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Design, Synthesis and Proticity Inclined Conformational Modulation in a Highly Fluorescent Bichromophoric Naphthalimide Derivative: Hint Directed from RICT Perspective” Ritika S Joshi, Oinam Romesh Meitei, Himank Kumar, Manojkumar Jadhao, and Sujit Kumar Ghosh J. Phys. Chem. A, Just Accepted Manuscript • DOI: 10.1021/acs.jpca.5b10669 • Publication Date (Web): 27 Jan 2016 Downloaded from http://pubs.acs.org on January 27, 2016

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The Journal of Physical Chemistry

Design,

Synthesis

Conformational Fluorescent

and

Proticity

Modulation

in

Bichromophoric

Inclined a

Highly

Naphthalimide

Derivative: Hint Directed from RICT Perspective Ritika Joshi, Oinam Romesh Meitei†, Himank Kumar, Manojkumar Jadhao and Sujit Kumar Ghosh*

Department of Chemistry, Visvesvaraya National Institute of Technology, Nagpur, Maharashtra, 440010, India

†

The present address of this author (O.R.M.) is as follows:

Lehrstuhl für Theoretische Chemie, Friedrich-Alexander Universität Erlangen-Nürnberg, Egerlandstraße 3, 91058 Erlangen, Germany

1 ACS Paragon Plus Environment


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ABSTRACT The present study embodies design, in silico DNA interaction, synthesis of benzothiazole containing

naphthalimide

derivative,

2-(6-chlorobenzo[d]thiazol-2-yl)-1H-benzo[de]

isoquinoline-1,3(2H)-dione (CBIQD) along with its systematic photophysics, solvatochromic behaviour and solvation dynamics using experimental and theoretical spectroscopic approach. Steady state dual emission and biexponential fluorescence decay reveals the formation of two different excited species. Ground and excited states optimised geometry and potential energy curve obtained from DFT and TD-DFT calculation ascertained existence of non-planar and planar conformation. On changing solvent polarity from nonpolar to protic polar, the feebly emissive emission band highly intensifies probably due to the reversal of n, π* - π, π* emissive state along with consequent modulation of their energy gap that is induced by Hbonding. Excluding non-polar solvents, in all other solvents stokes shift correlates linearly with orientation polarizability while in water the story remains intriguing. On photoexcitation, intermolecular H-bonding stimulates the pyramidalization tendency of imide ‘N’ with subsequent conformational change of GS non-planar geometry to a coplanar one through acceptor rehybridization generating Rehybridised Intramolecular Charge Transfer (RICT) state that caused a dramatic fluorescence upsurge. This allosteric modulation is promoted by excited state H-bonding dynamics especially in strong H-bond donor water. A close interplay between preferential solvation and proximity effect is evident in the emission behaviour in Benzene (Bn)-Ethanol (EtOH) binary mixture. Molecular docking analysis delineates considerable non-covalent sandwiched π-π stacking interactions of CBIQD with the pyrimidine rings as well as with imidazole rings of dG 6 and dG 2 base pairs of B-DNA double helix, which probably suffices the design strategy adopted. Overall a strategic design to synthesize highly fluorescent and potential bioactive agent is executed to revolutionize fluorophore field due its enormous progressive importance in biochemical applications.


