820 spectropolarimeter

HRESIMS data were collected on an MAT95XP machine (Thermo Fisher Scientific, Bremen, Germany). NMR spectra were acquired by an Avance-600 spectrometer (Bruker, Fällanden, Switzerland). Circular dichroism (CD) spectra were afforded by a Jasco 820 spectropolarimeter (Jasco Corporation, Kyoto, Japan). Optical rotations were obtained by an MCP-500 spectropolarimeter (Anton Paar, Graz, Austria). UV spectra were acquired using a UV-2600 spectrophotometer (Shimadzu, Kyoto, Japan). IR data were done with an Affinity-1 spectrometer (Shimadzu, Kyoto, Japan). Preparative HPLC was performed using an ODS-A column (250 × 20 mm, 5 μm, 12 nm, YMC Co., Ltd, Kyoto, Japan). An ODS-A/AQ column (250 × 10 mm, 5 μm, 12 nm, YMC CO., Ltd, Kyoto, Japan) was used for semipreparative HPLC separation and the CHIRALPAK IC column (250 × 10 mm, 5 μm) for chiral semipreparative HPLC separation. Silica gel (100–200 and 200-300 mesh, Qingdao Marine Chemical Inc., Qingdao, China), C₁₈ reversed-phase Silica gel (40–63 μm, Merck, Darmstadt, Germany), and Sephadex LH-20 gel (Pharmacia Fine Chemical Co. Ltd., Uppsala, Sweden) were used in the chromatography processes. Fractions were monitored by TLC, and spots were detected on heated TLC plates (Silica gel GF₂₅₄ plates, Qingdao Marine Chemical Inc., Qingdao, China) with 10% H₂SO₄ in EtOH under UV light.

Optical rotations were measured using an Anton Paar MCP-500 instrument (Anton Paar, Graz, Austria) and the circular dichroism (ECD) as well as the UV spectra were collected on a Jasco 820 spectropolarimeter (JASCO, Tokyo, Japan) in the 200–400 nm range (under N₂ protection). Infrared (IR) spectra were recorded on an IR Affinity-1 spectrophotometer (Shimadzu, Kyoto, Japan). All the 1D and 2D NMR data were recorded on a Avance-III 600 MHz HD spectrometer (Bruker, Bremen, Germany) with tetramethylsilane as an internal standard. HR-ESI-MS were collected on Bruker maXis high resolution mass spectrometer. A Shimadzu LC-20 AT equipped with an SPD-M20A PDA detector was used for HPLC analysis and preparative separations. An ACE 5 PFP-C₁₈ column (250 × 10.0 mm, 5 μm, 12 nm) was used for semipreparative HPLC separation, meanwhile, a CHIRAL-MD (2)-RH column (250 × 10.0 mm, 5 μm) was used for chiral-phase chromatography (Guangzhou FLM Scientific Instrument Co., Ltd., Guangzhou, China). Column chromatography material: commercial silica gel (200–300 mesh) was purchased from Qingdao Marine Chemical Plant (Qingdao, China); Sephadex LH-20 gel was purchased from Amersham Biosciences, Shanghai, China). All analytical grade solvents were purchased from Guangzhou Chemical Regents Company (Guangzhou, China). The natural sea salt was produced by Guangdong Yueyan saltern (Guangdong, China).

All circular dichroism (CD) measurements were performed using a JASCO-820 spectropolarimeter (JASCO). CD spectra were obtained using CD values in the wavelength range of 200–260 nm in a quartz cuvette with a 1 mm optical path length. CD thermal denaturation curves were obtained using CD values at a wavelength of 220 nm and a scan rate of +1 °C/min in a quartz cuvette with a 1 cm optical path length. Prior to the experiments, stock protein solutions were centrifuged at 10,000 rpm at 4 °C for 10 min to remove precipitates and equilibrated at the measurement temperature for 10 min. Thermal denaturation curves were obtained at a protein concentration of 10 μM to avoid thermal aggregation at high temperatures. The melting temperature (T_m) was determined by fitting the thermal denaturation curve to a two-state model using Origin 2020b (OriginLab; Northampton, MA, USA), as reported previously [15 (link),34 (link)].