Separation and purification method and structure of Serrawetin W2 structural analogue

Abstract

The invention discloses a separation and purification method and a structure of Serrawetin W2 structural analogues, and relates to the technical field of separation and purification of cyclic lipopeptide compounds, and the separation and purification method comprises the following steps: obtaining a crude extract of a strain YD25 through liquid fermentation, and carrying out primary separation through silica gel column chromatography; combining the parts with similar components to obtain a main compound component; performing HPLC (High Performance Liquid Chromatography) detection to determine a separated key target compound; the key components are further purified through fine column chromatography, and impurities are removed by selecting a proper mobile phase system and proportion; and further purifying by adopting gel column chromatography, and finally obtaining a high-purity monomeric compound through semi-preparative HPLC (High Performance Liquid Chromatography). The method has the beneficial effects that the cyclic lipopeptide serrawetin W2 structural analogue is obtained through multi-stage fermentation and separation and purification, the chemical structure of the cyclic lipopeptide serrawetin W2 structural analogue is identified, and an experimental basis is provided for research of cyclic lipopeptide compounds.

Description

Technical Field

[0001] The present invention relates to the technical field of separation of cyclolipopeptide compounds, and in particular to a separation and purification method of Serrawettin W2 structural analogs and a structure thereof. Background Art

[0002] The general structure of the cyclolipopeptide compound Serrawettin W2 consists of five amino acids (leucine, serine, threonine, phenylalanine, isoleucine) combined with a β-hydroxy fatty acid molecule (molecular weight 731.93). The biosynthetic pathway of serrawettin W2 is encoded by the swrA gene. The biosynthesis of serrawettin W2 was studied in Serratia marcescens, and a heteropolyketide synthase (PKS)-NRPS gene cluster was identified, which is presumed to be involved in the biosynthesis of serrawettin W2. The biosynthetic pathway of serrawettin W2 was determined based on the presence of PKS and NRPS encoding genes. First, C synthesized by the PKS swrEFG and other unidentified proteins 10 Fatty acid chains serve as precursors and are released as fatty acyl-CoA. Secondly, swrA encodes the core peptide chain, which contains a total of five modules. It is predicted that the swrA initiation module will catalyze the condensation of fatty acyl-CoA and leucine. After initiation, chain elongation is mediated by three other linear domains associated with serine, threonine, and phenylalanine. Finally, the oligopeptide is transferred to the active site isoleucine at the C-terminus of the last module.

[0003] Serrawettin W2 is a compound composed of five amino acids and a fatty acid chain. Its structural diversity is the first, fourth or fifth amino acid and the length of the fatty acid chain (C8, C 10 or C 12 ), the molecular weights of these structural analogs range from 703.3 to 759.3.

[0004] Laboratory collection of strains Serratia surfactantfaciens YD25 T(abbreviated as YD25) is a bacterium isolated from the rhizosphere soil of continuously planted tobacco in Yongding, Fujian Province, China. This bacterial strain can simultaneously produce two secondary metabolites: prodigiosin and serrawettin W2, and a series of structural analogs of serrawettin W2 were also found from the secondary metabolites of YD25. It was confirmed that there were 8 cyclolipopeptide substances in the crude metabolite extract, which were named sw-1 to sw-8 respectively. After separation and purification, compound sw-5 was obtained. Mass spectrometry and nuclear magnetic resonance confirmed that sw-5 was serrawettin W2, and the other seven cyclolipopeptides were structural analogs of serrawettin W2. The cyclolipopeptide compound serrawettin W2 isolated from the secondary metabolites of YD25 showed good antibacterial effect and also had a good inhibitory effect on HeLa cells, but other cyclolipopeptide compounds were difficult to separate during the separation and purification process because of their similar structures to serrawettin W2. Therefore, the specific structure and biological activity of these serrawettin W2 structural analogs are still unclear. Jeremy L. Motley et al. isolated serrawettin W2 and its three structural analogues from Serratia marcescens, and named them serrawettin W4, serrawettin W5 and serrawettin W6 respectively after mass spectrometry and nuclear magnetic resonance; Tanya Clements-Decker et al. identified three structural analogues by nuclear magnetic resonance, and named them serrawettin W2-FI10, serrawettin W2-FV8 and serrawettinW2-FV12:1 respectively after mass spectrometry and nuclear magnetic resonance. Summary of the invention

[0005] In order to solve the above technical problems, the present invention discloses a separation and purification method of Serrawettin W2 structural analogs and their structures.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] The separation and purification method of serrawettin W2 structural analogs comprises the following steps:

[0008] S1. A crude extract of strain YD25 is obtained from KB medium by liquid fermentation, and the crude extract is initially separated by silica gel column chromatography, and the composition of the crude extract is analyzed by TLC; parts with similar compositions are combined to obtain the main compound components;

[0009] S2. Perform HPLC detection on the obtained compound components to determine the separated key target compounds;

[0010] S3, through the fine column chromatography process, the key components are further purified, and the appropriate mobile phase system and ratio are selected to remove impurities and improve the purity of the target compound;

[0011] S4. Further purification was performed using dextran gel column chromatography technology, and finally a high-purity monomer compound was obtained by semi-preparative HPLC.

[0012] Furthermore, in step S1, a petroleum ether-ethyl acetate mixed solvent system is selected as the mobile phase for column chromatography separation.

[0013] Furthermore, in step S2, components containing main compounds are obtained after repeated reverse phase column chromatography, and the main compounds of each group of components are different substances.

[0014] Furthermore, in step S3, the fine column chromatography process includes silica gel column chromatography, gel column chromatography and reverse phase column chromatography, which are repeatedly used in multiple rounds.

[0015] Furthermore, in step S4, semi-preparative HPLC adopts an isocratic elution method, and the mobile phase program of semi-preparative HPLC is: methanol mass fraction is 85%, 0-45min isocratic elution, temperature is 35°C, and flow rate is 2mL / min.

[0016] The invention also discloses Serrawettin W2 structural analogs obtained by the separation and purification method.