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INTRODUCTION: The worth of small molecules that can simultaneously image and deliver therapeutic activity is momentous; as such an approach can provide an insight into its uptake, distribution, and therapeutic action.1–3 This harmony principally relies on the emission signals from the localized bioactive chromophores. Environmental perturbation could influence (decrease or increase) the emission quantum yield at the same wavelength or it may cause a considerable spectral shift, but both of which are extremely useful for monitoring its localization and other biological actions.4,5 In this milieu, the easy manipulation of extremely solvent sensitive 1, 8-naphthalimide core allows an exquisite control over the photophysical properties and directs the localization of the chromophores in biological systems. Naphthalimide derivatives are bestowed with numerous chemical and biochemical applications including potent photoinducible DNA-cleavers and effective intercalating agents.6,7 Some of them have also entered into clinical trials owing to their anti-cancer activity,8 while many other have found their usage as fluorescent probing of cells9 and in effective inhibition of enveloped viruses in blood and blood products.10 Although the spectroscopic and photophysical properties of N-aryl naphtalimides are fundamentally different from N-alkyl ones,11,12 a productive approach for controlling the properties of naphthalimide both in terms of modulating biological as well as photophysical properties is to extend the aromatic ring system with more easily delocalizable π-excessive or π-deficient heteroaromatics that may result in an increased intramolecular charge transfer (ICT). Among various heterocycles, benzothiazole has attractive biological, chemical and physical properties making it an appropriate building block in functionalization of chromophores.13–16 Usually, ICT arises from the presence of the electron withdrawing and donating moiety which may or may not be conjugated with a π spacer unit in the same skeleton. However, both diimide17–19 as well as benzothiazole20 are efficient π acceptors and



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the strength of diimide as an acceptor is at par with p-nitrophenyl.21 Hence it will be exciting to know how these two electron deficient units if interpolated together in a single skeleton, can affect the overall anticipated biological activity and the photophysical behavior as in: whether the acceptor characteristics of the aforesaid moieties remain restrained or become reversed. Several heterocyclic fused naphthalimide derivatives have been explored till date as potential DNA intercalator but little importance have been given to un-fused ones.22 On these premises, in the present article, we describe the design and synthesis of benzothiazole tethered 1, 8-napthalimide derivative (CBIQD), its steady state and time resolved photophysics in pure and mixed solvents so as to understand in greater detail, the molecular mechanism of the specific solvation and conformational relaxation, if any. The spectroscopic measurements in conjunction with the theoretical studies allow direct evidence regarding the structure of the ICT state and the various channels controlling the deactivation path. As far as literature reports are concerned and to the best of our knowledge, CBIQD has emerged as one of very rare representative naphthalimide derivative having enormous fluorescence in water.

EXPERIMENTAL SECTION: Materials: 1, 8-Naphthalic anhydride, 2-amino-6-chlorobenzothiazole is procured from Sigma Aldrich, USA. Analytical grade anhydrous toluene, triethylamine (Et3N), silica gel (60-120 mesh) and anhydrous sodium sulphate (Na2SO4) are purchased from SD Fine-Chem. Limited, India. The spectroscopic grade solvents cyclohexane (Cyhx), n-heptane (Hept), n-hexane (Hex), Benzene (Bn), 1,4-dioxane (Diox), tetrahydrofuran (THF), chloroform (Chloro), dichloromethane (DCM), dimethylsulfoxide (DMSO), acetonitrile (ACN), n-octanol


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(nOcOH), iso-propanol (iPrOH), n-butanol (nBuOH), ethanol (EtOH), methanol (MeOH) are purchased from Fisher Scientific, India. Millipore water is used wherever required. Instrumentation and methodology: Steady state UV-Vis absorption and emission study: JASCO V-630 Spectrophotometer with a matched pair of quartz cuvettes (path length 1 cm) and a JASCO FP-8300 Spectrofluorometer with a single 1.0 cm quartz cuvette having external slit width of 2.5 nm are used to collect absorption and fluorescence spectra, respectively. All the measurements are conducted at 298 K unless stated otherwise. Measurements are repeated in order to get reproducible results. An excitation wavelength corresponding to the absorption maxima of CBIQD in solvents is chosen and appropriate blanks are subtracted to correct the background. Time resolved fluorescence measurements: Fluorescence lifetimes are determined from time resolved intensity decay by the method of time-correlated single-photon counting using EL 340/L340 LED Driver ASSY (340 nm LED source) mounted on a PTI Pico Master TSCPC Spectrofluorometer with a PMH-100-4 detector. The lamp function is measured at the excitation wavelength of 340 nm (±10 nm) using SDS as a scatterer and 5 nm band width. The FelixGX software is used for fluorescence data collection and analysis. For all lifetime measurements the fluorescence decay curves are analyzed by single, bi and tri-exponential iterative fitting program such as: = exp −τ⁄τ

