A Novel Fluorescent Probe for the Detection of Cyanide Ions in Solutions and Studies on Its Biophysical Interactions with ctDNA and Proteases

A new cationic indolium based styryl dye (Ci) as a fluorescent probe was synthesized and its anions selectivity/sensitivity properties/molecular interactions with protease enzymes (pepsin/trypsin) and ctDNA has been studied by spectroscopic and computational methods. The fluorescence measurements at different temperatures indicated that quenching mechanism of enzymes by Ci was static. ΔH and ΔS data pointed out electrostatic/hydrophobic interactions with pepsin, and also hydrogen bonds/van der Waals forces with trypsin of Ci. According to Förster’s non-radiative energy transfer, binding distances (r) were calculated as 3.53/3.27 nm for pepsin/trypsin. It was also investigated that groove binding is effective in interaction with ctDNA. The results were supported with molecular docking analyzes which have same tendency. Ci has been demonstrated hypsochromic effect with a decrease in polarity of solvents and it showed highly selective colorimetric and fluorometric sensing behavior for cyanide in organic solvent and in aqueous solution. 1H NMR titration was performed to examine the interaction mechanism between Ci and cyanide. The LOD values of cyanide ion were reported as 4.87 × 10–9 M and 9.70 × 10–7 M in DMSO and DMSO/H2O binary mixture, respectively. In addition, sensitivity of Ci as a chemosensor to cyanide was investigated in bitter almond samples.


Introduction
Styrylindolium based dyes constitute an important class of merocyanine, they are extensively used in the elaboration of probes for analytes sensing and cell-imaging [1,2]. Besides, styrylindolium cations possess an extended π-conjugation within the indole ring and exhibit visible absorbance and fluorescence emission, and also their central conjugated iminium ion is considered as a good accepting electrophile [3]. The nucleophilic attack on the carbon-nitrogen double bond of the indole (imine) could modify the photophysical properties, for that reason, styrylindolium based molecules may serve in producing suitable colorimetric and fluorimetric receptors for nucleophilic analytes such as cyanide anions. Thus, the styrylindolium scaffold has been used as a building block for the synthesis of numerous sensors of biologically and environmentally important nucleophiles such as cyanide [4,5], fluoride [6], sulfide [7], amino acids [8]. In addition, several styrylindolium based derivatives for cell imaging have been reported [9,10].
Proteases are large and diverse group of hydrolases, classified according to the domain,the structure of the active site of the enzyme, and the specific reaction mechanism. They are structures that hydrolyze peptide bonds between amino acid residue of proteins [11]. They play important roles in physiological and pathological processes such as cell growth, migration, protein catabolism, blood clotting, protein activation, tissue development, tumor growth, cell regulation and signalling [12]. Pepsin, an aspartic protease enzyme, is involved in digestive processes in the mammal's stomach. It hydrolyzes proteins to peptides and amino acids, resulting in a decrease in its enzyme activity, which can cause nutrient absorption and side effects such as vomiting and nausea. It is widely used in various places such as food and drug production [13]. Pepsin has 326 amino acid residues containing five tryptophan (Trp), thirteen tyrosine (Tyr) and thirteen phenylalanine (Phe) in a single polypeptide enzymatic chain. Trypsin, which is produced in the pancreas and hydrolyses proteins, is a serine protease enzyme found in many vertebrates. It has 223 amino acid residues containing four Trp, ten Tyr and six Phe. It is involved in the digestion and deconstruction of protein in food and also some physiological processes such as immunity, reproduction [14]. The development of suitable fluorescent dyes has provided an opportunity to explore the physiological roles of biomolecules and keeping in view the sensing and biological significances of cationic styryl-based dyes which show a good affinity for DNA and a good cytotoxicity againist some cancer cell lines [15]. Here, a new cationic styrylindolium dye as a chemosensor (given as Ci or as dye in text) synthesized to determinate cyanide in aqueous media. Also its biophysical interaction and binding properties were investigated with ctDNA and pepsin/trypsin by multi spectroscopic and molecular docking methods in following sections.

