Supporting Information

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1 Supporting Information Chemoproteomics-Enabled Discovery of a Potent and Selective Inhibitor of the DNA Repair Protein MGMT Chao Wang +, Daniel Abegg +, Dominic G. Hoch, and Alexander Adibekian* ange_ _sm_miscellaneous_information.pdf

2 Table of Contents Materials and General Methods... S3 Experimental Details and Procedures... S3 Supplemental Figures... S29 Supplemental Tables... S41 X-ray crystallography data... S326 Copies of 1 H NMR, 13 C NMR and HMBC Spectra... S331 References... S397 S2

3 Materials and General Methods All commercial reagents were purchased from Sigma-Aldrich or Acros of the highest purity grade. All reagents were weighed and handled in air and used without further purification, unless otherwise noted. Liquid reagents were transferred with stainless steel syringes or cannula. Thin layer chromatography (TLC) was performed on plates of silica precoated with 0.25 mm Kieselgel 60 F254. Flash chromatography was performed using silica gel µm ( mesh) from SiliCycle. 1 H NMR, 13 C NMR and HMBC NMR spectra were recorded on ARX-300, AMX-400 and AM- 500 Bruker Avance spectrometers. 1 H and 13 C{1H} NMR chemical shifts (δ) are reported in ppm relative to TMS (0.00 ppm), with the solvent resonance used as internal reference, and coupling constants (J) are in Hertz (Hz). The following abbreviations were used to explain the multiplicities: s = singlet, d = doublet, t = triplet, q = quartet, p = pentad, m = multiplet, br = broad. The mass spectrometric data were obtained at the mass spectrometry facililty of the University of Geneva. DMEM/High glucose media with GlutaMAX (with and without sodium pyruvate), RPMI-1640 with GlutaMAX, phosphate buffered saline (PBS), MEM Non-Essential Amino Acids, Penicillin Streptomycin (Pen/Strep) and Trypsin-EDTA were obtained from Life Technologies. Protein concentration was determined using the Bradford assay (Bio-Rad). All the cloning was done using the Gateway technology (Thermo Fisher Scientific). All the plasmids were prepared using the QIAprep Spin Miniprep Kit (Qiagen). For all R reactions, Q5 High-Fidelity DNA Polymerase (New England BioLabs Inc) was used as recommended by manufacturer. R cleanup was performed using the GenElute R Clean-Up following the protocol of the producer. For the BP and the LR reactions the Gateway BP Clonase Enzyme Mix and the Gateway LR Clonase Enzyme mix (Thermo Fisher Scientific) were used according to the protocol of the manufacturer. Experimental Details and Procedures Synthesis of Chloromethyl Triazole Probe 1 (AA-CW159A) To a stirred solution of tetraethylene glycol (1.94 g, mmol, 1.0 eq.) in dry CH 2Cl 2 (18 ml) cooled in an ice bath, Et 3N (4.18 ml, 30 mmol, 3.0 eq.) was added, followed by the addition of MsCl (2.32 ml, 30 mmol, 3.0 eq.). The resulting reaction mixture was stirred at 0 C for 2 h and then at r.t. for 2 h. After that, the reaction mixture was concentrated and washed with 1 M HCl (50.0 ml) and the aqueous phase was extracted with CH 2Cl 2 (10.0 ml 3). The combined organic phases were washed with sat. aq. NaHCO 3 (30.0 ml) and dried over Na 2SO 4, then filtered and concentrated in vacuo to give the crude product as a yellow oil. The product was dissolved in dry DMF (70.0 ml) and NaN 3 (2.34 g, mmol, 3.5 eq.) was added, then the reaction mixture was stirred at 60 C overnight. After the TLC showed complete consumption of the starting compound, the reaction mixture was poured into water (100.0 ml) and extracted S3

4 with EtOAc (10.0 ml 3). The combined extracts were concentrated, dried and purified by column chromatography (silica gel, 10 % 20 % EtOAc in hexane) to give the previously reported [1] product AA-CW148 (2.35 g, 96 % yield) as a colorless oil. 1 H NMR (300 MHz, CDCl 3) δ (m, 12H), 3.39 (t, J = 5.1 Hz, 4H). A screw-cap tube was charged with 1-azido-2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethane (391.0 mg, mmol, 1.6 eq.), propargyl alcohol (56.1 mg, mmol, 1.0 eq.) and dry PhMe (3.0 ml) under N 2 atmosphere. The tube was screw-capped and left for 18 h at 125 C in an oil bath under stirring. After that, the reaction mixture was concentrated in vacuo and the resulting residue was purified by column chromatography (silica gel, 2 % 6 % MeOH in EtOAc) to afford the product AA-CW155A (90.0 mg, 30 % yield) as a slightly yellow oil. 1 H NMR (400 MHz, CDCl 3) δ 7.62 (s, 1H), 4.68 (s, 2H), 4.58 (t, J = 4.8 Hz, 2H), 3.88 (t, J = 4.8 Hz, 2H), (m, 4H), (m, 6H), 3.36 (t, J = 4.9 Hz, 2H); 13 C NMR (100 MHz, CDCl 3) δ , , 70.53, 70.48, 70.44, 70.27, 70.11, 69.97, 52.57, 50.65, 48.33; HRMS (ESI) m/z Calculated for C 11H 21N 6O 4 (M+H) + : , found: Under N 2 atmosphere, to a stirred solution of (1-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)- 1H-1,2,3-triazol-5-yl)methanol (28.3 mg, mmol, 1.0 eq.) in dry CH 2Cl 2 (3.0 ml) cooled in an ice bath, Et 3N (39.3 µl, mmol, 3.0 eq.) was added, followed by the addition of MsCl (22.3 µl, mmol, 3.0 eq.). The resulting reaction mixture was stirred at 0 C for 0.5 h and then at r.t. for 0.5 h. After that, n Bu 4NCl (79.0 mg, mmol, 3.0 eq.) was added and the reaction was stirred at r.t. overnight. The reaction mixture was concentrated in vacuo and purified by column chromatography (silica gel, 2 % MeOH in CH 2Cl 2) to give the product 1 (27.7 mg, 92 % yield) as a slightly yellow oil. 1 H NMR (400 MHz, CDCl 3) δ 7.66 (s, 1H), 4.80 (s, 2H), 4.61 (t, J = 5.0 Hz, 2H), 3.90 (t, J = 5.0 Hz, 2H), (m, 4H), (m, 6H), 3.39 (t, J = 4.9 Hz, 2H); 13 C NMR (100 MHz, CDCl 3) δ , , 70.72, 70.59, 70.55, 70.49, 70.13, 70.07, 50.70, 48.74, HRMS (ESI) m/z Calculated for C 11H 20ClN 6O 3 (M+H) + : , found: S4

5 General Procedure for Synthesis of Substituted Propargylic Alcohol Compounds To a stirred solution of terminal alkyne (1.0 eq.) in dry THF (3.0 ml) under N 2 atmosphere, EtMgBr (1.2 eq., 1 M in THF) was added at r.t. and the reaction was stirred for 0.5 h. Then paraformaldehyde (1.2 eq.) was added and the resulting reaction was stirred at r.t. overnight. After that, sat. NH 4Cl (3.0 ml) was added and the organic layer was separated. The aqueous layer was extracted with EtOAc. The combined organic layers were concentrated in vacuo, dried and purified by silica gel chromatography to afford the pure product. 3-Cyclohexylprop-2-yn-1-ol was prepared from ethynylcyclohexane (487.0 mg, mmol, 1.0 eq.), EtMgBr (5.4 ml, 1 M in THF, mmol, 1.2 eq.) and paraformaldehyde (162.0 mg, mmol, 1.2 eq.). The product was purified by column chromatography (silica gel, 50 % CH 2Cl 2 in hexane, then CH 2Cl 2) as a yellow oil (582.0 mg, 94 % yield). NMR data were consistent with literature values [2] ; 1 H NMR (400 MHz, CDCl 3) δ 4.26 (s, 2H), (m, 1H), (m, 2H), (m, 2H), (m, 7H). 3-(4-(Trifluoromethoxy)phenyl)prop-2-yn-1-ol was prepared from 1-ethynyl-4- (trifluoromethoxy)benzene (560.0 mg, mmol, 1.0 eq.), EtMgBr (3.6 ml, 1 M in THF, mmol, 1.2 eq.) and paraformaldehyde (108.0 mg, mmol, 1.2 eq.). The product was purified by column chromatography (silica gel, 50 % CH 2Cl 2 in hexane, then CH 2Cl 2) as a yellow oil (645.0 mg, 99 % yield); 1 H NMR (400 MHz, CDCl 3) δ 7.45 (d, J = 8.8 Hz, 2H), 7.15 (d, J = 8.1 Hz, 2H), 4.50 (s, 2H), 1.86 (s, 1H); 13 C NMR (100 MHz, CDCl 3) δ (q, J = 1.7 Hz), , , , (q, J = Hz), 88.05, 84.31, HRMS (ESI) m/z Calculated for C 10H 8F 3O 2 (M+H) + : , found: (Pyridin-3-yl)prop-2-yn-1-ol was prepared from 3-ethynylpyridine (495.0 mg, mmol, 1.0 eq.), EtMgBr (5.8 ml, 1 M in THF, mmol, 1.2 eq.) and paraformaldehyde (174.0 mg, mmol, 1.2 eq.). The product was purified by column chromatography (silica gel, 30 % EtOAc in hexane, then EtOAc) as a white solid (456.0 mg, 71 % yield). NMR data were S5

6 consistent with literature values [3] ; 1 H NMR (400 MHz, CDCl 3) δ 8.77 (d, J = 1.5 Hz, 1H), 8.52 (dd, J = 4.9 Hz, 1.6 Hz, 1H), 7.73 (dt, J = 7.9 Hz, 1.9 Hz, 1H), 7.27 (ddd, J = 7.9 Hz, 4.8 Hz, 0.8 Hz, 1H), 4.51 (s, 2H), 3.62 (br, 1H). General Procedure for Synthesis of 1,4-substituted Hydroxymethyl Triazoles Method A: A screw-cap tube was charged with substituted propargylic alcohol (1.0 eq.), alkyl azide (1.0 eq.) and dry PhMe (2.0 ml) under N 2 atmosphere. The reaction tube was screwcapped and left overnight under stirring at 125 C in an oil bath. Note: For compounds AA-CW256A and AA-CW262A, the reaction was carried out for 3 days. Method B [4] : A screw-cap tube was charged with Cp*Ru(PPh 3) 2Cl (2.0 mol %), evacuated and filled with N 2 (3 times). Then dry PhMe (2.0 ml), substituted propargylic alcohol (1.0 eq.) and alkyl azide (1.0 eq.) were added in sequence. The reaction tube was screw-capped and left overnight under stirring at 80 C in an oil bath. The reaction vessel was then cooled to r.t. and the reaction mixture was concentrated in vacuo. The resulting residue was purified by silica gel chromatography to afford the pure 1,4- substituted hydroxymethyl triazole. The correct configuration was assigned by 2D-HMBC analysis of the correlation between the triazole ring carbons and protons on neighboring carbon atoms (see attached HMBC spectra). Methyl 2-(5-(hydroxymethyl)-4-propyl-1H-1,2,3-triazol-1-yl)acetate was prepared from hex-2- yn-1-ol (50.0 mg, mmol) and methyl 2-azidoacetate (50.2 µl, mmol) using Method A. The product (31.0 mg, 29 % yield) was purified by column chromatography (silica gel, EtOAc, then 5 % MeOH in EtOAc) as a slightly yellow solid; 1 H NMR (400 MHz, CDCl 3) δ 5.23 (s, 2H), 4.67 (d, J = 5.0 Hz, 2H), 3.77 (s, 3H), 3.54 (t, J = 5.3 Hz, 1H), 2.59 (t, J = 7.6 Hz, 2H), (m, 2H), 0.91 (t, J = 7.4 Hz, 3H); 13 C NMR (100 MHz, CDCl 3) δ , , , 53.00, 52.52, 49.63, 26.75, 22.96, HRMS (ESI) m/z Calculated for C 9H 16N 3O 3 (M+H) + : , found: S6

