Chirascan spectropolarimeter

Optical rotations were measured on a Perkin-Elmer 241 Polarimeter (Perkin-Elmer, Bruker, Billerica, MA, USA) and UV data were obtained on a Shimadzu Biospec-1601 spectrophotometer. CD spectra were obtained on a Chirascan spectropolarimeter. ¹H and ¹³C NMR data were acquired using Bruker 600 and Varian Inova 600 spectrometers using solvent signals (MeOH-d₄; δ_H 4.87, 3.31/δ_C 49.15) as references. HRESIMS data were acquired using a LTQ Orbitrap XL Mass Spectrometer (Thermo, Waltham, MA, USA).

The circular dichroism spectra were acquired on a Chirascan spectropolarimeter. The samples were prepared in 10 mM phosphate buffer, pH 7.5, at a peptide concentration of 50 μM. Data were recorded at 25°C by step scan from 180 nm to 260 nm in a 0.5-mm-pathlength quartz cell using 0.2-nm-wavelength increments, a 1-nm bandwidth, and a response time of 1 s. Each spectrum was an average of three measurements and was subtracted from the buffer baseline. The data were converted to per residue molar ellipticity units (θ; in degrees · square centimeters · decimoles⁻¹ · number of residues⁻¹) and smoothed using Igor Pro software. Percent helicity was calculated as follows: (100 × CD₂₂₂)/(C × N × {40,000 × [1 − (2.5/N)]}), where CD₂₂₂ is the molar ellipticity (θ) at 222 nm (in millidegrees), N is the number of amino acids in the peptide, and C is the peptide molar concentration (in moles per liter).

Steady-state circular dichroism (CD) spectra were measured in the far-UV light range (185–260 nm) using a Chirascan spectropolarimeter. Spectra were recorded in a 60 µl quartz cell (Hellma) with a path length of 0.1 mm, at 20°C and a step resolution of 1 nm. The readings were the average of 1 s at each wavelength, and the recorded ellipticity values were the average of three determinations for each sample. CD spectroscopy of the protein (2.2 mg ml⁻¹) was performed in a buffer of 50 mM phosphate (pH 7). The average of three buffer spectra were subtracted from the averaged spectrum of the protein. CD values were converted to mean residue ellipticity (Q) in units of deg cm² dmol⁻¹ using the software Chirascan version 1.4 (Applied Photophysics). This baseline-corrected spectrum was used as input for computer methods to obtain predictions of secondary structure.

The circular dichroism spectra were acquired on a Chirascan spectropolarimeter. The samples were prepared in 10 mM phosphate buffer, pH 7.5, at a peptide concentration of 50 μM. Data were recorded at 25°C by step scan from 180 nm to 260 nm in a 0.5-mm path length quartz cell using 1-nm wavelength increments and a response time of 1 s. The data were converted to per residue molar ellipticity units [θ] (degree per square centimeter per decimole per residue) and smoothed using the Igor Pro software. The percentage of helicity was calculated as described previously (35 (link)).

Semi-preparative high-performance liquid chromatography (HPLC) was performed on an Agilent 1,260 G7111A Quaternary Pump equipped with a G7117C diode array detector (DAD). Agilent Technologies 5977A GC/MSD was used for monosaccharide configuration determination. ¹H and ¹³C nuclear magnetic resonance (NMR) data were acquired with a Bruker AVANCE 500 MHz spectrometer with a 5 mm triple resonance cryoprobe at 298 K. The referenced NMR solvent signals were used as acetone-d₆, δ_H 2.05/δ_C 29.8, 206.2, and methanol-d₄, δ_H 3.31, 4.87/δ_C 49.0, respectively. Heteronuclear multiple quantum correlation (HMQC) and heteronuclear multiple bond correlation (HMBC) experiments were optimized for 145 and 8 Hz, respectively. High-resolution electrospray ionization mass spectrometry (HRESIMS) data were recorded using an Agilent Accurate-Mass-Q-TOF LC/MS 6520 instrument equipped with an electrospray ionization (ESI) source. Fragmentor and capillary voltages were maintained at 125 and 3,500 V, respectively. Nitrogen was used as the nebulizing and drying gas (300°C) with a flow rate of 10 L/min, and the nebulizer pressure was set at 10 psi. Full-scan spectra were acquired across a scan range of m/z 100–1,000 at a rate of 1.03 spectra per second. Optical rotations were measured on an Anton-Paar MCP 200 polarometer. Circular dichroism (CD) spectra were recorded using a Chirascan spectropolarimeter.

Optical rotations were acquired on a JASCOP-1020 polarimeter. ECD data were measured on a Chirascan spectropolarimeter. IR spectra were measured on PerkinElmer infrared spectrophotometer. 1D and 2D NMR spectra were recorded on Bruker Avance 400 or 600 DRX spectrometers in acetone-d₆, methanol-d₄, DMSO-d₆ and chloroform-d. Column chromatography (CC) was performed on silica gel (200–300 mesh; Qingdao Marine Chemical Plant Branch., China), RP-C18 (ODS-A, 50 μm, YMC, Kyoto, Japan), or Sephadex LH-20 (100–200 mesh; Beijing Solarbio Technology Co., Ltd., China). Plates precoated with silica gel GF254 (Rushan, Shandong Sun Desiccant Co., Ltd.) were used for thin layer chromatography (TLC). An Agilent HPLC series 1260 and Shimadzu LC-20AR were used for analysis and isolation. For analysis, an Agilent Eclipse XDB-C18 column (4.6 × 150 mm, 5 μm) was used. The isolation was achieved on an Agilent semi-preparative Eclipse XDB-C18 column (9.4 × 250 mm, 5 μm). HPLC-MS data were acquired on an Agilent 1260 Series system coupled with an Agilent Accurate-Mass-Q-TOF MS 6520 system equipped with an Electrospray ionization (ESI) source.