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Near-infrared Fluorescent Probes with Large Stokes Shifts for Sensing Zn(II) Ions in Living Cells Shuwei Zhang, Rashmi Adhikari, Mingxi Fang, Nethaniah Dorh, Cong Li, Meghnath Jaishi, Jingtuo Zhang, Ashutosh Tiwari, Ranjit Pati, Fen-Tair Luo, and Haiying Liu ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.6b00490 • Publication Date (Web): 17 Nov 2016 Downloaded from http://pubs.acs.org on November 18, 2016
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ACS Sensors
Near-infrared Fluorescent Probes with Large Stokes Shifts for Sensing Zn(II) Ions in Living Cells Shuwei Zhang,† Rashmi Adhikari,† Mingxi Fang, † Nethaniah Dorh,† Cong Li,† Meghnath Jaishi,╫ Jingtuo Zhang,† Ashutosh Tiwari,*† Ranjit Pati,*╫ Fen-Tair Luo*‡ and Haiying Liu*† †
Department of Chemistry, Michigan Technological University, Houghton, MI 49931
╫
Department of Physics, Michigan Technological University, Houghton, MI 49931
‡
Institute of Chemistry, Academia Sinica, Taipei, Taiwan 11529, Republic of
KEYWORDS: Fluorescent probe, Near-infrared, Rhodol counterpart, Zinc(II) ions, Photoinduced electron transfer ABSTRACT: We report two new near-infrared fluorescent probes based on Rhodol counterpart fluorophore platforms functionalized with dipicolylamine Zn(II)-binding groups. The combinations of the pendant amines and fluorophores provide the probes with an effective three-nitrogen-atom and one-oxygen-atom binding motif. The fluorescent probes with large Stokes shifts offer sensitive and selective florescent responses to Zn(II) ions over other metal ions, allowing a reversible monitoring of Zn(II) concentration changes in living cells, and detecting intracellular zinc (II) ions released from intracellular metalloproteins.
Zinc (II), the second most abundant transition metal ion in the human body after iron, is often present in a tightly bound form in proteins. It plays several vital roles in a variety of physiological and pathological processes including cellular metabolism, gene expression, cell apoptosis, regulation of metalloenzymes, neural signal transmission and mammalian 1-3 reproduction. The disruption of zinc(II) homeostasis may contribute to Parkinson’s disease, Alzheimer's disease, amyotrophic lateral sclerosis, cerebral ischemia (ischemic stroke), epilepsy, prostate cancer, diabetes, immune dysfunction, and 1, 4-6 infantile diarrhea. Therefore, the development of fluorescent probes to effectively quantify and visualize Zn(II) concentration in biological systems is key to understanding of Zn(II) important roles in biological processes. Furthermore, the fluorescent probes with high sensitivity and spatial resolution of fluorescence would assist in identifying the molecular mechanisms that link zinc homeostasis and human pathophysiology. Several fluorescent probes for zinc(II) sensing have been developed by using different fluorophores 7, 8 9, 10 11 such as 2-aryl benzimidazoles, benzoxazoles, indoles, 12, 13 14 15 benzofurans, quinoline, dansyl, anthracene, 16, 17 18 19-25 24, coumarin, naphthalimide, fluorescein, Rhodamine, 26, 27 28-31 32 33 cyanine dye, BODIPY dyes, and porphyrins. They -10 can detect zinc (II) concentration in the range of 10 M in -4 1, 6, 34 the cytoplasm to 10 M in some vesicles. Most of these probes still suffer from some significant drawbacks such as auto-fluorescence and high light scattering. Near-infrared fluorescent probes have been developed to overcome these limitations by reducing background signals and photoinduced damages to biological samples. However, the nearinfrared (NIR) fluorescent probes for zinc (II) are still less common, as only a very few of them could be used to monitor the levels of endogenous zinc (II) in living cells and or-
28, 33, 35
ganisms. The search for readily accessible NIR fluorescent probes with excellent hydrophilic properties, high specificity, large Stokes shifts and dynamic responsive range is still a challenging task for imaging of zinc (II) concentration changes inside living cells. Fluorescent probes with large Stokes shifts possess advantages as spectral overlap between absorption and emission spectra can be eliminated. It allows for detection of fluorescence by avoiding measurement errors by excitation and scattered lights. In this paper, we report a rational design and synthesis of near-infrared fluorescent probes (A and B) with high Stokes shifts to image the changes in Zn(II) concentration in living cells by incorporating the di-2-picolylamine as a Zn-specific receptor into the near-infrared emissive Rhodol counterpart fluorophores (Figure 1). Fluorescent probes A and B display absorption peak at 576 nm and 586 nm, and weak fluorescence peak at 701 nm and 702 nm with large Stokes shifts of 125 nm and 116 nm in 10 mM HEPES buffer (pH 7.0), respectively. Near-infrared fluorescent probes A and B display sensitive and selective fluorescent responses to Zn(II) over most other metal ions with detection limits of 0.19 µM and 0.086 µM, allow for reversibly monitoring Zn(II) concentration in living cells, and sensitively detect intracellular levels of free zinc (II) ions which are released from intracellular metalloproteins when the cells were treated with 2,2’dithiodipyridine. First principles density functional calculations are carried out to gain electronic structure level understanding of the fluorescence behavior of probes A and B.
