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Zinc-specific intramolecular excimer formation in TQEN derivatives: fluorescence and zinc binding properties of TPEN-based hexadentate ligands† Yuji Mikata,*a,b Saaya Takeuchi,b Eri Higuchi,b Ayaka Ochi,b Hideo Konno,c Kazuma Yanaid and Shin-ichiro Satoe Zn2+-induced fluorescence enhancement of the TPEN (N,N,N’,N’-tetrakis(2-pyridylmethyl)ethylenediamine)based ligand, N,N-bis(1-isoquinolylmethyl)-N’,N’-bis(pyridylmethyl)ethylenediamine (N,N-1-isoBQBPEN, 1b), has been investigated. Upon Zn2+ binding, 1b shows a fluorescence increase (ϕZn = 0.028) at 353 and 475 nm. The fluorescence enhancement at longer wavelengths is due to intramolecular excimer formation of two isoquinolines and is specific for Zn2+; Cd2+ induces very small fluorescence at 475 nm (ICd/IZn = 10%). The excimer formation of the [Zn(1b)]2+ complex in the excited state is supported by the time-dependent DFT calculation. Neither long-wavelength fluorescence nor excimer formation is
Received 20th June 2014, Accepted 29th August 2014
observed in the Zn2+ complex of N,N’-1-isoBQBPEN (2b). The quinoline analog N,N-BQBPEN (1a) exhibits similar but significantly smaller excimer formation. Thermodynamic and kinetic comparisons of Zn2+
DOI: 10.1039/c4dt01847h
binding properties of ethylenediamine-based hexadentate ligands with pyridines and (iso)quinolines are
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comprehensively discussed.
Introduction Zinc is an indispensable element in living systems because of many important biological roles of this essential metal.1–6 Zinc exists in mobile and protein-bound forms in cells. Mobile zinc is a modulator of signaling processes and key element in gene expression. Many fluorescent zinc sensor molecules that reveal the distribution of unbound zinc in living cells have been extensively investigated in the recent two decades to understand zinc biology.4–18 Such fluorescent zinc probes often respond to cadmium because both Zn2+ and Cd2+ possess an occupied d10 shell and similar coordination preferences. The only difference in these two metals is the ionic radii of approximately 21 pm. So, new design strategies to visualize Zn2+ in high selectivity and sensitivity, especially
discriminating Zn2+ from Cd2+ by ionic radius, coordination environment or other ligand effects, are of considerable interest.19–26 We have previously reported the quinoline analog of the TPEN (N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine)based fluorescent zinc probe, TQEN (N,N,N′,N′-tetrakis(2-quinolylmethyl)ethylenediamine) (Chart 1).27 In recent years, many other quinoline-based fluorescent Zn2+ sensors have been developed.24,28–43 We also reported that the isoquinoline derivatives such as 1-isoTQEN (Chart 1)44 and 1-isoTQA45 (tris(1-isoquinolylmethyl)amine) exhibit high fluorescence intensity and zinc specificity in their long wavelength emission enhancement. The high Zn2+ specificity is hypothesized to be
a
KYOUSEI Science Center, Nara Women’s University, Nara 630-8506, Japan. E-mail: [email protected] b Department of Chemistry, Faculty of Science, Nara Women’s University, Nara 630-8506, Japan c National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, Ibaraki 305-8565, Japan d Graduate School of Chemical Sciences and Engineering, Hokkaido University, Sapporo 060-8628, Japan e Graduate School of Engineering, Hokkaido University, Sapporo 060-8628, Japan † Electronic supplementary information (ESI) available: The experimental procedure for synthesis of the compounds, Tables S1–S3, Scheme S1, Fig. S1–S31 and crystallographic data in CIF format. CCDC 998434–998437. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c4dt01847h
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Chart 1
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due to the formation of an intramolecular excimer by two adjacent isoquinoline rings upon Zn2+ binding. There are limited numbers of other isoquinoline-based fluorescent sensors in the literature.24,42,46 In this paper, we describe the spectral and theoretical studies of zinc-induced fluorescence enhancement of N,N-1isoBQBPEN (N,N-bis(1-isoquinolylmethyl)-N′,N′-bis(2-pyridylmethyl)ethylenediamine, 1b) and N,N′-1-isoBQBPEN (N,N′bis(1-isoquinolylmethyl)-N,N′-bis(2-pyridylmethyl)ethylenediamine, 2b) as benzene-depleted derivatives of 1-isoTQEN (Chart 1). These compounds are molecular hybrids of 1-isoTQEN and TPEN. Reduction of the molecular weight and aromatic ring size without losing necessary fluorescence properties would be an attractive improvement for application to cell study. The corresponding quinoline analogs, N,N-BQBPEN (N,N-bis(2-pyridylmethyl)-N′,N′-bis(2-quinolylmethyl)ethylenediamine, 1a) and N,N′-BQBPEN (N,N′-bis(2-pyridylmethyl)N,N′-bis(2-quinolylmethyl)ethylenediamine, 2a), are also examined. Replacement of two isoquinoline rings of 1isoTQEN with pyridines in the N,N-configuration does not affect the fluorescence of the Zn2+ complex. However, the N, N′-isomer completely loses long-wavelength emission. The time-dependent DFT calculation reveals that the excited-state intramolecular excimer, in which two isoquinoline moieties are in stacked parallel arrangement, is responsible for the long wavelength emission. The metal binding competition with TPEN demonstrates the thermodynamic and kinetic superiorities of 1-isoBQBPEN ligands.
Experimental General All reagents and solvents used for synthesis were from commercial sources and used as received. N,N-Dimethylformamide (DMF, Dojin) was of spectral grade (Spectrosol). All aqueous solutions were prepared using Milli-Q water (Millipore). 1H NMR (300 Hz) and 13C NMR (75.5 Hz) spectra were recorded on a Varian GEMINI 2000 spectrometer and referenced to internal Si(CH3)4 or solvent signals. UV-vis and fluorescence spectra were measured on a Jasco V-660 spectrophotometer and Jasco FP-6300 spectrofluorometer, respectively. Fluorescence quantum yields were measured on a HAMAMATSU photonics C9920-02 absolute PL quantum yield measurement system. Fluorescence lifetimes were measured on a HORIBA FluoroCube 5000U system. Caution: Perchlorate salts of metal complexes with organic ligands are potentially explosive. All due precautions should be taken. N,N-Bis(2-pyridylmethyl)-N′,N′-bis(2-quinolylmethyl)ethylenediamine (N,N-BQBPEN, 1a) To the dry CH3CN solution (40 mL) of 2-chloromethylquinoline hydrochloride (428 mg, 2.0 mmol) and N,N-bis(2-pyridylmethyl)ethylenediamine47 (4, Scheme S1†) (240 mg, 1.0 mmol) were added potassium carbonate (1.4 g, 10 mmol) and potassium iodide (332 mg, 2.0 mmol), and then refluxed for 2 days
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under N2. After the resulting solution was cooled to room temperature, the solid materials were filtered off and the filtrate was evaporated. Crystallization of the residue from CH3CN under ether diffusion conditions afforded N,NBQBPEN (1a) as yellow powder (160 mg, 0.3 mmol, 30%). 1 H NMR (CDCl3): δ 8.42 (ddd, J = 4.8, 1.8, 0.9 Hz, 2H), 8.02 (d, J = 8.2 Hz, 2H), 8.02 (d, J = 8.2 Hz, 2H), 7.75 (d, J = 8.2, 1.5 Hz, 2H), 7.61–7.69 (m, 4H), 7.48 (ddd, J = 8.2, 7.0, 1.2 Hz, 2H), 7.41 (ddd, J = 8.8, 7.3, 1.5 Hz, 2H), 7.33 (d, J = 7.6 Hz, 2H), 7.01 (ddd, J = 6.1, 4.9, 1.2 Hz, 2H), 3.97 (s, 4H), 3.76 (s, 4H), 2.80–2.83 (m, 4H). 13 C NMR (CDCl3): δ 160.1, 159.3, 148.6, 147.3, 136.1, 129.2, 128.9, 127.4, 127.2, 126.0, 122.6, 121.7, 120.9, 61.9, 61.0, 52.9, 52.5. ESI-MS m/z: 547.21 [M + Na+]. Anal Calcd for C34H32N6 (N,N-BQBPEN): H, 6.15; C, 77.83; N, 16.02. Found: H, 6.26; C, 77.61; N, 15.92. N,N-Bis(1-isoquinolylmethyl)-N′,N′-bis(2-pyridylmethyl)ethylenediamine (N,N-1-isoBQBPEN, 1b) To the dry CH3CN solution (50 mL) of 1-chloromethylisoquinoline44,48 (1.03 g, 5.80 mmol) and N,N-bis(2-pyridylmethyl)ethylenediamine (4, Scheme S1†) (691 mg, 2.9 mmol) was added potassium carbonate (4.0 g, 29 mmol), and then refluxed for 4 days. After the resulting solution was cooled to room temperature, the solid materials were filtered off and the filtrate was evaporated and washed with CH3CN to give N,N-1-isoBQBPEN (1b) as white powder (792 mg, 1.51 mmol, 52%). 1 H NMR (CDCl3): δ 8.38–8.43 (m, 4H), 7.98 (d, J = 8.5 Hz, 2H), 7.72 (d, J = 7.9 Hz, 2H), 7.49–7.57 (m, 4H), 7.46 (ddd, J = 9.1, 7.6, 1.5 Hz, 2H), 7.21 (d, J = 7.9 Hz, 2H), 7.15 (ddd, J = 8.2, 7.0, 1.2 Hz, 2H), 7.05 (ddd, J = 6.1, 4.9, 1.2 Hz, 2H), 4.22 (s, 4H), 3.63 (s, 4H), 2.87 (dd, J = 5.2, 2.4 Hz, 2H), 2.75 (dd, J = 5.2, 2.1 Hz, 2H). 13 C NMR (CDCl3): δ 159.3, 158.3, 148.7, 141.4, 136.2, 136.1, 129.7, 127.5, 126.7, 126.4, 122.8, 121.7, 120.6, 60.7, 60.6, 53.1, 51.6. ESI-MS m/z: 547.21 [M + Na+]. Anal Calcd for C34H32N6 (N,N-1-isoBQBPEN): H, 6.15; C, 77.83; N, 16.02. Found: H, 6.38; C, 77.71; N, 15.86. N,N′-Bis(2-pyridylmethyl)-N,N′-bis(2-quinolylmethyl)ethylenediamine (N,N′-BQBPEN, 2a) To the dry CH3CN solution (100 mL) of 2-chloromethylquinoline hydrochloride (1.72 g, 8.0 mmol) and N,N′-bis(2-pyridylmethyl)ethylenediamine49,50 (5, Scheme S1†) (959 mg, 4.0 mmol) were added potassium carbonate (5.5 g, 40 mmol) and potassium iodide (1.3 mg, 7.8 mmol), and then refluxed for 2 days. After the resulting solution was cooled to room temperature, the solid materials were filtered off and the filtrate was evaporated and washed with CH3CN to give N,N′BQBPEN (2a) as white powder (1.94 mg, 3.70 mmol, 93%). 1 H NMR (CDCl3): δ 8.45 (ddd, J = 4.8, 1.8, 0.9 Hz, 2H), 8.00 (dd, J = 8.5, 2.1 Hz, 2H), 7.72 (d, J = 7.9 Hz, 2H), 7.66 (ddd, J = 8.5, 7.0, 1.5 Hz, 2H), 7.57 (dd, J = 8.6, 3.1 Hz, 2H), 7.45–7.52 (m, 4H), 7.06 (ddd, J = 6.1, 4.9, 1.2 Hz, 2H), 3.93 (s, 4H), 3.81 (s, 4H), 2.82 (s, 4H).