Where, is a pre-exponential factor representing fractional contribution to the time-resolved decay of the component with a lifetime , average lifetimes for biexponential decays of fluorescence are calculated from decay times and pre-exponential factors using the following equation: =



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It is pertinent to mention here that the synthesized compound CBIQD exhibits two absorption bands of comparable intensity one at 343 nm (assigned as peak a) and the other at 326 nm (assigned as peak b) in alkanes (discussed in results and discussion section). The absorption intensity of ‘peak b’ is slightly higher than ‘peak a’ as well as the former is more sensitive to the solvent polarity; hence the steady state fluorescence measurement has been performed by exciting at ‘peak b’. Time resolved fluorescence measurement should also be performed by exciting at ‘peak b’. But due to unavailability of the tunable laser source in our lab, the TCSPC experiment has been performed by exciting the solution at the nearest available laser excitation source at 340 nm (± 10 nm), which coincidentally becomes very close to the ‘peak a’ of the compound in the tested solvents. However, any probable effect in the TCSPC measurement due to these excitation wavelength differences (‘peak a’ vs. ‘peak b’) has been verified using steady state emission and excitation study. In the steady state emission measurement, the position of fluorescence emission band(s) of CBIQD (both LE & CT band, wherever existing) in respective solvents remain unaltered while exciting at either of the two peaks (‘peak a’/ ‘peak b’) independently (SI: Figure S1). This indicates the emitting species of CBIQD is excitation independent (‘peak a’/ ‘peak b’). This is further confirmed by fluorescence excitation measurement. The excitation spectra of CBIQD, when monitored independently at their respective emission maxima, show resemblance with each other (In this context it should be noted that the emission maxima that are monitored obtained by exciting ‘peak a’ or ‘peak b’ individually). Hence from the above two experiments (steady state emission and excitation measurement), any inconsistency in time resolved fluorescence measurement data due to the excitation wavelength differences (‘peak a’/ ‘peak b’) is argued to be physically implausible.


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Quantum yield measurements: The fluorescence quantum yield ( ) of CBIQD in different solvents is estimated in absence of oxygen at 298K with Anthracene as reference standard23,24 using the following relation: =

where, represents the corrected fluorescence peak area, the absorbance at the excitation wavelength, ‘’ the refractive index of the solvent used, and the subscripts “” and “” refer to standard and unknown respectively. Quantum chemical calculations: The CBIQD molecule used in the study is fully optimized with 6-31G basis set, CAM-B3LYP coulomb attenuated hybrid exchange-correlation density functional.25 Geometry optimization is obtained using Conductor like Polarizable continuum model (CPCM).26 Time Dependent Density Functional Theory (TD-DFT) is used to optimize the geometry in the first singlet excited state using the 6-31G basis set and CAM-B3LYP functional.27 Vertical excitation energies are calculated using linear response TD-DFT with above mentioned basis set and functional.27,28 The energy in S1 state is obtained by linear addition of the vertical excitation energy to the GS energy.29 Remembering the reliability of the TD-DFT results that depends on the selected exchange-correlation (xc) functional, CAMB3LYP coulomb attenuated hybrid exchange-correlation density functional is chosen because of its fair accuracy in wide range of calculations available in literature.29–32 All calculations are performed using Gaussian 09 package.33 In silico studies: The computational molecular docking studies are carried out using Linux centos 6 operating system, 64 bit Intel ®core™ i5-2500 CPU @ 3.30 GHz, 4 GB RAM using Schrödinger suite 2014 by Maestro 9.9 (Maestro, version 9.9, Schrödinger, LLC, New York, NY, 2014). Ligand and DNA preparation: The synthesized ligand, CBIQD, is sketched in Maestro 9.9 and prepared by Ligprep (Ligprep, version 2.7, Schrödinger, LLC, New York, NY, 2014) 7 ACS Paragon Plus Environment


