CN113583067B - Low-toxicity pimamycin derivative, and preparation method and application thereof - Google Patents
Low-toxicity pimamycin derivative, and preparation method and application thereof Download PDFInfo
- Publication number
- CN113583067B CN113583067B CN202110604486.XA CN202110604486A CN113583067B CN 113583067 B CN113583067 B CN 113583067B CN 202110604486 A CN202110604486 A CN 202110604486A CN 113583067 B CN113583067 B CN 113583067B
- Authority
- CN
- China
- Prior art keywords
- derivative
- pimamycin
- toxicity
- low
- strain
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 231100000053 low toxicity Toxicity 0.000 title claims abstract description 18
- 238000002360 preparation method Methods 0.000 title claims description 8
- 239000000126 substance Substances 0.000 claims abstract description 4
- 239000003814 drug Substances 0.000 claims description 8
- 229940095731 candida albicans Drugs 0.000 claims description 4
- 230000001032 anti-candidal effect Effects 0.000 claims 1
- 238000000855 fermentation Methods 0.000 abstract description 21
- 230000004151 fermentation Effects 0.000 abstract description 21
- 238000000926 separation method Methods 0.000 abstract description 9
- 238000004128 high performance liquid chromatography Methods 0.000 abstract description 7
- 102000004531 Selenoprotein P Human genes 0.000 abstract description 6
- 108010042443 Selenoprotein P Proteins 0.000 abstract description 6
- 150000001875 compounds Chemical class 0.000 abstract description 5
- 238000011161 development Methods 0.000 abstract description 5
- 238000000034 method Methods 0.000 abstract description 5
- 230000003013 cytotoxicity Effects 0.000 abstract description 3
- 231100000135 cytotoxicity Toxicity 0.000 abstract description 3
- 241000936794 Streptomyces chattanoogensis Species 0.000 abstract description 2
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 2
- 238000000605 extraction Methods 0.000 abstract description 2
- 244000005700 microbiome Species 0.000 abstract description 2
- 230000008569 process Effects 0.000 abstract description 2
- 238000004366 reverse phase liquid chromatography Methods 0.000 abstract description 2
- 230000009466 transformation Effects 0.000 abstract description 2
- 241000364051 Pima Species 0.000 abstract 1
- 229930000044 secondary metabolite Natural products 0.000 abstract 1
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 24
- 239000000287 crude extract Substances 0.000 description 12
- 239000007788 liquid Substances 0.000 description 11
- NCXMLFZGDNKEPB-FFPOYIOWSA-N natamycin Chemical class O[C@H]1[C@@H](N)[C@H](O)[C@@H](C)O[C@H]1O[C@H]1/C=C/C=C/C=C/C=C/C[C@@H](C)OC(=O)/C=C/[C@H]2O[C@@H]2C[C@H](O)C[C@](O)(C[C@H](O)[C@H]2C(O)=O)O[C@H]2C1 NCXMLFZGDNKEPB-FFPOYIOWSA-N 0.000 description 11
- 210000004027 cell Anatomy 0.000 description 10
- 238000004519 manufacturing process Methods 0.000 description 8
- 229940079593 drug Drugs 0.000 description 7
- 238000002474 experimental method Methods 0.000 description 7
- 239000001963 growth medium Substances 0.000 description 7
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 6
- 230000000843 anti-fungal effect Effects 0.000 description 6
- 239000000284 extract Substances 0.000 description 6
- 241000187747 Streptomyces Species 0.000 description 5
- 229940121375 antifungal agent Drugs 0.000 description 5
- 230000001580 bacterial effect Effects 0.000 description 5
- APKFDSVGJQXUKY-KKGHZKTASA-N Amphotericin-B Natural products O[C@H]1[C@@H](N)[C@H](O)[C@@H](C)O[C@H]1O[C@H]1C=CC=CC=CC=CC=CC=CC=C[C@H](C)[C@@H](O)[C@@H](C)[C@H](C)OC(=O)C[C@H](O)C[C@H](O)CC[C@@H](O)[C@H](O)C[C@H](O)C[C@](O)(C[C@H](O)[C@H]2C(O)=O)O[C@H]2C1 APKFDSVGJQXUKY-KKGHZKTASA-N 0.000 description 4
- 238000005481 NMR spectroscopy Methods 0.000 description 4
- 241001052560 Thallis Species 0.000 description 4