[0017] The present invention also discloses the structure of the Serrawettin W2 analogs. The Serrawettin W2 analogs are structurally identified by nuclear magnetic resonance and mass spectrometry. The monomer compounds are all cyclolipopeptide serrawettin W2 compounds. All the compounds are divided into three categories according to the length of the fatty acid carbon chain, which are:

[0018] Compounds sw-9 and sw-11 with C8 fatty acid chains;

[0019] The fatty acid chain is C 10 Compounds sw-2, sw-3, sw-4, sw-5 and sw-10;

[0020] The fatty acid chain is C 12 Compounds sw-6, sw-7 and sw-8.

[0021] The beneficial effects of the present invention are that the metabolites of strain YD25 are accumulated by a multi-stage fermentation method; serrawettin W2 and its structural analogs are separated and purified by various means such as thin layer chromatography, column chromatography and semi-preparative liquid chromatography; and the structures are identified by high-resolution mass spectrometry, secondary mass spectrometry and nuclear magnetic resonance analysis.

[0022] The separation and purification of Serrawettin W2 and its structural analogues is carried out by performing a series of multi-stage separation and purification of the fermentation crude product through a variety of technical means such as silica gel column chromatography, Sephadex LH-20 gel column chromatography, C18 reverse phase column chromatography and semi-preparative liquid chromatography; at the same time, thin layer chromatography and liquid chromatography methods are used to track the target compounds to improve their separation accuracy and efficiency; and finally, sw-2 to sw-1110 monomer compounds are purified.

[0023] Serrawettin W2 and its structural analogs were identified. In the present invention, the 10 compounds sw-2 to sw-11 obtained after separation and purification were subjected to mass spectrometry and nuclear magnetic resonance detection, and the relative molecular mass of the compound was confirmed by HRESIMS. The chemical structure of the compound was inferred by MS / MS technology. The 1D-NMR spectrum ( 1 H spectrum, 13 C spectrum and DEPT spectrum) and 2D-NMR spectrum (HSQC, HMBC, 1 H- 1 The structures of the compounds were determined by H COSY and NOESY techniques, and they were identified as cyclolipopeptide serrawettin W2 compounds. All compounds were divided into three categories according to the length of the fatty acid carbon chain, namely:

[0024] Compounds sw-9 and sw-11 with C8 fatty acid chains;

[0025] The fatty acid chain is C 10 Compounds sw-2, sw-3, sw-4, sw-5 and sw-10;

[0026] The fatty acid chain is C 12 Compounds sw-6, sw-7 and sw-8.

[0027] The structural analogue of the cyclolipopeptide serrawettin W2 was obtained through multi-stage fermentation and separation and purification, and its chemical structure was identified, which provides an experimental basis for the research of cyclolipopeptide compounds and also provides a new resource for the development of new antibiotics. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The mass spectrum and fragmentation peak analysis diagram of sw-3 in the embodiment of the present invention;

[0029] Figure 2 The key sw-3 in the embodiment of the present invention is 1 H- 1 H COSY, HMBC and NOESY correlation diagrams;

[0030] Figure 3A and 3B They are respectively sw-3 in the embodiment of the present invention 1 H spectrum and 13 C spectrum;

[0031] Figure 4 It is the mass spectrum and fragment peak analysis diagram of SW-4 in the embodiment of the present invention;

[0032] Figure 5 The key of sw-4 in the embodiment of the present invention is 1 H- 1 H COSY, HMBC and NOESY correlation diagrams;

[0033] Fig. 6A and 6B They are respectively sw-4 in the embodiment of the present invention 1 H spectrum and 13 C spectrum;

[0034] Figure 7 The SW-7 mass spectrum and fragment peak analysis diagram in the embodiment of the present invention;

[0035] Figure 8 The key SW-7 in the embodiment of the present invention is 1 H- 1 H COSY, HMBC and NOESY correlation diagrams;

[0036] Fig. 9A and 9B They are respectively the sw-7 in the embodiment of the present invention 1 H spectrum and 13 C spectrum;

[0037] Fig.10 The SW-8 mass spectrum and fragment peak analysis diagram in the embodiment of the present invention;

[0038] Fig.11 The key of SW-8 in the embodiment of the present invention is 1 H- 1 H COSY, HMBC and NOESY correlation diagrams;

[0039] Fig. 12A and 12B They are respectively sw-8 in the embodiment of the present invention 1 H spectrum and13 C spectrum;

[0040] Fig.13 It is the mass spectrum and fragmentation peak analysis diagram of SW-10 in the embodiment of the present invention;

[0041] Fig.14 The key of sw-10 in the embodiment of the present invention is 1 H- 1 H COSY, HMBC and NOESY correlation diagrams;

[0042] Fig.15A and 15B are respectively sw-10 in the embodiment of the present invention 1 H spectrum and 13 C spectrum. DETAILED DESCRIPTION

[0043] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0044] First, the fermentation crude product of strain YD25 was initially separated using silica gel column chromatography, and then further separated using various column chromatography and semi-preparative HPLC. The components were combined and purity tested using TLC and HPLC, and finally a variety of single compounds were successfully separated and purified. Specifically:

[0045] The fermentation crude extract was analyzed by liquid chromatography, and the crude extracts with the same chemical components were combined to obtain a total of 518.8 g of fermentation crude extract as a sample. The fermentation crude product of strain YD25 was initially separated by silica gel column chromatography, and the process was as follows:

[0046] 1.1 Silica gel column chromatography

[0047] (1) Sample mixing: The collected 518.8 g fermentation crude product was subjected to subsequent purification steps. First, the fermentation crude product needed to be redissolved in a suitable organic solvent. Due to the large amount of sample, if there was an insoluble part, other organic reagents were appropriately added or ultrasonic assisted dissolution was used to ensure that the sample could be thoroughly and evenly dissolved. Subsequently, the dissolved sample was mixed with a certain amount of silica gel (100 to 200 mesh), and the dissolved sample was dripped onto the silica gel powder using a rubber-tipped dropper. Care was taken to avoid excessive dripping and penetration to the bottom of the plate. The sample was stirred thoroughly to ensure uniform distribution, and then dried naturally under ventilation at room temperature. After the organic solvent in the silica gel powder was completely volatilized, the sample was added again and mixed well, and this process was repeated until the sample was completely added. After drying, the total mass of the mixture of the sample and silica gel reached about 868.8 g. The blocky mixture was ground into powder to avoid interference of the large particle mixture with the separation of the compounds during the experiment. At this point, the sample was ready for the next step of chromatographic separation.

