Modulation of ESIPT fluorescence in o-hydroxy acetophenone derivatives: A comparative study in different bio-mimicking aqueous interfaces

Journal of Molecular Liquids 218 (2016) 549–557

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Modulation of ESIPT fluorescence in o-hydroxy acetophenone derivatives: A comparative study in different bio-mimicking aqueous interfaces Pynsakhiat Miki Gashnga, T. Sanjoy Singh, Sivaprasad Mitra ⁎ Centre for Advanced Studies, Department of Chemistry, North-Eastern Hill University, Shillong 793022, Meghalaya, India

a r t i c l e

i n f o

Article history: Received 2 December 2015 Received in revised form 23 February 2016 Accepted 1 March 2016 Available online xxxx Keywords: ESIPT Cyclodextrin Surfactant Protein Fluorescence modulation Binding constant

a b s t r a c t Excited state intramolecular proton transfer (ESIPT) reaction and photophysical behavior of two intramolecularly hydrogen bonded o-hydroxy acetophenone derivatives, namely 2-acetyl-4-methyl-6-nitrophenol (I) and 2acetyl-4-chloro-6-nitrophenol (II) are studied in the presence of different micro-heterogeneous environments ranging from cyclodextrin, surfactant as well as in model water soluble proteins like bovine and human serum albumin. The appearance of different emission profiles corresponding to a series of excited state isomers like primary enol structure (E), proton transferred keto form (K), or intermolecularly hydrogen bonded (with the solvent) open conformer is rationalized on the basis of varying degree of sequestration and the environment around the probe in the local micro-structure of the hydrophobic domain. The experimental observations are in complete agreement with the fluorescence behavior of these probes in homogeneous solvent medium as well as results based on density functional theory calculation published earlier (Chem. Phys. 342, 2007, 309). © 2016 Elsevier B.V. All rights reserved.

1. Introduction The transfer of a proton from one group to another in a pre-formed intramolecularly hydrogen bonded (HB) system under photon excitation, the so called excited state intramolecular proton transfer (ESIPT), has found tremendous application in diverse basic scientific domains like chemistry and biology [1–5], as well as in a series of technological advancements [6,7]. Since the first observation of ESIPT in methyl salicylate by Weller [8] almost six decades ago, extensive research in this field has developed the basic understanding and generalized picture of the mechanism of this process along with the discovery of many new systems with potential application in different fields [9–12]. The extremely fast ESIPT process (usually ranging within fs ~ sub-ps time domain) in isolated systems [13,14] or in noninteracting media [15,16] is considered to be due to an extensive electron density rearrangement of the donor-acceptor pair in the excited state. The large stability difference of the original hydrogen bonded structure (enol tautomer, E) in comparison with the resulting proton transferred species (usually called the keto tautomer, K) in the ground and excited states usually results a single absorption (form E) and emission (from K) spectra with considerable Stokes shift (~10,000 cm−1) [9, 10]. Fluorescence emission from excited primary enol conformer (E⁎) can also be observed in some cases in addition to the keto emission ⁎ Corresponding author. E-mail addresses: [email protected], [email protected] (S. Mitra).

http://dx.doi.org/10.1016/j.molliq.2016.03.001 0167-7322/© 2016 Elsevier B.V. All rights reserved.

and depends on the dynamics of ESIPT as well as its excited state lifetime. The extent of the intramolecular HB, and therefore the ratio of normal to ESIPT emission, can be modulated by suitable substitution in the vicinal site of the hydrogen bonded moiety [17,18]. Furthermore, as ESIPT is a direct consequence of a pre-formed HB between the donor and acceptor moiety, any perturbation due to the influence of interacting solvent (or medium) through intermolecular HB leads to the disappearance of ESIPT emission. Depending on the nature of the conformations generated either due to the interaction with the surrounding medium or due to the flexible rotation of groups containing hydrogen bonding partners, the ESIPT systems can show multiple emissions. These conformations often differ in their excited state energy as well as dynamics; and can be suitably manipulated with external controls to give desirable properties [19,20]. A few years ago, we reported the photophysical behavior of two substituted o-hydroxy acetophenone derivatives, namely 2-acetyl-4methyl-6-nitrophenol (I) and 2-acetyl-4-chloro-6-nitrophenol (II) (Chart 1, for structures) by fluorescence and computational studies [21]. Both the compounds were found to undergo ESIPT reaction (emission from K conformer) and show competitive emission from different excited state enol species (E1 and E2) depending on the nature of the solvent. The micro-heterogeneous host environment created by water soluble cyclodextrin (CD), surfactants and/or proteins provides excellent opportunity to bind with a series of small organic molecules; and therefore, can act as an efficient modulator to control the excited

