Ratiometric and intensity-based zinc sensors built on rhodol and rhodamine platforms

Transcription

1 Supporting Information Ratiometric and intensity-based zinc sensors built on rhodol and rhodamine platforms Elisa Tomat and Stephen J. Lippard* Department of Chemistry, Massachusetts Institute of Technology, Cambridge MA 02139, USA Scheme 1. Synthesis of sensors RF3 and RA1.

2 S2 Table S1. Photophysical properties of sensors RF3 and RA1. absorption λ max (nm), ε 10-4 (M -1 cm -1 ) emission λ max (nm), Φ ε Φ 10-4 unbound + Zn(II) unbound + Zn(II) unbound + Zn(II) RF3 514, 9.5(2) 495, 4.4(1) 540, 0.62(1) 523, 0.52(1) 5.9(1) 2.3(1) RA1 535, 4.5(1) 505, 0.13(2) 561, 0.70(1) 541, 0.78(1) 3.1(1) 0.88(5) Figure S1. Visible absorption spectra of RF3 (1.3 µm) before (blue) and after (red) addition of ZnCl 2 (300 µm) in neutral aqueous solution (50 mm PIPES buffer, 100 mm KCl, ph 7.0, 25 C). Figure S2. Normalized binding isotherm for the formation of a zinc complex of RF3 (50 mm PIPES buffer, 100 mm KCl, ph 7.0, 25 C, λ ex 463 nm).

3 S3 Figure S3. Benesi-Hildebrand plot for the zinc binding analysis of RF3, K d 22 ± 2 µm (50 mm PIPES buffer, 100 mm KCl, ph 7.0, 25 C, λ ex 463 nm). Figure S4. Selectivity chart for RF3 (1.0 µm in 50 mm PIPES buffer, 100 mm KCl, ph 7.0, 25 C, λ ex 463 nm). Light blue: 50 µm cation; dark blue: 500 µm cation; red: 500 µm zinc. Salts used: NaCl, CaCl 2, MgCl 2, MnCl 2, Fe(NH 4 ) 2 (SO 4 ) 2, CoCl 2, ZnCl 2, CdCl 2, HgCl 2. Addition of Ni(OAc) 2 and CuCl 2 caused quenching of the emission intensity and a ratio at wavelengths 523 and 540 nm could not be measured.

4 S4 Figure S5. Visible absorption spectra of RA1 (1.0 µm) before (purple) and after (orange) addition of ZnCl 2 (500 µm) in neutral aqueous solution (50 mm PIPES buffer, 100 mm KCl, ph 7.0, 25 C). Figure S6. Fit of the normalized fluorescence isotherm for RA1 to a 1:2 zinc binding model, K d ± 0.05 µm, K d2 2.2 ± 0.4 µm (50 mm PIPES buffer, 100 mm KCl, ph 7.0, 25 C, λ ex 490 nm).

5 S5 Figure S7. Selectivity charts for RA1 (1 µm in 50 mm PIPES buffer, 100 mm KCl, ph 7.0, 25 C, λ ex 490 nm). Orange: 50 µm cation; red: 500 µm cation; purple: 500 µm zinc. Salts used: NaCl, CaCl 2, MgCl 2, MnCl 2, Fe(NH 4 ) 2 (SO 4 ) 2, CoCl 2, Ni(OAc) 2, CuCl 2, CdCl 2, HgCl 2. After the second additions of Ni(OAc) 2 and CuCl 2 (final concentration: 500 µm), formation of a precipitate was observed and emission data points could not be recorded. Figure S8. Zinc response of RA1 recorded in aqueous solution buffered at ph values ranging from 5.5 to 9.0. Formation of a white precipitate was observed after the second zinc addition (final zinc concentration: 500 µm) at ph 8.5 and 9.0; intensity values in these conditions were not recorded.