[0048] (2) Column packing: There are two methods for column packing, wet packing and dry packing. Wet packing is to soak the column chromatography filler with eluent and then load it into the chromatography column. Wet packing is a commonly used method in experiments because of its simple operation and applicability to a variety of fillers. Dry packing is to directly load the dry column chromatography filler into the chromatography column and compact the filler with a vacuum pump. Compared with wet packing, dry packing can reduce nonspecific adsorption and improve column chromatography efficiency. Wet packing was selected in this experiment. In order to perform effective column chromatography separation, a silica gel column of appropriate size was selected based on the mass of the resulting mixture. In the process of preparing the column chromatography, first add about 4 to 6 times the mass of the sample into the container, and add an appropriate amount of petroleum ether to make the silica gel completely soaked and mixed evenly. In the process of filling the silica gel into the column, gently tap the left and right side walls of the column with a suction bulb, which helps to compact the silica gel layer and make the silica gel evenly distributed, avoiding possible fractures. After the silica gel is completely loaded into the column, level the silica gel layer and flush the silica gel column with a large amount of petroleum ether to ensure that the silica gel layer is compacted.

[0049] (3) Loading: There are two methods: wet loading and dry loading. Wet loading is to dissolve the sample and add it directly to the silica gel column; dry loading is to mix the sample with silica gel powder and dry it before adding it to the silica gel column. Wet loading is simple and convenient, while dry loading has better separation effect than wet loading. Therefore, dry loading is generally selected when conditions permit to improve the separation effect. After the column is loaded, the previously prepared sample silica gel mixed powder is slowly and evenly added to the silica gel chromatography column. During the loading process, the two sides of the column are also gently tapped to ensure that the sample is flat and evenly distributed in the column. Cover the sample layer with a layer of anhydrous sodium sulfate to absorb moisture in the air and mobile phase; add an appropriate silica gel layer and cotton as a buffer layer on the anhydrous sodium sulfate layer again, so that the sample layer will not be affected when the eluent is added, so that components of different polarities can be evenly separated from the silica gel column under the influence of the eluent and gravity.

[0050] (4) Column and collection: Considering the results obtained from the previous thin layer chromatography experiment and the characteristics of the sample containing a large amount of fatty acids, a mixed solvent system of petroleum ether and ethyl acetate was selected as the preliminary separation medium. During the separation process, the proportion of ethyl acetate in the eluent was increased according to the TLC test results to optimize the separation effect. After the elution was completed, the eluents of different gradients were collected and concentrated by a rotary evaporator. Finally, the obtained products were transferred to different sample bottles and appropriately labeled to prepare for subsequent analysis and application.

[0051] (5) Detection: After the crude separation step is completed, different samples are analyzed using HPLC technology to determine the main separated components. The spectral analysis can accurately identify the important components in the sample, and based on these results, the target components can be selected for further purification.

[0052] The important components are then carefully separated through a series of chromatographic techniques, including multiple applications of silica gel column chromatography, gel column chromatography and reverse phase column chromatography.

[0053] 1.2 Gel column chromatography

[0054] (1) Column packing: Gel column chromatography uses wet column packing. First, soak the Sephadex LH-20 gel in pure methanol eluent overnight to allow it to fully swell. Before formally loading the sample, use the eluent to balance the gel chromatography column for multiple column volumes to make the gel column compact and give full play to the effect of gel chromatography.

[0055] (2) Loading: Wet loading was selected as the loading method. In order to separate different compounds as much as possible, the sample was dissolved in as little methanol solution as possible. During the loading process, a rubber-tipped dropper was used to slowly add the solution. At the same time, the column valve was opened at a certain angle to maintain a certain flow rate, so that the sample layer was flush with the gel plane and the sample layer was as thin as possible.

[0056] (3) Column elution and collection: The elution volume varies for different samples, usually 2-3 retention volumes. Since the gel column runs quickly, the fractions need to be carefully collected in multiple batches.

[0057] (4) Detection: The collected fractions were detected by TLC and HPLC, and the same fractions were combined.

[0058] 1.3 Reverse phase column chromatography

[0059] (1) Column packing: Use wet column packing, soak the reverse phase filler in methanol and then pack it into the column. Use eluent to balance the gel chromatography column for multiple column volumes to make the reverse phase column more compact.

[0060] (2) Loading: Dry loading was selected as the loading method. The compound was dissolved in methanol and the sample was evenly added to the reverse phase column. Cotton was added on the sample layer as a buffer layer so that the sample layer would not be affected when the eluent was added.

[0061] (3) Column flow and collection: Since the pore size of the reversed-phase column packing material is relatively fine, the flow rate is slow when relying solely on gravity. Therefore, when performing reversed-phase column chromatography, a small pressurizer is used to apply appropriate pressure. Methanol-water is selected as the eluent, and the initial concentration is methanol: water = 75:25. After each elution of 5 mL, the eluate is collected and marked. After each flush of 3 retention volumes, the methanol ratio is appropriately increased, and different samples are collected and tested.

[0062] (4) Detection: The collected fractions were detected using a reverse phase plate and HPLC, and the same fractions were combined.

[0063] 1.4 Semi-preparative HPLC

[0064] (1) Sample preparation: The compound concentration was adjusted to 3 mg / mL using chromatographic methanol, and impurities were filtered using a 0.22 μm organic filter membrane.

[0065] (2) Determine the collection procedure: The semi-preparative HPLC procedure was optimized based on the analytical HPLC procedure to achieve the best purification effect. Since analytical HPLC uses gradient elution and semi-preparative HPLC uses isocratic elution, the mobile phase procedure of semi-preparative HPLC was initially determined to be 85% methanol (0-45 min) isocratic elution, the temperature was set to 35°C, and the flow rate was set to 2 mL / min. During the specific separation, the elution procedure was fine-tuned according to the different samples to achieve the purpose of separating pure compounds.

