CN111377987A - A group of rapamycin glucoside compounds and their enzymatic preparation and application - Google Patents

Abstract

The invention discloses rapamycin glucoside compounds, which are rapamycin 40-O- β -D glucoside, rapamycin 28-O- β -D glucoside or rapamycin 28,40-O- β -D glucoside, wherein the compounds are obtained by mediating glycosylation reaction of rapamycin, UDP-O- β -D-glucose and glycosyltransferase BSbGT-1. the invention also discloses application of the rapamycin glucoside compounds in preparing medicaments for preventing and treating animal or plant pathogenic bacteria, liver cancer and breast cancer.

Description

A group of rapamycin glucoside compounds and their enzymatic preparation and application

technical field

The invention relates to a group of rapamycin glucoside compounds and their enzymatic preparation and application, in particular to a group of rapamycin glucoside derivatives and a method for their enzymatic preparation, as well as the rapamycin glucoside derivatives Application in the preparation of medicines for the treatment and prevention of animal and plant pathogenic bacteria, liver cancer and breast cancer. It belongs to the technical field of microbial technology and its products and applications.

Background technique

Rapamycin (Rapamycin, also known as Sirolimus, Sirolimus) is a nitrogen-containing triene macrolide antibiotic produced by Streptomyces hygroscopicus. It was initially found to have good antifungal activity. In September, the FDA approved it as an immunosuppressant for the prevention and treatment of organ transplant rejection. The target of rapamycin is called TOR (Target of Rapamycin), which is an atypical serine/threonine protein kinase that exists in the cytoplasm and is an important signal transduction molecule that regulates cell proliferation, growth, differentiation and cell cycle. . For mammals, the target of rapamycin is called mTOR (mammalian Target of Rapamycin). Studies have shown that rapamycin acting on the mTOR target can inhibit tumor proliferation, arrest tumor cell cycle, and induce tumor cell apoptosis. Its structural formula is as follows:

Rapamycin structure

With the structure of rapamycin being solved, a variety of rapamycin analogs have entered different stages of clinical evaluation. The FDA approved Temsirolimus and everolimus in 2007 and 2009, respectively. (Everolimus) for the treatment of advanced renal cell carcinoma. Subsequently, the combination of everolimus and other antitumor drugs has been approved for marketing in the United States, the European Union, Japan, China and other countries for the treatment of pancreatic cancer, advanced breast cancer, prostate cancer and other tumors. Poor water solubility and stability, low bioavailability and blood toxicity are still important factors affecting its clinical application. Based on this, it is particularly necessary to modify the structural chemistry of rapamycin or discover structural analogs.

Glycosylation modification of natural medicines has an important influence on the water solubility, stability, and biological activity of the compounds themselves. At present, there are few reports on the glycosylation modification of rapamycin, which is limited to the rapamycin glycosides reported by Gantt in 2011 and the rapamycin glycoconjugates chemically synthesized by Moni et al. in 2013. Other types Glycosylation products have not been reported and the biological activities of the rapamycin glycoside compounds reported above are unknown. In addition, chemical synthesis has many by-products, difficult separation and purification, not green and environmentally friendly, and difficult to selectively protect. In view of this, there is an urgent need to find a more efficient alternative method for glycosylation modification of rapamycin, and to prepare a more abundant variety of rapamycin glycoside compounds.

SUMMARY OF THE INVENTION

Aiming at the deficiencies of the prior art, the problem to be solved by the present invention is to provide a group of rapamycin glucoside compounds and their enzymatic preparation methods, and the preparation of the rapamycin glucoside derivatives in the preparation of treatment and prevention animals and Application in medicine of plant pathogenic bacteria, liver cancer and breast cancer.

The group of rapamycin glucoside compounds according to the present invention is characterized in that the rapamycin glucoside compound is compound 1: rapamycin 40-O-β-D glucoside; compound 2: rapamycin 28-O-β-D glucoside; compound 3: rapamycin 28,40-O-β-D glucoside; the compound structural formula and its connection mode and corresponding names are as follows:

R group rapamycin glucoside nomenclature Compound 1 R₁=glucosyl,R₂=H Rapamycin40-O-β-Dglucoside Compound 2 R₁=H,R₂=glucosyl Rapamycin 28-O-β-D glucoside Compound 3 R₁=glucosyl, R₂=glucosyl Rapamycin28,40-O-β-D glucoside

Wherein: the rapamycin glucoside compound is preferably compound 1: rapamycin 40-O-β-D glucoside.