6 S6 Experimental Methyl 6-formylpyridine-2-carboxylate (Chem. Eur. J. 2004, 10, ) and 2 -carboxy-5-chloro-2,4-dihydroxybenzophenone (J. Am. Chem. Soc. 2003, 125, ) were prepared according to published procedures. All other materials were purchased commercially and used as received. Reverse-phase HPLC purifications were carried out on an Agilent Technologies 1200 Series HPLC system. Column chromatography purifications were performed using Whatman silica gel (60 Å, mesh). Analytical thin layer chromatography (TLC) sheets were purchased from Mallinkrodt Baker, Inc. Preparative TLC plates (silica gel, 20 cm 20 cm, 0.5 mm thickness) were obtained from Analtech or EMD Biosciences. Deuterated NMR solvents were purchased from Cambridge Isotope Labs and used as received. NMR spectra were acquired on a Varian 300 MHz or 500 MHz spectrometer at ambient probe temperature (283 K) and referenced to the residual solvent peaks. Low-resolution mass spectra were acquired on an Agilent 1100 Series LC/MSD Trap spectrometer. High-resolution mass spectra were provided by staff at the MIT Department of Chemistry Instrumentation Facility. Aqueous solutions were prepared using Millipore water. Molecular biology grade piperazine-n,n -bis(2-ethanesulfonic acid) (PIPES) and % KCl were obtained from Aldrich. Spectrophotometric measurements at ph 7.0 were conducted in 50 mm PIPES, 100 mm KCl aqueous buffer. In order to remove any adventitious metal ions, this buffer solution was treated with Chelex resin (Bio-Rad) according to the manufacturer s protocol. A 100 mm zinc(ii) stock solution was prepared using % ZnCl 2 (Aldrich). Stock solutions of the fluorescent dyes (1-3 mm) were prepared in DMSO or MeOH, partitioned in 10-µL or 20-µL aliquots and stored in the dark at -20 C. UV-vis spectra were recorded on a Varian Cary 50 Bio UV-visible spectrophotometer. Fluorescence spectra were recorded on a Quanta Master 4 L- format scanning spectrofluorimeter (Photon Technology International). The acquisition temperature was kept at 25 ± 0.5 C by circulating water baths. Sample solutions were placed in quartz cuvettes (Starna) with 1-cm path lengths. Fluorescence data relative to the titration of RF3 with ZnCl 2 were analyzed by the Benesi-Hildebrand method and fit to the following equation: F max F 0 F F 0 =1+ K d [Zn], where [Zn] [Zn] tot Fluorescence data relative to the titration of RA1 with ZnCl 2 were fit to a 1:2 RA1/Zn binding model by non-linear least-squares methods according to the following equations: F = I 1 [RA1] + I 2 [RA1Zn] + I 3 [RA1Zn 2 ] F = [RA1] tot I 1 + I 2 K 1 [Zn] + I 3 K 1 K 2 [Zn]2 1+ K 1 [Zn] + K 1 K 2 [Zn] 2, where [Zn] [Zn] tot

7 S7 Compound 1. 3-Benzyloxyaniline (730 mg, 3.67 mmol) and methyl 6- formylpyridine-2-carboxylate (605 mg, 3.67 mmol) were dissolved in MeOH (20 ml) under a nitrogen atmosphere and stirred at room temperature for 1 h. The reaction mixture was then cooled to 0 C before Na(OAc) 3 BH (1.17 g, 5.50 mmol) was introduced. Stirring was continued for 1.5 h at 0 C. The yellow/orange solution was poured in a saturated aqueous NaHCO 3 solution (50 ml) and stirred for 15 min before being transferred to a separatory funnel. After extraction with CH 2 Cl 2 (5 x 40 ml), the combined organic phases were dried over anhydrous Na 2 SO 4 and evaporated to give a light orange oil. Purification by column chromatography (silica, 1:1 hexanes/ethyl acetate) afforded 1.30 g of amine 1 as a yellow oil (77%). 1 H NMR (500 MHz, CD 2 Cl 2 ): δ 3.96 (3H, s), 4.50 (2H, br d), 4.82 (1H, br t), 4.99 (2H, s), (3H, m), (1H, m), (5H, m), 7.52 (1H, dd), 7.80 (1H, t), 7.98 (1H, dd). 13 C NMR (125 MHz, CD 2 Cl 2 ): δ 49.41, 52.80, 69.89, 99.80, , , , , , , , , , , , , , MS (ESI): calcd [M + H] +, 349.1; found, 349.1; calcd [2M + Na] +, 719.3; found, Compound 2. Amine 1 (540 mg, 1.55 mmol) and 2-(bromomethyl)pyridine hydrobromide (470 mg, 1.86 mmol) were combined in CH 3 CN (20 ml). After addition of diisopropylethylamine (DIPEA, 0.3 ml) and KI (257 mg, 1.55 mmol), the reaction mixture was heated to reflux under a nitrogen atmosphere. After 2 days, the reaction mixture was allowed to cool to room temperature, diluted with water (50 ml), and extracted with CH 2 Cl 2 (5 x 40 ml). The organic phase was dried over anhydrous Na 2 SO 4 and evaporated to give a red oil. Purification by column chromatography (silica, 1:2 to 1:4 hexanes/ethyl acetate) afforded 333 mg of amine 2 as a yellow oil (49%). 1 H NMR (500 MHz, CD 2 Cl 2 ): δ 3.94 (3H, s), 4.79 (2H, s), 4.86 (2H, s), 4.93 (2H, s), (3H, m), 7.02 (1H, t), 7.18 (1H, m), (6H, m), 7.44 (1H, dd), 7.63 (1H, t), 7.77 (1H, t), 7.97 (1H, dd), 8.56 (1H, dd). 13 C NMR (125 MHz, CD 2 Cl 2 ): δ 53.12, 57.91, 58.00, 70.22, , , , , , , , , , , , , , , , , , , , , MS (ESI): calcd [M + H] +, 440.2; found, Rhodafluor compound 3 and rhodamine compound Carboxy-5-chloro- 2,4-dihydroxybenzophenone (60 mg, 0.21 mmol) was dissolved in trifluoroacetic acid (2 ml) and concentrated sulfuric acid (3 drops). Compound 2 (100 mg, 0.23 mmol) was dissolved in trifluoroacetic acid (2 ml) and added dropwise to the benzophenone solution. The reaction mixture was heated to 75 C overnight, evaporated to give a dark red oil, and dried under vacuum for 3 h. The crude solid was then dissolved in 2 ml of MeOH/CH 2 Cl 2 (2:8) and loaded on 4 separate preparative TLC plates, which were allowed to air dry for 1 h before elution was carried out with 15% MeOH in CH 2 Cl 2. Compound 3 (41 mg, 33%) was collected from a bright orange band (R f = 0.2), and compound 4 (25 mg, 13 %) was collected from a pink band that remained in part on the baseline (R f 0.01). Compound 3: 1 H NMR (500 MHz, CD 3 OD): δ 4.00 (3H, s), 5.51 (2H, s), 5.74 (2H, s), (2H, m), (4H, m), 7.85 (2H, q), 7.99 (1H, t), 8.11 (1H, t), (2H, m), 8.38 (1H, d), 8.72 (1H, t), 9.22 (1H, d). 13 C NMR (125 MHz, CD 3 OD): δ 53.29, 57.99, 58.05, 99.48, , , , , , , , , , , , , 130,79, , ,