- APKFDSVGJQXUKY-INPOYWNPSA-N amphotericin B Chemical compound O[C@H]1[C@@H](N)[C@H](O)[C@@H](C)O[C@H]1O[C@H]1/C=C/C=C/C=C/C=C/C=C/C=C/C=C/[C@H](C)[C@@H](O)[C@@H](C)[C@H](C)OC(=O)C[C@H](O)C[C@H](O)CC[C@@H](O)[C@H](O)C[C@H](O)C[C@](O)(C[C@H](O)[C@H]2C(O)=O)O[C@H]2C1 APKFDSVGJQXUKY-INPOYWNPSA-N 0.000 description 4
- 229960003942 amphotericin b Drugs 0.000 description 4
- 238000001914 filtration Methods 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 206010017533 Fungal infection Diseases 0.000 description 3
- 208000037026 Invasive Fungal Infections Diseases 0.000 description 3
- 208000031888 Mycoses Diseases 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 238000010828 elution Methods 0.000 description 3
- 210000002919 epithelial cell Anatomy 0.000 description 3
- 239000000706 filtrate Substances 0.000 description 3
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 239000013612 plasmid Substances 0.000 description 3
- 238000011218 seed culture Methods 0.000 description 3
- 230000004083 survival effect Effects 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 241000222122 Candida albicans Species 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 2
- 206010061598 Immunodeficiency Diseases 0.000 description 2
- 102000008109 Mixed Function Oxygenases Human genes 0.000 description 2
- 108010074633 Mixed Function Oxygenases Proteins 0.000 description 2
- 101150082969 SELP gene Proteins 0.000 description 2
- 101150036293 Selenop gene Proteins 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 238000007664 blowing Methods 0.000 description 2
- 238000013375 chromatographic separation Methods 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 230000001419 dependent effect Effects 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 238000004821 distillation Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 239000003480 eluent Substances 0.000 description 2
- 238000001704 evaporation Methods 0.000 description 2
- 239000012065 filter cake Substances 0.000 description 2
- 238000011010 flushing procedure Methods 0.000 description 2
- HQVFCQRVQFYGRJ-UHFFFAOYSA-N formic acid;hydrate Chemical compound O.OC=O HQVFCQRVQFYGRJ-UHFFFAOYSA-N 0.000 description 2
- 108091008053 gene clusters Proteins 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 229910052739 hydrogen Inorganic materials 0.000 description 2
- 239000001257 hydrogen Substances 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 239000002054 inoculum Substances 0.000 description 2
- 238000011068 loading method Methods 0.000 description 2
- 239000011159 matrix material Substances 0.000 description 2
- 238000000655 nuclear magnetic resonance spectrum Methods 0.000 description 2
- 150000004291 polyenes Chemical class 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- 230000009885 systemic effect Effects 0.000 description 2
- 238000009210 therapy by ultrasound Methods 0.000 description 2
- YKSVGLFNJPQDJE-YDMQLZBCSA-N (19E,21E,23E,25E,27E,29E,31E)-33-[(2R,3S,4R,5S,6R)-4-amino-3,5-dihydroxy-6-methyloxan-2-yl]oxy-17-[7-(4-aminophenyl)-5-hydroxy-4-methyl-7-oxoheptan-2-yl]-1,3,5,7,37-pentahydroxy-18-methyl-9,13,15-trioxo-16,39-dioxabicyclo[33.3.1]nonatriaconta-19,21,23,25,27,29,31-heptaene-36-carboxylic acid Chemical compound CC(CC(C)C1OC(=O)CC(=O)CCCC(=O)CC(O)CC(O)CC(O)CC2(O)CC(O)C(C(CC(O[C@@H]3O[C@H](C)[C@@H](O)[C@@H](N)[C@@H]3O)\C=C\C=C\C=C\C=C\C=C\C=C\C=C\C1C)O2)C(O)=O)C(O)CC(=O)C1=CC=C(N)C=C1 YKSVGLFNJPQDJE-YDMQLZBCSA-N 0.000 description 1
- 208000029483 Acquired immunodeficiency Diseases 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 102400000888 Cholecystokinin-8 Human genes 0.000 description 1
- 101800005151 Cholecystokinin-8 Proteins 0.000 description 1
- 206010062016 Immunosuppression Diseases 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 235000019764 Soybean Meal Nutrition 0.000 description 1
- 244000015069 Zephyranthes candida Species 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 238000011483 antifungal activity assay Methods 0.000 description 1
