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C8cc08685k1.Pdf Electronic Supplementary Material (ESI) for ChemComm. This journal is © The Royal Society of Chemistry 2018 Supporting Information Minimalist Linkers Suitable for Irreversible Inhibitors in Simultaneous Proteome Profiling, Live-Cell Imaging and Drug Screening Cuiping Guo,Yu Chang, Xin Wang, Chengqian Zhang, Piliang Hao*, Ke Ding and Zhengqiu Li* School of Pharmacy, Jinan University, Guangzhou, China 510632 *Corresponding author ([email protected]) 1. General Information All chemicals were purchased from commercial vendors and used without further purification, unless indicated otherwise. All reactions requiring anhydrous conditions were carried out under argon or nitrogen atmosphere using oven-dried glassware. AR-grade solvents were used for all reactions. Reaction progress was monitored by TLC on pre-coated silica plates (Merck 60 F254 nm, 0.25 µm) and spots were visualized by UV, iodine or other suitable stains. Flash column chromatography was carried out using silica gel (Qingdao Ocean). All NMR spectra (1H-NMR, 13C-NMR) were recorded on Bruker 300 MHz/400 MHz NMR spectrometers. Chemical shifts were reported in parts per million (ppm) referenced with respect to appropriate internal standards or residual solvent peaks (CDCl3 = 7.26 ppm, DMSO-d6 = 2.50 ppm). The following abbreviations were used in reporting spectra, br s (broad singlet), s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), dd (doublet of doublets). Mass spectra were obtained on Agilent LC-ESI-MS system. All analytical HPLC were carried out on Agilent system. Water with 0.1% TFA and acetonitrile with 0.1% TFA were used as eluents and the flow rate was 0.5 mL/min. Antibodies against EGFR (ab52894) and BTK (ab137503) were purchased from Cell Signaling Technology (CST). Click reagents were purchased from Click Chemistry Tools (https://clickchemistrytools.com/). 2. Cell culture and Western blot Cell lines were obtained from the National Cancer Institute Developmental Therapeutics Program (NCI-60). Cells were cultured in Dulbeccoʼs modified Eagle medium (DMEM; Invitrogen, Carlsbad, CA) or RPMI 1640 Medium (Invitrogen, Carlsbad, CA) containing 10% heat-inactivated fetal bovine serum (FBS; Invitrogen), 100 units/mL penicillin, and 100 μg/mL streptomycin (Thermo Scientific) and maintained in a humidified 37 °C incubator with 5% CO2. To generate protein lysates, cells were washed twice with cold phosphate-buffered saline (PBS), harvested with 1× trypsin or by use of a cell scraper, and collected by centrifugation. Cell pellets were then washed with PBS and lysed with RIPA (Thermo Scientific™，#89900) lysis and extraction buffer (with Pierce™ Protease Inhibitor Tablets, Thermo Scientific™, #A32955). Protein concentration was determined by Pierce™ BCA Protein Assay Kit (Thermo Scientific™, #23252) and Synergy H1 Hybrid Multi-Mode Reader (BioTek). For Western blotting experiments, samples were resolved by SDS−polyacrylamide gels and transferred to poly membranes. Membranes were then blocked with 3% bovine serum albumin (BSA) in TBST (0.1% Tween in Tris-buffered saline) for 1 h at room temperature. After blocking, membranes were incubated with the corresponding primary antibody for another 1 hour. After incubation, membranes were washed with TBST (4×10 min) and then incubated with an appropriate secondary antibody. Finally, blots were washed again with TBST before being developed with SuperSignal West Dura Kit (Thermo Scientific), and finally imaged with Amersham Imager 600(GE Healthcare). Cell Counting Kit-8 (CCK-8, DOJINDO, #CK04) was used for cell proliferation assay. Proteome labeling, in-gel fluorescence scanning and cellular imaging experiments were performed as previously reported.[1-4] S1_1 Table S1. The probes used in current study AF-1 AF-2 IB-1 IB-2 IB-3 Table S2. Structures of the reporters TAMRA-alkyne TAMRA-N3 Biotin-alkyne Biotin-N3 Tetrazine-Cy5 Methyltetrazine-biotin S1_2 DBCO-Cy3 3. Chemical Synthesis Scheme S1 S2 was synthesized based on previously reported procedures from S1.