Supporting Information

Transcription

1 Supporting Information A Highly Selective Fluorescence Turn-on Sensor for Cysteine/Homocysteine and its Application in Bioimaging Meng Zhang, Mengxiao Yu, Fuyou Li,*, Minwei Zhu, Manyu Li, Yanhong Gao, Lei Li, Zhiqiang Liu, Jianping Zhang, Dengqing Zhang, Tao Yi and Chunhui Huang*, Department of Chemistry & Laboratory of Advanced Materials, Fudan University, Shanghai, 2433; Hospital of Obstetrics and Gynecology, Fudan University, Shanghai, 211, State Key Laboratory for Structural Chemistry of Unstable and Stable Species, Institute of Chemistry, Chinese Academy of Sciences, Beijing, 18; Hospital of Xinhua, Medicine School of Shanghai Jiaotong University, Shanghai, 292, P.R.China. To whom correspondence should be addressed. fyli@fudan.edu.cn; hch@chem.pku.edu.cn 1

2 Materials Acenaphthylene-1,2-dione and HEPES were purchased from Acros Organics, and all amino acids were used as received. All other chemical reagents were purchased from Sigma-Aldrich and were used as received. Millipore water was used to prepare all aqueous solutions. All spectroscopic measurements were performed in methanol HEPES (7:3, v/v, ph 7) solution. Synthesis of 2 O O NC CN O NC O N NC O N Malononitrile K 2 CO 3, 3-mercapto-propionic acid CH 3 CN, reflux CH 3 CN, reflux MeOH, r.t 3 1 S COOH 2 Scheme S1. The synthetic route to compound 2. 2-(2-Oxo-2H-acenaphthylen-1-ylidene)-malononitrile (3). Acenaphthylene-1,2-dione (1 mmol) and malononitrile (1 mmol) in CH 3 CN (3 ml) were refluxed for 3 hours. After cooling, the precipitate was filtrated and washed several times with CH 3 CN to yield an orange solid. (2.1 g, 91%). m.p C; 8-Oxo-8H-acenaphtho[1,2-b]pyrrole-9-carbonitrile (1) 1 2-(2-Oxo-2H-acenaphthylen-1-ylidene)-malononitrile (1 mmol) and K 2 CO 3 (1 mmol) in CH 3 CN (3 ml) were refluxed for 1 h. After cooling, the precipitate was filtrated and purified by recrystallization from CH 3 CN to yield a brown needle solid. (2. g, 87%). m.p C (lit. m.p C). 1 H NMR (4 Hz, DMSO-d 6 ) δ (ppm): (d, 2H), (d, Hz, 2H), (m, 2H); EI-MS, m/z: 23 (M + ); 3-(9-Cyano-8-oxo-8H-acenaphtho[1,2-b]pyrrol-3-ylsulfanyl)-propionic acid (2) 8-Oxo-8H-acenaphtho[1,2-b]pyrrole-9-carbonitrile (.13 g,.57 mmol) was added dropwise to a solution of 3-thiopropionic acid ( ml, 1.14 mmol) in 3 ml methanol, and then the mixture was stirred at room temperature. After a few minutes, the suspension became red and 2

3 was left to react for 2 hours. Then the suspension was filtered and concentrated. The residue was purified by chromatography using CH 2 Cl 2 to yield 2 (.84 g, 46%). m.p C; 1 H NMR (4Hz, DMSO-d 6 ) δ (ppm): (s, 1H), 8.68 (s, 1H), 8.57 (s, 1H), 8.5 (s, 1H), 7.95 (s, 1H), 7.77 (s, 1H), (t, J = 6.8 Hz, 2H), (t, J = 7.2 Hz, 2H); IR (KBr) ν/cm -1 : 336, 2231, 175, 1632, 1567, 1496; EI-MS, m/z: 334 (M + ); Anal. calcd. for C 18 H 1 N 2 O 3 S: C 64.66, H 3.1, N Found: C 64.85, H 3.5, N 8.5 %. CAUTION: Methanol is a toxic organic solvent. All reactions involving this reagent were carried out inside a ventilation cabinet. The operator wore personal protective equipment such as a laboratory coat, gloves and mask in the tests. Methods. NMR spectra were recorded at a Varion Gemin-4 4 MHz spectrometer. All chemical shifts are reported in the standard δ notation of parts per million. IR spectra were obtained on an IRPRESTIGE-21 spectrometer using KBr pellets. Mass spectra were recorded with a MA1212 mass spectroscope. The element analyses were performed on a VarioEL III O-Element Analyzer system. Electrospray ionization mass spectra (ESI-MS) were measured on a Micromass LCTTM system. Spectroscopic Measurements. UV-Vis spectra were recorded on a Shimadzu 3 spectrophotometer. Fluorescence spectra were measured on an Edinburgh LFS92 luminescence spectrometer with 1 W xenon lamp. Samples for absorption and emission measurements were contained in 1-cm 1-cm quartz cuvettes. The luminescence quantum yields in solution were measured by using rhodamine B (Φ F =.49 in ethanol) as a reference 2. The quantum yield Ф as a function solvent polarity is calculated using the following equation. Фsample=Фstd Isample Istd Astd Asample nsample nstd 2 Where subscript sample and std denote the sample and standard, respectively, Ф is quantum yield, I is the integrated emission intensity, A stands for the absorbance, n is refractive index. CAUTION: Methanol is a toxic organic solvent. All the spectroscopic experiments were performed in good ventilation condition. The operator wore personal protective equipment such as a laboratory coat, gloves and mask. 3

