Host-guest interaction of 3-hydroxyflavone and 7-hydroxyflavone with cucurbit [7]uril: A spectroscopic and calorimetric approach

Accepted Manuscript Host-guest interaction of 3-hydroxyflavone and 7-hydroxyflavone with cucurbit [7]uril: A spectroscopic and calorimetric approach

Sayeed Ashique Ahmed, Banibrata Maity, Soma Seth Duley, Debabrata Seth PII: DOI: Reference:

S1011-1344(16)30961-7 doi: 10.1016/j.jphotobiol.2017.02.006 JPB 10733

To appear in:

Journal of Photochemistry & Photobiology, B: Biology

Received date: Revised date: Accepted date:

26 October 2016 1 January 2017 7 February 2017

Please cite this article as: Sayeed Ashique Ahmed, Banibrata Maity, Soma Seth Duley, Debabrata Seth , Host-guest interaction of 3-hydroxyflavone and 7-hydroxyflavone with cucurbit [7]uril: A spectroscopic and calorimetric approach. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Jpb(2017), doi: 10.1016/j.jphotobiol.2017.02.006

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ACCEPTED MANUSCRIPT Host-Guest Interaction of 3-Hydroxyflavone and 7Hydroxyflavone with Cucurbit[7]uril: A Spectroscopic and Calorimetric Approach Sayeed Ashique Ahmeda, Banibrata Maitya, Soma Seth (Duley)b and Debabrata Seth*a Department of Chemistry, Indian Institute of Technology Patna, Patna-801103, Bihar, India

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a

Department of Chemistry, Nabadwip Vidyasagar College, West Bengal, India Abstract

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b

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E-mail: [email protected]; Fax: 91-612- 3028028

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The modulation of photophysical behaviour of small organic molecules in the presence of macrocycles is one of the most interesting areas of research. In this work we reported the interaction of two biologically active molecules 3-hydroxyflavone and 7-hydroxyflavone

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with macrocyclic host cucurbit[7]uril in aqueous medium. To investigate the change of photophysical properties of these two flavones, we have used steady state absorption,

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fluorescence, time resolved fluorescence emission spectroscopy and isothermal titration calorimetric technique. It is observed that on complexation with cucurbit[7]uril, the excited

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state proton transfer processes in both flavones have been facilitated. Isothermal titration calorimetric method was used in order to investigate the involvement of thermodynamic parameters in complexation between flavone with cucurbit[7]uril. The changes in

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thermodynamic properties due to the complexation of the flavones molecules with cucurbit[7]urils help to understand about the governing parameters involved in this

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complexation. The inclusion of flavone molecules inside the cavity of cucurbit[7]uril molecules was studied theoretically to decipher the molecular orientation of flavones in the presence of cucurbit[7]uril. The structure of HOMO and LUMO of the complexes between cucurbit[7]uril with flavones was reported. This study will be helpful to get the knowledge about the modulation of photophysical properties of the flavones molecules on addition of macrocyclic host cucurbit[7]urils. This study will be helpful for the use of cucurbit[7]urils as a potential drug delivery system. Keywords: 3-hydroxyflavone, 7-hydroxyflavone, cucurbit[7]uril, supramolecular interaction, ITC.

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ACCEPTED MANUSCRIPT 1. Introduction: Flavones and related compounds of this group are widely spread in plants [1]. These compounds are very much important to the survival of plants in the natural environment [2,3]. Flavonoids are the well known molecular systems which exhibits excited state proton transfer (ESPT) along with dual fluorescence behaviour [4]. Hence, flavones can be used as powerful tool for deeper understanding about the mechanistic studies on ESPT and related

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photo-physical prospects in a systematic way. The photo-physical properties of these compounds are highly influenced by the surrounding microenvironment, which indicate that

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flavones can be used as a fluorescent molecular probe [5]. Importance of these compounds are not restrict to plants as they have strong tendency to absorb in the UV region, they can

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protect the photosensitive biological targets such as DNA, proteins, lipids, etc [6]. Flavones and the compounds of flavonoid group show a broad range of therapeutic properties such as

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reducing agents, hydrogen-donating antioxidants, active against cancers, tumors, allergies, cardiac problems, inflammation, AIDS, etc. and having low systemic toxicity [8-11]. In 1936,