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Chart 1. Structure of the ESIPT probes investigated in this study.

state emission behavior of systems like I and II possessing multiple fluorescence peaks. The ability of these host systems to modulate excited state reactivity depends on their capacity to complex with guest organic substances. Changes in physico-chemical properties and reactivities result from such host–guest interactions. The effect on reactivity due to the formation of host–guest complexes varies widely depending on the guest, the host, and the type of excited state reaction and has been a topic of intense research activities [22–24]. Owing to the importance of these nano-cavities to control the dynamics of the excited state by restricting specific torsional motion leading to the design of new macromolecular architecture, we report herein the detail investigation of the interaction of I and II with α- and β-cyclodextrin (CD) which are known to form a hollow, truncated cone shaped hydrophobic interior; different surfactants like SDS, CTAB and TX-100usually form roughly spherical micellar aggregates under specific environmental conditions in solution above a minimum concentration known as critical micelles concentration (CMC); and, also water soluble model proteins like bovine and human serum albumin (BSA & HSA, respectively). 2. Experimental The synthesis and purification of ESIPT probes I and II are discussed elsewhere [21]. The water used as solvent in all the measurements was obtained from Elix10 water purification system (Millipore India Pvt. Ltd.). The best available grade of cyclodextrins, surfactants and proteins to provide the micro-heterogeneous host environment was obtained from commercial resources [22]. The chromophore concentration (~ 5 μM) was very low to avoid any aggregation and kept constant during spectral measurements; whereas, the final concentration of the heterogeneous media was adjusted by adding the required volume from the stock solution. All the solutions were prepared afresh and kept for 30 min for settling before the spectroscopic measurement. The experimental and data analysis procedure are also similar as described earlier [26]. All steady-state fluorescence emission data obtained from at least three separate experiments were averaged (Fobs) and corrected (F) for any possible change of absorption intensity (A) compared with the absorption in pure aqueous medium (A0) using Eq. (1) before processing further. FðλE ; λ F Þ ¼

A0 ðλE Þ  Fobs ðλE ; λ F Þ: AðλE Þ

ð1Þ

Relative fluorescence quantum yield of both the systems in varied experimental conditions was calculated by comparing the total

fluorescence intensity under the whole corrected fluorescence spectral range with the same procedure mentioned elsewhere [21]. The relative experimental error of the measured quantum yield was estimated to be within ± 10%. Fluorescence decay analysis was performed in a fluorescence spectrometer (QM-40, PTI, USA) equipped with a TCSPC fluorescence lifetime detection unit (PM-3). The sample was excited at 375 nm using a picosecond laser diode (PiL037X) obtained from A.L.S. GmbH. The fluorescence decay curves were analyzed by nonlinear least-square iterative convolution method based on Levenberg– Marquardt algorithm [21]. 3. Results and discussion 3.1. Spectroscopic behavior of I and II in homogeneous solvents To make a basis of our discussion on spectral properties of I and II in aqueous buffer and subsequent modulation in a series of different micro-heterogeneous systems discussed below, here we briefly incorporate the results observed in different non-polar as well as polar/protic organic solvents reported earlier [21] and summarized in Scheme I. The principal absorption peak at ~ 355 nm in all the solvents and the corresponding largely stokes shifted unstructured emission at 505 nm is due to the pro-ESIPT intramolecularly hydrogen bonded enol (E) and corresponding proton transferred keto (K) conformer, respectively. The non-ESIPT enol structure (E1 and/or E2) having the absorption at the same wavelength range as that of E, gives a distinct emission at ~430 nm only in non-polar media and also in polar solvents with relatively larger dielectric relaxation time like DMSO [21]. However, in all other cases, the fluorescence emission resembles that of K because of rapid conversion of E1/E2 → E → K in the excited state. An additional (solvent dependent) absorption peak within 440–485 nm range is also observed for both I & II and assigned to the facile formation of zwitterionic species (Z) in polar medium, possibly due to the presence of an electron withdrawing nitro group in ortho position of the phenolic moiety. However, upon excitation, Z also undergoes ESIPT to give an emission at ~500 nm, similar to that observed in structurally analogous 4-methyl-2,6-diformyl phenol (MFOH) [27] or classic ESIPT system methyl salicylate (MS) [28,29]. In aqueous medium, both I & II give a single absorption peak ~355 nm (Fig. 1). Excitation at this wavelength, the emission spectra show a major peak at ~ 505 nm along with a minor shoulder at ~ 440 nm. The excitation spectra corresponding to both these emissions match closely with the absorption spectral profile. While, the 505 nm emission can be unambiguously assigned to the ESIPT species, the appearance of 440 nm band in the aqueous medium seems contradictory to the predictions made from spectral observations