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using OPLS (Optimized potential for liquid simulations) 2005 force field to rectify molecular geometries, retain specific chirality and to get least energy conformations required for docking. The double helical B-DNA (PDB ID:1Z3F) acting as a receptor, is retained with the crystal ligand from the Protein Data Bank, and is prepared using protein preparation wizard by removing all the water molecules, adjusting the protonation states for crystal ligand, adding explicit hydrogen atoms to the structure and optimizing H-bonding network using force field 2005. A receptor grid is generated using 1.0 van der Waals (vdW) radius scaling factor and 0.25 partial charge cut off. Molecular docking and binding energy calculation: The molecular docking is carried out by glide34 docking (Glide, version 6.0, Schrödinger, LLC, New York, NY, 2014) using extra precision (XP) scoring function, without using any constraints with a 0.80 van der Waals (vdW) radius scaling factor and 0.15 partial charge cut off. XP docking utilizes the prepared flexible ligand and the grid generated around the rigid biological target (DNA). Five poses per ligand is generated and post docking minimization is carried out. The XP scoring function briefly explains for the major potential contributors to receptor-ligand binding affinity. Its performance for a series of designed, candidate naphthalimide derivatives (NI-1 to NI-8) is readily enumerated (SI: Figure S2 & Table S1) in terms of XP Gscore, Glide score and Emodel to pick the best pose of the ligand candidate against DNA (PDB ID: 1Z3F). Synthesis and characterization of CBIQD: The reaction is carried under anhydrous conditions using oven-dried glassware’s. Column chromatography is performed using silica gel (60-120 mesh). To a mixture of 1, 8-Naphthalic anhydride (0.99087g, 5mM) and 2amino-6-chloro-benzothiazole (0.55393g, 3mM), dried toluene (11.0 ml, 0.10349M) and dried triethylamine (3.67 ml, 0.002631M) in 3:1 (v/v) is added and refluxed. The progress of the reaction is monitored as indicated by the consumption of reactants on a TLC plate. After completion of reaction, toluene is distilled off. The resulting reaction mixture is extracted


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with CH2Cl2 (2 × 25 ml). The combined organic phase is dried over anhydrous Na2SO4. After the removal of solvent in vacuo, the crude is purified by column chromatography over silica gel

using petroleum ether/ethylacetate (5.7:1.0 v/v) as eluent to afford 2-(6-

chlorobenzo[d]thiazol-2-yl)-1H-benzo[de]

isoquinoline-1,3(2H)-dione

(CBIQD)

(0.95g,

52.2%) (Scheme1). NMR spectra are recorded with Bruker Avance II 400 MHz Spectrometer on a 400 MHz for proton and on 100 MHz for carbon in CDCl3 solvents using tetramethyl silane (TMS) as the internal reference. The coupling constant (J) is reported in hertz (Hz). The mass spectrum is recorded on Waters, Q-TOF Micromass mass spectrometer. 1H NMR (CDCl3, 400 MHz) δ: 8.62 (1H, d), 8.21 (2H, d), 7.70 (1H,d), 7.75 (2H, d), 7.53 (2H, dd), 7.26 (1H, s). 13C NMR (CDCl3, 100 MHz) δ: 122.83, 126.91, 128.19, 128.80, 130.86, 131.13, 131.62, 132.48, 133.83, 164.03, 167.75. HRMS (MS ES+) m/z [M+H] 364.2550 and [(M+2) +H] + 366.1747 in the ratio 3:1 characteristic of chloro compounds.

