CN104447917B - Low toxicity pimaricin derivant and its preparation method and application - Google Patents

Abstract

A low-toxic Pimaricin derivative, the molecular formula is C ₃₃ H ₄₉ NO ₁₀ , and the chemical structural formula is as follows: The low-toxicity pitamamycin derivative is obtained by constructing the producing strain Streptomyces chattanoogensis QZ02, fermenting and cultivating the mutant strain QZ02, treating bacteria, extracting with organic reagents, separating by reverse-phase chromatographic column chromatography and high-performance liquid chromatography. The compound of the invention has stable structure, good activity and extremely low toxicity, can be used as antifungal medicine and food preservative, and has good application prospect.

Description

Pimaricin derivative with low toxicity and its preparation method and application

technical field

The invention relates to a technology for separating secondary metabolites of microorganisms, in particular to a low-toxicity pitamamycin derivative and its preparation method and application.

technical background

With the increasing number of patients with immune system deficiency, the incidence and harm of systemic fungal infection are also increasing. Fungal pathogens have the characteristics of high transmission rate, high proliferation rate, and high lethality rate, and they are highly resistant to many drugs. At present, the antifungal drugs that can be used clinically to treat systemic fungal infections are very limited, and all of them have different degrees of side effects and toxicity. Among them, polyene antibiotics have extremely efficient antibacterial and antibacterial activities against a variety of fungal pathogens, and the development of pathogen resistance to them is very slow, and are currently recognized as the most effective antifungal drugs. However, while polyene antibiotics exhibit high antifungal activity, they also have severe hemolytic toxicity, which severely limits the clinical application value of polyene antibiotics.

Pimaricin is currently the most commonly used four polyene antibiotics (pimaricin, amphotericin B, nystatin and candicidin, etc.), with the lowest hemolytic toxicity. It is stable in nature and is widely used as food (such as cheese and other non-sterile food) antiseptic additives, antifungal veterinary drugs and for the treatment of keratitis. Therefore, on the basis of pimaricin, genetic engineering and other methods are used to modify and optimize the relevant functional groups of pimaricin, so as to further reduce its hemolytic toxicity and greatly overcome the toxic and side effects of polyene antibiotics.

Contents of the invention

One of the purposes of the present invention is to provide a low-toxicity pimaricin derivative; the second purpose is to provide the preparation method of the above-mentioned pimaricin derivative; the third purpose is to provide the application of the above-mentioned pimaricin derivative.

In order to realize the purpose of the present invention, the present invention has adopted following technical scheme:

A low-toxic Pimaricin derivative, the molecular formula is C ₃₃ H ₄₉ NO ₁₀ , and the chemical structural formula is as follows:

The preparation method of the above-mentioned low-toxic picamamycin derivatives is obtained through the construction of the production strain Streptomyceschattanoogensis QZ02, the fermentation and cultivation of the mutant strain QZ02, the treatment of bacteria, the extraction of organic reagents, reversed-phase column chromatography and high-performance liquid chromatography. Proceed as follows:

A. Construction of the mutant strain QZ02: the pimarmycin industrial high-yielding strain Streptomyces chattanogenesis L10 was used as the starting strain, and the P450 monooxygenase gene pimG in the pimarmycin biosynthesis gene cluster was inactivated and the P450 monooxygenase gene pimD was interrupted. The mutant strain QZ02 was obtained;

B. Fermentation culture of mutant strain QZ02: inoculate the spores of mutant strain QZ02 in the seed medium, cultivate at 220 rpm at 30° C. on a shaking table for 24 hours to obtain seed culture solution; then inoculate the seed culture solution at 10% inoculated in the fermentation medium, and cultured on a shaker at 220rpm at 30°C for 5 days to obtain the fermentation medium of the mutant strain QZ02;

C, thalline treatment: centrifuge the fermentation broth of the mutant strain QZ02, collect the fermented thallus, wash the thalline with water for 3 times, remove the washing liquid, and freeze-dry the thalline;