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C NMR (CDCl3): δ 160.2, 159.3, 148.7, 147.3, 136.1, 136.0, 129.2, 128.8, 127.4, 127.2, 125.9, 122.7, 121.8, 120.8, 61.8, 61.2, 52.7. ESI-MS m/z: 547.22 [M + Na+]. Anal Calcd for C34H32N6 (N,N′-BQBPEN): H, 6.15; C, 77.83; N, 16.02. Found: H, 6.25; C, 77.80; N, 15.94. 13
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N,N′-Bis(1-isoquinolylmethyl)-N,N′-bis(2-pyridylmethyl)ethylenediamine (N,N′-1-isoBQBPEN, 2b) To the dry CH3CN solution (40 mL) of 1-chloromethylisoquinoline (788 mg, 4.44 mmol) and N,N′-bis(2-pyridylmethyl)ethylenediamine (5, Scheme S1†) (530 mg, 2.22 mmol) was added potassium carbonate (3.1 g, 22 mmol), and then refluxed for 2.5 days. After the resulting solution was cooled to room temperature, the solid materials were filtered off and the filtrate was evaporated and washed with CH3CN to give N,N′-1-isoBQBPEN (2b) as white powder (500 mg, 0.95 mmol, 43%). 1 H NMR (CDCl3): δ 8.42 (ddd, J = 4.9, 1.5, 0.9 Hz, 2H), 8.34 (d, J = 5.8 Hz, 2H), 8.25 (d, J = 8.5 Hz, 2H), 7.71 (d, J = 7.9 Hz, 2H), 7.58 (ddd, J = 8.8, 7.6, 1.2 Hz, 2H), 7.38–7.48 (m, 6H), 7.15 (d, J = 7.9 Hz, 2H), 7.04 (ddd, J = 6.1, 4.9, 1.2 Hz, 2H), 4.17 (s, 4H), 3.72 (s, 4H), 2.80 (s, 4H). 13 C NMR (CDCl3): δ 159.4, 158.3, 148.6, 141.3, 136.2, 136.0, 129.7, 127.5, 126.8, 126.7, 126.4, 123.3, 121.7, 120.4, 61.2, 60.4, 52.6. ESI-MS m/z: 547.22 [M + Na+]. Anal Calcd for C34H32N6 (N,N′-1-isoBQBPEN): H, 6.15; C, 77.83; N, 16.02. Found: H, 6.07; C, 77.80; N, 15.83. N,N,N′-Tris(2-quinolylmethyl)-N′-(2-pyridylmethyl)ethylenediamine (TQMPEN, 3a) To the dry CH3CN solution (80 mL) of 2-chloromethylquinoline hydrochloride (2.68 g, 12.5 mmol) and N-(2-pyridylmethyl)ethylenediamine51 (6, Scheme S1†) (630 mg, 4.2 mmol) was added potassium carbonate (6.0 g, 43 mmol), and then refluxed for 3.5 days. After the resulting solution was cooled to room temperature, the solid materials were filtered off and the filtrate was evaporated and washed with CH3CN to give TQMPEN (3a) as light yellow powder (1.02 g, 1.8 mmol, 43%). 1 H NMR (CDCl3): δ 8.42 (ddd, J = 4.8, 1.8, 0.9 Hz, 1H), 7.92–8.01 (m, 5H), 7.85 (dd, J = 8.5, 2.4 Hz, 1H), 7.62–7.73 (m, 6H), 7.57 (d, J = 8.5 Hz, 2H), 7.45–7.52 (m, 4H), 7.42 (ddd, J = 9.4, 7.6, 1.8 Hz, 1H), 7.35 (d, J = 7.3 Hz, 1H), 7.02 (ddd, J = 6.1, 4.9, 1.2 Hz, 1H), 3.96 (s, 4H), 3.92 (s, 2H), 3.79 (s, 2H), 2.81–2.89 (m, 4H). 13 C NMR (CDCl3): δ 160.2, 159.4, 148.8, 147.3, 136.1, 129.3, 129.0, 127.4, 127.3, 126.0, 122.9, 121.8, 121.0, 120.8, 62.0, 61.8, 61.3, 53.0, 52.9. ESI-MS m/z: 597.24 [M + Na+]. Anal Calcd for C38H34N6 (TQMPEN): H, 5.96; C, 79.41; N, 14.62. Found: H, 6.05; C, 79.32; N, 14.73. N,N,N′-Tris(1-isoquinolylmethyl)-N′-(2-pyridylmethyl)ethylenediamine (1-isoTQMPEN, 3b) To the dry CH3CN solution (60 mL) of 1-chloromethylisoquinoline (1.70 g, 9.57 mmol) and N-(2-pyridylmethyl)ethylenediamine
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(6, Scheme S1†) (482 mg, 3.2 mmol) was added potassium carbonate (4.4 g, 32 mmol), and then refluxed for 3.5 days. After the resulting solution was cooled to room temperature, the solid materials were filtered off and the filtrate was evaporated and washed with CH3CN to give 1-isoTQMPEN (3b) as yellow powder (627 mg, 1.17 mmol, 37%). 1 H NMR (CDCl3): δ 8.39 (ddd, J = 4.8, 1.8, 0.9 Hz, 1H), 8.35 (d, J = 5.8 Hz, 2H), 8.31 (d, J = 5.8 Hz, 1H), 8.15 (d, J = 8.2 Hz, 1H), 7.89 (d, J = 8.5 Hz, 2H), 7.69 (dd, J = 8.1, 3.6 Hz, 3H), 7.34–7.58 (m, 8H), 7.07–7.14 (m, 3H), 7.02 (ddd, J = 5.8, 4.9, 0.9 Hz, 1H), 4.16 (s, 4H), 4.12 (s, 2H), 3.67 (s, 2H), 2.81–2.84 (m, 4H). 13 C NMR (CDCl3): δ 159.2, 158.2, 148.5, 141.3, 136.1, 135.9, 129.7, 127.4, 126.8, 126.7, 126.6, 126.4, 126.2, 123.3, 121.7, 120.5, 120.4, 61.0, 60.6, 60.2, 52.7, 52.0. ESI-MS m/z: 597.25 [M + Na+]. Anal Calcd for C38H34N6 (1-isoTQMPEN): H, 5.96; C, 79.41; N, 14.62. Found: H, 6.04; C, 79.11; N, 14.89. [Zn(1b)](ClO4)2 To an ethanol solution (0.8 mL) of N,N-1-isoBQBPEN (1b) (30.0 mg, 57 μmol) was added Zn(ClO4)2·6H2O (21.3 mg, 57 μmol) in ethanol (1.2 mL), and the solution was kept at 4 °C to give [Zn(1b)](ClO4)2 as white powder (37.8 mg, 50 μmol, 83%). Recrystallization from the filtrate afforded [Zn(1b)](ClO4)2·2CH3OH as colorless crystals. 1 H NMR (CD3OD): δ 8.31 (d, J = 8.2 Hz, 2H), 8.17 (br., 4H), 8.03 (dd, J = 7.6, 5.8 Hz, 4H), 7.96 (d, J = 6.4 Hz, 4H), 7.91 (d, J = 8.2 Hz, 2H), 7.81 (dd, J = 7.0, 8.2 Hz, 2H), 7.58 (d, J = 8.2 Hz, 2H), 7.48 (br., 2H), 5.30 (d, J = 18.0 Hz, 2H), 4.75 (d, J = 18.3 Hz, 2H), 4.46 (d, J = 17.1 Hz, 2H), 4.26 (d, J = 17.4 Hz, 2H), 3.43 (br., 2H), 3.32–3.27 (m, 2H). 13 C NMR (CD3OD): δ 156.6, 148.3, 141.8, 138.7, 138.1, 134.1, 130.4, 128.6, 127.1, 126.3, 125.7, 124.7, 62.3, 59.2, 58.2. ESI-MS m/z: 687.12 [(M − ClO4)+]. Anal Calcd for C34H34Cl2N6O9Zn ([Zn(1b)](ClO4)2·H2O): C, 50.60; H, 4.25; N, 10.41. Found: C, 50.89; H, 3.96; N, 10.52. [Zn(2b)](ClO4)2 To a solution of N,N′-1-isoBQBPEN (2b) (15.7 mg, 30 µmol) in CH2Cl2–DMF (1 : 1, 300 µL) was added Zn(ClO4)2·6H2O (11.2 mg, 30 µmol) in methanol (200 µL), and the solution was kept at 4 °C to give [Zn(2b)](ClO4)2·DMF as colorless crystals (12.2 mg, 14.2 µmol, 47%). 1 H NMR (CD3OD): δ 9.02 (br. d, 2H), 8.44 (br., 2H), 8.35 (d, J = 8.2 Hz, 1H), 8.30 (d, J = 8.9 Hz, 2H), 7.5–8.2 (m, 13H), 5.17 (d, J = 19.6 Hz, 2H), 5.03 (d, J = 19.2 Hz, 2H), 4.70 (d, J = 15.9 Hz, 2H), 4.56 (d, J = 17.4 Hz, 1H), 4.34 (d, J = 17.4 Hz, 1H), 4.09 (d, J = 15.9 Hz, 2H), 3.54 (d, J = 10.1 Hz, 1H), 3.20 (d, J = 10.4 Hz, 1H). 13 C NMR (CD3OD): δ 157.8, 155.8, 149.6, 148.4, 142.3, 141.8, 138.0, 137.8, 137.6, 134.1, 133.8, 130.4, 130.2, 128.6, 128.5, 126.84, 126.79, 126.3, 125.6, 125.5, 124.7, 124.4, 63.7, 60.5, 58.8. ESI-MS m/z: 687.14 [(M − ClO4)+].
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Anal Calcd for C37H39Cl2N7O9Zn ([Zn(2b)](ClO4)2·DMF): C, 51.56; H, 4.56; N, 11.37. Found: C, 51.38; H, 4.57; N, 11.40.
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X-ray crystallography Single crystals of N,N-1-isoBQBPEN (1b), N,N′-1-isoBQBPEN·4H2O (2b·4H2O), [Zn(1b)](ClO4)2·2CH3OH and [Zn(2b)](ClO4)2·DMF were covered by Paratone-N oil and mounted on a glass fiber. All data were collected at 123–153 K on a Rigaku Mercury CCD detector, with monochromatic MoKα radiation, operating at 50 kV/40 mA. Data were processed on a PC using the CrystalClear Software (Rigaku). Structures were solved by direct methods (SIR-92)52 and refined by full-matrix least-squares methods on F2 (SHELXL-97).53 Crystal data are summarized in Tables S1 and S2.† CCDC 998434–998437 contain the supplementary crystallographic data for this paper.
Results and discussion Ligand synthesis N,N-BQBPEN (1a), N,N′-BQBPEN (2a), TQMPEN (3a), N,N-1-isoBQBPEN (1b), N,N′-1-isoBQBPEN (2b) and 1-isoTQMPEN (3b) were synthesized as shown in Scheme S1.† Although the crystal structure of the zinc complex of 2a was reported previously,54 the synthesis of 2a has not been documented. All new compounds were characterized by 1H/13C NMR and elemental analysis. The structures of 1b and 2b were further confirmed by X-ray crystallographic analysis (Table S1† and Fig. 1). Zn2+-induced absorbance and fluorescence spectral changes in 1–3a To compare the fluorescence properties of 1–3a with TQEN, a 34 µM solution in aqueous DMF (DMF–H2O = 1 : 1) at 25 °C was used for spectral measurements. Upon addition of Zn2+, the absorption of 1a at 303 and 317 nm increased (Fig. 2a). A slight absorption maximum shift was observed from 303 nm to 305 nm. A distinct isosbestic point was observed at 285 nm during the titration and spectral changes saturated with 1 eq. of Zn2+, indicating the formation of the 1 : 1 complex of 1a with Zn2+ (Fig. S1a†). Upon excitation at 317 nm, 1a exhibits Zn2+-induced fluorescence enhancement at 383 nm (I/I0 = 11 at 1 equiv. of Zn2+) accompanied with a weak emission band at 450 nm (Fig. 2b). The fluorescence quantum yield of [Zn(1a)]2+ is small (ϕ =
Fig. 2 Absorption (a, c, e) and fluorescence (λex = 317 nm) spectra (b, d, f ) of 34 µM N,N-BQBPEN (1a) (a, b), N,N’-BQBPEN (2a) (c, d) and TQMPEN (3a) (e, f ) in DMF–H2O (1 : 1) at 25 °C in the presence of various concentration of Zn2+ ranging from 0 to 68 µM.