Synthesis and Spectral Characterization of Ci
which is synthesized following the steps presented in Scheme 1. Compound 1 (1,2,3,3-tetramethyl-3H-indol-1-ium iodide) was obtained from the previously reported method [16]. A mixture of compound 1 (1 eq) and 4-(dibutylamino)benzaldehyde (1 eq) and a catalytic amount of piperidine in 20 mL of ethanol was heated under reflux overnight. The solvent was evaporated after cooling down at room temperature. The column chromatography (MeOH/ DCM, 1/5) was used to purify the crude mixture. The pure form of Ci was obtained dark green powder. Yield 89%, m.p. 70-72 °C.
The structural analysis and spectral properties of compound Ci were done using FTIR, 1 H and 13 C NMR, HRMS methods. The related spectra are given in Fig. S1-S4 in Supplementary Information (SI). The synthesis reaction was monitored under UV light on silica gel 60F254 plates (Merck) with thin-layer chromatography (TLC). FTIR spectra were obtained from the Perkin Elmer Spectrum 100 FTIR spectrophotometer. 1 H and 13 C NMR spectra were recorded on Bruker-Spectrospin Avance Ultra-Shield spectrometer using DMSO-d 6 as solvent and TMS as standard reactive. The electron ionization (EI) mass spectrometry (Waters-LCT-Premier-XE-LTOF (TOF-MS) was used for high resolution mass spectra (HRMS) of compound Ci. The melting point data were measured from Electrothermal IA9200 instrument. 1

Spectroscopic Measurements of Dye/Enzymes
The fluorescence studies were performed on a Hitachi F-4500 (Japan) spectrofluorometer which contains 150 W xenon lamp and FLSolutions software. The both excitation and emission bandwiths were at 2.5 nm with a scan speed 20 nm/s. The pH values of the study solutions were measured on a Mettler Toledo (FiveEasy Plus) digital pHmeter. The interactions of Ci-pepsin/trypsin were studied by the fluorimetric titration method at 298, 303 and 310 K. Intrinsic fluorescence of enzymes was recorded by adding a 5-60 µL volume range of Ci stock solution on 2.5 × 10 -6 M pepsin and 5.0 × 10 -6 M trypsin solutions exciting at 280 nm. The fluorescence titration data were examined to the Stern-Volmer quenching model. ΔH and ΔS parameters were calculated from the van't Hoff equation. Based on Förster's theory, FRET parameters such as J(λ), r, R 0 were obtained between the dye and enzyme for molecular pairs. For inner filter effect, intrinsic fluorescence of protein was corrected in below equation [17].
A ex and A em are absorbances at wavelengths of excitation and emission; F cor and F obs are corrected and observed fluorescence intensities, respectively.
The FTIR spectra were recorded with a Thermo Scientific Nicolet iS5 which contains iD5 ATR from enzyme solutions (1.0 × 10 -5 M) with and without Ci compound. Analysis were obtained from a laminate-diamond crystal window at 4 cm −1 resolutions with 64 scans in the range of 1700-1500 cm −1 .
Far-UV CD spectra were performed from free enzyme (2.08 × 10 -6 M) and Ci/enzyme complex (1:1 mol ratio) on Jasco-J815 spectropolarimeter (Japan) under constant nitrogen flux using 0.1 cm quartz cell at 298 K. All spectra were taken from 205 to 260 nm wavelength range with scan speed of 50 nm/min and 0.1 nm step resolution. Each result was the average of three replicates.

UV-Vis Absorption Studies
UV-Vis absorption spectra were recorded with a UV-1700PharmaSpec spectrophotometer (Shimadzu) using the UV-Probe PC software program. The UV-Vis absorption spectra of Ci with and without DNA and pepsin/trypsin were obtained and compared with each other.

