SUPPORTING INFORMATION
A Stilbene that Binds Selectively to Transthyretin in Cells and Remains
Dark Until it Undergoes a Chemoselective Reaction
to Create a Bright
Blue Fluorescent Conjugate
Sungwook Choi,† Derrick Sek Tong Ong,‡ and Jeffery W. Kelly‡,§,∗
Department of New Drug Discovery and Development, Chungnam National University, Daejon, 305-764, Republic of Korea, Department of Chemistry, Molecular and Experimental Medicine, and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA
To whom correspondence should be addressed E-mail: [email protected], 858-784-9601
†
Department of New Drug Discovery and Development, Chungnam National University.
‡
Department of Chemistry and The Skaggs Institute for Chemical Biology.
§
S2
Figure S1. C18-RP-HPLC analysis of WT-TTR and K15A-TTR pre-incubated (18 h)
with (A) A1 and (B) A2. A
B
S3
Figure S2. RP-HPLC analysis showing the binding of compound A2 to the recombinant K15A-TTR homotetramer after immunocapture utilizing an anti-TTR antibody conjugated resin.
S4
Figure S3. (A) RP-HPLC analysis of modification of TTR in human blood plasma by A2. 48% of TTR out of a maximum of 50% was observed because only 2 of the 4 TTR subunits can be modified in each tetramer. (B) Mass spectrometry characterization of TTRs found in human blood plasma and their modification by A2.
A
S5
Figure S4. Fluorescence observed after an 18 h incubation of A1 with WT-TTR or K15A-TTR.
S6
Figure S5. Recombinant WT-TTR or K15A-TTR (3.6 μM, tetramer) was incubated with A2 or B1 (7.2 μM) for 18 h and analyzed by illumination with a hand-held UV lamp.
S7
Figure S6. Recombinant WT-TTR or K15A-TTR (10.8 μM) were incubated with A2 (21.6 μM) for 18 h before analysis by SDS-PAGE (right panel). Irradiation of the gels in a Gel Doc employing a standard amber filter (548-638 nm) reveals a fluorescence signal only from WT-TTR (left panel).
S8
Figure S7. HPLC analysis of photoisomerization of compounds (A) B1 alone in the buffer solution, (B) B1 in the presence of WT-TTR, (C) A2 alone in the buffer solution by UV irradiation for 10 sec.
A
B
S9
Figure S8. The fluorescence intensity of compounds (A) A2 and (B) B1 (respectively, 10 μM) in dichloromethane or water.
S10
Figure S9. Fluorescence changes of compounds (A) B1 and (B) A1 (respectively, 10 μM) in the dichloromethane in the presence of thiophenol.
S11
Figure S10. Detection of TTR in (A) HeLa cells that overexpressed WT-TTR using transient transfection and (B) Huh-7 (liver) cells using western blot analysis.
S12 Experimental Section
RP-HPLC analysis. WT-TTR was expressed in an E.coli expression system and purified as
described previously.1 The covalent TTR modifiers (A1 or A2, 5 μL of a 1.44 mM solution in DMSO, final concentration: 7.2 μM) were added to 1 mL of a solution of WT-TTR homotetramer (0.2 mg/mL, 3.6 μM) in 10 mM phosphate, 100 mM KCl and 1 mM EDTA (pH 7.0 phosphate buffer) in a 2 mL Eppendorf tube. The samples were vortexed, and incubated for 18 h at 25 °C. Reverse phase high performance liquid chromatography (RP-HPLC) was performed on a Waters 600E multi-solvent delivery system, using a Waters 486 tunable absorbance detector, a 717 autosampler, employing a ThermoHypersil Keystone Betabasic-18 column (150 Å pore size, 3 μm particle size). The “A” mobile phase comprises 0.1% TFA in 94.9% H2O + 5% CH3CN and
the “B” mobile phase is made up of 0.1% TFA in 94.9% CH3CN + 5% H2O. Linear gradients
were run from 100:0 to 0:100 A:B over 30 min.