0.010) and similar to the value reported for the [Zn(TQEN)]2+ complex (ϕ = 0.007).46 The fluorescence increase saturated with 1 equiv. of Zn2+, supporting the formation of the fluorescent 1 : 1 [ZnL]2+ complex (Fig. S1b†). Ligand 2a exhibited almost identical changes on absorbance spectra with 1a toward added Zn2+ (Fig. 2c and S1c†). In contrast, the fluorescence spectral change upon Zn2+ addition differs from that of 1a (Fig. 2d and S1d†). The fluorescence maximum was observed at 379 nm in I/I0 = 18 (at 1 equiv. of Zn2+) and the long wavelength emission is completely absent. This result suggests that the N,N-bis(quinolylmethyl) moiety, which 2a does not possesses, is responsible for the long wavelength emission. Ligand 3a also exhibited similar absorbance spectral changes responding to Zn2+ concentration (Fig. 2e and S1e†). Absorbance at 305 and 317 nm of 3a moved to 306 and 317 nm upon Zn2+ binding with an isosbestic point at 285 nm. The fluorescence profile induced by Zn2+ is almost identical to 1a and TQEN, exhibiting an increase at 383 nm with concomitant weak emission around 450 nm (Fig. 2f and S1f†). The fluorescence quantum yields of the [Zn(2a)]2+ (ϕ = 0.010) and [Zn(3a)]2+ complexes (ϕ = 0.009) are similar to those of [Zn(1a)]2+ and [Zn(TQEN)]2+. Zn2+-induced absorbance and fluorescence spectral changes in 1–3b
Fig. 1 ORTEP plot for (a) N,N-1-isoBQBPEN (1b) and (b) N,N’-1-isoBQBPEN (2b) in 50% probability. Hydrogen atoms were omitted for clarity. Atoms indicated with asterisks are generated by the symmetric operation.
16380 | Dalton Trans., 2014, 43, 16377–16386
A previous report showed that 1-isoTQEN (Chart 1), which has four isoquinolines in place of quinoline moieties of TQEN, exhibited (i) enhancement of the fluorescence intensity,
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(ii) red-shifts in excitation and emission wavelengths due to the intramolecular excimer formation, (iii) increased Zn2+/Cd2+ discriminating ability, (iv) reduction of background fluorescence induced by pH, and (v) strong metal binding affinity.44 In order to investigate the details of the above characteristics, especially the mechanism for Zn2+-specific excimer formation, the fluorescence properties of isoquinoline derivatives 1–3b were investigated. Upon addition of Zn2+, the absorption of 1b at 310 and 323 nm decreased and new peaks at 313 and 326 nm appeared (Fig. 3a). Distinct isosbestic points were seen at 311, 319 and 324 nm during the titration and spectral changes saturated with 1 equiv. of zinc ion, indicating the formation of the 1 : 1 complex of 1b and Zn2+ ion (Fig. S2a†). Fig. 3b and S2b† show the fluorescence spectral change of 1b with increasing amounts of Zn2+ ions. Two emission bands at 353 and 475 nm were observed similar to 1-isoTQEN. The intensity ratio of long and short emissions of [Zn(1b)]2+ (I475/ I353 = 2.0) is similar to [Zn(1-isoTQEN)]2+ (I477/I357 = 1.6) due to the efficient excimer interaction of two isoquinolines in N,N-configuration. This value is significantly larger than that of [Zn(1-isoTQA)]2+ (I470/I359 = 0.48), indicating the efficient steric effect of the bis(2-pyridylmethyl)amine moiety in 1b enforcing the opposite two isoquinolines in close proximity in the excited state, compared to single isoquinoline coordination in the [Zn-(1-isoTQA)]2+ complex. The spectral change of 1b with Zn2+ titration was stopped on 1 : 1 complex formation (I/I0 = 23 at 475 nm).
Paper Table 1 Fluorescence lifetime for Zn2+ complexes with N,N-1-isoBQBPEN (1b), N,N’-1-isoBQBPEN (2b) and 1-isoTQEN measured in DMF–H2O (1 : 1)
λex (nm)
λem (nm)
τ (ns)
N,N-1-isoBQBPEN (1b)
333
N,N′-1-isoBQBPEN (2b) 1-isoTQEN
333 333
353 475 352 357 477
0.1 8.8 2.0 2.5, 5.7 9.0
As observed for the N,N′-isomer of the bisquinoline derivatives 2a, the isoquinoline isomer 2b in the N,N′-configuration exhibited nearly identical spectral changes with 1b (Fig. 3c and S2c†) but only a single emission band at shorter wavelengths in the fluorescence spectrum upon Zn2+ binding (Fig. 3d and S2d†). The intense long wavelength emission observed in 1-isoTQEN and 1b is completely lost for the N,N′isomer 2b. This is because the N,N-bis((iso)quinolylmethyl) amine group is responsible for the long wavelength emission around 450 nm by intramolecular excimer formation. The feature of 450 nm emission of the Zn2+-1b complex is concentration-independent (Fig. S3†), indicating that this emission is due to intramolecular excimer formation. The water content in the solvent was found to be important for the fluorescent response of 1b. As shown in Fig. S4,† significant reduction in fluorescence intensity was observed in DMF as a solvent; however, DMF–H2O (4 : 1) as a solvent exhibited exactly the same spectral changes as those in DMF–H2O (1 : 1). The tris(isoquinoline) derivative 3b exhibited Zn2+-induced absorbance (Fig. 3e and S2e†) and fluorescence (Fig. 3f and S2f†) spectral changes similar to 1-isoTQEN and 1b because of the N,N-bis(1-isoquinolylmethyl)amine moiety. The slightly reduced long/short fluorescence intensity ratio of [Zn(3b)]2+ (I475/I354 = 1.5) may be a result of appended monomer emission due to the isolated isoquinoline chromophore at the N′-position. The fluorescence quantum yields of [Zn(1b)]2+ (ϕ = 0.028) and [Zn(3b)]2+ (ϕ = 0.033) are similar to that of [Zn(1isoTQEN)]2+ (ϕ = 0.034).46 The [Zn(2b)]2+ complex exhibits a slightly larger value (ϕ = 0.051) due to the strong emission at 350 nm. The fluorescence lifetime of Zn2+ complexes with 1b, 2b, and 1-isoTQEN depends significantly on the monitoring wavelengths (Fig. S5–S9† and Table 1). The long wavelength emission around 450 nm has a long fluorescence lifetime (τ = ∼9 ns), while the short-wavelength emission around 350 nm has a short lifetime (τ = 0.1–2.5 ns). These two emission bands originate from the same ground-state species because they exhibit identical excitation spectra (data not shown). These O2-independent lifetime values indicate that the nature of these emissions is fluorescence from singlet excited states. Fluorescence metal ion specificity of 1–3a and 1–3b
Fig. 3 Absorption (a, c, e) and fluorescence (λex = 326 nm) spectra (b, d, f) of 34 µM N,N-1-isoBQBPEN (1b) (a, b), N,N’-1-isoBQBPEN (2b) (c, d) and 1-isoTQMPEN (3b) (e, f ) in DMF–H2O (1 : 1) at 25 °C in the presence of various concentrations of Zn2+ ranging from 0 to 68 µM.
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Fig. 4 shows the metal ion specificity of the fluorescence response of 1–3a and 1–3b. The fluorescence enhancement was specific for Zn2+ at indicated wavelengths. The ICd/IZn
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replacement of (iso)quinolines with pyridine does not alter metal selectivity in these probes. The steric hindrance in quinoline derivatives due to the peri hydrogen reduces Zn2+/Cd2+ discrimination ability.19,27 It is of significant interest that Cd2+ induces only short wavelength emission by CHEF (chelation enhanced fluorescence) and PeT (Photoinduced Electron Transfer) inhibition mechanisms55,56 and long wavelength emission is absent for all Cd2+ complexes (Fig. S10†). The excimer-like interaction between two (iso)quinolines are blocked in the Cd2+ complex, probably due to the long Cd– N(iso)quinoline distance. The subtle difference in ionic radii between Zn2+ and Cd2+ is effectively highlighted with the long wavelength emission of the 1-isoTQEN family ligands by the excimer formation. pH effect on fluorescence intensity of 1–3a and 1–3b
Fig. 4 The relative fluorescence intensity of (a) 1–3a and (b) 1–3b in the presence of 1 equiv. of metal ions in DMF–H2O (1 : 1) at 25 °C. I0 is the emission intensity of free ligand.
values are 59–72% and 8–12% for quinoline (1–3a) and isoquinoline (1–3b) derivatives, respectively, and these are similar to the corresponding tetrakis(iso)quinoline derivatives.27,44 Thus,
Fig. 5 demonstrates the pH-profile of fluorescence zinc detection of 1–3a and 1–3b. The monitoring wavelengths are the same as those in the previous section. Protonation on the ligand nitrogen atoms prevents the zinc binding at low pH regions and the formation of Zn(OH)2 at high pH regions inhibits the interaction with the ligand. As the quinoline moiety of TQEN is replaced with pyridine, the Zn2+ detection range becomes wider (Fig. 5a–c). The N,N′-BQBPEN (2a) exhibits a distinct advantage in Zn2+ detection under high pH conditions compared with the corresponding N,N-isomer (1a), indicating the higher metal binding ability of 2a due to less hindered coordination geometry as found in the crystal structure.54 The trisquinoline, TQMPEN (3a), showed a pH-sensitive profile with a similar extent to TQEN. On the other hand, isoquinoline derivatives 1–3b exhibit no difference with a very wide pH window for Zn2+ response (Fig. 5d–f). Negligible proton-induced
Fig. 5 Effect of pH on the fluorescence intensity of (a) 1a at 383 nm, (b) 2a at 379 nm, (c) 3a at 383 nm, (d) 1b at 475 nm, (e) 2b at 352 nm and (f ) 3b at 475 nm in the absence (blue triangles) and presence (red circles) of 1 equiv. of zinc ions in DMF–H2O (1 : 1) at 25 °C.
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fluorescence was observed for all apo ligands including quinoline derivatives.