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Scheme I. Structure and primary spectroscopic parameters for different conformers of I (R = CH3) and II (R = Cl) under varying solvent environments.

in other solvents summarized above and discussed in detail elsewhere [21]. As in the case of non-polar solvents, this emission cannot be assigned to the non-ESIPT structures E1 or E2. Considering the polar nature of the aqueous environment, these conformers are expected to give only K emission. However, due to the strong hydrogen bonding ability of the water molecules, an open conformer – where the phenolic proton makes an intermolecular hydrogen bond with solvent water molecules (Scheme I) – can be envisaged consistent with the observations made for other related systems [30,31]. In a strong proton accepting solvent, this open conformer can even lead to the formation of an anion (A, Scheme I). This possibility is far more pertinent in the present systems, in comparison with other similar ESIPT probes discussed elsewhere, due to the presence of a strong electron withdrawing nitro group. Nevertheless, interaction with the polar protic solvent would give rise to nonESIPT emission at ~440 nm.

3.2. Spectroscopic behavior of I and II in the presence of α- and β-CD The absorption spectra of both I and II show insignificant changes by the addition of CD with ca. 1–2 nm red shift in peak maximum (Table 1) and little increase in absorption intensity. However, the emission profile shows a marked increase in intensity with a regular addition of CD in both cases. Some of the representative plots are shown in Fig. 2. Also, the spectral profile was found to be changed drastically in the CD environment (Fig. 2). The intensities of both the ESIPT as well as the non-ESIPT emission (505 and 440 nm, respectively) increase substantially in all the cases; however, the increase is more prominent for the later than the former. This happens to such an extent that the peak around 440 nm becomes the main emission and the 505 nm peak appears as a shoulder. In fact, the ratio of 440 nm to 505 nm band intensity increases monotonically till it levels off beyond 4– 6 mM CD concentration. This observation is similar for both the ESIPT systems discussed here with only notable exception in the case of II in β-CD; where it was found that the ESIPT emission undergoes a marked blue shift (~28 nm) along with a feeble emission at 440 nm as a shoulder even at [β-CD] = 30 mM (Fig. S1 in the Supplementary section). Nevertheless, the notable change in emission spectral profile in all the cases indicates the formation host–guest type of complex. The binding constant (K) and stoichiometry of the complex (n) were calculated from a non-linear regression (inset, Fig. 2) of the intensity ratio (I) variation with CD concentration using Eq. (2). The initial guess value for K was obtained from the linear Benesi–Hildebrand (BH) relation given in Eq. (3) following the similar method as discussed before [32,33]. I0 and Iα represent the corresponding intensity parameter in the absence and fully complexed CD concentration condition, respectively and ‘a’ is the ratio of the fluorescence intensities of the complexed and uncomplexed chromophore. I ¼ I0 

Fig. 1. Absorption (solid line), fluorescence emission (scattered points) and excitation (dashed line) spectra of I in aqueous medium.

