, Under argon was then added trimethylsilyl cyanide (4.6 mL, 36.0 mmol, 1.5 equiv) and water (1.7 mL, 96.0 mmol, 4.0 equiv). The reaction mixture was stirred at 100 °C for 2 h, until TLC (heptane:EtOAc 1:1, Rf 0.31) showed complete reaction. The reaction mixture was then cooled to RT, followed by addition of excess water (25 mL. OBS! Formation of toxic HCN gas), extraction with EtOAc (5 x 50 mL). The combined organic phases were washed with sat. brine (50 mL), dried over Na2SO4, filtered, and concentrated to dryness in vacuo. The crude was purified by flash chromatography (heptane:EtOAc, 40-55% gradient) to furnish 3-oxocyclohexane-1-carbonitrile (37-I1) as a slightly yellow oil (2.42 g, 19.6 mmol, 82%), vol.1, p.4

M. Hcl, mmol, 30.6 equiv) was stirred at 80 °C for 45 min, until TLC (heptane:EtOAc:AcOH 1:1:0.1, Rf 0.21) showed complete reaction. The reaction mixture was then diluted with water (50 mL), extracted with EtOAc (5 x 100 mL), and the combined organic phases dried over Na2SO4, filtered, and concentrated to dryness in vacuo

. Combiflash, MeCN:TFA 5:95:0.1, 0-10% gradient) to furnish 3-oxocyclohexane-1-carboxylic acid (37-I2) as a white solid (1.24 g, 8.72 mmol, 44%), A H2O:MeCN:TFA 95:5:0.1, buffer B H2O

, DMSO-d6) ? 3.68 (s, 1H), 2.77 (tq, J = 9

, DMSO-d6) ? 208.89, 175.14, 42.65, 42.09, 40.30, 26.92, 23.58. For step 3, a solution of 37-I2 (1.24 g, 8.72 mmol, 1.0 equiv) and benzyl bromide (1.14 mL, 9.59 mmol, 1.1 equiv) in dry MeCN (16 mL) was added DBU (1.30 mL, 8.72 mmol, 1.0 equiv). The reaction mixture stirred at RT for 1 h, Hz, 1H), 2.24-2.16 (m, 1H), 2.03-1.93 (m, 1H), 1.91-1.82 (m, 1H), 1.80-1.62 (m, 2H). 13 C NMR (151 MHz

. Etoh, 0 equiv) and the reaction mixture was stirred at 75 °C for 2 h. Then the mixture was adjusted to pH 14 using 40% aq. NaOH, followed by addition of water (150 mL), and extraction with EtOAc (3 x 100 mL). The combined organic layers were dried over Na2SO4 and concentrated in vacuo. The crude was purified by flash chromatography (heptane:EtOAc, 0-100% gradient) to furnish 4-bromo-6-methoxy-N1-methylbenzene-1,2-diamine

. Mhz, For step 5, a previously described procedure 27 was followed with no deviations, Chloroform-d) ? 6.54 (d, J = 2.0 Hz, 1H), 6.45 (d, J = 2.0 Hz, 1H), 4.11 (d, J = 67.7 Hz, 2H), vol.3

, Methyl (E)-3-(7-methoxy-1-methyl-1H-benzo, The compound was syn

, DIPEA (1.59 mL, 9.2 mmol, 2.5 equiv), and tri-o-phosphine (0.22 g, 0.74 mmol, 0.2 equiv) were added. Then Pd(OAc)2 (0.08 g, in vacuo. The crude was purified by flash chromatography (heptane:EtOAc, 0-100% gradient) to furnish 54 as a brown solid (0.31 g, 1.25 mmol, 32%). 1 H NMR (400 MHz, Chloroform-d) ? 7, dry DMF (5 mL), methyl acrylate (1.66 mL, 18.4 mmol, 5.0 equiv)

