4.1 General experimental procedures

Instrumental screening of all samples was done with HPLC-HRMS. Liquid chromatographic separation was achieved with a ReproSilGold C18-AQ column (150 × 2 mm i.d., 3 µm) equipped with a 5 × 2 mm i.d. ReproSilPur C18-AQ, 3 µm guard column (both Dr. Maisch HPLC GmbH, Ammerbuch, Germany) on a Shimadzu HPLC system (DGU-20A5R degasser; Nexera XT LC-20AD XR pump; Nexera XR, SIL-20AC XR autosampler, CTO-10SD VP oven unit; connected through a CBM-20A communication bus module, all Shimadzu, Tokyo, Japan), coupled to a high resolution Fourier transform mass spectrometer with a heated electrospray ionization source (LTQ Orbitrap XL, Thermo Scientific, Dreieich, Germany). The solvents used for LC separation were H2O + 0.1% FA (solvent A) and MeCN + 0.1% FA (solvent B). The gradient had a flow rate of 0.3 mL/min and started at 10% B for 1 min, was increased to 100% B over 20 min, held at 100% for additional 5 min, dropped to starting conditions within 0.1 min, and re-equilibrated for 4.9 min. The oven temperature was set to 40.0 °C. For ionization, HESI was applied. Capillary and vaporizer temperature were set to 350 °C each. Gas flows were 40 arbitrary units (AU) for sheath gas, and 20 AU and 5 AU for auxiliary gas flow and sweep gas flow, respectively. The source voltage was 3.5 kV, source current was 100 µA, capillary voltage was at 20 V and tube lens voltage at 120 V. The untargeted screening measurement ranged from m/z 150-1000. Data acquisition and evaluation was performed using the Xcalibur Tune Plus software, Version 3.1.66.10 (Thermo Fisher Scientific, Dreieich, Germany).

Furthermore, general screening was performed with TLC. For each analyte, a standard solution of 200 µg/mL in 80/20 MeCN/H2O (v/v) was prepared using pure substance. For chromatographic separation, 10  µL of the standard solution was applied to aluminum plates (5 × 10 cm) modified with RP-18 W nano silica gel with a fluorescent indicator at λ = 254 nm with a thin glass capillary. The mobile phase consisted of 30/70 MeCN/H2O (v/v), and the plates were developed for 10 min. Determination of the retention factors (Rf) was performed under UV-light at λ = 254 nm by dividing the distance travelled by the analyte by the distance of the solvent front. The Rf values were Rf = 0.41 (apicidin F, 1), Rf = 0.49 (apicidin J, 2), Rf = 0.10 (apicidin K, 3), and Rf = 0.21 (apicidin L, 4).

4.2 Chemicals

All solvents and reagents were purchased from Sigma-Aldrich (Deisenhofen, Germany), VWR (Darmstadt, Germany), Fisher Scientific Inc. (Schwerte, Germany), or Merck Schuchardt (Hohenbrunn, Germany) in gradient or analytical grade. Water for chromatography was purified with a PURELAB Flex 2 system (Veolia Water Technologies, Celle, Germany). d/l-2-aminooctanoic acid, l-glutamic acid, l-tryptophane, polyethylene glycol 3350, and resazurin sodium salt were purchased from Sigma-Aldrich/Merck KGaA (Darmstadt, Germany). l-lysin-monohydrate was purchased from Biosynth® GmbH (Berlin, Germany), l-phenylalanine was purchased from Flunka™ Biochemica, Honeywell Inc., (Offenbach, Germany). Copper sulfate pentahydrate, zinc sulfate heptahydrate, dimethylsulfoxid, and 2-[4-(2-hydroxyethyl)piper-azin-1-yl]ethanesulfonic acid were purchased from Carl Roth GmbH + Co. KG (Karlsruhe, Germany). Fetal calf serum, penicillium/streptomycin solution, and phosphate bovine serum was purchased from PAN™ Biotech (Aidenbach, Germany). Trypsin was purchased from Merck Biochrom GmbH (Berlin, Germany), Dulbecco’s modified eagle medium was purchased from Thermo Fisher Gibco (Darmstadt, Germany). Ethyl acetate and ammonium sulfate were purchased from Grüssing GmbH (Filsum, Germany).

