J 720w spectropolarimeter

In order to determine resistance to high temperatures, CDPs were incubated at 0.5 mM in PBS at 75° C or 100° C for 1 h, pelleted, and the supernatant analyzed by RPC. To determine resistance to proteolytic digestion, CDPs were mixed with 50 U of porcine pepsin (Sigma-Aldrich P7012) in Simulated Gastric Fluid^{43 (link)} at pH 1.05, or 50 U of porcine trypsin (Sigma-Aldrich 6567) in PBS, incubated for 30 m at 37° C, and analyzed by RPC. Oxidized and reduced forms (adding 10 mM DTT) were compared. CD spectra were measured with a Jasco J-720W spectropolarimeter using a 1.0 mm path length cell, with CDPs diluted into 20 mM phosphate buffer, pH 7.4, at a concentration of 15–25 µM.

Optical rotations were measured on a JASCO P-2200 polarimeter in a 0.5-dm cell. UV spectra were obtained with a Shimadzu BioSpec-mini spectrophotometer. The ECD spectra were measured on a JASCO J-720W spectropolarimeter in a 10-mm cell. The IR spectra were measured on a JASCO FT/IR-4100 Fourier transform infrared spectrometer using a KBr disk. The NMR spectra were measured on a JEOL ECA-500 spectrometer with deuterated solvent used as the internal reference. The ¹H NMR spectra were measured at 500 MHz, and the ¹³C NMR spectra were measured at 125 MHz. HRESIMS was conducted using a Q-Exactive Hybrid Quadrupole Orbitrap mass spectrometer. Diaion HP-20 (Mitsubishi Chemical Corporation, Tokyo, Japan), Silica gel (Chromatorex PEI MB 100–40/75, Fuji Silysia Chemical Ltd., Aichi, Japan), and ODS (Cosmosil 75C₁₈-PREP, Nacalai Tesque, Inc., Kyoto, Japan) were used for column chromatography. For preparative HPLC, a Waters 515 HPLC pump, equipped with an ERC RefractoMax520 differential refractometer detector and a Shimadzu SPD-10A UV-vis detector were used. For reversed-phase HPLC separations, a RP-C₁₈ silica gel column (YMC-Actus Triart C₁₈, 5 μm, 150×20 mm) was used, at a flow rate of 8.0 mL/min.