, -(hydroxymethyl)-4-methylphenyl)-3-(7-methoxy-1-methyl-1H-benzo, Methyl, vol.3, issue.3

, Starting from 54 (0.15 g, 0.61 mmol), 55 was obtained as a white solid (0.10 g, 0.27 mmol, 46%). 1 H NMR (400 MHz, Chloroform-d) ? 7.48 (s, 1H), 7.24 (s, 1H), vol.7

V. Ness-in, nitrophenyl)ethynyl)silane (58-I1) as a golden-brown oil (0.53 g, 2.42 mmol, 24%). 1 H NMR (400 MHz, Chloroform-d) ? 8.30 (t, The crude was purified by flash chromatography (heptane:EtOAc, 0-40%) to furnish trimethyl

, For step 2, to a solution of 58-I1 (0.53 g, 2.42 mmol, 1.0 equiv) in MeOH (25.0 mL) was added K2CO3 (0.60 g, 4.36 mmol, 1.80 equiv). The solution was stirred under nitrogen at RT for 22 h, until TLC (heptane:EtOAc 1:1, Rf 0.77) showed complete reaction. The reaction mixture was then concentrated in vacuo, the residue re-dissolved in EtOAc (15 mL), and the solution washed with water (10 mL), sat. brine (10 mL), dried over Na2SO4, filtered, and concentrated to dryness in vacuo to furnish 58 as a dark-brown oil (0.35 g, 2.38 mmol, 97%). 1 H NMR (400 MHz, Chloroform-d) ? 8.34 (t, J = 1.9 Hz, 1H), Hz, 1H), 0.27 (s, 9H). 13 C NMR (101 MHz, Chloroform-d), vol.8

, To a solution of 3-hydroxybenzonitrile (1.43 g, 12.0 mmol, 1.00 equiv) in acetone (30 mL) were added K2CO3 (2.49 g, 18.0 mmol, 1.50 equiv) and ethyl 2-bromoacetate (1.86 mL, 16.8 mmol, 1.40 equiv). The reaction mixture was stirred at reflux for 2 h, until TLC (DCM:MeOH 20:1, Rf 0.96) showed complete reaction. The reaction mixture was then filtered, and the filtrate concentrated to dryness in vacuo. The crude was purified by flash chromatography (heptane:EtOAc, 5-25% gradient) to furnish 60 as a colorless oil (1.00 g, 4.88 mmol, 41%). 1 H NMR (600 MHz

, Cells were harvested by centrifugation at 4,000 x g for 30 min. The cells were resuspended in lysis buffer (50 mM HEPES pH 7.5, cOmplete? Protease Inhibitor Cocktail (1 tablet/50 mL of buffer), 25 µg/mL DNase, 40 mM Mg2SO4, 150 mM NaCl, 5 mM imidazole, 5% glycerol, 0.5% TritonX-100, 3 mM DTT, 1 mg/mL Lysozyme) and lysed using a cell disruptor at 26 KPsi in 4 ?C. The cell lysate was spun down at 35,000 x g for 1 h at 4 ?C. The supernatant was filtered on a 0.45 µm filter and loaded onto a 5 mL HisTrap HP column (GE Healthcare), The column was washed with 5 column volumes of HisTrap binding buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 10 mM imidazole, 3 mM DTT) followed by eluting the protein using a gradient of HisTrap elution buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 1 M imidazole, 3 mM DTT

, mM TCEP) with a flow rate at 1 mL/min, and was eluted at 65 mL. Protein was concentrated to 12 mg/mL and, equilibrated with SEC buffer (25 mM HEPES pH 7.5, 150 mM NaCl, vol.1

. °c, The protein was analyzed on SDS page for purity, and the concentration was measured by absorbance

, Peptides and fluorescent peptide probes. Peptides 1 (LDEETGEFL-OH), 2 (Ac-LDEETGEFL-OH), and the FITC-Nrf2 peptide probe (FITC-?-DEETGEF-OH