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X-Ray crystallography of [Zn(1b)](ClO4)2 and [Zn(2b)](ClO4)2 Single crystals of [Zn(1b)](ClO4)2·2CH3OH and [Zn(2b)](ClO4)2·DMF were prepared and analyzed by X-ray crystallography (Fig. 6). Crystal data and bond distances around the metal centre are summarized in Tables S2† and 2, respectively. In the crystal structure of [Zn(1b)]2+, six nitrogen atoms of 1b coordinate to the Zn2+ center and no significant steric hindrance is found in the interaction between adjacent isoquinoline and pyridine rings. This structure is very close to the previously reported [Zn(1-isoTQEN)]2+ complex.44 The Zn–Npyridine distances (2.148(2) and 2.145(3) Å) are longer than the
Zn–Nisoquinoline distances (2.119(2) and 2.137(2) Å), indicating stronger metal coordination of isoquinoline nitrogens in comparison with pyridine nitrogen atoms. This is in good agreement with the difference in pKa values.57 The crystal structure of [Zn(2b)]2+ is also similar to the [Zn(1-isoTQEN)]2+ complex. In contrast to the structure of the quinoline counterpart [Zn(2a)]2+,54 two isoquinolines of [Zn(2b)]2+ are located at the ‘axial’ positions, in which the plane containing two aliphatic nitrogen atoms and a zinc center is defined as a ‘basal’ plane. Since the H-8 hydrogen atom of quinoline located at the ‘axial’ position of the [Zn(TQEN)]2+ complex exhibits significant steric hindrance (Zn–N = 2.4 Å),27 the pyridines occupy the ‘axial’ position in the [Zn(2a)]2+ complex. Even for tetrakis( pyridine) ([Zn(TPEN)]2+)27 and tetrakis(isoquinoline) ([Zn(1-isoTQEN)]2+), the bond distances of the ‘axial’ coordination (Zn–N4 and Zn–N6) is slightly longer than the ‘basal’ bond distances (Zn–N3 and Zn–N5) probably due to the structural requirement (Table 2). The strong coordination of isoquinolines in [Zn(2b)]2+ from the ‘axial’ positions shortens the bond distances, resulting in similar Zn–N distances for all aromatic nitrogen atoms. Theoretical calculations of [Zn(1b)]2+ and [Zn(2b)]2+ complexes Based on the crystal structures, electronic structures of [Zn(1b)]2+ and [Zn(2b)]2+ at the ground and excited states were calculated by time-dependent density-functional-theory (TDDFT) calculation (functional: ωB97XD;58 basis set: cc-pVDZ(C, N, H), 6-311++G(d,p) (Zn)). Although the optimized structure at the excited state for [Zn(1b)]2+ starting from the solid state structure (Fig. 7a) is almost identical with that at the ground state, the potential energy of the stacked excited state structure in which two isoquinoline rings are in parallel orientation (Fig. 7b) is 48 kJ mol−1 lower than the ground state-like (i.e., Franck Condon state), non-stacked excited structure. No minimum energy conformation was found in the parallelforced structure at the ground state. The absence of a parallelforced structure in the ground state means that the excited state with stacked isoquinoline rings has a so-called “excimer” character. The absorption and emission bands of [Zn(1b)]2+ were estimated in Tables S3† and 3, respectively. The molecular orbitals
Fig. 6 ORTEP plot for (a) [Zn(1b)](ClO4)2·2CH3OH and (b) [Zn(2b)](ClO4)2·DMF in 50% probability. Hydrogen atoms, counter anions and solvents were omitted for clarity.
Table 2 Selected bond distances (Å) for [Zn(TPEN)]2+, [Zn(1-isoTQEN)]2+, [Zn(1b)]2+ and [Zn(2b)]2+
Zn–N1 Zn–N2 Zn–N3 Zn–N4 Zn–N5 Zn–N6 a
[Zn(TPEN)]2+ a,b
[Zn(1-isoTQEN)]2+ c
[Zn(1b)]2+
[Zn(2b)]2+
2.19 2.20 2.09 2.19 2.10 2.21
2.1945(16)
2.189(2) 2.205(3) 2.119(2) 2.137(2) 2.148(2) 2.145(3)
2.228(3) 2.218(3) 2.155(3) 2.155(3) 2.153(3) 2.148(3)
2.1269(17) 2.1742(15)
b
c
Average values for two independent molecules. Ref. 27. Ref. 44.
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Fig. 7 Calculated S1 excited state structure of [Zn(1b)]2+ in (a) Franck Condon state and (b) parallel (excimer) configuration.
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State S1 S1
Dalton Transactions Calculated emission properties for [Zn(1b)]2+ and [Zn(2b)]2+
Wavelength (nm)
Oscillator strength
Orbital composition
[Zn(1b)]2+ with Franck Condon Conformation (See Fig. 7a) 340 0.070 LUMO → HOMO [Zn(1b)]2+ with Excimer Conformation (See Fig. 7b) 463 0.0055 LUMO → HOMO
CIa (%) 47 49
2+
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S1 a
335
[Zn(2b)] 0.079
LUMO → HOMO
48
Percentage of square of CI coefficient.
for each transition are indicated in Fig. S11–14.† The most striking point is that the longer wavelength emission centered at 463 nm was reasonably elucidated by computation from the LUMO–HOMO transition in a parallel-oriented structure (Fig. 7b). This excimer emission band perfectly matches the experimental observations. Moreover, the longer fluorescence lifetime of the excimer emission compared to the shorter wavelength emission is also predicted by the smaller value of the calculated oscillator strength (Table 3). No such long wavelength emissions or long fluorescence lifetimes was predicted for the non-parallel excited state of [Zn(1b)]2+ (Fig. 7a) and N,N′-isomer [Zn(2b)]2+, in which intramolecular excimer interaction of isoquinolines is not expected. Thermodynamic and kinetic competition of Zn2+ binding with TPEN We have demonstrated that 1-isoTQEN and its derivatives exhibit extremely high Zn2+ binding ability and resist metal ion removal by TPEN via competition experiments.44 Even for 2-quinolylmethylamine derivatives TQDACH (N,N,N′,N′-tetrakis(2-quinolylmethyl)-trans-1,2-diaminocyclohexane) and TQTACN (N,N′,N″-tris(2-quinolylmethyl)-1,4,7-triazacyclononane), Zn2+ is not transferred to TPEN by the strong metal binding affinity of the pre-organized, rigid aliphatic amine backbone of these ligands.24,42 Introduction of carboxylates in place of (iso)quinolines of (1-iso)TQEN significantly enhances the metal binding ability, resulting in removal of Zn2+ from the [Zn(TPEN)]2+ complex by apo (iso)quinoline-carboxylate ligands.59 The fluorescence intensity of Zn2+ complexes with 1–3b was stable in the presence of 1 equiv. of TPEN for at least 7 days at room temperature (Fig. 8b). In contrast, the quinoline derivatives 1–3a showed a gradual fluorescence quenching by TPEN in a structure-dependent manner (Fig. 8a). While the trisquinoline derivative 3a exhibited immediate loss of fluorescence upon addition of TPEN, the bisquinoline derivatives 1a and 2a showed different resistance properties toward TPEN. The N,N′isomer 2a releases Zn2+ at a slower rate than the N,N-isomer 1a. This is because the sterically unfavorable ‘axial’ quinoline coordination (i.e., N4 and N6 in Fig. 6) is absent in the [Zn(2a)]2+ complex, in which the two quinolines adopt ‘basal’ (i.e., those containing N3 and N5 in Fig. 6) coordination.54 As mentioned above, the close proximity between the peri hydrogen
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Fig. 8 Time dependent fluorescence intensity changes of (a) [Zn(1a)]2+ at 383 nm (red circles), [Zn(2a)]2+ at 379 nm (blue triangles) and [Zn(3a)]2+ at 383 nm (green squares) and (b) [Zn(1b)]2+ at 475 nm (red circles), [Zn(2b)]2+ at 352 nm (blue triangles) and [Zn(3b)]2+ at 475 nm (green squares) in the presence of 1 equiv. of TPEN at 25 °C in DMF– H2O (1 : 1).
atom at the 8-position of the ‘axial’ quinoline and ‘basal’ aromatic ring generates steric hindrance and weakens the metal– nitrogen bond of the ‘axial’ quinoline in the [Zn(1a)]2+ complex. The preliminary X-ray analysis of [Zn(1a)]2+ exhibits a five-coordinate structure due to the weak quinoline coordination (Fig. S15†). Based on the extremely slow metal exchange rate mentioned above, the kinetic metal binding competition of 1–3b versus TPEN was examined. In this experiment, to an equimolar mixture of ligand (1b, 2b or 3b) and TPEN in the apo form was added Zn2+ in portions and the fluorescence change was monitored. Under such conditions, the fluorescence intensity changes shown in Fig. 9 represent the relative Zn2+ capture rate of apo 1a (or 2b, 3b) versus apo TPEN. Thus, Fig. 9 can be regarded as a kinetic competition of Zn2+ binding of these ligands with TPEN. For the slow-binding ligands such as TQDACH and TQTACN, the fluorescence intensity was unchanged at 0–1 equiv. of Zn2+ until all TPEN was consumed, then the fluorescence started to increase and reached saturation at 2 equiv. of Zn2+.24,42 Similarly, a small slope during
Fig. 9 Kinetic competitive fluorescence intensity change of (a) 1b at 475 nm, (b) 2b at 352 nm, (c) 3b at 475 nm and (d) 1-isoTQEN at 475 nm in the presence (red circles) and absence (blue triangles) of 1 equiv. of TPEN with increasing amounts of zinc at 25 °C in DMF–H2O (1 : 1).
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0–1 equiv. of Zn2+ in Fig. 9d in the presence of TPEN suggested the slow Zn2+ binding rate of 1-isoTQEN. For 1–3b, however, such a bending point at 1 equiv. of Zn2+ was not observed because the Zn2+ capture rate of these ligands was as rapid as TPEN (Fig. 9a–c). The introduction of at least one pyridine ring into 1-isoTQEN significantly improved the metal binding rate of the ligand.
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Intracellular Zn2+ sensing with 1–3a and 1–3b PC-12 rat adrenal pheochromocytoma cells were applied to evaluate the ability of in vitro zinc sensing with 1–3a and 1–3b. The cells were incubated in growth media containing 100 µM of ligands and 2% (v/v) DMSO for 4 h and analyzed with a fluorescence microscope after washing of the cell and changing the media. [Zn( pyrithione)2] (final concentration: 90 µM) was added to the media during microscopic analysis and the fluorescence enhancement was monitored. As shown in Fig. 10, a small fluorescence inside the cells was observed in the presence of isoquinoline derivatives 1–3b after addition of [Zn( pyrithione)2] into the media. No cytotoxicity was observed up to 100 µM in the growth media. The cell-permeability and intracellular zinc-responding ability of the compounds strongly appeal the potential of these ligands as zinc-specific fluorescent sensor platforms.
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Conclusions The intramolecular excimer formation of the N,N-bis(1-isoquinolylmethyl)amine moiety of 1-isoTQEN derivatives upon Zn2+ binding plays a key role in the selective fluorescence enhancement at longer wavelengths. Such an excimer emission is completely specific to Zn2+. Cd2+ induces only moderate CHEFand PeT-based emissions at shorter wavelengths. The hexadentate tetrakis(heteroaromatic)ethylenediamine scaffold as a fluorescent Zn2+ probe has unique advantages in synthetic accessibility, stability of the Zn2+ complex, and excimer forming ability. Replacement of two of the quinoline heterocycles of TQEN with pyridines affords higher Zn2+ binding ability and proton insensitivity without losing any necessary fluorescence properties. Although suitable improvement to enhance fluorescence intensity and water-solubility is required, the present findings provide a new strategy for rational design of Zn2+-specific fluorescent probe molecules.
Acknowledgements Authors thank Ms Ikuko Hamagami and Ms Sakiko Akaji of Horiba, Ltd for their kind help in fluorescence lifetime measurements. Authors also thank Prof. Satoshi Tamotsu, Prof. Keiko Yasuda and Dr Masato Aoyama of Nara Women’s University for their kind help in cellular experiments. This work was supported by the Research for Promoting Technological Seeds, JST, Adaptable and Seamless Technology Transfer Program through Target-driven R&D, JST, Grant-in Aid for Scientific Research from the MEXT, Japan and the Nara Women’s University Intramural Grant for Project Research.
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Fig. 10 Differential interference contrast (DIC) and fluorescence (FL) micrographs of cultured PC-12 rat adrenal cells in the presence of 100 µM 1–3a and 1–3b for 4 h. [Zn( pyrithione)2] (90 µM) was added during microscopic analysis ( photograph taken after ∼4 min).