7 Methyl 2-(4-cyclohexyl-5-(hydroxymethyl)-1H-1,2,3-triazol-1-yl)acetate was prepared from 3- cyclohexylprop-2-yn-1-ol (69.1 mg, mmol) and methyl 2-azidoacetate (50.2 µl, mmol) using Method A. The product (23.0 mg, 18 % yield) was purified by column chromatography (silica gel, EtOAc, then 5 % MeOH in EtOAc) as a white solid; 1 H NMR (400 MHz, CDCl 3) δ 5.23 (s, 2H), 4.68 (d, J = 5.2 Hz, 2H), 3.78 (s, 3H), 3.39 (s, 1H), 2.65 (t, J = 12.1 Hz, 1H), (m, 4H), (m, 3H), (m, 3H); 13 C NMR (100 MHz, CDCl 3) δ , , , 53.03, 52.52, 49.56, 35.07, 32.88, 26.53, HRMS (ESI) m/z Calculated for C 12H 20N 3O 3 (M+H) + : , found: Methyl 2-(5-(hydroxymethyl)-4-(4-(trifluoromethoxy)phenyl)-1H-1,2,3-triazol-1-yl)acetate was prepared from 3-(4-(trifluoromethoxy)phenyl)prop-2-yn-1-ol (108.0 mg, mmol) and methyl 2-azidoacetate (50.2 µl, mmol) using Method A. The product (50.0 mg, 30 % yield) was purified by column chromatography (silica gel, 40 % EtOAc in hexane) as a white solid; 1 H NMR (400 MHz, CDCl 3) δ 7.77 (d, J = 8.8 Hz, 2H), 7.30 (d, J = 9.0 Hz, 2H), 5.31 (s, 2H), 4.80 (d, J = 5.4 Hz, 2H), 3.84 (s, 3H), 3.59 (t, J = 5.7 Hz, 1H). 13 C NMR (100 MHz, CDCl 3) δ , (q, J = 2.0 Hz), , , , , , (q, J = Hz), 53.31, 52.80, HRMS (ESI) m/z Calculated for C 13H 13F 3N 3O 4 (M+H) + : , found: Methyl 2-(5-(hydroxymethyl)-4-(naphthalen-1-yl)-1H-1,2,3-triazol-1-yl)acetate was prepared form 3-(naphthalen-1-yl)prop-2-yn-1-ol (94.9 mg, mmol) and methyl 2-azidoacetate (50.2 µl, mmol) using Method A. The product (17.0 mg, 11 % yield) was purified by column chromatography (silica gel, 50 % EtOAc in hexane) as a slightly brown solid; 1 H NMR (400 MHz, CDCl 3) δ (m, 3H), (m, 4H), 5.41 (s, 2H), 4.69 (s, 2H), 3.87 (s, 3H); 13 C NMR (100 MHz, CDCl 3) δ , , , , , , , , , , , , , 53.38, 53.22, HRMS (ESI) m/z Calculated for C 16H 16N 3O 3 (M+H) + : , found: S7

8 Methyl 2-(5-(hydroxymethyl)-4-(pyridin-3-yl)-1H-1,2,3-triazol-1-yl)acetate was prepared from 3-(pyridin-3-yl)prop-2-yn-1-ol (33.3 mg, mmol) and methyl 2-azidoacetate (25.1 µl, mmol) using Method A. The product (35.0 mg, 56 % yield) was purified by column chromatography (silica gel, 2 % MeOH in EtOAc) as a yellow solid; 1 H NMR (400 MHz, CDCl 3) δ 8.68 (s, 1H), 8.44 (d, J = 4.0 Hz, 1H), 8.02 (d, J = 7.9 Hz, 1H), 7.33 (dd, J = 7.7, 4.9 Hz, 1H), 5.36 (s, 2H), 4.81 (s, 2H), 3.80 (s, 3H); 13 C NMR (100 MHz, CDCl 3) δ , , , , , , , , 53.18, 52.55, HRMS (ESI) m/z Calculated for C 11H 13N 4O 3 (M+H) + : , found: (1-(2-Morpholinoethyl)-4-propyl-1H-1,2,3-triazol-5-yl)methanol was prepared from hex-2-yn-1- ol (25.3 mg, mmol) and 4-(2-azidoethyl)morpholine (39.0 mg, mmol) using Method B. The product (14.0 mg, 22 % yield) was purified by column chromatography (silica gel, 4 % 5 % MeOH in CH 2Cl 2) as a yellow solid; 1 H NMR (400 MHz, CDCl 3) δ 4.55 (s, 2H), (m, 2H), 3.69 (t, J = 4.7 Hz, 4H), (m, 2H), 2.68 (t, J = 7.6 Hz, 2H), 2.48 (t, J = 4.6 Hz, 4H), (m, 2H), 0.95 (t, J = 7.4 Hz, 3H); 13 C NMR (100 MHz, CDCl 3) δ , , 65.95, 59.37, 54.32, 50.99, 45.87, 26.95, 23.22, HRMS (ESI) m/z Calculated for C 12H 23N 4O 2 (M+H) + : , found: (4-Cyclohexyl-1-(2-morpholinoethyl)-1H-1,2,3-triazol-5-yl)methanol was prepared from 3- cyclohexylprop-2-yn-1-ol (34.6 mg, mmol) and 4-(2-azidoethyl)morpholine (39.0 mg, mmol) using Method B. The product (27.0 mg, 37 % yield) was purified by column chromatography (silica gel, EtOAc, then 5 % MeOH in EtOAc) as a yellow solid; 1 H NMR (400 MHz, CDCl 3) δ 4.78 (s, 2H), 4.34 (t, J = 6.8 Hz, 2H), 3.67 (t, J = 4.6 Hz, 4H), 2.81 (t, J = 6.8 Hz, 2H), 2.71 (tt, J = 12.2, 3.3 Hz, 1H), 2.49 (t, J = 4.6 Hz, 4H), (m, 5H), (m, 2H), (m, 4H); 13 C NMR (100 MHz, CDCl 3) δ , , 66.81, 58.57, 56.71, 53.86, 45.56, 34.79, 31.89, 26.65, HRMS (ESI) m/z Calculated for C 15H 27N 4O 2 (M+H) + : , found: S8

9 (1-(2-Morpholinoethyl)-4-(4-(trifluoromethoxy)phenyl)-1H-1,2,3-triazol-5-yl)methanol was prepared from 3-(4-(trifluoromethoxy)phenyl)prop-2-yn-1-ol (54.0 mg, mmol) and 4-(2- azidoethyl)morpholine (39.0 mg, mmol) using Method B. The product (38.0 mg, 41 % yield) was purified by column chromatography (silica gel, 2 % 3 % MeOH in CH 2Cl 2) as a slightly yellow solid; 1 H NMR (400 MHz, CDCl 3) δ 7.81 (d, J = 8.6 Hz, 2H), 7.30 (d, J = 8.3 Hz, 2H), 4.64 (s, 2H), (m, 2H), 3.73 (t, J = 4.5 Hz, 4H), (m, 2H), 2.54 (t, J = 4.3 Hz, 4H); 13 C NMR (100 MHz, CDCl 3) δ (q, J = 1.7 Hz), , , , , , (q, J = Hz), 65.93, 59.54, 54.30, 51.34, HRMS (ESI) m/z Calculated for C 16H 20F 3N 4O 3 (M+H) + : , found: (1-(2-Morpholinoethyl)-4-(naphthalen-1-yl)-1H-1,2,3-triazol-5-yl)methanol was prepared from 3-(naphthalen-1-yl)prop-2-yn-1-ol (45.5 mg, mmol) and 4-(2-azidoethyl)morpholine (39.0 mg, mmol) using Method B. The product (16.0 mg, 19 % yield) was purified by column chromatography (silica gel, 2 % 3 % MeOH in EtOAc) as a brown solid; 1 H NMR (400 MHz, CDCl 3) δ (m, 1H), (m, 2H), 7.64 (dd, J = 7.0 Hz, 1.0 Hz, 1H), (m, 3H), (m, 2H), 4.56 (s, 2H), 3.77 (t, J = 4.6 Hz, 4H), (m, 2H), 2.58 (t, J = 4.5 Hz, 4H); 13 C NMR (100 MHz, CDCl 3) δ , , , , , , , , , , , , 66.00, 59.41, 54.40, 51.47, HRMS (ESI) m/z Calculated for C 19H 23N 4O 2 (M+H) + : , found: (1-(4-Bromobenzyl)-4-propyl-1H-1,2,3-triazol-5-yl)methanol was prepared from hex-2-yn-1-ol (50.6 mg, mmol) and 1-(azidomethyl)-4-bromobenzene (1.0 ml, ~ 0.5 M in CH 2Cl 2, 0.5 mmol) using Method A. The product (32.0 mg, 21 % yield) was purified by column chromatography (silica gel, 50 % EtOAc in hexane) as a white solid; 1 H NMR (400 MHz, CDCl 3) δ 7.45 (d, J = 8.4 Hz, 2H), 7.11 (d, J = 8.4 Hz, 2H), 5.55 (s, 2H), 4.54 (d, J = 4.5 Hz, 2H), 2.68 (t, J = 4.6 Hz, 1H), 2.57 (t, J = 7.6 Hz, 2H), (m, 2H), 0.90 (t, J = 7.4 Hz, 3H); 13 C NMR (100 MHz, CDCl 3) δ , , , , , , 52.32, 51.70, 26.79, 23.12, HRMS (ESI) m/z Calculated for C 13H 17BrN 3O (M+H) + : , found: S9