The method for enzymatic preparation of rapamycin glucoside compounds of the present invention is:

In the buffer environment of 50 mM Tris-HCl, 10 mM MgCl ₂ , the final concentration of 200 μM rapamycin and the final concentration of 1 mM UDP-O-β-D glucose and 500 μg/ml glycosyltransferase BsGT- 1 Carry out the in vitro enzyme activity reaction experiment, incubate the reaction mixture at 37 ± 1 °C for 8 ± 0.5 hours, then add 3 times the volume of acetonitrile to terminate the reaction, 14000 r/min, 30 min to remove protein precipitation, spin the sample to dryness, and then Add acetonitrile to resuspend the product, centrifuge again at 14000r/min for 30min, and then separate and purify the product through semi-preparative liquid phase; the size of the chromatographic column is YMC-Pack Pro C18, 250mm×10.0mm, 5μm; mobile phase system: 55:45 Elution with acetonitrile water system, the peak time of rapamycin glucoside: compound 1: 11.1 min; compound 2: 8.4 min; compound ₃ : 5.2 min; CN was dissolved and identified by UHPLC-ESI-Q-TOF high-resolution mass spectrometry and nuclear magnetic resonance, respectively.

The above-mentioned glycosyltransferase BsGT-1 (CUB50191), the protein sequence of which has been published, was transformed into Escherichia coli BL21 (DE3) by pET28a-BsGT-1 recombinant plasmid to induce expression to obtain the protein.

In the above-mentioned method for enzymatic preparation of rapamycin glucoside compounds: the molar ratio of the mixture of rapamycin and UDP-O-β-Dglucose is preferably 1:5, which is mediated by glycosyltransferase BsGT-1. The reaction mixture for conducting the glycosylation reaction is preferably incubated at 37°C for 8 hours.

The application of the rapamycin glucoside compound of the present invention in the preparation of medicines for preventing and treating pathogenic bacteria of animals and plants, liver cancer and breast cancer.

Experiments have confirmed that the rapamycin glucoside compounds of the present invention have preventive and therapeutic effects on animal and plant pathogenic bacteria, liver cancer and breast cancer. Among them, the rapamycin glucoside compound 1 of the present invention has a minimum inhibitory concentration of 0.25 μg/ml against Candida albicans, and a minimum inhibitory concentration of 4 μg/ml against Gibberella tritici and Pythium melon; In the experiment, compound 1 has a semi-inhibitory effect on human hepatoma cell HepG2 and breast cancer cell MCF-7 when the concentration is 24.05 μg/ml and 19.56 μg/ml respectively; it is suggested that compound 1 of the present invention is used in the preparation of related medicines. The superiority of formulations. Therefore, the preferred compound 1 of the present invention: rapamycin 40-O-β-D glucoside is expected to be a highly effective candidate inhibitor for the prevention and treatment of animal and plant pathogenic bacteria, or for the preparation of prevention and treatment of liver cancer and Breast cancer-related pharmaceutical preparations.

The invention discloses a group of rapamycin glucoside compounds and an enzymatic preparation method thereof. In the preparation process, mild enzymatic synthesis is used to realize the conversion of rapamycin glucoside in one step in vitro; Fewer compounds are reacted, which is beneficial to the reuse of raw materials and the separation and purification of new products, and the conversion efficiency of the glycosylation reaction is high. Novel drugs with high antibacterial activity and good antitumor activity are more and more important in the prevention and treatment of drug-resistant plant and animal pathogenic bacteria and cancer. The novel rapamycin glucoside obtained by the present invention is expected to increase its application value in antifungal and antitumor activities, thereby producing better social benefit and economic value.

Description of drawings

Figure 1: HPLC analysis chromatogram of rapamycin glycosylation modification reaction solution by glycosyltransferase BsGT-1

Among them, the peak time of three kinds of rapamycin glucoside: compound 1: 11.1min; compound 2: 8.4min; compound 3: 5.2min; the peak time of Rapamycin standard substance (Rapamycin), 21.8min.

Figure 2: High resolution mass spectrum of compound 1: rapamycin 40-O-β-D glucoside, the quasi-molecular ion peak [M+Na] ⁺ is m/z 1098.5886.