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Szent-Gÿorgyi first brought the therapeutic activity of flavonoids in limelight [12]. Although various bioactive flavonoids have novel therapeutic activity, their applications are often limited due to weak water solubility. One of the ways to overcome the solubility problem in

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aqueous medium of flavonoids is by encapsulation of flavone in various encapsulating agent

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like cucurbit[n]urils, cyclodextrins, calixarenes, etc. From several decades the encapsulation of small organic drug molecule with macrocyclic host is one of the interesting areas of research. The changes in photophysical properties due to the complex formation can be

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investigated in a systematic way by using different spectroscopic techniques and the changes of thermodynamic parameters on complexation of small organic molecules with macrocyclic

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hosts can be clearly observed by using the isothermal titration calorimetry technique. The most frequently used macrocycles are cucurbit[n]urils, cyclodextrins, calixarenes, etc. [1328]. Recently, cucurbit[n]urils are received increasing attention among all macrocyclic hosts. It has enormous benefits over other macrocyclic hosts because of its unique molecular reorganization and high-binding affinity towards guest molecule. Cucurbit[n]urils are used in chemosensing, biochemical and medicinal purposes, etc. The structure of cucurbit[n]urils is highly symmetric in nature and it consist a hydrophobic cavity along with carbonyl rims having negative charge density on both side of the cavity [13]. In our present study, we have chosen highly water soluble macrocyclic host cucurbit[7]urils (CB7). Pumpkin shaped CB7 has seven glycoluril units, which are connected by a pair of methylene bridges. CB7 has a 2

ACCEPTED MANUSCRIPT portal diameter of 5.4 Å, height 9.1 Å and cavity diameter of 7.3 Å [29]. CB7 provides additional stability to the guest molecule as compared to the other macrocyclic host due to the presence of carbonyl portal ends. It provides charge-dipole or dipole-dipole interaction to the guest molecule; in addition to the usual hydrophobic interaction [13]. Spectroscopic techniques are very useful in order to investigate the changes in photophysical properties of dye molecules on complexation with macrocyclic hosts. In this present work we have studied

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the photophysical properties of 3-hydroxyflavone (3-HF) and 7-hydroxyflavone (7-HF) in presence of CB7 by using different spectroscopic techniques along with isothermal titration calorimetry (ITC) technique. The different forms of 3-HF and 7-HF which are exists within

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the medium are shown in scheme 1 and scheme 2. The effect of macrocyclic host on the

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photophysical properties of 3-HF and 7-HF has been investigated and the effect of the different forms of 3-HF and 7-HF on addition of highly water soluble macrocyclic host CB7

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was reported.

O

* O

OH

[Normal form]

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H+ -H

O

OH O

[Normal form]

ESPT

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O

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hv

* O

O O

[Anionic form]

O O

H

[Tautomer form]

Scheme 1. Normal, tautomeric, and anionic forms of 3-HF.

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ACCEPTED MANUSCRIPT HO

O

HO

O

hv

[Normal form]

[Normal form]

ESPT

O

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O

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H+ -H

O

*

O

O

O

O

OH

[Tautomer form]

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[Anionic form]

*

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Scheme 2. Normal, tautomeric, and anionic forms of 7-HF.

2. Materials, methods and instrumentation:

Cucurbit[7]urils (CB7), 3-hydroxyflavone (3-HF) and 7-hydroxyflavone (7-HF) were

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purchased from Sigma-Aldrich and used as received. Ground state absorption spectra were

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collected with the help of UV–Vis spectrophotometer (Model: UV-2550, Shimadzu). The steady-state fluorescence emission measurements were recorded with the help of Fluoromax4P spectrofluorometer (Horiba Jobin Yvon). Absorption and fluorescence measurement were

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performed by using quartz cuvette having a path length of 1 cm. The time resolved fluorescence emission measurements were carried out by using a time-correlated single-

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photon counting (TCSPC) technique using a time-resolved fluorescence spectrophotometer from Edinburgh Instruments (model: LifeSpec-II, U.K.). The time resolved fluorescence emission decays were collected by using a picoseconds diode laser with excitation wavelength at 375 nm and a light emitting diode with excitation wavelength 340 nm. The fluorescence transients were detected at magic angle (54.7°) polarization using a Hamamatsu MCP PMT (3809U) detector. The decays were analysed by using F-900 decay analysis software. The fluorescence emission decays were fitted by using the following multiexponential functions N