D. Organic reagent extraction: the lyophilized bacteria were ultrasonically extracted 3 times with methanol, each time for 30 minutes, centrifuged to obtain the extract, the extracts were combined, concentrated under reduced pressure to obtain the crude sample a;

E. Reversed-phase chromatographic column chromatography: Dissolve crude sample a in methanol with a weight ratio of 1.5-3 times, let it stand, filter out the precipitate, and then load the sample on reverse-phase chromatographic column chromatography, with a volume ratio of 0: 1～1:0 methanol aqueous solution for gradient elution, collect each part of the eluate, and detect by HPLC, the target compound is obtained by eluting with a 1:0 ratio of methanol aqueous solution, concentrate the eluent where the target compound is located, and obtain a crude sample b;

F. Separation by high performance liquid chromatography: the crude sample b was dissolved in a small amount of methanol, separated and purified by high performance liquid chromatography to obtain a derivative of Pimaricin.

The above-mentioned preparation method of pimaricin derivatives, wherein, the inactivation of the P450 monooxygenase gene pimG described in step A is realized by mutating the active site amino acid Cys344 of the gene pimG to Ala, and the P450 monooxygenase gene pimD The deletion was achieved by knocking out the internal 816bp base sequence of the gene pimD.

The preparation method of the above-mentioned pimaricin derivatives, wherein, the formula of the seed medium in step B is 1.75% of glucose, 1.5% of tryptone, and 1.0% of sodium chloride; the formula of the fermentation medium is 6.0% of glucose and 0.7% of yeast extract %, soybean meal powder 2.8%.

The preparation method of the above-mentioned Pimaricin derivatives, wherein, in step E, gradient elution is carried out with methanol aqueous solution with a volume ratio of 0:1 to 1:0, and the volume ratio gradient of methanol aqueous solution is 0:1, 3 :7, 4:6, 6:4, 1:0.

The preparation method of the above-mentioned Pimaricin derivatives, wherein, the high-performance liquid chromatography separation and purification described in step F uses methanol and 0.1% formic acid aqueous solution as the mobile phase, and the volume ratio of methanol and 0.1% formic acid aqueous solution is 54:46, The BDS HYPERSIL C18 reversed-phase semi-preparative column with a flow rate of 3ml/min and 250mm×10mm was used as the stationary phase, and the detection wavelength of the ultraviolet detector was 303nm. Each injection was 50-100μl, and the corresponding chromatographic peaks were collected and evaporated to dryness after multiple accumulations. .

Application of the above Pimaricin derivatives in the preparation of antifungal drugs and food preservatives.

The pimaricin derivative of the present invention is isolated and reported for the first time, and is determined to be a pimaricin derivative by nuclear magnetic resonance analysis, and its nuclear magnetic resonance data are as shown in Table 1:

Table 1

The application of the pimaricin derivatives of the present invention in the preparation of antifungal drugs and food preservatives is achieved in that, using Candida albicans as indicator bacteria, the in vitro resistance to described pimaricin derivatives and pimaricin Fungal activity was measured, and the MIC ₅₀ /MIC ₉₀ values of Pimaricin and the Pimaricin derivatives were 1.09±0.02/1.61±0.04 μg/ml and 2.10±0.19/4.43±0.41 μg/ml respectively, namely The antifungal activity of the described pimaricin derivatives is about 1/2 of that of pimaricin; taking defibrated horse whole blood as the research object, the hemolytic activity of pimaricin and the described pimaricin derivatives is measured in vitro Toxicity, it was found that the HC ₅₀ value of pimaricin was 114.0 μg/ml, and even when the concentration of the pimaricin derivative reached 800 μg/ml, the hemolysis rate caused by it was still less than 40%, that is, the horse The hemolytic toxicity of the mycin derivatives is much lower than that of pimamamycin. The picamamycin derivative described in the invention has a stable structure, good antifungal activity and extremely low hemolytic toxicity, can be used as a low toxicity antifungal drug and a food preservative, and has good application prospects.