Binding Stoichiometry of A2 to TTR in Human Blood Plasma. The plasma TTR binding
selectivity assay has been previously described.2 Briefly, to a 1 mL sample of human blood plasma in a 2 mL Eppendorf tube was added A2 (7.5 μL of a 1.44 mM solution in DMSO, final concentration: 10.8 μM) and then the plasma solution was incubated at 37 °C for 24 h on a rocker plate (30 rpm). A 1:1 (v/v) slurry of unfunctionalized sepharose resin in TSA (10 mM Tris, 140 mM NaCl, pH 8.0) buffer was added and the solution was incubated for 1 h at 4 °Con a rocker plate (18 rpm). The solution was then centrifuged and the supernatant was divided into two 400 μL aliquots, which were added to 200 μL of 1:1 (v:v) slurry of anti-TTR antibody conjugated sepharose resin in TSA. The solution was gently rocked (18 rpm) at 4 °C for 20 min, then centrifuged and the supernatant removed. The resin was washed three times by shaking for 1 min with 1 mL of TSA containing 0.05% saponin and then twice more with 1 mL of TSA. After centrifugation to remove the supernatant, 155 μL of triethylamine (100 mM, pH 11.5) was added to the sepharose resin to dissociate the TTR and bound test compound from the resin and the suspension was vortexed for 1 min. After centrifugation, the supernatant was analyzed by reverse phase HPLC on a Water 600 E multi-solvent delivery system, using a Waters 486 tunable absorbance detector, a 717 autosampler, and a ThermoHypersil Keystone Betabasic-18 column (150 Å pore size, 3 μm particle size). The “A” mobile phase comprises 0.1% TFA in 94.9% H2O
+ 5% CH3CN and the “B” mobile phase is made up of 0.1% TFA in 94.9% CH3CN + 5% H2O.
Linear gradients were run from 100:0 A:B to 0:100 A:B for 50 min.
S13
Photoisomerization. Photoisomerization reactions were performed using glass vials and an UV illuminator (Life Technologies, Gibco BRL UV transilluminator, TFX-35M, 6 bulbs, 15W each, 312 nm). The concentrations of A2 and B1 were 20 μM in 10 mM phosphate, 100 mM KCl and 1 mM EDTA (pH 7.0 phosphate buffer) and 5% DMSO cosolvent, respectively. The reaction times were 10 sec. The amounts of trans and cis were determined using reverse phase HPLC (isocratic mobile phase of 45% mobile phase A for A2, isocratic mobile phase of 80% mobile phase A for B1; mobile phase A = 0.1% TFA in 94.9% H2O + 5% CH3CN, mobile phase B = 0.1% TFA in 94.9% CH3CN + 5%
H2O).
Detection of protein labeling by SDS-PAGE. A solution of A2 (final concentration: 21.6 μM) was added to WT- or K15A-TTR (10.8 μM) in 10 mM phosphate, 100 mM KCl and 1 mM EDTA (pH 7.0 phosphate buffer) at 25 ºC. After 18 h, labeled (WT-TTR) or unlabeled (K15A-TTR) protein was solubilized in SDS gel loading buffer and boiled, and resolved by SDS-PAGE. Fluorescence images of the gels were then captured using Gel DocTM XR (BioRad) with a 548-630 nm filter. The gels were also stained with Coomassie Brilliant Blue, and images of the stained gels were captured.
Western blot analysis. Cells were lysed with complete lysis-M buffer containing complete Roche protease inhibitor cocktail (Roche, Nutley, NJ). Total cell protein was determined with Micro BCA assay reagent (Pierce, Rockford, IL) and each sample was diluted to the same protein concentration. Aliquots of cell lysates were separated in a 15 % SDS-PAGE gel and Western blot analysis was performed using appropriate antibodies. Mouse anti-TTR was generously provided by the Masliah laboratory (UCSD) and mouse monoclonal β-actin was acquired from Sigma (St. Louis, MO). Secondary goat anti-mouse HRP-conjugated antibodies were obtained from Pierce. Blots were visualized using SuperSignal West Pico Substrate (Pierce).