I (t )  A   Bi exp(t /  i )

(1)

i 1

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ACCEPTED MANUSCRIPT The parameter Bi and A represents the pre-exponential factor along with the characteristic lifetimes i and the background. The temperature was kept constant at 298 K throughout the experiment by using a peltier-controlled cuvette holder from Quantum Northwest (Model: TLC-50). Isothermal titration calorimetry measurements were performed with the help of iTC200 microcalorimeter from GE healthcare and during the measurements the cell temperature was kept constant at 298 K. Freshly prepared solutions were used throughout the

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measurements. Triple distilled water was used to prepare all solutions.

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3. Results and Discussion: 3.1 Ground state absorption studies:

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The aqueous solution of 3-HF (concentration: ~4.5× 10-6 (M)) shows a broad band with maximum at ~340 nm with a hump at ~300 nm [30]. The aqueous solution of 7-HF

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(concentration: ~4.5× 10-6 (M)) shows absorption maxima at ~ 311 nm (due to the neutral form of 7-HF) (Fig. 1) [31]. On addition of 3.8 × 10-4 (M) CB7 the changes in absorption

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spectra of 3-HF and 7-HF were shown in Fig.1. But with incremental addition of CB7 in the solution of 3-HF and 7-HF the observed change in absorbance values were irregular. Hence, we were unable to find out the value of binding constant from absorption data but changes

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observed in absorption spectra (Fig.1) may be due to interaction between 3-HF and 7-HF

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with CB7, respectively.

Fig. 1. The absorption spectra of (a) 3-HF and (b) 7-HF with varying concentration of CB7.

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ACCEPTED MANUSCRIPT 3.2 Fluorescence emission measurements: Aqueous solution of 3-HF exhibits dual emission maxima at ~418 nm and ~510 nm (λex = 340 nm) and these emission bands are due to normal and tautomeric band of 3-HF, respectively (Fig. 2(a)) [4,32]. In our study we are more interested on the fluorescence emission maxima at 510 nm. The more intense band at 510 nm originates from ESPT tautomer species and it indicates the presence of tautomeric forms of 3-HF in aqueous

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solution. It is very interesting that on addition of 3.8×10-4 (M) CB7 to the aqueous solution of 3-HF fluorescence intensity increased as compare to aqueous solution of 3-HF. It indicates

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that interaction takes place between 3-HF and CB7. The increase of fluorescence intensity of tautomer form (due to ESPT) on addition of CB7 is due to the presence of 3-HF in

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hydrophobic environment compared to polar bulk medium. Due to formation of the complex between 3-HF and CB7 the solvent mediated perturbation decreased [32]. Due to

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encapsulation of 3-HF inside the hydrophobic cavity of CB7 enhanced the stability of intramolecular hydrogen bonding which facilitate the ESPT process. The enhancement of the

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normal emission band (~418 nm) of 3-HF is due to decrease of various non-radiative processes. The aqueous solution of 7-HF shows broad emission band at ~536 nm due to the neutral and anionic form of 7-HF (λexc =340 nm) as shown in Fig. 2(b) [31]. This green

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fluorescence emission band arises due to excited state proton transfer. The fluorescence

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intensity increases on gradual addition of CB7 to the aqueous solution of 7-HF and also emission maxima blue shifted by 22 nm. This shift in fluorescence band clearly demonstrated that 7-HF present in hydrophobic environment as compared to the bulk solution and the

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solvent mediated perturbation is reduced due to the formation of 7-HF-CB7 complex. For this reason the fluorescence intensity gradually increases on addition of CB7. Now comparing the

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structural effect of the studied flavones on the complexation, the hydroxyl group present at three position of 3-HF can form five member ring on association with the carbonyl group present within the molecule due to internal tautomerisation and the charge is eliminated for which both forms of 3-HF can form complex with CB7 and the complexation is facilitated. Such type of ring formation is not possible in 7-HF for which only one form can form complex with CB7. The excitation spectra of 3-HF and 7-HF with and without CB7 have been shown in Fig. 3.

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Fig. 2. The emission spectral profiles of (a) 3-HF and (b) 7-HF with varying concentration of

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CB7 (λexc =340 nm).