Calculation Methods
The geometry optimizations of compound Ci were done using Becker's three parameterized Lee-Yang-Par (B3LYP) exchange functional with 6-31 + G(d,p) basis set [18][19][20]. Density Functional Theory (DFT) method as implemented in the Gaussian 09 package was used for calculations [21]. The electronic absorption spectra were obtained in DMSO using the PCM model using time-dependent DFT (TD-DFT) [22,23]. The molecular docking simulations were performed for Ci against pepsin/trypsin and DNA with PDB ID:1PSA, 1AQ7 and 1BNA, respectively. These target models were downloaded from the RCSB protein Data Bank [24] and minimized by using the protocols in Discovery Studio (DS) 3.5 [25]. Molecular electrostatic potential surface (MEPS) maps and frontier molecular orbitals (FMOs) were also generated from the optimized geometry to obtain important insights into the Ci. Afterwards, binding sites of the respective targets were defined via sub protocol of DS 3.5 for the docking process. Finally, the prepared Ci and target biomolecules have been reacted to the computational visualization technique, molecular docking using Auto Dock 4.2 [26]. Based on docking results, the best modes of Ci for each targets were visualized and evaluated of the potential of protein-ligand interactions with help of Discovery Studio (DS) 3.5. The high ranking docked complex for each target was chosen to examine of the binding mode and binding energy value in 200 conformations.

Photophysical Properties of Ci
The solvatochromic behavior of compound Ci was examined in five solvents which have different polarities such as toluene, THF, DCM, DMSO and methanol. The results showed that the absorption maximum of Ci was blue-shifted with the increase in the polarity of the solvent. As can be seen from Fig. 1, DMSO and methanol with the highest polarity among solvents caused a hypsochromic shift compared to the other three solvents.

Anions Interactions Studies in Organic Medium
To investigate the detection performance of compound Besides, for the emission studies, CN − caused a remarkable change in emission at 607 nm ( Fig. 2), while the other competing anions had no apparent effect on the emission spectra. As seen in Fig. 2, the obtained results were confirmed by a dramatic color change after the addition of 1 eq of anions in DMSO. The color of Ci changed from purple to colorless in the presence of CN − observed by naked eyes, while blue emission appeared under the UV light. However, there is no color change for the other anions. These results demonstrate that the synthesized compound could be efficiently used for the selective recognition of cyanide through the naked eye in organic medium.

Anions Interactions Studies in Aqueous Medium
Firstly, dye and CN − interaction was investigated in DMSO/ H 2 O binary solutions ranging from 10/90 to 1/99 (DMSO/ H 2 O v/v). The UV-Vis and fluorescence spectra showed good response in all of DMSO/H 2 O mixtures. As a result, we continued our research in a system containing a binary mixture of DMSO/H 2 O (1/99 v/v). By adding increasing concentrations of CN − , the UV-Vis and fluorescence spectra of Ci in an aqueous solution were evaluated. As illustrated in Fig. 4, the band with the highest absorbance at 554 nm diminished, and a new broad band was observed at 315-350 nm. Furthermore, the addition of cyanide resulted in a significant decrease in fluorescence intensity.

Determination of Cyanide in Real Sample
Finally, to investigate the sensor properties, the behavior of Ci towards the aqueous solution of bitter almond was examined. For this, the sample of 30 g of crushed bitter almond was placed in flask containg 0.5 g of NaOH and 100 mL of water, after stirring for 20 min final mixture solution was filtered [27]. After the addition of 20 µL of bitter almond, the maximum absorption peak of Ci showed a significant blue shift accompanied by a decrease in absorbance. Moreover, a dramatic change in fluorescence intensity was observed at 607 nm in Fig. 5.
The obtained results in UV-Vis and fluorescence spectra were confirmed by color change after the addition of 20 µL of bitter almond. The color of Ci changed from purple to pale yellow in the presence of CN − from bitter almond observed by naked eyes, while blue emission appeared under the UV lamp (λ ex =365 nm). These results are in line with those obtained in the CN − titration study of Ci in DMSO. As a result, Ci could be applied successfully for the detection of cyanide in bitter almond.

