In this paper, we have reported the synthesis of a novel chemo sensor (E)-2,4-dichloro-6-(((5-mercapto-1,3,4-thiadiazol-2-yl)imino)methyl)phenol (SB-1) and subsequently explored its role as a potential metal ion and pH sensor. The target probe was synthesized in excellent yield by the condensation reaction of 3,5-dichlorosalicylaldehyde and 5-amino-1,3,4-thiadiazole-2-thiol. The photophysical properties of SB-1 was investigated towards variety of metal ions in MeOH:H2O (v/v = 1:1, HEPES buffer, pH = 7.2) at room temperature. Absorption and emission spectral changes confirmed its selective “turn-off” behaviour towards Cu2+ ions along with visible colour change from original yellow to dark red.
Synthesis and characterization of SB-1
The desired probe SB-1 was synthesized using the protocol illustrated in Scheme 1. Ethanolic solutions of 2,5-dichlorosalicylaldehyde (1) and 5-amino-1,3,4-thiadiazole-2-thiol (2) were mixed with constant stirring together in a 100 mL round-bottom flask followed by the addition of 2-3 drops of acetic acid. The resulting mixture was refluxed for one hour. The completion of reaction was observed by TLC using ethylacetate : petroleum ether (50 : 50) as eluent. Deep yellow precipitate were filtered off and washed several times with cold water. The crude product was then purified by recrystallization from hot ethanol and dried over P4O10 to give the pure product SB-1 as dark red solid. The product SB-1 was characterized spectroscopically by IR, 1H and 13C NMR, and by mass spectra.
(E)-2,4-dichloro-6-(((5-mercapto-1,3,4-thiadiazol-2-yl)imino)methyl) phenol (SB-1)
Dark red solid; Yield: 87 %; m.p. = 195-197 ºC; IR (νmax cm-1)(KBr): 3224 (O-H), 3076 (Ar-H), 2964 (N=C-H), 1627 (C=N), 1465 (C=C), 1346 (C-N);1H NMR (CDCl3, 400 MHz): δ 14.17 (1H, brs, OH, D2O exchangeable), 12.13 (s, 1H, SH), 8.53 (s, 1H, ArH), 7.49 (1H, d, J = 2.4 Hz, ArH), 7.36 (1H, d, J = 2.5 Hz, ArH); 13C NMR (CDCl3, 400 MHz): δ = 158.20, 151.21, 146.70, 144.77, 133.74, 130.06, 129.45, 128.87, 127.84, 127.76, 126.11, 123.12, 120.68, 34.61, 12.48; HRMS (ESI) calcd. for C9H5Cl2N3OS2 [M + H]+ : 307.1994; Found: [M+H]+ = 353.1249.
Receptor-Spacer-Fluorophore paradigm for chemo sensor SB-1:
The “Receptor-Spacer-Fluorophore” paradigm for chemo sensor SB-1 is illustrated in Fig. 1. The construction of compound SB-1 involves the assembling of a salicylaldehyde ‘receptor’ unit (primarily responsible for the selective analyte binding) coupled to a thiadiazole ‘fluorophore’ subunit (which generate spectral/optical response due to receptor-analyte interaction) through an imine (-C=N) spacer. The role of spacer is to modulate the electronic interactions between receptor and fluorophore.
Effect of water percentage on fluorometric properties of SB-1
The chemo sensor SB-1 exhibited strong emission maximum in 100% methanolic solution (When no water is added). The emission intensity remained nearly constant on increasing the water fraction (fw) up to 40-50% (MeOH:H2O, v/v = 1:1), which clearly nullifies the possibility of “Aggregation-Induced Emission Enhancement (AIEE) Phenomenon”. Further increase in dilution (fw > 50 %) resulted in the precipitation of SB-1 from the solution due to its poor solubility in high water concentrations which may substantially affect the overall performance of the probe, and could intricately alter the measurements as well. Therefore, MeOH:H2O ratio kept at 1:1 during all the photophysical experiments.
