Synthetic honaucin A was used in all assays and measurements. For bioassays, lipopolysacchar-ides from Escherichia coli (O26:B6) as well as phorbol 12-myristate 13-acetate and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide were purchased from Sigma-Aldrich (St. Louis, MO). NRF2 luciferase reporter MCF7 stable cells were purchased from Signosis (Santa Clara, CA). RNA sequencing was carried out using an Illumina Hiseq2000 (San Diego, CA) following RNA quality assessment via Bioanalyzer 2100 (Agilent, Santa Clara, CA). For in vitro alkylation experiments, reaction products were separated via liquid chromatography using a Phenomenex Kinetex 5 μm EVO C-18 column. Highresolution mass analysis was accomplished with an Agilent 6230 ESI-TOF-MS operating in positive mode.
6230 esi tof ms
Manufactured by Agilent Technologies
The 6230 ESI-TOF-MS is a high-performance electrospray ionization time-of-flight mass spectrometer. It is designed to provide accurate mass measurement and high-resolution analysis of a wide range of molecules.
Automatically generated - may contain errors
Lab products found in correlation
Nrf2 luciferase reporter mcf7 stable cells, by Signosis (1 mentions)
Lipopolysaccharides from escherichia coli o26 b6, by Merck Group (1 mentions)
Kinetex 5 μm evo c18 column, by Phenomenex (1 mentions)
3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide, by Merck Group (1 mentions)
Phorbol 12 myristate 13 acetate, by Merck Group (1 mentions)
2100 bioanalyzer, by Agilent Technologies (1 mentions)
Hiseq 2000, by Illumina (1 mentions)
Bond elut c18 spe column, by Agilent Technologies (1 mentions)
Nanodrop 2000c spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Silica gel plate, by Merck Group (1 mentions)
Isolera one, by Biotage (1 mentions)
Chromaster system, by Hitachi (1 mentions)
Aeris peptide c18 column, by Phenomenex (1 mentions)
4 protocols using 6230 esi tof ms
1
Synthetic Honaucin A Bioassay and Chemical Analysis
2
Reaction of Honaucin A with N-Acetyl-L-cysteine
3
Characterization of Organic Compounds via Spectroscopic Techniques
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All chemicals, unless otherwise stated, were purchased from Sigma Aldrich and used as received. Cuprisorb resin was purchased from SeaChem Labs. Reaction progress was monitored by analytical thin-layer chromatography (TLC, Merck silica gel plates) with UV illumination or via CAM, ninhydrin, or KMnO4 staining. Column chromatography was performed on a Biotage Isolera One automated flash chromatography system. Nuclear magnetic resonance (1H and 13C NMR) spectra were recorded on Bruker 300 MHz and Jeol 500 MHz NMR spectrometers. Spectra were recorded in CDCl3 or D2O at 293 K and are reported in parts per million (ppm) on the δ scale relative to residual solvent as an internal standard (for 1H NMR: CDCl3 = 7.26 ppm, D2O = 4.79 ppm, for 13C NMR: CDCl3 = 77.0 ppm, CD3OD = 49.0 ppm). HRMS (high-resolution mass spectrometry) analysis was performed on an Agilent 6230 ESI-TOFMS in positive ion mode. UV-Vis spectra were collected in a quartz cuvette using a Thermo Scientific Nanodrop2000c spectrophotometer. IR spectroscopy was performed on a Nicolet 6700 FT-IR spectrophotometer. Size exclusion chromatography (SEC) was performed on a Hitachi Chromaster system equipped with an RI detector and two 5 μm, mixed bed, 7.8 mm I.D. × 30 cm TSK gel columns in series (Tosoh Bioscience) using an isocratic method with a flow rate of 0.7 mL min−1 in DMF (0.2% LiBr, 70 °C).
4
Solid-Phase Peptide Synthesis Protocol
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All peptides were synthesized using Fmoc/t-Bu Solid-Phase Peptide Synthesis (27 (link)). Six peptides were synthesized using Rink Amide resin on a Liberty Blue peptide synthesizer (CEM Corp); 31 peptides were synthesized on a Prelude peptide synthesizer (PTI); five peptides were synthesized manually as detailed in the Supplementary information . The remaining ten peptides were purchased from Shanghai Royobiotech at >95% purity. Synthesized peptides were purified using semipreparatory RP-HPLC using a Phenomenex Luna C18(2) or Phenomenex Aeris Peptide C18 column (10 mm × 250 mm, 5 μm) over a linear gradient of water and acetonitrile, with 0.1% TFA, at 4 ml/min and UV detection at 220 nm. Peptide identity was confirmed by high resolution mass spectrometry using an Agilent 6230 ESI-TOF MS. Peptide purity was characterized on an Agilent 1260 Infinity analytical RP-HPLC equipped with a Phenomenex Luna C18(2) column (4.6 mm × 250 mm, 5 μm) using a linear gradient of 0 to 50% acetonitrile with 0.1% TFA and in water with 0.1% TFA, over 15 min at 1.5 ml/min and visualized at 220 nm. Detailed methods are included in the Electronic Supplementary Information and characterization data are listed in Table S1 .
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