Sensing Mechanism
In view of the previously reported procedure [28], the sensing mechanism occurring after interaction of cyanide with indolium bearing molecules is a nucleophilic addition reaction. The cyanide nucleophile attacks the carbon atom of indolium moiety (C = N group) cut its electron-withdrawing ability, hence a change in photophysical properties is observed. To illustrate the sensing mechanism of Ci, 1 H NMR titration study was explored. 1 H NMR titration experiments of Ci with different equivalents of cyanide (0.5 and 1 eq) were carried out. The 1 H NMR spectra were taken for Ci and its cyanide adduct using DMSO-d 6 and partial spectra were specified in Fig. 6. As expected, after the addition of 1 eq of cyanide to the Ci, the nucleophilic attack of cyanide on the carbon atom of indolium moiety (C=N group) has occurred, and thus the proton signals shifted in high field in the spectrum (Scheme 2). It can be seen that the vinyl protons of Ci shifted from 8.29 ppm and 7.30 ppm to 6.70 ppm and 6.05 ppm (Hc and Hd) due to the breaking of conjugation. Besides, the proton signal of a methyl group at 3.95 ppm attached with C = N + (He) was shifted up field to 2.69 ppm. To get further information about the interaction of Ci with CN − via addition mechanism, density functional theory (DFT) calculations were performed at B3LYP/6-31 + G(d,p) level of theory. As shown in Fig. 7, conjugation between phenyl and indolium moieties through the styryl bridge disturbed after a nucleophilic attack by the cyanide (Ci + CN − ). Thus, electron transition from donor group dibutylamino attached phenyl ring to acceptor indolium moiety is interrupted, resulting in a hypsochromic shifted in absorption spectra. As considering the experimental results, TD-DFT calculations in DMSO showed that the peak at 488 nm (oscillator strength, f = 1.7115) seen in absorption spectra of Ci shifts to a lower one at 334 nm (f = 0.9358). In both cases, the transitions took place as from HOMO to LUMO with the highest contribution (~ %99).

Quantitative Analysis of Cyanide
The limit of detection (LOD), limit of quantification (LOQ) and the blank sample deviation of cyanide in the presence of Ci was calculated using the fluorescence titration data obtained above. Table 1 shows these data. The Pearson correlation coefficients of the linear calibration curves were found as 0.9966 in DMSO and 0.9967 in DMSO/H 2 O (1/99, v/v) mixture (Fig. S5 in SI). In a dynamic concentration range, the results indicated a good linear relationship between emission intensity and concentration CN − . According to calculation, the LOD values of Ci for cyanide were found as 4.9 × 10 -9 M and 9.7 × 10 -7 M in DMSO and DMSO/H 2 O (1/99, v/v) mixture respectively, which are much lower than the standard limits of CN − in the aqueous solution set by WHO [29].

Fluorescence Quenching of Enzymes with Ci
The effects of Ci on intrinsic fluorescence from tryptophan residues of pepsin/trypsin are presented with Fig. 8a, b, respectively, at given experimental conditions. The changes in fluorescence intensities in the range 290-450 nm were recorded in the spectra of both enzyme solutions excited at 280 nm. The fluorescence intensities were quenched regularly with a slight blue shift (~5 nm) for pepsin and with a red shift (~10 nm) for trypsin, The fixed concentrations of enzymes were titrated with different amount of Ci which added from 2.38 × 10 -6 M to 2.80 × 10 -5 M concentration range. This situation can be indicated that it may be due to the non-fluorescent complex formation between Ci and pepsin/trypsin. For fluorescent quenching data, the Stern-Volmer equation given below is used [17].
where F 0 and F are fluorescence intensities of Trp residues of enzymes in without and with of quencher. K sv , k q and τ 0 data are Stern-Volmer quenching constant , the quenching rate constant, and average lifetime of biomolecule in the absence of quencher (τ 0 = 10 -8 ) s, respectively. [Q] is concentration of Ci as quencher [30]. The Stern-Volmer graphs were obtained from titration data at 298, 303 and 310 K in Fig. 9a for pepsin and Fig. 9b for trypsin. Stern-Volmer graphs show good linearity means that the quenching mechanism should be static or dynamic which can be distinguished by their differing dependence on temperature and excited state lifetime. K sv constants were found the slopes of linear Stern-Volmer graphs and also k q values were calculated from K sv constants at three temperatures. Based on these data in Table 2, the values of K sv decrease as the temperature increases and it was concluded that the interactions of enzymes with dye are static quenching. The maximum diffusion quenching rate constant of diverse biomolecular extinguishers is 2.0 × 10 10 L/mol.s and the fact that the k q (2)  values in the table are much larger than this value indicates that there is a static quenching mechanism. This indicates the forming of a complex between Ci and pepsin/tripsin [31]. In static quenching interaction, when small molecules bind freely to a set of equal regions on a biomolecule, the equilibrium between free and bound molecules could be identified using fluorescence intensities the following equation [32].
where F 0 , F and [Q] are the same in Eq. (2). K is the binding constant, n is the number of binding sites per protein molecule, and [P] is the total protein concentration. In Fig. 10a Table 2.
According to the K values, it can be stated that Ci interacts with pepsin with a greater binding constant, that is, Ci holds pepsin more firmly than trypsin. The values of n, the stoichiometric binding numbers of the two systems, are approximately equal to 1, which indicates that the Ci interacts with one pepsin/trypsin molecule. A decrease in K values occurred with an increase in temperature. This situation, it shows that the stability of complexes decreases with increasing temperature [33].