10 (1-(4-Bromobenzyl)-4-(pyridin-3-yl)-1H-1,2,3-triazol-5-yl)methanol was prepared from 3- (pyridin-3-yl)prop-2-yn-1-ol (33.3 mg, mmol) and 1-(azidomethyl)-4-bromobenzene (0.5 ml, ~ 0.5 M in CH 2Cl 2, mmol) using Method B. The product (30.0 mg, 35 % yield) was purified by column chromatography (silica gel, EtOAc, then 3 % MeOH in EtOAc) as a white solid; 1 H NMR (400 MHz, CDCl 3) δ 8.71 (br, 1H), 8.47 (br, 1H), 8.08 (d, J = 7.8 Hz, 1H), 7.50 (d, J = 8.4 Hz, 2H), 7.38 (br, 1H), 7.23 (d, J = 8.4 Hz, 2H), 5.69 (s, 2H), 4.72 (s, 2H); 13 C NMR (100 MHz, CDCl 3) δ , , , , , , , , , , , 52.27, HRMS (ESI) m/z Calculated for C 15H 14BrN 4O (M+H) + : , found: (Benzo[d][1,3]dioxol-5-yl)-2-(5-(hydroxymethyl)-4-propyl-1H-1,2,3-triazol-1-yl)ethan-1-one was prepared from hex-2-yn-1-ol (15.2 mg, mmol) and 2-azido-1-(benzo[d][1,3]dioxol-5- yl)ethan-1-one (31.0 mg, mmol) using Method B. The product (17.0 mg, 37 % yield) was purified by column chromatography (silica gel, EtOAc, then 2 % MeOH in EtOAc) as a brown solid; 1 H NMR (400 MHz, CDCl 3) δ 7.62 (dd, J = 8.2 Hz, 1.6 Hz, 1H), 7.43 (d, J = 1.5 Hz, 1H), 6.91 (d, J = 8.2 Hz, 1H), 6.09 (s, 2H), 5.82 (s, 2H), 4.62 (s, 2H), 2.66 (t, J = 7.6 Hz, 2H), (m, 2H), 0.95 (t, J = 7.3 Hz, 3H); 13 C NMR (100 MHz, CDCl 3) δ , , , , , , , , , , 54.18, 52.72, 26.89, 23.03, HRMS (ESI) m/z Calculated for C 15H 18N 3O 4 (M+H) + : , found: (Benzo[d][1,3]dioxol-5-yl)-2-(4-cyclohexyl-5-(hydroxymethyl)-1H-1,2,3-triazol-1-yl)ethan-1- one was prepared from 3-cyclohexylprop-2-yn-1-ol (16.6 mg, mmol) and 2-azido-1- (benzo[d][1,3]dioxol-5-yl)ethan-1-one (25.0 mg, mmol) using Method A. The product (11.0 mg, 27 % yield) was purified by column chromatography (silica gel, 50 % EtOAc in hexane) as a brown solid; 1 H NMR (400 MHz, CDCl 3) δ 7.56 (dd, J = 8.2 Hz, 1.5 Hz, 1H), 7.37 (d, J = 1.4 Hz, 1H), 6.84 (d, J = 8.2 Hz, 1H), 6.02 (s, 2H), 5.74 (s, 2H), 4.56 (s, 2H), 2.64 (t, J = 11.9 Hz, 1H), (m, 4H), (m, 3H), (m, 3H); 13 C NMR (100 MHz, CDCl 3) δ , , , , , , , , , , 54.04, 52.73, 35.21, 32.93, 26.58, HRMS (ESI) m/z Calculated for C 18H 22N 3O 4 (M+H) + : , found: S10

11 1-(Benzo[d][1,3]dioxol-5-yl)-2-(5-(hydroxymethyl)-4-(pyridin-3-yl)-1H-1,2,3-triazol-1-yl)ethan- 1-one was prepared from 3-(pyridin-3-yl)prop-2-yn-1-ol (16.0 mg, mmol) and 2-azido-1- (benzo[d][1,3]dioxol-5-yl)ethan-1-one (25.0 mg, mmol) using Method A. The product (17.0 mg, 42 % yield) was purified by column chromatography (silica gel, 2 % 3 % MeOH in EtOAc) as a white solid; 1 H NMR (400 MHz, MeOD) δ 9.09 (br, 1H), 8.71 (br, 1H), 8.47 (d, J = 8.0 Hz, 1H), 7.83 (dd, J = 8.2, 1.5 Hz, 1H), 7.78 (br, 1H), 7.56 (d, J = 1.4 Hz, 1H), 7.04 (d, J = 8.2 Hz, 1H), 6.19 (s, 2H), 6.14 (s, 2H), 4.79 (s, 2H); 13 C NMR (100 MHz, MeOD) δ , , , , , , , , , , , , , 54.49, HRMS (ESI) m/z Calculated for C 17H 15N 4O 4 (M+H) + : , found: (1-(2-(3,5-Dimethylisoxazol-4-yl)ethyl)-4-propyl-1H-1,2,3-triazol-5-yl)methanol was prepared from hex-2-yn-1-ol (24.5 mg, mmol) and 4-(2-azidoethyl)-3,5-dimethylisoxazole (41.6 mg, mmol) using Method B. The product (19.0 mg, 29 % yield) was purified by column chromatography (silica gel, EtOAc) as a brown oil; 1 H NMR (400 MHz, CDCl 3) δ (m, 4H), 2.97 (t, J = 7.0 Hz, 2H), 2.57 (t, J = 7.6 Hz, 2H), 2.13 (s, 3H), 2.10 (s, 3H), (m, 2H), 0.92 (t, J = 7.4 Hz, 3H); 13 C NMR (100 MHz, CDCl 3) δ , , , , , 52.31, 47.77, 26.80, 23.55, 23.33, 13.75, 10.52, HRMS (ESI) m/z Calculated for C 13H 21N 4O 2 (M+H) + : , found: (4-Cyclohexyl-1-(2-(3,5-dimethylisoxazol-4-yl)ethyl)-1H-1,2,3-triazol-5-yl)methanol was prepared from 3-cyclohexylprop-2-yn-1-ol (34.6 mg, mmol) and 4-(2-azidoethyl)-3,5- dimethylisoxazole (41.6 mg, mmol) using Method B. The product (43.0 mg, 56 % yield) was purified by column chromatography (silica gel, EtOAc) as a brown solid; 1 H NMR (400 MHz, CDCl 3) δ 4.74 (s, 2H), 4.29 (t, J = 6.8 Hz, 2H), 3.13 (br, 1H), 2.89 (t, J = 6.8 Hz, 2H), (m, 1H), 2.13 (s, 3H), 2.03 (s, 3H), (m, 2H), (m, 1H), (m, 4H), (m, 3H); 13 C NMR (100 MHz, CDCl 3) δ , , , , , 56.36, 47.08, 35.10, 31.67, 26.69, 25.48, 23.78, 10.42, HRMS (ESI) m/z Calculated for C 16H 25N 4O 2 (M+H) + : , found: S11

12 (1-(2-(3,5-Dimethylisoxazol-4-yl)ethyl)-4-(4-(trifluoromethoxy)phenyl)-1H-1,2,3-triazol-5- yl)methanol was prepared from 3-(4-(trifluoromethoxy)phenyl)prop-2-yn-1-ol (130.0 mg, mmol) and 4-(2-azidoethyl)-3,5-dimethylisoxazole (100.0 mg, mmol) using Method B. The product (140.0 mg, 61 % yield) was purified by column chromatography (silica gel, 50 % EtOAc in hexane) as a brown solid; 1 H NMR (400 MHz, CDCl 3) δ 7.59 (d, J = 8.2 Hz, 2H), 7.20 (d, J = 8.2 Hz, 2H), 4.68 (br, 1H), 4.61 (s, 2H), 4.46 (t, J = 6.9 Hz, 2H), 2.94 (t, J = 6.9 Hz, 2H), 2.06 (s, 3H), 2.05 (s, 3H); 13 C NMR (100 MHz, CDCl 3) δ , , (q, J = 1.7 Hz), , , , , , (q, J = Hz), , 52.13, 47.99, 23.38, 10.42, HRMS (ESI) m/z Calculated for C 17H 18F 3N 4O 3 (M+H) + : , found: (1-(2-(3,5-Dimethylisoxazol-4-yl)ethyl)-4-(naphthalen-1-yl)-1H-1,2,3-triazol-5-yl)methanol was prepared from 3-(naphthalen-1-yl)prop-2-yn-1-ol (45.5 mg, mmol) and 4-(2-azidoethyl)- 3,5-dimethylisoxazole (41.6 mg, mmol) using Method B. The product (12.0 mg, 14 % yield) was purified by column chromatography (silica gel, 30 % 60 % EtOAc in hexane) as a brown solid; 1 H NMR (400 MHz, CDCl 3) δ 7.92 (t, J = 7.7 Hz, 2H), 7.86 (d, J = 7.8 Hz, 1H), (m, 3H), 7.42 (dd, J = 7.0 Hz, 0.9 Hz, 1H), 4.63 (t, J = 7.0 Hz, 2H), 4.47 (s, 2H), 3.12 (t, J = 7.0 Hz, 2H), 2.23 (s, 3H), 2.22 (s, 3H); 13 C NMR (100 MHz, CDCl 3) δ , , , , , , , , , , , , , , , 52.84, 48.16, 23.63, 10.64, HRMS (ESI) m/z Calculated for C 20H 21N 4O 2 (M+H) + : , found: General Procedure for Synthesis of 1,4-substituted Chloromethyl Triazoles To a solution of 1,4-substituted hydroxymethyl triazole (1.0 eq.) in dry CH 2Cl 2 (1.0 ml) cooled in an ice bath, Et 3N (1.5 eq.) was added, followed by the addition of MsCl (1.5 eq.). The reaction was stirred at 0 C for 0.5 h and then at r.t. for 0.5 h. After that, n Bu 4NCl (1.5 eq.) was added and the reaction was stirred at r.t. for 1 h. The reaction mixture was concentrated in vacuo and the resulting residue was purified with silica gel chromatography to afford the pure 1,4- substituted chloromethyl triazole. S12

13 Methyl 2-(5-(chloromethyl)-4-propyl-1H-1,2,3-triazol-1-yl)acetate (12) was prepared from methyl 2-(5-(hydroxymethyl)-4-propyl-1H-1,2,3-triazol-1-yl)acetate (12.0 mg, mmol). The product (12.6 mg, 97 % yield) was purified by column chromatography (silica gel, CH 2Cl 2, then EtOAc) as a colorless oil; 1 H NMR (400 MHz, CDCl 3) δ 5.24 (s, 2H), 4.61 (d, J = 5.0 Hz, 2H), 3.81 (s, 3H), 2.67 (t, J = 7.5 Hz, 2H), (m, 2H), 0.97 (t, J = 7.4 Hz, 3H); 13 C NMR (100 MHz, CDCl 3) δ , , , 53.10, 49.46, 31.98, 26.92, 22.74, HRMS (ESI) m/z Calculated for C 9H 15ClN 3O 2 (M+H) + : , found: Methyl 2-(5-(chloromethyl)-4-cyclohexyl-1H-1,2,3-triazol-1-yl)acetate (11) was prepared from methyl 2-(4-cyclohexyl-5-(hydroxymethyl)-1H-1,2,3-triazol-1-yl)acetate (10.5 mg, mmol). The product (11.1 mg, 98 % yield) was purified by column chromatography (silica gel, CH 2Cl 2, then EtOAc) as a colorless oil; 1 H NMR (400 MHz, CDCl 3) δ 5.23 (s, 2H), 4.63 (d, J = 5.0 Hz, 2H), 3.81 (s, 3H), 2.67 (tt, J = 12.0 Hz, 3.4 Hz, 1H), (m, 4H), (m, 2H), (m, 4H); 13 C NMR (100 MHz, CDCl 3) δ , , , 53.08, 49.36, 35.23, 32.62, 32.04, 26.47, HRMS (ESI) m/z Calculated for C 12H 19ClN 3O 2 (M+H) + : , found: Methyl 2-(5-(chloromethyl)-4-(4-(trifluoromethoxy)phenyl)-1H-1,2,3-triazol-1-yl)acetate (14) was prepared from methyl 2-(5-(hydroxymethyl)-4-(4-(trifluoromethoxy)phenyl)-1H-1,2,3- triazol-1-yl)acetate (13.8 mg, mmol). The product (12.9 mg, 88 % yield) was purified by column chromatography (silica gel, CH 2Cl 2, then EtOAc) as a white solid; 1 H NMR (400 MHz, CDCl 3) δ 7.77 (d, J = 8.7 Hz, 2H), 7.37 (d, J = 8.2 Hz, 2H), 5.34 (s, 2H), 4.76 (s, 2H), 3.86 (s, 3H); 13 C NMR (100 MHz, CDCl 3) δ , (q, J = 2.0 Hz), , , , , , (q, J = Hz), 53.30, 49.53, HRMS (ESI) m/z Calculated for C 13H 12ClF 3N 3O 3 (M+H) + : , found: S13