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Zinc-specific intramolecular excimer formation in TQEN derivatives: fluorescence and zinc binding properties of TPEN-based hexadentate ligands† Yuji Mikata,*a,b Saaya Takeuchi,b Eri Higuchi,b Ayaka Ochi,b Hideo Konno,c Kazuma Yanaid and Shin-ichiro Satoe Zn2+-induced fluorescence enhancement of the TPEN (N,N,N’,N’-tetrakis(2-pyridylmethyl)ethylenediamine)based ligand, N,N-bis(1-isoquinolylmethyl)-N’,N’-bis(pyridylmethyl)ethylenediamine (N,N-1-isoBQBPEN, 1b), has been investigated. Upon Zn2+ binding, 1b shows a fluorescence increase (ϕZn = 0.028) at 353 and 475 nm. The fluorescence enhancement at longer wavelengths is due to intramolecular excimer formation of two isoquinolines and is specific for Zn2+; Cd2+ induces very small fluorescence at 475 nm (ICd/IZn = 10%). The excimer formation of the [Zn(1b)]2+ complex in the excited state is supported by the time-dependent DFT calculation. Neither long-wavelength fluorescence nor excimer formation is
Received 20th June 2014, Accepted 29th August 2014
observed in the Zn2+ complex of N,N’-1-isoBQBPEN (2b). The quinoline analog N,N-BQBPEN (1a) exhibits similar but significantly smaller excimer formation. Thermodynamic and kinetic comparisons of Zn2+
DOI: 10.1039/c4dt01847h
binding properties of ethylenediamine-based hexadentate ligands with pyridines and (iso)quinolines are
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comprehensively discussed.
Introduction Zinc is an indispensable element in living systems because of many important biological roles of this essential metal.1–6 Zinc exists in mobile and protein-bound forms in cells. Mobile zinc is a modulator of signaling processes and key element in gene expression. Many fluorescent zinc sensor molecules that reveal the distribution of unbound zinc in living cells have been extensively investigated in the recent two decades to understand zinc biology.4–18 Such fluorescent zinc probes often respond to cadmium because both Zn2+ and Cd2+ possess an occupied d10 shell and similar coordination preferences. The only difference in these two metals is the ionic radii of approximately 21 pm. So, new design strategies to visualize Zn2+ in high selectivity and sensitivity, especially
discriminating Zn2+ from Cd2+ by ionic radius, coordination environment or other ligand effects, are of considerable interest.19–26 We have previously reported the quinoline analog of the TPEN (N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine)based fluorescent zinc probe, TQEN (N,N,N′,N′-tetrakis(2-quinolylmethyl)ethylenediamine) (Chart 1).27 In recent years, many other quinoline-based fluorescent Zn2+ sensors have been developed.24,28–43 We also reported that the isoquinoline derivatives such as 1-isoTQEN (Chart 1)44 and 1-isoTQA45 (tris(1-isoquinolylmethyl)amine) exhibit high fluorescence intensity and zinc specificity in their long wavelength emission enhancement. The high Zn2+ specificity is hypothesized to be
a
KYOUSEI Science Center, Nara Women’s University, Nara 630-8506, Japan. E-mail: [email protected] b Department of Chemistry, Faculty of Science, Nara Women’s University, Nara 630-8506, Japan c National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, Ibaraki 305-8565, Japan d Graduate School of Chemical Sciences and Engineering, Hokkaido University, Sapporo 060-8628, Japan e Graduate School of Engineering, Hokkaido University, Sapporo 060-8628, Japan † Electronic supplementary information (ESI) available: The experimental procedure for synthesis of the compounds, Tables S1–S3, Scheme S1, Fig. S1–S31 and crystallographic data in CIF format. CCDC 998434–998437. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c4dt01847h
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Chart 1
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due to the formation of an intramolecular excimer by two adjacent isoquinoline rings upon Zn2+ binding. There are limited numbers of other isoquinoline-based fluorescent sensors in the literature.24,42,46 In this paper, we describe the spectral and theoretical studies of zinc-induced fluorescence enhancement of N,N-1isoBQBPEN (N,N-bis(1-isoquinolylmethyl)-N′,N′-bis(2-pyridylmethyl)ethylenediamine, 1b) and N,N′-1-isoBQBPEN (N,N′bis(1-isoquinolylmethyl)-N,N′-bis(2-pyridylmethyl)ethylenediamine, 2b) as benzene-depleted derivatives of 1-isoTQEN (Chart 1). These compounds are molecular hybrids of 1-isoTQEN and TPEN. Reduction of the molecular weight and aromatic ring size without losing necessary fluorescence properties would be an attractive improvement for application to cell study. The corresponding quinoline analogs, N,N-BQBPEN (N,N-bis(2-pyridylmethyl)-N′,N′-bis(2-quinolylmethyl)ethylenediamine, 1a) and N,N′-BQBPEN (N,N′-bis(2-pyridylmethyl)N,N′-bis(2-quinolylmethyl)ethylenediamine, 2a), are also examined. Replacement of two isoquinoline rings of 1isoTQEN with pyridines in the N,N-configuration does not affect the fluorescence of the Zn2+ complex. However, the N, N′-isomer completely loses long-wavelength emission. The time-dependent DFT calculation reveals that the excited-state intramolecular excimer, in which two isoquinoline moieties are in stacked parallel arrangement, is responsible for the long wavelength emission. The metal binding competition with TPEN demonstrates the thermodynamic and kinetic superiorities of 1-isoBQBPEN ligands.
Experimental General All reagents and solvents used for synthesis were from commercial sources and used as received. N,N-Dimethylformamide (DMF, Dojin) was of spectral grade (Spectrosol). All aqueous solutions were prepared using Milli-Q water (Millipore). 1H NMR (300 Hz) and 13C NMR (75.5 Hz) spectra were recorded on a Varian GEMINI 2000 spectrometer and referenced to internal Si(CH3)4 or solvent signals. UV-vis and fluorescence spectra were measured on a Jasco V-660 spectrophotometer and Jasco FP-6300 spectrofluorometer, respectively. Fluorescence quantum yields were measured on a HAMAMATSU photonics C9920-02 absolute PL quantum yield measurement system. Fluorescence lifetimes were measured on a HORIBA FluoroCube 5000U system. Caution: Perchlorate salts of metal complexes with organic ligands are potentially explosive. All due precautions should be taken. N,N-Bis(2-pyridylmethyl)-N′,N′-bis(2-quinolylmethyl)ethylenediamine (N,N-BQBPEN, 1a) To the dry CH3CN solution (40 mL) of 2-chloromethylquinoline hydrochloride (428 mg, 2.0 mmol) and N,N-bis(2-pyridylmethyl)ethylenediamine47 (4, Scheme S1†) (240 mg, 1.0 mmol) were added potassium carbonate (1.4 g, 10 mmol) and potassium iodide (332 mg, 2.0 mmol), and then refluxed for 2 days
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under N2. After the resulting solution was cooled to room temperature, the solid materials were filtered off and the filtrate was evaporated. Crystallization of the residue from CH3CN under ether diffusion conditions afforded N,NBQBPEN (1a) as yellow powder (160 mg, 0.3 mmol, 30%). 1 H NMR (CDCl3): δ 8.42 (ddd, J = 4.8, 1.8, 0.9 Hz, 2H), 8.02 (d, J = 8.2 Hz, 2H), 8.02 (d, J = 8.2 Hz, 2H), 7.75 (d, J = 8.2, 1.5 Hz, 2H), 7.61–7.69 (m, 4H), 7.48 (ddd, J = 8.2, 7.0, 1.2 Hz, 2H), 7.41 (ddd, J = 8.8, 7.3, 1.5 Hz, 2H), 7.33 (d, J = 7.6 Hz, 2H), 7.01 (ddd, J = 6.1, 4.9, 1.2 Hz, 2H), 3.97 (s, 4H), 3.76 (s, 4H), 2.80–2.83 (m, 4H). 13 C NMR (CDCl3): δ 160.1, 159.3, 148.6, 147.3, 136.1, 129.2, 128.9, 127.4, 127.2, 126.0, 122.6, 121.7, 120.9, 61.9, 61.0, 52.9, 52.5. ESI-MS m/z: 547.21 [M + Na+]. Anal Calcd for C34H32N6 (N,N-BQBPEN): H, 6.15; C, 77.83; N, 16.02. Found: H, 6.26; C, 77.61; N, 15.92. N,N-Bis(1-isoquinolylmethyl)-N′,N′-bis(2-pyridylmethyl)ethylenediamine (N,N-1-isoBQBPEN, 1b) To the dry CH3CN solution (50 mL) of 1-chloromethylisoquinoline44,48 (1.03 g, 5.80 mmol) and N,N-bis(2-pyridylmethyl)ethylenediamine (4, Scheme S1†) (691 mg, 2.9 mmol) was added potassium carbonate (4.0 g, 29 mmol), and then refluxed for 4 days. After the resulting solution was cooled to room temperature, the solid materials were filtered off and the filtrate was evaporated and washed with CH3CN to give N,N-1-isoBQBPEN (1b) as white powder (792 mg, 1.51 mmol, 52%). 1 H NMR (CDCl3): δ 8.38–8.43 (m, 4H), 7.98 (d, J = 8.5 Hz, 2H), 7.72 (d, J = 7.9 Hz, 2H), 7.49–7.57 (m, 4H), 7.46 (ddd, J = 9.1, 7.6, 1.5 Hz, 2H), 7.21 (d, J = 7.9 Hz, 2H), 7.15 (ddd, J = 8.2, 7.0, 1.2 Hz, 2H), 7.05 (ddd, J = 6.1, 4.9, 1.2 Hz, 2H), 4.22 (s, 4H), 3.63 (s, 4H), 2.87 (dd, J = 5.2, 2.4 Hz, 2H), 2.75 (dd, J = 5.2, 2.1 Hz, 2H). 13 C NMR (CDCl3): δ 159.3, 158.3, 148.7, 141.4, 136.2, 136.1, 129.7, 127.5, 126.7, 126.4, 122.8, 121.7, 120.6, 60.7, 60.6, 53.1, 51.6. ESI-MS m/z: 547.21 [M + Na+]. Anal Calcd for C34H32N6 (N,N-1-isoBQBPEN): H, 6.15; C, 77.83; N, 16.02. Found: H, 6.38; C, 77.71; N, 15.86. N,N′-Bis(2-pyridylmethyl)-N,N′-bis(2-quinolylmethyl)ethylenediamine (N,N′-BQBPEN, 2a) To the dry CH3CN solution (100 mL) of 2-chloromethylquinoline hydrochloride (1.72 g, 8.0 mmol) and N,N′-bis(2-pyridylmethyl)ethylenediamine49,50 (5, Scheme S1†) (959 mg, 4.0 mmol) were added potassium carbonate (5.5 g, 40 mmol) and potassium iodide (1.3 mg, 7.8 mmol), and then refluxed for 2 days. After the resulting solution was cooled to room temperature, the solid materials were filtered off and the filtrate was evaporated and washed with CH3CN to give N,N′BQBPEN (2a) as white powder (1.94 mg, 3.70 mmol, 93%). 1 H NMR (CDCl3): δ 8.45 (ddd, J = 4.8, 1.8, 0.9 Hz, 2H), 8.00 (dd, J = 8.5, 2.1 Hz, 2H), 7.72 (d, J = 7.9 Hz, 2H), 7.66 (ddd, J = 8.5, 7.0, 1.5 Hz, 2H), 7.57 (dd, J = 8.6, 3.1 Hz, 2H), 7.45–7.52 (m, 4H), 7.06 (ddd, J = 6.1, 4.9, 1.2 Hz, 2H), 3.93 (s, 4H), 3.81 (s, 4H), 2.82 (s, 4H).
