Study of prototropic reactions of indole chalcone derivatives in ground and excited states using absorption and fluorescence spectroscopy

Journal Pre-proof Study of prototropic reactions of indole chalcone derivatives in ground and excited states using absorption and fluorescence spectroscopy

Manju K. Saroj, Ritu Payal, Sapan K. Jain, Ramesh C. Rastogi PII:

S0167-7322(19)32610-8

DOI:

https://doi.org/10.1016/j.molliq.2019.112164

Reference:

MOLLIQ 112164

To appear in:

Journal of Molecular Liquids

Received date:

14 June 2019

Revised date:

11 October 2019

Accepted date:

17 November 2019

Please cite this article as: M.K. Saroj, R. Payal, S.K. Jain, et al., Study of prototropic reactions of indole chalcone derivatives in ground and excited states using absorption and fluorescence spectroscopy, Journal of Molecular Liquids(2018), https://doi.org/10.1016/ j.molliq.2019.112164

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© 2018 Published by Elsevier.

Journal Pre-proof

Study of Prototropic Reactions of Indole Chalcone Derivatives in Ground and Excited States using Absorption and Fluorescence Spectroscopy Manju K. Saroj, Ritu Payal, Sapan K Jain# and Ramesh C. Rastogi* Department of Chemistry, University of Delhi, Delhi – 110007, India Abstract The prototropic behavior of biologically active intramolecular charge transfer (ICT) probes namely, indole chalcone (IC) derivatives in aqueous media of Ho/pH/H_ from -3.03 to 17.95, was studied using absorption and steady-state fluorescence spectroscopy. This

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study reveals the presence of monocation (MC), neutral (N) and monoanion (MA) prototropic species in ground and excited states. Prototropic equilibrium between the ionic species are found to be dependent on the nature of substituents. These molecules show the formation of

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MC via protonation of the carbonyl oxygen atom, conversely a predictable deprotonation

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takes place from the >NH group of indole ring to form the MA in the ground (S0) and the first excited singlet states (S1). The presence of –OH and –NH2 group enhances the prototropic

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behavior of these ICT probes leading to the formation of dianion species (DA). The highest occupied molecular orbitals (HOMO), lowest unoccupied molecular orbitals (LUMO) and

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molecular electrostatic potential (MEP) surfaces were generated from the optimized geometries of IC derivatives obtained using Density Functional Theory (DFT) calculations

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with restricted hybrid functional B3LYP using 6-31G (d) basis set to account for ICT. The MEP surfaces also show the possible protonation and deportation sites in these molecules.

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The dissociation constants, pKa and pKa*, for all the prototropic equilibria were determined in S0 and S1 states, respectively using different methods such as Henderson-Hasselbalch (HH), Photometric Titration (PT), Fluorimetric Titration (FT) and Förster Cycle (FC). For MC

N

equilibrium, the excited state pKa* values are found to be greater than the ground state pKa values, which suggests a more basic nature of these molecules in the excited state.

Keywords: Indole chalcones, Intramolecular charge transfer, Protonation, Dissociation constant, Prototropic equilibrium. _________________________________ *Corresponding author: Department of Chemistry, University of Delhi, Delhi – 110007, India, E-mail address: [email protected] (R.C. Rastogi) # presently at Department of Chemistry, Jamia Millia Islamia, Delhi-110025, India

Journal Pre-proof 1. Introduction Indole, the potent basic pharmacodynamic nucleus, has been reported to possess a wide variety of biologically active properties, and the substitution of indole ring into the chalcone moiety markedly influences the biological activity of the chalcone [1-5]. Indole based chalcones show anti-cancer [1], anti-inflammatory [2,3], neuroprotective [4] and anti-amoebic [5] properties. These compounds have also been investigated as β-amyloid imaging probes [6]. Indole chalcones are also used for the photogeneration of reactive oxygen species and photo-induced plasmid DNA cleavage [7,8].

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Multifunctional organic molecules can show both proton acceptor and donor behavior

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in acidic and basic media, respectively due to the presence of different acidic and basic centers, and can form different prototropic species [9] such as monoanion (MA), dianion

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(DA), monocation (MC), etc. Absorption and fluorescence spectroscopy has been extensively applied to study the prototropic behavior of a variety of organic molecules [10-15], which

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provides a better understanding of the prototropic structures in ground and excited states of

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photodynamic systems [16-20].

In our earlier work, Indole chalcone (IC) derivatives (Fig. 1) were investigated as an using

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interesting solvatochromic intramolecular charge transfer (ICT) probe by using

absorption and fluorescence spectroscopy in different solvent media [21]. The spectral

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characteristics of these molecules in different solvents were analyzed using microscopic and bulk solvent polarity parameters, which were also rationalized using Kamlet-Taft treatment.

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It was shown that these molecules exhibited ICT mechanism, which was highly influenced by the presence of different donor-acceptor groups and polarity of the solvents. Therefore, the ICT behavior of IC derivatives can play an important role in the formation of different prototropic species. The presence of various functional groups (such as >NH, >C=O, OH, NH2, etc.) makes them pH sensitive for protonation and deprotonation at various sites and responsible for the existence of various acid-base equilibria. In the present study, prototropic equilibria of IC derivatives in acidic and basic aqueous solutions have been investigated by absorption and fluorescence spectroscopy. The highest occupied molecular orbitals (HOMO), lowest unoccupied molecular orbitals (LUMO) and molecular electrostatic potential (MEP) have been quantum chemically evaluated to study the electron density and ICT behavior of these molecules using Density Functional Theory (DFT). The study reports the ground and excited state dissociation constants (pKa and pKa*) using methods such as Henderson-

Journal Pre-proof Hasselbalch (HH) [22,23], Photometric Titration (PT) [24], Fluorimetric Titration (FT) [25,26] and Förster Cycle (FC) [27-29]. 2. Experimental 2.1 Materials Materials and reagents for the synthesis of 3-(1H-indol-3-yl)-1-phenylprop-2-en-1one derivatives were purchased from Spectrochem Pvt. Ltd. and used without further purification. Analytical grade sulphuric acid ( 98.0%, Rankem), hydrochloric acid ( 35.4%, S.D. Fine), potassium hydroxide ( 84.0%, Merck), sodium hydroxide ( 98.0%, BDH),

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spectroscopic grade methanol (99.7%, Merck) and triple distilled water were used to prepare acidic and basic solutions.

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2.2 Instrumentation

The absorption spectra were recorded on UV-visible double-beam spectrophotometer

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(ANALYTIKA JENA UV WINASPECT SPECORD PC 250) using matching quartz cells of

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10 mm path length. The corrected fluorescence spectra of freshly prepared solutions were scanned in a rectangular quartz cell of 10 mm path length using spectrofluorometer (VARIAN CARY ECLIPSE). The slit widths used were 1 nm and 5 nm for absorption and

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fluorescence measurements, respectively. The concentration of the compounds was kept low,

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depending on the intensity of fluorescence to avoid aggregation and minimize the inner filter

2.3

Synthesis

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effect. pH values in the range of 1-12 were measured on Eutech make cyberscan pH-meter.

Synthesis and characterization of IC derivatives were done as described in our earlier work [21,30].

2.4 Preparation of Solutions Since the compounds are sparingly soluble in water, the stock solutions were prepared in methanol (1×10-2 mol L-1) and a calculated amount of freshly prepared stock solution of probe was added (3 min before the scan of spectra) to the freshly prepared solutions in the Ho/pH/H_ range of -3.03 to 17.95 to maintain the concentrations 2×10-5 mol L-1 and 1×10-5 mol L-1 for absorption and fluorescence spectra, respectively in which the percentage of methanol was not more than 1%. For the adjustment of pH, HCl-H2O and NaOH-H2O mixtures were used to prepare pH solutions in the range of pH 1-6 and pH 8-12, respectively. For the adjustment of acidity - basicity Scale, pH < 1 and pH >12, H2SO4-H2O and KOH-

Journal Pre-proof H2O mixtures were used according to Hammett's acidity scale (Ho) [31] and Yagil's basicity scale (H_), respectively [32]. 2.5 Determination of Ground State and Excited State Dissociation Constants The protonation and deprotonation dissociation constants in ground state (S0), pKa have been determined using HH [22,23,33-37] and PT methods [24]. The Henderson-Hasselbalch equation can be written as: 𝐴−𝐴𝑚𝑖𝑛

𝑝𝐻 = 𝑝𝐾𝑎 + 𝑙𝑜𝑔 𝐴

(1)

𝑚𝑎𝑥 −𝐴

where A is the absorbance of the solution at a given pH; Amax is the absorbance of the

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solution with the completely ionized probe and Amin is the absorbance of the solution. when 𝐴−𝐴𝑚𝑖𝑛

the total amount of probe is in the unionized form [38]. The plot of 𝑙𝑜𝑔 𝐴

𝑚𝑎𝑥 −𝐴

vs. pH

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produces a straight line with the intercept giving the ground state pKa for the equilibrium

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[33-36]. The ground state dissociation constant values, pKa’s (S0) were also calculated by PT method [24]. The change in absorbance value at a particular wavelength was plotted against

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pH resulting in the "S-shaped" (sigmoid) curve. The point of inflexion of the sigmoid curve, which was obtained from Microcal Origin 6.0 Professional, gave the pKa of the corresponding equilibrium (the concentration of the unionized molecular species equals the

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concentration of ionised species) [39].