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Fig. 3. The excitation spectral profiles of (a) 3-HF in absence and in presence of CB7 and (b) 3-HF in absence and in presence of CB7.

3.3 Determination of the binding constants and stoichiometry: To describe the complex formation between flavones with macrocyclic host, the following equilibrium has been considered

G+H

G:H

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ACCEPTED MANUSCRIPT where, G and H are the guest and host molecule, respectively. We are unable to get the binding constant value of 3-HF and 7-HF with macrocyclic host CB7 in ground state from absorbance spectra could not be obtained due to irregular pattern observed on gradual addition of CB7 to the aqueous solution of respective flavones. The binding constant value of the host-guest complex was frequently estimated from the changes of fluorescence spectra. The binding constant value (K1) of flavone complexes was estimated by using the nonlinear

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least-squares regression analysis, where the data are directly fitted by using the relevant equation. The equation 2 was used to estimate the binding constant of the 1:1 (flavone : CB7)

F water  F max K 1 [CB7] 1  K 1 [CB7]

(2)

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F 

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complexes:

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where, Fwater represents the fluorescence intensities of the flavone in aqueous medium (in absence of CB7). Fmax and F are the fluorescence intensity in presence of maximum concentrations of CB7 where 1:1 binding has been completed and the fluorescence intensities

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at different concentrations of CB7, respectively. The term [CB7] represents concentration of CB7 within the medium. By using the fluorescence data, it was observed that for both

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flavones 1:1 complex is formed with macrocyclic host CB7. We obtained well fitted curve for both 3-HF and 7-HF with CB7 having good correlation coefficients, R2 value 0.980 and

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0.994, respectively shown in Fig. 4. The binding constant values of 3-HF and 7-HF with macrocyclic host CB7 are found to be 2710 (± 230) M-1 and 23125 (± 1905) M-1,

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respectively.

Fig. 4. (a) The binding interaction between 3-HF with CB7 forming 1:1 complex and (b) the binding interaction between 7-HF with CB7 forming 1:1 complex. These plots were obtained from fluorescence data. 8

ACCEPTED MANUSCRIPT 3.4 Determination of the complex stoichiometry by Job’s plot: One of the most well known method to determine the stoichiometry of the host-guest complex is the Job’s method of continuous variation, where a series of solutions were prepared in which the total concentration {[Host]+[Guest]} of the solution was kept constant and varying the [Host] and [Guest] concentration. To perform the Job’s method of continuous variation for our studied systems, the concentration of the solution {[Dye] + [CB7]} was

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maintained at 1×10-5 M for both systems. Here the plot of ∆F.X3-HF {(difference of fluorescence intensity) × (mole fraction of the 3-HF)} against X3-HF mole fraction of 3-HF

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show maxima at ~0.55 and the plot of ∆F.X7-HF {(difference of fluorescence intensity) × (mole fraction of the 7-HF)} against X7-HF mole fraction of the 7-HF show maxima ~0.50

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(Fig. 5). From Job’s method we confirmed that 1:1 complex is formed between 3-HF and 7-

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HF with macrocyclic host CB7, respectively.

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Fig. 5. Job’s plot by continuous variation method for (a) 3-HF in presence of CB7 (b) 7-HF

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in presence of CB7.

3.5 Time resolved emission measurement: Time resolved fluorescence measurements serve a very sensitive indicator to investigate the effect of surrounding environment on the fluorescence molecule. Here, the measurement was performed to examine the effect of confinement in the cavity CB7 on the tautomer emission decay kinetics of 3-HF and 7-HF, respectively. The decay parameters are shown in Table 1. The emission decay of 3-HF in water was fitted by bi-exponential function (λex= 375nm, λem = 510 nm) and two components are observed with time constant value 0.17 ns (99%) along with a relatively small contribution from a long lived decay component 4.78 ns (1%) shown

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ACCEPTED MANUSCRIPT in Fig. 6(a). On addition of 3.8 ×10-4 (M) CB7 to the aqueous solution 3-HF, the emission decay was fitted by tri-exponential function with time constant value of 0.13 ns (29%), 1.39 ns (69%) and 8.89 ns (2%) (λex= 340, λem= 418 nm) shown in Fig. 6(d). Whereas, at λem = 510 nm (λex= 340) the emission decay profile was fitted by tri-exponential function with time constant value 0.1 ns (76%), 3.11 ns (4%) and 8.86 ns (20%) (Fig. 6(e). When, λex = 375 nm, on addition of 3.8 ×10-4 (M) CB7 to the aqueous solution 3-HF, the emission decay profile was fitted by tri-exponential function with time constant value 0.18 ns (77%), 2.51 ns (9%)