Effect of metal ions on photophysical properties of SB-1
The photophysical properties of probe SB-1 has been investigated by determining the changes in UV-Vis absorption and emission spectra of SB-1 (1×10-5 M) with the addition of different competitive metal ions in MeOH:H2O (v/v = 1:1) containing HEPES buffer (1×10-5 M, pH=7.2) at room temperature.
UV-Vis spectral analysis
The UV-Vis absorption spectrum of probe SB-1 showed two absorption maxima, one in the shorter wavelength region centred at 298 nm and a broad band in the longer wavelength region ranging from 434−439 nm (𝜆max = 437 nm) (Fig. 2a). The absorption spectrum remained unaltered upon addition of one equivalent of various metal ions such as Co2+, Cd2+, Mn2+, Hg2+, Pb2+, Zn2+, Sn2+, Ni2+, Sr2+, Mg2+, Cr3+, Al3+, Fe2+ metal ions (1×10-3 M) to the solution of probe SB-1 (1×10-5 M). However, addition of one equivalent of Cu2+ ion resulted in the significant changes in the absorption spectrum of SB-1 at long wavelength region. The broad band appeared in the range of 434−439 nm underwent hypochromic shift (significant reduction in absorption intensity) along with the slight bathochromic shift (~11 nm) of absorption maximum (𝜆max shifted to 448 nm) (Fig. 2a). However, the absorption maxima centered at 298 nm in shorter wavelength region remain unshifted with the addition of Cu2+ ions. Thereafter, the absorption spectrum of SB-1 was investigated with varying concentrations of Cu2+ ions (0-1 equiv.). The intensity of absorption maxima in the region 434−439 nm showed gradual diminishment with increasing concentration of Cu2+ ions (Fig. 2b). However, absorption intensity remained nearly constant and did not change after addition of 1 equiv. of Cu2+ ions which primarily suggest 1:1 binding mode between SB-1 and Cu2+ ions.
Fluorescence spectral analysis
The probe SB-1 (1×10-5 M) displayed a strong emission band at 579 nm upon excitation at λexc = 440 nm in MeOH:H2O (v/v = 1:1, HEPES buffer, pH = 7.2) (Fig. 3a) which is probably due to the presence of thiadiazole fluorophore in the compound SB-1. The sensing behaviour of SB-1 was thereafter scanned by the response of emission spectra of SB-1 to various metal ions. Addition of one equiv. of Co2+, Cd2+, Mn2+, Hg2+, Pb2+, Zn2+, Sn2+, Ni2+, Sr2+, Mg2+,Cr3+, Al3+, Fe2+ metal ions (1×10-3 M) did not change the emission intensity of SB-1 with the exception of Cu2+ ions. The emission intensity diminished remarkably upon addition of one equiv. of Cu2+ with a concomitant hypsochromic/blue shift (~19 nm) of emission maximum from 579 nm to 560 nm (Fig. 3a). These spectral changes confirm that SB-1 exhibited excellent selectivity for Cu2+ ions over other competitive metal ions.
Furthermore, the fluorescence titration curve of SB-1 was plotted with gradual addition of varying concentration of Cu2+ ions (0-1 equiv). The emission intensity of probe SB-1 showed diminishment with incremental amounts of Cu2+ ions (Fig. 3b). However, emission intensity did not change after the addition of 1 equiv. of Cu2+ ions indicating 1:1 binding mode between SB-1 and Cu2+ ions. This was further confirmed by the method of continuous variation.
Development of colorimetric sensing probes is advantageous as they allow rapid on-site detection of metal ions through visual inspection. Therefore, spectral changes observed in the UV-Vis absorption and emission behaviour of SB-1 upon addition of various metal ions were also inspected by carrying out the colorimetric experiments. Addition of various metal ions to the solution of SB-1 (1×10-5M) induced no visible colour change except Cu2+ ions. A prominent colour change was observed from original yellow to dark red in the presence of Cu2+ ions (Fig. 4). Therefore, SB-1 could be used as a potential colorimetric chemo sensor for the detection of Cu2+ ions.