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The excited state pKa* values for the different proton transfer reactions of IC

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derivatives in excited state were calculated by Fluorimetric Titration (FT) method [25,26]. The plots of relative fluorescence intensities (I/Io) vs. pH produced sigmoid curves which yielded the pKa* values from the corresponding inflection points. The excited state dissociation constants were also determined by FC method using the following equations: 𝑎 Based on absorption wavenumbers, 𝜐̅𝐻𝐴 and 𝜐̅𝐴𝑎− : 𝑎 𝑝𝐾𝑎 − 𝑝𝐾𝑎∗ = 2.1 × 10−3 (𝜐̅𝐻𝐴 − 𝜐̅𝐴𝑎− ) 𝑓

(2)

𝑓

Based on fluorescence wavenumbers, 𝜐̅𝐻𝐴 and 𝜐̅𝐴− : 𝑓

𝑓

𝑝𝐾𝑎 − 𝑝𝐾𝑎∗ = 2.1 × 10−3 (𝜐̅𝐻𝐴 − 𝜐̅𝐴− )

(3)

𝑎𝑣 Based on average of absorption and fluorescence wavenumbers, , 𝜐̅𝐻𝐴 and 𝜐̅𝐴𝑎𝑣− : 𝑎𝑣 𝑝𝐾𝑎 − 𝑝𝐾𝑎∗ = 2.1 × 10−3 (𝜐̅𝐻𝐴 − 𝜐̅𝐴𝑎𝑣− )

where

(4)

Journal Pre-proof 𝑎𝑣 𝜐̅𝐻𝐴 =

𝑓

𝑎 ̅𝐻𝐴 ̅𝐻𝐴 𝜐 +𝜐

2

𝜐̅𝐴𝑎𝑣− =

and

𝑓

𝑎 ̅𝐴 − +𝜐 ̅𝐴− 𝜐

2

HA and A- symbolize acid and its conjugated base, respectively. 2.6 Theoretical calculations The quantum chemical package Gaussian 09W [40] was used for all the theoretical calculations reported here. The DFT calculations with a restricted hybrid functional RB3LYP at 6-31G (d) basis set were performed. HOMO-LUMO and MEP surfaces were generated on the optimized geometries of the IC derivative. 3. Results and discussion

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The absorption band maxima show the large bathochromic shift (indicating   * type of transitions) with increase in solvent polarity, which indicates the charge-transfer (CT)

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interaction among the indole, phenyl and α, β-unsaturated ketone moieties of these IC derivatives (Fig. 1, Table 1) [21]. Absorption spectral data suggest the ground-state structure

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in all the molecules to be strongly sensitive to solvent environment. As the polarity of the

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solvent is increased from 1,4-doxane to water, the absorption profile becomes broad and structureless, suggesting larger solute-solvent interactions in highly polar solvents (Fig. S1). On increasing solvent polarity, the fluorescence spectra became structureless and an

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appreciable bathochromic shift was detected with a band enlargement in IC derivatives. Such

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a behavior indicated the stabilization of the highly dipolar excited state in polar solvents. Fluorescence intensity was much enhanced in polar solvents as compared to the non-polar

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solvents, which can be attributed to the less dissipation of energy due to increase in the rigidity of molecular structure in polar solvent. This was due to the specific solute-solvent interactions (solvent-carbonyl oxygen atom, solvent- >NH group of indole ring, etc.), which was established by Kamlet-Taft treatment of solvatochromic study in the earlier study [21]. A remarkable fluorescence enhancement of IC derivatives in protic polar solvents and a considerable quenching in the non-polar solvents indicates the key role of the proticity of solvents. The earlier solvatochromic study on IC derivatives [21] indicates the increase in excited state dipole moment of these derivatives (change in dipole moment = ~ 2D, Table S1), which suggests the charge transfer from donor to the acceptor (carbonyl) moieties leading to the ICT [41-44]. ICT in IC derivatives with electron donor and acceptor pairs can result in two possible structures, ICT ‘a’ and ICT ‘b’ as shown in Fig. 2a. The ICT ‘a’ structure is due to the delocalization of the electron lone pair on nitrogen atom of the indole

Journal Pre-proof ring towards the carbonyl group, whereas ICT ‘b’ structure can be ascribed to the shift of electron density from phenyl ring to the carbonyl group. HOMO and LUMO energies of Indole, formaldehyde, ether, methanol, methylamine and chloromethane are used to estimate the electronegativity of different functional groups >NH, >C=O, -OCH3, -OH, -NH2 and -Cl present in IC derivatives. According to Molecular orbital theory, the HOMO energy (EHOMO) is related to the ionisation potential (IP) by Koopmanns’ theorem and the LUMO energy (ELUMO) has been used to estimate the electron affinity (EA) [45]. If IP = -EHOMO and EA = -ELUMO, then the electronegativity ( ) as defined by Mulliken is average value of IP and EA [46]. Electronegativity values for >NH, >C=O, -OH,

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OCH3,

-NH2 and -Cl were calculated by using the values of EHOMO and ELUMO obtained from the Indole, formaldehyde, ether, methanol,

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optimised geometries (RB3LYP/6-31G (d)) of

methylamine and chloromethane, Table S2. Calculated electronegativity values of >C=O and

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>NH are 4.22 and 2.72, respectively, which shows that the carbonyl can withdraw electron density from the indole ring. Electronegativity values of -OCH3, -OH and -NH2 are 2.16, 2.57

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and 1.96, respectively, which are lower than that of >NH. Therefore, carbonyl group can predominantly withdraw the electron density from phenyl group as compared to the indole

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ring. In case of -Cl, the electronegativity is 3.70, which supports the electron donation from

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indole ring side instead of phenyl ring. Thus, two proposed ICT mechanisms are in conformity with the calculated electronegativity values of different functional groups present

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in IC derivatives (Fig 2a).

HOMO and LUMO were also visualized on the ground state optimized geometries of IC derivatives using DFT calculations (Figs 2b and S2). Molecular orbitals representing HOMO-1, HOMO, LUMO and LUMO+1 of selected IC derivatives are shown in Fig 2b. The ICT process is favourable in all IC derivatives, which is evident from a small HOMO-LUMO energy gap (3.85-4.05 eV, Table S3) [47]. On comparing HOMO and LUMO in all the IC derivatives, one can identify the depletion in electron density in indole ring. To consider   * type of transitions, electron densities on HOMO-1 and LUMO were compared for all the IC derivatives (Figs. 2b and S2). It is observed that there is an overall shift of electron density from the indole ring towards the rest of the molecule. Thus, there is predominant shift of the electron density from the indole ring in the direction of the carbonyl group in case of I, Me-I and Cl-I. Hence, nitrogen atom and carbonyl group become ‘positive’ and ‘negative’ centers, respectively in these derivatives. While in OMe-I and NH2-I, there is a general shift

Journal Pre-proof of electron density from the phenyl ring towards the rest of the molecule. Therefore, a net shift of the electron density from the phenyl ring in the direction of the carbonyl group is predicted. Whereas in OH-I, the shift of electron density towards the carbonyl group is contributed equally from both the indole and phenyl moieties, which is in accordance with the smallest difference between electronegativity values of indole and –OH functional groups. Therefore, R group (–OMe, –OH, –NH2, Fig. 2a) and carbonyl group may act as positive and negative centers, respectively [48,49]. Therefore, ICT 'a' structure can be expected to dominate the excited states of I, Me-I and Cl-I derivatives, whereas ICT 'b' structure may govern the excited states of the OMe-I, OH-I and NH2-I derivatives (Fig. 2a).