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C NMR (CDCl3): δ 160.2, 159.3, 148.7, 147.3, 136.1, 136.0, 129.2, 128.8, 127.4, 127.2, 125.9, 122.7, 121.8, 120.8, 61.8, 61.2, 52.7. ESI-MS m/z: 547.22 [M + Na+]. Anal Calcd for C34H32N6 (N,N′-BQBPEN): H, 6.15; C, 77.83; N, 16.02. Found: H, 6.25; C, 77.80; N, 15.94. 13
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N,N′-Bis(1-isoquinolylmethyl)-N,N′-bis(2-pyridylmethyl)ethylenediamine (N,N′-1-isoBQBPEN, 2b) To the dry CH3CN solution (40 mL) of 1-chloromethylisoquinoline (788 mg, 4.44 mmol) and N,N′-bis(2-pyridylmethyl)ethylenediamine (5, Scheme S1†) (530 mg, 2.22 mmol) was added potassium carbonate (3.1 g, 22 mmol), and then refluxed for 2.5 days. After the resulting solution was cooled to room temperature, the solid materials were filtered off and the filtrate was evaporated and washed with CH3CN to give N,N′-1-isoBQBPEN (2b) as white powder (500 mg, 0.95 mmol, 43%). 1 H NMR (CDCl3): δ 8.42 (ddd, J = 4.9, 1.5, 0.9 Hz, 2H), 8.34 (d, J = 5.8 Hz, 2H), 8.25 (d, J = 8.5 Hz, 2H), 7.71 (d, J = 7.9 Hz, 2H), 7.58 (ddd, J = 8.8, 7.6, 1.2 Hz, 2H), 7.38–7.48 (m, 6H), 7.15 (d, J = 7.9 Hz, 2H), 7.04 (ddd, J = 6.1, 4.9, 1.2 Hz, 2H), 4.17 (s, 4H), 3.72 (s, 4H), 2.80 (s, 4H). 13 C NMR (CDCl3): δ 159.4, 158.3, 148.6, 141.3, 136.2, 136.0, 129.7, 127.5, 126.8, 126.7, 126.4, 123.3, 121.7, 120.4, 61.2, 60.4, 52.6. ESI-MS m/z: 547.22 [M + Na+]. Anal Calcd for C34H32N6 (N,N′-1-isoBQBPEN): H, 6.15; C, 77.83; N, 16.02. Found: H, 6.07; C, 77.80; N, 15.83. N,N,N′-Tris(2-quinolylmethyl)-N′-(2-pyridylmethyl)ethylenediamine (TQMPEN, 3a) To the dry CH3CN solution (80 mL) of 2-chloromethylquinoline hydrochloride (2.68 g, 12.5 mmol) and N-(2-pyridylmethyl)ethylenediamine51 (6, Scheme S1†) (630 mg, 4.2 mmol) was added potassium carbonate (6.0 g, 43 mmol), and then refluxed for 3.5 days. After the resulting solution was cooled to room temperature, the solid materials were filtered off and the filtrate was evaporated and washed with CH3CN to give TQMPEN (3a) as light yellow powder (1.02 g, 1.8 mmol, 43%). 1 H NMR (CDCl3): δ 8.42 (ddd, J = 4.8, 1.8, 0.9 Hz, 1H), 7.92–8.01 (m, 5H), 7.85 (dd, J = 8.5, 2.4 Hz, 1H), 7.62–7.73 (m, 6H), 7.57 (d, J = 8.5 Hz, 2H), 7.45–7.52 (m, 4H), 7.42 (ddd, J = 9.4, 7.6, 1.8 Hz, 1H), 7.35 (d, J = 7.3 Hz, 1H), 7.02 (ddd, J = 6.1, 4.9, 1.2 Hz, 1H), 3.96 (s, 4H), 3.92 (s, 2H), 3.79 (s, 2H), 2.81–2.89 (m, 4H). 13 C NMR (CDCl3): δ 160.2, 159.4, 148.8, 147.3, 136.1, 129.3, 129.0, 127.4, 127.3, 126.0, 122.9, 121.8, 121.0, 120.8, 62.0, 61.8, 61.3, 53.0, 52.9. ESI-MS m/z: 597.24 [M + Na+]. Anal Calcd for C38H34N6 (TQMPEN): H, 5.96; C, 79.41; N, 14.62. Found: H, 6.05; C, 79.32; N, 14.73. N,N,N′-Tris(1-isoquinolylmethyl)-N′-(2-pyridylmethyl)ethylenediamine (1-isoTQMPEN, 3b) To the dry CH3CN solution (60 mL) of 1-chloromethylisoquinoline (1.70 g, 9.57 mmol) and N-(2-pyridylmethyl)ethylenediamine
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(6, Scheme S1†) (482 mg, 3.2 mmol) was added potassium carbonate (4.4 g, 32 mmol), and then refluxed for 3.5 days. After the resulting solution was cooled to room temperature, the solid materials were filtered off and the filtrate was evaporated and washed with CH3CN to give 1-isoTQMPEN (3b) as yellow powder (627 mg, 1.17 mmol, 37%). 1 H NMR (CDCl3): δ 8.39 (ddd, J = 4.8, 1.8, 0.9 Hz, 1H), 8.35 (d, J = 5.8 Hz, 2H), 8.31 (d, J = 5.8 Hz, 1H), 8.15 (d, J = 8.2 Hz, 1H), 7.89 (d, J = 8.5 Hz, 2H), 7.69 (dd, J = 8.1, 3.6 Hz, 3H), 7.34–7.58 (m, 8H), 7.07–7.14 (m, 3H), 7.02 (ddd, J = 5.8, 4.9, 0.9 Hz, 1H), 4.16 (s, 4H), 4.12 (s, 2H), 3.67 (s, 2H), 2.81–2.84 (m, 4H). 13 C NMR (CDCl3): δ 159.2, 158.2, 148.5, 141.3, 136.1, 135.9, 129.7, 127.4, 126.8, 126.7, 126.6, 126.4, 126.2, 123.3, 121.7, 120.5, 120.4, 61.0, 60.6, 60.2, 52.7, 52.0. ESI-MS m/z: 597.25 [M + Na+]. Anal Calcd for C38H34N6 (1-isoTQMPEN): H, 5.96; C, 79.41; N, 14.62. Found: H, 6.04; C, 79.11; N, 14.89. [Zn(1b)](ClO4)2 To an ethanol solution (0.8 mL) of N,N-1-isoBQBPEN (1b) (30.0 mg, 57 μmol) was added Zn(ClO4)2·6H2O (21.3 mg, 57 μmol) in ethanol (1.2 mL), and the solution was kept at 4 °C to give [Zn(1b)](ClO4)2 as white powder (37.8 mg, 50 μmol, 83%). Recrystallization from the filtrate afforded [Zn(1b)](ClO4)2·2CH3OH as colorless crystals. 1 H NMR (CD3OD): δ 8.31 (d, J = 8.2 Hz, 2H), 8.17 (br., 4H), 8.03 (dd, J = 7.6, 5.8 Hz, 4H), 7.96 (d, J = 6.4 Hz, 4H), 7.91 (d, J = 8.2 Hz, 2H), 7.81 (dd, J = 7.0, 8.2 Hz, 2H), 7.58 (d, J = 8.2 Hz, 2H), 7.48 (br., 2H), 5.30 (d, J = 18.0 Hz, 2H), 4.75 (d, J = 18.3 Hz, 2H), 4.46 (d, J = 17.1 Hz, 2H), 4.26 (d, J = 17.4 Hz, 2H), 3.43 (br., 2H), 3.32–3.27 (m, 2H). 13 C NMR (CD3OD): δ 156.6, 148.3, 141.8, 138.7, 138.1, 134.1, 130.4, 128.6, 127.1, 126.3, 125.7, 124.7, 62.3, 59.2, 58.2. ESI-MS m/z: 687.12 [(M − ClO4)+]. Anal Calcd for C34H34Cl2N6O9Zn ([Zn(1b)](ClO4)2·H2O): C, 50.60; H, 4.25; N, 10.41. Found: C, 50.89; H, 3.96; N, 10.52. [Zn(2b)](ClO4)2 To a solution of N,N′-1-isoBQBPEN (2b) (15.7 mg, 30 µmol) in CH2Cl2–DMF (1 : 1, 300 µL) was added Zn(ClO4)2·6H2O (11.2 mg, 30 µmol) in methanol (200 µL), and the solution was kept at 4 °C to give [Zn(2b)](ClO4)2·DMF as colorless crystals (12.2 mg, 14.2 µmol, 47%). 1 H NMR (CD3OD): δ 9.02 (br. d, 2H), 8.44 (br., 2H), 8.35 (d, J = 8.2 Hz, 1H), 8.30 (d, J = 8.9 Hz, 2H), 7.5–8.2 (m, 13H), 5.17 (d, J = 19.6 Hz, 2H), 5.03 (d, J = 19.2 Hz, 2H), 4.70 (d, J = 15.9 Hz, 2H), 4.56 (d, J = 17.4 Hz, 1H), 4.34 (d, J = 17.4 Hz, 1H), 4.09 (d, J = 15.9 Hz, 2H), 3.54 (d, J = 10.1 Hz, 1H), 3.20 (d, J = 10.4 Hz, 1H). 13 C NMR (CD3OD): δ 157.8, 155.8, 149.6, 148.4, 142.3, 141.8, 138.0, 137.8, 137.6, 134.1, 133.8, 130.4, 130.2, 128.6, 128.5, 126.84, 126.79, 126.3, 125.6, 125.5, 124.7, 124.4, 63.7, 60.5, 58.8. ESI-MS m/z: 687.14 [(M − ClO4)+].
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Anal Calcd for C37H39Cl2N7O9Zn ([Zn(2b)](ClO4)2·DMF): C, 51.56; H, 4.56; N, 11.37. Found: C, 51.38; H, 4.57; N, 11.40.
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X-ray crystallography Single crystals of N,N-1-isoBQBPEN (1b), N,N′-1-isoBQBPEN·4H2O (2b·4H2O), [Zn(1b)](ClO4)2·2CH3OH and [Zn(2b)](ClO4)2·DMF were covered by Paratone-N oil and mounted on a glass fiber. All data were collected at 123–153 K on a Rigaku Mercury CCD detector, with monochromatic MoKα radiation, operating at 50 kV/40 mA. Data were processed on a PC using the CrystalClear Software (Rigaku). Structures were solved by direct methods (SIR-92)52 and refined by full-matrix least-squares methods on F2 (SHELXL-97).53 Crystal data are summarized in Tables S1 and S2.† CCDC 998434–998437 contain the supplementary crystallographic data for this paper.
Results and discussion Ligand synthesis N,N-BQBPEN (1a), N,N′-BQBPEN (2a), TQMPEN (3a), N,N-1-isoBQBPEN (1b), N,N′-1-isoBQBPEN (2b) and 1-isoTQMPEN (3b) were synthesized as shown in Scheme S1.† Although the crystal structure of the zinc complex of 2a was reported previously,54 the synthesis of 2a has not been documented. All new compounds were characterized by 1H/13C NMR and elemental analysis. The structures of 1b and 2b were further confirmed by X-ray crystallographic analysis (Table S1† and Fig. 1). Zn2+-induced absorbance and fluorescence spectral changes in 1–3a To compare the fluorescence properties of 1–3a with TQEN, a 34 µM solution in aqueous DMF (DMF–H2O = 1 : 1) at 25 °C was used for spectral measurements. Upon addition of Zn2+, the absorption of 1a at 303 and 317 nm increased (Fig. 2a). A slight absorption maximum shift was observed from 303 nm to 305 nm. A distinct isosbestic point was observed at 285 nm during the titration and spectral changes saturated with 1 eq. of Zn2+, indicating the formation of the 1 : 1 complex of 1a with Zn2+ (Fig. S1a†). Upon excitation at 317 nm, 1a exhibits Zn2+-induced fluorescence enhancement at 383 nm (I/I0 = 11 at 1 equiv. of Zn2+) accompanied with a weak emission band at 450 nm (Fig. 2b). The fluorescence quantum yield of [Zn(1a)]2+ is small (ϕ =
Fig. 2 Absorption (a, c, e) and fluorescence (λex = 317 nm) spectra (b, d, f ) of 34 µM N,N-BQBPEN (1a) (a, b), N,N’-BQBPEN (2a) (c, d) and TQMPEN (3a) (e, f ) in DMF–H2O (1 : 1) at 25 °C in the presence of various concentration of Zn2+ ranging from 0 to 68 µM.