Selective behaviour of SB-1 towards Cu2+ ions
The selectivity of probe SB-1 towards Cu2+ ions over other metal ions was further validated by conducting the competitive experiments. The changes in fluorescence intensity of (SB-1 + Cu2+) complex in the presence of various interfering metal ions were investigated. It was observed that emission intensity of probe SB-1 in the presence of 10 equiv. of Cu2+ ions followed by the addition of 10 equiv. of other coexisting metal ions (Co2+, Cd2+, Mn2+, Hg2+, Pb2+, Zn2+, Sn2+, Ni2+, Sr2+, Mg2+,Cr3+, Al3+, Fe2+) remain as such. Therefore, these metal ions did not produce any considerable interference in the detection of Cu2+ ions confirming the excellent selectivity of SB-1 towards Cu2+ ions. These results are compiled in the bar diagram showed in Fig. 5.
Interference of various anions
Practical applicability of probe SB-1 was investigated by monitoring the effect of various anions on the emission spectra of (SB-1)-Cu2+ complex. It can be depicted from Fig. 6a that addition of various anions such as F-, Cl-, Br-, I-, SCN-, ClO3-, NO2-, S2-, CrO42-, C2O42-, and SO32-, (5 eq.) to (SB-1 + Cu2+) solution exhibited negligible or comparatively (in case of PO43-, and S2-) little changes in the emission spectra. However, addition of 5 equiv. of EDTA resulted in the significant increase in emission intensity indicating the decomplexation of SB-1 from (SB-1)-Cu2+ complex. This fluorometric change was also observed by the change in colour of solution from dark red to original yellow. Subsequent addition of Cu2+ ion to the solution of (SB-1 + Cu2+ + EDTA) diminish intensity again along with the colour change from yellow to dark red, thus confirming reversible nature of SB-1. This complexation-decomplexation mechanism has been illustrated in Scheme 2. Therefore, (SB-1)-Cu2+ complex could also be employed as a secondary sensor for the instant detection of EDTA anions through metal displacement approach. More interestingly, this switching phenomenon could be repeated several times by alternate Cu2+/EDTA additions without significant loss of any emission intensity (Fig. 6b).
The binding stoichiometry of probe SB-1 with Cu2+ ions was calculated using method of continuous variation i.e. Job’s plot (Fig. 7). Job’s plot was drawn on the basis of fluorescence titration measurement by altering the mole fraction of Cu2+ ions while total concentration of the resulting solution kept at constant value. The plot exhibited a maximum when the mole fraction of Cu2+ was 0.5, which strongly indicates 1:1 complex formation between probe SB-1 and Cu2+ ions.
Determination of binding constant
Binding constant measures how effectively a ligand complexes with the metal ion. The binding constant (Ka) was evaluated from the fluorescence titration data by using Benesi-Hildebrand equation [38, 39]. A graph was plotted between Io/(Io-I) and 1/[Cu2+] (M-1) where I = emission intensity at 560 nm and Io = emission intensity at 579 nm in absence of Cu2+ ions. The association constant for (SB-1 + Cu2+) complex was found to be 3.87×104 M-1 from the ratio of intercept/slope (linearly dependent coefficient R2 = 0.988) (Fig. 8). The high value of binding constant indicates strong complex formation between probe SB-1 and Cu2+ ions.
Limit of Detection
The limit of detection of probe SB-1 towards Cu2+ was calculated from fluorescence titration measurements. A curve of fluorescence intensity was plotted against [Cu2+] at 560 nm. The plot showed linear dependence and the detection limit, point at which curve cuts the ordinate axis, was found to be 1.01×10-7 M (Fig. 9). This value of limit of detection validates good sensitivity of probe SB-1 towards Cu2+ ions and falls well within in the range of tolerance limit of Cu2+ in drinking water.