[5] To a solution of compound S2 (50 mg, 0.095 mmol) in 3 mL DMF was added N-methylpropargylamine (0.0317 mL, 0.38 mmol), potassium carbonate (26 mg, 0.19 mmol) and potassium o o iodide (27 mg, 0.17 mmol) at 0 C. The mixture was stirred at 40 C for 3 h under N2 gas atomosphere and then quenched by addition of 10 mL water. The resulting mixture was extracted with ethyl acetate (3 × 20 mL), and the combined organic phase was washed with brine, dried over anhydrous Na2SO4. Upon solvent evaporation in vacuo, the residue was purified by flash column 1 (methanol:ethyl acetate = 1:10) to give product AF-1 as a yellow solid (23 mg, 48%). H NMR (300 MHz, CDCl3) δ 9.00 (s, 1H), 8.56 (s, 1H), 8.35 (s, 1H), 8.10 (s, 1H), 7.79 (dd, J = 3.0, 6.0 Hz, 1H), 7.46 (m, 1H), 7.09 (s, 1H), 7.0 (m, 1H), 6.96 (m, 1H), 6.23 (d, J = 15.0 Hz, 1H), 5.10 (s, 1H), 4.15 (d, J = 12.0 Hz, 1H), 4.02 (m, 2H), 3.90 (m, 1H), 3.37 (s, 2H), 3.28 (d, J = 6.0 Hz，2H), 2.43 (m, 13 1H), 3.26 (s, 3H), 2.29 (t, J = 3.0 Hz, 1H), 2.22 (m, 1H). C NMR (75 MHz, CDCl3) δ 163.98, 156.98, 154.63, 150.60, 148.16, 143.95, 135.46, 127.91, 125.63, 124.17, 121.89, 120.74, 116.49, 110.68, 109.61, 108.30, 79.48, 78.24, 77.23, 73.95, 73.12, 67.41, 56.55, 45.79, 42.11, 32.84. HR-MS (m/z) [M + H]+ calcd: 510.1703; Found: 510.1666. Scheme S2 o To a stirred solution of NaN3 (6 mg, 0.095 mmol) in 3 mL DMF was added S2 (50 mg, 0.095 mmol) at 0 C. The resulting mixture was stirred for 30 min and then at room temperature for 4 h. Subsequently, 5 mL water was added and the mixture was extracted with ethyl acetate (2 × 15 mL). The combined organic phase was washed with brine and then dried over anhydrous Na2SO4 and concentrated in vacuo. The residue was purified by flash column (methanol:CH2Cl2 = 3:50) to give AF-2 as a white solid (37 mg, 1 80%). H NMR (400 MHz, CDCl3) δ 9.10 (s, 1H), 8.65 (s, 1H), 8.13 (s, 1H), 7.93 (dd, J = 4.0, 8.0 Hz, 1H), 7.75 (s, 1H), 7.54 (m, 1H), 7.21 (s, 1H), 7.14 (t, J = 8.0 Hz, 1H), 7.01 (dt, J = 4.0 12.0 Hz, 1H), 6.32 (dt, J = 4.0, 12.0 Hz, 1H), 5.19 (t, J = 4.0 Hz, 1H), 4.20 (d, J = 12.0 Hz, 1H), 4.14 (d, J = 4.0 Hz, 2H), 4.11 (d, J = 4.0 Hz, 1H), 4.05 (m, 1H), 3.95 (m, 1H), 2.45 (m, 1H), 2.25 (m, 1H). S1_3 13 C NMR (100 MHz, CDCl3) δ 163.29, 156.96, 154.81, 150.57, 148.34, 139.60, 128.24, 125.66, 124.43, 121.95, 121.89, 116.83, 116.61, 110.18, 109.60, 108.72, 79.70, 73.18, 67.48, 51.49, 32.98, 29.92. HR-MS (m/z) [M + H]+ calcd: 484.1295; Found: 484.1290. Scheme S3 (IB-1). The intermediate S3 is commercially available and the cyclopropene-containing linker (S4) was synthesized based on previously published procedures.[6] To a solution of compound S4 (20.0 mg, 0.05 mmol) in 4 mL DMF was added HOBT (10.49 mg, 0.075 mmol), EDC (14.37 mg, 0.075 mmol), TEA (10 mg, 0.1 mmol) and S8 (6.2 mg, 0.05 mmol), successively. The reaction was stirred at room temperature overnight prior to addition of 3 mL water and then extracted with ethyl acetate (2 × 10 mL), the combined organic phase was washed with brine, dried over anhydrous Na2SO4 and concentrated. The residue was purified by flash 1 column (methanol:CH2Cl2 = 1 : 50) to give IB-1 as a light yellow solid (12.3 mg, 50 %). H NMR (300 MHz, CDCl3) δ 8.34 (s, 1H), 7.62 (d, J = 12.0 Hz, 2H), 7.37 (t, J = 6.0, 9.0 Hz, 2H), 7.14 (t, J = 9.0 Hz, 3H), 7.06 (d, J = 6.0 Hz, 2H), 6.53 (m, 1H), 6.32 (d, J = 3.0 Hz, 1H), 5.66 (s, 1H), 4.84 (m, 1H), 4.13 (d, J = 6.0 Hz, 1H), 3.67 (d, J = 6.0 Hz, 1H), 3.31 (m, 1H), 2.24 (m, 3H), 2.11 (s, 3H), 13 1.95 (m, 2H), 1.69 (m, 1H). C NMR (100 MHz, CDCl3) δ 166.07, 158.53, 157.84, 156.35, 156.18, 155.79, 154.25, 143.83, 129.97, 127.75, 124.05, 119.54, 119.30, 119.14, 115.54, 101.24, 98.59, 53.62, 52.70, 49.87, 46.16, 45.72, 42.13, 35.96, 31.91, 30.38, 29.26, 27.22, 22.46. ESI-MS (m/z) [M + H]+ calcd: 493.2; Found: 493.5. HR-MS (m/z) [M + H]+ calcd: 493.2347; Found: 493.2331. Scheme S4 (IB-2). Synthesis of S5 was based on previously published procedures from commercially available intermediate S3.[7] To a solution of 2 mL TFA and 6 mL DCM was added compound S5 (17.1 mg, 0.03 mmol), the reaction was then stirred at room temperature for 30 minutes. Upon solvent evaporation in vacuo, the crude product was dissolved in 3 mL DMF followed by addition of TCO-NHS (5.3 mg, 0.02 mmol) and TEA (10 mg, 0.1 mmol), the resulting mixture was stirred at room temperature for 1 hour and then quenched by addition of 3 mL water.
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