Synthesis
General Synthetic Methods. Unless otherwise indicated, all reactions were run under argon gas. Anhydrous solvents were obtained via passage through an activated alumina
S14
column and are from commercial suppliers. 1H and 13C NMR spectra were recorded on a Bruker 500 MHz spectrometer. Chemical shifts are reported relative to internal CDCl3
(Me4Si, δ 0.0) and DMSO-d6 (δ 2.50 for 1H and δ 39.52 for 13C). Reverse phase high
performance liquid chromatography (RP-HPLC) was performed on a Waters 600 E multi-solvent delivery system, using a Waters 486 tunable absorbance detector, a 717 autosampler, and a ThermoHypersil Keystone Betabasic-18 column (150 Å pore size, 3 μm particle size, mobile phase A = 0.1 % TFA in 94.9% H2O + 5 % CH3CN, mobile
phase B = 0.1 % TFA in 94.9 % CH3CN + 5 % H2O). Final compound purities were
determined by analytical RP-HPLC and were > 95 % in purity. All mass spectrometry data were collected at The Scripps Research Institute Center for Mass Spectrometry (ESI-MS; Agilent Technologies and LC/MSD TOF G1969A).
O2N OEt O OH O N OEt O OH O
Reagent s and conditions: (a) i. H210% Pd/C, MeOH. ii. (CHO)n, NaBH3CN, DMF (92% in 2 steps); (b)
BH3.THF (1M in THF), THF, reflux (78%); (c) NCS, PPH3, CH2Cl2(96%); (d) P(OEt)3, 150oC (98%);
4-(methoxymethoxy)-3,5-dimethylbenzaldehyde, NaOMe, DMF, EtOH (67%); (e) 2N NaOH, THF, EtOH (95%); (f) i. DCC, DMAP, CH2Cl2, 4-fluorophenol for A1 or 4-fluorobenzenethiol for A2, propylamine,
EDC, HOAT, DMAP for B1, ii. conc. HCl, THF, MeOH.
a) N OEt O OH N OEt O Cl b) c) d) N OEt O OMOM N OH O OMOM e) 1 2 3 4 5 N R O OH f ) R A1 F O F S N H A2 B1
(Dimethylamino)-5-(ethoxycarbonyl)benzoic acid (1). A mixture of 3-(ethoxycarbonyl)-5-nitrobenzoic acid (5 g, 20.905 mmol) and 10 % Pd/C (1.11 g, 1.045 mmol) in 100 mL of MeOH was stirred under H2 gas at room temperature. After 4 h, the
3-amino-5-S15
(ethoxycarbonyl)benzoic acid. To a solution of 3-amino-5-(ethoxycarbonyl)benzoic acid (2 g, 9.56 mmol) in 10 mL of DMF was added formaldehyde (~ 37 % wt % in H2O,
7.118 mL, 95.60 mmol) at room temperature. After 10 min, the solution was cooled to 0 °C and NaBH3CN (1.897 g, 28.68 mmol) was added. The mixture was stirred at room temperature for 3 h. The solution was diluted with EtOAc. The solution was washed with 5% citric acid and saturated brine and dried with Na2SO4. The solution was filtered and
concentrated to give 3-(Dimethylamino)-5-(ethoxycarbonyl)benzoic acid 1 (2.1 g, 92 % in two steps). 1H-NMR (500MHz, DMSO-d6) δ 7.78 (s, 1H), 7.44 (s, 1H), 7.41 (s, 1H),
4.32 (q, J=7.1 Hz, 2H), 2.99 (s, 6H), 1.33 (t, J=7.1 Hz, 3H); 13C-NMR (125MHz, DMSO-d6) δ 166.99, 165.54, 150.14, 131.76, 130.79, 116.89, 116.25, 115.59, 60.79,
39.84, 14.05; ESI-MS: m/z (MH+): 238.1074 (calc’d), 238.1076 (found).
