Synapt g2 qtof mass spectrometer

All peptides were assessed for purity by analytical C18 RP-HP-LCMS prior to use in biological assays. Buffers used were 0.1% trifluoroacetic acid in water (A) and 0.1% trifluoroacetic acid in acetonitrile (B). The standard method (1) consisted of a linear gradient of 5% to 95% B over 10 minutes on Agilent 1100 series HPLC-MSD and method (2) consisted of a linear gradient of 5% to 95% B over 8 minutes on Waters 2767 series HPLC-MSD. The C18 column (Phenomenex, Luna C18, 4.6 × 150mm) effluent was immediately mass analyzed in electrospray positive mode. Accurate mass measurements of final peptides were performed using C18 reversed-phase chromatography mass spectrometry (RPHPLCMS) and mass detected on a Waters Synapt G2 Q-Tof mass spectrometer tuned to a resolution (FWHM) of 25,000. Exact intact masses were calculated based on the monoisotopic m/z value of the base peak charge state. All peptides were analyzed using these methods. Supporting data of peptide 45 is shown (Supplementary Figure S2) as representative data sets of the all the molecules investigated (characterization of all peptides presented on Table 1S).

The LC-MS/MS analysis was carried out using a Waters Synapt G2 qTOF mass spectrometer. The Synapt G2 qTOF from Waters (Milford, USA) is a high-resolution quadrupole time-of-flight (qTOF) mass spectrometer capable of data independent analysis (DIA) using Waters ms E technology. When linked to a Waters Acquity UPLC, the system can achieve good chromatographic separation between compounds followed by simultaneous acquisition of both fragmented and unfragmented mass spectra of all compounds within each peak eluting off the column, together with UV spectra produced by the photodiode array (PDA) detector placed upstream of the qTOF. The acetone extracts were centrifuged at 12,000 rpm for 10 min before analysis. A waters HSS T3 column, 2.1 × 150 mm was used in obtaining the separation of the phytoconstituents. Two mobile phases (A) and (B) were used, where (A) consisted of 0.1% formic acid in water and (B) had acetonitrile 5 mM ammonium formate. A 5 μl volume of the extracts was injected into the analytical column for analysis. The sample flow rate was set at 0.4 mL/min. The MS spectra were acquired in the positive ion mode. The mass fragmentations were identified by using a spectrum database for organic compounds.

Melting points were determined with a Tektronix X-4 micromelting-point apparatus (Beijing, China). The optical rotations were recorded on a JASCO P-1020 digital polarimeter (Kyoto, Japan), and the UV spectra were measured with a Shimadzu UV-2201 UV-Vis recording spectrophotometer (Kyoto, Japan). The IR data (KBr disks) were measured by a Bruker IFS-55 spectrometer (Rheinstetten, Germany). The CD spectra were obtained using a Bio-Logic MOS-450 (Grenoble, France). The 1D and 2D NMR spectra were run on Bruker AVANCE-400 or AVANCE-600 NMR spectrometers (Rheinstetten, Germany). The HRESIMS data were obtained on a Waters Synapt G2 QTOF mass spectrometer (MA, USA). The analytical HPLC was performed using an Agilent 1200 (CA, USA) equipped with a DAD detector using an ODS column (5 μm, 250 × 4.60 mm; Phenomenex Luna, CA, USA). Semipreparative HPLC was performed using a Shimadzu LC-6AD (Kyoto, Japan) equipped with a UV SPD-20A detector using an ODS column (5 μm, 250 × 10 mm, Phenomenex Luna, CA, USA). Column chromatography was carried out using silica gel (100–200 mesh and 200–300 mesh, Qingdao, China), ODS (60–80 μm, Tokyo, Japan), and Sephadex LH-20 (Uppsala, Sweden). Analytical grade solvents were used for the extraction and chromatographic separation.

The ¹H and ¹³C NMR spectra were obtained in DMSO-d₆ on a Bruker Avance III HD 400 spectrometer at 400 and 100 MHz, respectively, using TMS as internal standard. The chemical shift values (δ) are reported in ppm units, and the coupling constants (J) are in Hertz (Hz). UV spectra were recorded on a Jasco V-530 spectrophotometer (Jasco, Tokyo, Japan). HRESIMS was performed on a Waters Synapt G2 QTOF mass spectrometer (Waters, Milford, MA, USA). TLC analyses were carried out on precoated silica gel 60 F₂₅₄ plates (Merck, Darmstadt, Germany). The developing system used was CHCl₃:MeOH solution, and visualization of TLC plates was performed under UV light (254 and 365 nm) or performed with anisaldehyde-H₂SO₄ spray reagent. The adsorbent used for open column chromatography was silica gel 60–230 mesh. HPLC was performed on a Waters 1525 Binary HPLC pump (Waters Corp., Milford, MA, USA) connected to a Waters 996 Photodiode Array detector using Zorbax RX-C18 (21.2 × 250 mm) and Waters Symmetry C18 (4.6 × 150 mm) columns with HPLC-grade methanol and water.