8 S , , , , , , , , , , , , HR-MS (ESI): calcd [M + H] +, ; found, Compound 4. 1 H NMR (300 MHz, CD 3 OD): δ 3.99 (6H, s), 5.33 (4H, s), 5.53 (4H, s), (4H, m), 6.99 (2H, d), 7.21 (1H, d), 7.73 (2H, t), 7.85 (2H, dd), 7.97(2H, t), (7H, m), 8.59 (2H, t), 9.19 (2H, d). 13 C NMR (125 MHz, CD 3 OD): δ 53.75, 56.47, 58.88, , , , , , , , , , , , , , , , , , , , , , , , MS (ESI): calcd [M] +, ; found, Sensor RF3. A portion of K 2 CO 3 (17 mg, 0.12 mmol) was dissolved in water (1 ml) and added to a solution of rhodafluor 3 (15 mg, mmol) in MeOH (2 ml). The bright orange solution was stirred at room temperature for 5 h. The reaction progress was monitored by reverse-phase analytical HPLC. Upon completion, the reaction mixture was neutralized with 0.1 N HCl and precipitation of a bright orange solid was observed. The desired product RF3 was collected on a medium-porosity fritted filter and dried under vacuum (11 mg, 70%); mp > 250 C, dec. 1 H NMR (500 MHz, 1% CF 3 CO 2 D in CD 3 OD): δ 5.23 (2H, s), 5.45 (2H, s), 6.52 (1H, d), 6.59 (1H, s), 6.72 (1H, d), 6.88 (1H, s), 7.20 (1H, d), (2H, m), 7.87 (1H, d), (5H, m), 8.64 (1H, t), 9.31 (1H, d). HR-MS (ESI): calcd for [C 33 H 23 ClN 3 O 6 ] + = [M + H] +, ; found, Sensor RA1. K 2 CO 3 (10 mg, 0.08 mmol) was dissolved in water (1 ml) and added to a solution of rhodamine 4 (14 mg, mmol) in MeOH (2 ml). The mixture was stirred at room temperature for 5 h. The reaction progress was monitored by reverse-phase analytical HPLC. Upon completion, the reaction mixture was neutralized with 0.1 N HCl and precipitation of a bright pink solid occurred. The desired product RA1 was collected on a medium-porosity fritted filter and dried under vacuum (8 mg, 65%); mp > 190 C, dec. 1 H NMR (500 MHz, 1% CF 3 CO 2 D in CD 3 OD): δ 5.36 (4H, s), 5.58 (4H, s), (4H, m), 7.10 (2H, d), 7.25 (1H, d), (2H, m), 7.87 (2H, d), 8.00 (2H, t), (7H, m), 8.62 (2H, t), 9.21 (2H, d). HR-MS (ESI): calcd for [C 46 H 35 N 6 O 7 ] + = [M + H] +, ; found,

9 Figure S9. 1 H NMR spectra (500 MHz, 1% CF 3 CO 2 D in CD 3 OD) of final products RF3 and RA1. S9

10 S10 (a) (b) (c) Figure S10. Hydrolysis of rhodafluor ester 3; (a) starting material 3, (b) reaction mixture 3 h after the start of the hydrolysis, and (c) reaction product RF3.

11 S11 (a) (b) (c) Figure S11. Hydrolysis of rhodamine ester 4; (a) starting material 4, (b) reaction mixture 2 h after the start of the hydrolysis, and c) reaction product RA1.