4.3 Fungal material

The filamentous fungus Fusarium fujikuroi IMI58289 (Commonwealth Mycological Institute, Kew, UK) and the mutants ΔAPF1, OE::APF2, ΔAPF3/OE::APF2, and ΔAPF9/OE::APF2 were used for the investigations [20]. For pre-cultivation, 300-mL Erlenmeyer flasks containing 100 mL Darken’s medium (DVK) [33] were inoculated with a 1 cm2 agar plug of the respective strain and shaken at 28 °C at 180 rpm for three days in the dark (New Brunswick Innova® 44 incubator shaker, Eppendorf, Hamburg, Germany). Standard culture was grown by inoculating each DVK agar plates with 500 µL of the liquid DVK pre-culture and cultivate for 5–10 days in an incubator (KB-115 refrigerated incubator, BINDER GmbH, Tuttlingen, Germany) at 28 °C and saturated humidity in the dark.

4.4 Micro-scale cultivation

For fungal cultivation, sterile flat bottom 96-well plates (Sarstedt AG & Co.KG, Nümbrecht, Germany) were prepared by adding 150 µL of medium with a Multipette® M4 equipped with a sterile 5 mL Combitip® advanced (both Eppendorf SE, Hamburg, Germany) to each well. Upon solidification of the media, the 96-well plate was inoculated with 50 µL of liquid pre-culture and grown for 5 days in the incubator at 28 °C in the dark. Extraction was carried out twice with MeOH/H2O (80/20, v/v). Here, 100 µL of extraction solution were added to the culture and agitated with the pipette tip to separate the mycelium from the agar and disrupt the fungal material. Then, the solution was transferred to a MULTI 96 filterplate (CHROMAFIL® PTFE, 8 mm, 0.45 µm pore size, Macherey–Nagel, Düren, Germany). This extraction step was repeated a second time before the filter was transferred to a NucleoVac 96 Vacuum Manifold (Macherey–Nagel, Düren, Germany) containing a Nunc™ 96-well polypropylene sample processing & storage microplate (Thermo Fisher Scientific Inc., Darmstadt, Germany). The samples were filtered under reduced pressure, the sample plate was covered with ThinSeal™ sealing films (EXCEL Scientific, Victorville, CA, USA) and submitted for HPLC-HRMS measurement.

4.5 Supplementation screening

For supplementation screening, the fungal strains were grown in 96-well plates containing solid DVK agar plugs as previously described. The supplementation was performed with single as well as combined amino acids. A control row without any supplementation was always included. For the initial experiments, low (5 mM) and high (50 mM) amino acid concentrations were prepared. This was done by weighting the respective amino acid on an analytical scale (Acculab Atilon Analytical Balance, Sartorius AG, Göttingen, Germany) and dissolving the powder into the hand-warm medium after autoclavation. For the optimization of the 2-aoa supplementation, standard agar plates were cast, and the fungal cultivation followed the standard procedure as presented previously.