Ethyl 3-(dimethylamino)-5-(hydroxymethyl)benzoate (2). To a solution of 1 (4 g, 16.86 mmol) in 40 mL of THF was added BH3⋅THF (1M in THF, 42.15 mL, 42.15
mmol) dropwise at 0 °C. The mixture was refluxed for 4 h and cooled to room temperature. The mixture was evaporated, diluted with EtOAc, and washed with saturated NaHCO3 and brine and dried with Na2SO4. The solution was filtered and
concentrated. The residue was subjected to chromatography over silica gel (Hexanes/EtOAc = 2/1) to give 2 (2.95 g, 78 %). 1H-NMR (500MHz, CDCl3) δ 7.35 (s,
1H), 7.32 (s, 1H), 6.91 (s, 1H), 4.68 (s, 2H), 4.36 (q, J=7.1 Hz, 2H), 2.99 (s, 6H), 1.39 (t, J=7.1 Hz, 3H); 13C-NMR (125MHz, CDCl3) δ 167.15, 150.71, 141.93, 131.36, 115.87,
114.91, 112.57, 65.41, 60.89, 40.54, 14.32; ESI-MS: m/z (MH+): 224.1281 (calc’d), 224.1283 (found).
Ethyl 3-(chloromethyl)-5-(dimethylamino)benzoate (3). To a solution of 2 (3.45 g, 15.452 mmol) and PPh3 (6.14 g, 23.17 mmol) in 70 mL of DCM was added NCS (3.19 g,
23.178 mmol) at 0 °C. The reaction mixture was stirred for 1.5 h. After removal of solvent under reduced pressure, the residue was dissolved in Et2O. The solution was
washed with brine and dried with MgSO4. The solution was filtered and concentrated.
The residue was subjected to chromatography over silica gel (Hexanes/EtOAc = 9.5/0.5) to give 3 (3.585 g, 96 %). 1H-NMR (500MHz, CDCl3) δ 7.38 (s, 1H), 7.35 (s, 1H), 6.89
S16
(s, 1H), 4.57 (s, 2H), 4.37 (q, J=7.1 Hz, 2H), 3.00 (s, 6H), 1.39 (t, J=7.1 Hz, 3H); 13 C-NMR (125MHz, CDCl3) δ 166.73, 150.63, 138.35, 131.59, 117.31, 116.20, 113.22,
60.95, 46.40, 40.43, 14.31; ESI-MS: m/z (MH+): 242.0942 (calc’d), 242.0944 (found).
(E)-Ethyl 3-(dimethylamino)-5-(4-(methoxymethoxy)-3,5-dimethylstyryl)benzoate (4). Compound 3 (1.224 g, 4.277 mmol) and triethylphosphite (1.512 mL, 8.555 mmol) were heated to 150 °C for 3 h. The reaction mixture was cooled to 0 °C and then 6 mL of DMF and 6 mL of EtOH were added. NaOMe (486 mg, 8.554 mmol) and 4-(methoxymethoxy)-3,5-dimethylbenzaldehyde (0.831 g, 4.277 mmol) were added to the solution. After 18 h, the mixture was diluted with EtOAc and the solution was washed with brine. After drying on Na2SO4, the organic layer was filtered and concentrated under
reduced pressure. The residue was subjected to chromatography over silica gel (Hexanes/EtOAc = 9/1) to give 4 (1.10 g, 67 %). 1H-NMR (500MHz, CDCl3) δ 7.56 (s,
1H), 7.30 (m, 1H), 7.20 (s, 2H), 7.06 (d, J=16.3 Hz, 1H), 6.99 (d, J=16.3 Hz, 1H), 6.97 (s, 1H), 4.96 (s, 2H), 4.39 (q, J=7.1 Hz, 2H), 3.62 (s, 3H), 3.03 (s, 6H), 2.32 (s, 6H), 1.42 (t, J=7.1 Hz, 3H); 13C-NMR (125MHz, CDCl3) δ 167.23, 154.65, 150.72, 138.36, 133.12,
131.48, 131.24, 128.70, 127.78, 127.07, 115.70, 114.53, 112.47, 99.11, 60.92, 57.39, 40.65, 16.99, 14.40; ESI-MS: m/z (MH+): 384.2169 (calc’d), 384.2173 (found).
