Cd2+-Selective Fluorescence Enhancement of Bisquinoline Derivatives with 2-Aminoethanol Skeleton

The development of fluorescent Cd2+ sensors requires strict selectivity over Zn2+ because of the high availability of Zn2+ in the natural environment. In this paper, bisquinoline-based fluorescent sensors with a 2-aminoethanol backbone were investigated. The weak coordination ability of quinoline compared to well-studied pyridine is suitable for Cd2+ selectivity rather than Zn2+. In the presence of 3 equiv. of metal ions, TriMeO-N,O-BQMAE (N,O-bis(5,6,7-trimethoxy-2-quinolylmethyl)-2-methylaminoethanol (3)), as well as its N,N-isomer TriMeO-N,N-BQMAE (N,N-bis(5,6,7-trimethoxy-2-quinolylmethyl)-2-methoxyethylamine (6)), exhibits Cd2+-selective fluorescence enhancement over Zn2+ in DMF-HEPES buffer (1:1, 50 mM HEPES, 0.1 M KCl, pH = 7.5) (IZn/ICd = 26–34%), which has similar selectivity in comparison to the corresponding ethylenediamine derivative TriMeOBQDMEN (N,N’-bis(5,6,7-trimethoxy-2-quinolylmethyl)-N,N’-dimethylethylenediamine) under the same experimental condition (IZn/ICd = 24%). The fluorescence mechanisms of N,O- and N,N-isomers of BQMAE are quite different, judging from the fluorescence lifetimes of their metal complexes. The Cd2+ complex with TriMeO-N,O-BQMAE (3) exhibits a long fluorescence lifetime similar to that of TriMeOBQDMEN via intramolecular excimer emission, whereas the Cd2+ complex with TriMeO-N,N-BQMAE (6) exhibits a short lifetime from monomer emission.


Introduction
Cadmium is a toxic metal and exists in vast areas of natural environments.Extensive use of cadmium in industry, including batteries and pigments, facilitates the exposure of cadmium to the air, water, and soils.As a result, cadmium-related health problems in humans are recent serious concerns [1,2].In this context, the detection and quantification of cadmium in the environment, especially in water, are of urgent demand [3][4][5].
The fluorescence method is a powerful tool for the detection of metal ions and biologically important small molecules [6][7][8][9][10][11][12][13][14][15].Its sensitivity, rapidness, and convenience allow us to visualize the target effectively.Specificity is the most important requisite for fluorescence sensing and owes to the rational molecular design of fluorescent probes.One of the most difficult and important problems in fluorescence detection is the discrimination between Cd 2+ and Zn 2+ because these two metal ions slightly differ in their ionic radii, which is only 21 pm [16][17][18][19].The weak basicity of the quinoline nitrogen atom compared to that of pyridine offers a suitable coordination environment around the larger and softer Cd 2+ center than Zn 2+ .Therefore, many quinoline-based fluorescent Cd 2+ sensors have been extensively developed [20][21][22][23][24][25].

Synthesis of Ligands
The newly designed six ligands (1-6) based on a 2-aminoethanol skeleton were prepared according to Scheme S1 using corresponding amines and methoxy-substituted chloromethylquinolines.N-alkylation of 2-methylaminoethanol followed by O-alkylation using n-butyllithium affords 1-3 in quite low yields (6-19%) due to the unoptimized experimental procedure for the second step.The synthesis of 4-6 was conducted in one step, starting from 2-methoxyethylamine in excellent yields (96% to quantitative yield).Structure and purity of ligands 3-6 were supported by 1 H/ 13 C NMR and elemental analysis.Structure of ligands 1 and 2 was confirmed by 1 H/ 13 C NMR.The complicated coupling pattern at the aliphatic proton region of 2 in 1 H NMR spectrum is still under investigation.
The metal binding affinity (K d ) was calculated from the non-linear fitting of titration curves with Cd 2+ and Zn 2+ , indicating that TriMeO-N,O-BQMAE (3) has two orders lower metal binding ability than TriMeOBQDMEN (Figures S3 and S4 and Table 1).This is because the coordination ability of the nitrogen atom is higher than that of oxygen, but two orders of difference are quite significant for such a small modification.