[0066] (3) Collection: The target peaks were separated on a semi-preparative HPLC, and the collection delay time was adjusted to 3 s to improve the collection efficiency and purity. By precisely controlling the collection window, cross contamination between different components can be effectively avoided, ensuring that each collected component has the highest possible purity.

[0067] (4) Detection: After the collected eluates are detected by HPLC, the same and single parts are combined, and the eluent is removed using a vacuum freeze dryer to obtain a pure compound.

[0068] The crude extract of strain YD25 obtained by liquid fermentation in KB medium was initially separated by silica gel column chromatography technology, and the composition of the crude extract was analyzed by TLC. A petroleum ether-ethyl acetate mixed solvent system was selected as the mobile phase for column chromatography separation. By TLC method, parts with similar compositions were combined, and finally 7 main compound components were obtained. Subsequently, these components were sent for HPLC detection to determine the key target compounds for separation. Next, the key components were further purified through a series of fine column chromatography processes, including silica gel column chromatography and gel column chromatography, and the appropriate mobile phase system and ratio were selected to remove impurities and improve the purity of the target compound. In the final stage of the purification process, dextran gel column chromatography technology was used for further purification, and finally a high-purity monomer compound was obtained by semi-preparative HPLC. Purity detection was also completed by HPLC to ensure that the obtained compounds met the required high purity standards.

[0069] Example

[0070] The present invention discloses a method for separating and purifying Serrawettin W2 structural analogs, comprising the following steps:

[0071] 2.1 Preliminary separation of metabolites

[0072] In the process of silica gel column chromatography separation, the first step of the experiment is to use pure petroleum ether as the eluent, the purpose is to effectively elute non-target components such as oils and fats from the sample to simplify the complexity of the subsequent separation process. This step is based on the high affinity of oils and other non-polar substances for petroleum ether, so that these components can be eluted first, thereby reducing interference for the purification of target compounds.

[0073] Select a larger chromatographic column with a size of 15×70cm and fix it firmly on the iron stand. Take 4000g of silica gel (300-400 mesh) and soak it in petroleum ether. Use the wet loading method to load it into the chromatography column. During the filling process, use a suction ball to continuously knock around the glass column to ensure that the silica gel is tight and the surface is flat. Due to the TLC experimental results and the high fatty acid content in the crude extract, the petroleum ether-ethyl acetate system is selected as the eluent, which is very effective for removing grease.

[0074] In the gradient elution process using a petroleum ether-ethyl acetate system as the eluent, the proportion of ethyl acetate is continuously increased as the elution proceeds. This process is intended to increase the polarity of the eluent, thereby more effectively separating different compounds in the fermentation crude. As the proportion of ethyl acetate increases, the polarity of the eluent gradually increases, which allows the compounds in the sample to be eluted from the silica gel column step by step according to their polarity and solubility. This gradient elution method can gradually separate the target compound from other impurities during the elution process, thereby achieving effective enrichment and purification of the target compound.

[0075] As the eluent is continuously added, it is necessary to ensure that the eluent is continuously collected. According to different degrees of separation and sample quantities, it is necessary to select appropriate containers to collect the eluent. Considering that this is the rough separation stage and the mass of the fermentation crude product is large, it is decided to use a 1L container to collect the eluent. During the elution process, the eluent will be divided into different components according to the results of silica gel column chromatography and thin layer chromatography. By observing the color development of silica gel column chromatography, the eluent can be quickly divided into different components. During the separation process, the components of some compounds can be quickly identified, and the samples can be merged according to their color development and polarity characteristics. The sample was recovered by rotary evaporation, and 7 crude separation components (components 1 to 7) were finally obtained, with sample masses of 6.2g, 7.9g, 3.9g, 146g, 112.1g, 22.8g and 203g, respectively. The elution conditions and masses of each component are shown in Table 1.

[0076] Table 1 Silica gel column chromatography separation of YD25 fermentation crude extract

[0077]

[0078] In order to further analyze the specific chemical composition of the seven components, a small amount of sample was taken from each component and redissolved and fixed to volume using chromatographic grade methanol. After that, the sample was filtered through an organic filter membrane to remove possible impurities or undissolved solid particles to ensure the cleanliness and consistency of the sample. The treated samples were sent to HPLC for further component analysis. Since the target compounds were mainly distributed between 7-13min, the analysis of the peak graph showed that the target compounds were mainly concentrated in the fifth and sixth components. After preliminary grouping and separation of the samples, the enrichment and purification of the target compounds mainly occurred in these two specific components. Subsequent experiments focused on the further purification and separation of the fifth and sixth components, using column chromatography, gradient elution or other appropriate chromatographic techniques to obtain high-purity target compounds from these two components.

[0079] 2.2 Purification of Component 5

[0080] In the crude fractionation stage of this study, 112.1 g of component 5 was obtained after the initial separation. In order to further purify this component, methanol was first used to completely dissolve it, and an ultrasonic oscillator was used to accelerate the dissolution process. After complete dissolution, considering that the sample still contained a large amount of oil impurities, it was decided to use wet column packing technology and select petroleum ether-ethyl acetate as the eluent system for elution again. Since the mass of component 5 is still large, a larger chromatography column (10×50cm) was still selected when selecting the chromatography column. Before elution, the silica gel was soaked in pure petroleum ether and stirred with a glass rod until no bubbles were generated to ensure the uniformity of the chromatography column. After that, the chromatography column was pre-equilibrated with eluent for at least two column volumes until the chromatographic baseline was stable. The sample was slowly added along the column wall using a rubber-tipped dropper to ensure that the sample layer was evenly dispersed. Usually, about 2-3 column volumes of eluent are used in the elution process, and a 100 mL container is used for collection, and about 90 mL of eluent is collected each time.

