Hp 1100 lc system

Unless otherwise indicated, all starting materials, chemicals, and anhydrous solvents were purchased from Sigma Aldrich (NSW, Australia) and were used without further purification. ¹H and ¹³C-NMR spectra were recorded on a Varian 500 MHz or a Varian Inova 600 MHz instruments in the indicated solvents. Chemical shifts are reported in ppm (δ). Signals are reported as s (singlet), br s (broad singlet), d (doublet), dd (doublet of doublets), t (triplet), or m (multiplet). High resolution mass spectra were collected using an LTQ Orbitrap XL ETD using flow injection, with a flow rate of 5 μL/min. Where indicated compounds were analyzed and purified by reverse phase HPLC, using an HP 1100 LC system equipped with a Phenomenex C-18 column (250 × 4.6 mm) for analytical traces and a Gilson GX-Prep HPLC system equipped with a Phenomenex C18 column (250 × 21.2 mm). H₂O and MeCN solutions were used as aqueous and organic buffers. All absorbance and fluorescence spectra were collected on a Synergy H4 Hybrid Multimode Microplate Reader using black-walled clear bottom 96-well plates. Data was processed using Microsoft Excel 2016 and all graphs were generated using GraphPad Prism 7 software. A mercury lamp (365 nm) was used as the UV light source in photoswitching experiments.

Samples obtained from FIH kinetic assays with 2CA and 3CA were diluted to a substrate concentration of 5 μm and then analyzed using a Waters® LCT Classic mass spectrometer by electrospray ionization in positive ion mode. An Agilent HP1100 LC system was used, and samples were run through a Phenomenex® Aeris Widepore 3.6-μm XB-C8 150 × 2.1-mm column at 0.5 ml/min in a gradient of phase A (95% H₂O, 5% CH₃CN, 0.01% formic acid) to phase B (95% CH₃CN, 5% H₂O, 0.01% formic acid). The LC/MS used the following method: 0–1.5 min, 100% A and then 1.5–12 min linear gradient from 0 to 100% B, 12–14 min 100% B, and 15.0–19.0 min 100% A for re-equilibration of the column. Data were collected and analyzed using MassLynx version 4.0.