4.6 Laboratory-scale supplemented culture and compound isolation procedure

For laboratory-scale cultivation of both strains, D/L-2-aminooctanoic acid was supplemented to the DVK medium at a final concentration of 20 mM. For the OE::APF2 strain, 150 supplemented agar plates were cast, while for ΔAPF9/OE::APF2, 150 standard agar plates and 200 supplemented agar plates were prepared and each inoculated with 500 µL of liquid DVK pre-culture. Cultivation was performed in the incubator for 5 days at 28 °C in the dark. Then, agar plates were scraped into a 5 L container and extracted three times with 2.5 L EtOAc. The extract was evaporated to dryness on a rotary evaporator at 40 °C (Laborota 4000 rotary evaporator, Heidolph, Schwabach, Germany, connected to a Duo Chiller RC-10 cooling system, VWR®, Darmstadt, Germany, with a PC510 vacuum pump equipped with a CVC 2 vacuum controller, both Vacuubrand™, Wertheim, Germany), remaining water was removed by lyophilization (Lyovac GT 2, Finn-Aqua Sanatasalo-Sohlberg GmbH, Cologne, Germany). The crude extract was re-constituted with 4 mL MeCN/H2O (80/20, v/v) and applied to a 5 g/20 mL Strata® C18-E (55 µm, 70 Å) SPE column (Phenomenex, Aschaffenburg, Germany). In detail, the SPE was conditioned with 50 mL MeCN, and equilibrated with 50 mL H2O before loading the sample. Here, after loading 1 mL of sample, 10 mL of H2O were applied as an additional washing step. Elution was performed by applying a gradient of MeCN and H2O which increased the ratio of MeCN in steps of 10% until 50/50 MeCN/H2O (v/v), followed by an elution with 100% MeCN. Elution was accelerated by application of reduced pressure (400 mbar) to the outlet of the SPE-chamber. The collected fractions were analyzed by TLC and HPLC-HRMS to identify the analyte-containing fractions, which were combined and once again brought to dryness as previously described. Next, the dried fractions were re-constituted in 4 mL of 80/20 MeCN/H2O (v/v) and further purified on a preparative HPLC–UV system (Degas® GPC degasser, Biotech I Kungsbacka AB, Onsala, Sweden, Jasco PU-2087 pump coupled to Jasco UV-2075-detector equipped with a Jasco AS-2057 Plus direct injection system interfaced by a Jasco LC-Net II / ADC, Jasco, Groß-Umstadt, Germany, and a Column Thermostat Jetstream 2 oven, VDS optilap Chromatographietechnik GmbH, Berlin, Germany). The chromatographic separation was achieved on a ReprosilPur 120 C18 (250 × 10 mm, 5 µm) semi-preparative column (Dr. Maisch GmbH, Ammerbuch, Germany) equipped with a SecurityGuard™ Gemini® C6-phenyl cartridge (4 × 2 mm) guard column (Phenomenex, Aschaffenburg, Germany) and a gradient consisting of H2O (solvent A) and MeCN (solvent B). The gradient started with 10% B, was held for 2 min, raised to 60% B within 11.5 min, held until 16 min, increased to 100% within 3 min, held for another 4 min, changed back to starting conditions within 0.01 min and re-equilibrated for 5 min. Each chromatographic run was monitored with a UV-detector set to λ = 225 nm, flow rate was set to 5 mL/min, oven temperature was at 40 °C. Data evaluation was done using ChromPass Software (version 1.8.6.1, Jasco, Groß-Umstadt, Germany). Apicidin F (1) and K (3) eluted after 15.4 and 15.2 min, and apicidin J (2) and L (4) eluted at 13.7 and 13.6 min. The compound purity was determined on a HPLC–DAD-ELSD system (Jasco PU-2087 pump coupled to a MD-2010 Plus diode array detector interfaced by a LC-Net II/ADC, Jasco, Groß-Umstadt, Germany, with a Column Thermostat Jetstream 2 oven, VDS optilap Chromatographietechnik GmbH, Berlin, Germany and an evaporative light scattering detector, Shimadzu, Tokyo, Japan), using a ReprosilGold C18-AQ column (150 × 2 mm i.d., 3 µm) equipped with a 5 × 2 mm i.d. guard column of the same material (both Dr. Maisch HPLC GmbH, Ammerbuch, Germany). The 30 min gradient was performed with H2O + 0.1% FA (solvent A) and MeCN + 0.1% FA (solvent B). The oven temperature was set to 40 °C, flow rate was at 0.3 mL/min. The gradient started at 10% B for one minute, was increased to 100% B over 20 min, held at 100% for additional 5 min, dropped to starting conditions within 0.1 min, and re-equilibrated for 4.9 min. The DAD spectrum was recorded between 195 and 650 nm, ELSD was set to 50 °C at 350 kPa with compressed air, and the gain was 10. The isolated compounds were analyzed at a concentration of 200–400 µg/mL in 80/20 MeCN/H2O (v/v). Their purity was determined by comparing 20 µL injection of the analyte solution with a previously injected 80/20 MeCN/H2O (v/v) blank solution. Here, the injection peak and solvent signals from the gradient were excluded from the evaluation. Data evaluation was done using ChromPass Software (version 1.8.6.1, Jasco, Groß-Umstadt, Germany).