Determination of binding sites
The binding ratio of SB-1 and Cu2+ has already been estimated using Job’s plot. Furthermore, the coordinating sites of SB-1 were determined by analyzing the changes observed in IR and 1H NMR spectra of SB-1 after the addition of Cu2+ ions.
FT-IR spectral behavior
IR spectrum of free probe SB-1 exhibited characteristic broad band at 3224.98 cm-1 due to hydroxyl (-OH) stretch, and the band at 1602 cm-1 corresponds to azomethine group (-C=N) (Fig. 10a). Upon binding with Cu2+ ions, the broad band disappeared completely whereas azomethine band shifted towards lower frequency region at 1572 cm-1 (Fig. 10b). These noticeable changes observed in the IR spectrum of SB-1 primarily indicate the involvement of -OH group through phenolic oxygen atom, and azomethine group through nitrogen atom.
1H-NMR spectra of SB-1 in presence of Cu2+ ion
Binding sites of probe SB-1 involved in the coordination sites with Cu2+ ions was further confirmed by the changes observed in 1H-NMR spectra in CDCl3/DMSO. Significant changes/shifts were observed in the peak positions after addition of Cu2+ ions. The broad singlet for phenolic -OH proton at 14.17 ppm in free probe SB-1 disappeared completely upon binding with Cu2+ ions, which validate the coordination between the Cu and oxygen atom (Fig. 11a and 11b). The azomethine proton appeared as a singlet at 8.53 ppm shifted to downfield region and appeared at 10.09 ppm. This is typically due to the transfer of electron density from the azomethine nitrogen to copper metal clearly indicating the participation of azomethine group. These notable changes in the 1H-NMR spectrum of probe SB-1 confirm that the coordinative sites of SB-1 for Cu2+ are the hydroxyl group from 3,5-dichlorosalicylaldehyde and azomethine group.
Effect of pH on absorbance characteristics of SB-1
Effect of pH on the UV-Vis absorption spectrum of SB-1 was investigated in MeOH:H2O (1:1, v/v). The solutions of different pH from 1-13 were prepared by adding appropriate amount of aq. HCl and aq. NaOH in the solution of probe SB-1. Absorption spectrum of SB-1 exhibited two bands, one centered at 298 nm, and another in the range form 434−439 nm. Almost No changes were observed when pH of the solution was shifted to basic region from 7 to 13 (Fig. 12).
Shifting of pH in acidic region from 7 to 2 resulted in the significant diminishment of absorption intensity of band at 298 nm. However, no changes were observed in the colour of the solution of probe SB-1. Additionally, when PH was further lowered up to 1, the solution turned to colourless from original yellow which might be due to the existence of tautomeric form II i.e. hydrazone form (Keto form) of probe SB-1 (Scheme 3). These colorimetric responses of probe SB-1 suggested that it could also act as a potential optical pH-sensor for selective detection of H+ ions in highly acidic medium when pH < 2.
A plausible mechanism based on the “Intramolecular Charge Transfer (ICT)” process has been illustrated in Scheme 4 for “turn-off” sensing of chemo sensor SB-1 with copper ions. The strong ground state fluorescence of SB-1 is probably due to the intramolecular charge transfer from salicylaldehyde moiety to fluorophoric thiadiazole moiety. Upon chelation with copper ions, ICT ability of phenolic hydroxyl restricted resulting in the diminishment of emission intensity with a blue shift.
Practical utilizations of chemo sensor SB-1
Sensing Experiments with Paper Strips
Rapid development of sensing paper strips is beneficial due to their portability, easy use, and availability. These strips provide instant on-site detection of a particular analyte and therefore negate the dependency on expensive and sophisticated instrumental techniques. Therefore, sensing behaviour of probe SB-1 were monitored using paper test strips. Whatman filter papers were dipped in 1×10-3M (MeOH:H2O (1:1, v/v)) solution of probe SB-1 for 10 minutes and then dried in air. The yellow colour of probe SB-1 immersed in test strips (Fig. 13a). The yellow-coloured test strip loaded with SB-1 was converted to dark red instantaneously when dipped in Cu2+ ion solution (1×10-3M, MeOH:H2O (1:1, v/v)) (Fig. 13b). The original yellow colour of test strip was reappeared back when dark red strip immersed in 1mM EDTA solution (Fig. 13c).