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tocol.37 Cells were serum starved for 3 h at 37 °C with 5% CO2 and then incubated with 2, 5 or 10 µM of probe A or B for 1 h at 37 °C with 5% CO2. To corresponding wells, 100 µM DTDP was added and cells were further incubated for 30 minutes at 37 °C with 5% CO2. Finally, cells were imaged at 20x or 60x using the inverted fluorescence microscope (Model AMF-4306; EVOS, AMG). Figure 1. Chemical structures of near-infrared fluorescent probes for Zn(II) ions.
Experimental Section Instrumentation 1
13
H NMR and C NMR spectra were collected by 400 MHz 1 Varian Unity Inova NMR spectrophotometer instrument. H 13 and C NMR spectra were recorded in CDCl3 solutions. Chemical shifts (δ) are given in ppm relative to solvent resid1 13 ual peaks ( H: δ 7.26 for CDCl3; C: δ 77.3 for CDCl3) as internal standards. High-resolution mass spectrometry data (HRMS) of the intermediates and fluorescent probes were measured with fast atom bombardment (FAB) ionization mass spectrometer, double focusing magnetic mass spectrometer or matrix assisted laser desorption/ionization time of flight mass spectrometer. Absorption spectra were taken on a Per-kin Elmer Lambda 35 UV/VIS spectrometer. Fluorescence spectra were recorded on a Jobin Yvon Fluoromax-4 spectrofluorometer.
Cell culture and Fluorescence imaging Breast cancer (MDA-MB-231) and normal endothelial (HUVEC-C) cell lines obtained from ATCC were cultured according to the published protocols.36 Cells were plated on 12-well culture plates at a density of 1 x 105 cells per mL for live cell imaging. After 24 h incubation at 37 °C with 5% CO2 incubator, the media was removed and cells were rinsed three times with PBS. Fresh serum free media with 2, 5 or 10 µM of probe A or B was added and then cells were further incubated for 1 h at 37 °C with 5% CO2. After the cells were rinsed three times with PBS, fresh serum free media with either 100 µM of Zn(II) or 100 µM Zn(II) plus sodium pyrithione (Pyr) was added. Cells were then incubated for 30 mins at 37 °C with 5% CO2. At this point, the cells were rinsed three times with PBS, and fresh serum free media was added before acquiring images using inverted fluorescence microscope (Model AMF-4306; EVOS, AMG). Fluorescence images were obtained at either 20x or 60x magnification and the exposure times were kept constant for each image series. 100 µM of TPEN (N,N,N’,N’-tetrakis (2-pyridylmethyl)ethylenediamine) was added to the wells containing 100 µM of Zn(II) + 100 µM of sodium pyrithione, and then cells were incubated for 10 mins at room temperature. Fluorescence images were acquired at 20x magnification using the inverted fluorescence microscope as described previously. Similar experiments with TPEN were also conducted by using 5 µM of each probe at either 10 µM or 30 µM of zinc (II) plus sodium pyrithione. The ability to detect intracellular levels of zinc(II) ions in HUVEC-C cells was evaluated using 2,2’-dithiodipyridine (DTDP) using a modified pro-