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Further, MEP surfaces of IC derivatives were considered to identify the positive and negative centres in the molecule. MEP surfaces express the positive, negative and neutral

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electrostatic potential regions in the molecule. MEP at any given point around a molecule provides information on the net electrostatic effect at that point caused by the total charge

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distribution of the molecular system [50]. MEP surfaces were plotted on the optimized geometries of IC derivatives as obtained by DFT calculation shown (Fig 3). Variations in the

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electrostatic potential at the MEP surface are represented by different colours, red representing most negative electrostatic potential regions and the regions of most positive

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electrostatic potential are indicated in blue. The increase in electrostatic potential of the surface is represented in the order, red< orange < yellow< green < blue. According to MEP

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surfaces, the most negative electrostatic potential in IC derivatives was found to be centred

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on most electronegative group i.e. carbonyl as surrounded by red colour in MEP surfaces. Similarly, the blue colour region on the >NH group of indole moiety indicated its electropositive character in IC derivatives. Therefore, MEP surfaces of IC derivatives suggest the carbonyl group as protonation site and >NH group of indole moiety as the deprotonation site in acidic and basic media, respectively. In OH-I and NH2-I, a positive electrostatic potential is observed (blue colour region) near -NH2 and -OH groups, which can be involved in deprotonation (Figs. 3e & 3f). Exceptionally, OMe-I is not showing any electropositive regions near the methoxy group, which can be due to the limitations of the quantum chemical calculations (Fig. 3d). 3.1 Prototropic Characteristics of IC derivatives The prototropic equilibrium for each group has been considered separately in terms of its ICT and probable protonation/deprotonation mechanism. In Group A molecules, charge transfer takes place from the indole ring to the carbonyl group. Group B and Group C

Journal Pre-proof molecules show charge transfer from phenyl ring to the carbonyl group, i.e. Group A: I, Me-I and Cl-I, Group B: OMe-I, and Group C: OH-I and NH2-I (Figs. 1 & 2a). The absorption and fluorescence spectra of IC derivatives have been studied in the Ho/pH/H– range from -3.03 to 17.95 in aqueous medium (Figs. 4 & 5). Absorption and fluorescence band maxima of IC derivatives along with their respective absorbance and fluorescence intensities have been summarized in Tables 2 & 3. Absorption and fluorescence spectra of I and OH-I in different acidic and basic aqueous media were shown in Figs 4 & 5, respectively. Absorption and fluorescence maxima of various prototropic forms of IC derivatives have been given in Table 4. The absorption spectra of IC derivatives show that the

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absorption band lying in the range of 390-410 nm (abs) is the most affected band in the Ho/pH/H– range from -3.03 to 17.95. Fluorescence spectra of different IC derivatives in acidic

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(Ho/pH from -3.03 to 6.00) and basic media (pH/H– from 7.91 to 17.95) were recorded at different excitation wavelengths obtained from their corresponding isosbestic points in

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absorption spectra (Table 4). 3.1.1 Group A: I, Me-I and Cl-I

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In Group A molecules, >NH group acts as donor and carbonyl group acts as acceptor in the absence of any strong electron donating group on phenyl ring. Thus, the charge

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delocalization that is ICT takes place from the >NH group to the carbonyl group, rendering positive charge on >NH group and negative charge on carbonyl group, which become acidic

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and basic centers, respectively. The acid-base spectral behavior of Group A: IC derivatives

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indicates the existence of following two-step equilibria: Equilibrium 1: Neutral (N)

Monocation (MC)

Equilibrium 2: Neutral (N)

Monoanion (MA)

3.1.1.1 Acid-Base Equilibria in Ground State The absorption band maxima (  abs = 391 nm) of I in different prototropic media, according to the Solvatochromic study [21], has been assigned to the neutral species in the pH range from 11.98 to 0.01 (Table 2). A new absorption band (𝜆1𝑎𝑏𝑠 = 471 nm) appears from Ho = -0.54 to -3.03 with simultaneous disappearance of the neutral species with the distinct isosbestic point at ~ 425 nm (Table 2 & Fig. 4a). This indicates the formation of monocationic species via protonation of carbonyl group, i.e. N

MC equilibrium in I. In this

monocationic species, positive charge is centered on the nitrogen atom of the indole ring after electronic rearrangement [51-53]. This is also corroborated from the previous studies of

Journal Pre-proof protonation of indole [51,54], indole-4-carboxylic [55], benzoylindoles [56] and chalcone analogs [57,58]. From H_ = 13.00 to 15.00, the absorption band maximum (𝜆2𝑎𝑏𝑠 ) at 465 nm becomes visible with disappearance of  abs at 391 nm with the isosbestic point at ~ 415 nm corresponding to the N

MA equilibrium (Tables 2 & 4, Fig. 4c). This shows the formation

of monoanionic species via deprotonation from >NH group of indole moiety in I (the only possible position for deprotonation), in which negative charge is centered on the oxygen atom of the carbonyl group after rearrangements) [52,53].

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Peculiar behavior: Absorbance of the characteristic absorption band of the anionic form (𝜆1𝑎𝑏𝑠 at 465 nm) starts decreasing beyond the H_ = 15.00 as a result of specific

MC and N

MA has been shown by Me-I and

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Similar spectral behavior i.e. N

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interaction of K+ and OH- ions with the Group A molecules [59-61].

Cl-I (Table 2) . The absorption band maxima,  abs of neutral species at 393 nm and 407 nm

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are completely replaced by the absorption band maxima 𝜆1𝑎𝑏𝑠 of cationic species at 480 nm and 475 nm of Me-I and Cl-I, respectively. Isosbestic points for N

MC equilibrium lie at

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~424 nm and ~430 nm for Me-I and Cl-I, respectively. The absorption band maxima, 𝜆2𝑎𝑏𝑠

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for monoanionic species of Me-I and Cl-I lie at 468 nm and 464 nm, respectively. The isosbestic points for this equilibrium are at ~425 nm and ~432 nm for Me-I and Cl-I,

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respectively (Table 4).

3.1.1.2 Acid-Base Equilibria in Excited State Similar equilibria, N

MC and N

MA exist in the excited state of I with the

same protonation and deprotonation mechanisms (Fig. 6a). The fluorescence band maxima (flu) of neutral species lie at 520 nm in the Ho/pH/H– range -0.54 to 13.00 (Table 2, Fig 4b & 4d). On decreasing the Ho from -1.52 to -3.03, a new red-shifted fluorescence band appears at 538 nm, which can be assigned to the monocationic species in the excited state (MC *) [52,62]. On increasing the H_ upto 17.95, a new red-shifted fluorescence band appears at 547 nm, which is attributed to the monoanionic species (MA*). Both MC* and MA* have lower fluorescence intensity compared to the N* form. Similarly, flu of neutral forms of Me-I and Cl-I lies at 521 nm and 527 nm, respectively. The flu of anionic species of Me-I and Cl-I in the excited state is centered at

Journal Pre-proof 542 nm and 556 nm (at H_ = 17.95), respectively whereas the same for monocationic species lies at 548 nm and 540 nm (at Ho = -3.03), respectively (Table 2). Peculiar behavior: A low intensity fluorescence band is observed for Group A molecules at ~650 nm in pH ~1.02 to 0.01 (Fig. 4b, shown in square). This fluorescence band appears due to the complex formation between the N*(keto) and MC*(enol) forms i.e. the interaction of hydroxyl group of MC* with the carbonyl group of N*. As the acidity of the medium increases beyond pH = 0.01, the N* form gets completely protonated (at the carbonyl group) and hence, it is no more available for complex formation thereby resulting in the disappearance of band at 650 nm [63]. Therefore, the complex only exists in a narrow range

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of acidity (pH=1.02 to 0.01). 3.1.2 Group B: OMe-I

MC and N

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OMe-I also shows two equilibria, N

MA in ground as well as in

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excited states (Fig. 6b, Table 3). The ICT in OMe-I takes place from the phenyl ring to the carbonyl because of the presence of strong electron-donating methoxy group on para position

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of phenyl ring, which is different from the Group A molecules (Fig. 2a). Consequently, the carbonyl group turns basic and offeres protonation (same as Group A) and consequently, positive charge is generated on oxygen atom of methoxy group (monocationic species, MA &

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MA*).