Synthesis of Ligands
The newly designed six ligands (1-6) based on a 2-aminoethanol skeleton were prepared according to Scheme S1 using corresponding amines and methoxy-substituted chloromethylquinolines.N-alkylation of 2-methylaminoethanol followed by O-alkylation using n-butyllithium affords 1-3 in quite low yields (6-19%) due to the unoptimized experimental procedure for the second step.The synthesis of 4-6 was conducted in one step, starting from 2-methoxyethylamine in excellent yields (96% to quantitative yield).Structure and purity of ligands 3-6 were supported by 1 H/ 13 C NMR and elemental analysis.Structure of ligands 1 and 2 was confirmed by 1 H/ 13 C NMR.The complicated coupling pattern at the aliphatic proton region of 2 in 1 H NMR spectrum is still under investigation.
The metal binding affinity (Kd) was calculated from the non-linear fitting of titration curves with Cd 2+ and Zn 2+ , indicating that TriMeO-N,O-BQMAE (3) has two orders lower metal binding ability than TriMeOBQDMEN (Figures S3 and S4 and Table 1).This is because the coordination ability of the nitrogen atom is higher than that of oxygen, but two orders of difference are quite significant for such a small modification.

Fluorescence Spectral Changes of N,N-BQMAE Derivatives 4-6
The metal ion-induced fluorescence spectral changes of N,N-BQMAE derivatives 4-6 were next examined (Figure 2).Absorbance spectral changes and excitation spectrum for 6 are shown in Figure S2.Because of the synthetic inconvenience of N,O-BQMAE derivatives 1-3 and disappointing fluorescence response of 1 and 2 toward metal ions due to the weak metal binding ability of these compounds, the other N3O1 ligands with a bis(2-quinolylmethyl)amine moiety were explored.As shown in Figure 2, even in the unsubstituted quinoline derivative N,N-BQMAE (4), metal-induced fluorescence enhancement was clearly observed, which is largely different from the N,O-isomer 1 and 2. In the presence of a large excess of metal ions to assure the complete complexation, Zn 2+ induces a slightly higher fluorescence intensity of 4 than Cd 2+ (Figure S5).Other methoxysubstituted derivatives, including TriMeO-N,O-BQMAE (3) and TriMeOBQDMEN in the previous section, show complete metal binding in the presence of 3 equiv.of metal ions, and exhibit consistent preference of Cd 2+ over Zn 2+ (Figures S3, S4, S6 and S7).As the number of methoxy substituents increases, the fluorescence Cd 2+ selectivity is improved (Figure 2b,d,f, and Table 1), and the metal binding affinity is enhanced (Figures S5-S7 and Table 1).The three trimethoxy-substituted derivatives, namely, TriMeO-N,O-BQMAE (3) (K d(Zn) = ~10 −4 ), TriMeO-N,N-BQMAE (6) (K d(Zn) = ~10 −5 ), and TriMeOBQDMEN (K d(Zn) = ~10 −6 ) exhibit approximately one order different metal binding affinities in this order (Table 1).Since the fluorescence metal ion selectivity of these compounds is essentially the same (I Zn /I Cd = ~30%), this ligand library is a nice set of fluorescent sensors with different metal binding affinities, which is useful for convenient determination of Cd 2+ concentration.