RESULTS AND DISCUSSIONS: Design and synthesis: With the aim of synthesizing a dual therapeutic and imaging agent, we have strategically designed a bichromophoric naphthalimide conjugate on account of its potential promising pharmacological profile, ease of synthesis and excellent fluorescent properties which can monitor its uptake, in vivo localization and its interaction pattern without the use of costaining, in future. For CBIQD, the bichromophoric design is adopted by annealing “minimal intercalator” naphthalimide22 with other bioactive fluorophore, benzothiazole, assuming benzothiazole skeleton could function as sequence recognition moiety whereas the rigid planar naphthalimide core could serve as a DNA intercalator with its extended hetero-aromatic structure that may reinforce the DNA binding capacity via better stacking interactions with nucleic acid base pairs. Moreover, the absence of amino group at the 5th position of the naphthalimide core is strategically adopted to avoid N-acetylation that



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caused unpredictable toxicity of amonafide during metabolism.35 The above said design is finalized considering synthetic feasibility (Scheme 1) and on triumph of in silico molecular docking (SI: Figure S2 & Table S1) with DNA (PDB ID: 1Z3F) to ensure the possibility of its future bioactive interaction in vitro/in vivo. The one-pot synthesis of the designed molecule (CBIQD) involves a simple condensation reaction between 1,8-naphthalic anhydride and 2-amino-6-chloro-benzothiazole under refluxing conditions as discussed in the experimental section earlier. To warrant the possibility of future bioactivity of any emerging drug molecule, molecular docking exercise is becoming an important approach to elucidate the interaction and binding phenomena between potential ligand and its respective macromolecular target.36,37 The molecular docking studies of CBIQD with DNA is accomplished by Glide docking protocol of Schrӧdinger suite and the binding interaction pattern is compared with a typical intercalator Ellipticine, which is chosen for its GC specificity. It is reported that naphthalimide derivatives show higher sequence specificity towards GC base pairs.38–40 Hence from the several ligand-DNA complexes deposited in the Protein Data Bank, one structure with PDB ID 1Z3F is selected. 1Z3F, a hexamer d(CGATCG)2 complexed with Ellipticine is preferred because of its GC rich content. This B-DNA is used as a target for docking with prepared ligand, CBIQD. From the visual inspection of docking pose of CBIQD with the B-DNA 1Z3F, the bichromophoric CBIQD, takes up an orientation very similar to that of Ellipticine and hence it may act in a similar fashion as the crystal ligand does (Figure 1). The naphthalimide (NI) core of CBIQD intercalates itself by non-covalent, sandwiched π-π stacking interactions with dG 6 and dG 2 base pairs steps of the B chain and A chain respectively. The aromatic ring 1 and ring 2 of the tricycle planar NI chromophore specifically shows stacking interactions with the pyrimidine rings of dG 6 and dG 2 base pairs as well as with imidazole ring of the


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dG 2 residues of the double helix (Figure 1a and 1b). The propensity of interaction of the above “minimal intercalator” might increase as the thiazole ring of the benzothiazole (BT) fragment is also involved in stacking with the imidazole ring of dG 6 base pair leading to significant pi-electron overlap. Some part of the BT moiety protrudes out which preferably supports our design strategy where we assumed intercalation by the NI moiety and sequence recognition by BT fragment. CBIQD is inserted optimally in the Watson-Crick minihelix in the region of the large groove. The insertion of most DNA intercalator occurs into GC intercalation site41,42 as these base pairs get un-stacked easily as is the case with this bichromophoric molecule under study. The planarity of a probe molecule is an essential prerequisite for an intercalation to occur. It is pertinent to mention that the optimized ground state geometry of CBIQD is non-planar with around 90° dihedral angles between the NI and BT ring, as observed from the DFT calculation (detail discussed later on) using Gaussian 09 program as well as from the ligand preparation wizard of Schrӧdinger module (Ligprep, version 2.7, Schrӧdinger, LLC, New York, NY, 2011). Interestingly, this ground state non-planar geometry of CBIQD attains a coplanar conformation as it makes its way into the DNA groove (dihedral angle between the NI core and the hetero-aromatic BT ring in CBIQD becomes 16.2° as depicted from the docking exercise in Figure 1a. Although CBIQD is most stable with its non-planar geometry in the ground state but in presence of DNA, the π-π stacking interactions and the rotatable bond penalty favors an energetic advantage for the formation of a stable CBIQD-DNA complex whereby it is enforcing CBIQD to attain a planar geometry in the complex formed. This specific host-guest interaction may modify the physical, chemical and biological properties of the synthesized small molecule and it may result in a hindered or suppressed function of the nucleic acid under physiological conditions. The geometrical alteration described above has paved way for an opportunity to decode the photophysical behaviour of



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the synthesized molecule and this may give an insight into various dynamical processes (described in detail in the next section).