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3.1.2.1 Acid-Base Equilibria in Ground State The neutral form of OMe-I exists in pH/Ho range from 11.98 to -0.54 and shows

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 abs at 396 nm. On increasing the acidity, a new absorption band (𝜆1𝑎𝑏𝑠 ) appears at the expense of the absorption band of neutral species from Ho ~ -0.95 to -3.03 at 497 nm with isosbestic point at ~ 432 nm corresponding to MC

N. The anionic form of OMe-I appears

at H_ = 13.00 marked by the appearance of a new absorption band, 𝜆2𝑎𝑏𝑠 at 466 nm with simultaneous decrease in absorbance of neutral species. This N

MA equilibrium has

isosbestic point at ~421 nm. Peculiar behavior: The absorption band at 466 nm corresponds to MA of OMe-I having the maximum absorbance at H_ = 15.00 and beyond this, absorbance starts decreasing as a result of specific interaction of K+ and OH- ions similar to Group A molecules. 3.1.2.2 Acid-Base Equilibria in Excited State

Journal Pre-proof A similar existence of three species N*, MA* & MC* (* indicates the excited species) was observed in the excited state and shown by the fluorescence spectra of OMe-I, that follows the same protonation and deprotonation processes as in the ground state. The neutral form of OMe-I exists in the pH/Ho range from 11.98 to -0.54 having the fluorescence band, flu at 517 nm. The fluorescence band maxima for MC* & MA* of OMe-I are at 575 nm and 542 nm, respectively. 3.1.3 Group C: OH-I and NH2-I OH-I and NH2-I are homologous to OMe-I having electron donating group at para

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position of phenyl ring and show formation of similar ICT structure in the excited state

MA

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(Fig. 2a). In these molecules, three equilibria namely, MC

N, N

MA and

DA exist in the Ho/pH/H– range from -3.03 to 17.95 in ground as well as in excited

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states (Figs. 5a-d, & Table 3). The equilibria and corresponding prototropic species have been

e-

shown in Fig. 6c. 3.1.3.1 Acid-Base Equilibria in Ground State

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OH-I and NH2-I exist as neutral forms in Ho/pH range (-0.95 to 8.98) and (-0.95 to 10.93) with their absorption band maxima (  abs ) at 393 nm and 399 nm, respectively (Table

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3). Below Ho = -0.95, new absorption bands, 𝜆1𝑎𝑏𝑠 at 495 nm and 467 nm have been observed

MC

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(up to Ho = -3.03) for OH-I (Fig. 5a) and NH2-I, respectively for the cationic species. This N equilibrium for OH-I and NH2-I with isosbestic points at ~432 nm and ~ 434 nm,

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respectively is marked by protonation at the carbonyl group and positive charge at oxygen and nitrogen atoms of OH and NH2 groups, respectively (Fig. 6c). The protonation of OH and NH2 groups does not occur as they tend to become electron deficient due to ICT mechanism (=OH+ and =NH2+).

At pH/H_ value above 9.96, OH-I and NH2-I behave differently as compared to the Groups A & B molecules because of the presence of new acidic centers, OH and NH2 groups.  abs shows a bathochromic shift for OH-I (393 nm to 407 nm) and NH2-I (399 nm to 406 nm) indicating the formation of MA species caused by deprotonation from OH and NH2 groups (Fig. 5c, Table 3). In highly basic medium (beyond pH = 13.00), a similar trend is marked by the appearance of a new absorption band, 𝜆2𝑎𝑏𝑠 at 470 nm and 468 nm for OH-I and NH2-I, respectively (Table 3). These bands can be assigned to the dianionic species (DA),

Journal Pre-proof which are formed due to deprotonation from the >NH group of the indole ring. Hence, MA DA equilibrium occurs in the ground state for OH-I and NH2-I and shows isosbestic points at ~ 432 nm and ~ 428 nm, respectively (Table 4). The absorption band maxima (𝜆2𝑎𝑏𝑠 ) arising from DA shows a continuous increase in absorbance from H_= 13.00 to 17.95 unlike Groups A & B molecules. 3.1.3.2 Acid-Base Equilibria in Excited State In excited state, similar protonation and deprotonation mechanisms occur in OH-I and NH2-I, as in ground state. The neutral form of OH-I shows fluorescence band maxima, flu

f

at 518 nm, which exists in pH/Ho range from -0.54 to 6.82. A bathochromically shifted band

oo

is observed at 571 nm in Ho range from -1.52 to -3.03, indicating the formation of monocationic species (Figs. 5b & 5d). The fluorescence band maxima, 𝜆𝑓𝑙𝑢 for neutral form

pr

of OH-I shows a blue shift of 13 nm from pH 7.91 to 11.98 marking the presence of MA in state

e-

excited

(Table 3). On further increasing pH, a red shift in the fluorescence band is observed and a

Pr

clear fluorescence band is observed at 532 nm on attaining H_ = 14.00. This corresponds to the DA formed by deprotonation from >NH group (indole ring). flu of DA is found to show

al

continuous increase in fluorescence intensity till H_ = 17.95, which is also the maximum

rn

H_ value used in present study, indicating incomplete formation of DA. A similar prototropic spectral behavior has been shown by NH2-I. The fluorescence

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band maxima of MC are observed at 548 nm, whereas that of N is observed at 510 nm. The MA and DA of NH2-I show fluorescence band maxima at 507 nm and 535 nm, respectively. The continuous increase in fluorescence intensity till H_ = 17.95 indicates the incomplete formation of DA of NH2-I (Fig. 6c). 3.2 Ground State dissociation constants The absorption spectra were recorded in acidic and basic media with variation in pH by 0.2-0.5 units in the range from -3.03 to -0.54 for acidic medium (the expected region for the pKa of MC N

N) and 12.0 to 15.00 for basic medium (the expected region for the pKa of

MA) (see supporting data, Tables S4 & S5). The ground state (S0) protonation and deprotonation dissociation constants, pKa of IC

derivatives for different prototropic equilibria have been determined using two methods: HH [22,23] and PT methods [24] (Figs. 7 & 8, Table 4). The Henderson-Hasselbalch method was

Journal Pre-proof applied on the absorbance at a particular wavelength given in Tables S2 & S3 for different equilibria in the Ho/pH/H_ range of IC derivatives. The calculated values of log

A  A m in in the region from -3.03 to -0.54 for acidic A m ax  A

medium and from 12.00 to 15.00 for basic medium were plotted against Ho/pH/H_ values (eq. 1, Tables S4 & S5). These plots for I derivative are shown in Figs. 7a & 7b. A straight line obtained with slope approximately close to unity gives the ground state pKa value, which is equal to the intercept at abscissa [33-36].

f

Employing the PT method, absorbance of IC derivatives measured at a particular

oo

selected wavelength was plotted against Ho/pH/H_ values. These plots resulted in "S-shaped" curves and the inflection point of the sigmoid curves gave the pKa value of IC derivatives due

pr

to the same concentration of both the molecular species and ionic species [39]. These PT

e-

curves are shown in Figs. 7c & 7d.

Ground state dissociation constants, pKa ’s (S0) obtained from both the methods are

MC

Pr

found to be in good agreement for different prototropic equilibria. The pKa (S0) values for N are found to be in the range of Ho ~ -2.10 to -2.80, and for N

MA are found to

al

be in the range of H_ ~13.37 to 13.55 for I, Me-I, Cl-I and OMe-I, which indicates similarity

rn

in protonation and deprotonation sites (Table 5). The pKa(S0) values for MA

DA are 13.97 and 13.69 for OH-I and

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NH2-I, respectively. However, the formation of DA starts from pH = 11.98, which suggests that the pKa(S0) value for N

MA is less than 11.98 for these molecules.