[0081] After TLC detection, the same components were combined to obtain a total of six independent components. Subsequently, these components were detected by HPLC, and it was found that the target compound was not found in the first three components 5-1, 5-2 and 5-3, while the target compound was mainly concentrated in the last three components. Since component 5-5 mainly contained sw-5 compound, and sw-5 has been proved to be the isolated compound serrawettin W2. Therefore, the focus was placed on the further separation of components 5-4 and 5-6.

[0082] In order to purify these two components, silica gel column chromatography and gel column chromatography were performed on the two components. Silica gel column chromatography was eluted with eluents of various systems such as petroleum ether-ethyl acetate and methanol-chloroform, and gel column chromatography was eluted with pure methanol and eluents of methanol-chloroform system. After multiple silica gel column chromatography and gel column chromatography on components 5-4 and 5-6, TLC detection and component merging were performed after each column chromatography, and samples other than the target compound were discarded. All samples containing the target compound were merged, and component 5-4 was separated to obtain the key component 5-4-2. Component 5-6 was separated to obtain six components, and 5-6-2 was separated to obtain the key components 5-6-2-2, 5-6-2-3, 5-6-2-4 and 5-6-2-5. 5-6-2-3 was further separated, 5-6-3 was merged into 5-4-2, and 5-6-4 was separated to obtain 5-6-4-1. 5-4-2 was separated into important components 5-4-2-2 and 5-4-2-3. During the separation process, impurities in the sample were continuously removed, and the same components were combined to enrich the desired compounds.

[0083] Given that the target compounds are a series of cyclolipopeptides with similar structures and similar relative molecular weights and structural features, there are challenges in using silica gel and gel column chromatography for separation. Silica gel columns rely on the polarity differences of compounds for separation, while gel columns distinguish compounds based on their relative molecular weights, but these methods may not be effective enough in separating cyclolipopeptides with similar structures. In the later stages of purification of such compounds, it may be difficult to achieve complete separation of the target compounds by relying solely on silica gel and gel columns, and other more sophisticated separation techniques need to be considered to ensure the purification effect. Based on the characteristics and quality of the samples, open reversed-phase column chromatography was selected for the target components. The process used wet column packing technology and dry loading methods. The elution system used was a methanol-water system with an initial ratio of 1:1. Pump pressure was used for elution, and the ratio was adjusted after flushing two column volumes of eluent, and approximately 5 mL of sample was collected each time into a 20 mL collection bottle. 5-4-2-2, 5-4-2-3, and 5-6-2-3 were subjected to reversed-phase column chromatography, detected by HPLC, and combined. After repeated reverse phase column chromatography, eight groups of components containing main compounds were obtained, and the main compounds of each group of components were different substances. Subsequently, these eight component samples were subjected to semi-preparative HPLC to obtain pure compounds that met the purity requirements of the experiment. After separating the eight groups, a total of eight compounds were obtained and named. The compounds and masses corresponding to each component are shown in Table 2 below.

[0084] Table 2 Main compounds and masses corresponding to the eight components

[0085]

[0086] 2.3 Purification of Component 6

[0087] The same strategy as component 5 was adopted when treating component 6, that is, multiple rounds of silica gel column chromatography and gel column chromatography were used to remove other compounds except the target compound, and then the components containing the target compound were separated. First, the important component 6-3 was obtained after one round of silica gel column chromatography in a petroleum ether-ethyl acetate system, and then component 6-3-2 was obtained after multiple rounds of repeated silica gel column chromatography and gel column chromatography. This component was further separated to obtain 6-3-2-2 and 6-3-2-3.

[0088] The two separated components are mainly composed of different compounds. In order to separate and purify the two components, an open reverse phase column chromatography method was adopted, and an eluent consisting of methanol and water mixed in a ratio of 3:1 was selected for separation and purification. After HPLC analysis of the separated components, two important components were found, which were marked as component 6-3-2-2-1 and component 6-3-2-3-1, as shown in Table 3 below.

[0089] Table 3 Main compounds and their masses corresponding to components 6-3-2-2-1 and 6-3-2-3-1

[0090]

[0091] Strain YD25 was fermented on a large scale, and the cells were centrifuged and extracted with anhydrous ethanol to obtain 518.8g of crude extract. The crude extract was roughly divided by silica gel column chromatography. The sample was mixed with 100-200 mesh silica gel, and the sample was ground into powder after drying. The silica gel column was loaded by wet loading. The silica gel column was continuously rinsed while loading. After the silica gel was compacted, the sample was loaded by dry loading to ensure the level of the sample layer and the silica gel layer. Petroleum ether was used as the initial eluent. The proportion of ethyl acetate was continuously increased during the elution process to increase the polarity of the eluent, so that different compounds in the fermentation crude product were eluted from the silica gel column step by step according to polarity and solubility. The sample was combined into seven crude components (components 1 to 7) by silica gel column color development and TLC detection. The target compounds were analyzed by HPLC test results. The mass was 112.1g and 22.8g respectively, and finally confirmed that the focus was on separating and purifying these two components.

[0092] Since component 5 and component 6 still contain a large amount of impurities after crude fractionation, petroleum ether-ethyl acetate system is still selected as eluent. Component 5 has a larger mass and component 6 has a smaller mass, so the two components are separated by chromatography columns of different specifications. Through multiple rounds of repeated cross-use of silica gel column chromatography, gel column chromatography and open reverse phase column chromatography, eight components are finally separated from component 5 and two components are separated from component 6. These 10 components are purified by semi-preparative HPLC to obtain 10 different compounds. After collecting and combining the different compounds, the eluent is removed by a vacuum freeze dryer to obtain pure compounds.