Binding Modes Between Ci and Enyzmes
The bonding modes non-covalent interactions between proteins and small molecules are commonly described in terms of hydrophobicity, hydrogen bonds, electrostatic and van der Waals forces. These can be clarified by looking at the magnitude and sign of the thermodynamic parameters of ΔH (enthalpy change) and ΔS (entropy change) [34]. Both the parameters can be determined from the van't Hoff equation.
where K is bonding constant at corresponding temperature and R is the gas constant. Here, the log K values were plotted against 1/T at three temperatures according to Eq. (4). ΔH and ΔS parameters were calculated from the slope and intercept of van't Hoff graphs from their linear equations, log (4) log K = −ΔH∕2.303 RT + ΔS∕2.303R K = 734.72 / 2.303RT + 2.2956 / 2.303R for Ci -pepsin and log K = 1921.5 / 2.303 RT -1.7692/ 2.303R for Ci-trypsin (Fig. S6 in SI). Then from using Eq. (5), the Gibbs energy change (∆G) value was calculated in following. Table 2 indicates the calculated the thermodynamic parameters of dye-enzyme systems. The histogram graphs of these parameters were given in Fig. 11 at 298 K. Because it appears, negative ΔG indicates that the binding mechanism of Ci with enzyme is spontaneous, and the negative ΔH indicates that the binding is exothermic. Positive values for ΔS and negative values for ΔH indicate that electrostatic forces play an active role in Ci-pepsin interactions. However, positive values for ΔS are often considered proof of hydrophobic interaction, since water molecules regularly dispersed round the ligand and protein cause an at random alignment in consequence of the hydrophobic interaction [35]. Within in the interaction between Ci and trypsin, the negative values of ΔS and ΔH indicate that van der Waals forces and hydrogen bonds play significant roles in the bonding mechanism.

FRET Measurements of Dye-Enzyme Pairs
Based on the Förster theory [36], the energy transfer efficiency (E), the critical distance (R 0 ) at 50% energy transfer and the distance (r) between the donor (pepsin/trypsin) and acceptor (dye) pair were found from experimental results by using Eqs. (6) and (7) in below [17].  where n is the refractive index of the medium; k 2 is the orientation factor; and J is called overlap integral. In order to find the J value, the fluorescence spectrum of the enzyme and the absorption spectrum of the dye are found by overlapping. Q D is the quantum yield of the donor. The spectral overlaps (dye/enzyme = 1:1) were given in Figs. S7 and S8 in SI at experimental conditions for pepsin and trypsin, respectively. The value of J is calculated by the following equation.
where F D (λ) is the normalized donor emission; ε A (λ) is the molar extinction coefficient of acceptor at given a wavelength. Here, n = 1.336 is refractive index of working medium; the fluorescence quantum yields, Q D are 0.150 for pepsin [37] and 0.146 for trypsin [38]. The E, J, R 0 and r results obtained were listed in Table 3. The consequences indicated that the dye-enzyme distance, r, (3.53/3.27 nm) was less than 8 nm, and also the r value was greater than R 0 , confirming that non-radiation energy transfer occurred in the static quenching mechanism. Furthermore, the short binding distances indicated Ci and tryptophan residues of digestive enzymes produced strong interactions.