3.3 Excited State dissociation constants 3.3.1 Fluorimetric Titration (FT) The excited state pKa* values for the different proton transfer reactions of IC derivatives in excited state were calculated by using Fluorimetric Titration method (FT) [25,26] and are given in Table 5. A decrease in fluorescence intensity was observed for MC equilibria for all the IC derivatives with the only exception of MA

N and N

MA

DA equilibrium in case

of OH-I and NH2-I. Using this gradual fluorescence quenching behaviour of IC derivatives in acidic as well as in basic media, the pKa* (for S1 state) values for these equilibria were

Journal Pre-proof determined from sigmoid curves of relative fluorescence intensity (I/Io) vs. Ho/pH/H_. The fluorimetric titration curves for I in acidic and basic media are shown in Figs. 7e & 7f, where the inflection points give the pKa* values [39]. These sigmoid curves for I in acidic and basic media with a single inflection point indicate the single protonation and deprotonation in these molecules except for OH-I and NH2-I, where two equilibria exist in basic media [64]. The small variation in fluorescence intensity during the formation of MA of OH-I and NH2-I does not allow the use of Fluorimetric Titration method to determine the pKa* values. The plots of fluorescence intensity vs. pH/H_ show that the formation of DA in OH-I and NH2-I is not complete till H_ = 17.95, which is the most basic solution used in the study.

f

Therefore, the pKa* value for the MA

oo

DA equilibrium could not be evaluated by

fluorimetric titrations, however approximate pKa* values i.e. 16.80 and 17.22 for OH-I and

pr

NH2-I, respectively as obtained by using Microcal Origin 6.0 Professional software are

The pKa* values for MC

e-

shown in Figs. 8a & 8b [39].

N equilibrium were found to be less negative (-0.04 to

Pr

1.94) than those in the ground state (-2.10 to -2.80), which suggests that the IC derivatives become more basic in the excited state than the ground state (Table 4). This is because of the

al

charge transfer from the donor to the acceptor group (carbonyl group) i.e. ICT in excited state, which makes the carbonyl group more basic in excited state in comparison to the

rn

ground state. The pKa* values for N

MA equilibrium in the excited state are closer to the

Jo u

ground state values, indicating that the lifetimes of the species are very short and that the prototropic equilibria are not well established in the excited state (Table 5) [65]. 3.3.2 Förster Cycle (FC) Method The pKa* values calculated with the help of the FC by using absorption and fluorescence spectral band maxima for neutral, MC, MA and DA species are given in Table 4 (Eqs. 2-4) [27,28]. The agreement among the pKa*(abs), pKa*(flu) and pKa*(av) values is poor because the FC method strictly applies only when solvent relaxation is same in both the ground and the excited states of the species participating in the equilibrium [27,66]. The pKa*(FT) values are closer to pKa*(flu) (Eq. 3) than pKa*(abs) (Eq. 2) and, pKa*(av) values (Eq. 4). It can be seen that the prototropic equilibrium, MA

DA is not

complete for OH-I and NH2-I in the excited state, thus the FC method provides a good method for estimating the values of pKa*.

Journal Pre-proof 3

Conclusions IC derivatives have been found to show protonation and deprotonation in highly

acidic and basic media, respectively. Structures of anionic as well as cationic species are found to be very much dependent on ICT processes of these molecules. The proposed ICT Structure as well as protonation/deprotonation sites are found to be in fair agreement with the theoretically calculated HOMO-LUMO transitions and MEP surfaces. Absorption and fluorescence spectra of IC derivatives in acid-base media (Ho/pH/H_) show similar prototropic species in ground and excited states. IC derivatives show protonation at carbonyl group and deprotonation from the >NH in highly acidic and basic media, respectively. I, Me-

oo

f

I, Cl-I and OMe-I molecules exist as three distinct prototropic species, MC, N and MA. While OH-I and NH2-I molecules show the formation of an additional species i.e. DA in the

pr

Ho/pH/H_ range -3.03 to 17.95. DA prototropic species formed by OH-I and NH2-I molecules are highly fluorescent, which can be attributed to their highly stable and more rigid

e-

structures. The ground and excited state dissociation constants have been calculated for

MC

Pr

different prototropic equilibria for IC derivatives in the So and S1 states. The pKa* values for N equilibrium are found to be less negative than the ground state pKa values, which

Acknowledgements

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suggests that the IC derivatives become more basic in the excited state.

rn

The financial support from University of Delhi under the Scheme “To strengthen

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Research & Development Doctoral Research Program” is gratefully acknowledged. Manju K. Saroj is thankful to the University Grants Commission, New Delhi for the financial assistance. We greatly appreciate the efforts received from the unknown reviewers for meticulous analysis of our manuscript and very constructive suggestions and queries.

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Journal Pre-proof Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☒The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

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Pr

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pr

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None

Journal Pre-proof Figures Captions Structures of indole chalcone derivatives.

Fig. 2.

(a) Possible Intramolecular Charge transfer structures of IC derivatives. (b) Optimized geometries and molecular orbitals of I, OMe-I and NH2-I.

Fig. 3.

Molecular Electrostatic Potential surfaces of (a) I, (b) Me-I, (c) Cl-I (d) OMe-I (e) OH-I and (f) NH2-I. The red and blue colours correspond to negative and positive potential regions, respectively.

Fig.4.

Absorption and fluorescence spectra of I in different acidic (a & b) and basic (c & d) aqueous media.

Fig. 5.

Absorption and fluorescence spectra of OH-I in different acidic (a & b) and basic (c & d) aqueous media.

Fig. 6.

Possible prototropic species of (a) I, Me-I & Cl-I (b) OMe-I (c) OH-I and NH2-I in ground and excited states in Ho/pH/H_ range from -3.03 to 17.95.

Fig. 7.

Plots to determine pKa(S0) by using HH (a & b) and PT methods (c & d); and plots of relative fluorescence intensity (I/Io) vs. Ho/pH/H_ to determine pKa*(S1)

Pr

e-

pr

oo

f

Fig. 1.

N and N

Plots of fluorescence intensity vs. pH/H_ using fluorimetric titration method for DA of (a) OH-I and (b) NH2-I (broken line represents the first

rn

equilibrium, MA derivative curve).

Jo u

Fig. 8.

al

by using fluorimetric titration method (e & f) for equilibria, MC MA of I (broken line represents the first derivative curve).

Journal Pre-proof

Enone moiety 5'

O

4' 6'

2"

9' 3'

1"

1

3

3"

2

7' 8' 1'

N

6"

2'

4" 5"

R

H

Indole moiety

Group Molecules

oo

f

Phenyl moiety

R

Abbreviation

H

I

3-(1′H-Indol-3′-yl)-1-4''-tolyl-prop-2-en-1-one

CH3

Me-I

1-(4′′-Chloro-phenyl)-3-(1H′-indol-3′-yl)-prop-2-en-1-one

Cl

Cl-I

B

3-(1′H-Indol-3′-yl)-1-(4′′-methoxyphenyl)-prop -2-en-1-one

OCH3

OMe-I

C

1-(4′′-Hydroxy-phenyl)-3-(1′H-indol-3′-yl)-prop-2-en-1-one

OH

OH-I

3-(1′H-Indol-3′-yl)-1-(4''-aminophenyl)-prop-2-en-1-one

NH2

NH2-I

3-(1′H-Indol-3′-yl)-1-phenylprop-2-en-1-one

Jo u

rn

al

Pr

e-

pr

A

Fig. 1

Jo u

rn

al

Pr

e-

pr

Fig. 2a

oo

f

Journal Pre-proof

Journal Pre-proof Optimized geometry

HOMO-1

HOMO

LUMO

I

f o

OMe-I

l a

NH2-I

e

o r p

r P

n r u

o J

Fig. 2b

LUMO+1

Journal Pre-proof

(a)

(b)

(c)

f o

(d)

(e)

l a

e

o r p

r P

n r u

o J

Fig. 3

(f)

Journal Pre-proof

0.9

0.8

0.7

= -3.03 = -2.76 = -2.51 = -2.28 = -2.06 = -1.85 = -1.70 = -1.62 = -1.52 = -1.38 = -0.95 = -0.54

Ho = -3.03

(b)

Ho = -2.06 Ho = -1.70

50

Ho = -1.52 Ho = -0.95 Ho = -0.54 Ho = 0.01

40

pH = 1.02

Fluorescence intensity

Ho Ho Ho Ho Ho Ho Ho Ho Ho Ho Ho Ho

(a)