- 239000003429 antifungal agent Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 229960004348 candicidin Drugs 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 235000019249 food preservative Nutrition 0.000 description 1
- 239000005452 food preservative Substances 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000001506 immunosuppresive effect Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 150000002611 lead compounds Chemical class 0.000 description 1
- 239000003120 macrolide antibiotic agent Substances 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229960000988 nystatin Drugs 0.000 description 1
- VQOXZBDYSJBXMA-NQTDYLQESA-N nystatin A1 Chemical compound O[C@H]1[C@@H](N)[C@H](O)[C@@H](C)O[C@H]1O[C@H]1/C=C/C=C/C=C/C=C/CC/C=C/C=C/[C@H](C)[C@@H](O)[C@@H](C)[C@H](C)OC(=O)C[C@H](O)C[C@H](O)C[C@H](O)CC[C@@H](O)[C@H](O)C[C@](O)(C[C@H](O)[C@H]2C(O)=O)O[C@H]2C1 VQOXZBDYSJBXMA-NQTDYLQESA-N 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- -1 pimamycin Chemical compound 0.000 description 1
- 239000002459 polyene antibiotic agent Substances 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 230000002028 premature Effects 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 230000024053 secondary metabolic process Effects 0.000 description 1
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000004455 soybean meal Substances 0.000 description 1
- 239000012086 standard solution Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000820 toxicity test Toxicity 0.000 description 1
- 238000002054 transplantation Methods 0.000 description 1
- 239000012137 tryptone Substances 0.000 description 1
- 238000000825 ultraviolet detection Methods 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 239000000273 veterinary drug Substances 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H17/00—Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals
- C07H17/04—Heterocyclic radicals containing only oxygen as ring hetero atoms
-
- A23L3/3562—
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/10—Antimycotics
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
- C07H1/06—Separation; Purification
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/44—Preparation of O-glycosides, e.g. glucosides
- C12P19/60—Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin
- C12P19/62—Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin the hetero ring having eight or more ring members and only oxygen as ring hetero atoms, e.g. erythromycin, spiramycin, nystatin
- C12P19/626—Natamycin; Pimaricin; Tennecetin
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- General Engineering & Computer Science (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biomedical Technology (AREA)
- Pharmacology & Pharmacy (AREA)
- Oncology (AREA)
- Virology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Communicable Diseases (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Tropical Medicine & Parasitology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention relates to the technical field of transformation and separation of microorganism secondary metabolites, and discloses a low-toxicity pimamycin derivative with a molecular formula of C 33 H 49 NO 11 The chemical structural formula is as follows:the strain Streptomyces chattanoogensis QZ is taken as a chassis to construct a SelP protein anaplerotic strain, and the low-toxicity pimamycin derivative Pima 9 is obtained through the processes of strain fermentation, thallus treatment, organic reagent extraction, medium-pressure reversed phase chromatography coarse separation, high-performance liquid chromatography separation and the like. The compounds developed by the invention have lower cytotoxicity and better development prospect.
Description
Technical Field
The invention relates to the technical field of micro-secondary metabolite transformation and separation, in particular to development, preparation and application of a low-toxicity pimamycin derivative.