The final yield of each compound (isolated with and without supplementation with dl-2-aminooctanoic acid) was 7.5 mg of apicidin F (1) at ≥ 98% purity from 150 supplemented agar plates, 7.9 mg of apicidin J (2) at ≥ 98% purity from 150 supplemented agar plates, 13.3 mg of apicidin K (3) at ≥ 98% purity from 200 supplemented and 150 standard agar plates, and 25.2 mg of apicidin L (4) at ≥ 98% purity from 200 supplemented and 150 standard agar plates.

4.7 Structure elucidation of apicidin L

The structure elucidation was performed using NMR-spectroscopy and HRMS fragmentation experiments. The 1H, 13C, and 2D NMR spectra were recorded on a DD2 600 MHz NMR spectrometer (Agilent Technologies, Santa Clara, CA, USA). The signals are reported in parts per million (ppm) and are referenced to the solvent. For structure elucidation, additionally to the 1D NMR experiments, 2D NMR experiments such as H,H-correlated spectroscopy (H,H-COSY), heteronuclear multiple-quantum correlation (HMQC), heteronuclear multiple bond correlation (HMBC), and rotating Overhauser enhancement spectroscopy (ROESY) were performed. The pulse programs were taken from the software library. NMR measurements were performed with apicidin L (4) dissolved in C5D5N to show allocation of nitrogen-coupled protons. -, 1 The obtained data was evaluated using MestReNova 14.2 (Mestrelab Research S.L., Santiago de Compostela, Spain). The complete dataset can be found in the Additional file 1.

The HRMS fragmentation experiments were performed on a Thermo Scientific LTQ Orbitrap XL system (Thermo Scientific, Dreieich, Germany) using direct injection with a syringe pump. apicidin L (4) was dissolved in 80/20 MeCN/H2O (v/v) containing 1% acetic acid at a concentration of 15 µg/mL. The injection flow rate was 5 µL/min. The ionization was performed in positive mode with a heated electrospray ionization (HESI) applying the following parameters: sheath gas flow was at 5 AU, aux gas flow and sweep gas flow were not applied. The HESI was turned off. Capillary temperature was set to 275 °C, while source voltage was at 4.0 kV, capillary voltage was at 43 V and tube lens voltage was set to 150 V. Fragmentation experiments were performed with collision-induced dissociation (CID) at an isolation width of 1.8 Da. For MS2, the relative fragmentation energy was at 11%, while for MS3 and MS4 10% to 13% were applied depending on the parent ion to be fragmented. Data evaluation was performed using the Xcalibur software (version 3.1, Thermo Fisher Scientific, Dreieich, Germany).