Absorbance

0.6

0.5

0.4

pH = 2.91 pH = 3.91 30

pH = 4.97 pH = 6.82

20

ro of

0.3

pH = 2.05

0.2

10

400

450

550

0 450

re

600

H_ = 12.00 H_ = 12.50 H_ = 13.00 H_ = 13.32 H_ = 13.75 H_ = 14.00 H_ = 14.11 H_ = 14.33 H_ = 14.51 H_ = 14.69 H_ = 14.85 H_ = 15.00

(c)

ur

na

0.8

0.6

Jo

Absorbance

500

Wavelength/nm

0.4

500

550

600

650

700

w avelength/nm pH = 6.95 pH = 7.91

45

Fluorescence intensity

1

350

lP

0 300

-p

0.1

(d)

40

pH = 8.98 pH = 9.96 pH = 10.93

35

pH = 11.98 H_ = 13.00 H_ = 13.50

30

H_ = 14.00 H_ = 14.51 H_ = 15.00 H_ = 15.44 H_ = 16.00

25

H_ = 16.58 H_ = 16.90 H_ = 17.39

20

H_ = 17.95 15

10 0.2 5

0 300

0 450 350

400

450

500

550

500

550

600

600

Wavelength/nm

w avelength/nm

Fig. 4

650

700

Journal Pre-proof Ho = -3.03

(a)

Ho = -2.76

80

Ho = -3.03

(b)

Ho = -2.06

Ho = -2.51 1

Ho = -1.70

Ho = -2.28

Ho = -1.52

70

Ho = -2.06

Ho = -0.95

Ho = -1.85

Ho = -0.54

Ho = -1.70 0.8

60

Fluorescence intensity

Ho = -1.62 Ho = -1.52

Absorbance

Ho = -1.38 Ho = -0.95 0.6

Ho = -0.54

pH = 1.02 50

pH = 2.05 pH = 2.91 pH = 3.91

40

pH = 4.97 pH = 6.82

30

ro of

0.4

Ho = 0.01

20 0.2

0 300

350

400

450

500

550

600

650

0 450

re

Wavelength/nm

-p

10

1

250

H_ = 12.00

(c)

lP

H_ = 12.50

500

550

600

pH = 8.98 pH = 9.96

H_ = 13.32 H_ = 13.75

0.8

pH = 10.93 pH = 11.98 H_ = 13.00

200

na

H_ = 14.00

ur

0.6

Jo

0.5

0.4

H_ = 14.51 H_ = 14.69 H_ = 14.85 H_ = 15.00

Fluorescence intensity

H_ = 14.33

Absorbance

H_ = 13.50 H_ = 14.00

H_ = 14.11

0.7

700

pH = 6.95 pH = 7.91

(d)

H_ = 13.00

0.9

650

w avelength/nm

H_ = 14.51 H_ = 15.00 150

H_ = 15.44 H_ = 16.00 H_ = 16.58 H_ = 16.90 H_ = 17.39 H_ = 17.95

100

0.3

0.2

50

0.1

0 300

350

400

450

500

550

600

0 450

500

550

600

w avelength/nm

Wavelength/nm

Fig. 5

650

700

Journal Pre-proof

HO

pKa* (I) = 13.37 pKa* (Me-I) = 13.24 pKa* (Cl-I) = 14.57

O

R

-H

max (I) = 538 nm max (Me-I) = 548 nm max (Cl-I) = 540 nm

N N* H max (I) = 520 nm

f o

r u o

pKa (I) = -2.10 pKa (Me-I) = -2.13 pKa (Cl-I) = -2.79

J

 O

pKa (I) = 13.44 pKa (Me-I) = 13.37 pKa (Cl-I) = 13.53

+H

N H

MC

max (I) = 471 nm max (Me-I) = 480 nm max (Cl-I) = 475 nm

Acidic medium

R

-H

R

where R = H, Me & Cl Absorption

l a n

MA* N  max (I) = 547 nm  max (Me-I) = 542 nm  max (Cl-I) = 556 nm

o r p

e

r P Fluorescence

Fluorescence

HO

+H

R

max (Me-I) = 521 nm max (Cl-I) = 527 nm

Absorption

MC*

Absorption

N H

O

-H

+H

Fluorescence

pKa* (I) = -0.56 pKa* (Me-I) = -0.86 pKa* (Cl-I) = - 1.01

O -H

N R N  H max (I) = 391 nm max (Me-I) = 393 nm max (Cl-I) = 407 nm

Neutral medium Fig. 6 (a)

+H

N

MA

max (I) = 465 nm max (Me-I) = 468 nm max (Cl-I) = 464 nm

Basic medium

R

Journal Pre-proof

pKa* = -0.14

pKa* = 13.39

O

N H

Fluorescence

 max = 575 nm

HO

pKa = -2.38

r u o +H

N H

MC  max = 497 nm

Acidic medium

OMe

l a n

J

-H

+H

N*

MA*

N

OMe

 max = 517 nm

f o

 max = 542 nm

o r p

e

r P  O

N N H  max = 396 nm

Neutral medium Fig. 6 (b)

OMe

Fluorescence

-H

OMe

Absorption

MC*

Absorption

N H

O

-H

Absorption

+H

Fluorescence

HO

pKa = 13.48

O -H  OMe

+H N

MA  max = 466 nm

Basic medium

OMe

Journal Pre-proof

XH

-H

HO +H MC

XH

max (OH-I) = 495 nm max (NH2-I) = 467 nm

Acidic medium

N*

Absorption

pKa (OH-I) = -2.12 pKa (NH2-I) = -2.50

N H

XH

+H

N H

max (OH-I) = 518 nm max (NH2-I) = 510 nm

Fluorescence

Absorption

max (OH-I) = 571 nm max (NH2-I) = 548 nm

N H

-H

l a n

r P

where X=O for OH-I X=NH for NH2 -I

DA*

X

O

pKa (OH-I) = 13.97 pKa (NH2-I) = 13.69

-H 

XH

+H

max (OH-I) = 393 nm max (NH2-I) = 399 nm

Neutral medium

N

max (OH-I) = 532 nm max (NH2-I) = 535 nm

o r p

e

O

N

O

f o



N H

+H

max (OH-I) = 505 nm max (NH2-I) = 507 nm

r u o

J

X

MA*

Fluorescence

MC*

O

-H

Fluorescence

N H

pKa* (OH-I) = ~16.80 pKa* (NH2-I) = ~17.22 -H

O -H

X N MA H max (OH-I) = 406 nm max (NH2-I) = 406 nm

Less basic medium Fig. 6c

Absorption

O

+H

Absorption

HO

pKa* (OH-I) = ~9.10 pKa* (NH2-I) = ~9.17

Fluorescence

pKa* (OH-I) = 0.04 pKa* (NH2-I) = 1.94

+H

N DA max (OH-I) = 470 nm max (NH2-I) = 468 nm

X

Strong Basic medium

Journal Pre-proof

1.5

pKa = -2.48

0.5 0 -2.5

-2

-1.5

-1

-0.5

0

Ho/pH

-1 -1.5

0 12

12.5

13

13.5

Absorbance

0.8

R = 0.9902

1 0.8 0.6

(d)

pKa = 13.44

pr

0.4

15

-1

1.2

pKa = -2.10

14.5

pH/H_

1.4

0.6

14

-1.5

(c)

0.2

0.4 0.2

-4

1.4

-3

-2

Ho/pH

-1

(e)

1.2

0

al

1 0.8

pKa* = -0.56

rn

0.6

0 -4

-2

Jo u

0.4 0.2

8

1

Pr

-5

e-

0

0

I/I o

pKa = 13.48

0.5

-0.5

R = 0.9758

1

Absorbance

-0.5

1

f

-3

(b)

oo

1

log(A-A min)/(A max-A)

(a)

0

2

10

12

14

16

18

pH/H_

1.4

(f)

1.2 1

I/I o

log(A-A min)/(A max-A)

1.5

0.8

pKa* = 13.37

0.6 0.4 0.2 0

4

6

8

Ho/pH

4

6

8

10

12

14

pH/H_

Fig. 7

16

18

20

Journal Pre-proof

250

pKa* =

Fluorescence Intensity

100

(a)