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and 7.96 ns (14%) (when λem = 418 nm) and the emission decay profile was fitted by triexponential function with time constant value 0.11 ns (22%), 1.42 ns (77%) and 4.90 ns (1%)

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when λem = 510 nm (Fig. 6(a)). The emission wavelengths for λex= 340 nm are 418 nm and

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510 nm and the emission wavelengths for λex= 375nm were taken at 418 nm and 510 nm (emission maximum for aqueous medium) and it is observed that on addition of 4.5×10-4 (M)

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CB7 to the aqueous solution of 3-HF the average life time value of 3-HF was increased (Table 1). Sengupta and co-worker observed three components for the aqueous solution of 3HF on addition of γ-cyclodextrin. They described that the components arise from populations

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differing in the extent of H-bonding of 3-HF with the microenvironment [33]. In our study, we also observed tri-exponential decay of 3-HF on addition of CB7 and we are presuming

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that the tri-exponential decay of 3-HF observed due to different extent of H-bonding within the medium. The decrease in population of the fast component on addition of macrocyclic

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host CB7 to the aqueous solution of 3-HF may be due to decrease of non-radiative decay processes. The increase in average life time value of 3-HF on addition of CB7 is due to supramolecular complex formation.

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The emission decay of 7-HF was fitted by bi-exponential function ( λex = 375 nm), one is fast component with time constant of 0.18 ns (99.7%) another one long lived component with

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time constant of 5.55 ns (0.3%) when λem = 536 nm (Fig. 6(b)). These components arise may be due to the different form of 7-HF exist within the medium. On addition of 4.5 × 10-4 (M) CB7 to the aqueous solution of 7-HF, the emission decay profile fitted by tri-exponential function having three distinct components with time constant values 0.49 ns (76%), 4.08 ns (21%) and 13.87 ns (3%) (Fig. 6(c)) (λex= 375, λem= 514nm). The emission decay profile was fitted by tri-exponential function having three distinct components with time constant values 0.45 ns (57%), 1.22 ns (42%) and 8.87 ns (1%) (Fig. 6(f)) (λex=340, λem=514nm). Such multiexponential decay may be observed due to the presence of heterogeneity of 7-HF in the micro-environments. The average life time value of 7-HF is found to be increased due to addition of CB7. The enhancement of fluorescence life time is due to the reduction of non10

ACCEPTED MANUSCRIPT radiative decay processes and hydrophobic effect due to supramolecular complex formation of 7-HF with CB7. For 7-HF such heterogeneous fluorescence decay also suggested that

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different extent of H-bonding of 7-HF within the aqueous medium on addition CB7.

Fig. 6. The fluorescence emission decays of (a) 3-HF in presence and absence of CB7 and (b,c) 7-HF in absence and presence of CB7 (λex= 375 nm). The fluorescence emission decays

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of (d, e) 3-HF and (f) 7-HF in presence and absence of CB7 (λex= 340 nm).

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ACCEPTED MANUSCRIPT Table 1: The fluorescence lifetime values of 3-HF (4.5 ×10-6 (M)) and 7-HF (4.5 ×10-6 (M))

Sr.

System

λex

λem

τ1

(nm)

(nm)

(ns)

(ns)

375

510

0.17 0.99

4.78

0.01

375

510

0.11 0.22

1.42

375

418

0.18 0.77

2.51

340

510

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in the presence of CB7.

τ2

0.10 0.76

340

418

a1

τ3

a2

a3

⟨τ⟩c

χ2

No.