(E)-3-(dimethylamino)-5-(4-(methoxymethoxy)-3,5-dimethylstyryl)benzoic acid (5). To a solution of 4 (1.046 g, 2.728 mmol) in THF and EtOH (8/4 mL) was added 2 N NaOH (2.728 mL, 5.456 mmol) at room temperature. The reaction mixture was stirred overnight at room temperature and diluted with EtOAc and acidified with 5% citric acid. The solution was washed with saturated brine and dried with Na2SO4. The solution was
filtered and concentrated. Compound 5 (0.94 g, 97 %) was obtained without further purification (TLC indicated a pure compound). 1H-NMR (500MHz, DMSO-d6) δ 7.47 (s,
1H), 7.31 (s, 2H), 7.12-7.16 (m, 4H), 4.94 (s, 2H), 3.51 (s, 3H), 2.98 (s, 6H), 2.25 (s, 6H);
13
C-NMR (125MHz, DMSO-d6) δ 167.75, 154.18, 150.47, 137.92, 132.56, 131.62,
130.63, 128.12, 127.43, 126.86, 114.89, 114.16, 111.62, 98.51, 56.71, 40.03, 16.55; ESI-MS: m/z (MH+): 356.1856 (calc’d), 356.1854 (found).
S17
(E)-4-fluorophenyl 3-(dimethylamino)-5-(4-hydroxy-3,5-dimethylstyryl)benzoate (A1). To a solution of 5 (80 mg, 0.225 mmol) in DCM (3 mL) were added DCC (70 mg, 0.338 mmol), DMAP (5.5 mg, 0.045 mmol), and 4-fluorophenol (61.5 mg, 0.450 mmol) at room temperature. The reaction mixture was stirred overnight and filtered through Celite. The filtrate was washed with brine, and dried with Na2SO4. The solution was
filtered and concentrated. The residue was subjected to chromatography over silica gel (Hexanes/EtOAc = 8/1) to give (E)-4-fluorophenyl 3-(dimethylamino)-5-(4-(methoxymethoxy)-3,5-dimethylstyryl)benzoate.
MOM deprotection; to a solution of (E)-4-fluorophenyl 3-(dimethylamino)-5-(4-(methoxymethoxy)-3,5-dimethylstyryl)-benzoate (81 mg, 0.180 mmol) in i-PrOH and THF (1/2 mL) was added 0.5 mL of concentrated HCl. The reaction mixture was stirred overnight. After removal of solvent under reduced pressure, the residue was dissolved in EtOAc. The solution was washed with brine and dried with Na2SO4. The solution was
filtered and concentrated. The compound was purified by column chromatography (5:1 Hexane:EtOAc) to give A1 (81 % in 2 steps). 1H-NMR (500MHz, DMSO-d6) δ 8.40 (s,
1H), 7.61 (s, 1H), 7.29-7.36 (m, 4H), 7.16-7.24 (m, 5H), 7.08 (d, J=16.4 Hz, 1H), 3.01 (s, 6H), 2.18 (s, 6H); 13C-NMR (125MHz, DMSO-d6) δ 165.15, 160.50, 158.58, 153.31, 150.61, 146.72, 138.77, 129.61, 129.32, 127.80, 126.75, 124.66, 124.24, 123.71, 123.64, 116.11, 115.92, 114.93, 114.80, 111.21, 40.02, 16.54; ESI-MS: m/z (MH+): 406.1813 (calc’d), 406.1829 (found). (E)-S-4-fluorophenyl 3-(dimethylamino)-5-(4-hydroxy-3,5-dimethylstyryl)benzo- thioate (A2). To a solution of 5 (80 mg, 0.225 mmol) in DCM (3 mL) were added DCC (70 mg, 0.338 mmol), DMAP (5.5 mg, 0.045 mmol), and 4-fluorobenzenethiol (37 μL, 0.338 mmol) at room temperature. The reaction mixture was stirred overnight and filtered through Celite. The filtrate was washed with brine, and dried with Na2SO4. The solution
was filtered and concentrated. The residue was subjected to chromatography over silica gel (Hexanes/EtOAc = 8/1) to give (E)-S-4-fluorophenyl 3-(dimethylamino)-5-(4-(methoxymethoxy)-3,5-dimethylstyryl)benzothioate.
