2795 separations module

Commercially available reagents and solvents were used as received. Compounds ISRIB-A1 and ISRIB-A2 were prepared as previously reported (Sidrauski et al., 2013b). Compound ISRIB-A7 was available commercially from Specs (The Netherlands). ¹H NMR spectra were recorded on a Varian INOVA-400 400 MHz spectrometer and a Bruker Avance 300 300 MHz spectrometer. Chemical shifts are reported in δ units (ppm) relative to residual solvent peak. Coupling constants (J) are reported in hertz (Hz). LC-MS analyses were carried out using Waters 2795 separations module equipped with Waters 2996 photodiode array detector, Waters 2424 ELS detector, Waters micromass ZQ single quadropole mass detector, and an XBridge C18 column (5 µm, 4.6 × 50 mm). Microwave reactions were carried out in a CEM Discover microwave reactor.

The crude venom of C. albostriatus was obtained by electronic stimulation, and preserved at −80°C after lyophilization. The lyophilized venom was dissolved in ddH₂O to a final concentration of 5 mg/ml and subjected to the first round of semi-preparative RP-HPLC purification (C18 column, 10 mm × 250 mm, 5 μm, Welch, Shanghai, China) using linear acetonitrile gradient ranging from 10 to 55% with an increasing rate of 1% per minute (Waters e2695 Separations Module, Waters, CA, United States). The fraction containing Ca2a was then collected, lyophilized, and subjected to a second round of analytical RP-HPLC purification (C18 column, 4.6 mm × 250 mm, 5 μm, Welch, Shanghai, China). The acetonitrile gradient was increased ranging from 20 to 40% at an increasing rate of 1% per minute (Waters 2795 Separations Module, Waters, CA, United States). Fractions were lyophilized and stored at −20°C before use. The purity of the toxin was tested by MALDI-TOF MS analysis (AB SCIEX TOF/TOF^TM 5800 system, Applied Biosystems, United States).