Description of drawings

Figure 1 is a schematic diagram of the construction of the strain Streptomyces chattanoogensis QZ02 for producing Pimaricin derivatives;

Figure 2 is the hydrogen nuclear magnetic resonance spectrum ( ¹ H NMR) of the Pimaricin derivatives;

Fig. 3 is a carbon nuclear magnetic resonance spectrum ( ¹³ C NMR) of a derivative of Pimaricin.

Specific implementation method

The present invention will be further described in detail through examples below, but these examples do not imply any limitation to the present invention.

Example 1

Prepare 500ml of seed medium and 5L of fermentation medium according to the seed medium and fermentation culture formula, respectively, pack them in 250ml triangular bottles, fill each bottle with 50ml, sterilize at 115°C for 30min, insert the spores of the mutant strain QZ02 into the above seeds In the culture medium, at 220 rpm, at 30° C., on a shaker for 24 hours to obtain a seed culture solution. The seed culture solution was added to the fermentation medium according to 10% inoculum amount, and cultured on a shaker at 220 rpm at 30° C. for 5 days to obtain a fermentation culture of the mutant strain QZ02. Centrifuge the 5L fermentation culture obtained from the fermentation culture, collect the fermented cells, wash the cells with water for 3 times, remove the washing liquid, and freeze-dry the cells overnight; Centrifuge to obtain the extract, repeat the ultrasonic extraction 3 times, combine the extracts, filter the extracts, concentrate under reduced pressure to obtain about 4 g of crude sample a; dissolve the crude sample a in 12 ml of methanol, let it stand, filter out the precipitate, and then put it on the reverse phase Chromatographic column chromatography, followed by gradient elution with methanol aqueous solution with a volume ratio of 0:1, 3:7, 4:6, 6:4, 1:0, detected by HPLC, found that the Pimaricin derivative It mainly exists in the eluate obtained by eluting with methanol aqueous solution at a ratio of 1:0, and it is concentrated to obtain about 2 g of crude sample b; the crude sample b is dissolved in a small amount of methanol, separated and purified by high-performance liquid chromatography, and it is obtained as Methanol and 0.1% formic acid aqueous solution are mobile phase, ratio is 54: 46, and flow rate is 3ml/min, and the BDS HYPERSIL C18 reversed-phase semi-preparative column of 250mm * 10mm is stationary phase, and the detection wavelength of ultraviolet detector is 303nm, every injection 50 μl, the corresponding chromatographic peaks were collected, accumulated several times and then evaporated to dryness to obtain the pimarmycin derivative of the present invention.

Example 2

According to the seed medium and fermentation culture formula, prepare 1L seed medium and 10L fermentation medium respectively, pack them into 250ml triangular bottles, fill each bottle with 50ml, and sterilize at 115°C for 30min, insert the spores of the mutant strain QZ02 into the above seeds In the culture medium, at 220 rpm, at 30° C., on a shaker for 24 hours to obtain a seed culture solution. The seed culture solution was added to the fermentation medium according to 10% inoculum amount, and cultured on a shaker at 220 rpm at 30° C. for 5 days to obtain a fermentation culture of the mutant strain QZ02. Centrifuge the 10L fermentation culture obtained from the fermentation culture, collect the fermented cells, wash the cells with water for 3 times, remove the washing liquid, and freeze-dry the cells overnight; Centrifuge to obtain the extract, repeat the ultrasonic extraction 3 times, combine the extracts, filter the extracts, concentrate under reduced pressure to obtain about 9 g of crude sample a; dissolve the crude sample a in 25 ml of methanol, let it stand, filter out the precipitate, and then divide it into two Perform gradient elution with methanol-water solutions with a volume ratio of 0:1, 3:7, 4:6, 6:4, and 1:0 in sequence, collect and concentrate each part of the eluate, and pass through HPLC detects, finds that described Pimaricin derivative mainly exists in the eluate obtained by elution of the methanol aqueous solution of 1:0 ratio, it is concentrated to obtain about 4g crude sample b; The crude sample b is dissolved in a small amount of methanol Among them, through separation and purification by high performance liquid chromatography, it uses methanol and 0.1% aqueous formic acid as mobile phase, the ratio is 54:46, the flow rate is 3ml/min, and the BDS HYPERSIL C18 reversed-phase semi-preparative column of 250mm×10mm is the stationary phase. The detection wavelength of the ultraviolet detector is 303nm, 50 μl is injected each time, the corresponding chromatographic peaks are collected, accumulated for many times and then evaporated to dryness to obtain the Pimaricin derivative of the present invention.

Example 3

Determination of Antifungal Activity and Hemolytic Toxicity of Pimaricin Derivatives in Vitro

The antifungal activity in vitro was measured using Candida albicans as indicator bacteria, and the MIC ₅₀ and MIC ₉₀ values of the corresponding antibiotics were obtained by measuring the growth of Candida albicans under different antibiotic concentrations. First, the overnight culture solution of Candida albicans was inoculated in LB medium at a ratio of 1/10000, and distributed in 96-well plates at 200 μl/well. The DMSO solutions of the Pimaricin derivatives and Pimaricin at different concentrations (0-800 μg/ml) were prepared, and added to the above LB medium at a ratio of 2%. The 96-well plate was placed in an incubator at 34°C and cultured statically for 12 hours, and the OD ₆₆₀ under different concentrations of antibiotics was measured using a microplate reader. The measured data can be plotted against the antibiotic concentration to draw a bacteriostatic curve, and the MIC ₅₀ and MIC ₉₀ values can be obtained. The hemolytic toxicity of antibiotics in vitro is measured by the ability of antibiotics to lyse red blood cells. Mix 0.1ml antibiotic DMSO solution of different concentrations with 0.9ml PBS buffer containing 2.5% defibrinated horse whole blood, incubate at 37°C for 1h, centrifuge at 5000rpm for 5min, take the supernatant, and measure the OD ₅₄₅ of the sample with a microplate reader. Among them, the result obtained by mixing 0.1ml DMSO with 0.9ml PBS buffer containing 2.5% defibrated horse whole blood was used as a blank control, and the result obtained by mixing 0.1ml DMSO with 0.9ml deionized water containing 2.5% defibrated horse whole blood was 100% hemolysis. The HC ₅₀ value can be obtained by drawing the hemolysis curve of the above results against the antibiotic concentration.

For the above experiments, three groups of parallel experiments were carried out with different concentrations of antibiotics, and the experimental results are shown in Table 2.

Table 2

Above-mentioned experimental result shows, the antifungal activity of described Pimaricin derivative is about 50% of Pimaricin activity, and the hemolytic toxicity of described Pimaricin derivative is far lower than Pimaricin, even Its concentration reaches 800μg/ml, and the hemolysis rate caused by it is still less than 40%. It is one of the most effective polyene antibiotics at present. Therefore, the picamamycin derivatives of the present invention have stable structures, good antifungal activity and extremely low hemolytic toxicity, and can be used as antifungal drugs and food preservatives, and have good application prospects.

The construction process of Pimaricin derivatives producing bacterial strain Streptomyces chattanoogensis QZ02 is shown in Figure 1, and the proton nuclear magnetic resonance spectrum ( ¹ H NMR) of Pimaricin derivatives in the present invention is shown in Figure 2, and in the present invention The carbon nuclear magnetic resonance spectrum ( ¹³ C NMR) of the picamamycin derivative is shown in FIG. 3 .