MOM deprotection; to a solution of (E)-S-4-fluorophenyl 3-(dimethylamino)-5-(4-(methoxymethoxy)-3,5-dimethylstyryl)benzothioate (80 mg, 0.172 mmol) in i-PrOH and
S18
THF (1/2 mL) was added 0.5 mL of concentrated HCl. The reaction mixture was stirred overnight. After removal of solvent under reduced pressure, the residue was dissolved in EtOAc. The solution was washed with brine and dried with Na2SO4. The solution was
filtered and concentrated. The compound was purified by column chromatography (6:1 Hexane:EtOAc) to give A2 (73 % in 2 steps). 1H-NMR (500MHz, DMSO-d6) δ 8.42 (s,
1H), 7.57-7.60 (m, 2H), 7.42 (s, 1H), 7.37 (t, J=8.9 Hz, 2H), 7.23 (s, 2H), 7.16-7.20 (m, 2H), 7.07 (d, J=16.4 Hz, 1H), 7.02 (s, 1H), 3.00 (s, 6H), 2.19 (s, 6H); 13C-NMR (125MHz, DMSO-d6) δ 189.74, 163.84, 161.87, 153.38, 150.65, 139.07, 137.30, 137.23,
136.94, 129.59, 127.72, 126.81, 124.50, 124.25, 122.76, 116.52, 116.35, 114.72, 112.26, 108.06, 39.92, 16.53; ESI-MS: m/z (MH+): 422.1584 (calc’d), 42.1599 (found).
(E)-3-(dimethylamino)-5-(4-hydroxy-3,5-dimethylstyryl)-N-propylbenzamide (B1). To a solution of 5 (80 mg, 0.225 mmol), EDC (88 mg, 0.45 mmol), HOAT (61.2 mg, 0.45 mmol), and DMAP (5.5 mg, 0.045 mmol) in 2 mL of DMF was added propylamine (37.8 μL, 0.45 mmol) at room temperature. The reaction mixture was stirred overnight and diluted with EtOAc. The solution was washed with brine and dried with Na2SO4.
The solution was filtered and concentrated.
MOM deprotection; to a solution of crude (E)-3-(dimethylamino)-5-(4-(methoxymethoxy)-3,5-dimethylstyryl)-N-propylbenzamide in 2 mL of THF and 1 mL of MeOH was added 0.5 mL of concentrated HCl. The reaction mixture was stirred overnight. The solution was evaporated under reduced pressure and the remained water was lyophilized. The residue was subjected to chromatography over silica gel (Hexanes/EtOAc = 6/4) to B1 (68 % in 2 steps). 1H-NMR (500MHz, DMSO-d6) δ
8.35-8.38 (m, 2H), 7.34 (s, 1H), 7.18 (s, 2H), 7.10 (d, J=16.4 Hz, 1H), 6.97-7.02 (m, 3H), 3.22 (q, J=6.7 Hz, 2H), 2.96 (s, 6H), 2.19 (s, 6H), 1.55 (m, 2H), 0.90 (t, J=7.1 Hz, 3H); 13 C-NMR (125MHz, DMSO-d6) δ 166.63, 153.17, 150.42, 137.96, 135.66, 128.55, 127.96,
126.54, 125.42, 124.28, 112.60, 112.43, 109.77, 40.86, 40.12, 22.36, 16.55, 11.40; ESI-MS: m/z (MH+): 352.2151 (calc’d), 352.4699 (found).
S19
(1) Lashuel, H. A.; Wurth, C.; Woo, L.; Kelly, J. W. Biochemistry 1999, 38, 13560-73.
(2) Purkey, H. E.; Dorrell, M. I.; Kelly, J. W. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 5566-5571.