16.80

200 150 100 50 0 8

10

12

14

16

18

(b)

pKa* = 17.22

80 60 40 20 0

20

4

6

8

pH/H_

10

12

pH/H_

Pr

e-

pr

oo

f

Fig. 8

al

6

rn

4

Jo u

Fluorescence Intensity

300

14

16

18

20

Journal Pre-proof Table 1 Absorption and fluorescence band maxima (in nm) of IC derivatives in different solvents λabs sol λsol

NH2-I

Cl-I

abs flu abs flu abs flu abs flu sol λsol λsol λsol λsol λsol λsol λsol λsol λsol

446 367 445 366 437 362 444 366 440 366 453 376 458 373 450 366 440 366 445 370 444 367 464 386 455 367 452 370 442 362 445 366 444 364 458 378 460 368 458 367 452 365 450 367 447 366 464 377 474 385 464 383 456 376 456 382 458 382 483 393

oo

479 390 472 388 461 382 464 384 463 386 492 400

f

467 390 464 385 455 390 458 385 457 386 477 402

473 373 467 372 459 370 462 372 458 370 485 382

pr

480 389 476 394 465 390 469 388 465 395 490 399 480 391 476 388 466 392 470 394 467 397 492 400 491 388 486 388 475 388 480 387 471 395 506 396 499 386 497 385 489 385 494 385 478 395 508 392 509 397 503 397 493 395 499 397 494 402 515 401 520 391 521 392 513 393 517 393 502 399 524 407

Absorption band maxima. Fluorescence band maxima.

rn

b

OMe-I

Jo u

a

OH-I

e-

DMF tertButanol DMSO Acetonit rile 2Propanol 1Butanol Ethanol Methan ol Ethylen e glycol Water

Me-I b flu λabs flu

Pr

1,4Dioxane THF Ethyl acetate DCM

I a

al

b

Solvent

Journal Pre-proof Table 2 Absorption and fluorescence band maxima (in nm) of I, Me-I & Cl-I in different Ho/pH/H_ aqueous media Cl-I I

471(0. 91)

-2.06

-

-1.52

391( 0.39 )

1.02

6.82

7.91

8.98

9.96

10.93

11.98

13.00

13.50

471(0. 33)

M C

538(5.51)

M C

471(0. 22)

N, M C

538(7.85)

M C

471(0. 10)

N, M C

520(12.39)

N

471(0. 06)

N

520(28.19)

N

-

N

520(28.27)

N

-

N

520(39.61) , 650 (10.5)

N

-

N

520(43.62)

N

-

N

520(35.00)

N

-

N

520(39.99)

N

-

N

520(40.78)

N

-

-

465(0. 26) 465(0. 53)

N

520(38.29)

N

N

520(37.43)

N

520(26.73)

N

524(21.65)

-

N, M A N, M A

-

480(0 .89)

M C

548( 5.4)

M C

-

475(0 .76)

M C

480(0 .34)

N, M C

548( 2.8)

M C

407( 0.21 )

475(0 .18)d

N, M C

540( 03.0 ) 540( 03.7 )

480(0 .16)

N, M C

548( 6.4)

M C

407( 0.29 )

475(0 .12)d

N, M C

540( 06.8 )

480(0 .11)

N, M C

-

N

-

N

-

N

-

N

-

N

-

N

-

N

393( 0.25 ) 393( 0.28 ) 393( 0.33 ) 393( 0.38 ) 393( 0.34 ) 393( 0.27 ) 393( 0.25 ) 393( 0.36 ) 393( 0.38 ) 393( 0.34 ) 393( 0.30 ) 393( 0.33 ) 393( 0.33 ) 393( 0.27 )

al

0.01

M C

rn

-0.54

391( 0.42 ) 391( 0.50 ) 391( 0.45 ) 391( 0.49 ) 391( 0.42 ) 391( 0.40 ) 391( 0.38 ) 391( 0.28 ) 391( 0.47 ) 391( 0.52 ) 391( 0.29 ) 391( 0.37 )

-

Jo u

-0.95

M C

A S

 flu A

-

N

-

N

-

N

-

N

-

N

-

N

-

N

-

N

-

N

468(0 .25) 468(0 .36)

N, M A N, M A

14.00

-

465(0. 53)

M A

528(9.00)

-

-

468(0 .52)

M A

14.51

-

465(1. 01)

M A

534(6.44)

-

-

468(0 .65)

M A

15.00

-

465(1. 13)

M A

534(5.79)

-

-

468(0 .74)

M A

15.44

-

465(0. 99)

M A

536(4.84)

-

-

468(0 .70)

M A

16.00

-

465(0. 93)

M A

540(5.79)

-

-

468(0 .50)

M A

1  abs 𝜆𝑎𝑏𝑠

/

S

f

-

𝜆𝑎𝑏𝑠

𝜆1𝑎𝑏𝑠 / 𝜆2𝑎𝑏𝑠

521( 17.3 ) 521( 35.0 ) 521( 32.6 ) 521( 26.0 ) 521( 41.2 ) 521( 48.6 ) 521( 56.5 ) 521( 67.6 ) 521( 56.5 ) 521( 50.4 ) 521( 45.6 ) 521( 22.0 ) 526( 17.3 ) 532( 12.2 ) 537( 11.0 ) 542( 12.0 ) 542( 14.1 )

pr

-3.03

𝜆fluc

A S

e-

A Sb

Pr

𝜆𝑎𝑏𝑠

𝜆1𝑎𝑏𝑠 / 𝜆2𝑎𝑏𝑠 a

Me-I

oo

Ho/p H/H _

N

N

N

N

N

N

N

N

N

N

-

-

-

𝜆2𝑎𝑏𝑠

407( 0.28 ) 407( 0.33 ) 407( 0.31 ) 407( 0.30 ) 407( 0.15 ) 407( 0.18 ) 407( 0.30 ) 407( 0.33 ) 407( 0.20 ) 407( 0.26 ) 407( 0.25 ) 407( 0.24 ) 407( 0.19 )

464(0 .10) 464(0 .14) 464(0 .17) 464(0 .23) 464(0 .25) 464(0 .39)

A S

N, M A N, M A N, M A N, M A N, M A N, M A

-

-

464(0 .53)

M A

-

-

464(0 .43)

M A

M A

-

464(0 .46)

M A

M A

-

464(0 .44)

M A

𝜆flu

527( 10.2 ) 527( 11.5 ) 527( 11.8 ) 527( 14.2 ) 527( 24.4 ) 527( 21.2 ) 527( 24.3 ) 527( 19.0 ) 527( 27.8 ) 527( 32.7 ) 527( 32.3 ) 527( 27.9 ) 527( 23.7 ) 527( 21.9 ) 527( 14.6 ) 556( 08.1 ) 556( 08.6 )

A S M C M C M C

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N M A M A

Journal Pre-proof

17.95 a

-

465(0. 57)

M A

547(12.04)

M A

468(0 .38)

-

M A

542( 12.4 )

M A

1abs = Absorption band maxima of MC; 2abs Absorption band maxima of MA.

b

Assigned species, MA = monoanion, N = neutral, MC = monocation. Excited at wavelength of isosbestic point (Table 5). d Shoulder absorption bands.