, FAM-LDEETGEFL-NH2) and Cy5-Nrf2 (Cy5-LDEETGEFL-NH2) peptide probes were synthesized inhouse by solid-phase peptide synthesis as previously described, 94-96 starting from a Rink amide MBHA resin preloaded with Fmoc-Leu. The FAM-Nrf2 probe was generated by coupling 5(6)-carboxyfluorescein (5(6)-FAM) to the N-terminal amino group of the Fmoc-deprotected and resin-attached LDEETGEFL peptide using HATU as coupling reagent and collidine as base, The FAM-Nrf2

, The Cy5-Nrf2 probe was synthesized by coupling Sulfo-Cy5-NHS ester (Lumiprobe, Germany) to the N-terminal amino group of the peptide LDEETGEFL-NH2 in solution followed by HPLC purification. The reaction was performed in a 1:1.15 molar ratio of peptide (1.29 mg) and dye in PBS buffer (pH 8.4) with 10% DMSO for 3 h at room temperature. All final peptides (both purchased and in-house synthesized) were characterized for purity, /5/5) for 2 h, evaporation in vacuo, cold ether precipitation, and HPLC purification

, FITC-Nrf2) and the Keap1 Kelch domain were determined as Kd values by saturation binding experiments, where increasing concentrations of Keap1 Kelch (0-300 nM) were added to a fixed concentration of peptide probe (3 nM). The assay was performed in a 1×HBSTET buffer, Fluorescence polarization (FP) assay. The binding affinities between fluorescent peptide probes (Cy5-Nrf2, FAM-Nrf2

, The g-factor was adjusted at each experiment so that a series of three blank wells containing probe but no Keap1 Kelch defined the baseline FP value. The Cy5-Nrf2, FAM-Nrf2, and FITC-Nrf2 probes were measured at excitation/emission values of 635/670 nm, 470/525 nm, and 470/535 nm, respectively. The FP values were fitted to the one-site specific binding equation: Y = Bmax × X/(Kd + X), with Bmax being the maximal FP value, X the Keap1 Kelch concentration, and Y the variable FP values. The Kd values were derived from the resulting binding saturation curve as being equal to the Keap1 Kelch concentration, where the curve is half-saturated, Tween20, 3 mM EDTA, 1 mM TCEP, pH = 7.4) using black flat-bottom 384-well plates (Corning Life Sciences

, Waters ACQUITY H-class UPLC system with a C18 reverse phase column (Acquity UPLC BEH C18, 2.1 mm × 50 mm, 1.7 µm), a Sample Manager FTN and a TUV dual wavelength detector, using a linear gradient of the binary solvent system of buffer A (milliQ H2O:MeCN:formic acid, 95:5:0.1 v/v%) to buffer B (MeCN:formic acid, p.100

, Incubation times were 1-3 h before analysis by LC-MS. LC-MS spectra were obtained with the LC-MS system described above but here using a Poroshell C18 reverse phase column (Agilent, Poroshell, 300SB-C18, 2.1×75 mm) and a linear gradient of the binary solvent system from 0 to 60% B over 6 min. The samples were monitored by TIC, UV, and ELS. The spectra were analyzed using the Agilent Mas-sHunter Qualitative Analysis software version B, Compounds were tested for reactivity towards the Keap1 Kelch domain using a similar protocol. The com-TCEP. Final compound concentrations were 800 µM

. Johnston, Hydrogen peroxide (31-1000 µM) and 3-methyltoxoflavin (0.2-400 µM) were used as positive controls. Hank's Balanced Salt Solution (HBSS) from Sigma-Aldrich (cat# H9269horseradish peroxidase (Sigma-Aldrich, cat# P2088-5KU) with 20 mL HBSS, and 40 µL was added to each well followed by 45 min incubation at room temperature and assay quenching by addition of 10 µL NaOH (1 M) per well

, Absorbance values from blank wells (i.e. wells without redox-active compounds

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