Materials Unless specifically indicated, all reagents and solvents were obtained from commercial suppliers and used without further purification. 3-((Bis(pyridin-2ylmethyl)amino)methyl)-4-hydroxybenzaldehyde (1)38, 9(2-carboxyphenyl)-6-(diethylamino)-1,2,3,4tetrahydroxanthylium perchlorate (2)39-40 and 4-(2carboxyphenyl)-7-(diethylamino)-2-methylchromenylium perchlorate (3)41 were prepared according to reported procedures. Synthesis of fluorescent probes A and B Fluorescent probe A: 3-((Bis(pyridin-2ylmethyl)amino)methyl)-4-hydroxybenzaldehyde (1) (0.30 g, 0.90 mmol) and 9-(2-carboxyphenyl)-6-(diethylamino)1,2,3,4-tetrahydroxanthylium perchlorate (2) (0.43 g, 0.90 mmol) were dissolved in acetic acid (10mL) in a 25 mL round bottom flask. The reaction mixture was heated at 100 °C and stirred for 3 h. When the solvent was evaporated under reduced pressure, the crude product was purified by silica gel chromatography using CH2Cl2/CH3OH/ CH3COOC2H5/CH3COOH (20/2/10/0.32, v/v/v/v) as eluent to obtain fluorescent probe A as deep blue solid (0.12 g, 16.8%). 1 H NMR (400 MHz, CDCl3) δ 8.51 (d, J = 4.0 Hz, 2H), 8.04 (d, J = 8.0 Hz, 1H), 7.62-7.58 (m, 3H), 7.54-7.51 (m, 2H), 7.31-7.26 (m, 3H), 7.23 (s, 1H), 7.15-7.11 (m, 3H), 6.86 (d, J = 8.0 Hz, 1H), 6.65-6.62 (m, 2H), 6.50-6.47 (m, 1H), 3.85-3.78 (m, 6H), 3.42-3.37 (m, 4H), 2.78-2.64 (m, 2H), 2.15-2.11 (m, 1H), 1.8313 1.79 (m, 1H), 1.63-1.59 (m, 2H), 1.17 (t, J = 4.0 Hz, 6H); C NMR (100 MHz, CDCl3) δ 169.9, 158.3, 158.1, 154.7, 151.6, 148.8, 137.4, 133.6, 133.3, 131.4, 129.6, 129.4, 127.7, 127.3, 125.3, 123.5, 123.3, 122.6, 116.9, 111.7, 97.0, 59.0, 57.0, 45.2, 27.5, 24.3, 22.3, + + 12.8. HRMS (ESI): calculated for C44H43N4O4 [M-ClO4] , 691.3279; found, 691.3275. Fluorescent probe B: 3-((Bis(pyridin-2ylmethyl)amino)methyl)-4-hydroxybenzaldehyde (1) (0.12 g, 0.26 mmol) and 4-(2-carboxyphenyl)-7-(diethylamino)methylchromenylium perchlorate (3) (0.09 g, 0.26 mmol) were dissolved in acetic acid (4 mL) in a 10 mL round bottom flask. The reaction mixture was heated at 100 °C and stirred for 2 h. After the solvent was evaporated under reduced pressure, the crude product was purified by silica gel chromatography using CH2Cl2/CH3OH/CH3COOC2H5/ CH3COOH (30/3/10/0.4, v/v/v/v) as eluent to obtain fluorescent probe B 1 as deep blue solid (0.04 g, 19.3%). H NMR (400 MHz, CDCl3) δ 8.57 (d, J = 4.0 Hz, 2H), 7.93 (d, J = 4.0 Hz, 1H), 7.65-7.61 (m, 3H), 7.56-7.52 (m, 2H), 7.37-7.30 (m, 3H), 7.26-7,24 (m, 2H), 7.19-7.16 (m, 3H), 6.91 (d, J = 8.0 Hz, 1H), 6.55 (d, J = 8.0 Hz, 1H), 6.47-6.45 (m, 1H), 6.41-6.37 (m, 1H), 3.89 (s, 4H), 3.81 13 (s, 2H), 3.40-3.34 (m, 4H), 1.18 (t, J = 4.0 Hz, 6H); C NMR (100 MHz, CDCl3) δ 170.2, 158.8, 158.3, 153.3, 152.7, 149.8, 148.9, 137.3, 134.4, 132.3, 129.7, 129.5, 128.8, 128.3, 127.3, 127.2, 124.2, 123.5, 122.6, 118.3, 117.4, 109.5,105.9, 100.5, 97.9, 94.6, 59.1, 57.2,
