-limiting embodiments with reference to the following drawings:

Fig. 1 is the preparation flow chart of 6-Cl-Bacillamide C of the present invention;

Fig. 2 is the high performance liquid chromatogram of crude extract in embodiment 1;

Fig. 3 is the high performance liquid chromatogram of 6-Cl-Bacillamide C after semi-preparative HPLC purification in Example 2;

Fig. 4 is the first order mass spectrum of 6-Cl-Bacillamide C;

FIG. 5 is a secondary mass spectrum of 6-Cl-Bacillamide C. FIG.

Detailed ways

The present invention will be described in detail below with reference to specific embodiments. The following examples will help those skilled in the art to further understand the present invention, but do not limit the present invention in any form. It should be noted that, for those skilled in the art, several changes and improvements can be made without departing from the inventive concept. These all belong to the protection scope of the present invention.

Example 1

This embodiment provides a preparation method of a precursor-guided Bacillamide halogenated analog as shown in Figure 1, which specifically includes the following steps:

Configuration of artificial seawater: sodium chloride 26.518g/L, magnesium chloride 2.447g/L, magnesium sulfate 3.305g/L, calcium chloride 1.141g/L, potassium chloride 0.725g/L, sodium bicarbonate 0.202g/L, Sodium bromide 0.083g/L, dissolve the above components in order with ultrapure water to avoid the formation of precipitation, pH 7.0-7.2.

Configuration of LB medium: 10g/L tryptone, 5g/L yeast extract, 10g/L sodium chloride, solid medium plus 1.5% agar powder, dissolve with artificial seawater, 121°C, 20min high temperature sterilization for use.

Preparation of 6-chloro-tryptophan precursor mother solution: first dissolve with formic acid, then dilute to volume with artificial seawater, prepare a 250 mM mother solution for later use, and then filter and sterilize it with a 0.22 μm filter membrane for later use.

Activation of strains: Bacillus atrophicus B. atrephaeus C89 stored at -80°C was streaked on LB solid medium, and cultured in a constant temperature incubator at 28°C overnight.

Preparation of seed solution: After single colony grows on the above-mentioned strain activation plate, select a single colony and add it to 5-10 mL of LB liquid medium, and cultivate at 28° C. and 150 rpm for 12-16 hours.

Strain fermentation: inoculate the seed liquid into 500 mL of LB liquid medium according to the inoculum amount of 1%, start the fermentation at 28 ° C and 150 rpm, and add 6-chloro- The tryptophan precursor continued to ferment, and the fermentation was terminated after 48 hours.

Example 2

The separation and purification of the fermentation product specifically includes the following steps:

Extraction and filtration under reduced pressure: add an equal volume of ethyl acetate in the fermentation medium to the conical flask at the end of the fermentation, and extract overnight; then use qualitative filter paper with a pore size of 20 μm to filter the fermentation broth under reduced pressure to collect the bacterial liquid supernatant; The supernatant of the bacterial liquid was extracted with an equal volume of ethyl acetate, and the crude extract was obtained after rotary evaporation; the crude extract in the rotary evaporation flask was dissolved with methanol, and then transferred to the sample bottle and dried, and the crude extract was detected by high performance liquid chromatography. Paste, the detection conditions are UV detection wavelength: 220nm; mobile phase is A phase: ultrapure water, B phase: HPLC grade acetonitrile; column temperature: 25 ℃ Elution conditions: 0-30min, B phase 10-100%; 30 -35min, B phase 100-10%. The chromatographic column was ZORBAX Eclipse XDB-C18 (4.6×150 mm, 5 μm). The flow rate was 1 mL/min, the injection volume was 20 μL, and the sample concentration was 4 mg/mL. Fig. 2 is the high performance liquid chromatogram of crude extract, as can be seen from Fig. 2, the peak time of 6-Cl-Bacillamide C is 11.871min).

Gel column chromatography: after dissolving the above dried crude extract in methanol, it was filtered through a 0.22 μm filter membrane, and the filtered liquid was subjected to gel column chromatography. The gel column chromatography column packing was Sephadex LH-20, and the mobile phase was methanol. The specific steps of gel column chromatography are as follows: first equilibrate the gel column with methanol; then slowly drop the sample to be separated along the wall of the gel column with a rubber tip dropper. Add an eluent reservoir to the upper end of the column; open the valve at the lower end of the gel column to start elution, and start collecting the effluent with a test tube when the sample flows to the bottom 15-20 cm of the gel column.

Analysis of above-mentioned chromatographic solution by high performance liquid chromatography: utilize high performance liquid chromatography to detect the effluent in the above-mentioned test tube, merge the test tube components of the same peak shape, and detect the conditions used for ultraviolet detection wavelength: 220nm; mobile phase is A phase: Ultrapure water, phase B: HPLC grade acetonitrile; column temperature: 25°C The elution conditions are: 0-30 min, phase B 10-100%; 30-35 min, phase B 100-10%. The chromatographic column was ZORBAX Eclipse XDB-C18 (4.6×150 mm, 5 μm). The flow rate was 1 mL/min, the injection volume was 20 μL, and the sample concentration was 4 mg/mL.