Furthermore, in order to enlighten the docking results presented above, a novel Glide XP scoring function with XP descriptors is implemented to estimate DNA-ligand binding affinities. There is a reward of -0.3 for the low molecular weight of CBIQD as it interacts with above mentioned DNA. Several such instances provide ample evidence for the success of small molecule inhibitors as DNA targeting drugs.43 Hence the molecular docking exercise described above not only shows the base pair specific interaction in the GC rich DNA but also indicates a strong interaction towards it. This indicates that the synthesized small molecule may act as an inhibitor in emerging cancer therapeutics.

Photophysics: Steady state measurements of newly synthesized CBIQD: The UV- Vis absorption spectra of CBIQD are studied in solvents spanning from lower to higher polarity regions. It is observed that both absorption and emission spectra strongly depends on solvent polarity. The synthesized CBIQD exhibits two absorption bands in the region of 300-400 nm, one at 343 nm (peak a) and the other at 326 nm (peak b) in alkanes. Increase in the solvent polarity from non-polar to protic polar, modifies the unstructured absorption band into a structured one (centered at 344 nm in water) with a positive solvatochromism of ‘peak b’ and hence it may be ascribed as a π, π* band. The band that appears at 343 nm (peak a) is possibly of n, π* type44–47 and it remains insensitive to the solvent polarity. However, this insensitivity in solvatochromism of ‘peak a’ (Figure 2a, SI: Table S2) is unusual for a pure n, π* transition. Hence a mixed contribution of π, π* transition cannot be neglected.48 Besides, the comparable molar extinction coefficient (e.g. 5.219 x 103 L.mol-1cm-1 at 331 nm vs. 4.615 x 103 L.mol1

cm-1 at 344 nm in Dioxane) or absorption intensity of both the peaks reasserts the existence


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of n, π* absorption masked by an overlapping π, π* absorption.38 The same trend of positive solvatochromism and change in band structure has been detected in dioxane-water mixtures as well. These alterations clearly indicate specific ground state interaction of the probe molecule with polar solvents. Considering the GS dipole moment as 6.39 D (obtained from DFT calculations) and the profound coplanarity between the imide N and π system of naphthalene ring, the probe molecule is ought to form H-bond with carbonyl oxygen which is manifested as red shifted

#!$ !"

band in water.

CBIQD exhibits dual emission while exciting at ‘peak b’ in respective solvents. In alkanes, a feebly emissive shorter wavelength emission band around 367 nm credited as LE or FC state gradually intensifies without any appreciable shift up to MeOH but in water it is 16 nm red shifted (367 nm to 383 nm). The plausible cause of rise in intensity could be reversal of the emissive n, π* and π, π* states due to solvent proximity effect49,50 (discussed later on). Furthermore, a long wavelength fluorescence band that shows a substantial 23 nm red shift in

#!$ %#

(from 374 nm in dioxane to 397 nm in water) and tremendous fluorescence

amplification especially in water (Figure 2b; Inset, SI: Table S2) is correlated with the formation of a new emitting ICT species. In water, the lack of mirror symmetry of the absorption and emission spectra point towards a different geometry of the emitting state, i.e., to the presence of a possible completely planar ICT excited state.