CB7(0. 38mM)

CB7(0. 38mM)

1.121

0.09

7.96

0.14

1.48

1.103

3.11

0.04

8.86

0.20

1.97

1.097

0.13 0.29

1.39

0.69

8.89

0.02

1.18

1.116

0.18 0.997

5.55

0.003

-

-

0.20

1.102

0.49 0.76

4.08

0.21

13.87 0.03

1.65

1.350

0.45 0.57

1.22

0. 42

8.87

0.50

1.205

CB7(0. 38mM) 7-HF in water

375

536

7

7-HF+

375

514

CB7(0. 38mM) 340

CB7(0. 38mM)

514

0.01

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7-HF+

⟨τ⟩c = a1× τ1 + a2×τ2 + a3×τ3

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3-HF+ 5

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1.17

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CB7(0. 38mM)

1.025

0.01

3-HF+ 4

0.22

4.90

3-HF+ 3

-

0.77

3-HF+ 2

-

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3-HF in water

(ns)

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1

(ns)

3.6 Study of binding thermodynamics of 3-HF and 7-HF with CB7 using Isothermal titration calorimetric measurements: Isothermal titration calorimetric method is the useful method in order to investigate the involvement of thermodynamic parameters in complexation between guest and macrocyclic host molecule and to know the stoichiometry of the host-gust complex. It is the direct method where the changes of heat are measured due to complex formation between dyes with macrocyclic host at constant temperature. The equilibrium involving the complexation between guest and macrocyclic host can be represented as follows:

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ACCEPTED MANUSCRIPT m(Dye) + n(Macrocycle)  (Dyem.Macrocylen) where, m and n may be 1, 2,3 etc.. It is observed from ITC measurement that both flavones form 1:1 complex with CB7. Here the data obtained from isotherm was fitted by one set of sites binding model for both flavones (Fig.6). The binding constant value (K) of 3-HF and 7-HF with CB7 are 2.1(  0.07) × 102 M-1 and 8.05(  2.38) × 103 M-1, respectively (Table 2). On comparing the binding constant values

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obtained from ITC with fluorescence, it is observed that the binding constant values obtained from ITC are significantly different from that obtained from fluorescence measurement. This

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difference is observed because data obtained from ITC measurement describe the ground

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state complex formation phenomenon of flavone with CB7 and hence the binding constant value obtained from this measurement represents the binding interaction of the guest with macrocyclic host in the ground state. Whereas, the binding constant values obtained from

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fluorescence technique depict the binding interaction in the excited state of the guest molecule. The fluorescence measurement and ITC measurement shows the similar value of

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binding constant if the association involve two-state transition between free and bound molecules by following a lock and key or the rigid body mechanism and if the spectroscopic signal change flashes the total population of free and bound molecules [32]. On comparing

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the binding constant value obtained from fluorescence data and ITC measurement, it is

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observed that binding constant values obtained from fluorescence measurement are higher than the binding constant value obtained from ITC measurement. It indicates that the binding strength in ground state is weaker as compared to the binding strength in excited state for

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both 3-HF and 7-HF. The changes of enthalpy and entropy values are found to be positive for both flavone and also observed that both the processes are endothermic in nature (Table 2 and

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Fig. 7). From these data it is clear that both complexation processes are spontaneous as (∆G<0) and both complexation processes are entropy driven processes because of a major contribution from enthalpy ( H < TS ). The Gibb’s free energy value are -3.42 kcal.mol-1 (-0.81 kJ.mol-1) and -5.33 kcal.mol-1(-1.27 kJ.mol-1) for 3-HF and 7-HF with CB7, respectively. The complexation process of flavones with CB7 is entropically favourable but not enthalpically. It is observed that the sign of enthalpy change and entropy change were found to be positive for both flavones. According to Ross and Subramanian when the sign of enthalpy change and entropy changes are positive, it represents that hydrophobic association takes place [35]. In our system hydrophobic interaction takes place between flavone and CB7 where the potential withdrawal of more hydrophobic part of the flavone from bulk solution 13

ACCEPTED MANUSCRIPT inside the hydrophobic cavity of CB7 takes place. The value of ∆S is found to be positive for both flavones may be due to the release of high energy water molecules from the cavity of CB7 on penetration of the dye molecule inside the hydrophobic cavity of CB7 [36]. It provides significant entropy gain in terms of orientational and translational diffusion. Here release of high energy water molecules from hydrophobic cavity of CB7 acts as the driving

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force for the host-guest formation between dye and CB7.

Fig. 7. (a) ITC isotherm for the injection of 4.3 mM CB7 solution in 8.6 μM 3-HF solution at 298 K and data points represent integrated heats of interaction as a function of molar ratio

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and the solid line represents the line of best fit. (b) ITC isotherm for the injection of 4.3 mM CB7 solutions in 8.6 μM 7-HF solutions at 298 K and data points represent integrated heats

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of interaction as a function of molar ratio and the solid line represents the line of best fit.