Semi-preparative high-performance liquid chromatography is used for crude and secondary preparation of the above combined samples: the chromatographic conditions are external detection wavelength: 220 nm; mobile phase is A phase: ultrapure water, B phase: HPLC grade acetonitrile; column temperature: 25°C; Elution conditions are 0-11min, B phase 10-44%; 11-16min, B phase 44-60%; 16.01-20min, 100-100%; 20.01-25min, 100-10%; Column is Durashell C18- AM, (4.6×250mm, 5μm). Figure 3 is a chromatogram of a sample purified by gel column chromatography and semi-preparative high performance liquid chromatography. It can be seen from Figure 3 that the peak time of 6-Cl-Bacillamide C is 11.023 min.

The structure of the obtained compound is shown below, its molecular formula is C ₁₈ H ₁₉ ClN ₄ O ₂ S, the primary mass spectrum of the compound is shown in FIG. 4 , and the secondary mass spectrum is shown in FIG. 5 .

Figure 5 is the secondary mass spectrum of 6-Cl-Bacillamide C. The first three peaks in Figure 5 are the fragment ion peaks formed by the further bond cleavage of the molecular ion of the 6-Cl-Bacillamide C molecule in the secondary mass spectrum. The peak does not appear in HPLC separation; the last peak is the molecular ion peak of 6-Cl-Bacillamide C, which can be detected in HPLC.

The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the above-mentioned specific embodiments, and those skilled in the art can make various variations or modifications within the scope of the claims, which do not affect the essential content of the present invention.

Claims (10)

1. the preparation method of the Bacillamide halogenated analog of a precursor guidance, is characterized in that, comprises the following steps: S1. Bacillus atrophicus B.atrephaeus C89 of marine origin is activated by strain, and the seeds are cultivated for subsequent fermentation; S2, prepare precursor compound: configure 6-chloro-tryptophan mother liquor with artificial seawater, filter and sterilize for use; S3, adding the precursor compound prepared in step S2 at the initial stage of the logarithmic growth phase of B.atrephaeus C89 fermentation and then continuing the fermentation; S4. After fermentation of B.atrephaeus C89, the fermentation broth can be separated and purified to obtain Bacillamide chlorinated analogs: 6-Cl-Bacillamide C

2. the preparation method of the Bacillamide halogenated analog of precursor guidance as claimed in claim 1, is characterized in that, in step S1, the substratum that is used for bacterial classification activation is LB solid substratum, is used for seed culture, fermentation The medium is LB liquid medium. 3. the preparation method of the Bacillamide halogenated analog of precursor guidance as claimed in claim 2, is characterized in that, described LB liquid medium is dissolved in 10g tryptone, 5g yeast extract, 5g yeast extract in every 1L artificial seawater. 10g sodium chloride; the LB solid medium is prepared by dissolving 10g tryptone, 5g yeast extract, 10g sodium chloride and 15-20g agar powder into each 1L of artificial seawater. 4. the preparation method of the Bacillamide halogenated analog of precursor guidance as claimed in claim 3, is characterized in that, described artificial seawater composition is: sodium chloride 26-27g/L, magnesium chloride 2-3g/L, sulfuric acid Magnesium 3-4g/L, calcium chloride 1-2g/L, potassium chloride 0.5-1g/L, sodium bicarbonate 0.1-0.2g/L, sodium bromide 0.05-0.1g/L, use ultrapure water one by one Dissolve ingredients and adjust pH to 7.0-7.2. 5. the preparation method of the Bacillamide halogenated analog of precursor guidance as claimed in claim 1, is characterized in that, in step S3, the precursor compound addition time is 5-7h after fermentation starts, and the fermentation end time is after 48h . 6 . The method for preparing a precursor-directed halogenated analog of Bacillamide according to claim 5 , wherein the precursor compound is added at a working concentration of 0.2-0.6 mM. 7 . 7. the preparation method of the Bacillamide halogenated analog of precursor guidance as claimed in claim 1, is characterized in that, in step S4, described separation and purification step is: add a certain volume of ethyl acetate extraction overnight after fermentation finishes immediately , the supernatant filtered under reduced pressure was continuously extracted with ethyl acetate, and the extracted organic phase solution was rotary evaporated to dryness to obtain a crude extract containing 6-Cl-Bacillamide C. 8. the preparation method of the Bacillamide halogenated analog of precursor guidance as claimed in claim 7, is characterized in that, described separation and purification step also comprises: the crude extract is dissolved with methanol, utilizes gel filtration chromatography to analyze crude The extract is separated. 9. the preparation method of the Bacillamide halogenated analog of precursor guidance as claimed in claim 8 is characterized in that, gel filtration chromatography used packing material is Sephadex LH-20, and mobile phase is methanol, The eluate was analyzed by high performance liquid chromatography (HPLC) to determine the composition of the difference peak of 6-Cl-Bacillamide C. 10. the preparation method of the Bacillamide halogenated analog of precursor guidance as claimed in claim 9 is characterized in that, utilizes semi-preparative high performance liquid chromatography to further separate the composition containing 6-Cl-Bacillamide C difference peak, namely 6-Cl-Bacillamide C can be obtained.

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