Figure 3: High resolution mass spectrum of compound 2: rapamycin 28-O-β-D glucoside, the quasi-molecular ion peak [M+Na] ⁺ is m/z 1098.5913.

Figure 4: High resolution mass spectrum of compound 3: rapamycin 28,40-O-β-D glucoside, the quasi-molecular ion peak [M+Na] ⁺ is m/z 1260.6433.

Figure 5:1H NMR spectrum of compound ¹ : rapamycin 40-O-beta-D glucoside.

Figure 6: ^13C NMR spectrum of compound 1: rapamycin 40-O-beta-D glucoside.

Figure 7: HSQC two-dimensional nuclear magnetic resonance spectrum of compound 1: rapamycin 40-O-beta-D glucoside.

Figure 8: HMBC two-dimensional nuclear magnetic resonance spectrum of compound 1: rapamycin 40-O-beta-D glucoside.

Figure 9: ¹ H- ¹ H COSY two-dimensional nuclear magnetic resonance spectrum of compound 1: rapamycin 40-O-β-D glucoside.

Figure 10: ^1H NMR spectrum of compound 2: rapamycin 28-O-beta-D glucoside.

Figure 11: ^13C NMR spectrum of compound 2: rapamycin 28-O-beta-D glucoside.

Figure 12: HSQC two-dimensional nuclear magnetic resonance spectrum of compound 2: rapamycin 28-O-beta-D glucoside.

Figure 13: HMBC two-dimensional nuclear magnetic resonance spectrum of compound 2: rapamycin 28-O-beta-D glucoside.

Figure 14: ¹ H- ¹ H COSY two-dimensional nuclear magnetic resonance spectrum of compound 2: rapamycin 28-O-beta-D glucoside.

Figure 15: ^1H NMR spectrum of compound 3: rapamycin 28,40-O-beta-D glucoside.

Figure 16: ^13C NMR spectrum of compound 3: rapamycin 28,40-O-beta-D glucoside.

Figure 17: HSQC two-dimensional nuclear magnetic resonance spectrum of compound 3: rapamycin 28,40-O-beta-D glucoside.

Figure 18: HMBC two-dimensional nuclear magnetic resonance spectrum of compound 3: rapamycin 28,40-O-beta-D glucoside.

Figure 19: ¹ H- ¹ H COSY two-dimensional nuclear magnetic resonance spectrum of compound 3: rapamycin 28,40-O-β-D glucoside.

Detailed ways

The content of the present invention will be described in detail below with reference to specific embodiments. The examples described below are only preferred embodiments of the present invention, and are not intended to limit the present invention in any form. within the scope of the technical solution of the invention.

The reagents, plasmids, strains, cells, or experimental equipment used in the present invention are all commercially available products.

Example 1 Expression of glycosyltransferase BsGT-1, in vitro glycosylation reaction, and preparation of rapamycin glucoside

The inventor's experimental screening confirmed the glycosyltransferase BsGT-1 (CUB50191), which can efficiently glycosylate rapamycin, and its protein sequence has been published.

The recombinant plasmid containing pET28a-BsGT-1 was transformed into Escherichia coli BL21 (DE3), and the resulting transformant was transferred to 2 ml of LB medium supplemented with kanamycin (final concentration of 40 μg/ml), 200 r/min, at 37 Cultivate at ℃ for 12 hours, then transfer 2ml of seed liquid to 100ml of LB medium (final concentration of kanamycin is 40μg/ml), and expand the culture at 37℃. When the OD value reaches about 0.6-0.8, add The final concentration of isopropyl-β-D-thiogalactoside (IPTG) was 0.1 mM, and then transferred to a 16 °C shaker, 200 r/min, cultured for 24 hours, centrifuged at 8000 r/min for 5 min, and the cells were collected. The collected cells were resuspended with 10 ml of Tris-HCl buffer (50 mM Tris-HCl, pH 7.5), and the resuspended cells were placed in an ice-water mixture for sonication for 5 min (sonication for 5 seconds, pause for 10 seconds). The sonicated cells were centrifuged (13000 r/min, 4°C for 30 min) to separate the supernatant and the precipitate, and then the protein expression was detected by polyacrylamide gel electrophoresis.

