Investigations into the biosynthesis of the antifungal strobilurins

Prestrobilurin A, formed via benzoate and cinnamate, has been identified as the first enzyme free intermediate in strobilurin biosynthesis.


Selection of Fungal Strains
Bolinea lutea strains F23523 and F24150 were obtained as a gift from Novartis, Switzerland in 2007. Two strains of Strobilurus tenacellus, one gifted by Prof. Zeeck in Germany and other obtained from CBS-KNAW, Fungal Diversity Centre, Netherland were selected for preliminary screening.

Strobilurus tenacellus: Maintenance and Fermentation
S. tenacellus culture was received on malt extract agar (MEA) medium in a glass ampule. For maintenance it was transferred to an agar plate on MEA medium (50 g·L -1 water). The strain grew well on the agar plates and was inoculated into liquid shake flask culture in M2 medium (10 g malt extract, 4 g yeast extract, 4 g glucose L -1 ) as seed culture and incubated for 5 days at 22 °C and 150 rpm. Production cultures in MEA medium (150 mL) in 500 mL conical flask were inoculated with the seed culture (10 mL) and grown at 22 °C and 150 rpm. After 7-10 days of shake flask incubation the liquid culture was collected and filtered under vacuum.

Bolinea lutea: Agar Plate Preparation and Storage
Both strains of B. lutea were preserved as frozen culture. They were grown on agar plates on YGM medium containing yeast extract (0.4%), glucose (0.4%) and malt extract (1%) in water supplemented with 2% each of oatmeal and agar. The autoclaved agar solution was then melted in a microwave and poured into sterile petri dishes. A sterile loop was used to inoculate the agar plates from the frozen glycerol stock culture. The plates were left to grow at 25 °C for 5 days or when a substantial amount of biomass is spread throughout the plate. The mature agar slants could either be used to inoculate seed culture or stored for future use (viable up to 6 months in refrigerator at 4 °C). For long storage the mycelia from mature plates were also Crude B. lutea extract (50-70 mg) was loaded on a silica gel coated TLC glass plate (20 × 20 cm). The plate was developed with 10% EtOAc in petroleum ether (4 consecutive elutions) to afford 5 bands. A yellow pure band at Rf 0.84 corresponded to strobilurin A 2, while strobilurin B 3 appeared at Rf 0.56 as a bright yellow band. Strobilurin H 45 is a minor metabolite which appeared in mixture with strobilurin B 3 which could not be separated by preparative TLC. The target bands were carefully scraped off and extracted with EtOAc for further characterization.
A series of known concentrations of strobilurin A 2, B 3, G 16 and H 45 (0.03-1 mg·mL -1 in MeOH) were detected by diode array detector (200-400 nm). Calibration curves were plotted by area under HPLC peaks vs. a series of standard of known strobilurin concentrations. Crude extraction method was used to identify the compounds via LCMS analysis (retention times, UV characteristics and ESI-MS) and then peak integrations were performed to compare peak area with the corresponding standard curves for quantification.

General Procedure for Precursor Feeding
Conical flasks (500 mL), each containing 150 mL of the specified production medium were inoculated with the mycelia from the seed cultures (5-10 mL) as described earlier. The flasks were incubated at 25 °C and 150 rpm in a shaker. The selected proposed precursors were supplied as a pulse feed on days 2, 3 and 4 of cultivation as MeOH or DMSO solution. Controls in parallel were run with each experiment for systematic comparison. After fermentation for an appropriate duration the flasks were collected for extraction. Metabolites of interest were isolated following standard protocol.

Feeding and Incorporation of [2, 3-13 C 2 ]-Cinnamic Acid (23) and thiolester (24).
Following the above procedure [2, 3-13 C 2 ]-cinnamic acid 23 and its SNAC thiolester 24 were fed separately to the cultures of B. lutea as MeOH solution (0.05 mM). After 8-10 days of cultivation LCMS screening of the crude extract found all observed strobilurins enriched with precursors identified by their difference in m/z value as compared to control. The two major enriched metabolites eluting at Rt 41.7 min and 43.5 min were purified by preparative HPLC. Their structures were confirmed as strobilurin A and strobilurin B by NMR spectroscopy. The 13C NMR signals at 131.3and 130.3 ppm respectively showed incorporations of 70%.

