Polyhydroxy pyridone compound as well as preparation method and application thereof

Abstract

The invention relates to the technical field of compounds, and particularly discloses a polyhydroxy pyridone compound as well as a preparation method and application thereof. The method comprises the following steps: heating fermentation liquor, then carrying out solid-liquid separation to remove denatured protein and other insoluble substances in the fermentation liquor, heating, evaporating and concentrating the obtained filtrate, adding a poor solvent, cooling, uniformly stirring, completely crystallizing, filtering, suspending and washing crude crystals with an aqueous solution of the poor solvent, and drying to obtain the polyhydroxy pyridone compound. By adopting the preparation method, the polyhydroxy pyridone compound with the purity of more than 95% and the yield of more than 95% can be obtained.

Description

Technical Field

[0001] This application relates to the field of compound technology, and in particular to a polyhydroxypyridinone compound, its preparation method, and its application. Background Technology

[0002] Pyridones are a class of heterocyclic molecules with broad biological activities, playing an important role in medicinal chemistry and natural product chemistry. Studies have shown that these compounds generally exhibit antibacterial, anti-inflammatory, and antitumor pharmacological activities. For example, 5-hydroxy-2-pyridone derivatives have been shown to significantly inhibit methicillin-resistant Staphylococcus aureus (MRSA); 3,4-dihydroxypyridones exhibit good antioxidant properties; 2-pyridones are used to synthesize antibacterial drugs (such as quinolones), antiviral drugs, or antitumor drugs; 4-pyridones bind to metal ions such as iron and aluminum to treat iron overload diseases, similar to deferoxamine; and 6-amino-2-pyridones can be used as dye intermediates in the synthesis of azo dyes.

[0003] Pyridone compounds generally exhibit antibacterial, anti-inflammatory, and antitumor pharmacological activities, and can be used as precursors in the synthesis of bioactive substances, as well as as raw materials or intermediates in the development and preparation of novel antibiotics, drugs, and dyes. This invention further isolates and purifies polyhydroxyaminopyridones from the fermentation broth of *Indigofera tinctoria*, discovering that this compound has certain applications in dyeing. This not only achieves resource utilization of the fermentation broth but also helps solve environmental problems and enhances the economics of the process. Summary of the Invention

[0004] The purpose of this application is to overcome the shortcomings of the prior art and provide a polyhydroxypyridinone compound, its preparation method, and its applications. This application isolates a polyhydroxypyridinone compound from the fermentation broth of *Gynostemma pentaphyllum*, which can be used as a precursor in the synthesis of bioactive substances, and as a raw material or intermediate in the development and preparation of novel antibiotics, drugs, and dyes.

[0005] To achieve the above objectives, the technical solution adopted in this application is as follows: This application provides a polyhydroxypyridinone compound, the structural formula of which is shown in Formula (I); the polyhydroxypyridinone compound of this application is named 5-amino-1,3,4-trihydroxy-1,2,3,6-tetrahydropyridin-2,6-dione.

[0006] Formula (I).

[0007] This application provides a polyhydroxypyridinone compound that can be used as a precursor for the synthesis of bioactive substances, and as a raw material or intermediate for the development and preparation of novel antibiotics, drugs and dyes.

[0008] This application also provides a method for preparing the above-mentioned polyhydroxypyridinone compounds, comprising the following steps: 1) The engineered bacteria IN10-06 were fermented to obtain a fermentation broth. The fermentation broth was then heated and subjected to solid-liquid separation to obtain a filtrate. 2) After heating and evaporating the filtrate to obtain a concentrated solution, a poor solvent is added to the concentrated solution, the solution is cooled and stirred to induce crystallization. After the crystallization is complete, the solution is filtered, and the crude crystals are suspended and washed with an aqueous solution containing the poor solvent and then dried to obtain polyhydroxypyridinone compounds. The engineered bacterium IN10-06 has the accession number CCTCCNO: M2024514 and is deposited at the China Center for Type Culture Collection.

[0009] The polyhydroxypyridinone compounds of this application were discovered in the fermentation broth of the strain with the above-mentioned accession number. Pure products were obtained through separation and purification. This application utilizes the difference in solubility of substances to purify them under different conditions such as temperature and solvent. The fermentation broth was heated and then solid-liquid separation was performed to remove denatured proteins and other insoluble substances from the fermentation broth. The filtrate was then heated, evaporated, concentrated, and then a poor solvent was added. After cooling and stirring until crystallization was complete, the filtrate was filtered. The crude crystals were then suspended and washed with an aqueous solution of the poor solvent and dried to obtain the polyhydroxypyridinone compounds. Using the above preparation method, polyhydroxypyridinone compounds with a purity greater than 95% and a yield greater than 95% can be obtained.