1 þ aK½CDn 1 þ K½CDn

1 1 1 1  ¼ þ : I  I0 I∞  I0 KðI∞  I0 Þ ½CD

ð2Þ

ð3Þ

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Table 1 Steady state spectral properties of conformers I and II in various heterogeneous environments. I

II

Medium

λabsa (nm)

λflb (nm)

ϕfc/10−2

ΔνSSd (cm−1)

λabsa (nm)

λflb (nm)

ϕfc/10−2

ΔνSSd (cm−1)

SDS CTAB TX-100 α-CD β-CD HSA BSA

363 361 360 362 362 360,440 360,440

436, 508 438, 510 436, 509 434, 507 435, 506 508 510

0.20 0.18 0.21 0.21 0.39 0.40 0.35

4612, 7863 4869, 8092 4841, 8131 4582, 7900 4635, 7861 3042 3120

362 361 363 364 363 440 440

435, 509 436, 510 435, 510 438, 505 436, 507 508 510

0.27 0.13 0.23 0.19 0.33 0.39 0.31

4635, 7977 4765, 8092 4559, 7940 4641, 7670 4612, 7824 3042 3120

a b c d

Absorption maxima. Fluorescence maxima. Fluorescence yield. Stokes shift.

The calculated values, given in Table 2, indicate that although the stoichiometry of complex formation is 1:1 in all the cases, the binding is relatively stronger in the case of I. Furthermore, α-CD gives a tighter complex in comparison with β-CD for both I and II. The increase in non-ESIPT emission intensity inside CD cavity is rather unusual, as the formation of open conformer (having an intermolecular hydrogen bonding with water solvent) is not expected inside the hydrophobic cavity. Rather, it is quite common to observe the increase in proton transferred keto tautomer emission in the presence of CDs for a series of ESIPT probes [23,34] and even in some of the derivatives of the present systems [32,35]. In view of this, the unique photophysical behavior of I and II in the CD environment is quite interesting and needs special attention. Considering the CD cavity to be non-polar in nature, the appearance of non-ESIPT emission in both I and II is expected, in analogy with the spectral behavior of these systems in non-polar solvents discussed in the previous section. In fact, the formation and abundance of non-ESIPT structures (E1 & E2) inside the CD cavity are rationalized on the basis of their lower dipole moment (μ = 1.085 D) in comparison with both the pro-ESIPT enol structure (E, μ = 5.843 D) and its ESIPT counterpart (K, μ = 7.249 D) obtained from DFT calculations using the B3LYP/6-31 ++G (d,p) protocol. On the other hand, extremely weak binding of II in β-CD (K ~ 2.2 M−1) indicates the absence of any true host–guest complex in this case. The probe mainly resides on the rim of the CD structure and the shift in ESIPT fluorescence towards the blue side of the spectrum may be due to the overall decrease in the solvent polarity in the presence of cyclodextrin (general solvent effect).

3.3. Fluorescence modulation in the presence of surfactant solutions The absorption maximum of the aqueous solutions of both I and II remains practically unaffected; however, the emission spectra show a marked effect by the addition of surfactants (SDS, CTAB & TX-100) (Figs. 3 & S2). In general, the non-ESIPT 440 nm shoulder gets intensified and appears as a proper emission peak on addition of the surfactants; however, the ESIPT fluorescence at 505 nm still remains the main emission band with a substantial increase in intensity in the presence of surfactants. A notable difference is observed in the case of II/CTAB system, where the non-ESIPT peak appears to be more intense in the presence of surfactant. The significant change in fluorescence emission spectral profile of both I and II in the presence of surfactants indicates that the probe molecules penetrate into the micelles; and therefore, experience an environment characteristically different from that in pure aqueous medium. Typically, the micellar cross section in solution is characterized by the formation of three distinct sublayers [36,37]: (i) the hydrophobic core consisting of the non-polar tail of the surfactant molecule; (ii) head-group region forming a compact Stern layer with characteristic charge (for example, anionic and cationic for SDS and CTAB, respectively while that in TX-100 is non-ionic); and (iii) a wide Gouy–Chapman (GC) layer consisting of counter ions (opposite to the head group). The GC layer is normally followed by a pure aqueous phase at the infinite distance from the core. The fluorescence emission of a probe in micellar medium is normally characterized by the environment in its immediate vicinity, i.e. location of the probe in micellar subdomain.