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Results and Discussion Synthetic approach to fluorescent probes A and B Dipicolylamine (DPA), a zinc binding ligand with three nitrogen atoms for zinc(II) chelation, was incorporated into near-infrared emissive Rhodol counterpart fluorophores where an oxygen atom from a hydroxyl group can involve in zinc(II) chelation to enhance zinc(II) binding strengths, we conducted a condensation reaction of 3-((bis(pyridin-2ylmethyl)amino)methyl)-4-hydroxybenzaldehyde with 9-(2carboxyphenyl)-6-(diethylamino)-1,2,3,4tetrahydroxanthylium perchlorate (2), and 4-(2carboxyphenyl)-7-(diethylamino)-methylchromenylium perchlorate (3) in acetic acid solution at 100 °C, respectively, affording fluorescent probes for Zn(II) (A and B) (Scheme 1). We intentionally controlled one carbon chain length between zinc-binding ligand and the fluorophores in order to manipulate the sensitive fluorescence responses of the probes to Zn(II) ions via effective photo-induced electron transfer effect of a tertiary amine from zinc-binding ligand as an electron donor to the fluorophores. The fluorescent 1 13 probes were characterized by H NMR, C NMR and highresolution mass spectrometry.
tween two bulky groups in a trans configuration in a double bond (Scheme 1).
Optical responses of fluorescent probes A and B to Zn(II) ions The optical responses of the fluorescent probes to Zn(II) ions were investigated in aqueous HEPES buffer (pH 7.0) containing 1% EtOH solution. Probe A displays a main absorption peak at 576 nm and a shoulder peak at 430 nm. Upon gradual addition of Zn(II) to 10 µM solution of fluorescent probe A, the main and shoulder absorption peaks of the probe A undergo red shifts by 26 and 20 nm, respectively , and absorbance of the probe A increases as the molar absorptivity of probe A increases from 1.77 x 104 M-1cm-1 in the absence of Zn(II) ion and to 2.06 104 M-1cm-1 in the presence of 10 µM Zn(II) ions (Figure 2). Fluorescent probe B exhibits a main broad absorption peak at 586 nm and a shoulder peak at 435 nm in the same buffer solution. Gradual addition of Zn(II) to 10 µM fluorescent probe B solution triggers red shifts of the main and shoulder absorption peaks by 38 and 21 nm, respectively, and results in enhancement of the absorbance as the molar absorptivity of probe B increases from 1.80 x 104 M-1cm-1 in the absence of Zn(II) ion and to 2.10 104 M-1cm-1 in the presence of 10 µM Zn(II) ions (Figure 2). Blank 1.0 µM
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44.7, 30.0, 12.8. HRMS (ESI): calculated for C41H39N4O4 [M+ ClO4] , 651.2966; found, 651.2966.
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Scheme 1. Synthetic route to fluorescent probes A and B.