Reckoning geometry optimizations and potential energy curve (PEC) for photophysical proposition: The optimized geometry obtained from DFT and TD-DFT quantum chemical calculations27,28 shows that CBIQD adopts a non-planar geometry in the GS wherein the naphthalimide (NI) ring lies perpendicular to the benzothiazole (BT) ring (θ = 90.62°) (Figure 3b) whereas in the ES it exhibits a planar conformation (θ = 179.98°) (Figure 3a). The GS non-planar orientation of these NI and BT rings could be due to electrostatic repulsion of the



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diffuse electron cloud of thiazole sulphur and carbonyl oxygen. TD-DFT predicts that the most probable transition of CBIQD at 326 nm in heptane is likely to be S0S1 transition. The potential energy curve (PEC) that is obtained as a function of change in dihedral angle (Figure 3c) illustrates the existence of non-planar orientation of CBIQD in S0 (θ = 90°) state, while a planar conformation in S1 state (θ = 180°), as the most stable structures. Moreover the non-existence of energy barrier for these two states in PEC indicates that the above conformational change does not require any additional supply of energy. The coplanar arrangement of the probe molecule in the ES is expected to favor ICT process.51 Hence it becomes reasonable to assume S1 to be an ICT excited state as reflected in the fluorescence decay analysis discussed later on. However, in view of the geometry optimizations in GS and ES, LE state is believed to possess a slight variation of geometry from that of the GS with ascending polarity of solvents. Such geometric alterations of probe molecule are highly interesting particularly for its possible influence in the solvatochromic properties by virtue of its H-bonding ability.

Quantification of dipolar asymmetry: Generally, Stokes’ shift (&'̅ can be correlated with ∆) values of the solvents using the following Lippert-Mataga equation: '̅! − '̅ =

*+ ,*- ./! 0

∈,3

4 ,3

1∈3 − 4 35+constant

where, &'̅ is the Stokes shift, μ% and µg are ground and excited state dipole moments, 9 is the velocity of light, ℎ is the Planck’s constant, ‘;’ is the Onsager cavity radius swept out by the fluorophore. However, a non-linear Lippert Plot (Figure 4) obtained, is regarded as an evidence of specific solvent effects in the spectral shifts of CBIQD for the medium and higher solvents studied. Hence, efforts are made to establish a relationship between '̅!" , ' => and orientation polarizability Δ) (Figure 2c). In NP solvents both '̅!" and '̅> values are deviated toward higher energies, for protic polar solvents like alcohols; the '̅!" data points 14 ACS Paragon Plus Environment

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are marginally deviated while '̅> values are markedly lower and follow linear correlation with Δ) as compared to aprotic polar solvents. This lowering of '̅!" and '̅> values can be attributed to the specific intermolecular interaction between CBIQD and solvent molecules which causes an extra stabilization for both GS and ES of the dye. However for water, the isolated '̅!" and '̅> having further lower values indicates a much greater stabilization of the GS as well as ES relative to other solvents. This implies that the FC and the subsequent relaxed states are not identical and it becomes moderately polar (as compared to FC), which is reflected in time resolved measurements also. Thus, it is assumed that the emissive state (LE) is not attained by the direct excitation in solvents except alkanes.

Solvation and solvation dynamics: Interplay between preferential solvation and proximity effect: To rationalize the specific effect of polarity and / H-bonding, the emission characteristics, Δ'̅ and fluorescence intensity, of CBIQD are monitored in various Bn–EtOH and Bn–ACN binary solvent mixtures independently [Figure 5(a & b)]. The addition of ACN and EtOH to the Bn solution of probe molecule alters the fluorescence emission differently such that, even in presence of small amounts (5%) of polar solvents, the

%# #!$

respectively. However, the positive drift of

shows 2 nm and 5 nm bathochromic shift

%# #!$

from 378 nm to 381 nm and 379 nm to 390

nm is continued up to 50% ACN and 65% EtOH respectively and interestingly from there hypsochromism dominates for both the cases. On the other hand, addition of more polar solvent (ACN/EtOH) in Bn solution leads to fluorescence enrichment up to 60% ACN and 65% EtOH and then decreases noticeably. The above predispositions can be accounted in terms of (a) prefential solvation52,53 and (b) proximity effect.54 The observed bathochromic shift corresponds to an increase in ET(30) in the immediate vicinity of the probe molecule due to strong preferential solvation by the more polar component (ACN or EtOH) of solvent 15 ACS Paragon Plus Environment