Table 2: The binding constant values for the binding interaction of 3-HF with CB7 and 7-HF with CB7.

System

K1:1 (M-1)

∆H (kJ.mol-1)

T∆S (kJ.mol-1)

∆G (kJ.mol-1) 14

ACCEPTED MANUSCRIPT 3-HF + CB7

210 (±8)

90.71 (±2.97)

91.53

-0.82

7-HF + CB7

8.05 (±2.38) × 103

0.12 (±0.002)

1.38

-1.26

O

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HO

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Based on the above study the probable structures of the complexes are shown below:

O

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OH O

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(a)

O

(b)

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Fig. 8. The probable structure of the complexes (a) 3HF-CB7; (b) 7HF-CB7.

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3.7 Excited State Vs Ground State Guest/Host Association: The binding constant values of 3-HF and 7-HF with CB7 obtained form ITC measurements are different from the binding constant value obtained from fluorescence measurement to

considered;

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explain such difference the following factors are very much important and must be

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(a) The inclusion rate of the guest inside the cavity of macrocycle. (b) The exclusion rate of the guest from the cavity of the macrocycle. (c) The decay rate of the excited state of the probe. Now considering the short lifetime the decay rate must be faster than the rate of inclusion of the guest molecule inside the cavity of the host molecule or the rate of exclusion of the guest molecule from the cavity of the host molecule. Under this situation the guest molecules which remain inside the cavity of macrocyclic will absorb light to remain in excited state. Under such conditions the rate of exclusion, the rate of inclusion and the host-guest binding interaction will depend on the ground state binding interaction. Again the electronic excitation of the guest by the light absorption may also destabilize the complex for which the 15

ACCEPTED MANUSCRIPT ground state binding constant values are different than the binding constant value in excited state. For long lifetime in excited state, the guest molecules present outside the cavity of macrocylic host may enter inside the cavity of the host molecule during the excited state lifetime period. For which the rate of inclusion and the rate of exclusion in excited state can notably differ from the ground state. For such reason the ground state binding constant value expected to be different from the excited state binding constant value. The binding constant

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value obtained from fluorescence measurement is totally different than the binding constant value obtained from ITC measurement. The fluorescence measurement deals with local changes surrounding the guest molecule whereas ITC measurement associated with a global

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change of the host within the medium.

4. Computational Study

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The inclusion of flavone molecules inside the cavity of CB7 molecules were studied experimentally which is further explained by finding the orientation of interaction and the

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encapsulation of the dye to CB7 from optimized geometry. To find out the exact orientation of interaction of the dye with the CB7, we optimized the inclusion (1:1) complex (flavone: CB7) by using B3LYP/STO-3G level of theory by docking the dye to hydrophobic cavity of

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CB7. The optimized geometry of (1:1) inclusion complex is presented in the Fig. 9 and 10

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where it is found that the electron rich moiety (fragment 2) lies outside of the CB7 cavity due to dipole-dipole interaction which is consistent with the experimental observation. The ESPT transition is takes place favourably in the flavonoides which is hindered by carboxyl moiety

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of CB7 cavity and it is clear that the proton transfer of (-OH) group of 3-hydroxyflavone takes place during penetration inside the CB7 cavity. The geometries of the complexes was optimized in gaseous phase and the interaction energy in between the 3-HF and CB7

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molecule is found -0.63686 kcal/mol. The interaction energy in between the 7-HF and CB7 molecule is found -3.06475 kcal/mol. The negative values of binding energy EBE  indicate that the encapsulation of flavone to the CB7 cavity is thermodynamically feasible. The binding energy has been calculated by using equation 3.

CB7  Flavone  CB7 : Flavone

EBE  ECB7. flavone  ECB7  E flavone

(3)

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Fig. 9: Optimized geometry of 3HF, CB7 and the 1:1 inclusion complex (CB7:3HF) at B3LYP/STO-3G level of theory at Gaussian 09 suite of program [37].

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Fig. 10: Optimized geometry of 7HF and the 1:1 inclusion complex (CB7:7HF) at B3LYP/STO-3G level of theory at Gaussian 09 suite of program [37].