Optical properties of fluorescent probes A and B in different solvents. All optical data of fluorescent probes were obtained with 10 µM concentration of fluorescent probe A or B in different solvents. Fluorescent probes A and B were very stable in 10 mM HEPES buffer (pH 7.0) containing 1% EtOH solution. These fluorescent probes possess advantageous photophysical properties including a large absorption extinction coefficient, excellent photostability, and large Stokes shifts with near-infrared emission (Table 1 and Figure S7). Probes A and B display large Stokes shifts of 111 nm and 101 nm with absorption and emission peaks at 581 nm, 594 nm and 692 nm, 695 nm in ethanol solution, respectively. However, the fluorescent probes exhibit smaller Stoke shifts in less polar solvents such as dichloromethane and toluene. The fluorescent probes possess low fluorescence quantum yields in different solvents (Table 1). Probe B shows slightly longer absorption and emission wavelengths in all solvents because of its slightly better π-conjugation than probe A although they have very similar structures (Table 1). The slightly better π-conjugation of the fluorescent probe B is due to less steric hindrance be-
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Figure 2. UV-vis absorption spectra of 10 µM probes A (a) and B (b) upon gradual addition of Zn(II) from 1 µM to 20 µM in 10 mM HEPES buffer solutions (pH 7.0). Fluorescent probe A displays a very weak fluorescence peak at 701 nm with a fluorescence quantum yield of 0.06% in HEPES buffer (pH 7.0) containing 1% EtOH solution in the absence of Zn(II) ions. This is due to fluorescence quenching effect via photo-induced electron transfer from the tertiary amine of a Zn(II)-binding dipicolylamine ligand as an electron donor to the Rhodol counterpart fluorophore (Table 1). Gradual addition of Zn(II) to 10 µM solution of fluorescent probe A causes significant fluorescence increases because binding of Zn(II) to the dipicolylamine ligand effectively suppresses the fluorescence quenching effect, and recovers the probe fluorescence (Figure 3). The probe A shows a linear fluorescence response to Zn(II) from 0.3 µM to 4.0 µM with a detection limit of 0.19 µM (Figure S8). The 1:1 stoichiometry was further confirmed by a good non-linear fitting of the titration data at 701 nm by assuming a 1:1 association between fluorescent probe A and Zn(II) (Figure S10). Fluorescent probe B displays a slight higher fluorescence background with fluorescence quantum yield of 0.10% in HEPES buffer than the probe A (Figure 3), and possesses a linear fluorescence response to Zn(II) from 0.3 µM to 4.0 µM with a lower detection limit of 0.086 µM when compared to the probe A (Figure S9). However, probe B displays similar fluo-
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ACS Sensors rescent responses to zinc (II) ions with 1:1 stoichiometry which is similar as probe A. This 1:1 binding stoichiometry between probe B and Zn(II) was verified by Job’s plot that is based on non-linear fitting of the titration curve using 1:1 binding model (Figure S11). Blank
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Zn(II) for both the probes A and B. The dihedral angle between the two pyridinal planes of DPA in probes A and B are calculated to be 115.12ᴼ and 102.12ᴼ respectively. Upon the addition of Zn(II) ion, the dihedral angle changes to 132.90ᴼ and 129.90ᴼ in the so formed Zn(II)/probe A and B complexes, respectively, suggesting a significant conformational change upon binding. The calculated Zn-N and Zn-O bonding distances in Zn(II)/probes A and B complexes are explicitly shown above in Figures 4b and 4d. Further analysis reveals that the addition of Zn(II) ion in the probes A and B is also accompanied by the redistribution of charge between Zn(II) ions and the associated ligands. The Mulliken charges on three nitrogen atoms and an oxygen atom in probes A and B before binding to Zn(II) are found 0.57 e, 0.08 e, -0.07 e, 0.20 e and 0.68 e, -0.07 e, -0.09 e, -0.21 e respectively. Upon the addition of Zn(II), the charge configuration in these species are found 0.43 e, 0.21 e, 0.03 e, -0.34 e and 0.44 e, 0.18 e, 0.04 e, -0.32 e respectively. On the other hand, the charge on Zn ion in fluorescent complex A and B after binding are found to be 1.21 e and 1.12 e, respectively.
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Figure 3. Fluorescence spectra of 10 µM fluorescent probes A (a) and B (b) upon gradual addition of Zn(II) from 1 µM to 20 µM in 10 mM HEPES buffer solution (pH 7.0) after excitation at 595 nm. Fluorescence responsive curves of fluorescent probes A (c) and B (d) to Zn(II) ions.
Table 1. Optical properties of fluorescent probes A and B.
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671
2.68
0.49
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625
668
0.62