Fig. 2. Fluorescence emission spectra of I (a) and II (b) with increasing concentration of β- and α-CD, respectively (λexc = 355 nm in both the cases). [β-CD]/mM = 0, 2.25, 4.9, 7.1, 13.3, 16.9, 21.5 and 29.8 in (a); and [α-CD]/mM = 0, 0.4, 4.2, 6.7 and 19.1 in (b). Inset shows the variation of the non-ESIPT to ESIPT emission intensity ratio with CD concentration.

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Table 2 Physico-chemical parameters for the interaction of I and II with different micro-heterogeneous host media. I

II

Medium

Binding constant, K (M−1)

SDS CTAB TX-100 α-CD β-CD HSA BSA

90.5 121.5 125.7 1025.3 390.2 9.76 × 103 3.31 × 104

a

cmca value (mM)

Stoichiometry ratio

Binding constant, K (M−1)

1:1 1:1 1:1 1:1

55.3 192.7 270.2 251.5 2.2 5.24 × 103 1.79 × 104

8.1(8.0) 1.1(0.9) 0.32(0.3)

cmca value (mM)

Stoichiometry ratio

8.2(8.0) 0.92(0.9) 0.31(0.3) 1:1 1:1 1:1 1:1

Estimated from fluorescence intensity variation with surfactant concentration. Numbers in the parenthesis are the literature values.

Usually, organic fluorophores do not penetrate into the hydrocarbon core of the micellar structure; rather, the preferred location is mostly in the micelle–water interface characterized by GC layer and/or micellar Stern/palisade layer. The extent of penetration is mostly dependent on the charge condition in different layers and the probe molecule under consideration. The formation of a definite spectral peak at ~ 440 nm for both I and II in comparison with a shoulder in pure aqueous medium indicates that the probes experience a relatively non-polar environment under micellar condition. So, the location of the probes can be viewed either in the GC layer or preferably, into the more hydrophobic Stern layer considering the fact that the later provides superior confinement with better hydrophobic environment. However, the estimated binding constant of the probes in

the surfactant medium is moderately low (see below). Moreover fluorescence results suggest the probe location with considerable access towards the aqueous environment as indicated by the presence of ESIPT emission (~ 505 nm) also; confirming that the binding of the probe occurs mainly at the GC layer. The anomalous observation of II in the presence of CTAB in comparison with I can be explained on the basis of electronic effect of the chloro-substitution in the former. The electron withdrawing effect of this group makes the aromatic nucleus to be electron deficient and therefore, penetrates more in GC layer with negatively charged counter-ions of CTAB. Under this condition, II experiences more non-polar environment and lesser accessibility of the solvent water molecules which results to the increase in non-ESIPT emission under micellar condition.

Fig. 3. Fluorescence spectral modulations of I and II in the presence of varying concentrations of SDS and CTAB surfactants.

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Fig. 4. Birds eye view representation (solid line) of the change in ESIPT fluorescence peak intensity (scattered points) for I (510 nm) and II (508 nm) with varying concentrations of CTAB and SDS, respectively. The break point in each case indicates the onset of micelle formation.

The variation in fluorescence intensity for some of the represented cases of I and II against surfactant concentration is shown in Fig. 4. In almost all the cases, the intensity variation shows a sharp break point close the total surfactant concentration characterized by cmc, further confirming that the change in fluorescence behavior can be rationalized in terms of binding of the probes in the micellar sub-domains. Considering the aggregation number of the micelle formation to be constant and the total surfactant concentration (Dt) used in the experiment to be well above the critical micelle concentration (cmc), the probe-micelle association constant (K) can be related with the change in fluorescence intensity (F) by the following relation [37,38]: F  F0 ¼ K  ½Dt : Fm  F0