Fluorescence Spectral Changes of N,N-BQMAE Derivatives 4-6
The metal ion-induced fluorescence spectral changes of N,N-BQMAE derivatives 4-6 were next examined (Figure 2).Absorbance spectral changes and excitation spectrum for 6 are shown in Figure S2.Because of the synthetic inconvenience of N,O-BQMAE derivatives 1-3 and disappointing fluorescence response of 1 and 2 toward metal ions due to the weak metal binding ability of these compounds, the other N3O1 ligands with a bis(2-quinolylmethyl)amine moiety were explored.As shown in Figure 2, even in the unsubstituted quinoline derivative N,N-BQMAE (4), metal-induced fluorescence enhancement was clearly observed, which is largely different from the N,O-isomer 1 and 2. In the presence of a large excess of metal ions to assure the complete complexation, Zn 2+ induces a slightly higher fluorescence intensity of 4 than Cd 2+ (Figure S5).Other methoxysubstituted derivatives, including TriMeO-N,O-BQMAE (3) and TriMeOBQDMEN in the previous section, show complete metal binding in the presence of 3 equiv.of metal ions, and exhibit consistent preference of Cd 2+ over Zn 2+ (Figures S3, S4, S6 and S7).As the number of methoxy substituents increases, the fluorescence Cd 2+ selectivity is improved (Figure 2b,d,f, and Table 1), and the metal binding affinity is enhanced (Figures S5-S7 and Table 1).The three trimethoxy-substituted derivatives, namely, TriMeO-N,O-BQMAE (3) (Kd(Zn) = ~10 −4 ), TriMeO-N,N-BQMAE (6) (Kd(Zn) = ~10 −5 ), and TriMeOBQDMEN (Kd(Zn) = ~10 −6 ) exhibit approximately one order different metal binding affinities in this order (Table 1).Since the fluorescence metal ion selectivity of these compounds is essentially the same (IZn/ICd = ~30%), this ligand library is a nice set of fluorescent sensors with different metal binding affinities, which is useful for convenient determination of Cd 2+ concentration.,d,f) in the presence of 3 equiv.of metal ions.I0 is the emission intensity of free ligand.

Effect of pH and Estimation of LOD
The effect of pH on the fluorescence intensity of the Cd 2+ complex with TriMeO-N,O-BQMAE (3) and TriMeO-N,N-BQMAE (6) was examined (Figure 3).Protonation to the ligand at low pH region and coordination of hydroxide ion to Cd 2+ at high pH region prevent the binding of Cd 2+ to the probes.The pH window for fluorescent detection of Cd 2+ is wider for 6 (Figure 3b) than 3 (Figure 3a), reflecting the difference in metal binding affinity of the ligands (Table 1).For 6, fluorescence intensity is fairly stable between pH = 4 and pH = 11.The limit of detection (LOD) for 6 was estimated as low as 17 nM (Figure S8), which is comparable to the other tetradentate fluorescent Cd 2+ probes in the literature [24,30,31].This value (LOD = 17 nM) is lower than the environmental limit of water in Japan (3 ppb, 27 nM).Accordingly, the limit of quantitation (LOQ) is calculated to be 57 nM.The above properties of TriMeO-N,N-BQMAE (6) demonstrate the potential of this ligand for practical uses.,d,f) in the presence of 3 equiv.of metal ions.I 0 is the emission intensity of free ligand.

Effect of pH and Estimation of LOD
The effect of pH on the fluorescence intensity of the Cd 2+ complex with TriMeO-N,O-BQMAE (3) and TriMeO-N,N-BQMAE (6) was examined (Figure 3).Protonation to the ligand at low pH region and coordination of hydroxide ion to Cd 2+ at high pH region prevent the binding of Cd 2+ to the probes.The pH window for fluorescent detection of Cd 2+ is wider for 6 (Figure 3b) than 3 (Figure 3a), reflecting the difference in metal binding affinity of the ligands (Table 1).For 6, fluorescence intensity is fairly stable between pH = 4 and pH = 11.The limit of detection (LOD) for 6 was estimated as low as 17 nM (Figure S8), which is comparable to the other tetradentate fluorescent Cd 2+ probes in the literature [24,30,31].This value (LOD = 17 nM) is lower than the environmental limit of water in Japan (3 ppb, 27 nM).Accordingly, the limit of quantitation (LOQ) is calculated to be 57 nM.The above properties of TriMeO-N,N-BQMAE (6) demonstrate the potential of this ligand for practical uses.