4 TPA cross-section Determination. Two-photon absorption cross-sections were determined by the method of two-photon induced fluorescence using Rhodamine B as a standard with known two-photon absorption cross-sections. 3 A regenerative amplifier (Spitfire, Spectra Physics) which was seeded by a mode-locked Ti:Sapphire laser (Tsunami, Spectra Physics) produced laser beam centered at 795 nm and pulse width of about 12 fs. The laser was used to drive an optical parameter amplifier (OPA-8CF, Spectra Physics) and deliver our desired wavelength range of 69~15 nm. Then it was focused into a quartz cuvette with an optical path length of 1 mm. The two-photon induced fluorescence was collected with a right angle and sent to a polychromator (Spectropro-55i, Acton) equipped with a liquid-nitrogen-cooled CCD detector (SPEC-1-4B/LbN, Roper Scientific). To reject the interference of stray laser light, a 1-mm long saturated aqueous solution of CuSO 4 was placed in front of the entrance slit. The samples were dissolved in methanol at a concentration of 1-4 M, and Rhodamine B in ethanol at the same concentration. Two-photon absorption cross-section (δ) was obtained as follows, F Φ C n δ S δ S R R S = R. (1) FR Φ SCSnR Where subscripts S and R denote the sample and the reference, respectively, F represents the intensity of two-photon induced fluorescence, Φ stands for fluorescence quantum yield, C for concentration, and n for refractive index of the solvents. Cell Culture. The ACCM and Caov-3 cell lines were provided by Institute of Biochemistry and Cell Biology (China). The PANC and HeLa cell lines were provided by Peking University Health Science Center (China). Cells were grown in H-DMEM (Dulbecco s Modified Eagle s Medium, High Glucose) supplemented with 1 % FBS (Fetal Bovine Serum) in an atmosphere of 5 % CO 2, 95 % air at 37 o C. Cells ( / L) were plated on 18 mm glass coverslips and allowed to adhere for 24 hours. 4

5 Fluorescence Imaging. Confocal fluorescence imaging was performed with a Zeiss LSM51 Axioskop 2FS laser scanning microscope with a 4x water-immersion objective lens. Excitation of 1-loaded cells at 543 nm was carried out with a HeNe laser. Emission was collected using a 56 nm longpass filter. Two-photon fluorescence imaging was realized with an OLYMPUS BX61W1 laser scanning microscopy with 4x water-immersion objective lens. Two-photon fluorescence was excited with a Ti:sapphire femtosecond laser source (Coherent Chamelon Ultra) set at 88 nm and with output power of 2 W, which corresponds to an average power of approximately 5 mw in the focal plane. Fluorescence emission from 555 to 655 nm was collected. For fixed cell imaging, immediately before the experiments, cells were washed with the PBS buffer and then incubated with 1 µm 1 in ethanol HEPES (7:3, v/v, ph 7) for 1 min at 25 C. Fluorescence imaging was then carried out after washing cells with the PBS buffer. For living cell imaging, cells were incubated with 5 μm 1 in DMSO-PBS (1:49, v/v, ph 7) for 1 min at 25 C. For the control experiment, living Caov-3 cells were pretreated with a DMSO-PBS (1:49, v/v, ph 7) solution of 5 μm N-ethylmaleimide for 2 hrs in an atmosphere of 5 % CO 2, 95 % air at 37 o C, and then incubated with 5 μm 1 in DMSO-PBS (1:49, v/v, ph 7) for 1 min at 25 C. Fluorescence imaging was then carried out after washing cells with the PBS buffer. 5

6 Fluorescence Intensity (a.u.) Figure S1. Normalized single-photon absorption ( ) and fluorescence ( ) spectra of 2 (1 µm) in methanol δ (GM) Figure S2. Two-photon absorption spectrum of 2 (1 µm) in methanol. 6

7 ph Figure S3. Influence of ph on absorbance at 58 nm for 1 (1 µm) in methanol water solution (7:3, v/v) ph Figure S4. Influence of ph on absorbance at 58 nm for 1 (1 µm) in the presence of Cys (2 µm) in methanol-water solution (7:3, v/v). 7

8 GSH Cys Hcy only Time (min) Figure S5. Time-course of absorbance at 58 nm for 1 (1 µm) in the absence and presence of 2 equiv of Hcy, Cys and GSH in 1 min (25 C). 8

9 Ala Arg Asn Asp Gln His Iso Leu Lys Met 9

10 Gly Pro Ser Thr Try Tyr Val GSH Figure S6. Absorption spectra of 1 (1 µm) upon addition of various amino acids and reduced glutathione (1 µm) in methanol HEPES solution (7:3, v/v, ph 7). 1