[0010] This application isolates a polyhydroxypyridinone compound from fermentation broth, which can be used to synthesize bioactive substances from precursor materials.

[0011] In a preferred embodiment of the method for preparing the polyhydroxypyridinone compounds described in this application, step 1) of the fermentation broth preparation method specifically includes the following steps: a. Inoculate the engineered bacteria IN10-06 into the culture medium, add kanamycin and culture to obtain seed culture; b. Transfer the seed culture to a basic fermentation medium containing an inducer for two-stage fermentation. Control the residual sugar to be less than 5 g / L during fermentation. Stop fermentation when the pH rebounds to 7.0-7.5 or the dissolved oxygen rebounds to 70-80% to obtain the fermentation broth. The two-stage fermentation includes: a first-stage fermentation and a second-stage fermentation. The first stage of fermentation includes the following conditions: The pre-induction culture temperature is 30~37℃, OD 600≥50℃ cooling down to 20~25℃; initial rotation speed 300~500rpm; dissolved oxygen 30~35%; ventilation ratio 0.8~1.5 vvm; pH 6.9~7.0; The second stage of fermentation includes the following conditions: Temperature: 20~25℃; Rotation speed: 800~1000rpm; Ventilation ratio: 1.2~1.5 vvm; pH: 6.9~7.0.

[0012] In some specific embodiments, the first stage of fermentation includes: an aeration ratio of 0.8 to 1.0 vvm during fermentation from 0 to 12 hours; an aeration ratio of 1.2 to 1.4 vvm during fermentation from 12 to 36 hours; and an aeration ratio of 1.5 vvm after fermentation from 36 hours.

[0013] In the technical solution of this application, the above-mentioned two-stage fermentation is adopted. In the first stage, the optimal cell growth conditions are obtained by adjusting the conditions to grow more cells. In the second stage, the optimal enzymatic reaction conditions are obtained by adjusting the conditions to induce the production of more polyhydroxypyridinone compounds and increase the yield.

[0014] In some specific embodiments, the inducer includes arabinose.

[0015] In a preferred embodiment of the method for preparing the polyhydroxypyridinone compounds described in this application, the basic fermentation medium comprises the following components at the following concentrations: Yeast powder 5~10 g / L, 10% liquid glucose 20~40 g / L, ammonium sulfate 10~20 g / L, potassium dihydrogen phosphate 0.2~1 g / L, magnesium sulfate heptahydrate 0.2~0.4 g / L, manganese sulfate monohydrate 0.01~0.02 g / L, biotin 2~4 μg / L, vitamin B1 0.2~0.4 mg / L, monosodium glutamate 1~2 g / L, solvent is water, pH=7.0~7.5.

[0016] In a preferred embodiment of the method for preparing the polyhydroxypyridinone compounds described in this application, the fermentation further includes feeding with a fed culture medium, stopping the feeding when the residual sugar is >15 g / L, and restarting the feeding when the residual sugar is <5 g / L; The fed-batch culture medium contains the following components at the following concentrations: Glucose 400~600 g / L, magnesium sulfate heptahydrate 2~4 g / L, ammonium chloride 40~80 g / L, solvent is water, pH=7.0~7.5.

[0017] In a preferred embodiment of the preparation method of the polyhydroxypyridinone compounds described in this application, in step 1), the heating temperature is 60~80℃ and the holding time is 1~2h; In step 1), the heating evaporation concentration factor is 20~50X.

[0018] The heating temperature and holding time described above in this application can achieve complete denaturation and precipitation of the protein, thereby improving the purity of the product. Furthermore, the method in this application uses a heating evaporation concentration factor of 20 to 50X, which can simultaneously improve the purity and yield of the product.

[0019] In a preferred embodiment of the preparation method of the polyhydroxypyridinone compounds described in this application, in step 2), the unsuitable solvent includes one of methanol, ethanol, isopropanol, and acetonitrile; Preferably, the unsuitable solvent is isopropanol.

[0020] The above-mentioned unsuitable solvents are unsuitable solvents for polyhydroxypyridinone compounds. This application uses the above-mentioned unsuitable solvents to better precipitate crystals and to purify the compounds by utilizing the differences in solubility of substances in different solvents, so as to obtain polyhydroxypyridinone compounds with higher purity, greater than 95%.

[0021] More preferably, the amount of isopropanol added is 1 to 5 times the amount of the concentrate.

[0022] This application uses the above-mentioned range of undesirable solvents to achieve better crystallization. When the amount of undesirable solvents added exceeds the above range, it will lead to the situation where oil is produced but crystals do not crystallize.