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Both theoretical and experimental observations imply that the formation of (1:1) inclusion complex in between dye and CB7 molecules. Molecular orbital picture of the CB7:3HF complex have shown in Fig. 11.

HOMO

LUMO

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ACCEPTED MANUSCRIPT Fig. 11: Molecular orbital picture of CB7:3HF inclusion complex. From HOMO it was observed that,  delocalization is located over the C-C and C-O of fragment 2 in contrast LUMO where it is located throughout the entire dye and which envisaged the total electronic distribution over the molecular skeleton. No specific bonding is observed in the hydroxyl group (-OH) which confirms the deprotonation of 3-hydroxyflavone takes place during formation of inclusion complex with the CB7. From the optimized

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geometry of the complex the energy of HOMO and LUMO are found at -1.93 eV and at 1.34 eV. The electron densities are located over the flavonoid moiety not over the rest CB7

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moiety. The theoretical calculation established a good agreement with the experimental observations. Molecular orbital picture of the CB7:7HF complex have shown in Fig. 12.

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From MO viewpoint, it is found that HOMO is located over the CB7 moiety due to  orbital contribution of the carbonyl groups on CB7 moiety but the LUMO is attributed over the

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entire guest molecule (7HF). The delocalized  * contribution from 7HF is also observed from the LUMO. The corresponding HOMO and LUMO energies are found at -2.34 eV and

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HOMO

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1.93 eV.

Fig. 12: Molecular orbital picture of CB7:7HF inclusion complex. Conclusion:

In this study, interaction of weakly water soluble flavones i.e. 3-HF and 7-HF with highly water soluble macrocyclic host CB7 has been prudently studied by using different types of spectroscopic techniques along with isothermal titration calorimetric technique. On addition of CB7 to aqueous solution of flavones very interesting results are observed in fluorescence emission spectra. The fluorescence intensity of tautomers band gradually increases on gradual addition of CB7 for both the flavones; it implies that the interaction of flavone with CB7 18

ACCEPTED MANUSCRIPT facilitated the stability of tautomers band by formation of the complex. From Job’s plot it is found that 1:1 complex is formed for both the flavones with CB7. The binding constant value of 3-HF with CB7 is found to be 2710 (± 230) M-1 and for 7-HF is found to be 23125 (± 1905) M-1 by using fluorescence data. The fluorescence life time value gradually increases due to the formation of complex between flavone and CB7. From isothermal measurement, it is observed that complexation process between flavones with CB7 is endothermic in nature. The binding constant value of 3-HF and 7-HF with CB7 are found to be 210 (±8) M-1 and

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8.05 (±2.38) × 103 M-1, respectively. By using density functional theory geometry of the CB7: flavone complex was optimized. It was observed that in the case of CB7:3HF complex

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HOMO was located on the 3HF molecules whereas in CB7:7HF complex HOMO was

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located over the CB7 moiety.

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Acknowledgements

S.A.A. B.M. and D.S. are thankful to Indian Institute of Technology Patna (IIT Patna), India for the research facilities. S.A.A. and B.M. are thankful to IIT Patna for research fellowships.

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We are thankful to Dr. Aninda Chatterjee for helpful discussion.

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Burant, J. C., Iyengar, S. S., Tomasi, J., Cossi, M., Rega, N., Millam, J. M., Klene, M., Knox, J. E., Cross, J. B., Bakken, V., Adamo, C., Jaramillo, J., Gomperts, R., Stratmann, R. E., Yazyev, O., Austin, A. J., Cammi, R., Pomelli, C., Ochterski, J. W., Martin, R. L., Morokuma, K., Zakrzewski, V. G., Voth, G. A., Salvador, P., Dannenberg, J. J., Dapprich, S., Daniels, A. D., Farkas, Ö., Foresman, J. B., Ortiz, J. V., Cioslowski, J., Fox, D. J., Gaussian, Inc., Wallingford CT.

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Graphical Abstract

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ACCEPTED MANUSCRIPT Highlights:

Interaction of biologically active flavones with CB7 was reported.

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Emission properties of drug molecule was modulated several folds in the presence of CB7.

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The stoichiometry of the complexes was found to be 1:1 for both the drug molecules.

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Thermodynamics of the binding was studied by using isothermal titration calorimetry.

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The inclusion of flavone molecules inside the cavity of CB7 molecules was studied

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

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theoretically.

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