FTIR Studies of Dye-Enzymes
The basic of the FTIR method is to study the transition between the vibrational level of the basis state molecule due to the absorption of light in the infrared zone and the alter in the secondary structure of the protein after binding to the ligand. Amide bonds induce temporary vibrations in the peptide bands emerging from these infrared spectra [39]. Among the amide bands which are vibration bands, amide I (1600-1700 cm −1 ) is mainly associated with the stretching band C = O, and amide II (1500-1600 cm −1 ) is the stretching C-N combined with N-H. As can be seen from Fig. S9a, b, the addition of Ci changed the position of the absorption peak of the amide bands of pepsin/trypsin. The peak in the amide I and amid II band shifted from 1651.54 cm −1 to 1652.24 cm −1 and from 1519.51 cm −1 to 1524.97 cm −1 for pepsin; and from 1660.05 cm −1 to 1652.74 cm −1 and from 1601.90 cm −1 to 1594.82 cm −1 for trypsin, respectively [40]. Shifts in the bands indicate an alter in the secondary structure of the enzyme after binding process.

Circular Dichroism (CD) Studies of Dye-Enzymes
CD is a sensitive spectroscopic technique for monitoring changes in protein structure as it interacts with small molecules. Far UV spectra (260-200 nm) were used to define the secondary structure of the protein. Two negative elliptic bands at 208 nm (π → π *) and 222 nm (n → π *) are characteristic of the typical α-helix conformation of proteins [41,42]. The CD spectra of pepsin/trypsin in the absence and presence of Ci are shown in Fig. 12. CD results were transformed into the mean residual ellipticity (MRE) in deg cm 2 /dmol, according to the following equation [43].
In equation n, the number of amino acid residues contained in the protein (n, 326 for pepsin and for trypsin 223); C is the molar concentration of protein; θ obs is the  observed ellipticity and l is the path length of cuvette in cm. α-helical contents of free enzyme and dye/enzyme complex was calculated from MRE values at 208 nm using the following equation [44].
As shown in Fig. 12, the intensity of both bands diminished with the addition of Ci, indicating a change in the secondary structure of the enzyme and a diminish in the α-helix content that was shown in Table 4. The results recommended a lessening of α-helical structure percent from 53.55 and 73.90 in free pepsin/trypsin to 52.29 and 70.05 at a molar proportion of chemical to color of 1:1, respectively. As a result, it was clarified that the binding of Ci to pepsin/trypsin resulted in a change in the secondary structure of the enzyme with loss of helical stability.

Absorption Measurements of Dye-Enzymes
The UV-Vis absorption method is frequently used to examine the complex formation and conformational changes in protein structure that occur in protein-ligand interaction [45]. Absorption spectra of enzymes in the absence and presence of Ci were recorded at 298 K in Fig.  S10a, b for pepsin and trypsin, respectively. It can be seen from the spectra with the addition of dye, the maximum peak of absorbance of enzyme at 275 nm which seems to be because of the aromatic amino acid residues increased sharply. The result indicated that there was an alter in the structure of enzyme and the micro environment round the tryptophan residues in dye-enzyme complex.

Binding Property of Ci with DNA
The UV-Vis absorption is an effective method that shows the changes in absorption and peak position when a complex is formed between DNA and a small molecule, as in proteins. In this case, absorption spectrum of DNA shows hyperchromic and hypochromic impacts associated with its double helix structure. Absorption hyperchromism and blue shift indicate that non-covalent interactions such as electrostatic binding and groove binding damage the secondary structure of the DNA double strand. On the other hand, hypochromism and red absorption shift indicate contraction of DNA in helix axis and structural change of DNA with intercalating binding [46]. Figure 13 shows absorption spectra of 1.0 × 10 -5 M Ci with and without ctDNA were recorded at 298 K and pH 7.4 in PBS. It can be seen the absorption peak at 275 nm of Ci displayed gradually increased and a blue shift (~5 nm) the increasing amount (from 0 to 2.97 × 10 -5 M) of ctDNA. This could be attributed to an interaction between DNA and Ci which binds to the DNA helix via groove binding mode.