[0023] In a preferred embodiment of the preparation method of the polyhydroxypyridinone compounds described in this application, the cooling and crystallization temperature in step 2) is 2~12℃, and the stirring time is 1~3h.

[0024] Under these crystallization conditions, the solubility of the substance is the worst, the amount of precipitation is the largest, and the yield is also the highest.

[0025] In a preferred embodiment of the preparation method of the polyhydroxypyridinone compound described in this application, in step 2), the mass ratio of the undesirable solvent to water in the aqueous solution containing the undesirable solvent is (1~5):1; And / or, the mass ratio of the crude crystals to the aqueous solution containing a poor solvent is 1:(10~30).

[0026] In the technical solution of this application, the undesirable solvent and water in the aqueous solution of the undesirable solvent are in the above mass ratio range, which can simultaneously improve the yield and purity of the product.

[0027] This application also provides the application of the above-mentioned polyhydroxypyridinone compounds in dyes.

[0028] Compared with the prior art, this application has the following beneficial effects: This application provides a polyhydroxypyridinone compound, its preparation method, and its application. The method involves heating the fermentation broth and then performing solid-liquid separation to remove denatured proteins and other insoluble substances from the fermentation broth. After obtaining the filtrate, the filtrate is heated, evaporated, concentrated, and then a poor solvent is added. The mixture is then cooled, stirred until homogeneous, and crystallized completely. After filtration, the crude crystals are suspended and washed with an aqueous solution of the poor solvent and then dried to obtain the polyhydroxypyridinone compound. Using the above preparation method, polyhydroxypyridinone compounds with a purity greater than 95% and a yield greater than 95% can be obtained. Attached Figure Description

[0029] Figure 1 The images show the liquid phase (A) and ultraviolet absorption spectrum (B) of the fermentation broth in Example 1. Figure 2 The liquid phase (A) and ultraviolet absorption spectrum (B) of the pure 5-amino-1,3,4-trihydroxy-1,2,3,6-tetrahydropyridine-2,6-dione in Example 1 are shown. Figure 3 The hydrogen spectrum of Example 1; Figure 4 The carbon spectrum of Example 1; Figure 5 The HSQC diagram for Example 1; Figure 6 The HMBC diagram for Example 1; Figure 7 The infrared spectrum of Example 1; Figure 8 This is the liquid chromatography-mass spectrum of Example 1; Figure 9 Images of cotton fabrics dyed with different concentrations of 5-amino-1,3,4-trihydroxy-1,2,3,6-tetrahydropyridine-2,6-dione. Detailed Implementation

[0030] To better illustrate the purpose, technical solution, and advantages of this application, the following description will be provided in conjunction with the accompanying drawings and specific embodiments.

[0031] Unless otherwise specified, the experimental methods used in the following examples and comparative examples are conventional methods, and the materials and reagents used are commercially available unless otherwise specified.

[0032] In the following embodiments, the detection chromatographic conditions include: Mobile phase: gradient elution of methanol and water, as shown in Table 1 below.

[0033] Table 1 Wavelength: 330nm; Flow rate: 0.6 ml / min, column temperature: 35℃, injection volume: 10 μL, run time: 20 min; Sample preparation solution: water; Column: ALCOEN Hadesll C18 4.6*250mm 5μm, 100A.

[0034] A 1 mg / mL stock solution of 5-amino-1,3,4-trihydroxy-1,2,3,6-tetrahydropyridine-2,6-dione standard was prepared, and then serially diluted to different concentrations for high-performance liquid chromatography (HPLC). A standard curve was plotted with the prepared sample concentration on the x-axis and the corresponding peak area on the y-axis. The obtained standard curve result was Y = 184806X + 15878, R0 2 =0.9996.

[0035] In the following embodiments, the engineered bacterium IN10-06 (Escherichiacoli IN10-06) with accession number CCTCCNO: M2024514 is derived from patent number 2024104844582 and was provided by Nanjing Hegu Life Biotechnology Co., Ltd.

[0036] In the following examples, the basal fermentation medium comprises the following components at the following concentrations: Yeast powder 10 g / L, 10% liquid glucose 30 g / L, ammonium sulfate 20 g / L, potassium dihydrogen phosphate 0.5 g / L, magnesium sulfate heptahydrate 0.3 g / L, manganese sulfate monohydrate 0.02 g / L, biotin 3 μg / L, vitamin B1 0.3 mg / L, monosodium glutamate 2 g / L, solvent: water, pH=7.0.