11 .4 Hcy Figure S7. Absorption spectral change of 1 (1 μm) upon addition of Hcy ( 33 μm) in methanol HEPES solution (7:3, v/v, ph 7). Each spectrum is acquired 1 min after Hcy addition. Fluorescent intensity (a.u) Cys 2 μμ Fluorescence intensity (a. u) R 2 = [Cysteine] 1-5 M Figure S8. Fluorescence spectral change of 1 (5 μm) upon addition of Cys (2.5 2 μm) in methanol HEPES solution (7:3, v/v, ph 7). Inset:The fluorescent intensity (58 nm) plot vs concentration of Cys (5 5 μm) (λ ex = 465 nm). Each spectrum is acquired 1 min after Cys addition. 11

12 Fluorescence Intensity (a.u.) Hcy Fluorescence intensity (a.u) R 2 = [Homocysteine] 1-5 M Figure S9. Fluorescence spectral change of 1 (5 μm) upon addition of Hcy ( 2 μm) in methanol HEPES solution (7:3, v/v, ph 7) (λ ex = 465 nm). Inset: The fluorescent intensity (58 nm) plot vs concentration of Hcy (5 3 μm). Each spectrum is acquired 1 min after Hcy addition. Figure S1. Fluorescence photograph of 1 (1 μm) in the absence and presence of Cys and Hcy (1 μm) in methanol HEPES solution (7:3, v/v, ph 7) under irradiation of a 365 nm UV lamp. 12

13 Figure S11. Two-photon absorption and fluorescence spectra of 1-Cys in methanol HEPES solution (7:3, v/v, ph 7). Fluorescence Intensity (a.u.) Cys 1+Cys+other amino acids and GSH 1+various amino acids and GSH Figure S12. Fluorescence spectra of 1 (5 μm) containing Cys (1 μm) or/and other selected amino acids (1 mm) in methanol HEPES solution (λ ex = 465 nm). 13

14 Fluorescence Intensity (a.u.) Hcy 1+Hcy+other amino acids and GSH 1+ various amino acids and GSH Figure S13. Fluorescence spectra of 1 (5 μm) containing Hcy (1 μm) or/and other selected amino acids (1 mm) in methanol HEPES solution (λ ex = 465 nm). 14

15 a 4 3 F f / F i b 4 3 F f / F i c 4 3 F f / F i Figure S14. (a) Fluorescence responses of 1 (5 μm) to various amino acids (25 μm for Cys and Hcy, 125 μm for all other amino acids and GSH). (b) Fluorescence responses of 1 (5 μm) to25 μm Cys with or without other various amino acids and GSH (125 μm). (c) Fluorescence responses of 1 (5 μm) to 25 μm Hcy with or without other various amino acids and GSH (125 μm). Bars represent the final fluorescence intensity (F f ) over the initial intensity (F i ) at 588 nm (λ ex = 465 nm).condition: in methanol HEPES solution (7:3, v/v, ph 7). 1, Cys; 2, Hcy; 3, Gly; 4, Ala; 5, Val; 6, Leu; 7, Thr; 8, Gln; 9, Asn; 1, Met; 11, Ser; 12, Pro; 13, Tyr; 14, Try; 15, Asp; 16, Glu; 17, Lys; 18, Arg; 19, His; 2, Iso; 21, GSH; 22, the mixture of GSH and all other amino acids except Cys and Hcy. 15

16 Fluorescence Intensity (a.u.) Cys Cys + NEM +1 Cys + NEM Cys Wavelength / nm Figure S15. The fluorescence spectra of 1 (1 μm) in the presence and absence of Cys (3 μm) and N-ethylmaleimide (NEM) (1 mm) in DMSO-PBS (1:49, v/v, ph 7) solution (λ ex = 543 nm). Cys solution was pretreated with NEM for 2 hrs at 25 o C, and then 1 was added. Figure S16. Confocal fluorescence and brightfield images of Caov-3 cells. (a) Fluorescence image of cells incubated with 5 μm 1 for 1 min at 25 o C (λ ex = 543 nm). (b) Brightfield image of cells shown in panel a. The overlay image of (a) and (b) is shown in (c). Figure S17. Confocal fluorescence and brightfield images of Caov-3 cells. The Caov-3 cells were pretreated with 5 μm N-ethylmaleimide for 2 hrs in an atmosphere of 5 % CO 2, 95 % air at 37 o C, and then incubated with 5 μm 1 for 1 min. (a) Fluorescence image of cells (λ ex = 543 nm). (b) Brightfield image of cells shown in panel a. The overlay image of (a) and (b) is shown in (c). 16

17 References: (1) Xiao, Y.; Liu, F.; Qian, X.; Cui, J. Chem. Commun., 25, 2, 239. (2) Casey, K. G.; Quitevis, E. L. J. Phys. Chem. 1998, 92, 659. (3) Xu, C.; Webb, W. W. J. Opt. Soc. Am. B 1996, 13,