Fluorescent Sensors for Hg2+ and Cu2+ Based on Condensation Products of 4H-1,2,4 Triazole-4-Amine and Carboxylated Benzoic Acids

Mercury (Hg) causes serious health issues in its all forms. Deficiency as well as excess of copper ion (Cu2+) in human body is hazardous. A series of four compounds have been derived from carboxylated benzoic acids (benzoic acid, isophthalic acid, terephthalic acid and phthalic acid) and 4H-1,2,4 triazole-4-amine and characterized. Fluorescence detection of Hg2+ was recorded by the derivates with benzoic acid and isophthalic acid while the derivatives of terephthalic acid and phthalic acid detect Cu2+ by fluorescence “off” mode. Metal ions like Li+, Na+, K+, Zn2+, Al3+, Mg2+, Mn2+, Co2+, Ni2+, Cu2+, Cd2+, Pb2+ and Hg2+ found not to interfere. The stoichiometry of binding is 1:1 for the benzoic acid derivative with Hg2+ while it is 1:2 for the other three derivatives. The binding constants are ca. 10–4.5 between the sensors and Hg2+ or Cu2+ and detection limits are around 10–5.5 M. DFT calculation provided optimized geometries of the sensors and confirmed the stoichiometry of binding with Hg2+/Cu2+.


Introduction
Most of the heavy metals in ionic form are hazardous to the environment and human health and one of them is Mercury ion (Hg 2+ ) [1]. The relatively high stability of Hg 2+ ion enable it to reach human body through water, soil, air and food chain [2]. Hg 2+ ions severely affect many living organisms [3]. Power plants release considerable amount of Hg 2+ into the atmosphere due to burning of large quantity of coal and hence releasing Hg 2+ [4]. Batteries, pesticides and paper mills are other major sources of Hg 2+ pollution. Compact Fluorescent Lamps contain Hg 2+ ions and have become a matter of great environmental concern as unscientific disposal of CFLs acts as source of Hg 2+ pollution [5]. Hg 2+ is more toxic in metaloorganic form and can lead to deterioration of brain, hamper fetus growth [6], can damage immune system, kidney, heart, and lung of human being [7]. Hence monitoring system for Hg 2+ in water has been a matter of utmost importance with respect to the environmental and human health.
The common strategy for developing sensor for Hg 2+ is to synthesize molecules containing S so as to form Hg-S bonds which leads to structural change observed spectroscopically or reaction giving new spectroscopically active molecules (chemodosimeters) [8][9][10][11][12][13]. The occurance of mercury in sulfur rich environment is a disadvantage of S based fluorescent sensors for Hg 2+ [14]. Direct chelation of S based molecules to Hg 2+ and subsequent fluorescence enhancement are not known much [15][16][17]. Schiff bases, due to easy synthetic procedure, have gained interest as sensor for metal ions. The number of Schiff bases not having S but act as Hg 2+ fluorescence sensor are rare. Examples can be cited are Schiff base obtained from-α-naphthaldehyde with naphthylamine [18], Nphenylthiosemicarbazide and 2-naphthaldehyde [19] etc. Amide armed calix [4]-aza-crown based sensor containing S is reported but Hg 2+ binds to N atoms [20], sensor based on a pentaaza macrocycle conjugated to a hemicyanine dye where Hg 2+ binds to carboxylate is known [21]. Hence development of fluorescence sensor for Hg 2+ without having S must be of interest.
Copper is the third most abundant metal in the globe. It is involved in many critical life processes like -photosynthesis, respiration, superoxide dismutase etc. Excess accumulation of Cu 2+ in human body may cause severe health problems like Parkinson's, Alzheimer's, Menke's and Wilson's disease [22][23][24][25]. Retarded growth and defective nervous system is related to deficiency of copper [26]. Industries like electroplating, electric gadgets, alloy etc. are the sources of major copper pollutions. Therefore development of simple but sensitive methods to detect Cu 2+ is of great importance. Schiff bases which are simple to synthesis and highly sensitive as fluorescent sensor for Cu 2+ are known [27,28].
In this paper we report the synthesis and characterization of a series of four compounds derived from 4H-1,2,4 triazole-4-amine and carboxylated benzoic acids. The two compounds obtained from 4H-1,2,4 triazole-4-amine with benzoic acid and isophthalic acid act as fluorescent sensor for Hg 2+ while the other two compounds obtained from 4H-1,2,4 triazole-4-amine with phthalic acid and terephthalic acid are found to act as fluorescent sensor for Cu 2+ based on fluorescent quenching. DFT structure optimization of the sensors as well as their Hg 2+ /Cu 2+ complexes reported and found to be in conformity to experimental results.