0.010) and similar to the value reported for the [Zn(TQEN)]2+ complex (ϕ = 0.007).46 The fluorescence increase saturated with 1 equiv. of Zn2+, supporting the formation of the fluorescent 1 : 1 [ZnL]2+ complex (Fig. S1b†). Ligand 2a exhibited almost identical changes on absorbance spectra with 1a toward added Zn2+ (Fig. 2c and S1c†). In contrast, the fluorescence spectral change upon Zn2+ addition differs from that of 1a (Fig. 2d and S1d†). The fluorescence maximum was observed at 379 nm in I/I0 = 18 (at 1 equiv. of Zn2+) and the long wavelength emission is completely absent. This result suggests that the N,N-bis(quinolylmethyl) moiety, which 2a does not possesses, is responsible for the long wavelength emission. Ligand 3a also exhibited similar absorbance spectral changes responding to Zn2+ concentration (Fig. 2e and S1e†). Absorbance at 305 and 317 nm of 3a moved to 306 and 317 nm upon Zn2+ binding with an isosbestic point at 285 nm. The fluorescence profile induced by Zn2+ is almost identical to 1a and TQEN, exhibiting an increase at 383 nm with concomitant weak emission around 450 nm (Fig. 2f and S1f†). The fluorescence quantum yields of the [Zn(2a)]2+ (ϕ = 0.010) and [Zn(3a)]2+ complexes (ϕ = 0.009) are similar to those of [Zn(1a)]2+ and [Zn(TQEN)]2+. Zn2+-induced absorbance and fluorescence spectral changes in 1–3b
Fig. 1 ORTEP plot for (a) N,N-1-isoBQBPEN (1b) and (b) N,N’-1-isoBQBPEN (2b) in 50% probability. Hydrogen atoms were omitted for clarity. Atoms indicated with asterisks are generated by the symmetric operation.
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A previous report showed that 1-isoTQEN (Chart 1), which has four isoquinolines in place of quinoline moieties of TQEN, exhibited (i) enhancement of the fluorescence intensity,
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(ii) red-shifts in excitation and emission wavelengths due to the intramolecular excimer formation, (iii) increased Zn2+/Cd2+ discriminating ability, (iv) reduction of background fluorescence induced by pH, and (v) strong metal binding affinity.44 In order to investigate the details of the above characteristics, especially the mechanism for Zn2+-specific excimer formation, the fluorescence properties of isoquinoline derivatives 1–3b were investigated. Upon addition of Zn2+, the absorption of 1b at 310 and 323 nm decreased and new peaks at 313 and 326 nm appeared (Fig. 3a). Distinct isosbestic points were seen at 311, 319 and 324 nm during the titration and spectral changes saturated with 1 equiv. of zinc ion, indicating the formation of the 1 : 1 complex of 1b and Zn2+ ion (Fig. S2a†). Fig. 3b and S2b† show the fluorescence spectral change of 1b with increasing amounts of Zn2+ ions. Two emission bands at 353 and 475 nm were observed similar to 1-isoTQEN. The intensity ratio of long and short emissions of [Zn(1b)]2+ (I475/ I353 = 2.0) is similar to [Zn(1-isoTQEN)]2+ (I477/I357 = 1.6) due to the efficient excimer interaction of two isoquinolines in N,N-configuration. This value is significantly larger than that of [Zn(1-isoTQA)]2+ (I470/I359 = 0.48), indicating the efficient steric effect of the bis(2-pyridylmethyl)amine moiety in 1b enforcing the opposite two isoquinolines in close proximity in the excited state, compared to single isoquinoline coordination in the [Zn-(1-isoTQA)]2+ complex. The spectral change of 1b with Zn2+ titration was stopped on 1 : 1 complex formation (I/I0 = 23 at 475 nm).
Paper Table 1 Fluorescence lifetime for Zn2+ complexes with N,N-1-isoBQBPEN (1b), N,N’-1-isoBQBPEN (2b) and 1-isoTQEN measured in DMF–H2O (1 : 1)
λex (nm)
λem (nm)
τ (ns)
N,N-1-isoBQBPEN (1b)
333
N,N′-1-isoBQBPEN (2b) 1-isoTQEN
333 333
353 475 352 357 477
0.1 8.8 2.0 2.5, 5.7 9.0
As observed for the N,N′-isomer of the bisquinoline derivatives 2a, the isoquinoline isomer 2b in the N,N′-configuration exhibited nearly identical spectral changes with 1b (Fig. 3c and S2c†) but only a single emission band at shorter wavelengths in the fluorescence spectrum upon Zn2+ binding (Fig. 3d and S2d†). The intense long wavelength emission observed in 1-isoTQEN and 1b is completely lost for the N,N′isomer 2b. This is because the N,N-bis((iso)quinolylmethyl) amine group is responsible for the long wavelength emission around 450 nm by intramolecular excimer formation. The feature of 450 nm emission of the Zn2+-1b complex is concentration-independent (Fig. S3†), indicating that this emission is due to intramolecular excimer formation. The water content in the solvent was found to be important for the fluorescent response of 1b. As shown in Fig. S4,† significant reduction in fluorescence intensity was observed in DMF as a solvent; however, DMF–H2O (4 : 1) as a solvent exhibited exactly the same spectral changes as those in DMF–H2O (1 : 1). The tris(isoquinoline) derivative 3b exhibited Zn2+-induced absorbance (Fig. 3e and S2e†) and fluorescence (Fig. 3f and S2f†) spectral changes similar to 1-isoTQEN and 1b because of the N,N-bis(1-isoquinolylmethyl)amine moiety. The slightly reduced long/short fluorescence intensity ratio of [Zn(3b)]2+ (I475/I354 = 1.5) may be a result of appended monomer emission due to the isolated isoquinoline chromophore at the N′-position. The fluorescence quantum yields of [Zn(1b)]2+ (ϕ = 0.028) and [Zn(3b)]2+ (ϕ = 0.033) are similar to that of [Zn(1isoTQEN)]2+ (ϕ = 0.034).46 The [Zn(2b)]2+ complex exhibits a slightly larger value (ϕ = 0.051) due to the strong emission at 350 nm. The fluorescence lifetime of Zn2+ complexes with 1b, 2b, and 1-isoTQEN depends significantly on the monitoring wavelengths (Fig. S5–S9† and Table 1). The long wavelength emission around 450 nm has a long fluorescence lifetime (τ = ∼9 ns), while the short-wavelength emission around 350 nm has a short lifetime (τ = 0.1–2.5 ns). These two emission bands originate from the same ground-state species because they exhibit identical excitation spectra (data not shown). These O2-independent lifetime values indicate that the nature of these emissions is fluorescence from singlet excited states. Fluorescence metal ion specificity of 1–3a and 1–3b
Fig. 3 Absorption (a, c, e) and fluorescence (λex = 326 nm) spectra (b, d, f) of 34 µM N,N-1-isoBQBPEN (1b) (a, b), N,N’-1-isoBQBPEN (2b) (c, d) and 1-isoTQMPEN (3b) (e, f ) in DMF–H2O (1 : 1) at 25 °C in the presence of various concentrations of Zn2+ ranging from 0 to 68 µM.
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Fig. 4 shows the metal ion specificity of the fluorescence response of 1–3a and 1–3b. The fluorescence enhancement was specific for Zn2+ at indicated wavelengths. The ICd/IZn
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replacement of (iso)quinolines with pyridine does not alter metal selectivity in these probes. The steric hindrance in quinoline derivatives due to the peri hydrogen reduces Zn2+/Cd2+ discrimination ability.19,27 It is of significant interest that Cd2+ induces only short wavelength emission by CHEF (chelation enhanced fluorescence) and PeT (Photoinduced Electron Transfer) inhibition mechanisms55,56 and long wavelength emission is absent for all Cd2+ complexes (Fig. S10†). The excimer-like interaction between two (iso)quinolines are blocked in the Cd2+ complex, probably due to the long Cd– N(iso)quinoline distance. The subtle difference in ionic radii between Zn2+ and Cd2+ is effectively highlighted with the long wavelength emission of the 1-isoTQEN family ligands by the excimer formation. pH effect on fluorescence intensity of 1–3a and 1–3b
Fig. 4 The relative fluorescence intensity of (a) 1–3a and (b) 1–3b in the presence of 1 equiv. of metal ions in DMF–H2O (1 : 1) at 25 °C. I0 is the emission intensity of free ligand.
values are 59–72% and 8–12% for quinoline (1–3a) and isoquinoline (1–3b) derivatives, respectively, and these are similar to the corresponding tetrakis(iso)quinoline derivatives.27,44 Thus,
Fig. 5 demonstrates the pH-profile of fluorescence zinc detection of 1–3a and 1–3b. The monitoring wavelengths are the same as those in the previous section. Protonation on the ligand nitrogen atoms prevents the zinc binding at low pH regions and the formation of Zn(OH)2 at high pH regions inhibits the interaction with the ligand. As the quinoline moiety of TQEN is replaced with pyridine, the Zn2+ detection range becomes wider (Fig. 5a–c). The N,N′-BQBPEN (2a) exhibits a distinct advantage in Zn2+ detection under high pH conditions compared with the corresponding N,N-isomer (1a), indicating the higher metal binding ability of 2a due to less hindered coordination geometry as found in the crystal structure.54 The trisquinoline, TQMPEN (3a), showed a pH-sensitive profile with a similar extent to TQEN. On the other hand, isoquinoline derivatives 1–3b exhibit no difference with a very wide pH window for Zn2+ response (Fig. 5d–f). Negligible proton-induced
Fig. 5 Effect of pH on the fluorescence intensity of (a) 1a at 383 nm, (b) 2a at 379 nm, (c) 3a at 383 nm, (d) 1b at 475 nm, (e) 2b at 352 nm and (f ) 3b at 475 nm in the absence (blue triangles) and presence (red circles) of 1 equiv. of zinc ions in DMF–H2O (1 : 1) at 25 °C.
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fluorescence was observed for all apo ligands including quinoline derivatives.