[0037] Example 1: Fermentation production and purification of polyhydroxypyridinone compounds 1) The glycerol culture of engineered bacteria IN10-06 (Escherichiacoli IN10-06), deposited at the China Center for Type Culture Collection (CCTCCNO: M2024514), was inoculated into a test tube containing LB liquid medium and cultured with shaking. Then, 1% of the fermentation medium volume was transferred to an Erlenmeyer flask containing basal fermentation medium, and kanamycin was added to a final concentration of 50 μg / mL. The culture conditions were: temperature 35℃, rotation speed 200 rpm, time 8 h, until the OD of the bacterial culture was reached. 600 =3.5, to obtain seed liquid; 2) Prepare 50L of basic fermentation medium and place it in a 100L fermenter for autoclaving. After sterilization, wait for the basic fermentation medium to cool to 25℃, then use ammonia to adjust its pH to 7.0 and add 0.5g / L of arabinose to the fermenter. Transfer the seed liquid obtained in step (1) to the fermenter through flame inoculation and start fermentation. Control the temperature at around 25℃ throughout the fermentation process, with an initial aeration ratio of 0.8vvm, an initial rotation speed of 200rpm, and dissolved oxygen rotation speed correlation at 35%. When the rotation speed reaches 900rpm, cancel the dissolved oxygen rotation speed correlation and control the dissolved oxygen at 35% by increasing the aeration ratio (1.0vvm). Control the pH at 7.0 throughout the fermentation process. When the dissolved oxygen rapidly rebounds to above 50%, add feed medium for feeding. The initial feeding rate is 3g / L / h, and the residual sugar is controlled to be less than 5g / L throughout the fermentation process. Stop feeding after 72h and wait for the pH to rebound to 7. Fermentation was terminated after dissolved oxygen rebounded to 70% or 0.5%, yielding the fermentation broth. The yield of polyhydroxypyridinone compounds was measured at 3.5 g / L (see Figure 1 for the liquid phase (A) and UV absorption spectrum (B) of the fermentation broth in Example 1). Figure 1 (As shown).

[0038] 3) Take 5L of fermentation broth (3.5g / L of polyhydroxypyridinone compounds, initial pH 7.0), heat the fermentation broth to 70℃ and keep it at that temperature for 2h, centrifuge at 5000g for 10min to remove insoluble matter, and concentrate by rotary evaporation at 80℃ for 30X to obtain a concentrated solution. Add 2X isopropanol to the concentrated solution, cool it to 8℃ and stir for 2h. After complete crystallization, centrifuge at 5000g to obtain crude crystals. Add the obtained crude crystals to an aqueous solution containing a poor solvent (isopropanol:water = 2:1) at 8℃ and stir for 2h to separate the solid and liquid. Dry the solid to obtain the polyhydroxypyridinone compounds (5-amino-1,3,4-trihydroxy-1,2,3,6-tetrahydropyridine-2,6-dione, pure product). A total of 16.95g of pure product was obtained, with a yield of 96.86% and a purity of 97.7%.

[0039] Pure 5-amino-1,3,4-trihydroxy-1,2,3,6-tetrahydropyridine-2,6-dione liquid phase (A) and ultraviolet absorption spectrum (B) are shown below. Figure 2 As shown.

[0040] The characterization analysis of polyhydroxypyridinone compounds (pure 5-amino-1,3,4-trihydroxy-1,2,3,6-tetrahydropyridine-2,6-dione) is as follows: The structural formula of polyhydroxypyridinone compounds is shown in (I).

[0041] (I) 1H NMR (400 MHz, DMSO- d 6) δ 10.84 (s, 1H), 7.07 (s, 2H), 5.38 (s, 2H), 4.47 (s, 1H).

[0042] 13 C NMR (101 MHz, DMSO) δ 168.83, 165.75, 133.53, 114.25, 67.70.

[0043] ESI-MS: m / z: 175.0 [M+H] + .

[0044] Table 2. Carbon and Hydrogen Data Attribution δ168.83, 165.75: carbonyl carbon (C=O, pyridinide core structure); δ133.53: olefin carbon associated with intracyclic double bond; δ114.25: aliphatic carbon associated with C-NH2 (amino substitution effect); δ67.70: aliphatic carbon of CH-OH.

[0045] The correlation between δ4.47 (¹H) and δ67.70 (¹³C) confirms the CH-O fragment; δ10.84 (OH) is long-range coupled with the carbonyl carbon (δ168.83 / 165.75), supporting a hydrogen bond network; and the correlation between δ5.38 (NH2) and δ114.25 (C-NH2) verifies the amino position. (3350 cm⁻¹) -1 3185 cm -1 The OH / NH stretching vibration (hydrogen bonding) is located at 1676 cm⁻¹. -1 C=O stretching vibration (carbonyl group), 1570 cm⁻¹ -1 NH bending or C=C vibration, 1328 cm -1 CN or CO stretching vibrations. The correlation between δ4.47 (¹H) and δ67.70 (¹³C) confirms direct CH coupling, supporting the CH-O segment.