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∘ mixture leading to more negative Gibbs energy of solvation ∆AB>C . Notably, the

fluorescence enhancement in Bn-ACN mixture is not that much pronounced as compared to Bn-EtOH mixtures and is likely due to polarity change. While in the latter case, the appearance of a small plateau (Figure 5b) is a strong evidence of specific solvation due to H-bonding. Under the influence of H-bonding of EtOH, the n, π* state of CBIQD is destabilized, whereas the emissive π, π* state is stabilized such that electronic energy gap reaches a maximum at 65% alcoholic composition. This phenomenon diminishes n, π*- π, π* vibronic coupling (as opposed to the proximity effect), leading to fluorescence enhancement but after this juncture decrease in intensity could be due to alteration of intermolecular H-bonding between solute and solvent by the intramolecular H-bonded network pre-existing in EtOH. In higher alcoholic concentration (>65%), the microenvironment around the probe mainly consists of associated alcoholic clusters owing to its self-aggregation and thereby reducing H-bond ability of bare EtOH molecules. However, it is pertinent to mention that even though the fluorescence enhancement and red drift of emission maxima in both the solvent mixtures individually has reached at its highest point respectively but they do not match with the emission maxima values in pure ACN and EtOH solvents as observed in the solvatochromism studies. This is probably because of the difference in the solvation pattern or dynamics in the cybotactic region of the probe molecule.

Conformational changes modulated by excited state H-bonding dynamics (ESHBD): The allosteric modulation that is typically used for the conformational change in protein has been adopted exclusively for CBIQD as the non-covalent H-bonding interaction in the ES results in a significant conformational change through acceptor rehybridization with increase in solvent polarity. CBIQD comprises of Donor (BT) and Acceptor (NI) units linked by a N-C single bond (visualized from quantum chemical calculations), where very limited


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degrees of freedom exists and hence internal rotation around the central bond seems to be the first choice of vibrational mode of relaxation involving a change in the electronic structure of the ES.55 As there is a high degree of co-planarity within the NI motif, it is apt to form Hbond with protic polar solvents in GS itself, owing to specific sites possessing high bond dipoles which increase its (NI) electron withdrawing capacity.56–58 In water, the solvent molecule forms H-bond with the carbonyl oxygen of CBIQD both in GS and ES. As a consequence, the NI moiety becomes electron deficient (as compared to its non H-bonded form). This electron deficiency of the NI moiety prompts the photoinduced intramolecular charge transfer (from BT moiety) to occur. Now, as the ICT from BT to NI goes on increasing, it eventually increases the electron density on imide N which is a crucial part of the acceptor (NI) and the N-C bond that connects NI and BT fragments. This urges an increase in the pyramidalization tendency of imide N resulting in a rehybridised intramolecular charge transfer state; so that there is a greater overlap of the respective atomic orbitals of the BT and NI fragments which further accelerates the ICT process. Hence, the Hbonding with the acceptor in the GS is a prior requirement for the formation of ICT state which not only induces but also stimulates the ICT process in water. For this planar conformation of CBIQD that is formed in water due to the formation of H-bonding and subsequent acceptor rehybridization in the ES, the term Rehybridization by Intramolecular Charge Transfer (RICT) is hereby suggested. The pre-existing H-bond is strengthened in tandem with arising ICT state so that majority of the molecules become H-bonded in water. Thus a dynamic imbalance is established such that the highly polar and fully H-bonded coplanar RICT state becomes the highly populated emissive state, as opposed to most of the TICT probes cited in literature,59,60 while the less polar, non-planar conformation is dominated by the former and the molecule exhibits dual emission from both these states. Contrary to this phenomena, in all other solvents (except water) reverse situation survives.



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Here, we presume that the non-planar ICT state of CBIQD is governing the spectral features over the planar ICT state. The prime difference is the extent of charge transfer that is promoted as a function of the change in the dihedral angle between the two rings (90°

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