Background
The invention relates to a microorganism secondary metabolism and separation technology, in particular to a pimamycin derivative and a preparation method thereof
Systemic fungal infections, also known as invasive fungal infections (Invasivefungalinfections (IFIs)), occur mainly in some immunocompromised or immunocompromised populations, such as: cancer chemotherapy patients, patients who have received immunosuppression due to organ transplantation, intensive care patients, premature infants, aids patients, and acquired immunodeficiency patients. It is counted that over 50 tens of thousands die annually from an invasive fungal infection, and this figure is still continuously rising due to the lack of effective antifungal agents. Among the current drugs for treating systemic fungal infections, polyene drugs are known as the "gold standard" of antifungal drugs because they have good antifungal activity and rarely develop resistance. However, the water solubility is relatively poor and the cytotoxicity is relatively strong, which severely limits the development and application of polyene antibiotics.
Pimamycin is listed by the international health organization as one of four important polyene macrolide antibiotics (amphotericin B, nystatin, pimamycin, and candicidin). Meanwhile, the antibiotics with relatively simple structure, low toxicity and stable chemical property are also used in the four compounds. Thus, are useful as food preservatives, antifungal veterinary drugs, and in the treatment of corneal fungal infections.
Disclosure of Invention
The invention aims to provide development, preparation and application of a low-toxicity pimaricin derivative.
The technical scheme adopted by the invention is as follows:
a low-toxicity pimamycin derivative with molecular formula of C 33 H 49 NO 11 The chemical structural formula is as follows:
a preparation method of a low-toxicity pimaricin derivative is characterized in that a SelP in-vivo anaplerotic strain is constructed by taking a Streptomyces chattanoogensisQZ strain as a chassis, and the low-toxicity pimaricin derivative is prepared through the processes of strain fermentation, thallus treatment, organic reagent extraction, medium-pressure reversed-phase chromatography crude separation, high-performance liquid chromatography separation and the like. The method comprises the following specific steps:
A. the construction of a production strain QZ02: integrating 2-OG dependent hydroxylase in a streptomyces chattanogensQZ 02 biosynthesis gene cluster, obtaining a gene sequence of SelP through NCBI, synthesizing a corresponding sequence by a chemical synthesis method, and constructing a strong promoter kasOp of a streptomyces system at the upstream of the gene * Integrating the strain into the genome of Streptomyceschattanoogensis QZ02 through an integrated plasmid to obtain a production strain QZ02:: selP;
B. fermenting and culturing a production strain QZ02: inoculating spores of a SelP modified strain into a seed culture medium, performing shake culture at a speed of 200-220rpm and a temperature of 28-32 ℃ for 24 hours to obtain fermentation seed liquid, transferring the seed liquid into the fermentation culture medium according to an inoculum size of 10-15% V/V, and performing shake culture at a speed of 200-220rpm and a temperature of 28-32 ℃ for 5 days to obtain a fermentation culture of a production strain;
C. and (3) thallus treatment: centrifuging the fermentation culture to separate fermentation liquor from thalli, and evaporating the fermentation liquor to obtain fermentation liquor extract; washing the thalli with water and freeze-drying;
D. treating the fermentation liquid extract: extracting the fermentation liquid extract by using an equal amount of methanol, filtering to obtain an extracting solution, and concentrating under reduced pressure to obtain a first crude extract;
E. and (3) thallus treatment: ultrasonically extracting the freeze-dried thalli with equal amount of methanol for 20min, extracting overnight, filtering to remove filter cakes, concentrating the filtrate by reduced pressure distillation, and combining the filtrate with the crude extract to obtain a first total crude extract;
F. medium pressure reversed phase chromatographic separation: fully dissolving the first total crude extract in a small amount of methanol, standing and centrifuging after ultrasonic treatment to remove redundant sugar, loading the sample into a medium-pressure reversed phase chromatographic system, eluting with methanol-water systems with different concentrations, flushing with a standard of baseline after eluting, collecting eluent, detecting with LC-Mass, and concentrating more components of a target product to obtain a second crude extract;
G. high performance liquid chromatography separation: and (3) fully dissolving the second crude extract with a small amount of methanol, and separating by a high performance liquid chromatography system to obtain a low-toxicity pimaricin derivative pure product.
Further, the formula of the seed culture medium in the step B comprises 20g/L of glucose, 20g/L of tryptone and 10g/L of sodium chloride; the fermentation medium formula comprises 65g/L glucose, 10g/L yeast extract and 28g/L soybean meal.