14 Methyl 2-(5-(chloromethyl)-4-(naphthalen-1-yl)-1H-1,2,3-triazol-1-yl)acetate (13) was prepared from methyl 2-(5-(hydroxymethyl)-4-(naphthalen-1-yl)-1H-1,2,3-triazol-1-yl)acetate (11.3 mg, mmol). The product (11.9 mg, 99 % yield) was purified by column chromatography (silica gel, CH 2Cl 2, then EtOAc) as a brown solid; 1 H NMR (400 MHz, CDCl 3) δ (m, 3H), (m, 2H), (m, 2H), 5.45 (s, 2H), 4.63 (s, 2H), 3.89 (s, 3H); 13 C NMR (100 MHz, CDCl 3) δ , , , , , , , , , , , , , 53.26, 49.84, HRMS (ESI) m/z Calculated for C 16H 15ClN 3O 2 (M+H) + : , found: Methyl 2-(5-(chloromethyl)-4-(pyridin-3-yl)-1H-1,2,3-triazol-1-yl)acetate (10) was prepared from methyl 2-(5-(hydroxymethyl)-4-(pyridin-3-yl)-1H-1,2,3-triazol-1-yl)acetate (9.2 mg, mmol). The product (9.8 mg, 99 % yield) was purified by column chromatography (silica gel, CH 2Cl 2, then EtOAc) as a slightly yellow solid; 1 H NMR (400 MHz, CDCl 3) δ 8.95 (br, 1H), 8.69 (d, J = 4.2 Hz, 1H), 8.09 (d, J = 7.9 Hz, 1H), 7.45 (dd, J = 7.8 Hz, 4.8 Hz, 1H), 5.35 (s, 2H), 4.77 (s, 2H), 3.86 (s, 3H); 13 C NMR (100 MHz, CDCl 3) δ , , , , , , , , 53.35, 49.59, HRMS (ESI) m/z Calculated for C 11H 12ClN 4O 2 (M+H) + : , found: (2-(5-(Chloromethyl)-4-propyl-1H-1,2,3-triazol-1-yl)ethyl)morpholine (8) was prepared from (1-(2-morpholinoethyl)-4-propyl-1H-1,2,3-triazol-5-yl)methanol (7.1 mg, mmol). The product (6.5 mg, 85 % yield) was purified by column chromatography (silica gel, CH 2Cl 2, then 5 % MeOH in EtOAc) as a colorless oil; 1 H NMR (400 MHz, CDCl 3) δ 4.73 (s, 2H), 4.49 (br, 2H), 3.70 (br, 4H), 2.91 (br, 2H), 2.67 (t, J = 7.6 Hz, 2H), 2.53 (br, 4H), (m, 2H), 0.97 (t, J = 7.4 Hz, 3H); 13 C NMR (100 MHz, CDCl 3) δ , , 66.77, 58.23, 53.82, 46.07, 32.13, 26.98, 22.81, HRMS (ESI) m/z Calculated for C 12H 22ClN 4O (M+H) + : , found: S14

15 4-(2-(5-(Chloromethyl)-4-cyclohexyl-1H-1,2,3-triazol-1-yl)ethyl)morpholine (9) was prepared from (4-cyclohexyl-1-(2-morpholinoethyl)-1H-1,2,3-triazol-5-yl)methanol (13.6 mg, mmol). The product (8.9 mg, 62 % yield) was purified by column chromatography (silica gel, CH 2Cl 2, then 5 % MeOH in EtOAc) as a colorless oil; 1 H NMR (400 MHz, CDCl 3) δ 4.78 (s, 2H), 4.36 (t, J = 6.7 Hz, 2H), 3.69 (t, J = 4.5 Hz, 4H), 2.85 (t, J = 6.7 Hz, 2H), 2.77 (tt, J = 12.2 Hz, 3.2 Hz, 1H), 2.52 (t, J = 4.3 Hz, 4H), (m, 4H), (m, 3H), (m, 3H); 13 C NMR (100 MHz, CDCl 3) δ , , 66.80, 58.46, 53.88, 45.69, 37.35, 34.96, 31.51, 26.63, HRMS (ESI) m/z Calculated for C 15H 26ClN 4O (M+H) + : , found: (2-(5-(Chloromethyl)-4-(4-(trifluoromethoxy)phenyl)-1H-1,2,3-triazol-1-yl)ethyl)morpholine (15) was prepared from (1-(2-morpholinoethyl)-4-(4-(trifluoromethoxy)phenyl)-1H-1,2,3-triazol- 5-yl)methanol (26.7 mg, mmol). The product (17.9 mg, 64 % yield) was purified by column chromatography (silica gel, CH 2Cl 2, then EtOAc) as a yellow oil; 1 H NMR (400 MHz, CDCl 3) δ 7.78 (d, J = 8.8 Hz, 2H), 7.36 (d, J = 8.0 Hz, 2H), 4.89 (s, 2H), 4.57 (t, J = 6.2 Hz, 2H), 3.69 (t, J = 4.5 Hz, 4H), 2.97 (t, J = 6.1 Hz, 2H), 2.56 (t, J = 4.3 Hz, 4H); 13 C NMR (100 MHz, CDCl 3) δ (q, J = 1.7 Hz), , , , , , (q, J = Hz), 66.82, 58.26, 53.85, 46.36, HRMS (ESI) m/z Calculated for C 16H 19ClF 3N 4O 2 (M+H) + : , found: (2-(5-(Chloromethyl)-4-(naphthalen-1-yl)-1H-1,2,3-triazol-1-yl)ethyl)morpholine (2) was prepared from (1-(2-morpholinoethyl)-4-(naphthalen-1-yl)-1H-1,2,3-triazol-5-yl)methanol (10.8 mg, mmol). The product (9.3 mg, 82 % yield) was purified by column chromatography (silica gel, CH 2Cl 2, then EtOAc) as a yellow solid; 1 H NMR (300 MHz, CDCl 3) δ (m, 3H), (m, 4H), 4.75 (s, 2H), 4.69 (t, J = 6.3 Hz, 2H), 3.73 (t, J = 4.5 Hz, 4H), 3.08 (t, J = 6.2 Hz, 2H), 2.63(t, J = 4.3 Hz, 4H); 13 C NMR (100 MHz, CDCl 3) δ , , , , , , , , , , , , 66.81, 58.31, 53.92, 46.47, HRMS (ESI) m/z Calculated for C 19H 22ClN 4O (M+H) + : , found: S15

16 1-(4-Bromobenzyl)-5-(chloromethyl)-4-propyl-1H-1,2,3-triazole (3) was prepared from (1-(4- bromobenzyl)-4-propyl-1h-1,2,3-triazol-5-yl)methanol (9.3 mg, mmol). The product (7.9 mg, 80 % yield) was purified by column chromatography (silica gel, CH 2Cl 2, then EtOAc) as a slightly yellow solid; 1 H NMR (400 MHz, CDCl 3) δ 7.50 (d, J = 8.4 Hz, 2H), 7.13 (d, J = 8.4 Hz, 2H), 5.58 (s, 2H), 4.38 (s, 2H), 2.65 (t, J = 7.5 Hz, 2H), (m, 2H), 0.96 (t, J = 7.4 Hz, 3H); 13 C NMR (100 MHz, CDCl 3) δ , , , , , , 51.84, 31.78, 26.91, 22.76, HRMS (ESI) m/z Calculated for C 13H 16BrClN 3 (M+H) + : , found: (1-(4-Bromobenzyl)-5-(chloromethyl)-1H-1,2,3-triazol-4-yl)pyridine (4) was prepared from (1-(4-bromobenzyl)-4-(pyridin-3-yl)-1H-1,2,3-triazol-5-yl)methanol (9.4 mg, mmol). The product (9.2 mg, 93 % yield) was purified by column chromatography (silica gel, CH 2Cl 2, then EtOAc) as a slightly yellow solid; 1 H NMR (400 MHz, CDCl 3) δ 8.94 (br, 1H), 8.68 (d, J = 4.1 Hz, 1H), 8.09 (d, J = 7.9 Hz, 1H), 7.53 (d, J = 8.3 Hz, 2H), 7.45 (dd, J = 7.8 Hz, 4.8 Hz, 1H), 7.21 (d, J = 8.3 Hz, 2H), 5.68 (s, 2H), 4.56 (s, 2H); 13 C NMR (100 MHz, CDCl 3) δ , , , , , , , , , , , 52.13, HRMS (ESI) m/z Calculated for C 15H 13BrClN 4 (M+H) + : , found: (Benzo[d][1,3]dioxol-5-yl)-2-(5-(chloromethyl)-4-propyl-1H-1,2,3-triazol-1-yl)ethan-1-one (5) was prepared from 1-(benzo[d][1,3]dioxol-5-yl)-2-(5-(hydroxymethyl)-4-propyl-1H-1,2,3- triazol-1-yl)ethan-1-one (5.4 mg, mmol). The product (5.0 mg, 87 % yield) was purified by column chromatography (silica gel, CH 2Cl 2, then EtOAc) as an orange solid; 1 H NMR (400 MHz, CDCl 3) δ 7.65 (dd, J = 8.2 Hz, 1.6 Hz, 1H), 7.47 (d, J = 1.5 Hz, 1H), 6.95 (d, J = 8.2 Hz, 1H), 6.11 (s, 2H), 5.87 (s, 2H), 4.59 (s, 2H), 2.72 (t, J = 7.5 Hz, 2H), (m, 2H), 1.00 (t, J = 7.4 Hz, 3H); 13 C NMR (100 MHz, CDCl 3) δ , , , , , , , , , , 54.27, 32.47, 27.00, 22.80, HRMS (ESI) m/z Calculated for C 15H 17ClN 3O 3 (M+H) + : , found: S16