Feeding of (2E, 4Z, 6E)-3-fluorophenyl-4-methylheptatrienoic acid (30) and thiolester (31)
(E,Z,E) 3-fluorophenyl-4-methylhepta-trienoic acid 30 and its SNAC thiolester 31were fed (150 mg·L -1 ) to the whole cell culture of B. lutea following the above feeding procedures. After 8-10 days of fermentation the culture was extracted accordingly. The crude extract was centrifuged and then subjected to LCMS analysis. Initial LCMS analysis suggested that a new peak eluted at 23.1 min with difference of 18 in m/z value from natural analogue strobilurin A 2 is 3fluorostrobilurin A 36 The peak was targeted for purification using the method above. The structure was confirmed as 3-fluorostrobilurin A by 1D, 2D NMR and HRMS investigations. (15-18 mg·L -1 culture, inoculated with 150 mg of 30.

Production of [14-C 2 H 3 ]-3-Fluorstrobilurin A
Following the above procedure(E,Z, E) [4-C 2 H 3 ]-3-fluorophenyl-4-methylhepta-trienoic acid 30, was fed to B. lutea (500 mL culture) at a oncentration of 100 mg·L -1 . The cultures were allowed to grow for 8-10 day at 25 °C and 150 rpm. The culture was collected and extracted using the standard procedure. The crude extract was dissolved in HPLC MeOH (10 mg·mL -1 ) and centrifuged to remove solids before injection. 50 µL of this solution was injected for LCMS analysis. Initial analysis showed a new peak eluted at 24.2 min. The new peak was isolated as mixture with strobilurin A 2 (1:3). Repurification afforded a mixture of enriched-compound and strobilurin A again in 3:2 ratio (0.9 mg). The mixture was subjected to spectroscopic analysis and structure of the new compound was identified as [

3-Fluorocinnamic Acid
Following the above procedure 3-fluorocinnamic acid (150 mg·L -1 ) was fed to B. lutea. Fluorineenriched metabolites were observed by preliminary LCMS analysis. The enriched metabolite 3fluorostrobilurin A 36 was purified and its structure was confirmed by spectroscopic data. Other fluorinated strobilurins, 3-fluorostrobilurin B 55 and 3-fluorostrobilurin C 58 were only observed by LCMS analysis and could not be isolated due to very low yield for NMR confirmation.

Nicotinic Acid
Feeding of nicotinic acid acid (50 mg·L -1 ) was carried out following the above procedures. 3aza-strobilurin A 60 was produced as shown by LCMS analysis. The enriched metabolite 60 was purified as pale yellow oil (10 mg·L

Ethyl 2-methyl-3-oxobutanoate
Ethyl acetoacetate (5 mL, 39.534 mmol) was added dropwise to a mixture of THF (30 mL) and NaH (60%, 1.7 g, 42.500 mmol, pre-washed with n-hexane) at 0 °C under an atmosphere of nitrogen. Iodomethane (2.3 mL, 36.945 mmol) was added to stir for 0.5 h at room temperature and then the mixture was stirred for 20 h at 50 °C. Water (40 mL) was added to quench the reaction and the aqueous layer was extracted with EtOAc (3 × 40 mL). The organic layers were combined, dried over MgSO 4 , filtered and concentrated in vacuo. The crude oil was purified by flash chromatography (SiO 2 , 10% EtOAc in petroleum ether 40-60 °C) giving ethyl 2-methyl-3oxobutanoate as a colourless oil (3.207 g, 56%

Figure S12
Comparison of 13 C-NMR spectrum of [7-13 C]-strobilurin A to that of strobilurin A.