ð4Þ

Here, F0 and Fm are the fluorescence intensities of I and/or II at a particular wavelength in water and under fully micellized condition. The values of K are obtained from the slopes of the linear variation of (F − F0) / (Fm − F0) against [Dt] and incorporated in Table 2. Some of the representative figures are shown in the Supplementary section (Fig. S3). The higher K values in TX-100 can be explained by the combined effect of micellar size and the chain length of the hydrocarbon tail. While, the average radius of SDS micelle is quite small (R ~ 30 Ǻ) in comparison with both CTAB and TX-100 micelles (R ~ 50 Ǻ) [39–41], the hydrocarbon chain length in TX-100 is significantly longer (~35.3 Ǻ) than both SDS (~15 Ǻ) and CTAB (~20 Ǻ) [42]. Furthermore,

the predicted cmc values obtained from the break point of intensity variation curves (Fig. 3) given in Table 2 are found to be in very good agreement with the literature reports. 3.4. Spectral properties of I and II in the presence of serum albumins The spectroscopic behavior of I and II was monitored in the presence model water soluble proteins like bovine and human serum albumin (BSA and HSA, respectively) in buffer solution of pH = 7.4 following the same method as discussed earlier [25]. Interestingly, while a considerable fraction of I exists in zwitterionic form (with absorption at ~440 nm) along with the E absorption at ~360 nm; the absorption spectra of II shows the peak only at ~440 nm. This observation is consistent with our earlier report [21], where it was shown that the formation of zwitterionic species in the ground state is favored in the case of II due to the presence of more electron-withdrawing chloro-substitution. Furthermore, with increasing protein concentration, the intensity of ~ 360 nm absorption for I decreases continuously (till it vanishes at ~ 10 μM protein concentration) with a concomitant increase at ~ 440 nm indicating the full E → Z conversion in the ground state. However, for II, the absorption spectra remain practically unaffected even at ~30 μM protein concentration (Fig. 5). As only the Z conformer is present for both I and II in the presence of added proteins, we will monitor the fluorescence behavior only by excitation at ~ 440 nm. As discussed earlier for DMSO, excitation at 440 nm in aqueous buffer also gives the emission from K conformer at ~510 nm. The intensity of

Fig. 5. Absorption spectral profiles of I and II in the presence of increasing concentration of BSA. [BSA] = 0–54 μM and increasing by 6 μM in each step.

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555

Fig. 6. Variation of ESIPT fluorescence of II in the presence of increasing concentration of BSA and HSA. The concentration of the protein increases by 6 μM in each step in the arrow direction.

this emission increases continuously with the increase in protein concentration with a very little (2–3 nm) blue shift in the emission maximum (Fig. 6). The change in fluorescence intensity as well as position indicates that both the probes bind in the protein hydrophobic domain. The association constant (K) for this binding can be determined from the modified form (Eq. (5)) of BH equation given earlier (Eq. (3)) [26]. ΔI max 1 1 ¼1þ  : ΔI K ½protein

ð5Þ

Here, ΔImax and ΔI represent the maximal and relative increase in fluorescence intensity, respectively. The linear nature of (ΔImax / ΔI) against (1 / [protein]) concentration confirms the stoichiometry of binding to be 1:1 in all the cases. Some of the representative plots are shown in the inset of Fig. 6. The binding constant in each case is given by the reciprocal of the slope of the straight line and also incorporated in Table 2. Both the systems show moderate to strong binding (K ~ 103–104 M−1) with the albumins and can explain the large modulation of fluorescence spectral behavior in the presence of proteins. The intrinsic tryptophan fluorescence of both the serum albumin proteins

quenches in the presence of I and II. Representative quenching data of HSA/I and BSA/II systems are shown in Fig. S4 (a) and (b), respectively in the Supplementary section. In all the cases, the Stern–Volmer plot of the quenching data is linear (inset of Fig. S4) and confirms that the probes bind preferentially in Sudlow's site 1 of the protein. However, no trace of ~ 440 nm enol emission appears even in the presence of the highest albumin concentration in contrast to what has been observed in CD cavities (discussed above). Therefore, it is believed that in spite of the association in the ligand binding domain of the protein, the probes still have sufficient exposure to aqueous medium to give K emission at ~505 nm. 3.5. Time-resolved fluorescence decay behavior of I and II in different environments As in the cases of different organic solvents [21], the fitting of timeresolved fluorescence traces for both I and II needed at least a three exponential decay function both in the homogeneous aqueous (buffer) medium as well as in the presence of all the micro-heterogeneous systems studied here. Some of the representative decay traces along

Fig. 7. Representative time-resolved fluorescence decay traces (open circle) along with the simulated data with three exponential decay function (solid line) and instrument response function (IRF) for I in aqueous medium (a, λmon = 505 nm) and II in the presence of certain heterogeneous systems (b, λmon = 505 nm). For (a), the upper panels show the distribution of weighted residual and also the values of reduced chi-square (χ2), Durbin–Watson (DW) parameter in different fitting models.

