Spectroscopic Study on the Reaction of Singlet-Excited Nile Blue with Certain Antioxidants

The photoinduced interaction of nile blue (NB) with various antioxidant molecules was investigated by uorescence quenching technique and lifetime measurements. The various substituted catecholic compounds are employed as quenchers to evaluate their antioxidant activity. The formations of ground state complex between NB and quencher molecules observed from the UV-Visible absorption spectroscopy. The bimolecular quenching rate constants (k q ) values depend on presence of substituent and its electronic properties of quencher molecules. Fluorescence quenching experiments have been performed at three different temperatures to assess the thermodynamic parameters. Time resolved uorescence measurements suggest that the uorescence quenching of NB with antioxidant molecules undergoes static quenching mechanism. The bond dissociation enthalpy (BDE) values reveal the discharge of H T from the antioxidant molecules. The electronic properties play an important role in the antioxidant activity of quencher molecules. The mechanism of uorescence quenching between NB and quencher molecules are analysed based on the uorescence quenching experiments, cyclic voltammetry experiments and BDE calculations.


Introduction
Nile blue (NB) is an attractive molecule (Structure 1) in the class of cationic oxazine dye. Nile blue derivatives have been shown to be potentially effective photosensitizers for photodynamic therapy of malignant tumours due to its absorption of light in the red region of spectrum. NB e ciently react with tumour cells than normal tissues and retard the tumour growth [1,2]. NB and its derivatives are favorable materials for optical and photonic devices [3 5]. NB employed as a reagent to assess the antioxidant activity of commercial wine and fruit juice samples [6]. NB have strong binding a nity with DNA and show extensive biological applications [7,8].
Catechols and catecholamines are potentive antioxidants and scavenge reactive oxygen species [9]. The antioxidant activity found to have direct relationship with their biological actions [10][11]. The antioxidant effect depends on the chemical composition, solubility in lipids, ability to scavenge free radicals, donation of hydrogen atoms and free radicals related with chain reactions. It has been observed that presence of two hydroxyl groups in catechol molecule is responsible for the antioxidant activity. The protective effects of antioxidants vary depending on the structure of molecules [12]. The presence of catechol compounds in olive oil is liable for higher antioxidant activity in biological environment [13]. The neurological disorder, Parkinson disease arises due to the non-production of dopamine treated by administering Levodopa [14].
Our prime intention is to investigate the interaction of antioxidant molecules with appropriate biological targets and in this perspective study on the uorescence quenching of NB with catechol and phenol molecules is of utmost important in pharmaceutical point of view. We are interested in understanding the release of H • from antioxidant molecules as it may possibly solve biological related problems [15].
Besides, presence of substituent in catechol molecule in uences the release of H • from aromatic hydroxyl groups (-OH). Fluorescence quenching of 9-aminoacridine and acri avine by various antioxidants such as estrogens, avonoids, phenol and its derivatives, catechols and uracils were thoroughly investigated and found to undergo pronounced charge transfer process [16][17][18][19][20][21]. Recently, NB employed as a uorescent probe to evaluate the antioxidants of uracil molecules by abstracting the release of H • from antioxidants via ground state complex formation [22]. Although reports on the antioxidant activity of catechol was available, but comparison on the antioxidant activity of quencher molecules with different substitution based on steady state measurements and density functional studies are not reported so far. Thus, the present work focus on the photo induced interaction of NB with various quencher molecules using steady state and lifetime measurements. Bond dissociation enthalpy calculations reveal the e ciency of antioxidants with respect to position of the substituent. The structure of the catechol derivatives chosen for this study is depicted in structure 2.

Materials
Nile blue, catechol, pyrogallol, 4-t-butyl catechol, dopamine, levodopa and 4-aminophenol were purchased from Sigma-Aldrich and used without further puri cation. All the stock and aliquot solutions were prepared using double distilled water.
The emission spectra were measured by exciting the NB at 636 nm and emission maximum of NB is observed at 669 nm in phosphate buffered at pH 7.4. In order to avoid the quenching by singlet oxygen, samples were degassed with pure nitrogen gas for 15 min. Fluorescence lifetime measurements were carried out in a time correlated single photon counting (TCSPC) spectrometer. The data were analysed through software provided by IBH (DAS -6). The kinetic trace examined by non-linear square tting of mono exponential method.

Cyclic voltammetry measurements
The reduction potential and oxidation potential of NB and quencher molecules were measured with potassium chloride (0.1 M) as the supporting electrolyte. The reduction potential for NB observed at -3.88 V versus SCE [22]. The experimental setup consist a platinum working electrode, a glassy carbon-counter electrode and a silver reference electrode. Irreversible peak potential of quencher molecules measured at different scan rates (0.05 V/s). All samples bubbled in presence of nitrogen gas for 5 min at room temperature for deaeration.

BDE calculation
All the organic quencher molecules were optimized by B3LYP [23][24][25][26] method with 6-31G** [27-29] basis set. Harmonic vibrational frequency calculations were carried out on these optimized geometries to con rm that they are saddle point with all positive frequencies. RB3LYP method applied for quencher molecules and UB3LYP method applied for radicals. All these computational methods have been used as employed in the Gaussian 16 software package [30].

UV-Visible absorption spectra
The absorption of NB is characterized by a strong band at 636 nm. UV Visible spectral studies have been performed to reveal the presence of ground state interaction between NB and quencher molecules. All the quencher molecules show no absorption bands in the range of 600-700 nm. Interestingly, addition of quencher molecules decreases the absorbance of NB followed with an observable red shift (longer wavelength). This shows the existence of ground state complex formation between NB and quencher molecules [31]. Figure 1 indicates UV-Visible absorption study of NB with increasing concentration of dopamine in phosphate buffered media at pH 7.4. It is worthy to note that similar behaviour noticed for other quencher molecules.