Fluorescence Lifetimes of N,O-and N,N-BQMAE Derivatives 3-6 and TriMeOBQDMEN
The fluorescence lifetimes (τ) for Cd 2+ and Zn 2+ complexes with N,O-and N,N-BQMAE derivatives 3-6 were measured in the presence of 2 equiv. of metal ions, in which most of the ligands form metal complexes.For comparison, TriMeOBQDMEN was also re-examined in DMF-HEPES buffer (1:1, 50 mM HEPES, 0.1 M KCl, pH = 7.5).Table 2 summarizes the results.As discussed previously for TriMeOBQDMEN based on the investigation in DMF-H2O solution [29], the fluorescent Cd 2+ selectivity of this ligand is explained by the difference in fluorescence lifetime in Cd 2+ and Zn 2+ complexes, in which a very long (τ = ~30 nsec) lifetime was observed exclusively for the Cd 2+ complex.From the crystal structure of the Cd 2+ complex with BQDMEN ([Cd2(µ-Cl)2(BQDMEN)2] 2+ ), this long lifetime was assigned to be an intramolecular excimer emission from the highly stacked

Fluorescence Lifetimes of N,O-and N,N-BQMAE Derivatives 3-6 and TriMeOBQDMEN
The fluorescence lifetimes (τ) for Cd 2+ and Zn 2+ complexes with N,Oand N,N-BQMAE derivatives 3-6 were measured in the presence of 2 equiv. of metal ions, in which most of the ligands form metal complexes.For comparison, TriMeOBQDMEN was also re-examined in DMF-HEPES buffer (1:1, 50 mM HEPES, 0.1 M KCl, pH = 7.5).Table 2 summarizes the results.As discussed previously for TriMeOBQDMEN based on the investigation in DMF-H 2 O solution [29], the fluorescent Cd 2+ selectivity of this ligand is explained by the difference in fluorescence lifetime in Cd 2+ and Zn 2+ complexes, in which a very long (τ = ~30 nsec) lifetime was observed exclusively for the Cd 2+ complex.From the crystal structure of the Cd 2+ complex with BQDMEN ([Cd 2 (µ-Cl) 2 (BQDMEN) 2 ] 2+ ), this long lifetime was assigned to be an intramolecular excimer emission from the highly stacked two quinoline rings with interligand interaction.Interestingly, in DMF-HEPES buffer (1:1, 50 mM HEPES, 0.1 M KCl, pH = 7.5), this long fluorescence lifetime was preserved for the TriMeOBQDMEN-Cd 2+ complex (τ = 32 nsec) (Table 2).Here again, the Zn 2+ complex with TriMeOBQDMEN exhibits a shorter lifetime (τ = 20 nsec) than Cd 2+ .These results strongly support that the fluorescence lifetime is a critical measure for the mechanism of fluorescence and structure of the metal complexes, which is independent of the solvent system used for measurements.
Similar distinct difference in fluorescence lifetimes of Cd 2+ and Zn 2+ complexes was found for TriMeO-N,O-BQMAE (3) (τ Cd = 30 nsec and τ Zn = 22 nsec).This is because the ligand structure of N,O-BQMAE resembles that of BQDMEN (Scheme 1) and rationally suggests the specific formation of a bis(µ-chloro) dinuclear cadmium complex for 3. On the other hand, the N,N-BQMAE derivatives did not exhibit any differences in fluorescence lifetimes between Cd 2+ and Zn 2+ complexes.Even in the trimethoxy-substituted TriMeO-N,N-BQMAE ( 6), the lifetimes for Cd 2+ and Zn 2+ complexes are completely the same (τ Cd = 18 nsec and τ Zn = 17 nsec), and these values are too short to consider the excimer formation discussed above.These values (τ = ~20 nsec) are in good agreement with those for monomer complexes such as TriMeOTQTACN [28] (N,N',N"-tris( 5 1) may reflect such differences.Although extensive trials of crystallization of Cd 2+ complexes of 1-6 have been unsuccessful so far, the proton NMR indicates the coordination of quinoline and methoxy moieties to the metal center for N,N-BQMAE (4)-Cd 2+ complex, affording the chemical shift changes (Figure S9).The characteristic peak splitting of the methylene signal around 4.4-4.7 ppm also supports the coordination of 2-aminomethylquinoline moiety to the metal center.
At the present stage, the details in the fluorescent Cd 2+ /Zn 2+ discrimination mechanism of TriMeO-N,N-BQMAE ( 6) is still unknown, but the dynamic equilibrium of mononuclear ([Cd(L)Cl] + ) and µ-chloro-bridged dinuclear ([Cd 2 (µ-Cl) 2 (L) 2 ] 2+ ) cadmium complexes needs to be considered.To answer these critical questions, related projects using other ligand systems with different skeletons are now in progress in our laboratory.