17 1-(Benzo[d][1,3]dioxol-5-yl)-2-(5-(chloromethyl)-4-cyclohexyl-1H-1,2,3-triazol-1-yl)ethan-1- one (6) was prepared from 1-(benzo[d][1,3]dioxol-5-yl)-2-(4-cyclohexyl-5-(hydroxymethyl)-1H- 1,2,3-triazol-1-yl)ethan-1-one (8.2 mg, mmol). The product (7.4 mg, 86 % yield) was purified by column chromatography (silica gel, 50 % EtOAc in hexane) as a white solid; 1 H NMR (400 MHz, CDCl 3) δ 7.65 (dd, J = 8.2 Hz, 1.4 Hz, 1H), 7.46 (d, J = 1.4 Hz, 1H), 6.94 (d, J = 8.2 Hz, 1H), 6.11 (s, 2H), 5.85 (s, 2H), 4.61 (s, 2H), 2.71 (t, J = 11.9 Hz, 1H), (m, 4H), (m, 3H), (m, 3H); 13 C NMR (100 MHz, CDCl 3) δ , , , , , , , , , , 54.16, 35.28, 32.67, 32.53, 26.52, HRMS (ESI) m/z Calculated for C 18H 21ClN 3O 3 (M+H) + : , found: (Benzo[d][1,3]dioxol-5-yl)-2-(5-(chloromethyl)-4-(pyridin-3-yl)-1H-1,2,3-triazol-1-yl)ethan-1- one (7) was prepared from 1-(benzo[d][1,3]dioxol-5-yl)-2-(5-(hydroxymethyl)-4-(pyridin-3-yl)- 1H-1,2,3-triazol-1-yl)ethan-1-one (7.1 mg, mmol). The product (4.6 mg, 61 % yield) was purified by column chromatography (silica gel, CH 2Cl 2, then EtOAc) as a white solid; 1 H NMR (400 MHz, CDCl 3) δ 9.05 (br, 1H), 8.77 (br, 1H), 8.15 (d, J = 7.7 Hz, 1H), 7.69 (d, J = 7.5 Hz, 1H), (m, 2H), 6.98 (d, J = 8.1 Hz, 1H), 6.13 (s, 2H), 5.97 (s, 2H), 4.74 (s, 2H); 13 C NMR (100 MHz, CDCl 3) δ , , , , , , , , , , , , , , , 54.36, HRMS (ESI) m/z Calculated for C 17H 14ClN 4O 3 (M+H) + : , found: (2-(5-(Chloromethyl)-4-propyl-1H-1,2,3-triazol-1-yl)ethyl)-3,5-dimethylisoxazole (18) was prepared from (1-(2-(3,5-dimethylisoxazol-4-yl)ethyl)-4-propyl-1H-1,2,3-triazol-5-yl)methanol (15.5 mg, mmol). The product (13.3 mg, 80 % yield) was purified by column chromatography (silica gel, CH 2Cl 2, then EtOAc) as a yellow oil; 1 H NMR (400 MHz, CDCl 3) δ 4.39 (t, J = 7.0 Hz, 2H), 4.34 (s, 2H), 3.01 (t, J = 7.0 Hz, 2H), 2.64 (t, J = 7.5 Hz, 2H), 2.17 (s, 3H), 2.13 (s, 3H), (m, 2H), 0.96 (t, J = 7.4 Hz, 3H); 13 C NMR (100 MHz, CDCl 3) δ , , , , , 47.80, 31.55, 26.88, 23.42, 22.90, 13.75, 10.59, HRMS (ESI) m/z Calculated for C 13H 20ClN 4O (M+H) + : , found: S17

18 4-(2-(5-(Chloromethyl)-4-cyclohexyl-1H-1,2,3-triazol-1-yl)ethyl)-3,5-dimethylisoxazole (17) was prepared from (4-cyclohexyl-1-(2-(3,5-dimethylisoxazol-4-yl)ethyl)-1H-1,2,3-triazol-5- yl)methanol (14.7 mg, mmol). The product (15.4 mg, 99 % yield) was purified by column chromatography (silica gel, CH 2Cl 2, then EtOAc) as a slightly yellow oil; 1 H NMR (400 MHz, CDCl 3) δ 4.74 (s, 2H), 4.31 (t, J = 6.7 Hz, 2H), 2.93 (t, J = 6.7 Hz, 2H), (m, 1H), 2.15 (s, 3H), 2.03 (s, 3H), (m, 2H), (m, 1H), (m, 4H), (m, 3H); 13 C NMR (100 MHz, CDCl 3) δ , , , , , 47.17, 37.07, 35.25, 31.35, 26.66, 25.50, 23.78, 10.38, HRMS (ESI) m/z Calculated for C 16H 24ClN 4O (M+H) + : , found: (2-(5-(Chloromethyl)-4-(4-(trifluoromethoxy)phenyl)-1H-1,2,3-triazol-1-yl)ethyl)-3,5- dimethylisoxazole (19) was prepared from (1-(2-(3,5-dimethylisoxazol-4-yl)ethyl)-4-(4- (trifluoromethoxy)phenyl)-1h-1,2,3-triazol-5-yl)methanol (13.5 mg, mmol). The product (13.0 mg, 92 % yield) was purified by column chromatography (silica gel, CH 2Cl 2, then EtOAc) as a slightly yellow solid; 1 H NMR (400 MHz, CDCl 3) δ 7.73 (d, J = 8.7 Hz, 2H), 7.36 (d, J = 8.2 Hz, 2H), 4.51 (s, 2H), 4.48 (t, J = 7.1 Hz, 2H), 3.10 (t, J = 7.1 Hz, 2H), 2.22 (s, 3H), 2.18 (s, 3H); 13 C NMR (100 MHz, CDCl 3) δ , , (q, J = 1.7 Hz), , , , , (q, J = Hz), , 48.00, 32.20, 23.42, 10.68, HRMS (ESI) m/z Calculated for C 17H 17ClF 3N 4O 2 (M+H) + : , found: (2-(5-(Chloromethyl)-4-(naphthalen-1-yl)-1H-1,2,3-triazol-1-yl)ethyl)-3,5-dimethylisoxazole (16) was prepared from (1-(2-(3,5-dimethylisoxazol-4-yl)ethyl)-4-(naphthalen-1-yl)-1H-1,2,3- triazol-5-yl)methanol (8.3 mg, mmol). The product (8.2 mg, 94 % yield) was purified by column chromatography (silica gel, CH 2Cl 2, then EtOAc) as a slightly brown solid; 1 H NMR (400 MHz, CDCl 3) δ (m, 3H), (m, 4H), 4.59 (t, J = 7.1 Hz, 2H), 4.34 (s, 2H), 3.17 (t, J = 7.1 Hz, 2H), 2.26 (s, 3H), 2.25 (s, 3H); 13 C NMR (100 MHz, CDCl 3) δ , , , , , , , , , , , , , , , 48.25, 32.36, 23.49, 10.76, HRMS (ESI) m/z Calculated for C 20H 20ClN 4O (M+H) + : , found: S18

19 Synthesis of AA-CW236 Derivatives Under N 2 atmosphere, to a stirred solution of 4-methoxyiodobenzene (702.0 mg, mmol, 1.0 eq.), Pd(PPh 3) 2Cl 2 (21.1 mg, 0.03 mmol, 1.0 mol %) and CuI (11.4 mg, mmol, 2.0 mol %) in Et 3N/CH 3CN (1:1, 4.0 ml) as well as propargyl alcohol (210 µl, mmol, 1.2 eq.) were added and the resulting reaction was stirred at r.t. After stirring for 3 h, the reaction mixture was poured into water and extracted with CH 2Cl 2, then the combined organic phases were concentrated in vacuo and purified by column chromatography (silica gel, CH 2Cl 2) to afford the previously reported [5] product AA-CW659 (483.0 mg, 99 % yield) as a slightly yellow solid. 1 H NMR (400 MHz, CDCl 3) δ 7.37 (d, J = 8.8 Hz, 2H), 6.83 (d, J = 8.8 Hz, 2H), 4.48 (s, 2H), 3.80 (s, 3H). A screw-cap tube was charged with Cp*Ru(PPh 3) 2Cl (8.0 mg, mmol, 2.0 mol %), evacuated and filled with N 2 (3 times). Then, dry PhMe (2.0 ml), 3-(4-methoxyphenyl)prop-2- yn-1-ol (81.1 mg, mmol, 1.0 eq.) and 4-(2-azidoethyl)-3,5-dimethylisoxazole (83.1 mg, mmol, 1.0 eq.) were added in sequence. The reaction tube was screw-capped and left overnight under stirring at 80 C in an oil bath. After that, the reaction mixture was concentrated in vacuo and purified by column chromatography (silica gel, 80 % - 90 % EtOAc in hexane) to give the product AA-CW661A (97.4 mg, 59 % yield) as a slightly brown solid. 1 H NMR (400 MHz, CDCl 3) δ 7.45 (d, J = 8.7 Hz, 2H), 6.88 (d, J = 8.7 Hz, 2H), 4.58 (s, 2H), 4.42 (t, J = 7.1 Hz, 2H), 3.78 (s, 3H), 2.91 (t, J = 7.1 Hz, 2H), 2.07 (s, 3H), 2.06 (s, 3H); 13 C NMR (100 MHz, CDCl 3) δ , , , , , , , , , 55.31, 52.35, 47.93, 23.45, 10.50, HRMS (ESI) m/z Calculated for C 17H 21N 4O 3 (M+H) + : , found: To a stirred solution of (1-(2-(3,5-dimethylisoxazol-4-yl)ethyl)-4-(4-methoxyphenyl)-1H-1,2,3- triazol-5-yl)methanol (39.4 mg, mmol, 1.0 eq.) in dry CH 2Cl 2 (2.0 ml) cooled in an ice bath, Et 3N (25.1 µl, mmol, 1.5 eq.) was added, followed by the addition of MsCl (14.2 µl, mmol, 1.5 eq.). The reaction was stirred at 0 C for 0.5 h and then at r.t. for 0.5 h. After S19

20 that, n Bu 4NCl (50.0 mg, mmol, 1.5 eq.) was added and the reaction was stirred at r.t. for 1 h. The reaction mixture was concentrated in vacuo and purified by column chromatography (silica gel, CH 2Cl 2, then EtOAc) to give the product AA-CW236-2 (41.3 mg, 99 % yield) as a white solid. 1 H NMR (400 MHz, CDCl 3) δ 7.58 (d, J = 8.7 Hz, 2H), 6.99 (d, J = 8.7 Hz, 2H), 4.49 (s, 2H), 4.42 (t, J = 7.1 Hz, 2H), 3.83 (s, 3H), 3.04 (t, J = 7.1 Hz, 2H), 2.18 (s, 3H), 2.14 (s, 3H); 13 C NMR (100 MHz, CDCl 3) δ , , , , , , , , , 55.37, 47.88, 32.67, 23.40, 10.65, HRMS (ESI) m/z Calculated for C 17H 20ClN 4O 2 (M+H) + : , found: A screw-cap tube was charged with Cp*Ru(PPh 3) 2Cl (8.0 mg, mmol, 2.0 mol %), evacuated and filled with N 2 (3 times). Then, dry PhMe (2.0 ml), 3-phenylprop-2-yn-1-ol (66.1 mg, mmol, 1.0 eq.) and 4-(2-azidoethyl)-3,5-dimethylisoxazole (83.1 mg, mmol, 1.0 eq.) were added in sequence. The reaction tube was screw-capped and left overnight under stirring at 80 C in an oil bath. After that, the reaction mixture was concentrated in vacuo and purified by column chromatography (silica gel, 60 % 70 % EtOAc in hexane) to give the product AA-CW666A (59.3 mg, 40 % yield) as a white solid. 1 H NMR (400 MHz, DMSO-d 6) δ 7.71 (d, J = 7.9 Hz, 2H), 7.49 (t, J = 7.5 Hz, 2H), 7.40 (t, J = 7.4 Hz, 1H), 5.69 (t, J = 5.0 Hz, 1H), 4.59 (d, J = 4.9 Hz, 2H), 4.54 (t, J = 6.8 Hz, 2H), 2.95 (t, J = 6.8 Hz, 2H), 2.09 (s, 6H); 13 C NMR (100 MHz, DMSO-d 6) δ , , , , , , , , , 51.45, 47.89, 23.15, 10.52, HRMS (ESI) m/z Calculated for C 16H 19N 4O 2 (M+H) + : , found: To a stirred solution of 1-(2-(3,5-dimethylisoxazol-4-yl)ethyl)-4-phenyl-1H-1,2,3-triazol-5- yl)methanol (12.4 mg, mmol, 1.0 eq.) in dry CH 2Cl 2 (2.0 ml) cooled in an ice bath, Et 3N (8.7 µl, mmol, 1.5 eq.) was added, followed by the addition of MsCl (4.9 µl, mmol, 1.5 eq.). The reaction was stirred at 0 C for 0.5 h and then at r.t. for 0.5 h. After that, n Bu 4NCl (17.3 mg, mmol, 1.5 eq.) was added and the reaction was stirred at r.t. for 1 h. The reaction mixture was concentrated in vacuo and purified by column chromatography (silica gel, CH 2Cl 2, then EtOAc) to give the product AA-CW236-3 (12.2 mg, 93 % yield) as a white solid. 1 H NMR (400 MHz, CDCl 3) δ 7.68 (d, J = 7.1 Hz, 2H), 7.49 (t, J = 7.4 Hz, 2H), 7.43 (t, J = 7.3 Hz, 1H), 4.51 (s, 2H), 4.47 (t, J = 7.1 Hz, 2H), 3.08 (t, J = 7.1 Hz, 2H), 2.21 (s, 3H), 2.17 (s, 3H); 13 C NMR (100 MHz, CDCl 3) δ , , , , , , , , , 47.94, 32.48, 23.46, 10.67, HRMS (ESI) m/z Calculated for C 16H 18ClN 4O (M+H) + : , found: S20