Apicidin F (1) Yellow-white powder (7.5 mg from 150 agar plates). TLC: 0.41 (MeCN/H2O, 30/70, v/v). UV (MeCN/H2O + 0.1% FA) λmax (log ε): 230, 290 nm. 1H NMR (CD3OD, 600 MHz) δ 7.57 (1H, d, J = 7.9 Hz, H-5), 7.43 (2H, d, J = 18.8 Hz, H-5‴, H-9‴), 7.36 (1H, d, J = 8.2 Hz, H-8), 7.32 (2H, s, H-6‴, H-8‴), 7.29–7.23 (1H, m, H-7), 7.23–7.15 (1H, m, H-7‴), 7.06 (1H, t, J = 7.5 Hz, H-6), 7.02 (1H, s, H-9), 5.26 (1H, t, J = 7.7 Hz, H-2‴), 5.17 (1H, d, J = 5.8 Hz, H-2″), 4.61 (1H, d, J = 16.1 Hz, H-2), 4.30 (1H, dt, J = 15.6, 8.1 Hz, H-2′), 4.00 (3H, s, H-10), 3.81 (2H, d, J = 13.5 Hz, H-6″), 3.28–3.16 (4H, m, H-3, H-3‴), 3.04 (2H, dd, J = 13.5, 7.5 Hz, H-3‴), 2.93 (2H, t, J = 13.3 Hz, H-6″), 2.19 (2H, t, J = 7.6 Hz, H-7′), 1.96 (2H, d, J = 14.5 Hz, H-3″), 1.72–1.66 (2H, m, H-3′), 1.62 (2H, d, J = 12.2 Hz, H-4′), 1.56 (2H, s, H-6′), 1.49 (3H, d, J = 15.6 Hz, H-2″, H4″), 1.44 (2H, dt, J = 12.9, 5.6 Hz, H-3″), 1.36–1.25 (2H, m, H-5′), 1.17 (2H, dd, J = 30.7, 11.8 Hz, H-5″). 13C NMR (CD3OD, 150 MHz) δ 175.87 (C, C-1′), 175.44 (C, C-1), 175.00 (C, C-1‴), 173.49 (C, C-1″), 138.49 (C, C-4‴), 133.86 (C, C-8a), 130.42 (CH, C-5‴, C-9‴), 129.60 (CH, C-6‴, C-8‴), 127.89 (CH, C-7‴), 125.01 (C, C-4a), 123.62 (CH, C-7), 123.04 (CH, C-9), 120.88 (CH, C-6), 119.74 (CH, C-5), 109.34 (CH, C-8), 107.72 (C, C-4), 66.21 (CH3, C-10), 59.43 (CH, C-2), 56.51 (CH, C-2′), 52.12 (CH, C-2″), 51.45 (CH, C-2‴), 45.20 (CH2, C-6″), 37.81 (CH2, C-3‴), 30.68 (CH2, C-3′), 29.86 (CH2, C-5′), 26.85 (CH2, C-4′), 26.68 (CH2, C-3), 26.40 (CH2, C-5″), 26.21 (CH2, C-6′), 25.23 (CH2, C-3″), 20.62 (CH2, C-4″). Positive ion HRESIMS (25 µg/mL): m/z 646.3230 (calcd for C35H43N5O7 [M + H]+ 646.3236, Δm: 0.9 ppm), purity ≥ 98%.

Apicidin J (2) White powder (7.9 mg from 150 agar plates). TLC: 0.49 (MeCN/H2O, 30/70, v/v). UV (MeCN/H2O + 0.1% FA) λmax (log ε): 220, 291 nm. 1H NMR (C5D5N, 600 MHz) δ 9.71 (1H, d, J = 7.0 Hz, NH), 8.57 (1H, d, J = 10.0 Hz, NH‴), 7.89 (1H, d, J = 10.0 Hz, NH′), 7.75 (1H, d, J = 7.9 Hz, H-5), 7.53 (1H, d, J = 8.2 Hz, H-8), 7.46 (3H, dd, J = 9.9, 2.8 Hz, H-5‴, H-9 H-9‴), 7.34 (3H, dt, J = 10.8, 7.5 Hz, H-7, H-6‴, H-8‴), 7.30–7.23 (2H, m, H-7‴), 7.17 (1H, t, J = 7.4 Hz, H-6), 5.58 (1H, dd, J = 8.7, 7.0 Hz, H-2‴), 5.26 (1H, s, OH′), 5.00 (1H, dd, J = 7.8, 1.5 Hz, H-2″), 4.78 (1H, t, J = 7.8 Hz, H-2′), 4.69–4.62 (1H, m, H-2), 4.08 (2H, ddd, J = 20.2, 13.9, 9.3 Hz, H-3, H-5″), 3.93 (3H, s, H-10), 3.89–3.81 (3H, m, H-3), 3.55 (4H, dd, J = 13.6, 8.6 Hz, H-3‴), 3.37–3.33 (2H, m, H-5″), 3.30 (2H, dd, J = 13.6, 7.0 Hz, H-3‴), 2.42 (2H, t, J = 7.5 Hz, H-7′), 2.39–2.32 (2H, m, H-3″), 2.10 (2H, ddd, J = 19.8, 11.9, 8.9 Hz, H-4″), 2.06–1.94 (4H, m, H-3′, H-6′), 1.74–1.66 (4H m,, H-3′, H-6′), 1.57 (2H, dtd, J = 12.2, 8.0, 4.0 Hz, H-4″), 1.47–1.37 (2H, m, H-3″), 1.36–1.26 (4H, m, H-4′, H-5′). 13C NMR (C5D5N, 150 MHz) δ 176.07 (C, C-1′), 175.23 (C, C-1), 174.04 (C, C-1‴), 172.48 (C, C-1″), 138.66 (C, C-4‴), 133.51 (C, C-8a), 130.27 (CH, C-5‴, C-9‴), 129.54 (CH, C-6‴, C-8‴), 127.68 (CH, C-7‴), 124.81 (C, C-4a), 123.47 (CH, C-7), 123.29 (CH, C-9), 120.79 (CH, C-6), 120.03 (CH, C-5), 109.44 (CH, C-8), 108.65 (C, C-4), 66.30 (CH3, C-10), 61.59 (CH, C-2), 58.75 (CH, C-2″), 56.04 (CH, C-2′), 54.36 (CH, C-2‴), 47.31 (CH2, C-5″), 37.17 (CH2, C-3‴), 35.30 (CH2, C-7′), 30.83 (CH2, C-3′), 29.68 (CH2, C-5′), 26.56 (CH2, C-4′), 26.48 (CH2, C-6′), 25.89 (CH2, C-3), 25.63 (CH2, C-3″), 25.35 (CH2, C-4″). Positive ion HRESIMS (25 µg/mL): m/z 632.3071 (calcd for C34H41N5O7 [M + H]+ 632.3079, Δm: 1.3 ppm), purity ≥ 98%.