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X-Ray crystallography of [Zn(1b)](ClO4)2 and [Zn(2b)](ClO4)2 Single crystals of [Zn(1b)](ClO4)2·2CH3OH and [Zn(2b)](ClO4)2·DMF were prepared and analyzed by X-ray crystallography (Fig. 6). Crystal data and bond distances around the metal centre are summarized in Tables S2† and 2, respectively. In the crystal structure of [Zn(1b)]2+, six nitrogen atoms of 1b coordinate to the Zn2+ center and no significant steric hindrance is found in the interaction between adjacent isoquinoline and pyridine rings. This structure is very close to the previously reported [Zn(1-isoTQEN)]2+ complex.44 The Zn–Npyridine distances (2.148(2) and 2.145(3) Å) are longer than the
Zn–Nisoquinoline distances (2.119(2) and 2.137(2) Å), indicating stronger metal coordination of isoquinoline nitrogens in comparison with pyridine nitrogen atoms. This is in good agreement with the difference in pKa values.57 The crystal structure of [Zn(2b)]2+ is also similar to the [Zn(1-isoTQEN)]2+ complex. In contrast to the structure of the quinoline counterpart [Zn(2a)]2+,54 two isoquinolines of [Zn(2b)]2+ are located at the ‘axial’ positions, in which the plane containing two aliphatic nitrogen atoms and a zinc center is defined as a ‘basal’ plane. Since the H-8 hydrogen atom of quinoline located at the ‘axial’ position of the [Zn(TQEN)]2+ complex exhibits significant steric hindrance (Zn–N = 2.4 Å),27 the pyridines occupy the ‘axial’ position in the [Zn(2a)]2+ complex. Even for tetrakis( pyridine) ([Zn(TPEN)]2+)27 and tetrakis(isoquinoline) ([Zn(1-isoTQEN)]2+), the bond distances of the ‘axial’ coordination (Zn–N4 and Zn–N6) is slightly longer than the ‘basal’ bond distances (Zn–N3 and Zn–N5) probably due to the structural requirement (Table 2). The strong coordination of isoquinolines in [Zn(2b)]2+ from the ‘axial’ positions shortens the bond distances, resulting in similar Zn–N distances for all aromatic nitrogen atoms. Theoretical calculations of [Zn(1b)]2+ and [Zn(2b)]2+ complexes Based on the crystal structures, electronic structures of [Zn(1b)]2+ and [Zn(2b)]2+ at the ground and excited states were calculated by time-dependent density-functional-theory (TDDFT) calculation (functional: ωB97XD;58 basis set: cc-pVDZ(C, N, H), 6-311++G(d,p) (Zn)). Although the optimized structure at the excited state for [Zn(1b)]2+ starting from the solid state structure (Fig. 7a) is almost identical with that at the ground state, the potential energy of the stacked excited state structure in which two isoquinoline rings are in parallel orientation (Fig. 7b) is 48 kJ mol−1 lower than the ground state-like (i.e., Franck Condon state), non-stacked excited structure. No minimum energy conformation was found in the parallelforced structure at the ground state. The absence of a parallelforced structure in the ground state means that the excited state with stacked isoquinoline rings has a so-called “excimer” character. The absorption and emission bands of [Zn(1b)]2+ were estimated in Tables S3† and 3, respectively. The molecular orbitals
Fig. 6 ORTEP plot for (a) [Zn(1b)](ClO4)2·2CH3OH and (b) [Zn(2b)](ClO4)2·DMF in 50% probability. Hydrogen atoms, counter anions and solvents were omitted for clarity.
Table 2 Selected bond distances (Å) for [Zn(TPEN)]2+, [Zn(1-isoTQEN)]2+, [Zn(1b)]2+ and [Zn(2b)]2+
Zn–N1 Zn–N2 Zn–N3 Zn–N4 Zn–N5 Zn–N6 a
[Zn(TPEN)]2+ a,b
[Zn(1-isoTQEN)]2+ c
[Zn(1b)]2+
[Zn(2b)]2+
2.19 2.20 2.09 2.19 2.10 2.21
2.1945(16)
2.189(2) 2.205(3) 2.119(2) 2.137(2) 2.148(2) 2.145(3)
2.228(3) 2.218(3) 2.155(3) 2.155(3) 2.153(3) 2.148(3)
2.1269(17) 2.1742(15)
b
c
Average values for two independent molecules. Ref. 27. Ref. 44.
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Fig. 7 Calculated S1 excited state structure of [Zn(1b)]2+ in (a) Franck Condon state and (b) parallel (excimer) configuration.
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State S1 S1
Dalton Transactions Calculated emission properties for [Zn(1b)]2+ and [Zn(2b)]2+
Wavelength (nm)
Oscillator strength
Orbital composition
[Zn(1b)]2+ with Franck Condon Conformation (See Fig. 7a) 340 0.070 LUMO → HOMO [Zn(1b)]2+ with Excimer Conformation (See Fig. 7b) 463 0.0055 LUMO → HOMO
CIa (%) 47 49
2+
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S1 a
335
[Zn(2b)] 0.079
LUMO → HOMO
48
Percentage of square of CI coefficient.
for each transition are indicated in Fig. S11–14.† The most striking point is that the longer wavelength emission centered at 463 nm was reasonably elucidated by computation from the LUMO–HOMO transition in a parallel-oriented structure (Fig. 7b). This excimer emission band perfectly matches the experimental observations. Moreover, the longer fluorescence lifetime of the excimer emission compared to the shorter wavelength emission is also predicted by the smaller value of the calculated oscillator strength (Table 3). No such long wavelength emissions or long fluorescence lifetimes was predicted for the non-parallel excited state of [Zn(1b)]2+ (Fig. 7a) and N,N′-isomer [Zn(2b)]2+, in which intramolecular excimer interaction of isoquinolines is not expected. Thermodynamic and kinetic competition of Zn2+ binding with TPEN We have demonstrated that 1-isoTQEN and its derivatives exhibit extremely high Zn2+ binding ability and resist metal ion removal by TPEN via competition experiments.44 Even for 2-quinolylmethylamine derivatives TQDACH (N,N,N′,N′-tetrakis(2-quinolylmethyl)-trans-1,2-diaminocyclohexane) and TQTACN (N,N′,N″-tris(2-quinolylmethyl)-1,4,7-triazacyclononane), Zn2+ is not transferred to TPEN by the strong metal binding affinity of the pre-organized, rigid aliphatic amine backbone of these ligands.24,42 Introduction of carboxylates in place of (iso)quinolines of (1-iso)TQEN significantly enhances the metal binding ability, resulting in removal of Zn2+ from the [Zn(TPEN)]2+ complex by apo (iso)quinoline-carboxylate ligands.59 The fluorescence intensity of Zn2+ complexes with 1–3b was stable in the presence of 1 equiv. of TPEN for at least 7 days at room temperature (Fig. 8b). In contrast, the quinoline derivatives 1–3a showed a gradual fluorescence quenching by TPEN in a structure-dependent manner (Fig. 8a). While the trisquinoline derivative 3a exhibited immediate loss of fluorescence upon addition of TPEN, the bisquinoline derivatives 1a and 2a showed different resistance properties toward TPEN. The N,N′isomer 2a releases Zn2+ at a slower rate than the N,N-isomer 1a. This is because the sterically unfavorable ‘axial’ quinoline coordination (i.e., N4 and N6 in Fig. 6) is absent in the [Zn(2a)]2+ complex, in which the two quinolines adopt ‘basal’ (i.e., those containing N3 and N5 in Fig. 6) coordination.54 As mentioned above, the close proximity between the peri hydrogen
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Fig. 8 Time dependent fluorescence intensity changes of (a) [Zn(1a)]2+ at 383 nm (red circles), [Zn(2a)]2+ at 379 nm (blue triangles) and [Zn(3a)]2+ at 383 nm (green squares) and (b) [Zn(1b)]2+ at 475 nm (red circles), [Zn(2b)]2+ at 352 nm (blue triangles) and [Zn(3b)]2+ at 475 nm (green squares) in the presence of 1 equiv. of TPEN at 25 °C in DMF– H2O (1 : 1).
atom at the 8-position of the ‘axial’ quinoline and ‘basal’ aromatic ring generates steric hindrance and weakens the metal– nitrogen bond of the ‘axial’ quinoline in the [Zn(1a)]2+ complex. The preliminary X-ray analysis of [Zn(1a)]2+ exhibits a five-coordinate structure due to the weak quinoline coordination (Fig. S15†). Based on the extremely slow metal exchange rate mentioned above, the kinetic metal binding competition of 1–3b versus TPEN was examined. In this experiment, to an equimolar mixture of ligand (1b, 2b or 3b) and TPEN in the apo form was added Zn2+ in portions and the fluorescence change was monitored. Under such conditions, the fluorescence intensity changes shown in Fig. 9 represent the relative Zn2+ capture rate of apo 1a (or 2b, 3b) versus apo TPEN. Thus, Fig. 9 can be regarded as a kinetic competition of Zn2+ binding of these ligands with TPEN. For the slow-binding ligands such as TQDACH and TQTACN, the fluorescence intensity was unchanged at 0–1 equiv. of Zn2+ until all TPEN was consumed, then the fluorescence started to increase and reached saturation at 2 equiv. of Zn2+.24,42 Similarly, a small slope during
Fig. 9 Kinetic competitive fluorescence intensity change of (a) 1b at 475 nm, (b) 2b at 352 nm, (c) 3b at 475 nm and (d) 1-isoTQEN at 475 nm in the presence (red circles) and absence (blue triangles) of 1 equiv. of TPEN with increasing amounts of zinc at 25 °C in DMF–H2O (1 : 1).
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0–1 equiv. of Zn2+ in Fig. 9d in the presence of TPEN suggested the slow Zn2+ binding rate of 1-isoTQEN. For 1–3b, however, such a bending point at 1 equiv. of Zn2+ was not observed because the Zn2+ capture rate of these ligands was as rapid as TPEN (Fig. 9a–c). The introduction of at least one pyridine ring into 1-isoTQEN significantly improved the metal binding rate of the ligand.
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Intracellular Zn2+ sensing with 1–3a and 1–3b PC-12 rat adrenal pheochromocytoma cells were applied to evaluate the ability of in vitro zinc sensing with 1–3a and 1–3b. The cells were incubated in growth media containing 100 µM of ligands and 2% (v/v) DMSO for 4 h and analyzed with a fluorescence microscope after washing of the cell and changing the media. [Zn( pyrithione)2] (final concentration: 90 µM) was added to the media during microscopic analysis and the fluorescence enhancement was monitored. As shown in Fig. 10, a small fluorescence inside the cells was observed in the presence of isoquinoline derivatives 1–3b after addition of [Zn( pyrithione)2] into the media. No cytotoxicity was observed up to 100 µM in the growth media. The cell-permeability and intracellular zinc-responding ability of the compounds strongly appeal the potential of these ligands as zinc-specific fluorescent sensor platforms.
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Conclusions The intramolecular excimer formation of the N,N-bis(1-isoquinolylmethyl)amine moiety of 1-isoTQEN derivatives upon Zn2+ binding plays a key role in the selective fluorescence enhancement at longer wavelengths. Such an excimer emission is completely specific to Zn2+. Cd2+ induces only moderate CHEFand PeT-based emissions at shorter wavelengths. The hexadentate tetrakis(heteroaromatic)ethylenediamine scaffold as a fluorescent Zn2+ probe has unique advantages in synthetic accessibility, stability of the Zn2+ complex, and excimer forming ability. Replacement of two of the quinoline heterocycles of TQEN with pyridines affords higher Zn2+ binding ability and proton insensitivity without losing any necessary fluorescence properties. Although suitable improvement to enhance fluorescence intensity and water-solubility is required, the present findings provide a new strategy for rational design of Zn2+-specific fluorescent probe molecules.
Acknowledgements Authors thank Ms Ikuko Hamagami and Ms Sakiko Akaji of Horiba, Ltd for their kind help in fluorescence lifetime measurements. Authors also thank Prof. Satoshi Tamotsu, Prof. Keiko Yasuda and Dr Masato Aoyama of Nara Women’s University for their kind help in cellular experiments. This work was supported by the Research for Promoting Technological Seeds, JST, Adaptable and Seamless Technology Transfer Program through Target-driven R&D, JST, Grant-in Aid for Scientific Research from the MEXT, Japan and the Nara Women’s University Intramural Grant for Project Research.
References
Fig. 10 Differential interference contrast (DIC) and fluorescence (FL) micrographs of cultured PC-12 rat adrenal cells in the presence of 100 µM 1–3a and 1–3b for 4 h. [Zn( pyrithione)2] (90 µM) was added during microscopic analysis ( photograph taken after ∼4 min).
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