21 Synthesis of the Clickable Probe 20 (AA-CW538) Under N 2 atmosphere, to a stirred solution of 4-iodophenol (825.0 mg, mmol, 1.0 eq.), Pd(PPh 3) 2Cl 2 (26.3 mg, mmol, 1.0 mol %) and CuI (14.3 mg, mmol, 2.0 mol %) in Et 3N/Dioxane (1:1, 3.0 ml) as well as propargyl alcohol (262.0 µl, mmol, 1.2 eq.) were added and the resulting reaction was stirred at r.t. After stirring for 2 h, the reaction mixture was poured into water and extracted with CH 2Cl 2, then the combined organic phases were concentrated in vacuo and purified by column chromatography (silica gel, 40 % EtOAc in hexane) to afford the product AA-CW514 (402.0 mg, 72 % yield) as a white solid. 1 H NMR (400 MHz, MeOD) δ 7.25 (d, J = 8.6 Hz, 2H), 6.74 (d, J = 8.6 Hz, 2H), 4.37 (s, 2H); 13 C NMR (100 MHz, MeOD) δ , , , , 85.21, 84.62, HRMS (ESI) m/z Calculated for C 9H 9O 2 (M+H) + : , found: To a stirred solution of 4-(3-hydroxyprop-1-yn-1-yl)phenol (124.5 mg, mmol, 1.0 eq.) in isopropanol (5.0 ml), a 2 M solution of NaOH (84.0 mg, mmol, 2.5 eq.) in water (1.05 ml) was added. After vigorously stirring for 5 min., Ac 2O (198.5 µl, mmol, 2.5 eq.) was added and the resulting reaction mixture was stirred at r.t. overnight. After that, the reaction mixture was poured into water and extracted with CH 2Cl 2, then the combined organic phases were concentrated in vacuo and purified by column chromatography (silica gel, 20 % EtOAc in hexane) to afford the product AA-CW521 (160.0 mg, 82 % yield) as a slightly yellow oil. 1 H NMR (400 MHz, CDCl 3) δ 7.46 (d, J = 8.5 Hz, 2H), 7.05 (d, J = 8.5 Hz, 2H), 4.88 (s, 2H), 2.29 (s, 3H), 2.12 (s, 3H); 13 C NMR (100 MHz, CDCl 3) δ , , , , , , 85.62, 83.06, 52.76, 21.12, HRMS (ESI) m/z Calculated for C 13H 13O 4 (M+H) + : , found: A screw-cap tube was charged with Cp*Ru(PPh 3) 2Cl (2.4 mg, mmol, 2.0 mol %), evacuated and filled with N 2 (3 times). Then dry PhMe (1.5 ml), 3-(4-acetoxyphenyl)prop-2- yn-1-yl acetate (41.8 mg, mmol, 1.2 eq.) and 4-(2-azidoethyl)-3,5-dimethylisoxazole (25.0 mg, mmol, 1.0 eq.) were added in sequence. The reaction tube was screw-capped and left overnight under stirring at 80 C in an oil bath. After that, the reaction mixture was concentrated in vacuo and purified by column chromatography (silica gel, 60 % 70 % EtOAc in hexane) to give the product AA-CW532A (36.7 mg, 61 % yield) as a colorless oil. 1 H NMR (400 MHz, CDCl 3) δ 7.73 (d, J = 8.7 Hz, 2H), 7.18 (d, J = 8.7 Hz, 2H), 4.95 (s, 2H), 4.52 (t, J = 6.7 Hz, 2H), 2.98 (t, J = 6.7 Hz, 2H), 2.30 (s, 3H), 2.12 (s, 3H), 2.06 (s, 3H), 2.04 (s, 3H); S21

22 13 C NMR (100 MHz, CDCl 3) δ , , , , , , , , , , , 52.68, 48.02, 23.64, 21.15, 20.66, 10.41, HRMS (ESI) m/z Calculated for C 20H 23N 4O 5 (M+H) + : , found: To a stirred solution of 4-(5-(acetoxymethyl)-1-(2-(3,5-dimethylisoxazol-4-yl)ethyl)-1H-1,2,3- triazol-4-yl)phenyl acetate (36.7 mg, mmol, 1.0 eq.) in CH 2Cl 2 (2.0 ml), pyrrolidine (76.9 µl, mmol, 10.0 eq.) was added and the resulting reaction mixture was stirred at r.t. for 20 min. The reaction mixture was quenched with 1 M HCl and extracted with CH 2Cl 2, the combined organic phases were concentrated in vacuo and purified by column chromatography (silica gel, CH 2Cl 2, then EtOAc) to give the product AA-CW534 (32.5 mg, 99 % yield) as a white solid. 1 H NMR (400 MHz, CDCl 3) δ 7.50 (d, J = 8.4 Hz, 2H), 6.94 (d, J = 8.5 Hz, 2H), 4.95 (s, 2H), 4.53 (t, J = 6.6 Hz, 2H), 2.99 (t, J = 6.6 Hz, 2H), 2.13 (s, 3H), 2.08 (s, 6H); 13 C NMR (100 MHz, CDCl 3) δ , , , , , , , , , , 52.88, 48.10, 23.59, 20.71, 10.46, HRMS (ESI) m/z Calculated for C 18H 21N 4O 4 (M+H) + : , found: To a stirred solution of (1-(2-(3,5-dimethylisoxazol-4-yl)ethyl)-4-(4-hydroxyphenyl)-1H-1,2,3- triazol-5-yl)methyl acetate (15.0 mg, mmol, 1.0 eq.) in dry DMF (0.6 ml), K 2CO 3 (7.0 mg, mmol, 1.2 eq.) was added, followed by the addition of propargyl bromide (4.4 µl, 80 wt. % in PhMe, mmol, 1.2 eq.). The resulting reaction mixture was stirred at r.t. for 6 h. After that, the reaction mixture was diluted with EtOAc and washed with water. The organic phase was concentrated in vacuo and purified by column chromatography (silica gel, 50 % EtOAc in hexane) to give the product AA-CW536 (15.1 mg, 91 % yield) as a colorless oil. 1 H NMR (400 MHz, CDCl 3) δ 7.66 (d, J = 8.4 Hz, 2H), 7.07 (d, J = 8.3 Hz, 2H), 4.94 (s, 2H), 4.74 (d, J = 1.3 Hz, 2H), 4.53 (t, J = 6.6 Hz, 2H), 3.00 (t, J = 6.6 Hz, 2H), 2.55 (s, 1H), 2.14 (s, 3H), 2.09 (s, 3H), 2.06 (s, 3H); 13 C NMR (100 MHz, CDCl 3) δ , , , , , , , , , , 78.30, 75.82, 55.85, 52.81, 47.98, 23.69, 20.71, 10.43, HRMS (ESI) m/z Calculated for C 21H 23N 4O 4 (M+H) + : , found: S22

23 To a stirred solution of (1-(2-(3,5-dimethylisoxazol-4-yl)ethyl)-4-(4-(prop-2-yn-1-yloxy)phenyl)- 1H-1,2,3-triazol-5-yl)methyl acetate (15.0 mg, mmol, 1.0 eq.) in MeOH (0.5 ml), K 2CO 3 (10.5 mg, mmol, 2.0 eq.) was added and the resulting reaction was stirred at r.t. After stirring for 0.5 h, the reaction mixture was diluted with water and extracted with CH 2Cl 2. The combined organic phases were concentrated in vacuo and dried over MgSO 4 to give the product AA-CW537 as a white solid. 1 H NMR (400 MHz, CDCl 3) δ 7.54 (d, J = 8.5 Hz, 2H), 7.04 (d, J = 8.5 Hz, 2H), 4.72 (d, J = 2.2 Hz, 2H), 4.61 (s, 2H), 4.49 (t, J = 6.9 Hz, 2H), 2.99 (t, J = 6.9 Hz, 2H), 2.54 (t, J = 2.1 Hz, 1H), 2.15 (s, 3H), 2.11 (s, 3H). HRMS (ESI) m/z Calculated for C 19H 21N 4O 3 (M+H) + : , found: Then to a stirred solution of (1-(2-(3,5- dimethylisoxazol-4-yl)ethyl)-4-(4-(prop-2-yn-1-yloxy)phenyl)-1h-1,2,3-triazol-5-yl)methanol (AA-CW537) in dry CH 2Cl 2 (2.0 ml) cooled in an ice bath, Et 3N (7.9 µl, mmol, 1.5 eq.) was added, followed by the addition of MsCl (4.4 µl, mmol, 1.5 eq.). The reaction was stirred at 0 C for 0.5 h and then at r.t. for 0.5 h. After that n Bu 4NCl (15.8 mg, mmol, 1.5 eq.) was added and the reaction was stirred at r.t. for 1 h. The reaction mixture was concentrated in vacuo and purified by column chromatography (silica gel, CH 2Cl 2, then EtOAc) to give the product 20 (11.6 mg, 82 % yield for two steps) as a white solid. 1 H NMR (400 MHz, CDCl 3) δ 7.62 (d, J = 8.8 Hz, 2H), 7.10 (d, J = 8.8 Hz, 2H), 4.75 (d, J = 2.4 Hz, 2H), 4.49 (s, 2H), 4.45 (t, J = 7.1 Hz, 2H), 3.07 (t, J = 7.1 Hz, 2H), 2.55 (t, J = 2.4 Hz, 1H), 2.21 (s, 3H), 2.16 (s, 3H); 13 C NMR (100 MHz, CDCl 3) δ , , , , , , , , , 78.24, 75.87, 55.87, 47.93, 32.59, 23.45, 10.67, HRMS (ESI) m/z Calculated for C 19H 20ClN 4O 2 (M+H) + : , found: Cell culture and preparation of lysates MCF7, MDA-MB-231, HeLa, Caco-2 and A431 cells were maintained in DMEM media. HCC 1187 and RPMI 8402 cells were cultured in RPMI-1640 media. All media were supplemented with 10% fetal calf serum (FCS), non-essential amino acids and penicillin streptomycin. Cells were grown at 37 C under 5% CO 2 atmosphere. Adherent cells were allowed to grow to confluence and were harvested by scraping, centrifuged at x g for five min. at 4 C and resuspended in PBS. Cells in suspension were centrifuged at x g for five min. at 4 C, washed one time with PBS and resuspended in PBS after centrifugation. Cells were lysed by sonication to form cell lysates and protein concentration was determined using the Bradford assay. Preparation of proteomes for SDS-PAGE experiments Lysate (2 mg/ml, 50 µl) was treated with indicated compound (1 µl of 50x stock in DMSO) or DMSO for one hour at r.t. Sample was then treated with either 10 µm AA-CW159A or 30 µm S23

24 AA-CW538 (1 µl of 50x stock in DMSO) for one hour at r.t. Click chemistry was initiated by the addition of TAMRA alkyne (Jena Bioscience, 10 µm, 25x stock in DMSO) or TAMRA azide (Sigma-Aldrich, 50 µm, 25x stock in DMSO), tris(2-carboxyethyl)phosphine hydrochloride (TCEP, Alfa Aesar, 1 mm, fresh 50x stock in water), tris[(1-benzyl-1h-1,2,3-triazol-4- yl)methyl]amine (TBTA, Sigma-Aldrich, 100 µm, 16x stock in DMSO:tButanol 1:4), and copper(ii) sulfate (1 mm, 50x stock in water) to the lysate and incubated in the dark for one hour at r.t. SDS-PAGE reducing loading buffer (4x) was added and proteins were separated using a 10-12% SDS-PAGE gel. Gels were visualized at 625 nm using a Hitachi FMBIO II Multi-View fluorescence scanner, then stained using Coomassie. Images were quantified with ImageJ. Cloning and site-directed mutagenesis The hmgmt-wt gene was amplified from a cdna library derived from HeLa cells and flanked with Attb-sites using the following primer: forward 5 -GGG GAC AAG TTT GTA CAA AAA AGC AGG CTC CAT GGA CAA GGA TTG TGA AAT G-3 and reverse 5 -GGG GAC CAC TTT GTA CAA GAA AGC TGG GTT CAG TTT CGG CCA GCA G-3. The R reaction was cleaned up and the R product recombined into the pdonr 221. This pentr was used for R based site directed mutagenesis to generate the mutated pentr vector hmgmt-c145a-pentr (forward primer: 5 CTC ATC CCG GCC CAC AGA GTG 3 ; reverse primer: 5 CAC TCT GTG GGC CGG GAT GAG 3 ). Afterwards the R reaction was incubated with DpnI (New England BioLabs Inc, 37 C, 12 hours) to degrade hmgmt-wt-pentr. The two entry vectors were used for transferring the hmgmt-wt and hmgmt-c145a genes into the pcdna 6.2 N- EmGFP-DEST vector via the LR reaction to merge the emgfp gene to the 5 of each hmgmt gene. The pentr and pexp plasmids were propagated via transformation into E. coli DH5α strain using heat shock. Transfection and Western blot HeLa or MCF7 cells were seeded in a Petri dish, left to attach and grow to 80% confluence. Cells were transfected with either GFP-hMGMT-WT, GFP-hMGMT-C145A or GFP plasmid using Lipofectamine 2000 (Invitrogen). After 24 hours, lysate was prepared and treated as described above. Click chemistry, reducing SDS-PAGE and visualization were performed as described above. Proteins were transferred to a nitrocellulose membrane using a Trans-Blot Turbo transfer system (Bio-Rad). Membrane was blocked with 5% milk in Tris-Buffered Saline with 0.1% Tween (TBST) for 30 min. Mouse monoclonal antibody against GFP (Roche, 1/1000 in 5% milk in TBST) was added and incubated overnight at 4 C. Membrane was washed three times for five min. with TBST and incubated with anti-mouse HRP antibody (GE Healthcare, 1/2000 in 5% milk in TBST) for one hour at r.t. Membrane was washed three times for five min. with TBST and visualized with a Fusion Solo (Vilber Lourmat) after ECL (Witec AG). Gel-based MGMT kinetics Recombinant human MGMT (Cayman Chemical, 20 ng in PBS, 25 µl) was treated with indicated concentrations of coumpounds (0.5 µl of 50x stock in DMSO) for 10, 20, 30, 40, 50 or 60 min. Samples were then treated with 10 µm AA-CW159A (0.5 µl of 50x stock in DMSO) for one hour. Click chemistry, reducing SDS-PAGE and visualization were performed as described above. Images were quantified with ImageJ and the kinectics values were calculated using GraphPad Prism (V6.03). In-situ labeling experiments MCF7 cells were seeded ( cells/ml) in a 6 cm Petri dish and left two days to attach and grow. The plates were washed one time with PBS and AA-CW236 or DMSO was added at indicated concentrations (1000x stock in DMSO) in culture media and incubated for S24

25 10 hours. Lysate was prepared as described above and treated with 30 µm AA-CW538 (50x stock in DMSO) for one hour. Click chemistry, reducing SDS-PAGE and visualization were performed as described above. Visualization of DNA alkylation MCF7 cells were seeded ( cells/ml) in a 24-well plate containing a coverslip and left two days to attach and grow. Cells were incubated for 12 hours with either 1 µm AA-CW236, 1 µm lomeguatrib (1000x stock in DMSO) or DMSO. Media was replaced with fresh compound and after 30 min, water or 300 µm temozolomide (10x fresh solution in water) was added and incubated for 8 hours. Cells were washed two times with PBS and fixed with methanol at 20 C for 30 min. Cells were again washed two times with PBS and coverslip was transferred to a wet box. Cells were treated with a 4 C solution of 70 mm NaOH, 140 mm NaCl and 40% ethanol for five min. [6] and then washed three times with PBS. Blocking was performed with a 5% bovine serum albumin (BSA) solution in PBS for 30 min. Cells were washed one time with PBS 0.1% BSA and mouse monoclonal O 6 -methyl-2-deoxyguanosine-directed antibody (Axxora, 1/500 in PBS 0.1% BSA) was added and incubated for two hours at r.t. Cells were washed three times with PBS 0.1% BSA and incubated for one hour with anti-mouse IgG alexa fluor 488 (Jackson, 1/200 in PBS 0.1% BSA). Cells were washed three times with PBS 0.1% BSA, one time with PBS, one time with water and then mounted on a slide with ProLong Gold antifade mountant with DAPI (Life Technologies). Cells were visualized with an LSM 700 confocal microscope (Zeiss). Cell viability assay Caco-2 cells were seeded in a 96-well plate ( cells/ml) and were allowed to attach overnight. Cells were pre-incubated with DMSO or 3 µm AA-CW236 for 8 hours (1000x stock in DMSO) followed by the addition of indicated concentrations of temozolomide (10x stock in water) and incubated for 5 days. Cells were imaged using a SpectraMax i3 plate reader equipped with a SpectraMax MiniMax imaging cytometer (Molecular Devices) and counted with ImageJ. SILAC labeling of MCF7 cells MCF7 cells were passaged 6 times in DMEM without L-lysine and L-arginine (Thermo Scientific) supplemented with 10% dialyzed FCS (Thermo Scientific), non-essential amino acids, penicillin streptomycin and either 100 mg/l 13 C 6 L-lysine-HCl and 13 C 6 15 N 4 L-arginine- HCl (heavy) or L-lysine-HCl and L-arginine-HCl (light) (Thermo Scientific). AA-CW159A enrichment for mass spectrometry MCF7 lysate (2 mg/ml, 0.5 ml) was treated with DMSO or 10 µm AA-CW159A (100x stock in DMSO) for one hour at r.t. Protein was subjected to click chemistry. Photocleavable () biotin alkyne (Click Chemistry Tools, 20 µm, 50x stock in DMSO), tris(2-carboxyethyl)phosphine hydrochloride (TCEP) (1 mm, 50x fresh stock in water), tris[(1-benzyl-1h-1,2,3-triazol-4- yl)methyl]amine (TBTA) (100 µm, 16x stock in DMSO:tButanol 1:4), and copper(ii) sulfate (1 mm, 50x stock in water) were added to the proteome and left to react for one hour at r.t. Protein was precipitated by adding MeOH (4 vol.), CHCl 3 (1 vol.) and water (3 vol.) to the reaction mixture and the turbid mixture was centrifuged for five min. at x g at 4 C yielding a protein layer between the aqueous and organic layers. The protein layer was isolated, dried and solubilized in 2% SDS in PBS via sonication. Tube was centrifuged at x g for five min. and soluble fraction was transferred to a new tube. PBS was added to give a final SDS concentration of 0.2%. 70 µl of streptavidin agarose beads (ProteoChem) were added and the mixture was rotated for four hours at r.t. Beads were washed with 1% SDS in PBS (1x 10 ml), S25

26 PBS (3x 10 ml), and water (3x 10 ml). Beads were resuspended in 6 M urea in PBS (500 μl), reduced with 10 mm neutralized TCEP (20x fresh stock in water) for 30 min. at r.t., and alkylated with 55 mm iodoacetamide (400 mm fresh stock in water) for 30 min. at r.t. in the dark. Beads were pelleted by centrifugation (1 400 x g, two min.) and resuspended in 150 μl of 2 M Urea, 1 mm CaCl 2 (100x stock in water) and trypsin (Promega, 1 μl of 0.5 μg/μl) in 50 mm NH 4HCO 3. The digestion was performed for 12 hours at 37 C. Samples were acidified to a final concentration of 5% acetic acid, desalted over a self-packed C18 spin column and dried. Samples were analyzed by LC-MS/MS (see below) and the MS data was processed with MaxQuant (see below). AA-CW159A-competitive SILAC experiment MCF7 light and heavy lysates (2 mg/ml, 0.5 ml) were treated with indicated concentration of AA-CW236 (100x stock in DMSO) or DMSO for one hour. Lysate was then treated with 10 µm AA-CW159A (100x stock in DMSO) for one hour at r.t. Protein was subjected to click chemistry. Photocleavable () biotin alkyne (Click Chemistry Tools, 20 µm, 50x stock in DMSO), tris(2- carboxyethyl)phosphine hydrochloride (TCEP) (1 mm, 50x fresh stock in water), tris[(1-benzyl- 1H-1,2,3-triazol-4-yl)methyl]amine (TBTA) (100 µm, 16x stock in DMSO:tButanol 1:4), and copper(ii) sulfate (1 mm, 50x stock in water) were added to the proteome and left to react for one hour at r.t. Light and heavy proteomes were mixed together at a ratio of 1:1. Protein was precipitated by adding MeOH (4 vol.), CHCl 3 (1 vol.) and water (3 vol.) to the reaction mixture and the turbid mixture was centrifuged for five min. at x g at 4 C yielding a protein layer between the aqueous and organic layers. The protein layer was isolated, dried and solubilized in 2% SDS in PBS via sonication. Tube was centrifuged at x g for five min. and soluble fraction was transferred to a new tube. PBS was added to give a final SDS concentration of 0.2%. 140 µl of streptavidin agarose beads (ProteoChem) were added and the mixture was rotated for four hours at r.t. Beads were washed with 1% SDS in PBS (1x 10 ml), PBS (3x 10 ml), and water (3x 10 ml). Beads were resuspended in 6 M urea in PBS (500 μl), reduced with 10 mm neutralized TCEP (20x fresh stock in water) for 30 min. at r.t., and alkylated with 55 mm iodoacetamide (400 mm fresh stock in water) for 30 min. at r.t. in the dark. Beads were pelleted by centrifugation (1 400 x g, two min.) and resuspended in 150 μl of 2 M Urea, 1 mm CaCl 2 (100x stock in water) and trypsin (Promega, 1 μl of 0.5 μg/μl) in 50 mm NH 4HCO 3. The digestion was performed for 12 hours at 37 C. Samples were acidified to a final concentration of 5% acetic acid, desalted over a self-packed C18 spin column and dried. Samples were analyzed by LC-MS/MS (see below) and the MS data was processed with MaxQuant (see below). IAA-competitive dimethyl labeling experiment MCF7 lysate (2 mg/ml, 5 ml) was treated with 200 nm of AA-CW236 or lomeguatrib (100x stock in DMSO) or DMSO for one hour. Sample was then incubated with 10 µm iodoacetamide alkyne (Setareh Biotech, 100x stock in DMSO) for one hour. Photocleavable () biotin azide (Click Chemistry Tools, 20 µm, 50x stock in DMSO), tris(2-carboxyethyl)phosphine hydrochloride (TCEP) (1 mm, 50x fresh stock in water), tris[(1-benzyl-1h-1,2,3-triazol-4- yl)methyl]amine (TBTA) (100 µm, 16x stock in DMSO:tButanol 1:4), and copper(ii) sulfate (1 mm, 50x stock in water) were added to the proteome and left to react for one hour at r.t. Protein was precipitated by adding MeOH (4 vol.), CHCl 3 (1 vol.) and water (3 vol.) to the reaction mixture and the turbid mixture was centrifuged for 10 min. at x g at 4 C yielding a protein layer between the aqueous and organic layers. The protein layer was isolated, dried and solubilized in 2% SDS in PBS via sonication. Tube was centrifuged at x g for five min. and soluble fraction was transferred to a new tube. PBS was added to give a final SDS concentration of 0.2%. 500 µl of streptavidin agarose beads were added and the mixture was rotated for four hours at r.t. Beads were washed with 1% SDS in PBS (2x 10 ml), PBS (3x 10 ml), and water (3x 10 ml). Beads were resuspended in 6 M urea in PBS S26

27 (500 μl), reduced with 10 mm neutralized TCEP (20x fresh stock in water) for 30 min. at r.t., and alkylated with 55 mm iodoacetamide (400 mm fresh stock in water) for 30 min. at r.t. in the dark. Beads were pelleted by centrifugation (1 400 x g, two min.) and resuspended in 300 μl of 2 M Urea, 1 mm CaCl 2 (100x stock in water) and trypsin (Promega, 2 μl of 0.5 μg/μl) in 50 mm NH 4HCO 3. The digestion was performed for 12 hours at 37 C. Beads were washed with 1% SDS in PBS (1x 10 ml), PBS (3x 10 ml), and water (3x 10 ml). Beads were transferred to a microtube in 500 μl of 100 mm tetraethylammonium bromide (TEAB) buffer, centrifuged (1 400 x g, 2 min.) and resuspended in 500 μl of 100 mm TEAB buffer. Dimethyl labeling was performed as reported previously [7]. Briefly, 20 μl of either 4% formaldehyde or 4% formaldehyde-d 2 solution was added together with 20 μl of 0.6 M NaBH 3CN (freshly made in water) and left to react for one hour at r.t. with rotation. Beads were washed three times with PBS and mixed at a ratio 1:1. Beads were resuspended in 2 ml PBS and photocleavage was performed with irradiation at 365 nm in a CL-1000 UV crosslinker (UVP) for one hour at 4 C. Beads were centrifuged and supernatant was transferred to a new tube. Beads were resuspended in 1 ml PBS and incubated for one hour at 37 C with shaking, centrifuged and supernatants were combined. Beads were resuspended in 1 ml PBS with 1 M NaCl and incubated for 6 hours with shaking, centrifuged and supernatants were combined. Peptides were desalted over a self-packed C18 spin column and dried. Samples were analyzed by LC- MS/MS (see below) and the MS data was processed with MaxQuant (see below) and Proteome Discoverer (see below). LC-MS/MS analysis Peptides were resuspended in water with 0.1% formic acid (FA) and analyzed using Proxeon EASY-nLC 1000 nano-uhplc coupled to QExactive Plus Quadrupole-Orbitrap mass spectrometer (Thermo Scientific). The chromatography column consisted of a 30 cm long, 75 μm i.d. microcapillary capped by a 5 μm tip and packed with ReproSil-Pur 120 C18-AQ 2.4 μm beads (Dr. Maisch GmbH). LC solvents were 0.1% FA in H 2O (Buffer A) and 0.1% FA in MeCN (Buffer B). Peptides were eluted into the mass spectrometer at a flow rate of 300 nl/min. over a 240 min. linear gradient (3-35% Buffer B) at 50 C. Data was acquired in data-dependent mode (top-20, NCE 30, R = ) after full MS scan (R = , m/z ). Dynamic exclusion was set to 10 s, peptide match to prefer and isotope exclusion was enabled. MaxQuant analysis The MS data was analyzed with MaxQuant [8] (V ) and searched against the human proteome (Uniprot, 89,649 entries) and a common list of contaminants (included in MaxQuant). The first peptide search tolerance was set at 20 ppm, 10 ppm was used for the main peptide search and fragment mass tolerance was set to 0.02 Da. The false discovery rate for peptides, proteins and sites identification was set to 1%. The minimum peptide length was set to 6 amino acids, minimal number of peptides per protein was set to 2 and peptide requantification was enabled. Oxidized methionine was set as variable modification and carbamidomethylation of cysteine as fixed modification. For competitive ABPP-SILAC experiments 13 C 6 L-lysine and 13 C 6, 15 N 4 L-arginine were set as heavy isotope labels. For IAA-competitive capture-andrelease experiments, iodoacetamide alkyne adduct ( Da) and carbamidomethylation on cysteine and oxidation of methionine were set as variable modifications. Dimethyl labels were searched as N-terminal and lysine modification for light Da and heavy Da. Only modified peptides with PEP value 1% and present in 4 out of 6 injections were considered. Proteome Discoverer analysis The MS data was analyzed with Proteome Discoverer (V ) using the Sequest HT algorithm [9] and searched against the human proteome (Uniprot, 89,649 entries). Dimethyl S27

28 labels were searched as N-terminal and lysine modification for light Da and heavy Da. Iodoacetamide alkyne adduct ( Da), carbamidomethylation on cysteine and oxidation of methionine were set as dynamic modification. Maximum precursor mass was set to Da, precursor mass tolerance to 10 ppm and fragment mass tolerance to 0.02 Da. Only modified peptides with PEP value 1% were considered. hmgmt docking studies AA-CW236, 8, 9 and 11 were covalently docked to the crystal structure of human MGMT (PDB: 1EH6) on Cys 145 using the GOLD suite (v5.3, CCDC). The default GOLD fitness function was used to determine the best binding configuration. The distance for hydrogen bonding was set to 3 Å and the minimun H-bond geometry weight to 0.5. S28

29 Supplemental Figures kda ( ) (+) IAA (10 mm) (+) (+) AA-CW159A (10 µm) kda ( ) (+) IAA (10 mm) (+) (+) AA-CW159A (10 µm) Figure S1: Gel-based fluorescence image of MCF7 cell lysate pretreated or not with 10 mm iodoacetamide and labeled with 10 µm AA-CW159A (left). The Coomassie stain image is shown on the right side. S29

30 kda AA-CW159A Mock Overexpr. kda AA-CW159A Mock Overexpr GFP-hMGMT 50- -GFP-hMGMT Figure S2: Gel-based fluorescence labeling of mock-transfected MCF7 cell lysate and lysate with overexpressed GFP-hMGMT and labeled with 10 µm AA-CW159A (left). The Coomassie stain image is shown on the right side. S30

31 Mock DMSO DMSO DMSO kda kda kda Figure S3: Coomassie stain images corresponding to the fluorescence gels in Figure 2B. S31

32 19 14 A B Mock (nm) (nm) kda 250- kda GFP-hMGMT GFP-hMGMT Mock (nm) kda kda (nm) C 25- Figure S4: Fluorescence gels of mock-transfected MCF7 cell lysate and lysates with overexpressed GFP-hMGMT. Lysates were treated with indicated concentrations of 19 (A, top) or 14 (B, top) followed by 10 µm AA-CW159A. Corresponding Coomassie stain images are shown (A and B, bottom). Determination of the IC 50 values of compounds 19 and 14 against GFP-hMGMT (C). S32

33 AA-CW236 - lomeguatrib Figure S5: Determination of the binding constants K I and k inact for AA-CW236, O 6 - benzylguanine and lomeguatrib against human MGMT. S33

34 Scheme S1: Synthesis of clickable probe 20 (AA-CW538) S34

35 MCF7 breast adenocarcinoma MDA-MB-231 breast adenocarcinoma HCC 1187 breast carcinoma HeLa cervix carcinoma Caco-2 colon adenocarcinoma A431 squamous cell carcinoma RPMI 8402 acute lymphoblastic leukemia ( ) (+) ( ) (+) ( ) (+) ( ) (+) ( ) (+) ( ) (+) ( ) (+) AA-CW236 kda Figure S6: Coomassie stain image corresponding to the fluorescence gel in Figure 3C. S35

36 kda AA-CW AA-CW236 (nm) kda AA-CW AA-CW236 (nm) endog. MGMT Figure S7: Gel-based profiling of AA-CW236 activity in-situ. MCF7 cells were treated with various concentrations of AA-CW236 for 10 hours, lysed and treated with 30 µm AA-CW538. Fluorescence gel is shown on the left side and the Coomassie stain image on the right side. S36

37 L o g 2 D M L R a tio L o m e g u a trib /D M S O D N M 2 P G K 1 R a tio G N B M G M T M R P S 1 8 C R A R S L o m e g u a trib n M Figure S8: Scatter plot with DML ratios from the competitive experiment with 200 nm lomeguatrib (n = 6) followed by 10 µm IAA alkyne. The complete list of peptides and ratios is shown in Table S5. S37

38 A (nm) endog. MGMT- AA-CW236 B C endog. MGMT- endog. MGMT- AA-CW236-2 AA-CW236-3 D AA-CW236 AA-CW236-2 AA-CW (nm) Figure S9: (A) Fluorescence gels of MCF7 lysates treated with indicated concentrations of AA-CW236, AA-CW236-2 or AA-CW236-3 followed by 30 µm AA- CW538. (B) Chemical structures of AA-CW236, AA-CW236-2 and AA-CW (C) Determination of IC 50 values for compounds AA-CW236, AA-CW236-2 and AA-CW236-3 against endogenous hmgmt. (D) Corresponding Coomassie stain images. S38