Apicidin K (3) White powder (13.3 mg from 200 supplemented and 150 standard agar plates). TLC: 0.10 (MeCN/H2O, 30/70, v/v). UV (MeCN/H2O + 0.1% FA) λmax (log ε): 235, 291 nm. 1H NMR (C5D5N, 600 MHz) δ 9.97 (1H, d, J = 6.9 Hz, NH), 8.55 (1H, d, J = 10.1 Hz, NH‴), 7.73 (1H, d, J = 8.0 Hz, H-5), 7.54 (1H, d, J = 8.2 Hz, H-8), 7.47 (2H, d, J = 7.2 Hz, H-5‴, H-9‴), 7.42 (1H, s, H-9), 7.33 (3H, q, J = 7.3 Hz, H-7, H-6‴, H-8‴), 7.30–7.25 (1H, m, H-7‴), 7.17 (1H, t, J = 7.5 Hz, H-6), 5.85 (1H, dt, J = 10.1, 7.5 Hz, H-2‴), 5.48 (1H, d, J = 5.7 Hz, H-2″), 5.16 (1H, s, OH′), 4.79 (1H, q, J = 8.6 Hz, H-2′), 4.55 (1H, dt, J = 9.8, 6.7 Hz, H-2), 4.33 (1H, d, J = 13.3 Hz, H-6″), 4.15 (2H, dd, J = 14.5, 9.9 Hz, H-3), 3.92 (3H, s, H-10), 3.80 (2H, t, J = 6.5 Hz, H-8′), 3.76 (2H, dd, J = 14.5, 6.7 Hz, H-3), 3.55 (2H, dd, J = 13.8, 7.3 Hz, H-3‴), 3.40 (2H, dd, J = 13.8, 7.6 Hz, H-3‴), 3.30–3.22 (2H, m, H-6″), 2.28 (2H, dd, J = 11.2, 6.7 Hz, H-4″), 2.01 (2H, d, J = 13.4 Hz, H-3″), 1.95 (2H, dt, J = 18.0, 7.1 Hz, H-3′), 1.62 (4H, p, J = 7.1 Hz, H-3′, H-7′), 1.42–1.28 (8H, m, H-3″, H-4″, H-5″, H-6′), 1.28–1.18 (4H, m, H-4′, H-5′), 1.09 (2H, tdd, J = 13.2, 9.3, 4.0 Hz, H-5″). 13C NMR (C5D5N, 150 MHz) δ 177.18 (C, C-1′), 174.74 (C, C-1‴), 174.36 (C, C-1), 172.53 (C, C-1″), 138.87 (C, C-4‴), 133.45 (C, C-8a), 130.26 (CH, C-5‴, C-9‴), 129.28 (CH, C-6‴, C-8‴), 127.40 (CH, C-7‴), 124.63 (C, C-4a), 123.38 (CH, C-7), 123.34 (CH, C-9), 120.70 (CH, C-6), 119.88 (CH, C-5), 109.37 (CH, C-8), 108.63 (C, C-4), 66.16 (CH3, C-10), 62.46 (CH2, C-8′), 61.96 (CH, C-2), 55.53 (CH, C-2′), 51.58 (CH, C-2″), 50.93 (CH, C-2‴), 44.75 (CH2, C-6″), 38.00 (CH2, C-3‴), 34.03 (CH2, C-7′), 30.85 (CH2, C-3′), 29.81 (CH2, C-5′), 26.64 (CH2, C-4′), 26.56 (CH2, C-6′), 26.04 (CH2, C-3), 25.02 (CH2, C-3″), 20.42 (CH2, C-4″). Positive ion HRESIMS (25 µg/mL): m/z 632.3432 (calcd for C34H43N5O6 [M + H]+ 632.3443, Δm: 1.7 ppm), purity ≥ 98%.