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Table 3 Average fluorescence decay time (bτf N), radiative (κr) and total nonradiative (Σκnr) decay rate constants for I and II in various heterogeneous environments.a I Medium

Water α-CD (7 mM) β-CD (10 mM) SDS (10 mM) CTAB (5 mM) TX-100 (1 mM) BSA (30 μM) HSA (30 μM) a

II κr /107

Σκnr/109

440 nm

505 nm

s−1

s−1

440 nm

505 nm

s−1

s−1

0.55 1.15 1.28 1.33 0.99 1.87 1.42 1.49

0.38 0.98 0.60 0.33 0.77 1.21 – –

0.31 0.18 0.30 0.15 0.18 0.11 0.25 0.27

1.82 0.87 0.78 0.75 1.01 0.53 0.70 0.67

0.16 0.31 0.78 0.41 0.45 1.40 1.26 1.22

0.22 0.19 0.18 0.76 0.85 0.90 – –

1.69 0.61 0.42 0.66 0.29 0.16 0.25 0.32

6.23 3.22 1.28 2.43 2.22 0.71 0.79 0.82

bτf N, ns

κr/107

Σκnr/109

bτf N, ns

bτf N = Σaiτi / Σai , κr = ϕf / bτf N and Σκnr = (1 − ϕf) / bτf N.

with the deconvolution curve are shown in Fig. 7 and all the decay parameters are listed in the Supplementary section (Table S1). The amplitude weighted average decay time (b τf N) and the calculated radiative (κr) and total non-radiative (Σκnr) decay rate constants are given in Table 3. It is seen that the total non-radiative process contributes maximum in the total decay of the excited state in both the cases and consistent with the observation of very low quantum yield in these systems [21]. However, the magnitude of Σκnr significantly reduces due to the binding of the probes in the microdomain of the host provided by the heterogeneous environments, which results in the increase of fluorescence intensity in the steady state experiments. It is to be noted that analysis of binding behavior of the ESIPT probes with different heterogeneous hosts described before was done by monitoring the change in fluorescence intensities. However, fluorescence intensities of both enol and keto isomers change by the addition of CD, surfactants, and also BSA/HSA in a non-straightforward way. This may be an indication that it is not just the population change but also a significant degree of variation in radiative as well as non-radiative decay rates (Table 3), which affect the fluorescence intensities.

4. Conclusion The modulation of ESIPT behavior and competitive fluorescence from different ground and excited state conformers of two substituted o-hydroxy acetophenone derivatives were studied in presence of cyclodextrins, surfactants and also in model water soluble proteins like bovine and human serum albumins. The change in fluorescence properties and ESIPT behavior was ascribed due to the sequestration of the probe in hydrophobic sub-domain provided by the microheterogeneous media and resulting difference in the intermolecular interactions of the liquid environment surrounding the probe. The experimental results are consistent with the observations made in neat homogeneous solvent environments. Detailed analysis of the spectroscopic data indicates that both the probes form 1:1 complex with CD, surfactant as well as protein nano-cavities. Binding constant of the probe with different heterogeneous microstructures as well as critical micelle concentration was obtained from the variation of fluorescence intensity on increasing concentration of the hydrophobic component in the aqueous medium. This study basically accentuates the fundamental issues of controlling the spectroscopy and dynamics of ESIPT systems leading towards the development of complex molecular architecture in liquid environment.

Acknowledgment S. Mitra thanks CSIR for a research project. Thanks are also due to DST, Govt. of India for supporting the Chemistry Department, NEHU through FIST.

Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.molliq.2016.03.001.

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