Ultimate 3000 series uhplc

NMR spectra were recorded with an Avance III 700 spectrometer (Bruker BioSpin, Rheinstetten, Germany) with a 5 mm TCI cryoprobe (¹H 700 MHz, ¹³C 175 MHz). NMR data were referenced to selected chemical shifts of acetone-d₆ (¹H: 2.05 ppm, ¹³C: 29.32 ppm), pyridine-d₅ (¹H: 7.22 ppm, ¹³C: 123.87 ppm) and CDCl₃ (¹H: 7.27 ppm, ¹³C: 77.00 ppm), respectively. Optical rotations were taken with an MCP 150 polarimeter (Anton Paar, Graz, Austria). UV/Vis spectra were taken with a UV-2450 UV/Vis spectrophotometer (Shimadzu, Kyoto, Japan), while ECD spectra were collected with a JD-815 spectrophotometer (Jasco, Pfungstadt, Germany). ESI-MS spectra were recorded with an UltiMate^® 3000 Series uHPLC (Thermo Fisher Scientific, Waltman, MA, USA) utilizing a C18 Acquity^® UPLC BEH column (2.1 × 50 mm, 1.7 µm; Waters, Milford, MA, USA) connected to an amaZon^® speed ESI-Iontrap-MS (Bruker). HPLC parameters were set as follows: solvent A: H₂O + 0.1% formic acid, solvent B: acetonitrile (ACN) + 0.1% formic acid; gradient: 5% B (0.5 min), 5−100% (19.5 min), 100% (5 min), flowrate 0.6 mL/min, and DAD detection 190−600 nm. HR-ESI-MS spectra were obtained with an Agilent 1200 Infinity Series HPLC (Agilent Technologies, Böblingen, Germany; conditions same as for ESI-MS spectra) connected to a maXis^® ESI-TOF-MS (Bruker).

To locate the position of the double bond in the fatty acid moiety of hybridorubrin C (3), a sample was epoxidized with meta‐chloroperoxybenzoic acid (mCPBA) followed by MS/MS analysis, as recently published:^[14] at first, 10 μg of 3 was incubated with 10 μg of mCPBA in 10 μL of CH₂Cl₂ at room temperature. The reaction was quenched after 10 min with 490 μL of CH₂Cl₂:MeCN (1:1). The same procedure was applied to an authentic reference sample of cis‐octadecenoic acid [C18:1(9)].
Then, 1 μL of the samples was injected into an UltiMate 3000 Series uHPLC (Thermo Fisher Scientific) equipped with a C18 Kinetex column (1,7 μm, 150×2.1 mm, Phenomenex, Torrance, CA/USA) and the following gradient of H₂O+0.1 % formic acid (A) and MeCN+0.1 % formic acid (B): 1 % B for 2 min, increasing to 100 % B over 18 min, keeping 100 % B for further 4 min, flow rate 0.3 mL min⁻¹. This HPLC was connected to a maXis HD UHR‐ESI‐QTOF‐MS (Bruker) with the following parameters: scan range: m/z 50–1500, ion polarity: negative, capillary voltage: 4500 V, nebulizer pressure: 4.0 bar, dry heater: 200 °C, dry gas: 9.0 L min⁻¹, collision energy: 20.3–50.7 eV. Results are depicted in Figure S45.

The extracts obtained were dissolved in Acetone:MeOH (1:1) and adjusted to a concentration of 4.5 mg mL⁻¹. An injection volume of 2 µL was applied to an UltiMate^® 3000 Series uHPLC (Thermo Fisher Scientific^®, Waltman, MA, USA). Mass spectrometry was performed with a connected amaZon^® speed ESI Iontrap MS (Amazon, Bruker). HRESIMS measurements were performed with sample concentrations of 1 mg mL⁻¹ with an Agilent 1200 series HPLC-UV system in combination with an ESI-TOF-MS (Maxis, Bruker). The conditions were identical to the methods described before [26 (link)].

NMR spectra were recorded with an Avance III 700 spectrometer with a 5 mm TCI cryoprobe (¹H 700 MHz, ¹³C 175 MHz) and an Avance III 500 spectrometer (¹H 500 MHz, ¹³C 125 MHz; both Bruker, Billerica, MA/USA). NMR data were referenced to selected chemical shifts of acetone-d₆ (¹H: 2.05 ppm, ¹³C: 29.32 ppm), pyridine-d₅ (¹H: 7.22 ppm, ¹³C: 123.87 ppm) and DMSO-d₆ (¹H: 7.27 ppm, ¹³C: 77.00 ppm), respectively. Optical rotations were taken with a MCP 150 polarimeter (Anton Paar, Graz, Austria); UV spectra were taken with a UV-2450 UV/VIS spectrophotometer (Shimadzu, Kyoto, Japan); ECD spectra were collected with a JD 815 spectrophotometer (Jasco, Pfungstadt, Germany).
ESI-MS spectra were recorded with an UltiMate® 3000 Series uHPLC (Thermo Fisher Scientific, Waltman, MA/USA) utilizing a C18 Acquity® UPLC BEH column (2.1 × 50 mm, 1.7 μm; Waters, Milford, MA/USA) connected to an amaZon® speed ESI Iontrap MS (Bruker). HPLC parameters were set as follows: solvent A: H₂O + 0.1% formic acid, solvent B: acetonitrile + 0.1% formic acid; gradient: 5% B (0.5 min), 5–100% (19.5 min), 100% (5 min), flowrate 0.6 mL/min, and DAD detection 190–600 nm. HR-ESI-MS spectra were obtained with an Agilent 1200 Infinity Series HPLC (Agilent Technologies, Böblingen, Germany; conditions same as for ESI MS spectra) connected to a maXis® ESI-TOF-MS (Bruker).

Electrospray mass (ESI-MS) spectra were recorded with an UltiMate^® 3000 Series uHPLC (Thermo Fisher Scientific, Waltman, MA, USA) utilizing a C18 Acquity^® UPLC BEH column (2.1 × 50 mm, 1.7 µm; Waters, Milford, USA) connected to an amaZon speed^® ESI-Iontrap-MS (Bruker, Billerica, MA, USA). HPLC parameters were set as follows: solvent A: H₂O+0.1 % (v/v) formic acid, solvent B: acetonitrile (ACN)+0.1 % (v/v) formic acid, gradient 5 % B for 0.5 min, increasing to 100 % B in 19.5 min, keeping 100% B for further 5 min, flowrate 0.6 mL/min, and DAD detection 190–600 nm.
High-resolution electrospray mass (HR-ESI-MS) spectra were obtained with an Agilent 1200 Infinity Series HPLC (Agilent Technologies, Santa Clara, CA, USA) connected to a maXis^® electrospray time-of-flight mass spectrometer (ESI-TOF-MS; Bruker; HPLC conditions same as for ESI-MS spectra).
Nuclear magnetic resonance (NMR) spectra were recorded with an Avance III 500 spectrometer (Bruker, ¹H NMR: 500 MHz, and ¹³C NMR: 125 MHz). UV/Vis spectra were taken with a UV/Vis spectrophotometer UV-2450 (Shimadzu, Kyoto, Japan), and electronic circular dichroism (ECD) spectra (Figure S2) were recorded on a J-815 spectropolarimeter (JASCO, Pfungstadt, Germany).