Instrumentation

Analytical HPLC was performed on a gradient Agilent 1260 Infinity quaternary HPLC system. The column was an Agilent Zorbax SB-C18 (2.1 × 50 mm; 1.8 μm) eluted with a 0.6 mL min−1 gradient of 10–100% MeCN/H2O (0.01% TFA) over 11 min. Preparative HPLC was performed on a gradient Shimadzu HPLC system comprising two LC-20AP preparative liquid pumps with static mixer, SPD-M10AVP diode array detector and CBM-20A system controller with standard Rheodyne injection port. The columns used in the purification of the metabolites were either an Agilent Zorbax SB-C18 column (50 × 150 mm; 5 µm) eluted isocratically at 60 mL min−1 or an Agilent Zorbax SB-C18 column (21.2 × 250 mm; 5 µm) eluted isocratically at 20 mL min−1, eluted isocratically with MeCN/H2O with either 0.01% TFA or 0.1% TFA modifier, as described for each separation. LCMS was performed on an Agilent 1260 Infinity series HPLC equipped with an Agilent 6120 Infinity series single quadrupole mass detector in both positive and negative ion modes. High resolution electrospray ionisation mass spectra (HRESIMS) were obtained using an Agilent 1260 Infinity series HPLC equipped with an Agilent 6230 LC/TOF. Ozone was produced using a GL-3189A ozone generator with an ozone output of 10 mg min−1. Optical rotations were recorded on a Jasco P-2000 polarimeter using a 10 mm quartz cell. UV-vis spectra were recorded on a Jasco V-730 UV-Visible spectrophotometer. IR spectra were recorded using a Jasco FT/IR-4700 with an ATR ProONE (ZnSe crystal) attachment. NMR data were recorded in DMSO-d6 on a Bruker Avance II DRX-600K spectrometer. All NMR spectra were recorded at 25 °C, processed using Bruker Topspin 4 software and referenced to residual solvent signals (DMSO-d6: δH 2.49/δC 39.5).

Strain taxonomy and identification

A. gemini was isolated from a piece of rotting wood collected from woodland in tropical northern Australia in October 1975. Multi-locus phylogenetic analysis using marker sequences extracted from the assembled genome identified the fungal isolate as the founding member of a new genus Austroacremonium belonging to the taxonomic class Sordariomycetes with the type species named as A. gemini [8]. A. gemini is only known from the ex-type isolate MST-FP2131.

Media optimisation

A preserved culture of A. gemini (MST-FP2131) was recovered onto MEA plates, which were incubated for 7 d at 24 °C. Squares of agar from the MEA recovery plates were used as inoculum for solid media optimisation on various agar and grains. After incubating for 7 d at 24 °C, two discs (each 2 cm diameter) were cut from the agars and were extracted with MeOH (2 mL), while subsamples of the grains (5 g) were extracted with MeOH (10 mL). The MeOH extracts were analysed by analytical HPLC to assess their secondary metabolite production and relative yields.

Preparative cultivation & isolation

A. gemini (MST-FP2131) was grown on sterilised (121 °C for 40 min) jasmine rice in 85 × 250-mL Erlenmeyer flasks each containing 50 g of rice. Agar squares from a 7-d culture on Petri plates were used as inoculum for the flasks. The cultures were incubated at 24 °C for 14 d and the grains were pooled and extracted with acetone (2 × 4 L). The combined extracts were reduced in vacuo to produce an aqueous slurry (1 L). The slurry was partitioned against EtOAc (2 × 4 L) and the combined EtOAc layer was dried in vacuo to give a crude extract (42 g). The extract was redissolved in 90% MeOH/H2O (500 mL) and defatted with hexane (2 × 500 mL) to provide an enriched extract (38 g). The enriched extract was adsorbed onto silica gel (85 g) and dry-loaded onto a silica gel column (100 g; 300 × 50 mm). The column was washed once with hexane, then eluted with 50% CHCl3/hexane, 75% CHCl3/hexane and 100% CHCl3, followed by a stepwise gradient of 1, 2, 4, 8, 16, 32 and 100% MeOH/CHCl3 (500 mL each step), to yield 11 fractions (Fr 1–11). A subsample of Fr 9 (2.3 g) was purified by isocratic preparative HPLC (Zorbax C18, isocratic 70% MeCN/H2O containing 0.1% TFA, 60 mL min–1) to yield geministatin A (1) (tR 18.42 min; 203 mg). Fr 7 (430 mg) was purified by isocratic preparative HPLC (Zorbax C18, isocratic 90% MeCN/H2O containing 0.1% TFA, 20 mL min–1) to yield geministatin B (2) (tR 24.68 min; 7.9 mg).

Chemical degradation studiesPreparation of geministatin B (2)

A solution of geministatin A (1; 50 mg) in acetone (4 mL) was treated with aqueous HCl (10 M; 2 mL) and incubated at 25 °C for 24 h. The reaction mixture was diluted with H2O (50 mL), adsorbed onto C18 silica (5 g), washed with H2O (50 mL) and eluted with MeCN (50 mL). The MeCN eluate was purified by preparative HPLC (Zorbax C18; isocratic 100% MeCN, 20 mL min–1) to yield geministatin B (2; tR 11.19 min; 20.0 mg, 40%).