The present invention uses nickel filler to purify the protein with His tag, and the supernatant of the protein after ultrasonication is added to the nickel filler that has been equilibrated in advance with a buffer (50mM Tris-HCl, pH 7.5), and then at a temperature of 4°C. Incubate overnight for 12 hours, then add the incubated mixture to the purification column, wait for the filler to settle naturally, rinse and equilibrate with the buffer, and use different concentrations of imidazole in Tris-HCl buffer (10mM, 50mM, 100mM, 150 mM and 200 mM imidazole, 50 mM Tris-HCl, pH 7.5) were eluted into 2 ml centrifuge tubes, respectively, and then the proteins were collected and detected by SDS-PAGE. The collected relatively purified protein was concentrated with a 30kDa ultrafiltration tube, and the concentrated protein sample was subjected to the next step of molecular sieve purification to obtain a highly purified protein for glycosylation reaction.

Utilize the separated and purified glycosyltransferase BsGT-1 to catalyze rapamycin and a glucose glycosyl donor to prepare a rapamycin glucoside compound. The preparation method and reaction conditions are as follows:

The in vitro enzyme activity reaction system includes glycosyltransferase BsGT-1 (500 μg/ml), Tris-HCl buffer (50 mM Tris-HCl, 10 mM MgCl ₂ ), rapamycin (final concentration 200 μM, dissolved in DMSO) and UDP-O-β-Dglucose (final concentration 1mM, dissolved in water) was used for in vitro enzyme activity experiments. The reaction mixture was incubated at 37 °C for 8 hours, then 3 times the volume of acetonitrile was added to terminate the reaction, 14000 r/min, 30 min to remove protein precipitates, the samples were rotary evaporated to dryness, and acetonitrile was added to resuspend the product, and centrifuged again at 14000 r/min After 30 min, the product was separated and purified by semi-preparative liquid phase.

The preparation of the above rapamycin glucoside used YMC column (YMC-Pack Pro C18, 250mm×10.0mm, 5μm), mobile phase system: acetonitrile water system elution (55:45), three kinds of rapamycin glucose The peak time of the glycoside: compound 1: 11.1 min; compound 2: 8.4 min; compound 3: 5.2 min; the peak time of Rapamycin standard substance (Rapamycin), 21.8 min. The three separated compounds were evaporated to dryness, dissolved in CD ₃ CN, and identified by UHPLC-ESI-Q-TOF high-resolution mass spectrometry and nuclear magnetic resonance respectively.

Example 2 Structure identification of compound 1, compound 2 and compound 3

The structures of compound 1, compound 2 and compound 3 were determined by high-resolution mass spectrometry and nuclear magnetic resonance analysis. According to Figure 2 and Figure 3, UHPLC-ESI-Q-TOF high-resolution mass spectrometry gave compound 1 and compound 2 quasi-molecular ion peaks [M+Na] ⁺ are m/z 1098.5886 and 1098.5913, respectively, confirming that both 1 and 2 are monoglucosides of rapamycin. Meanwhile, the monosaccharide can also be confirmed from the hydrogen nuclear magnetic resonance spectrum ( ¹ H NMR, Fig. 5, Fig. 10) and carbon spectrum ( ¹³ C NMR, Fig. 6, Fig. 11) of compounds 1 and 2. Two-dimensional nuclear magnetic resonance spectroscopy determined the linkage of the sugar and rapamycin backbones of compounds 1 and 2 (Figures 7-9, Figures 12-14).

In the HMBC spectrum of compound 1, H-40 to C-1' correlation and H-1' to C-40 correlation, thus confirming that -OH at C-40 in compound 1 is glycosylated, while in compound 2 C Glycosylation at -28 was also identified through key HMBC correlations from H-28 to C-1' and from H-1' to C-28. The larger coupling constant (J1', 2', 7.8 Hz) between the anomeric protons H-1' and H-2' on the sugar and the chemical shift value of 4.46 ppm in the upfield of the anomeric proton, revealing the sugar donor and rapamycin receptors are linked by β-D glucosidic bonds. Therefore, it can be determined that isomers 1 and 2 are rapamycin 40-O-β-D-glucoside and rapamycin 28-O-β-D-glucoside, respectively. The quasi-molecular ion peak [M+Na] ⁺ of compound 3 is m/z 1260.6433, which is predicted to have a disaccharide group (Fig. 4). The HMBC spectrum of compound 3 showed two sets of correlations present in compounds 1 and 2, which confirmed that compound 3 was glycosylated at both C-28 and C-40, and was therefore identified as rapamycin 28,40-O - β-D-diglucoside. Figures 5–19 provide evidence for the NMR identification of new compounds.