Further, the elution system used in step F is a methanol-water system, i.e., elution is performed according to a gradient of 0, 5%, 10%, 20%, 30%, 40%, 50%, 100%, with the baseline flush being the criterion for sufficient elution.
Further, the high performance liquid chromatography system used in the step G uses 0.1% formic acid water and acetonitrile as mobile phases, and the ratio of acetonitrile to 0.1% formic acid water is 3:7, the flow rate is 4mL/min, the ultraviolet detection wavelength is 303nm, the sample injection amount and the sample concentration are 25-50 mu L, the retention time of the compound is 4-6min, the corresponding chromatographic peaks are collected, accumulated and evaporated to dryness.
The application of the low-toxicity pimamycin derivative in preparing antifungal medicines.
The nuclear magnetic resonance data of the pimaricin derivative prepared by the invention are shown in the table in figure 1.
The beneficial effects of the invention are as follows:
(1) The minimum inhibition concentration of the pimaricin derivative prepared by the invention is 32 mug/mL, and the pimaricin derivative has better antifungal activity;
(2) The cell survival rate of the pimamycin derivative is 68.12%, and compared with the pimamycin, the cell viability is reduced;
(3) Can be developed into lead compounds of antifungal drugs and has certain application potential.
Drawings
FIG. 1 is a chart of nuclear magnetic resonance data for a pimamycin derivative;
FIG. 2 shows a plasmid map of pSET 152-SelP;
FIG. 3 nuclear magnetic resonance hydrogen spectrum of pimamycin derivative [ ] 1 HNMR) map;
FIG. 4 nuclear magnetic resonance spectrum of pimamycin derivative [ ] 13 CNMR) map.
Detailed Description
The following describes the detailed embodiments of the low toxicity pimaricin derivatives, the preparation method and the application of the low toxicity pimaricin derivatives according to the invention with reference to the accompanying drawings.
The low-toxicity pimamycin derivative is prepared by the following steps:
A. the construction of a production strain QZ02: QZ02 originated from a preliminary study of the university of Shanghai transportation, and reference to the strain herein may be made to chinese patent document CN104447917B. The Streptomyces chattanoogensisQZ chassis strain is integrated with 2-OG dependent hydroxylase in a Selvamic in biosynthesis gene cluster to obtain a production strain QZ02:: selP.
B. Fermenting and culturing a production strain QZ02: inoculating the spores of SelP in a seed culture medium, shaking at 28-32 ℃ at 200-220rpm for 24 hours to obtain fermentation seed liquid, transferring the seed liquid into the fermentation culture medium according to 10-15% of V/V inoculum size, and shaking at 28-32 ℃ at 200-220rpm for 5 days to obtain fermentation culture of the production strain.
C. And (3) thallus treatment: centrifuging the fermentation culture to separate fermentation liquor from thalli, and evaporating the fermentation liquor to obtain fermentation liquor extract; the cells were washed with water and lyophilized.
D. Treating the fermentation liquid extract: extracting the fermentation liquid extract by using an equal amount of methanol, filtering to obtain an extracting solution, and concentrating under reduced pressure to obtain a first crude extract.
E. And (3) thallus treatment: ultrasonic extracting the freeze-dried thallus with equal amount of methanol for 20min, extracting overnight, filtering to remove filter cake, concentrating the filtrate by distillation under reduced pressure, and mixing with the crude extract to obtain first total crude extract.
F. Medium pressure reversed phase chromatographic separation: fully dissolving the first total crude extract in a small amount of methanol, standing and centrifuging after ultrasonic treatment to remove redundant sugar, loading the sample into a medium-pressure reversed phase chromatographic system, eluting with methanol-water systems with different concentrations, flushing with a standard of baseline after eluting, collecting eluent, detecting with LC-Mass, and concentrating more components of a target product to obtain a second crude extract.
G. High performance liquid chromatography separation: and (3) fully dissolving the second crude extract with a small amount of methanol, and separating by a high performance liquid chromatography system to obtain a low-toxicity pimaricin derivative pure product.