Apicidin L (4) White powder (25.2 mg from 200 supplemented and 150 standard agar plates). TLC: 0.21 (MeCN/H2O, 30/70, v/v). UV (MeCN/H2O + 0.1% FA) λmax (log ε): 235, 287 nm. 1H NMR (C5D5N, 600 MHz) δ 9.96 (1H, d, J = 6.7 Hz, NH), 8.55 (1H, d, J = 10.1 Hz, NH‴), 7.86 (1H, d, J = 10.0 Hz, NH′), 7.74 (1H, d, J = 7.9 Hz, H-5), 7.53 (1H, d, J = 8.1 Hz, H-8), 7.50–7.43 (3H, m, H-9, H-5‴, H-9‴), 7.36–7.30 (3H, m, H-7, H-6‴, H-8‴), 7.25 (1H, t, J = 8.1 Hz, H-7‴), 7.18 (1H, t, J = 7.0 Hz, H-6), 5.61 (1H, ddd, J = 10.2, 8.7, 6.8 Hz, H-2‴), 5.10 (1H, s, OH′), 5.03–4.95 (1H, m, H-2″), 4.75 (1H, ddd, J = 10.0, 8.4, 6.8 Hz, H-2′), 4.48 (1H, dt, J = 9.6, 7.0 Hz, H-2), 4.24–4.14 (2H, m, H-3), 4.11 (2H, dd, J = 9.6, 3.9 Hz, H-5″), 3.91 (3H, s, H-10), 3.87 (2H, dd, J = 14.5, 7.3 Hz, H-3), 3.81 (2H, t, J = 6.5 Hz, H-8′), 3.54 (2H, dd, J = 13.6, 8.7 Hz, H-3‴), 3.35 (2H, dt, J = 10.1, 8.1 Hz, H-5″), 3.28 (2H, dd, J = 13.6, 6.8 Hz, H-3‴), 2.38–2.30 (2H, m, H-3″), 2.11 (2H, dt, J = 18.0, 6.8 Hz, H-4″), 2.06–1.89 (2H, m, H-3′), 1.66–1.60 (4H, m, H-3′, H-7′), 1.60–1.51 (2H, m, H-4″), 1.50–1.31 (4H, m, H-6′, H-3″), 1.30–1.18 (4H, m, H-4′, H-5′). 13C NMR (C5D5N, 150 MHz) δ 176.65 (C, C-1′), 175.13 (C, C-1), 173.85 (C, C-1‴), 172.39 (C, C-1″), 138.64 (C, C-4‴), 133.44 (C, C-8a), 130.16 (CH, C-5‴, C-9‴), 129.42 (CH, C-6‴, C-8‴), 127.53 (CH, C-7‴), 124.68 (C, C-4a), 123.36 (CH, C-7), 123.28 (CH, C-9), 120.69 (CH, C-6), 119.93 (CH, C-5), 109.35 (CH, C-8), 108.72 (C, C-4), 66.15 (CH3, C-10), 62.46 (CH, C-8′), 62.40 (CH2, C-2), 58.63, (CH, C-2″), 55.80 (CH, C-2′), 54.28 (CH, C-2‴), 47.18 (CH2, C-5″), 37.15, (CH2, C-3‴), 34.05 (CH2, C-7′), 30.87 (CH2, C-3′) 29.86 (CH2, C-5′), 26.67 (CH2, C-4′), 26.58 (CH2, C-6′), 26.01 (CH2, C-3), 25.52 (CH2, C-3″), 25.35 (CH2, C-4″). Positive ion HRESIMS (25 µg/mL): m/z 618.3280 (calcd for C34H43N5O6 [M + H]+ 618.3287, Δm: 1.1 ppm), purity ≥ 98%.