[0046] Carbonyl signals from ¹³C NMR (δ 168.83, 165.75) and IR 1676 cm⁻¹ were obtained. -1 The pyridinide skeleton was confirmed; broad peaks were observed at δ7.07(OH) and δ10.84(OH) in ¹H NMR and at 3350-3185 cm⁻¹ in IR. -1 Amino and hydroxyl substitutions were determined, with δ133.53 (¹³C) and δ5.38 / 4.47 (¹H) supporting unsaturated and substituted structures on the ring.

[0047] The proton NMR spectrum of polyhydroxypyridinone compounds is as follows: Figure 3 As shown, the carbon spectra of polyhydroxypyridinone compounds are as follows: Figure 4 As shown, the HSQC of polyhydroxypyridinone compounds is as follows: Figure 5 As shown, the HMBC of polyhydroxypyridinone compounds is as follows: Figure 6 As shown, the extra-liquid phase mass spectra of polyhydroxypyridinone compounds are as follows: Figure 7 As shown, the liquid chromatography-mass spectra of polyhydroxypyridinone compounds are as follows: Figure 8 As shown.

[0048] Example 2 Similar to Example 1, the difference in Example 2 lies in step 3), which is different: Take 5L of fermentation broth (yield of 3.5g / L of polyhydroxypyridinone compounds, initial pH 7.0), heat the fermentation broth to 80℃ and keep it at that temperature for 1h, centrifuge at 5000g for 10min to remove insoluble matter, and concentrate by rotary evaporation at 80℃ for 50X to obtain a concentrated solution. Add 5X isopropanol to the concentrated solution, cool to 2℃ and stir for 2h. After complete crystallization, centrifuge at 5000g to obtain crude crystals. Add the obtained crude crystals to an aqueous solution containing a poor solvent (isopropanol:water = 5:1) at 2℃ and stir for 2h. Separate the solid and liquid, and dry the solid to obtain pure 5-amino-1,3,4-trihydroxy-1,2,3,6-tetrahydropyridine-2,6-dione. A total of 17.31g of pure product was obtained, with a yield of 98.91% and a purity of 95.2%.

[0049] Example 3 Similar to Example 1, the difference in Example 3 is that step 3) is different, specifically: Take 5L of fermentation broth (yield of 3.5g / L of polyhydroxypyridinone compounds, initial pH 7.0), heat the fermentation broth to 60℃ and keep it at that temperature for 2h, centrifuge at 5000g for 10min to remove insoluble matter, and concentrate by rotary evaporation at 80℃ for 20X to obtain a concentrated solution. Add 1X isopropanol to the concentrated solution, cool to 12℃ and stir for 1h. After complete crystallization, centrifuge at 5000g to obtain crude crystals. Add the obtained crude crystals to an aqueous solution containing a poor solvent (isopropanol:water = 1:1) at 12℃, stir for 2h, and then separate the solid and liquid. Dry the solid to obtain pure 5-amino-1,3,4-trihydroxy-1,2,3,6-tetrahydropyridine-2,6-dione, yielding 16.63g of pure product, with a yield of 95.02% and a purity of 99.2%.

[0050] Example 4 Similar to Example 1, Example 4 differs in that step 3) is different, specifically: Take 5 L of fermentation broth (yield of 3.5 g / L of polyhydroxypyridinone compounds, initial pH 7.0), heat the fermentation broth to 70 °C and keep it at that temperature for 2 h, centrifuge at 5000 g for 10 min to remove insoluble matter, and concentrate by rotary evaporation at 80 °C for 30X to obtain a concentrated solution. Add 2X ethanol to the concentrated solution, cool to 8 °C and stir for 3 h. After complete crystallization, centrifuge at 5000 g to obtain the crude product. Add the obtained crude product crystals to an aqueous solution (ethanol:water = 2:1) containing a poor solvent at 8 °C, stir for 2 h, and then separate the solid and liquid. Dry the solid to obtain pure 5-amino-1,3,4-trihydroxy-1,2,3,6-tetrahydropyridin-2,6-dione. A total of 16.95 g of pure product was obtained, with a yield of 95.01% and a purity of 97.8%.