Molecular Docking Results
Optimized Ci was performed at G09 on the DFT/ B3LYP/6-31 + G (d,p) level. To the same level, the frontier molecular orbitals (HOMO and LUMO) and molecular electrostatic potential surface (MEPS) of the respective compound are formed in Fig. 14. The difference in energy between HOMO and LUMO orbital is a measure of electron conductivity. Therefore, it is the parameter used to determine molecular and electrical transport characteristics. Using the energy values of HOMO and LUMO, the Fig. 11 The representation of thermodynamic parameters of pepsin/trypsin interaction with Ci at 298 K chemical potential, electronegativity, electrophilic index, hardness and softness properties of the substance can be calculated [47]. As appeared in figure, the energy difference between HOMO and LUMO is 2.625 eV. The ionization energy using the orbital energies of HOMO and LUMO is I = -E HOMO = 8.210 eV and the electron affinity is A = -E LUMO = 5.585 eV. Hardness was found to be η = (I − A)/2 = 1.313; chemical potential was found to be μ = -(I + A)/2 = -6.898; electrophilic index was found to be ω = μ 2/2η = 18.12 eV and softness was found to be ζ = 1/2η = 0.38 eV.  MEPS is used to examine the interaction of a molecular system with its environment, biological recognition studies, the reactivity of chemical systems in nucleophilic and electrophilic reactions, and the study of hydrogen bond interactions. Also MEPS is an isoelectronic density surface mapping method, is a visualization method used to understand the surface and relative polarization [48]. The charges of the structure from negative to positive are defined from red to blue. The Ci is completely dominated by shades of blue originating from the nitrogen atoms in the main structure. Ci was docked with pepsin, trypsin, and DNA to define the interplay against the targets via Auto Dock software. Then, DS 3.5 was used to view the binding affinity of the respective compound to each active surface of the targets. The docking results were interpreted according to binding energy, types of non-bonding interaction and also the conformation of the Ci in active area of each target.
Considering its interactions with the targets, Ci created two weak carbon H-bonds with Glu13, and Gly217, and nine hydrophobic interactions with the amino acids Ala115, Leu112, Ile120, Tyr75, Phe111 and Ile30 residues of pepsin target in Fig. 15 (top-left side). As a result of the interaction of the second order target, trypsin with Ci, the related compound exhibits similar H-bonds and hydrophobic interactions with Ser195, Phe41, Tyr151, Ile73, Tyr39 and His40 residues in binding site of the current target protein, respectively in Fig. 15 Table S1.
The docking results displayed that the Ci had the best affinity, inhibition constant and RMSD values for pepsin (-7.50 kcal/ mol, 114.93 nM and 0.0002). Subsequently, the same compound showed the second and third best binding affinity (-6.90 and -6.80 kcal/mol, 399.21 and 13,979 nM and 0.0940 and 0.4260) with trypsin and DNA targets. The results are given in Table S2.

Conclusion
In this study, the Ci which is containing indolium core, have been synthesized and characterized. Its anions selectivity/sensitivity and interactions with protease enzymes (pepsin/trypsin) and ctDNA were studied using spectroscopic methods as well as computational calculations. The fluorescence titration assays showed that binding process is static quenching mechanism to form of Ci-pepsin/trypsin complexes. ΔH and ΔS parameters indicated that the binding modes in Ci-enzyme complex involved predominantly the electrostatic/hydrophobic interactions between dye and pepsin, and also hydrogen bonds/van der Waals forces between dye and trypsin. The absorption studies indicated that Ci interacts with ctDNA in a mode of groove binding. Furthermore, biological activity of Ci was further confirmed by molecular docking study on ctDNA and pepsin/trypsin which displayed good tendency with experimental data. The Ci indicated hypsochromic effect with a decrease in solvent polarity, and it showed highly selective colorimetric and fluorometric sensing behavior for CN − in organic and aqueous media. The LOD values of CN − in the presence of Ci were 4.87 × 10 -9 M and 9.70 × 10 -7 M in DMSO and DMSO/H 2 O binary mixture, respectively. Moreover, Ci showed sensitivity to the CN − in bitter almond.

Competing Interests
The authors declare no competing interests.
Ethics Approval Not applicable.

Conflicts of Interest
The authors declare that they have no competing interests.