Example 3 Antifungal activity test of rapamycin glucoside

Screening method: half-dilution method

Cell lines: Candida albicans; Fusarium graminearum and Pythium aphanidermatum

Action time: 48 hours

experimental method:

1. Use YPD liquid medium (dissolve 10g yeast paste, 20g peptone in 900ml water, high pressure 115℃, 15min, add 100ml 20g glucose) to cultivate Candida albicans, when the OD ₆₀₀ to 0.3, carry out dilution coating, dilute to 10 The antifungal test was carried out at a concentration of ⁵ -10 ⁶ cells/ml. The initial drug concentration was set to 32 μg/ml, and it was doubling and half-diluted in a 96-well plate. The inhibition rates of the corresponding concentrations of compounds 1, 2, and 3 were as follows:

The inhibition rates of compound 1 against Candida albicans were 96.08% (32μg/ml), 95.20% (16μg/ml), 94.75% (8μg/ml), 94.26% (4μg/ml), 94.26% (2μg/ml) , 94.01% (1μg/ml), 92.83% (0.5μg/ml), 88.98% (0.25μg/ml), 32.67% (0.125μg/ml), 19.71% (0.0625μg/ml);

The inhibition rates of compound 2 against Candida albicans were 94.87% (32μg/ml), 93.82% (16μg/ml), 92.82% (8μg/ml), 86.86% (4μg/ml), 26.97% (2μg/ml) , 11.97% (1 μg/ml);

The inhibitory rate of compound 3 against Candida albicans at 32μg/ml was only 39.88%, and its MIC>32μg/ml.

2. Use PDA solid medium to cultivate three plant pathogenic bacteria, before solid medium solidifies, add the drug concentration of different initial 32 μg/ml to carry out doubling dilution, then, inoculate three plant pathogenic bacteria, grow. After 48 hours, the expansion of the colony was observed (the lowest drug concentration that did not expand the colony was the MIC for inhibiting fungi), see Table 1.

Table 1. MIC values of rapamycin and its glucosides against fungi

Conclusion: It can be seen from Table 1 that the minimum inhibitory concentration of rapamycin glucoside compound 1 against Candida albicans is 0.25 μM, and the minimum inhibitory concentration against plant pathogens: Gibberella tritici and Pythium melon fruit is 4μM, the minimum inhibitory concentration of rapamycin glucoside compound 2 against Candida albicans, Gibberella triticale and Pythium cucurbita were 4μM, respectively.

8 μM, 8 μM. In conclusion, rapamycin glucoside compounds 1 and 2 are expected to be candidate inhibitors against animal pathogenic bacteria and plant pathogenic bacteria.

Example 4 Antitumor activity test of rapamycin glucoside

Screening method: tetrazolium salt (methyl-thiazol-tetozolium, MTT) reduction method

Cell lines: HepG2 liver cancer cells and MCF-7 breast cancer cells

Action time: 48 hours

experimental method:

Trypsinize cells in logarithmic growth phase, add a certain amount of culture medium to stop the reaction, collect cells by centrifugation, and resuspend cells with 1 ml of culture medium. Take another sterile gun tank, mix the cell suspension with the fresh culture medium well and add it to a 96-well plate (the edge wells are filled with sterile PBS). Put the inoculated cell culture plate into the incubator for culture, adhere to the wall and grow until the cell monolayer covers the 96-well bottom plate. The wells were 100 μl, and 4 replicate wells were set up in parallel experiments. 5% CO ₂ , incubated at 37°C for 48 hours, aspirated the supernatant, washed 2-3 times with PBS, then added 100 μl of MTT solution (5 mg/ml, 0.5% MTT medium), continued to culture for 4 hours, discarded the supernatant, After washing 2-3 times with PBS, 100 μl of dimethyl sulfoxide (DMSO) was added to each well, and it was shaken at low speed for 10 min on a shaker to fully dissolve the crystals. The absorbance value of each well was measured at OD492nm of microplate reader.

The inhibition rates of three rapamycin glucoside compounds on human liver cancer HepG2 were measured at different drug concentrations:

The inhibition rates of compound 1 on HepG2 hepatoma cells were 4.19% (0.1μg/ml), 5.63% (1μg/ml), 19.90% (5μg/ml), 36.26% (20μg/ml), 66.77% (50μg/ml) ), 74.84% (100μg/ml), 79.09% (200μg/ml); the inhibition rates of MCF-7 breast cancer cells were 4.83% (0.1μg/ml), 6.93% (1μg/ml), 13.85% ( 5 μg/ml), 41.25% (20 μg/ml), 72.77% (50 μg/ml), 72.65% (100 μg/ml).