Materials and methods
The chemicals have been procured either from Sigma Aldrich or LOBA Chemie. Except for Pb(NO 3 ) 2 , CdCl 2 and HgCl 2 the other metal salts are sulfates. Double distilled water obtained from quartz double distillation plant were used to prepare metal salt solutions (0.01 M). The FT-IR spectra were recorded as KBr pallet in Perkin Elmer RXI spectrometer. NMR spectra ( 1 H and 13 C) were obtained using Bruker Ultra Shield 300 MHz spectrophotometer using CDCl 3 or DMSO-d 6 as solvent. The UV/Visible spectra were recorded in Shimadzu UV 1800, fluorescence in HITACHI 2700 and HRMS in WATERS Q-TOF premier mass spectrometer. (Scheme 1)

Computational Details
All the structures were fully optimized at M06-2X/6-311 + + G** level of theory without any symmetry constraints [29]. At the same level of theory frequency calculations were performed to understand the nature of the stationary states. All these structures were found to be local minima with all real frequencies. Effective core potential basis set of Stuttgart/Dresden (SDD) were used for Hg atom. Natural bond orbital analyses (NBO) [30][31][32] were performed to understand the electronic feature of these systems. All these calculations were performed using Gaussian16 suite of program [33].
The solutions of AM1, AM2, AM3 and AM4 were titrated with the metal ions-Li + , Na + , K + , Mg 2+ , Mn 2+ , Co 2+ , Ni 2+ , Cu 2+ , Zn 2+ , Cd 2+ , Al 3+ , Pb 2+ and Hg 2+ . The fluorescence intensity of AM1 and AM2 were quenched by Hg 2+ while other metal ions did not affect the fluorescence intensity significantly. In case of AM3 and AM4 the fluorescence intensity was found to be quenched by Cu 2+ while other metal ions had no significant affect. Figure 1A and Fig. 1B show the effect of increasing concentration of Hg 2+ on fluorescence intensity quenching of AM1 and AM2 respectively. Figure 1C and Fig. 1D show the gradual quenching effect of Cu 2+ concentration on fluorescence intensity of AM3 and AM4 respectively. were studied. For this purpose one equivalent of a metal ion was added into the solution of AMX (X = 1-4) and allowed to equilibrate for 5 min. Then one equivalent of Hg 2+ (for X = 1,2) or Cu 2+ (for X = 3,4) was added and again allowed to equilibrate for about 5 min. The fluorescence spectrum was then recorded. It was observed that Hg 2+ or Cu 2+ could quench the fluorescence intensity of the respective sensor solution even in presence of another metal ion to the same extent when Hg 2+ or Cu 2+ was added to the sensor solution in absence of the other metal ion. This has been illustrated in Fig. 3(A to D) through bar diagrams.

Determination of binding constants and stoichiometry of binding
The binding constants and the stoichiometry of binding between the sensors (AM1 to AM4) and Hg 2+ or Cu 2+ were determined from the fluorescence titration data of the sensor against Hg 2+ or Cu 2+ . The log[(I 0 -I s )/(I s -I f )] was plotted versus log of the concentration of Hg 2+ or Cu 2+ . The binding constant and the stoichiometry of binding were obtained as reported [34]. Here, I 0 is the fluorescence intensity of AM1 and AM2 or AM3 and AM4 prior to addition of Hg 2+ or Cu 2+ respectively, I s is the fluorescence intensity of AM1 and AM2 or AM3 and AM4 at an intermediate added concentration of Hg 2+ or Cu 2+ respectively, I f is the fluorescence intensity of AM1 and AM2 or AM3 and AM4 in presence of one equivalent of Hg 2+ or Cu 2+ respectively. The binding constant was found to be 10 4.3 for AM1 towards Hg 2+ with 1:1 binding ratio; for AM2 towards Hg 2+ it was calculated as 10 4.2 ; the binding constant for AM3 and Cu 2+ system is 10 4.5 while the binding constant was calculated as 10 4.1 M for AM4 and Cu 2+ system. In case of the three sensors AM2 to AM4 the sensor:metal ratio was found to be 1:2.
The sensor AM1 has been synthesized from monocarboxylic derivative of benzene while the sensors AM2, AM3 and AM4 were synthesized from dicarboxylic derivatives of benzene by reacting with 4H-1,2,4-triazole-4-amine. Hence AM1 has one NNO binding sites while the others i.e. AM2, AM3 and AM4 have two NNO binding sites each. Therefore the binding ratio of sensor to metal is 1:1 in case of AM1 while it is 1:2 in case of the other three sensors. The fluorescence quenching of sensors due to interaction with Hg 2+ or Cu 2+ is due to decrease in conjugation upon binding.

