Rp c18 column

NMR spectra were recorded on a Bruker Avance 600 MHz spectrometer. HRESIMS data were obtained using a Xevo G2 Q-TOF mass spectrometer (Waters, Milford, MA, USA). CD spectra were measured on a J-715 spectropolarimeter (JASCO Corporation, Tokyo, Japan). Optical rotations were recorded on a Rudolph IV Autopol automatic polarimeter (Hackettstown, NJ, USA). The UV spectra were recorded on a UV-1800 spectrophotometer (Shimadzu, Kyoto, Japan). For column chromatography (CC), Sephadex LH-20 (GE Healthcare Bio-Science AB, Pittsburgh, PA, USA), silica gel (200–300 mesh, 300–400 mesh, Tsingtao Marine Chemical Co. Ltd., Tsingtao, China) and RP-C18 (ODS-A, 50 µm, YMC, Kyoto, Japan) were used. Preparative HPLC was run with a P3000 pump (CXTH, Beijing, China) and a UV3000 ultraviolet-visible detector (CXTH, Beijing, China), using a preparative RP-C18 column (5 µm, 20 mm × 250 mm, YMC, Kyoto, Japan). IR data were measured on a Bruker Tensor 27 spectrometer.

The synthesized peptide was purified using the analytical HPLC Thermo Separation system and UV detection (210 nm) with a YMC-Pack RP C18 column (4.6 × 250 mm, 5 μm), and a gradient elution of 0–40% B in A (A = 0.1% TFA in water; B = 0.1% TFA in acetonitrile/H₂O, 4:1) over 30 min (flow rate 1 mL/min). The main fraction, corresponding to the peptide, was collected, lyophilized, and confirmed by MS/MS experiment.

A JASCO P-1020 digital polarimeter (Tokyo, Japan) was used to detect optical rotations of the isolated compounds in MeOH. A Lambda 35 UV/Vis spectrophotometer (Perkin Elmer, Waltham, United States) was used to collect UV data of the isolated compounds. A scientific LTQ Orbitrap XL spectrometer (Thermo Scientific, Waltham, United States) was used to acquire HRESIMS. An Agilent DD2 500 MHz spectrometer (Agilent Technologies, Santa Clara, United States; 500 and 125 MHz for ¹H and ¹³C, respectively) with tetramethylsilane (TMS) as an internal standard was used to obtain NMR spectra. HPLC was conducted on an Agilent 1,260 system using an RP-C18 column (5 mm, 10 × 250 mm, flow rate 2 ml/min, YMC, Kyoto, Japan) with MeOH (HPLC grade) as mobile phase. Silica gel (100–200 mesh and 200–300 mesh, Qingdao Marine Chemical Factory, Qingdao, China), octadecylsilyl (ODS) reversed-phase gel (30–50 μm, YMC CO., LTD., Japan), and Sephadex LH-20 (GE Healthcare, United States) were used for chromatographic separation.

The synthesized peptide was purified using the analytical HPLC Thermo Separation system (Thermo Fisher Scientific, Waltham, MA, USA ) with UV detection (210 nm) with a YMC-Pack RP C18 column (4.6 × 250 mm, 5 μm), with a gradient elution of 0–40% B in A (A = 0.1% TFA in water; B = 0.1% TFA in acetonitrile/H₂O, 4/1, v/v) over 30 min (flow rate 1 mL/min). The main fraction, corresponding to the peptide, was collected and lyophilized.

The synthesized peptide was purified with the use of the analytical HPLC Thermo Separation system and UV detection (210 nm) with a YMC-Pack RP C18 column (4.6 × 250 mm, 5 μm), and a gradient elution of 0–40% of B in A (A = 0.1% TFA in water; B = 0.1% TFA in acetonitrile/H2O, 4: 1) over 30 min (flow rate 1 ml/min). The main fraction, corresponding to the peptide, was collected, lyophilized and confirmed in the MS/MS experiment.

1D NMR and 2D NMR spectra were measured on a Bruker Avance 600 MHz spectrometer. HRESIMS was carried out on a Xevo G2 Q-TOF mass spectrometer with an electrospray ionization source (Waters, Milford, MA, USA). Optical rotations were measured with a P-1020 digital polarimeter (JASCO Corporation, Tokyo, Japan). CD spectra were measured on a J-715 spectropolarimeter (JASCO Corporation). Optical rotations were recorded on a Rudolph IV Autopol automatic polarimeter (Hackettstown, NJ, USA). The UV spectra were recorded on a UV-1800 spectrophotometer (Shimadzu, Kyoto, Japan). The high-performance liquid chromatography (HPLC) analysis was performed on a 1200 system (Agilent, Santa Clara, CA, USA). Thin-layer chromatography (TLC) plates (5 × 10 cm) were performed on GF₂₅₄ (Qingdao Marine Chemical Co. Ltd., Qingdao, China) plates. For column chromatography (CC), RP-C18 (ODS-A, 50 µm, YMC, Kyoto, Japan), silica gel (200–300 mesh, 300–400 mesh, Qingdao Marine Chemical Co. Ltd., Qingdao, China) and Sephadex LH-20 (GE Healthcare Bio-Science AB, Pittsburgh, PA, USA) were used. Semi-preparative HPLC was run with a P3000 pump (CXTH, Beijing, China) and a UV3000 ultraviolet-visible detector (CXTH, Beijing, China), using a preparative RP-C18 column (5 µm, 20 × 250 mm, YMC, Kyoto, Japan).

The synthesized peptide was purified using an analytical HPLC Thermo Separation system with UV detection (210 nm) with a YMC-Pack RP C18 column (4.6 × 250 mm, 5 μm), with a gradient elution of 0–40% B in A (A = 0.1% TFA in water and B = 0.1% TFA in acetonitrile/H₂O, 4:1 v/v) over 30 min (flow rate 1 mL/min). The main fraction, corresponding to the peptide, was collected and lyophilized.