Application as Logic Gate Circuit
Ion-induced logic gate circuits possess remarkable applications in electronics and nano-molecular devices. In present work, we report a simple ‘NOR’ logic gate at sub micromolar level. The yellow solution of SB-1 changed to dark red upon addition of Cu2+ ions and the original yellow colour reappeared when EDTA2- ions was added to SB-1 + Cu2+ solution. Moreover, EDTA2- ions alone as chemical input did not induce any colour change in solution of SB-1. These colorimetric responses of probe SB-1 prompted us to mimic a universal ‘NOR’ logic gate. A ‘NOR’ gate could be understood as a combination of two separate logic gates ‘OR’ and ‘NOT’. It is also known as ‘Negated OR gate’ due to negation of results of OR gate by a NOT function. The presence and absence of chemical inputs (In1 = Cu2+ and In2 = EDTA2) were assigned with binary digits ‘1’ (ON-state) and ‘0’ (OFF-state), respectively. The output signals were observed at 560 nm. Higher emission intensity of SB-1 was assigned as 1 (ON-state) and the low intensity as 0 (OFF-state). When neither input was present (In1 = In2 = 0), emission intensity of SB-1 at 560 nm was high indicating ‘ON’ state. Addition of Cu2+ (In1 = 1, In2 = 0) quenched the emission intensity significantly indicating ‘OFF’ state. Presence of EDTA2- alone (In1 = 0, In2 = 1) did not alter the emission intensity of SB-1 indicating ‘ON’ state. Finally, when both the inputs were present (In1 = In2 = 0), system was present in ‘ON’ state. All these combinations construct a ‘truth table’ shown in Fig. 14b which finally led to formation of a ‘NOR’ logic gate (Fig. 14c).
The optimized structure of SB-1 and its tautomeric form (II, Keto/hydrazone form) was investigated by quantum chemical calculations by DFT using the B3LYP/6311G basis set and represented in Fig. 15. HOMO-LUMO analysis of SB-1 (I, Enol/Azo form) revealed that HOMO is primarily confined over the salicylaldehyde moiety while LUMO is localized over thethiadiazole ring and azo linkage. Therefore, charge transfer occurs from thethiadiazole ring to the salicylaldehyde moiety with an energy separation (ΔE) of -3.4406 eV. On the other side, the HOMO–LUMO analysis of Keto form (II) implies that charge transfer occurs between the salicylaldehyde and thiadiazole rings with an energy separation of -3.0866 eV (Fig. 15). Therefore, it can be concluded that more electron density is available in Enol form (Azo form) for binding with Cu2+ ions due to the possibility of less charge transfer between LUMO and HOMO. In Keto form (II), due to the low energy gap (ΔE = -3.4406 eV), there is more possibility of charge transfer resulting in the reduction of electron density available for Cu2+ binding. Therefore, it is clear from the above studies that chemo sensor SB-1 exists in its tautomeric form I (Enol/Azo form) in its ground state for metal ion binding.
Molecular docking analysis:
The binding modes of SB-1 and (SB-1)-Cu to TRK are shown in Fig. 16 and 17. Analysis of the close contacts in the structure of the complex predicted by Vina revealed that SB-1 forms two hydrogen bonds with the target protein. As illustrated from the Fig. 16(c) the hydrogen-bond interaction is with Thr670 and Cys673. In addition, the test compound exhibited additional stabilization through hydrophobic and Van der Waals interactions with nearby amino acid residues, Leu644 and Val654. The docking results reveal that compound SB-1 fit in the groove region with the minimum binding affinity of -8.0 kcal/mol.