The plasmid map of SelP of the streptomyces chattanogensis QZ02 is shown in figure 2, and the hydrogen nuclear magnetic resonance spectrum of the pimamycin derivative is shown in the invention 1 HNMR) as shown in FIG. 3, the nuclear magnetic resonance carbon spectrum of the present invention 13 CNMR) is shown in fig. 4.
The invention is illustrated in more detail by the following examples.
Embodiment one: antifungal Activity assay
A. Candida albicans SC5314 (ATCMYA-2876) was used as an experimental standard strain. Clinical strains of Candida albicans (CCC-593, CCC-495, CCC-487, CCC-515, CCC-496, CCC-2260, CCC-2251, CCC-2242, CCC-2233, CCC-2224) as indicator bacteria.
B. Selecting candida albicans single colony to prepare bacterial suspension in a culture medium, wherein the final concentration of the bacterial suspension is about 0.5-2.5X10 4 CFU/mL. 200. Mu.L of the bacterial liquid was added to the first well of the flat bottom 96-well plate, and 100. Mu.L of the bacterial liquid to be tested was added to each well.
C. Adding the drug to be tested or positive control (amphotericin B) into the first hole, blowing and mixing uniformly, taking 100 mu L of uniform suspension, adding into the second hole, blowing and mixing uniformly, repeating until reaching the last hole, carrying out 2-time gradient dilution, and discarding redundant bacterial liquid.
D. The 96-well plate was incubated at 37℃for 24h and the minimum inhibitory concentration was measured.
E. Each group of experiments comprises 3 groups of parallel experiments, each experiment is repeatedly carried out for 3 times, and the accuracy of the test results is ensured.
Embodiment two: cytotoxicity detection
A. The oral epithelial cells are cultured outside the complete culture matrix, and after the cells uniformly adhere to the wall and grow to be full, the cells are digested and collected, and the cells are resuspended in the complete culture matrix.
Each well of the 96-well plate was seeded with 5X 10 3 100. Mu.L of cell suspension of individual cells. Cell incubator (37 ℃,5% CO) 2 ) Culturing, and replacing serum-free DMEM medium overnight when the cell is attached to about 80%.
C. And (3) carrying out gradient half-dilution to obtain to-be-detected medicines or amphotericin B standard solutions with different concentrations, and respectively adding serum-free DMEM culture medium for 24 hours.
D. CCK8 experiments were performed according to the instructions of the commercial kit to test the inhibition of oral epithelial cells by the compounds.
E. Each group of experiments comprises 3 groups of parallel experiments, each experiment is repeatedly carried out for 3 times, and the accuracy of the test results is ensured.
The effective concentration of the pimamycin measured by the method is 2 mug/mL, the effective concentration of the amphotericin B is 1 mug/mL, the concentration of the pimamycin derivative is more than 32 mug/mL, and the antibacterial activity is not obvious. In toxicity test of oral epithelial cells, the cell survival rate of the pimamycin effect is 54.18 percent, the cell survival rate of the pimamycin derivative effect is 68.12 percent, and the toxicity is reduced in a same ratio. The compound can be used as a precursor for development of pimaricin derivatives, and has a certain application prospect.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (2)
1. A low-toxicity pimamycin derivative has a molecular formula of C 33 H 49 NO 11 The chemical structural formula is as follows:
2. use of a low toxicity pimamycin derivative as claimed in claim 1 for the preparation of an anti-candida albicans medicament.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110604486.XA CN113583067B (en) | 2021-05-31 | 2021-05-31 | Low-toxicity pimamycin derivative, and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110604486.XA CN113583067B (en) | 2021-05-31 | 2021-05-31 | Low-toxicity pimamycin derivative, and preparation method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113583067A CN113583067A (en) | 2021-11-02 |
| CN113583067B true CN113583067B (en) | 2023-12-05 |
Family
ID=78243232
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110604486.XA Active CN113583067B (en) | 2021-05-31 | 2021-05-31 | Low-toxicity pimamycin derivative, and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113583067B (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104370984A (en) * | 2014-10-28 | 2015-02-25 | 上海交通大学 | High-efficiency and low-toxicity pimaricin derivative as well as preparation method and application thereof |