Materials and Methods
All reagents and solvents used for the preparation of probe molecules were obtained from commercial sources and used as received.DMF (N,N-dimethylformamide) used for spectroscopic measurements was of spectral grade (Dojin, Spectrosol).All aqueous solutions were prepared using Milli-Q water (Millipore, Merck, Germany). 1 H/ 13 C NMR spectra were recorded on a JEOL (Akishima, Japan) AL-400 (400/100 MHz) spectrometer and referenced to internal Si(CH 3 ) 4 or solvent signal.Elemental analyses were recorded on J-Science (Kyoto, Japan) JM-10 micro corder.UV-vis and fluorescence spectra were measured on a Jasco (Hachioji, Japan) V-660 spectrophotometer and Jasco (Hachioji, Japan) FP-6300 spectrofluorometer, respectively.Fluorescence quantum yields were measured on a HAMAMATSU photonics (Hamamatsu, Japan) C9920-02 absolute PL quantum yield measurement system.Fluorescence lifetimes were measured on a HORIBA fluorescence lifetime system Tempro equipped with a 370, 400, 460, or 490 nm bandpath filter.TriMeOBQD-MEN [29] was prepared as described previously.CAUTION: Perchlorate salts of metal complexes with organic ligands are potentially explosive.All due precautions should be taken.
This material (200 mg, 0.925 mmol) was dissolved in THF (4 mL) and cooled on ice, then n-butyllithium (1.6 M solution in hexane, 869 µL, 1.39 mmol) was added slowly through a syringe.After stirring for 30 min on ice, 2-chloromethylquinoline (205 mg, 1.15 mmol) in THF (2 mL) was added slowly through a syringe.The reaction mixture was warmed to room temperature and refluxed for 2 h.After the reaction, solvent was removed under reduced pressure, and organic materials were extracted with dichloromethane-water.The organic layer was dried and evaporated, and the residue was purified by alumina column chromatography using chloroform containing 0.1% methanol (R f = 0.25) as an eluent.The product was further purified by recycling GPC to afford N,O-bis(2-quinolylmethyl)-2methylaminoethanol (N,O-BQMAE (1)) as yellow oil (21.3 mg, 0.0596 mmol, 6%). 1 The mixture of 2-methylaminoethanol (62.8 mg, 0.836 mmol), 6-methoxy-2-chloromethylquinoline (154 mg, 0.742 mmol), potassium carbonate (103 mg, 0.745 mmol), and potassium iodide (142 mg, 0.855 mmol) in dry acetonitrile (20 mL) was refluxed for 23 h.After the reaction mixture was cooled to room temperature, solvent was removed under reduced pressure, and organic materials were extracted with dichloromethane-water.The organic layer was dried and evaporated to afford N-(6-methoxy-2-quinolylmethyl)-2methylaminoethanol as yellow oil in quantitative yield.

Conclusions
The newly designed six bisquinoline-based N3O1 tetradentate ligands with 2-aminoethanol backbone were examined as a fluorescent Cd 2+ sensor.This ligand library includes N,O-BQMAE derivatives 1-3 and N,O-BQMAE derivatives 4-6, which differ in the position of two quinoline moieties and the number of methoxy substituents on the quinoline rings.Judging from the moderately good fluorescent Cd 2+ selectivity over Zn 2+ (I Zn /I Cd = ~30%) and wide range of metal binding affinity (two orders differences in K d ), the trimethoxy derivatives TriMeO-N,O-BQMAE (3) and TriMeO-N,N-BQMAE (6), as well as previously reported TriMeOBQDMEN, are regarded as a potential set of fluorescent ligands with different metal binding affinities useful for convenient quantification of Cd 2+ in solution.

Table 1 .
Fluorescent and Thermodynamic Properties for Cd 2+ and Zn 2+ Complexes of BQMAE Derivatives

Table 2 .
Fluorescence Lifetimes for Cd