B. glumae strain 411gr-6 was cross-hatch streaked on 2 kg casamino acid-peptone-glucose (CPG; casamino acid 1 g, peptone 10 g, glucose 10 g and agar 18 g, in 1 L distilled water) agar medium and incubated at 28 °C for 3–4 days. Bacterial cells were harvested and extracted with 2 L MeOH, which had a potent inhibitory effect on spore germination against Colletotrichum orbiculare. After concentration in vacuo, the residue (6.0 g) was suspended in distilled water and extracted with CHCl3. The antifungal organic layer was evaporated to dryness. The residue (872.0 mg) was applied to Diaion HP-20 resin columns (Mitsubishi Chemical, Tokyo, Japan) and eluted from columns using stepwise gradients of aqueous Me2CO (0, 20, 40, 60, 80 and 100%, v/v). The antifungal 60% Me2CO fraction (54.4 mg) was further purified using a semi-preparative Varian Prostar 210 (Palo Alto, CA, USA) HPLC system equipped with a reversed-phase C18 column (ODS-H80, 250 × 10 mm, 4 μm, YMC, Kyoto, Japan). The column was eluted at a flow rate of 2 ml min−1 for 30 min using 50% aqueous CH3CN containing 0.1% formic acid. The effluent was monitored at 365 nm. Final purification using a reversed-phase HPLC system gave phencomycin (0.8 mg, tR=13.8), 1 (1.0 mg, tR=11.4) and 2 (2.0 mg, tR=18.4).

A new phencomycin derivative, 4-hydroxyphencomycin (1) was isolated as a yellow powder; UV (MeOH) λmax (logɛ) 209 (4.61), 270 (4.57), 370 (3.91) nm; 1H NMR (CDCl3, 500 MHz): 14.48 (1H, br s, 11-OH), 9.01 (1H, d, J=8.0, H-2), 8.49 (1H, d, J=7.0, H-7), 8.47 (1H, d, J=8.0, H-9), 8.07 (1H, dd, J=7.0, 8.0), 7.46 (1H, d, J=8.0, H-3), 4.13 (3H, s, 12-OCH3); 13C NMR (CDCl3, 125 MHz): 165.7 (C-11), 165.5 (C-12), 156.7 (C-4), 141.5 (C-2), 140.7 (C-9a and C-10a, overlapped signals), 139.0 (C-5a), 134.4 (C-4a and C-7, overlapped signals), 132.4 (C-9), 132.2 (C-8), 130.5 (C-6), 115.9 (C-1), 110.3 (C-3), 53.1 (12-OCH3). HR-ESI-MS analysis identified an [M+H]+ ion at m/z 299.0675 (calcd m/z: 299.0668), consistent with a molecular formula of C15H10N2O5, one more oxygen than phencomycin.6 The 12 degrees of unsaturation, implied by the molecular formula and UV absorbance spectrum, were accounted for a phenazine ring substituted by two carbonyl groups. The 13C NMR spectrum of 1 showed resonances for only 13 carbon atoms, because of the overlapped carbon signals at δ 134.4 and 140.7. 1H NMR spectrum of 1 suggested that the three substituents, including a hydroxyl, carboxyl and carbomethoxy group, could be present at 1, 4 and 6, or 1, 6 and 9 positions. The substituents and their location on the phenazine ring were determined by analysis of the 1H–1H COSY and HMBC spectra of 1 (Figure 2).

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