| CN104447917A (en) * | 2014-10-28 | 2015-03-25 | 上海交通大学 | Low-toxicity pimaricin derivative as well as preparation method and application thereof |
| CN106191095A (en) * | 2016-07-21 | 2016-12-07 | 上海交通大学 | The genetic engineering modified method of pimaricin A bacterial strain |
| CN110551165A (en) * | 2019-08-16 | 2019-12-10 | 上海交通大学 | High-efficiency low-toxicity tetramycin B derivative and preparation and application thereof |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6045815A (en) * | 1997-08-15 | 2000-04-04 | Board Of Regents, The University Of Texas System | Parenteral pimaricin as treatment of systemic infections |
-
2021
- 2021-05-31 CN CN202110604486.XA patent/CN113583067B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104370984A (en) * | 2014-10-28 | 2015-02-25 | 上海交通大学 | High-efficiency and low-toxicity pimaricin derivative as well as preparation method and application thereof |
| CN104447917A (en) * | 2014-10-28 | 2015-03-25 | 上海交通大学 | Low-toxicity pimaricin derivative as well as preparation method and application thereof |
| CN106191095A (en) * | 2016-07-21 | 2016-12-07 | 上海交通大学 | The genetic engineering modified method of pimaricin A bacterial strain |
| CN110551165A (en) * | 2019-08-16 | 2019-12-10 | 上海交通大学 | High-efficiency low-toxicity tetramycin B derivative and preparation and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113583067A (en) | 2021-11-02 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110863021B (en) | Preparation method and application of cytochalasin compound | |
| CN113583067B (en) | Low-toxicity pimamycin derivative, and preparation method and application thereof | |
| CN108530379B (en) | Natural Rakicidins compound Rakicidin G and extraction method thereof | |
| CN102965300B (en) | Micromonospora strain, its preparation method and application | |
| CN115477693B (en) | Two cyclic peptide compounds derived from marine fungi, preparation method thereof and application thereof in preparation of anti-inflammatory drugs | |
| CN109678917B (en) | Amikenarycin and preparation method and application thereof | |
| US11851692B2 (en) | Method for preparing an antimycin compound produced by Streptomyces sp.4-7 | |
| CN114907367B (en) | Macrolide compound FW-Z, fermentation strain, fermentation method and application thereof | |
| CN115109023B (en) | Macrolide compound FWYZ52-A, fermentation strain, fermentation method and application thereof | |
| CN113134006B (en) | Application of ursolic acid derivative in preparing antitumor drugs | |
| CN111807946B (en) | Pratennsinon A compound and preparation and application thereof | |
| CN115557963A (en) | Polyketone compounds with anti-inflammatory activity and preparation method and application thereof | |
| CN111808112B (en) | Pratensilin D compound and preparation and application thereof | |
| CN110452940B (en) | Separation and extraction method of secondary metabolite of streptomycete | |
| CN116103161A (en) | Plant endophytic fungus for producing eupatorium and application thereof | |
| CN114149445A (en) | Preparation method of xanthone compound and application of xanthone compound in resisting drug-resistant bacteria | |
| CN113265436A (en) | Method for efficient biotransformation of anthraquinone products from traditional Chinese medicine sources by using streptomyces coeruleorubidus DM | |
| CN101585809B (en) | Novel compound with antibacterial and antitumor activities | |
| CN118388505B (en) | Carboxylic lactone mycin compound and preparation method and application thereof | |
| CN115433153B (en) | Pair of polyketides with anti-inflammatory activity, preparation method and application thereof | |
| CN113135975B (en) | Ursolic acid derivative and preparation method and application thereof | |
| CN114989190B (en) | Macrolide compound kongjuemycin, preparation method and application thereof | |
| LU503011B1 (en) | Preparation method of Trienomycin A, Trienomycin A and application thereof | |
| CN103012559A (en) | Peptaibol compound and application thereof | |
| CN113143934B (en) | Application of ursolic acid derivative in preparing medicine for preventing or treating cardiovascular diseases |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |