(Supplementary Information)

Transcription

1 Foldamer-mediated Structural Rearrangement Attenuates Aβ Oligomerization and Cytotoxicity (Supplementary Information) Sunil Kumar* 1, Anja Henning-Knechtel 2, Ibrahim Chehade 2, Mazin Magzoub 2, and Andrew D. Hamilton* 1 *Correspondence: sk6327@nyu.edu, andrew.hamilton@nyu.edu Addresses: 1 Department of Chemistry, New York University, New York, NY 10003, USA. 2 Biology Program, Division of Science, New York University Abu Dhabi, Abu Dhabi, United Arab Emirates.

2 Figure S1. Raw plots (a) and the normalized plots (b) for the aggregation of 5 μm Aβ42 probed using amyloid kinetic assay. A stock solution of 1 mm Aβ42 was diluted to 5 μm Aβ42 in phosphate buffer to initialize the aggregation reaction. All the amyloid assays of Aβ42 and Aβ40 aggregation in the absence and presence of small molecule ligands were conducted at least in triplicate. Buffer: 150 mm KCl, 50 mm NaPi, ph 7.4.

3 Figure S2. Chemical structures of the compounds used in the study.

4 Figure S3. Induction of a secondary structure in Aβ42 derived by 5. Time-dependent CD spectra of 15 μm Aβ42 recorded at 0 (black) and 4 h (light red) and in the presence of 5 (light blue) at an equimolar ratio after 4 h. Buffer: 150 mm KCl, 50 mm NaPi, ph 7.4.

5 Figure S4. The effect of small molecules on cell viability. Mouse neuroblastoma cells were incubated with 5 μm of each of the indicated small molecule ligands identical to the work shown in Figure 4 (main text). The cell viability was assessed using MTS assay after incubating the small molecules with the cells for 48 h. Error bars represent the S.D. in the mean of four replicates.

6 Figure S5. Effect of 5 on the cellular uptake and intracellular localization of Aβ42. a, N2a cells were treated with 5 μm Aβ42 (doped with 1 μm AβTR) in the presence of 5 at an equimolar ratio in phenol red- and serum-free medium for 24 h, and then stained with 50 nm MitoTracker Green for 15 min prior to confocal imaging. b, N2a cells were treated with 5 μm Aβ42 in the presence of 5 μm of 5 (doped with 1 μm 5F) in phenol red- and serum-free medium for 24 h, and then stained with 50 nm MitoTracker Red for 15 min prior to confocal imaging.

7 Figure S6. Representative plots for the kinetic pathways of seed catalyzed Aβ aggregation probed using amyloid kinetic assays. A stock solution of 1 mm Aβ42 was diluted to 5 μm Aβ42 in the phosphate buffer containing Aβ42 seeds (10%, v/v) to initialize the aggregation reaction. The seed catalyzed amyloid kinetic assays of Aβ42 (10%, v/v) in the absence and presence of 5 were conducted at least in triplicate. Buffer: 150 mm KCl, 50 mm NaPi, ph 7.4.

8 Figure S7. Effect of 5 on seed induced Aβ40 aggregation. The seed induced amyloid kinetic assay was initialized by adding 20 μm Aβ40 in solution containing Aβ40 seeds (25%, v/v) and 5 (20 μm). Buffer: 150 mm KCl, 50 mm NaPi, ph 7.4.

9 Figure S8. TEM image analysis of seed catalyzed Aβ42 aggregation. a, The seed catalyzed amyloid kinetic assays of 5 μm Aβ42 in phosphate buffer containing Aβ42 seeds (10%, v/v) to initialize the aggregation reaction. b, The seed catalyzed amyloid kinetic assays of Aβ42 (10%, v/v) aggregation in phosphate buffer containing Aβ42-5 complex seeds (10%, v/v). Buffer: 150 mm KCl, 50 mm NaPi, ph 7.4.

10 Scheme S1. Synthetic route for the preparation of 6. a, Dichloromethane (anhydrous), triethylamine (anhydrous), 2-chloro-1-methylpyridinium iodide, 50 C, 6 h. b, Ethyl acetate, H2 (g), Pd/C, 16 h, r.t. c, Compound 1, dichloromethane (anhydrous), triethylamine (anhydrous), 2-chloro-1-methylpyridinium iodide, 50 C, 16 h. d, Ethyl acetate, H2 (g), Pd/C, 16 h, r.t. e, Compound 3, dichloromethane (anhydrous), triethylamine (anhydrous), 2-chloro-1-methylpyridinium iodide, 50 C, 16 h. f, Dichloromethane (anhydrous), trifluoroacetic acid, triethylsilane, 6 h, r.t.

11 Scheme S2. Synthetic route for the preparation of 7. a, Dichloromethane (anhydrous), triethylamine (anhydrous), 2-chloro-1-methylpyridinium iodide, 50 C, 6 h. b, Ethyl acetate, H2 (g), Pd/C, 16 h, r.t. c, Compound 1, dichloromethane (anhydrous), triethylamine (anhydrous), 2-chloro-1-methylpyridinium iodide, 50 C, 16 h. d, Ethyl acetate, H2 (g), Pd/C, 16 h, r.t. e, Compound 1, dichloromethane (anhydrous), triethylamine (anhydrous), 2-chloro-1- methylpyridinium iodide, 50 C, 16 h. f, Dichloromethane (anhydrous), trifluoroacetic acid, triethylsilane, 6 h, r.t.

12 Scheme S3. Synthetic route for the preparation of 5F. a, Pd/C, ethyl acetate, H2 (g), 12 h, r.t. b, 1,1 -Thiocarbonyldi-2(1H)-pyridone, dichloromethane (anhydrous), 12 h, r.t. c, (5- (Aminoacetamido) Fluorescein, triethylamine (anhydrous), pyridine (anhydrous), 16 h, r.t. d, Dichloromethane, triisopropylsilane, trifluoroacetic acid, in dark, 4 h, r.t.

13 Synthesis of compounds 1, 2, 3, 6a, and 7a have been reported somewhere else 1,2. Standard Protocol for Reduction of Nitro-oligoquinolines. To a solution of nitroquinoline ( mmol) in tetrahydrofuran (10 ml), Pd/C (10% wt.) was added and the reaction started with constant stirring at room temperature in the atmosphere of H2 (g). The progress of the reaction was monitored by TLC. Generally, the reduction reactions were completed in 16 h. The reaction mixture was filtered and dried which result in a yellow solid with quantitative yield. The product was used in the next step without further characterization. Standard Protocol for Amide Coupling. To a solution of nitroquinoline carboxylic acid (1.2 mmol) in dichloromethane (10 ml), triethylamine (4.0 mmol) and 2-chloromethyl-1-methyl pyridinium iodide (1.2 mmol) were added and the reaction was refluxed for 20 min. at 50 C under inert atmosphere. To this solution, aminooligoquinoline (1.0 mmol) was added and reaction started with constant stirring at 50 C under inert atmosphere. The reaction mixture was stirred for 6 h. after which the volatiles were removed on rotovap. Flash chromatography (0 to 35% ethyl acetate in hexane, v/v) yielded the desired product as a yellow to brown solid. Standard Protocol for Deprotection of Oligoquinolines. To a solution of oligoquinoline (0.04 mmol), a cocktail solution (2 ml, dichlormethane: trifluoroacetic acid: triethylsilane, 80:15:5, v/v) was added and the solution was stirred at room temperature for 6 h. The reaction mixture was dried and washed with cold diethyl ether (4 3 ml) that results in a yellow to brown solid. The tert-butyl boc deprotected compounds were purified on a reverse phase HPLC column (Water Gen-Pak FAX, mm) with ACN/H 2 O/TFA buffer system. Buffer A: 10% ACN, 90% H 2 O, and 0.1% TFA (v/v); buffer B: 95% ACN, 5% H 2 O, and 0.1% TFA (v/v). Elution conditions: % buffer B in buffer A during 25 min at a flow rate of 2.0 ml/min.

14 Compound 6b 1 H NMR (600 MHz, Chloroform-d) δ (s, 1H), (s, 1H), (m, 2H), (dd, J = 8.3, 1.5 Hz, 1H), (dd, J = 8.4, 1.3 Hz, 1H), (dd, J = 8.3, 1.3 Hz, 1H), (s, 1H), (s, 1H), (t, J = 8.0 Hz, 1H), (t, J = 8.0 Hz, 1H), (dt, J = 8.4, 2.4 Hz, 1H), (dd, J = 8.3, 7.5 Hz, 1H), (s, 1H), (s, 2H), (dd, J = 8.9, 5.9 Hz, 1H), (dd, J = 8.9, 6.0 Hz, 1H), (dd, J = 8.9, 6.5 Hz, 1H), (dd, J = 9.0, 6.6 Hz, 1H), (q, J = 7.1 Hz, 1H), (m, 1H), (dq, J = 16.1, 7.1 Hz, 1H), (s, 3H), (m, 2H), (m, 2H), (s, 9H), (ddt, J = 22.5, 13.7, 7.6 Hz, 2H), (d, J = 6.8 Hz, 3H), (d, J = 6.8 Hz, 3H), (t, J = 7.5 Hz, 3H), (t, J = 7.5 Hz, 3H). MS-ESI (m/z): calculated for C47H50N6O11 (M+H): , found Compound 6c 1 H NMR (600 MHz, Chloroform-d) δ (s, 1H), (s, 1H), (s, 1H), (dd, J = 7.5, 1.2 Hz, 1H), (dd, J = 8.3, 1.4 Hz, 1H), (dd, J = 8.4, 1.4 Hz, 1H), (dd, J = 7.6, 1.2 Hz, 1H), (dd, J = 7.5, 1.3 Hz, 1H), (dd, J = 8.3, 1.2 Hz, 1H), (m, 2H), (dd, J = 8.3, 1.3 Hz, 1H), (s, 1H), (dd, J = 8.3, 7.6 Hz, 1H), (dd, J = 8.3, 7.6 Hz, 1H), (m, 2H), (m, 2H), (t, J = 7.9 Hz, 1H), (s, 1H), (s, 1H), (s, 1H), (s, 2H), (s, 2H), (s, 2H), (m, 2H), (m, 2H), (dq, J = 27.6, 7.1 Hz, 4H), (s, 3H), (m, 1H), (dq, J = 13.0, 6.5 Hz, 1H), (ddd, J = 13.3, 7.5, 5.6 Hz, 1H), (m, 1H), (s, 9H), (s, 9H), (m, 3H), (s, 3H), (d, J = 6.8 Hz, 3H), (d, J = 6.8 Hz, 3H), (t, J = 7.4 Hz, 3H), (t, J = 7.5 Hz, 3H). MS-ESI (m/z): calculated for C63H67N8O15 (M+H): , found Compound 7b

15 1 H NMR (600 MHz, Chloroform-d) δ (s, 1H), (s, 1H), (dd, J = 8.4, 1.4 Hz, 1H), (dd, J = 8.4, 1.2 Hz, 1H), (dd, J = 8.4, 1.3 Hz, 1H), (s, 1H), (m, 2H), (m, 1H), (dd, J = 7.5, 1.4 Hz, 1H), (m, 1H), (s, 1H), (s, 2H), (s, 2H), (q, J = 7.1 Hz, 2H), (s, 3H), (m, 1H), (m, 4H), (s, 9H), (s, 9H), (d, J = 6.8 Hz, 3H), (t, J = 7.5 Hz, 3H). MS- ESI (m/z): calculated for C48H50N6O13 (M+H): , found Compound 7c 1 H NMR (600 MHz, Chloroform-d) δ (s, 1H), (s, 1H), (s, 1H), (dd, J = 7.6, 1.3 Hz, 1H), (dd, J = 8.2, 1.4 Hz, 1H), (dd, J = 7.5, 1.3 Hz, 1H), (dd, J = 7.7, 1.3 Hz, 1H), (dd, J = 4.7, 1.2 Hz, 1H), (dd, J = 4.7, 1.3 Hz, 1H), (dd, J = 8.3, 1.3 Hz, 1H), (s, 1H), (dd, J = 8.3, 7.6 Hz, 1H), (dd, J = 7.4, 1.4 Hz, 1H), (dd, J = 8.3, 7.5 Hz, 1H), (m, 1H), (d, J = 1.5 Hz, 1H), (t, J = 7.9 Hz, 1H), (s, 1H), (s, 1H), (s, 2H), (d, J = 16.8 Hz, 2H), (m, 3H), (s, 3H), (m, 2H), (m, 2H), (s, 9H), (s, 9H), (d, J = 6.8 Hz, 3H), (s, 3H), (d, J = 6.8 Hz, 3H), (t, J = 7.5 Hz, 3H), (t, J = 7.5 Hz, 3H). MS-ESI (m/z): calculated for C63H67N8O15 (M+H): , found Compound 6 1 H NMR (600 MHz, DMSO-d6) δ (br, s, 2H), (s, 1H), (s, 1H), (s, 1H), (dd, J = 7.5, 1.3 Hz, 1H), (dd, J = 8.2, 1.4 Hz, 1H), (dd, J = 7.5, 1.2 Hz, 1H), (dd, J = 7.6, 1.2 Hz, 1H), (d, J = 1.3 Hz, 1H), (d, J = 1.3 Hz, 1H), (dd, J = 8.4, 1.3 Hz, 1H), (s, 1H), (d, J = 8.0 Hz, 1H), (t, J = 7.9 Hz, 1H), (dd, J = 7.5, 1.4 Hz, 1H), (m, 1H), (t, J = 8.0 Hz, 1H), (s, 1H), (s, 1H), (s, 1H), (s, 2H), (d, J = 52.7 Hz, 2H), (m, 2H), (m, 2H), (s, 3H), (dq, J = 13.0, 6.4 Hz, 1H), (dp, J = 13.1, 6.5 Hz, 1H), (m, 2H), (m, 2H), (d, J = 6.8

16 Hz, 3H), (p, J = 2.6 Hz, 3H), (dt, J = 9.0, 7.4 Hz, 6H). MS-ESI (m/z): calculated for C55H50N8O15 (M+H): , found Anal. Calcd for C55H50N8O15: C, 62.08; H, 4.83; N, 10.53; O, Found: C, 61.74; H, 4.90; N, HPLC retention peak: 21 min. Compound 7 1 H NMR (600 MHz, DMSO-d6) δ (br, s, 2H), (s, 1H), (s, 1H), (s, 1H), (dd, J = 7.6, 1.2 Hz, 1H), (dd, J = 8.0, 1.7 Hz, 1H), (d, J = 7.5 Hz, 1H), (dd, J = 7.6, 1.2 Hz, 1H), (dd, J = 8.3, 1.3 Hz, 1H), (dd, J = 8.4, 1.3 Hz, 1H), (dd, J = 8.4, 1.3 Hz, 1H), (s, 1H), (d, J = 7.9 Hz, 1H), (t, J = 8.0 Hz, 1H), (dd, J = 7.5, 1.7 Hz, 1H), (m, 1H), (t, J = 8.0 Hz, 1H), (s, 1H), (s, 1H), (s, 1H), (m, 4H), (m, 4H), (q, J = 6.7 Hz, 1H), (dp, J = 13.0, 6.5 Hz, 1H), (tt, J = 13.3, 7.5 Hz, 1H), (m, 1H), (m, 1H), (dt, J = 13.6, 7.5 Hz, 1H), (d, J = 6.8 Hz, 3H), (d, J = 6.8 Hz, 3H), (t, J = 7.5 Hz, 3H), (t, J = 7.5 Hz, 3H). MS-ESI (m/z): calculated for C55H50N8O15 (M+H): , found Anal. Calcd for C55H50N8O15: C, 62.08; H, 4.83; N, 10.53; O, Found: C, 61.72; H, 4.89; N, HPLC retention peak: 20 min. Compound 5-NH2 1 H NMR (600 MHz, Chloroform-d) δ (s, 1H), (s, 1H), (s, 1H), (d, J = 7.5 Hz, 1H), (d, J = 7.5 Hz, 1H), (d, J = 7.6 Hz, 1H), (d, J = 8.3 Hz, 1H), (d, J = 8.4 Hz, 1H), (d, J = 8.4 Hz, 1H), (t, J = 8.0 Hz, 1H), (d, J = 9.8 Hz, 2H), (d, J = 8.1 Hz, 1H), (t, J = 7.9 Hz, 1H), (s, 1H), (t, J = 7.8 Hz, 1H), (s, 1H), (s, 1H), (d, J = 7.3 Hz, 1H), (s, 2H), (s, 2H), (m, 2H), (dt, J = 47.9, 7.9 Hz, 2H), (s, 3H), (dq, J = 13.4, 6.3 Hz, 1H), (dq, J = 13.3, 6.6 Hz, 1H), (dt, J = 13.7, 6.8 Hz, 1H), (m, 1H), (s, 9H), (s, 9H), (td, J = 14.2, 7.1 Hz,

17 4H), (d, J = 6.7 Hz, 3H), (dt, J = 19.8, 7.5 Hz, 6H), (t, J = 7.0 Hz, 3H). MS-ESI (m/z): calculated for C63H68N8O13 (M+H): , found Compound 5-NCS To a solution of 5-NH2 (23 mg, mmol) in dichloromethane (10 ml), 1,1 -Thiocarbonyldi- 2(1H)-pyridone (14 mg, mmol, 3 equivalent) was added into the flask and the reaction solution stirred for 12 h at room temperature under inert atmosphere. The progress of the reaction was monitored by TLC. Flash chromatography (0 to 40% ethylacetate in hexane) yielded the desired product as yellow solid (21 mg, 89%). 1 H NMR (600 MHz, Chloroform-d) δ (s, 1H), (s, 1H), (s, 1H), (d, J = 7.5 Hz, 1H), (d, J = 7.5 Hz, 1H), (d, J = 8.4 Hz, 1H), (d, J = 7.6 Hz, 1H), (d, J = 8.6 Hz, 1H), (d, J = 8.2 Hz, 2H), (t, J = 7.9 Hz, 1H), (s, 1H), (t, J = 8.1 Hz, 1H), (s, 1H), (s, 1H), (d, J = 7.6 Hz, 2H), (s, 1H), (d, J = 7.3 Hz, 1H), (d, J = 33.6 Hz, 2H), (m, 2H), (d, J = 32.7 Hz, 2H), (m, 1H), (m, 1H), (s, 3H), (p, J = 9.1, 8.2 Hz, 1H), (dt, J = 12.8, 6.5 Hz, 1H), (s, 9H), (s, 9H), (m, 4H), (d, J = 5.9 Hz, 3H), (m, 6H), (t, J = 6.3 Hz, 3H). MS-ESI (m/z): calculated for C64H66N8O13S (M+H): , found Compound 5F To a solution of 5-NCS (20 mg, mmol) in pyridine (5 ml, anhydrous), N, N- diisopropylethylamine (0.005 ml, 0.05 mmol) was added and the solution was stirred for 10 min. To this solution, 5-(aminoacetamido) fluorescein (13.9 mg, mmol) was added and the reaction was started in dark with continuous stirring under inert atmosphere. The reaction solution was stirred overnight in dark. The product was purified using column chromatography (0 20% methanol in dichloromethane with 1 % triethylamine, v/v) as an orange solid (20 mg, 75%). The compound (tert-butyl 5F) was used in the next step without further characterization. To a solution of tert-butyl 5F (16 mg, mmol) in dichloromethane (4 ml), triethylsilane (0.1 ml, excess) was added, followed by the addition of trifluoroacetic acid (0.4 ml, excess) and the reaction solution was stirred in dark at room temperature for 4 h. The solution was then dried and

18 the orange solid was washed with cold diethyl ether (3 5ml), which afforded the desired product (5F) as an orange solid (13 mg, 72%). 1 H NMR (600 MHz, DMSO-d6) δ (br, 2H), (s, 1H), (s, 1H), (s, 1H), (s, 1H), (s, 1H), (s, 1H), (s, 1H), (s, 1H), (s, 1H), (d, J = 7.6 Hz, 1H), (s, 1H), (d, J = 8.2 Hz, 2H), (m, 2H), (dd, J = 17.0, 8.5 Hz, 2H), (s, 1H), (s, 1H), (d, J = 8.3 Hz, 1H), (s, 1H), (m, 2H), (d, J = 9.1 Hz, 2H), (s, 2H), (s, 2H), (m, 2H), (dt, J = 37.1, 7.6 Hz, 2H), (m, 2H), (d, J = 14.4 Hz, 2H), (s, 3 H), (m, 1H), (m, 1H), (m, 4H), (m, 6H), (t, J = 7.0 Hz, 3H), (d, J = 7.1 Hz, 3H). MS-ESI (m/z): calculated for C78H67N10O19S (M+H): , found HPLC retention peak: 21 min.

19 References 1. Kumar, S.; Birol, M.; Schlamadinger, D. E.; Wojcik, S. P.; Rhoades, E.; Miranker, A. D. Nat Commun 2016, 7, Kumar, S.; Birol, M.; Miranker, A. D. Chem. Commun. 2016, 52,