Jo u

rn

al

Pr

e-

pr

oo

f

c

408( 0.19 )

464(0 .42)

M A

556( 09.4 )

M A

Journal Pre-proof Table 3 Absorption and fluorescence band maxima (in nm) of OMe-I, OH-I and NH2-I in different Ho/pH/H_ aqueous media NH2-I OMe-I

0.01

1.02

6.82

7.91

8.98

9.96

10.93

11.98

13.00

13.50

14.00

489(0. 09) -

N

-

N

-

N

-

N

-

N

-

N

-

N

-

N

-

466(0. 23) 466(0. 33)

N

N, M A N, M A

466(0. 60)

M A

14.51

-

466(0. 73)

M A

15.00

-

466(0. 72)

M A

15.44

-

466(0. 67)

M A

16.00

-

466(0. 54)

M A

466(0. 36)

M A

17.95

575( 6.6)

M C

575( 11.0 ) 517( 14.6 ) 517( 32.0 ) 517( 36.3 ) 517( 37.0 ) 517( 86.0 ) 517( 80.9 ) 517( 74.5 ) 517( 68.9 ) 517( 90.4 ) 517( 57.4 ) 522( 80.9 ) 523( 68.8 ) 522( 56.2 ) 522( 29.4 ) 530( 18.2 ) 532( 16.4 ) 542( 18.1 ) 542( 22.0

M C

N N N

N

N

N

-

495(1. 13)

MC

495(0. 60)

393( 0.25 ) 393( 0.34 ) 393( 0.39 ) 393( 0.51 ) 393( 0.42 ) 393( 0.39 ) 393( 0.41 ) 393( 0.49 ) 393( 0.45 ) 407( 0.76 ) 407( 0.80 ) 407( 0.70 ) 407( 0.79 ) 407( 0.68 ) 426( 0.54 )

571(5 .8)

M C

N, MC

571(6 .5)

M C

495(0. 32)

N, MC

571(9 .2)

M C

495(0. 14)

N, MC

-

N

-

N

N

N

N

-

-

-

N

-

N

𝜆𝑎𝑏𝑠

𝜆1𝑎𝑏𝑠 / 𝜆2𝑎𝑏𝑠

A S

-

467(0. 69)

MC

548(3 .9)

M C

467(0. 27)

N, MC

548(2 .0)

M C

467(0. 13)

N, MC

532(6 .4)

-

467(0. 13)

N, MC

532(0 6.4)

-

-

N

532(2 0.8)

-

-

N

532(1 8.3)

-

-

N

532(2 3.5)

-

-

N

510(6 3.6)

N

-

N

510(5 9.8)

N

-

N

510(6 9.6)

N

-

N

507(7 2.4)

M A

-

N

507(7 9.1)

M A

-

MA

507(8 0.7)

M A

-

MA

513(8 0.2)

-

468 (0.32)

MA ,DA

518(6 1.8)

-

468 (0.68)

MA ,DA

528(5 0.5)

-

399( 0.35 ) 399( 0.38 ) 399( 0.38 ) 399( 0.38 ) 399( 0.38 ) 399( 0.39 ) 399( 0.40 ) 399( 0.58 ) 399( 0.56 ) 399( 0.46 ) 399( 0.54 ) 406( 0.62 ) 406( 0.57 ) 406( 0.47 ) 406( 0.38 )

f

M C

A S

oo

-0.54

497(0. 21)

575( 5.9)

 flu

518(1 2.6)

N

518(3 8.8)

N

518(4 2.0)

N

e-

-0.95

497(0. 46)

S

𝜆𝑎𝑏𝑠

A S

518(5 9.0)

N

-

N

518(7 4.2)

N

-

N

505(5 5.7)

M A

-

N

505(4 4.5)

M A

-

MA

505(2 3.3)

M A

-

MA

505(2 2.1)

M A

-

MA

505(2 0.2)

M A

470(0. 17)

MA ,DA

510(2 3.2)

-

470(0. 26)

MA ,DA

516(2 4.5)

-

470(0. 63)

MA ,DA

532(3 8.1)

D A

Pr

-1.52

396( 0.22 ) 396( 0.29 ) 396( 0.36 ) 396( 0.27 ) 396( 0.39 ) 396( 0.27 ) 396( 0.34 ) 396( 0.38 ) 396 0.49 ) 396( 0.40 ) 396( 0.45 ) 396( 0.42 ) 396( 0.39 ) 396( 0.39 ) 396( 0.31 )

M C N, M C N, M C N, M C

c

𝜆1𝑎𝑏𝑠 / 𝜆2𝑎𝑏𝑠

al

-2.06

497(1. 02)

 flu A

rn

-3.03

A Sb

Jo u

𝜆𝑎𝑏𝑠

𝜆1𝑎𝑏𝑠 / 𝜆2𝑎𝑏𝑠 a

OH-I

pr

Ho/p H/H _

 flu

A S

-

-

470(0. 79)

DA

532(5 8.4)

D A

-

468 (0.86)

DA

535(2 8.9)

D A

-

-

470(0. 95)

DA

532(7 4.6)

D A

-

468 (0.92)

DA

535(3 1.6)

D A

-

-

470(0. 97)

DA

532(8 9.4)

D A

-

468 (0.93)

DA

535(3 5.4)

D A

M A

-

470(1. 03)

DA

532(1 16.4)

D A

-

468(0. 96)

DA

535(4 2.0)

D A

M A

-

470(1. 25)

DA

532(2 45.7)

D A

-

468(0. 81)

DA

535(1 00.6)

D A

Journal Pre-proof )

1abs = Absorption band maxima of MC; 2abs Absorption band maxima of MA.

b

Assigned species, MA = monoanion, N = neutral, MC = monocation. Excited at wavelength of isosbestic point (Table 5).

oo pr ePr al rn Jo u

c

f

a

Journal Pre-proof Table 4 Spectral characteristics of prototropic species of indole chalcone derivatives Molecules

Species

Ho/pH/H_

Wavenumber/ cm-1

Wavelength/nm λabs max

λFlu max

ν̅flu

MC N MA

-3.03 6.82 17.95

471 391 465

538 520 547

21231 25575 21505

18587 19231 18282

Me-I

MC N MA

-3.03 6.82 17.95

480 393 468

548 521 542

20833 25445 21368

18248 19194 18450

Cl-I

MC N MA

-3.03 6.82 17.95

475 407 464

540 527 556

21053 24570 21552

18519 18975 17986

OMe-I

MC N MA

-3.03 6.82 17.95

497 396 466

20121 25253 21459

17391 19342 18450

OH-I

MC N MA DA

-3.03 6.82 ~9.00 17.95

NH2-I

MC N MA DA

-3.03 6.82 ~9.00 17.95

oo

e-

pr

575 517 542 571 518 505 532

20202 25445 24570 21277

17513 19305 19802 18797

467 399 406 468

548 510 507 535

21413 25063 24631 21368

18248 19608 19724 18692

al

Pr

495 393 407 470

Jo u

rn

f

ν̅abs

I

39

Journal Pre-proof Table 5 Ground and excited states’ dissociation constants (pKa and pKa*) of different prototropic equilibria of indole chalcone derivatives Isosbestic point

Ground State dissociation Constants pKa (PT)b

MC

N

425

-2.48

-2.10

-0.56

-7.34

-11.22

-3.45

Me-I

MC

N

424

-2.38

-2.13

-0.86

-7.96

-11.81

-4.11

Cl-I

MC

N

430

-2.51

-2.79

-1.01

-6.63

-9.51

-3.75

OMe-I

MC

N

432

-2.22

-2.38

-0.14

-9.80

-13.14

-6.47

OH-I

MC

N

432

-2.14

-2.12

0.04

-9.50

-13.12

-5.88

NH2-I

MC

N

434

-2.13

-2.50

1.94

-7.76

-10.16

-5.36

I

N

MA

415

13.48

13.44

13.37

8.17

4.89

11.44

Me-I

N

MA

425

13.38

13.37

13.24

8.31

4.81

11.81

Cl-I

N

MA

432

13.55

13.53

14.57

8.80

6.14

11.45

OMe-I

N

MA

421

13.51

13.48

13.39

8.56

5.51

11.60

OH-I

N

MA

-

-

-

-

9.10

7.66

10.54

NH2-I

N

MA

-

-

-

-

9.17

8.59

9.74

OH-I

MA

DA

432

13.90

13.97

~16.80

12.52

12.13

12.90

NH2-I

MA

DA

428

13.69

~17.22

12.28

12.79

11.77

Pr

pr

oo

I

f

pKa (HH)a

13.80

Jo u

Calculated from Henderson-Hasselbalch method using Eq. (1). Calculated from the Photometric Titration (PT) method. c Calculated from Fluorimetric Titration (FT) Method. d Calculated from Förster Cycle method using Eqs. (2), (3) and (4). b

Excited State Dissociation Constants pKa* pKa* pKa* pKa* c d d (FT) (av) (abs) (flu) d

e-

 /nm

rn

a

Equilibria

al

Molecules

40

Journal Pre-proof Highlights  Indole chalcones exhibit intramolecular charge transfer (ICT) properties in ground and excited states. 

ICT was confirmed by Density Functional Theory calculations.



An ICT dependent protonation-deprotonation mechanism has been suggested.



Hydroxyl/Amino groups induce distinct spectral behavior in indole chalcones upon

Jo u

rn

al

Pr

e-

pr

oo

f

deprotonation.

41

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6ab

Figure 6c

Figure 7

Figure 8