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Study On Isolation, Identification, Bioactivity And Biosynthesis Of Maituolaimycin Inhibiting HIV PR And CVB6

Posted on:2005-03-08Degree:DoctorType:Dissertation
Country:ChinaCandidate:Z P WangFull Text:PDF
GTID:1101360152980058Subject:Biochemical Engineering
Abstract/Summary:PDF Full Text Request
In the backgrounds of a mutant Streptomyces regensis 099 with strongactivity against Bacillus subtilis, this dissertation was mainly in the activeconstituent maituolaimycin research of separation and purification fromfermentation broth by bioassay, structure determination, bioactivitiesscreening, biosynthesis pathway and structure mechanics by SYBYLsoftware. Under the direction of bioassays against Bacillus subtilis, the novelactive constituent maituolaimycin was obtained from the fermentation ofthe mutant strain 099. Multi separation and purification methods, includingpreparative HPLC and macroporous resin chromatography were performed.The HRFAB-MS, HRNMR spectrometers analysis methods elucidated thestructure of maituolaimycin, a novel macrylide with formula C32H44O9N2,molecule weight at 600.3 and a rare unit of 1,4-dioxaspiro [2.5] oct-6-enein the 14-membered cyclic structure and a unit of 2-oxa-5-amicazabiocyclo[3.2.2] nonane. The bioactivity screen of maituolaimycin approached at anti-HIV PRactivity with IC50 at 39.8μg/ml, anti-Coxsackievirus B6(CVB6) activitywith IC50 at 231.1μg/ml,Safe Index at 2.88 stronger than positive controlribavirin (PBV)with IC50 at 569μg/ml,Safe Index at 1.75. The acutetoxicity of LD50 at 1707mg/kg (ip.) indicated maituolaimycin low toxicity.The 13C stable isotopes labeled sodium acetate (CH3-13COONa, 13CH3-COONa, 13CH3-13COONa) feeding experiments were performed. The 13CNMR of Maituolaimycin indicated that the abundance of C-1, C-3, C-5, C-7, C-9, C-12, C-14, C-19, C-22, C-28 were enriched. ESI-MS analysis testconfirmed the amounts of adding isotope. These proved origin of carbonskeleton in Maituolaimycin carbon skeleton were from six-moleculeacetate and four-molecule propionate. The rests part may from methionineand glucose. After aimed separation of primary metabolite fatty acid, therelationship model of primary metabolite fatty acid and secondarymetabolite maituolaimycin were developed. Knowing maituolaimycin's novel structure, the structure stimulationsof maituolaimycin with SYBYL software was performed. The molecularmechanics investigated that the 14-membered circle twinkled with pyranring exists in a chair conformation. The tetrahydropyran exist in a boatconformation. Minimize Energy gives the lowest energy was 128KJ/mol. Multi quantitative methods of maituolaimycin were developed. TheHPLC analysis measurement condition was as follows: mobile,acenonitril:water: isopronal (65: 35:2) ; 0.3 ml/min flow rate; 254nm detectwavelength; the maituolaimycin peak was a single peak after ESI-MS andPDAD detect. This analysis measurement behaved good linear, precise,accuracy and recovery to analysis form and uniform samples.
Keywords/Search Tags:Maituolaimycin, Bio-assayed isolation, Structure elucidation, Anti-HIV PR, Anti-CVB6, HPLC-ESI-MS, LD50
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