[0051] Example 5 Similar to Example 1, Example 5 differs in that step 3) is different, specifically: Take 5 L of fermentation broth (yield of 3.5 g / L of polyhydroxypyridinone compounds, initial pH 7.0), heat the fermentation broth to 70 °C and keep it at that temperature for 2 h, centrifuge at 5000 g for 10 min to remove insoluble matter, and concentrate by rotary evaporation at 80 °C for 30X to obtain a concentrated solution. Add 2X methanol to the concentrated solution, cool to 8 °C and stir for 3 h. After complete crystallization, centrifuge at 5000 g to obtain the crude product. Add the obtained crude product crystals to an aqueous solution containing a poor solvent (methanol:water = 2:1) at 8 °C and stir for 2 h. Separate the solid and liquid, and dry the solid to obtain pure 5-amino-1,3,4-trihydroxy-1,2,3,6-tetrahydropyridin-2,6-dione. A total of 17.01 g of pure product was obtained, with a yield of 95.53% and a purity of 98.4%.

[0052] Comparative Example 1 Similar to Example 1, the difference in Comparative Example 1 lies in step 3), which is different: Take 5L of fermentation broth (yield of 3.5g / L of polyhydroxypyridinone compounds, initial pH 7.0), heat the fermentation broth to 50℃ and keep it at that temperature for 2h, centrifuge at 5000g for 10min to remove insoluble matter, concentrate by rotary evaporation at 80℃ for 30X to obtain a concentrated solution, add 2X isopropanol to the concentrated solution, cool to 8℃ and stir for 2h, after complete crystallization, centrifuge at 5000g to obtain the crude product; add the obtained solid to an aqueous solution containing a poor solvent (isopropanol:water = 2:1) at 8℃, stir for 2h, and then separate the solid and liquid. Dry the solid to obtain pure 5-amino-1,3,4-trihydroxy-1,2,3,6-tetrahydropyridine-2,6-dione, a total of 16.85g of pure adenine was obtained, with a yield of 96.29% and a purity of 92.4%.

[0053] Comparative Example 2 Similar to Example 1, the difference in Comparative Example 2 lies in step 3), which is different: Take 5 L of fermentation broth (yield of 3.5 g / L of polyhydroxypyridinone compounds, initial pH 7.0), heat the fermentation broth to 70 °C and keep it at that temperature for 2 h, centrifuge at 5000 g for 10 min to remove insoluble matter, and concentrate by rotary evaporation at 80 °C for 10X to obtain a concentrated solution. Add 2X isopropanol to the concentrated solution, cool to 8 °C and stir for 2 h. After complete crystallization, centrifuge at 5000 g to obtain the crude product. Add the obtained solid to an aqueous solution containing a poor solvent (isopropanol:water = 2:1) at 8 °C, stir for 2 h, and then separate the solid and liquid. Dry the solid to obtain pure 5-amino-1,3,4-trihydroxy-1,2,3,6-tetrahydropyridine-2,6-dione. A total of 15.06 g of pure product was obtained, with a yield of 86.06% and a purity of 99.2%.

[0054] Comparative Example 3 Similar to Example 1, the difference in Comparative Example 3 is that step 3) is different, specifically: Take 5 L of fermentation broth (yield of polyhydroxypyridinone compounds 3.5 g / L, initial pH 7.0), heat the fermentation broth to 70 °C and keep it at that temperature for 2 h, centrifuge at 5000 g for 10 min to remove insoluble matter, and concentrate by rotary evaporation at 80 °C for 100X to obtain a concentrated solution. Add 2X isopropanol to the concentrated solution, cool to 8 °C and stir for 2 h. After complete crystallization, centrifuge at 5000 g to obtain the crude product. Add the obtained solid to an aqueous solution containing a poor solvent (isopropanol:water = 2:1) at 8 °C, stir for 2 h, and then separate the solid and liquid. Dry the solid to obtain pure 5-amino-1,3,4-trihydroxy-1,2,3,6-tetrahydropyridine-2,6-dione. A total of 17.21 g of pure product was obtained, with a yield of 98.34% and a purity of 89.1%.

[0055] Comparative Example 4 Similar to Example 1, the difference in Comparative Example 4 lies in step 3), which is different: Take 5L of fermentation broth (3.5g / L of polyhydroxypyridinone compounds, initial pH 7.0), heat the fermentation broth to 70℃ and keep it at that temperature for 2h, centrifuge at 5000g for 10min to remove insoluble matter, and concentrate by rotary evaporation at 80℃ for 30X to obtain concentrated liquid. Add 10X isopropanol to the concentrated liquid and then cool it to 8℃ and stir for 2h. The substances in the solution are oily and no solid can be precipitated.

[0056] Comparative Example 5 Similar to Example 1, the difference in Comparative Example 5 lies in step 3), which is different: Take 5L of fermentation broth (yield of polyhydroxypyridinone compounds 3.5g / L, initial pH 7.0), heat the fermentation broth to 70℃ and keep it at that temperature for 2h, centrifuge at 5000g for 10min to remove insoluble matter, and concentrate by rotary evaporation at 80℃ for 30X to obtain a concentrated solution. Add 2X isopropanol to the concentrated solution, cool to 8℃ and stir for 2h. After complete crystallization, centrifuge at 5000g to obtain the crude product. Add the obtained solid to an aqueous solution containing a poor solvent (isopropanol:water = 10:1) at 8℃, stir for 2h, and then separate the solid and liquid. Dry the solid to obtain pure 5-amino-1,3,4-trihydroxy-1,2,3,6-tetrahydropyridine-2,6-dione. A total of 16.99g of pure product was obtained, with a yield of 97.09% and a purity of 90.7%.

[0057] Comparative Example 6 Similar to Example 1, the difference in Comparative Example 6 lies in step 3), which is different: Take 5L of fermentation broth (3.5g / L of polyhydroxypyridinone compounds, initial pH 7.0), heat the fermentation broth to 70℃ and keep it at that temperature for 2h, centrifuge at 5000g for 10min to remove insoluble matter, and concentrate by rotary evaporation at 80℃ for 30X to obtain concentrated liquid. Add 2X of acetone to the concentrated liquid and cool it to 8℃ and stir. It was found that oil was produced but no crystals were precipitated and no solid could be obtained.

[0058] Comparative Example 7 Similar to Example 1, the difference in Comparative Example 6 lies in step 3), which is different: Take 5L of fermentation broth (yield of polyhydroxypyridinone compounds 3.5g / L, initial pH 7.0), heat the fermentation broth to 70℃ and keep it at that temperature for 2h, centrifuge at 5000g for 10min to remove insoluble matter, and concentrate by rotary evaporation at 80℃ for 30X to obtain a concentrated solution. Add 2X isopropanol to the concentrated solution, cool to 30℃ and stir for 2h. After complete crystallization, centrifuge at 5000g to obtain the crude product. Add the obtained solid to an aqueous solution containing a poor solvent (isopropanol:water = 2:1) at 30℃, stir for 2h, and then separate the solid and liquid. Dry the solid to obtain the polyhydroxypyridinone compounds (5-amino-1,3,4-trihydroxy-1,2,3,6-tetrahydropyridine-2,6-dione, pure product). A total of 16.95g of pure product was obtained, with a yield of 96.86% and a purity of 97.7%.

[0059] The results of preparing pure 5-amino-1,3,4-trihydroxy-1,2,3,6-tetrahydropyridine-2,6-dione in Examples 1-5 and Comparative Examples 1-7 are shown in Table 3.

[0060] Application Example 1 The polyhydroxypyridinone compound (pure 5-amino-1,3,4-trihydroxy-1,2,3,6-tetrahydropyridine-2,6-dione) sample prepared in Example 1 was uniformly dispersed in 500 mL of distilled water. The pH was adjusted to 5 with acetic acid, and fabric samples were placed in the water for dyeing. The temperature was raised to 130°C and maintained for 1 hour. After cooling, the fabric samples were rinsed with water and the color was measured. The liquor ratio was 1:50. Figure 9 Figures showing cotton fabrics dyed with different concentrations of 5-amino-1,3,4-trihydroxy-1,2,3,6-tetrahydropyridine-2,6-dione.

[0061] Table 3 A comparison of the 5-amino-1,3,4-trihydroxy-1,2,3,6-tetrahydropyridine-2,6-dione products obtained under different purification conditions in Examples 1-5 and Comparative Examples 1-6 revealed that the purification conditions in Examples 1-5 are within the scope of this invention and can produce products with high purity and yield. In Example 4, replacing isopropanol with ethanol still yielded a product with high purity and a yield of 95%.

[0062] Comparing Example 1 and Comparative Example 1, it can be found that insufficient heating temperature leads to incomplete protein denaturation and precipitation, ultimately affecting product purity. Comparing Example 1, Comparative Examples 2 and 3, it can be found that while a lower rotary evaporation concentration factor increases the final product purity, it also reduces the final product yield; while a higher rotary evaporation concentration factor increases the yield, it also decreases the product purity. In Comparative Example 4, exceeding the acceptable solvent range results in oil precipitation without crystallization. Comparing Example 1 and Comparative Example 5, it can be found that exceeding the acceptable ratio of isopropanol and water in the suspension washing of crude crystals with an unsuitable solvent increases the yield but decreases the purity. In Comparative Example 6, acetone was used instead of isopropanol, resulting in oil precipitation without crystallization during the crystallization process, making it impossible to obtain solids. Comparing Example 1 and Comparative Example 7, it can be found that the yield decreases when the cooling crystallization and suspension washing temperatures are high.

[0063] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application and are not intended to limit the scope of protection of this application. Although this application has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of this application without departing from the substance and scope of the technical solutions of this application.

Claims

1. A polyhydroxypyridinone compound, characterized in that, The structural formula of the polyhydroxypyridinone compounds is shown in formula (I); Formula (I).

2. The method for preparing the polyhydroxypyridinone compound as described in claim 1, characterized in that, Includes the following steps: 1) The engineered bacteria IN10-06 were fermented to obtain a fermentation broth. The fermentation broth was then heated and subjected to solid-liquid separation to obtain a filtrate. 2) After heating and evaporating the filtrate to obtain a concentrated solution, a poor solvent is added to the concentrated solution, the solution is cooled and stirred to induce crystallization. After the crystallization is complete, the solution is filtered, and the crude crystals are suspended and washed with an aqueous solution containing the poor solvent and then dried to obtain polyhydroxypyridinone compounds. The engineered bacterium IN10-06 has the accession number CCTCCNO: M2024514 and is deposited at the China Center for Type Culture Collection.

3. The method for preparing polyhydroxypyridinone compounds as described in claim 2, characterized in that, In step 1), the method for preparing the fermentation broth specifically includes the following steps: a. Inoculate the engineered bacteria IN10-06 into the culture medium, add kanamycin and culture to obtain seed culture; b. Transfer the seed culture to a basic fermentation medium containing an inducer for two-stage fermentation. Control the residual sugar to be less than 5 g / L during fermentation. Stop fermentation when the pH rebounds to between 7.2 and 7.5 or the dissolved oxygen rebounds to 70-80% to obtain the fermentation broth. The two-stage fermentation includes: a first-stage fermentation and a second-stage fermentation. The first stage of fermentation includes the following conditions: The pre-induction culture temperature is 30~37℃, OD 600 ≥50℃ cooling down to 20~25℃; initial rotation speed 300~500rpm; dissolved oxygen 30~35%; ventilation ratio 0.8~1.5 vvm; pH 6.9~7.0; The second stage of fermentation includes the following conditions: Temperature: 20~25℃; Rotation speed: 800~1000rpm; Ventilation ratio: 1.2~1.5 vvm; pH: 6.9~7.

0.

4. The method for preparing polyhydroxypyridinone compounds as described in claim 3, characterized in that, The basic fermentation medium comprises the following components at the following concentrations: Yeast powder 5~10 g / L, 10% liquid glucose 20~40 g / L, ammonium sulfate 10~20 g / L, potassium dihydrogen phosphate 0.2~1 g / L, magnesium sulfate heptahydrate 0.2~0.4 g / L, manganese sulfate monohydrate 0.01~0.02 g / L, biotin 2~4 μg / L, vitamin B1 0.2~0.4 mg / L, monosodium glutamate 1~2 g / L, solvent is water, pH=7.0~7.

5.

5. The method for preparing the polyhydroxypyridinone compound as described in claim 3, characterized in that, The fermentation process also includes feeding with a fed medium. When the residual sugar is >15g / L, the sugar feeding is stopped, and when the residual sugar is <5g / L, the feeding is restarted. The fed-batch culture medium contains the following components at the following concentrations: Glucose 400~600 g / L, magnesium sulfate heptahydrate 2~4 g / L, ammonium chloride 40~80 g / L, solvent is water, pH=7.0~7.

5.

6. The method for preparing the polyhydroxypyridinone compound as described in claim 2, characterized in that, In step 1), the heating temperature is 60~80℃, and the holding time is 1~2h; And / or, in step 1), the heating evaporation concentration factor is 20~50X.

7. The method for preparing the polyhydroxypyridinone compound as described in claim 2, characterized in that, In step 2), the undesirable solvent includes one of methanol, ethanol, isopropanol, and acetonitrile; Preferably, the unsuitable solvent is isopropanol.

8. The method for preparing the polyhydroxypyridinone compound as described in claim 2, characterized in that, In step 2), the cooling temperature for crystallization is 2~12℃, and the stirring time is 1~3h.

9. The method for preparing the polyhydroxypyridinone compound as described in claim 2, characterized in that, In step 2), the mass ratio of the undesirable solvent to water in the aqueous solution containing the undesirable solvent is (1~5):1; And / or, the mass ratio of the crude crystals to the aqueous solution containing a poor solvent is 1:(10~30).

10. The use of the polyhydroxypyridinone compounds as described in claim 1 in dyes.

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