Preparation Of Tautomycin And Their Derivatives And Their Biological Activities

Tautomycin was discovered as a new antibiotic in the screening of agricultural antibiotics against Sclerotinia sclerotiorum at the end of1960’s by Shanghai Institute of Pesticide. It was produced with Streptomyces spiroverticillatus. It’s MIC against S. sclerotiorum was0.5μg/mL. It has strong inhibition activity against Ser/Thr protein phosphatase. Its IC50s for PP1and PP2A were0.21and0.94nM, respectively. Tautomycin owns the maleic anhydride structure which is the important group in the action against protein phosphatase. Most natural products with maleic anhydride or its derivatives have the antifungal activity. What’s the relationship between the maleic anhydride or its derivatives and the antifungal activity? What’s the mechanism of antifungal activities for tautomycin? In the paper, analysis methods, fermentation, isolation and purification, structure modification of tautomycin, and other compounds with maleic anhydride and their derivatives, their antifungal activities against S. sclerotiorum and Botrytis cinerea were investigated. The effects of groups in tautomycin on the antifungal activity and key structure against fungi were also studied.The following major progresses were achieved in this work:Analysis methods of tautomycin in the broth were investigated, such as HPLC, bioassay. HPLC was good to quantify tautomycin and its optimized conditions were:column:C18, wavelength:220nm, mobile phase:CH3CN/H2O=40/60, velocity:1mL/min, inject volume:20μL, the sample dissolved in0.5M NaHCO3. The bioassay with S. sclerotiorum could be used as the method for analysis of tautomycin, but it is time-consuming and inaccurate.Optimization of the feeding process for tautomycin production by S. spiroverticillatus was studied by feeding glucose and/or maleic anhydride. The feeding of glucose was based on the reducing sugar content (lower than8g/L) at the cultivation time of40h. After adding2%(w/v) of glucose, the biomass increased from21g/L to28g/L, and tautomycin from572.06mg/L to837.6mg/L. And1723.1mg/L of tautomycin content (increased201.2%) was obtained by feeding0.2% (w/v) of maleic anhydride solution at pH range of4-7in the broth. In the experiments of the15-L fermenter, tautomycin content reached the highest (1714.7mg/L),199.7%higher than that of the control using the feeding process of adding both glucose and maleic anhydride.The isolation and purification of tautomycin was investigated. An effective method was achieved and high purified sample was obtained. In HPLC analysis, the purity was100.0%. The sample was identified as tautomycin through HPLC-MS.The derivatives of tautomycin were prepared with chemical modification, including tautomycin monmethyl ester, tautomycin dimethyl ester, tautomycin diammonium salt, tautomycin alcohol, tautomycin acid, tautomycin acid monomethyl ester, tautomycin acid trimethyl ester. Their antifungal activities proved the key structure in tautomycin against fungi was the maleic anhydride.The hydrolysis of tautomycin with lipase to produce tautomycin acid was studied. The optimized hydrolysis conditions were:temperature35℃, pH7.0, concentration of tautomycin15000μg/mL, reaction time3h. The Km and Vm were determined as86.1μmol/mL and219.1μmol/mL·min, respectively, using Lineweaver-Burk. The sample obtained from hydrolysis was identified as tautomycin acid.Using tautomycin as the substrate, a tautomycin derivative was obtained through derivation with combinatorial chemistry, such as ammonolysis and base hydrolysis. The compound was preliminarily identified as tautomycin maleimide. The antifungal activities (MIC) against S. sclerotiorum and B. cinerea were0.05ug/mL and60μg/mL, respectively.The precursors of tautomycin acid were synthesized using maleic anhydride as starting material. They all had the antifungal activities.5dialkylmaleic anhydrides were prepared through Grignard Reaction using the dimethyl maleic anhydride as the starting material. The effect of the length of carbon chain on the antifungal activity was determined. The results showed that the short chain of the alkyl group was beneficial for the antifungal activity. So dimethyl maleic anhydride had the best antifungal activity in them. Its antifungal activities (EC100) against S. sclerotiorum and B. cinerea were140μg/mL and180μg/mL. Using the compounds with maleic anhydride as the starting material,4maleimides were synthesized with aniline or butylamine. In them, two are new compounds. They all could effectively inhibit S. sclerotiorum and B. cinerea.The modes of inhibiting fungi by tautomycin and related compounds were preliminarily investigated, too. The results showed that as to S. sclerotiorum,0.3μg/mL tautomycin could effectively inhibit producing sclerotia, while the concentrations of dimethyl maleic anhydride, diphenylmaleic anhydride and dimethyl maleate reached60μg/mL,120μg/mL and1.2μL/mL, respectively. Furthermore, the latter three compounds could only delay the germination of sclerotia, while tautomycin could totally inhibit the germination of sclerotia. As to B. cinerea, dimethyl maleic anhydride had no effect to the germination of spores, but it induced the germ tubes to deform.900μg/mL dimethyl maleic anhydride on leaves of tomato could effectively prevent against B. cinerea.437.5μg/mL tautomycin could totally inhibit the germination of spores, and500μg/mL tautomycin on leaves of tomato could completely prevent against B. cinerea.