[0093] The present invention also discloses a structural identification of Serrawettin W2-type structural analogs, wherein the relative molecular mass of the compound is determined by mass spectrometry, and then the structure of the compound is determined by nuclear magnetic resonance, and the two are mutually verified to jointly identify the structure of the compound. The specific process is as follows:

[0094] 3.1 Mass spectrometry analysis

[0095] Take a small amount of sample, dissolve it with chromatographic methanol, and filter it with a 0.22μm organic filter membrane. Send the sample to be tested to the Kunming Institute of Botany for mass spectrometry detection. Triple TOF 6600+ ultra-high performance liquid chromatography-mass spectrometer was used for mass spectrometry analysis, and the mass spectrometry conditions were set as follows: electrospray ion source (ESI); positive ion mode. The injection volume was 5μL, the detection wavelength was 215nm, and the flow rate was 0.3mL / min; the mobile phase system was ultrapure water and chromatographic grade methanol system, and the elution gradient was 85% methanol isocratic elution for 15min.

[0096] 3.2 Nuclear magnetic resonance analysis

[0097] First, after preliminary separation by semi-preparative HPLC, the sample is further tested for purity using analytical HPLC to ensure that the sample purity meets the conditions for NMR analysis. To reduce signal interference caused by water, the sample must be completely dehydrated in a vacuum freeze dryer. Select the most suitable deuterated solvent to dissolve the sample, and test the solubility of the sample in different deuterated reagents. Take 5-10 mg of the sample and dissolve it in the selected deuterated solvent, and carefully transfer the dissolved sample to the NMR tube. The different samples were sent to the School of Chemistry and Chemical Engineering of Shaanxi Normal University and the Xi'an Institute of Modern Chemistry for testing.

[0098] A total of 10 compounds were obtained from the crude extract of isolated strain YD25, as shown in Table 4.

[0099] Table 4 Structures of 10 compounds

[0100]

[0101]

[0102] The isolated compounds are all white powdery solids, easily soluble in chloroform, methanol and DMSO, and insoluble in water. In TLC experiments, the substance can be found by phosphomolybdic acid colorimetry, and in HPLC, the presence of the substance can be detected at 215nm ultraviolet wavelength. After mass spectrometry and nuclear magnetic resonance analysis, all compounds were divided into three categories according to the length of the fatty acid carbon chain, namely compounds sw-9 and sw-11 with a fatty acid chain of C8, and compounds sw-12 with a fatty acid chain of C 10 Compounds sw-2, sw-3, sw-4, sw-5 and sw-10, the fatty acid chain is C 12 Compounds sw-6, sw-7 and sw-8 were obtained. Since sw-3 and sw-5 were previously isolated compounds, HPLC and MS / MS were performed to confirm the compounds.

[0103] 3.3 Fatty acid chain is C 10 or C 12 Compounds

[0104] (1) Compound sw-3

[0105] According to the MS / MS results, [M+H] + The peaks of the fragments 714.4660, 601.3815, 454.2995, 353.2454 and 266.2134 were analyzed and the missing amino acids were inferred to be Ile / Leu, Phe, Thr, Ser and Ile / Leu. Sw-3 may be composed of five amino acids and one C 10 The results of mass spectrometry analysis and previous research data confirmed that the molecular formula of SW-3 may be C 10 OH-Leu-Ser-Thr-Phe-Leu. The mass spectrometry detection results and the schematic diagram of each fragment peak in the mass spectrum are shown in Figure 1 shown.

[0106] pass Figure 3A and 3B middle 1 H and 13 C NMR confirmed the relationship between H and C signals in compound sw-3. 13 In the CNMR spectrum, at δ C There are carbon signals of amide bonds at 171.20, 174.59, 171.65, 171.78, 171.52 and 171.40. C There are five amino acid α-C signals at 52.28, 57.40, 59.60, 55.59 and 51.72, indicating that this compound is a cyclic peptide containing five amino acids.1 H- 1 The H COSY spectrum and HMBC spectrum show the presence of Leu, Ser, Thr, and Phe in the compound; 1 H- 1 The correlation between H-33 / H-34 / H-35 / H-36 / H-37 in the H COSY spectrum and the correlation between H-34 / H-35 and C-33 (δ C 51.72) showed the presence of Leu, and the molecular formula of compound sw-3 was determined to be C based on the results of mass spectrometry and NMR. 10 OH-Leu-Ser-Thr-Phe-Leu, sw-3 critical 1 H- 1 H COSY, HMBC and NOESY correlation diagrams Figure 2 shown.

[0107] (2) Compound sw-4

[0108] According to the MS / MS results, [M+H] + After analyzing the compound structures corresponding to fragment peaks 753.4480, 640.3650, 454.2874, 353.2412 and 266.2097 and the mass difference of each fragment peak, it is inferred that the missing amino acids are Ile / Leu, Trp, Thr, Ser and Ile / Leu in order, and fragment peak 266.2097 may be C 10 OH-Ile / Leu. Based on the MS / MS analysis results and the relative molecular mass, it is inferred that the molecular formula of sw-4 may be C 10 OH-Ile / Leu-Ser-Thr-Trp-Ile / Leu. The mass spectrometry detection results and the schematic diagram of each fragment peak in the mass spectrum are shown in Figure 4 shown.

[0109] pass Fig. 6A and 6B middle 1 H and 13 C NMR confirmed the relationship between H and C signals in compound sw-4. 13 In the CNMR spectrum, at δ C The carbon signals of amide bonds are present at 172.66, 173.53, 171.27, 171.45, 173.15 and 171.62, and the NH signals of amide bonds are present at δ H 8.67, 8.08 and 7.70, in δ CThere are five amino acid α-C signals at 51.72, 57.69, 58.25, 57.90 and 53.18, indicating that this compound is a cyclic peptide containing five amino acids. 1 H- 1 The H COSY spectrum and HMBC spectrum show the presence of Leu, Ser, and Thr in the compound; 1 H- 1 The H COSY spectrum shows that H-24 / H-25 is correlated, and the H-24 / H-25 and C-34 (δ C 173.15) related, in δ C There were eight signals at 127.49, 136.27, 111.04, 118.49, 121.93, 119.36, 109.9, and 123.83, indicating the presence of Trp; 1 H- 1 The correlation between H-35 / H-36 / H-37 / H-38 / H-39 in the H COSY spectrum and the correlation between H-35 / H-36 and C-40 (δ C 171.62) showed the presence of Ile, which corresponds to the mass spectrometry data. According to the mass spectrometry and NMR results, the molecular formula of compound sw-4 was determined to be C 10 OH-Leu-Ser-Thr-Trp-Ile, sw-4 critical 1 H- 1 HCOSY, HMBC and NOESY correlation diagrams Figure 5 shown.