The inhibitory rate of compound 2 on HepG2 liver cancer cells at 100μg/ml was 28.37%, and its IC ₅₀ value was considered to be greater than 100μg/ml; the inhibition rates on MCF-7 breast cancer cells were 2.49% (1μg/ml) and 10.57%, respectively (5 μg/ml), 13.02% (20 μg/ml), 18.41% (50 μg/ml), 51.31% (100 μg/ml), 65.12% (200 μg/ml), 73.09% (300 μg/ml).

The inhibitory rate of compound 3 on HepG2 liver cancer cells and MCF-7 breast cancer cells at 100 μg/ml was 16.90% and 26.65%, respectively, and its IC ₅₀ value was considered to be greater than 100 μg/ml.

In this regard, the inventors obtained the IC ₅₀ values of three rapamycin glucoside compounds on HepG2 liver cancer cells and MCF-7 breast cancer cells through nonlinear fitting curves, as shown in Table 2:

Table 2. IC50 values of rapamycin glucoside on human hepatoma cells HepG2

Conclusion: It can be seen from Table 2 that rapamycin glucoside compound 1 has a semi-inhibitory effect on HepG2 liver cancer cells and MCF-7 breast cancer cells at concentrations of 24.05 μg/ml and 19.56 μg/ml, and has anti-tumor effects. The activity was close to that of the rapamycin parent drug (10.70 μg/ml and 8.64 μg/ml) and exhibited better water solubility. Therefore, the compound is expected to be a candidate drug for the preparation of liver cancer cells and breast cancer cells.

Claims (8)

1. A group of rapamycin glucoside compounds, characterized in that, the rapamycin glucoside compounds are compound 1: rapamycin 40-O-β-D glucoside; compound 2: rapamycin or Compound 3: Rapamycin 28,40-O-β-D glucoside. 2 . The rapamycin glucoside compound according to claim 1 , wherein the rapamycin glucoside compound is compound 1: rapamycin 40-O-β-D glucoside. 3 . 3. the method for the described rapamycin glucoside enzymatic preparation of claim 1 or 2, the step is: In the buffer environment of 50 mM Tris-HCl, 10 mM MgCl ₂ , the final concentration of rapamycin was 200 μM and the final concentration of 1 mM UDP-O-β-D-glucose and 500 μg/ml of glycosyltransferase BsGT -1 Carry out the in vitro enzymatic activity reaction, incubate the reaction mixture at 37±1°C for 8±0.5 hours, then add 3 times the volume of acetonitrile to stop the reaction, 14000r/min, 30min to remove the protein precipitate, spin the sample to dryness, and then Add acetonitrile to resuspend the product, centrifuge again at 14000r/min for 30min, and then separate and purify the product through semi-preparative liquid phase; the size of the chromatographic column is YMC-Pack Pro C18, 250mm×10.0mm, 5μm; mobile phase system: 55:45 Elution with acetonitrile water system, the peak time of rapamycin glucoside: compound 1: 11.1 min; compound 2: 8.4 min; compound ₃ : 5.2 min; CN was dissolved and identified by UHPLC-ESI-Q-TOF high-resolution mass spectrometry and nuclear magnetic resonance, respectively. 4. the method for enzymatic preparation of rapamycin glucoside compounds according to claim 3, is characterized in that: the mol ratio that described rapamycin and UDP-O-β-D-glucose mix is 1:5 , which was incubated with the reaction mixture of glycosyltransferase BsGT-1-mediated glycosylation reaction at 37°C for 8 hours. 5. the application of the rapamycin glucoside compound described in claim 1 or 2 in the preparation of the medicine for preventing and treating animal pathogenic bacteria. 6. The application of the rapamycin glucoside compound described in claim 1 or 2 in the preparation of a medicine for preventing and treating phytopathogenic bacteria. 7. The application of the rapamycin glucoside compound according to claim 1 or 2 in the preparation of a medicament for preventing and treating liver cancer. 8. The application of the rapamycin glucoside compound of claim 1 or 2 in the preparation of a medicament for preventing and treating breast cancer.

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