Preparation of geministatin C (3)

A solution of geministatin A (1; 20 mg) in MeOH (2 mL) was heated at 80 °C in a sealed vial for 4 h. The reaction mixture was purified by preparative HPLC (Zorbax C18; isocratic 95% MeCN/H2O, 20 mL min–1) to yield geministatin C (3; tR 10.60 min; 5.9 mg, 30%).

Preparation of geministatin D (4)

A solution of geministatin A (1; 20 mg) in 95% acetone/H2O (2 mL) was heated at 80 °C for 4 h. The reaction mixture was purified by preparative HPLC (Zorbax C18; isocratic 95% MeCN/H2O, 20 mL min–1) to yield geministatin D (4; tR 8.69 min; 4.2 mg, 21%).

Preparation of geministatin E (5)

A solution of geministatin B (2; 20.0 mg) in MeOH (2 mL) was heated at 80 °C in a sealed vial for 30 min. The reaction mixture was purified by preparative HPLC (Zorbax C18; isocratic 100% MeCN, 20 mL min–1) to yield geministatin E (5; tR 8.44 min; 6.8 mg, 34%).

Preparation of dehydromerulinic acid A (6)

A solution of geministatin B (2; 12.1 mg) in 95% acetone/H2O (2 mL) was heated at 80 °C in a sealed vial for 1 h. The reaction mixture was purified by preparative HPLC (Zorbax C18, isocratic 95% MeCN/H2O containing 0.01% TFA, 20 mL min–1) to yield dehydromerulinic acid A (6; tR 11.41 min; 8.3 mg, 69%).

Ozonolysis of geministatin A (1)

Geministatin A (1; 150 mg) was dissolved in MeOH (40 mL) and ozone was bubbled through the solution for 3 min at a rate of 10 mg min–1. The reaction mixture was purified by preparative HPLC (Zorbax C18; isocratic 40% MeCN/H2O containing 0.01% TFA, 20 mL min–1) to yield geministatin A ozonolysis product (7; tR 5.69 min; 11.7 mg, 7.8%).

Description of physicochemical propertiesGeministatin A (1)

White powder; [α]D24 ‒28.7 (c 1.00, MeOH); UV (MeCN) λmax (log ε) 215 (4.49), 265 (4.14), 305 (3.86) nm; IR (ATR) νmax 3673, 2987, 2883, 1795, 1634, 1529, 1385, 1219, 1162, 1112, 945, 905, 818, 644 cm–1; 1H and 13C NMR see Table 1 and Table S2. HR-ESI(‒)-MS m/z 699.3389; calcd for C38H51O12‒ [M ‒ H]–, 699.3386.

Table 1 1H (600 MHz) and 13C (150 MHz) NMR data for 1 and 2 in DMSO-d6Geministatin B (2)

White powder; UV (MeCN) λmax (log ε) 215 (4.55), 265 (4.19), 305 (3.95) nm; IR (ATR) νmax 3346, 3041, 2987, 2880, 2584, 1798, 1633, 1530, 1388, 1288, 1213, 1165, 1092, 939, 873, 820, 765, 639 cm–1; 1H and 13C NMR see Table 1 and Table S3; HR-ESI(‒)-MS m/z 537.2863; calcd for C32H41O7‒ [M ‒ H]‒, 537.2858.

Geministatin C (3)

White powder; [α]D24 ‒26.5 (c 1.00, MeOH); UV (MeCN) λmax (log ε) 215 (3.99), 265 (3.63), 305 (3.20) nm; IR (ATR) νmax 3036, 2986, 2878, 1801, 1682, 1633, 1598, 1487, 1354, 1286, 1230, 1196, 1152, 1115, 931, 822, 609 cm–1; 1H and 13C NMR see Table S4; HR-ESI(‒)-MS m/z 563.3224; calcd for C31H47O9‒ [M ‒ H]‒, 563.3226.

Geministatin D (4)

White powder; [α]D24 –34.7 (c 0.250, MeOH); UV (MeCN) λmax (log ε) 215 (3.91), 265 (3.38), 305 (3.12) nm; IR (ATR) νmax 3964, 3681, 3032, 2985, 2881, 1767, 1527, 1392, 1325, 1197, 1156, 1115, 1053, 928, 874, 822, 745, 653, 603 cm–1; 1H and 13C NMR see Table S5; HR-ESI(‒)-MS m/z 549.3075; calcd for C30H45O9‒ [M ‒ H]‒, 549.3069.

Geministatin E (5)

Pale yellow oil; UV (MeCN) λmax (log ε) 215 (4.38), 265 (4.08), 305 (3.67) nm; IR (ATR) νmax 3101. 2986, 2880, 1963, 1841, 1634, 1535, 1406, 1356, 1289, 1220, 1183, 1126, 1056, 981, 889, 784, 641 cm–1; 1H and 13C NMR see Table S6; HR-ESI(‒)-MS m/z 401.2702; calcd for C25H37O4‒ [M ‒ H]‒, 401.2697.

Ozonolysis Product (7)

White powder; [α]D24 ‒32.7 (c 0.500, MeOH); UV (MeCN) λmax (log ε) 215 (4.68), 265 (4.33), 305 (4.06) nm; IR (ATR) νmax 3679, 2984, 2891, 1771, 1632, 1523, 1388, 1217, 1159, 1109, 873, 819, 736, 645 cm–1; 1H and 13C NMR see Table S8; HR-ESI(‒)-MS m/z 639.2302; calcd for C30H39O15‒ [M ‒ H]‒, 639.2294.