[0110] (3) Compound sw-7

[0111] According to the MS / MS results, [M+H] + The fragment peak 120.0807 represents the presence of Phe in the substance. After analyzing the compound structures corresponding to the fragment peaks 742.4712, 629.3868, 482.3179, 381.2931 and 294.2421 and the mass difference of each fragment peak, it is speculated that the missing amino acids are Ile / Leu, Phe, Thr, Ser and Ile / Leu in order. The fragment peak 294.2421 may be C 12 OH-Ile / Leu. Sw-7 may be composed of five amino acids and one C 12 The molecular formula of SW-7 is inferred from the MS / MS analysis results and the relative molecular mass: C 12 OH-Ile / Leu-Ser-Thr-Phe-Ile / Leu. The mass spectrometry detection results and the schematic diagram of each fragment peak in the mass spectrum are shown in Figure 7 shown.

[0112] pass Fig. 9A and 9B middle 1 H and 13 C NMR determined the relationship between H and C signals in compound sw-11. 13 In the CNMR spectrum, at δ C There are carbon signals of amide bonds at 173.05, 172.79, 172.61, 172.94, 173.21 and 172.94. C There are five amino acid α-C signals at 53.13, 58.79, 61.03, 57.01, and 52.12, and three amide bond hydrogen signals at δ H 4.51, 4.31 and 4.07, indicating that this compound is a cyclic peptide containing five amino acids. 1 H- 1 The H COSY and HMBC spectra show the presence of Leu, Ser, and Thr in the compound; 1 H- 1 The H COSY spectrum shows that H-26 / H-27 is correlated, and the H-26 / H-27 and C-34 (δ C 173.21) related, in δ C There are four signals at 139.08, 130.29, 129.49 and 127.74, indicating the presence of Phe; 1 H- 1 The correlation between H-35 / H-36 / H-37 / H-38(H-39) in H COSY spectrum and H-35 / H-36 and C-40(δ C 172.94) showed the presence of Leu, which indicated the presence of two amino acids Leu in sw-7, which was consistent with the mass spectrometry data. Based on the mass spectrometry and NMR results, the molecular formula of compound sw-7 was determined to be C 12 OH-Leu-Ser-Thr-Phe-Leu, sw-7 critical 1 H- 1 H COSY, HMBC and NOESY correlation diagrams Figure 8 shown.

[0113] (4) Compound sw-8

[0114] According to the MS / MS results, [M+H] +The fragment peak 120.0810 represents the presence of Phe in the substance. After analyzing the compound structures corresponding to the fragment peaks 742.4732, 629.3879, 482.3197, 381.2742 and 294.2429 and the mass difference of each fragment peak, it is inferred that the missing amino acids are Ile / Leu, Phe, Thr, Ser and Ile / Leu in order. The fragment peak 294.2429 may be C 12 OH-Ile / Leu. Sw-8 may be composed of five amino acids and one C 12 The MS / MS analysis results and the relative molecular mass indicate that SW-8 is an isomer of SW-7, and the molecular formula may be C 12 OH-Ile / Leu-Ser-Thr-Phe-Ile / Leu. The mass spectrometry detection results and the schematic diagram of each fragment peak in the mass spectrum are shown in Fig.10 shown.

[0115] pass Fig. 12A and 12B middle 1 H and 13 C NMR confirmed the relationship between H and C signals in compound sw-8. 13 In the CNMR spectrum, at δ C There are carbon signals of amide bonds at 172.80, 172.52, 172.43, 172.91, 173.01 and 172.66, and 5 amide bond NH signals δ H 8.37, 8.21, 8.16, 7.8 and 7.72, in δ C There are five amino acid α-C signals at 53.13, 58.79, 61.03, 57.01 and 52.12, indicating that this compound is a cyclic peptide containing five amino acids. 1 H- 1 The H COSY and HMBC spectra show the presence of Ile, Ser, Thr, and Phe in the compound; 1 H- 1 The correlation between H-35 / H-36 / H-37 / H-38(H-39) in H COSY spectrum and H-35 / H-36 and C-40(δ C 172.66) showed the presence of Leu, and the molecular formula of sw-8 is C 12 OH-Ile-Ser-Thr-Phe-Leu. sw-8 critical 1 H- 1 H COSY, HMBC and NOESY correlation diagrams Fig.11 shown.

[0116] (5) Compound SW-10

[0117] According to the MS / MS results, [M+H] + After analyzing the compound structures corresponding to fragment peaks 739.4307, 640.3634, 454.2863, 353.2403 and 266.2093 and the mass difference of each fragment peak, it is inferred that the missing amino acids are Ile / Leu, Trp, Thr, Ser and Ile / Leu in order, and fragment peak 266.2093 may be C 10 OH-Ile / Leu. Sw-10 may be composed of these five amino acids and a C 10 The MS / MS analysis results and the relative molecular mass indicate that the molecular formula of SW-10 may be C 10 OH-Ile / Leu-Ser-Thr-Trp-Ile / Leu. The mass spectrum and the schematic diagram of each fragment peak in the mass spectrum are shown in Fig.13 shown.

[0118] pass Fig.15A and 15B middle 1 H and 13 C NMR confirmed the relationship between H and C signals in compound sw-10. 13 In the CNMR spectrum, at δ C There are carbon signals of amide bonds at 172.39, 175.44, 169.90, 170.48, 172.78 and 171.11. C There are amino acid α-C signals at 51.85, 56.97, 56.46 and 49.87, and four amide bond hydrogen signals at δ H 7.59, 7.88, 7.33 and 8.46, indicating that this compound is a cyclic peptide containing five amino acids. 1 H- 1 The H COSY and HMBC spectra show the presence of Leu, Ser, and Thr in the compound; 1 H- 1 The H COSY spectrum shows that H-24 / H-25 is correlated, and the H-24 / H-25 and C-34 (δ C 172.78) related, in δ C There are eight signals at 126.22, 134.82, 116.61, 117.19, 120.73, 118.26, 109.87, and 122.44, indicating the presence of Trp; 1 H- 1The correlation between H-35 / H-36 / H-37 (H-38) in the H COSY spectrum and the correlation between H-35 / H-36 and C-39 (δ C 171.11) showed the presence of Val, which corresponds to the mass spectrometry data. According to the mass spectrometry and NMR results, the molecular formula of compound sw-10 was determined to be C 10 OH-Leu-Ser-Thr-Trp-Val, sw-10 key 1 H- 1 HCOSY, HMBC and NOESY correlation diagrams Fig.14 shown.