Claims (7)

1. A low-toxic Pimaricin derivative, the molecular formula is C ₃₃ H ₄₉ NO ₁₀ , and the chemical structural formula is as follows: 2. the preparation method of the described low-toxicity Pimaricin derivatives of claim 1 is characterized in that, through producing strain Streptomyces chattanoogensis QZ02 construction, mutant strain QZ02 fermentation culture, thalline treatment, organic reagent extraction, reversed-phase chromatographic column layer Analysis and high performance liquid chromatography separation, the specific steps are as follows: A. Construction of the mutant strain QZ02: the pimarmycin industrial high-yielding strain Streptomyces chattanogenesis L10 was used as the starting strain, and the P450 monooxygenase gene pimG in the pimarmycin biosynthesis gene cluster was inactivated and the P450 monooxygenase gene pimD was interrupted. The mutant strain QZ02 was obtained; B. Fermentation culture of mutant strain QZ02: inoculate the spores of mutant strain QZ02 in the seed medium, cultivate at 220 rpm at 30° C. on a shaking table for 24 hours to obtain seed culture solution; then inoculate the seed culture solution at 10% inoculated in the fermentation medium, and cultured on a shaker at 220rpm at 30°C for 5 days to obtain the fermentation medium of the mutant strain QZ02; C, thalline treatment: centrifuge the fermentation broth of the mutant strain QZ02, collect the fermented thallus, wash the thalline with water for 3 times, remove the washing liquid, and freeze-dry the thalline; D. Organic reagent extraction: the lyophilized bacteria were ultrasonically extracted 3 times with methanol, each time for 30 minutes, centrifuged to obtain the extract, the extracts were combined, concentrated under reduced pressure to obtain the crude sample a; E. Reversed-phase chromatographic column chromatography: Dissolve crude sample a in methanol with a weight ratio of 1.5-3 times, let it stand, filter out the precipitate, and then load the sample on reverse-phase chromatographic column chromatography, with a volume ratio of 0: 1～1:0 methanol aqueous solution for gradient elution, collect each part of the eluate, and detect by HPLC, the target compound is obtained by eluting with 1:0 methanol aqueous solution, concentrate the eluate where the target compound is located, and obtain a crude sample b; F. Separation by high performance liquid chromatography: the crude sample b was dissolved in a small amount of methanol, separated and purified by high performance liquid chromatography to obtain a derivative of Pimaricin. 3. the preparation method of Pimaricin derivative according to claim 2 is characterized in that: the inactivation of P450 monooxygenase gene pimG described in step A is by the active site amino acid Cys344 mutation of gene pimG to Ala To realize, interrupting the P450 monooxygenase gene pimD is realized by knocking out the internal 816bp base sequence of the gene pimD. 4. the preparation method of pimaricin derivative according to claim 2 is characterized in that: the formula of seed culture medium described in step B is glucose 1.75%, tryptone 1.5%, sodium chloride 1.0%; Fermentation medium The formula is 6.0% of glucose, 0.7% of yeast extract and 2.8% of soybean cake powder. 5. according to the preparation method of claim 2 described pimaricin derivatives, it is characterized in that: step E is that the volume ratio described in step E is the aqueous methanol solution of 0:1～1:0 to carry out gradient elution, the methanol aqueous solution The volume ratio gradient is 0:1, 3:7, 4:6, 6:4, 1:0 in turn. 6. the preparation method of Pimaricin derivative according to claim 2 is characterized in that: the high performance liquid chromatography separation and purification described in step F is to be mobile phase with methanol and 0.1% formic acid aqueous solution, methanol and 0.1% The volume ratio of the formic acid aqueous solution is 54:46, the flow rate is 3ml/min, the 250mm×10mm BDS HYPERSILC18 reversed-phase semi-preparative column is used as the stationary phase, and the detection wavelength of the ultraviolet detector is 303nm. The chromatographic peaks were added several times and then evaporated to dryness. 7. the application of the described Pimaricin derivatives of claim 1 in the preparation of antifungal drugs and food preservatives.