4.8 Crystallization attempts of apicidin L

Initially, standard solutions of pure apicidin L (4) were prepared at a concentration of 200 µg/mL in MeCN/H2O. The applied attempts included slow solvent evaporation, hanging drop vapor diffusion from a 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and polyethylene glycol solution, and supplementation of zinc- and copper-salts. None of these investigations resulted in formation of a single crystal but amorphous, non-defractable structures. In case of supplementation of Cu(II), a precipitate was obtained.

4.9 Cytotoxicity assays

All four compounds (1–4) were taken for cytotoxicity testing and compared to apicidin (5). Here, two cell lines were used, human liver hepatocellular carcinoma cell line HepG2 and human lung carcinoma cell line A549. Cell viability was determined using the resazurin assay. About 1 × 104 cells were seeded in 96-well plates in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10 mM HEPES, 100 IU/mL penicillin, 100 µg/mL streptomycin, 2 mM l-glutamine and 10% (v/v) fetal calf serum (FCS) and cultivated in 5% CO2 at 37 °C for 24 h. After starvation for 24 h in medium without serum, the compounds of interest were applied in concentrations from 0.01 to 100 µM. The substances were incubated for 24 h. Subsequent measurement in a microplate reader (Infinite M200Pro, Tecan Austria GmbH, Männedorf, Switzerland) allowed the determination of cell viability. The data was normalized to the negative control (1% DMSO). T-2 toxin at 10 µM served as positive control. Statistical evaluation of ANOVA and post hoc Tukey’s test was performed with OriginPro 2024, version 10.1.0.170. Additionally, cytotoxicity against rat skeletal myoblasts (L6) was also determined by validated protocols at the Swiss TPH [34].

4.10 Antiparasitic activity

Furthermore, the whole set of isolated compounds was tested in vitro against T. brucei rhodesiense (bloodstream trypomastigotes) and P. falciparum (intraerythrocytic forms). All assays were performed using validated protocols at the Swiss TPH [34].

4.11 In vitro HDAC inhibition assay against HDAC1-3 and HDAC6

In vitro inhibitory activity against HDAC1-3 and 6 was determined using our previously published assay protocol [35, 36]. For compounds and controls, three-fold serial dilutions of the respective DMSO-stock solution in assay buffer (50 mM Tris–HCl, pH 8.0, 137 mM NaCl, 2.7 mM KCl, 1.0 mM MgCl2•6 H2O, 0.1 mg/mL BSA) were prepared and 5.0 µL of these serial dilutions were transferred into OptiPlate-96 black micro-plates (PerkinElmer). Then, 35 µL of the fluorogenic substrate ZMAL (Z-Lys(Ac)-AMC[37], 21.43 µM in assay buffer) and 10 µL enzyme solution (HDAC1-BPS Bioscience, Catalog# 50051; HDAC2-BPS Bioscience, Catalog# 50052; HDAC3/NcoR2-BPS Bioscience, Catalog# 50003; HDAC6-BPS Bioscience, Catalog# 50,006) were added. Next, the total assay volume (50 µL, max. 1% DMSO) was incubated at 37 °C for 90 min. Subsequently, 50 µL trypsin solution (0.4 mg/mL trypsin in buffer: 50 mM Tris- HCl, pH 8.0, 100 mM NaCl) was added, followed by additional 30 min of incubation at 37 °C. Fluorescence (excitation: 355 nm, emission: 460 nm) was measured using a FLUOstar OPTIMA microplate reader (BMG LABTECH). The IC50 values were determined by generating normalized dose–response curves using the build-in "log(inhibitor) vs. response (three parameters)" equation provided by GraphPad Prism (GraphPad Prism 9.0, San Diego, USA). Compounds were tested in duplicates, reported mean IC50 values, including standard deviation, are calculated from at least two independent experiments.