[0119] In the present invention, the structures of the compounds were analyzed by mass spectrometry and nuclear magnetic resonance, and identification was carried out by combining nuclear magnetic spectroscopy data and comparing relevant literature materials. The identification results are as follows: the 10 compounds separated are all cyclolipopeptide substances, which are composed of a fatty acid carbon chain and five amino acids. SW-5 is serrawettin W2, and the other compounds are structural analogs thereof. The specific structures of the 10 compounds were identified by modern spectral techniques such as 1D-NMR, 2D-NMR and HRESIMS (MS / MS).

[0120] However, there are still problems in the separation and purification process. After MS / MS and NMR detection, it was concluded that compound sw-2 and compound sw-6 are mixtures, both of which are composed of two isomers, so it is difficult to separate them by traditional separation and purification methods. Currently, a variety of chiral columns have been used to try to separate sw-2, but unfortunately it has not been separated. More methods will be tried to separate them in the future. Sw-2 and sw-6 have similar structures and only differ in carbon chain length. Because the structures and properties of these compounds are very similar, sw-2 and sw-6 are treated as one compound in the activity experiment. It is currently known that Tanya Clements-Decker et al. have isolated sw-6-A from Serratia NP2 and named it serrawettin W2-FV12:1.

[0121] The five compounds sw-3, sw-5, sw-7, sw-8 and sw-9 have similar structures. Specifically, sw-3 and sw-5 are isomers, and there is only one amino acid difference between them. Similarly, there is also one amino acid difference between sw-7 and sw-8. In addition, there are differences in the length of the fatty acid carbon chain between sw-5, sw-8 and sw-9, which are C 10 , C 12and C8. The difference between sw-3 and sw-7 is mainly the difference in the carbon chain length of the fatty acid. In the subsequent activity experiments, their effects on biological activity will be inferred based on these structural differences.

[0122] The present invention successfully separated serrawettin W2 and 9 structural analogs thereof by using multiple column chromatography and semi-preparative HPLC techniques, and performed structural identification on these compounds.

[0123] (1) Isolation and purification of Serrawettin W2 and its structural analogs

[0124] The crude extract was separated and purified by various chromatographic separation methods. Ten monomer compounds were obtained by separation and purification of the crude extract of strain YD25, namely sw-2 to sw-11.

[0125] (3) Structural identification of Serrawettin W2 and its analogs

[0126] The compounds were tested by mass spectrometry and nuclear magnetic resonance. The structures of 10 compounds were analyzed by mass spectrometry and nuclear magnetic resonance (NMR), and identification was carried out by combining NMR spectroscopy data and comparing relevant literature. It was confirmed that all 10 compounds were cyclolipeptide compounds, namely serrawettin W2 and its structural analogs.

[0127] Of course, the above description is not a limitation of the present invention, and the present invention is not limited to the above examples. Changes, modifications, additions or substitutions made by technicians in this technical field within the essential scope of the present invention should also fall within the protection scope of the present invention.

Claims

1. A method for separating and purifying Serrawettin W2 structural analogs, characterized in that: The following steps are involved: S1. A crude extract of strain YD25 is obtained from KB medium by liquid fermentation, and the crude extract is initially separated by silica gel column chromatography, and the composition of the crude extract is analyzed by TLC; parts with similar compositions are combined to obtain the main compound components; S2. Perform HPLC detection on the obtained compound components to determine the separated key target compounds; S3, through the fine column chromatography process, the key components are further purified, and the appropriate mobile phase system and ratio are selected to remove impurities and improve the purity of the target compound; S4. Further purification was performed using dextran gel column chromatography technology, and finally a high-purity monomer compound was obtained by semi-preparative HPLC.

2. The method for separating and purifying a Serrawettin W2 structural analogue according to claim 1, characterized in that: In step S1, a petroleum ether-ethyl acetate mixed solvent system is selected as a mobile phase for column chromatography separation.

3. The method for separating and purifying a Serrawettin W2 structural analogue according to claim 2, characterized in that: In step S2, after repeated reverse phase column chromatography, components containing main compounds are obtained, and the main compounds of each group of components are different substances.

4. The method for separating and purifying a Serrawettin W2 structural analogue according to claim 3, characterized in that: In step S3, the fine column chromatography process includes silica gel column chromatography, gel column chromatography and reverse phase column chromatography, which are repeatedly used in multiple rounds.

5. The method for separating and purifying a Serrawettin W2 structural analogue according to claim 4, characterized in that: In step S4, semi-preparative HPLC adopts isocratic elution, and the mobile phase program of semi-preparative HPLC is: methanol mass fraction is 85%, 0-45min isocratic elution, temperature is 35°C, and flow rate is 2mL / min.

6. Serrawettin W2 structural analogs obtained by the separation and purification method according to claim 5.

7. A structure of the Serrawettin W2 analogue according to claim 6, characterized in that: Serrawettin W2 analogs were structurally identified by nuclear magnetic resonance and mass spectrometry. The monomer compounds were all cyclolipopeptide serrawettin W2 compounds. All compounds were divided into three categories according to the length of the fatty acid carbon chain, namely: Compounds sw-9 and sw-11 with C8 fatty acid chains; The fatty acid chain is C 10 Compounds sw-2, sw-3, sw-4, sw-5 and sw-10; The fatty acid chain is C 12 Compounds sw-6, sw-7 and sw-8.

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