UV/Visible spectral analysis of sensor and metal ion interactions
UV/Visible spectral titrations were done for the sensors AM1 and AM2 with Hg 2+ and for AM3 and AM4 with Cu 2+ . Figure 4A, B shows the UV-Visible spectral on the spectrum of AM1 is not significant (Fig. 4A). AM2 shows a shoulder at 250 nm and upon addition of Hg 2+ the shoulder gradually becomes a well defined peak at 230 nm (Fig. 4B). AM3 shows a peak at 280 nm and with the addition of Cu 2+ the absorbance decreases without any change in peak position (Fig. 4C). AM4 shows one peak at 260 nm which on gradual addition of Cu 2+ decreases its absorbance with a shift in peak position to 240 nm (Fig. 4D). AM1 binds to one Hg 2+ while the other three sensors bind to two of either Hg 2+ or Cu 2+ (Scheme 2). Hence the effect of Hg 2+ on the sensor AM1 is minimum while the others show significant change. The log[(A s -A 0 )/(A max -A s )] was plotted versus log of the concentration of the respective metal ion to determine the binding constant and the stoichiometry of binding as reported 33 . This could not be done for AM1 as there was no significant change in its UV/Vis spectrum upon interaction with Hg 2+ . Here, A 0 is the absorbance of the sensor before adding Hg 2+ or Cu 2+ , A s is the absorbance at an intermediate concentration of added Hg or Cu, A max is the absorbance of the sensor in presence of one equivalent Hg 2+ or Cu 2+ . The binding constant values and stoichiometric ratio obtained are quite similar to those obtained from the fluorescence data.

Determination of detection limit
The detection limits for the sensors towards Cu 2+ and Hg 2+ have been determined from fluorescence data by plotting (I 0 -I s ) versus log of the respective metal ion concentration [35]. The results have been summarized in Table 1. The detection limits for different systems are found to be 10 -5.5 M for  Figure 5 shows the optimized geometries and shape of highest occupied molecular orbital (HOMO) of the molecules. The HOMO of all these molecules has contribution from the lone pair orbital of the nitrogen atom. Thus, these nitrogen atoms will be involved in the bond formation with the metal cations. Figure 6 shows the optimized geometries of the metal complexes of these molecules. All these metals are bonded to the ring nitrogen, oxygen atom of the carbonyl group as well as a weak interaction with the N-H moiety. The calculated binding energies of the each metal cation is significant (12.3 kcal/mol for AM1, 10.6 kcal/mol for AM2, 11.1 kcal/ mol for AM3 and 9.6 kcal/mol for AM4) suggesting that the binding of these metal cations will be favorable.

Conclusions
The compounds derived from 4H-1,2,4-triazole-4-amine with benzoic acid and isophthalic acid could sense Hg 2+ by fluorescence "on-off" mode with stoichiometry of binding 1:1 and 1:2 respectively. The binding constant and detection limits were 10 4.3 and 10 -5.5 M for the first sensor while these are 10 4.2 and 10 -5.4 M respectively. The compounds derived from 4H-1,2,4-triazole-4-amine with terephthalic acid and phthaleic acid could sense Cu 2+ by fluorescence "on-off" mode. The binding constant and detection limits were 10 4.5 and 10 -5.0 M for the first sensor while these are 10 4.2 and 10 -5.2 M respectively. No interference was observed from other metal ions.