Effect of quenchers in emission spectra of NB
The emission spectra of NB were measured in absence and presence of quencher molecules by exciting at 636 nm. It has been observed that on increasing the concentration of quencher molecules, the emission intensity of NB decreases. Figure Table 1). The observed results specify the existence of static quenching between NB and quencher molecules.

Lifetime measurements
The uorescence quenching of NB with quencher molecules were carried for understanding the decay mechanism through excited state lifetime measurements. The uorescence quenching shall be either dynamic or static [33]. The lifetime of NB was recorded with different concentrations of quencher molecules. The decay curve properly t well with single exponential decay. The excited state lifetime of NB was observed and found to be 1.74 ns [34]. The lifetime of NB in presence and absence of quencher molecules was noted. Interestingly, the lifetime of the NB molecule remains unaffected. The decay process was plotted and looks like single decay curve. The excited state lifetime measurement of NB in absence and presence of catechol was shown in Fig. 4 and indicate the existence of static quenching between NB and quencher molecules. The presence of static quenching embraces the possibility of ground state complex formation. Similar behaviour observed for NB in presence of other quencher molecules. Hence, the quenching pursues static mechanism.

Mechanism of uorescence quenching
The uorescence quenching of NB with quencher molecules can be rationalised by various mechanisms.
The possibility of energy transfer mechanism can be eliminated as the absorption spectrum of catechol and phenol molecules unsuccessfully overlay with the uorescence spectrum of NB. The prospect of either electron transfer or proton transfer mechanism, were evaluated by employing Rehm -Weller expression, shown as follows,  Table 2.

ΔG = RT ln K ln K = ΔH/RT + ΔS/R
The ΔG value is negative and signi es the interaction process is spontaneous. The ΔH and ΔS value point out the non-bonded (Van der Waals) interactions and hydrogen bond formation [37]. Thus, quencher molecules are destined to NB due to Van der Waals interaction and hydrogen bond formation.
The ∆H and ∆S values predict the possibility of charge transfer and hydrogen bonding interaction. NB possesses high reduction potential and quencher molecules own oxidation potential. The charge transfer occurs between NB and quencher molecules. The charge transfer process might be one of the promising evidence for the quenching mechanism of the non-radiative processes.

Bond Dissociation Enthalpy calculation
Density functional theory (DFT) calculations were carried out to understand the radical scavenging performance of chosen phenolic quencher molecules, as shown in strucutre 2. Bond dissociation enthalpy (BDE) found to be as a common descriptor for radical scavenging activity of quencher molecules. The radical scavenging of quencher molecules was de ned as where Q-H and ROO • correspond to the quencher molecule and peroxyl radical, respectively.
The BDE of O-H bond in the quencher molecule may act as parameters to envisage the pathway of scavenging free radicals by quencher molecule. The lowest BDE, indicates the most preferred mechanism of scavenging the ROO • radical. The lower the BDE value, weaker the O-H bond strength and greater the free radical scavenging ability of organic quencher molecules. The weakest O-H bond are recognized for the quencher molecules and compared with corresponding k q values (shown in Fig. 5).
The calculated BDE values are shown in Fig. 6. The quencher molecules with smallest BDE value, exhibit highest k q values and correlate well between them. Intriguingly, analogous trend observed in the steady state measurements.
Among the quenchers, dopamine shows lower BDE (70.63 kcal/mol) value and found that outmost high antioxidant activity. This is owing to the electron rich ole n, electron releasing (-NH 2 ) group and especially, intramolecular hydrogen bond between two -OH group in dopamine [38]. On comparing L-DOPA and pyrogallol, the former show less BDE value due to presence of electron releasing -NH 2 functional group and intramolecular hydrogen bond. Pyrogallol consists of three hydroxyl groups and one of the hydroxyl group in pyrogallol molecule show less BDE value and it account for high antioxidant activity than 4-aminophenol (70.36 kcal/mol). The trend indicates the presence of electron releasing species at C-4th position has great impact in radical scavenging potential of quencher molecules. Thus 4t-butyl catechol show less BDE than catechol molecule. The observed behaviour is owing to the presence of electron releasing methyl group at the 4th position and intramolecular hydrogen bond. The existence of substituent at C-4th position plays an important role in assessing the BDE of quencher molecules. The interpretations disclose the prominence of H in determining the antioxidant activity. The present research reveals the activity of radical scavenging con des on the position and electron releasing property of substituent in quencher molecules.

Conclusion
The uorescence quenching of NB in presence of antioxidant molecules investigated by using steady state, lifetime measurements and BDE calculations. The formation of ground state complexes between NB and quencher molecules were con rmed using UV-Visible spectroscopy and lifetime measurements. The calculated bimolecular quenching rate constant (k q ) depend on the substituent in quencher molecules. The effect on uorescence spectra of NB in existence of quencher molecules were carried out at diverse temperatures on the way to estimate the thermodynamic parameters. The BDE value was deliberated to examine the discharge of Hin the quencher molecules. Based on the uorescence quenching experiments and BDE calculations, it suggests that the hydrogen atom transfer between the NB and quencher molecules as one of the possible quenching mechanism. The present study demonstrates a new way in designing novel molecules with great antioxidant activity.

Declarations
Con icts of interest/Competing interests: Authors have no con ict of interest.       Comparison of bimolecular quenching rate constant with BDE value Figure 6 Structure of quencher molecules with BDEs at B3LYP/6-31G**/LANL2DZ level of theory (energies are given in kcal/mol)

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