Inclusion of an Anthracene-based Fluorophore within Molecular

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Inclusion of an Anthracene-Based Fluorophore Within Molecular Containers: A Comparative Study of the Cucurbituril and Cyclodextrin Host Familiesontrolled Self-Assembled Surface in Switchable Wettability of ZnO Nanostructured Films Aniruddha Ganguly, Soumen Ghosh, and Nikhil Guchhait J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.6b04178 • Publication Date (Web): 27 Apr 2016 Downloaded from http://pubs.acs.org on April 28, 2016

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The Journal of Physical Chemistry

Inclusion of an Anthracene-based Fluorophore within Molecular Containers: A Comparative Study of the Cucurbituril and Cyclodextrin Host Families Aniruddha Ganguly,* Soumen Ghosh and Nikhil Guchhait* Department of Chemistry, University of Calcutta, 92 A. P. C. Road, Calcutta-700009, India *To whom correspondence should be addressed. Tel.: +91-33-2350-8386. Fax: +91-332351-9755. E-mail: [email protected] (A.G.), [email protected] (N.G.)

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ABSTRACT: In this article, the binding interaction of a promising chloride channel blocker 9-MA with two different classes of molecular containers, viz. β-cyclodextrins (βCD and methyl β-CD) and cucurbit[7]uril, having comparable cavity dimension, has been thoroughly demonstrated via the inspection of the modulation of the excited state properties of the emissive molecule. The spectral data suggest that CB7 encapsulates the probe more efficiently in a 1:2 fashion whereas the efficacies of the β-CDs are relatively less and the corresponding stoichiometry is 1:1. Interestingly, despite being thermodynamically much more favorable than the probe–β-CD complexation equilibria, the fraction of the probe-CB7 complex formed is appreciably petite with respect to that of the probe–β-CD complexes. This apparent inconsistency has been addressed on the basis of the proposition that since the formation of a 1:2 complex is entropically disadvantageous, it is anticipated that the activation barrier of the corresponding reaction is reasonably high, thus only a small fraction of the reactants are able to surpass the energy barrier to form the products. This proposition has been thoroughly corroborated using fluorescence lifetime measurements at different temperatures.

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INTRODUCTION Inclusion of chromophoric guest molecules into the cavities of macrocyclic hosts has been a topic of burgeoning research in supramolecular chemistry with an objective to explore the remarkable modulations in the properties of the encapsulated guests as well as the prospect of the system to act as a minimally invasive therapeutic delivery vehicle with properties tunable via external stimuli.1-3 Though the formation of such host–guest complexes is an outcome of relatively weaker van der Waals’ interactions, the cooperativity of the contacts involved between the constituent units often results in appreciably strong binding.4,5 Cyclodextrins (CDs), being the most widely used hosts, are a class of cyclic oligosaccharides that are synthesized from starch via a simple enzymatic conversion.

These

semi-natural

compounds

commonly

comprise

several

D-

glucopyranoside units linked together by α-1,4-glycosidic bonds; α-, β- and γ-CDs, consisting of six, seven and eight monomer units, respectively, are the most common ones.1,2,4 CDs are pictured as shallow truncated cones with the diameter of the primary hydroxyl rim of the cavity reduced compared with the secondary one. The interior of the cone is considerably less hydrophilic than the aqueous environment and thus able to host other hydrophobic molecules, whereas the exterior is sufficiently hydrophilic to ensure water solubility of both the host and the complex.5,6 Of them, β-CD has always grabbed much attention owing to its appropriate cavity size capable of encapsulating a wide variety of guests as compared to α- or γ-CD, as well as due to its low price and easy accessibility.7,8 But, one severe impediment limiting the application of β-CD is its relatively low water solubility;9 which has led towards the development of several


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functionalized (mostly alkylated) β-CD derivatives, viz. methyl β-CD, 2-hydroxypropylβ-CD etc. with enhanced water solubility.2,10 Cucurbit[n]urils, a comparatively fresh genre of water soluble macrocycles, are synthesized by condensation of glycoluril with formaldehyde under controlled conditions in presence of acid as catalyst.1,3 Structurally, CBn homologues are highly symmetric (consisting an equatorial symmetry plane; i.e. both cavity openings in CBs are identical, unlike to that in CDs) pumpkin-shaped macrocyclic hosts with negatively charged carbonyl rims and a hydrophobic cavity having very low polarizability.3,11,12 Despite the remarkable resemblance in cavity dimension with the CDs (the cavity dimension of CB6, CB7 and CB8 being analogous to that of α-, β- and γ-CDs respectively), the structural differences between the constituent monomers lead to differential binding behavior of the guest with these two classes of macrocycles.12,13 Though for neutral guests, the classic “Hydrophobic effect”, i.e. a composite effect derived from an interplay between the release of “high-energy water” upon complexation of non-polar organic residues and concomitant differential dispersion interactions inside the cavity, is the prevailing factor in both the cases, for cationic guests, ion-dipole interactions between positive charges on the guest and carbonyl oxygens lining the CB cavity openings becomes significant, whereas the bristling hydroxyl groups in CDs generally do not engage in strong interactions with the included guest irrespective of its charge characteristic.12,14,15 Though the differential interaction of cationic (and/or pH responsive) dyes with CD and CB homologues has been a topic of intense research,13,16-19 equivalent studies involving neutral (and/or pH insensitive) molecules are surprisingly limited.20 Thus, in the present contribution, we have tried to unravel the differential binding interaction of 9-methyl


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anthroate (9-MA, vide Scheme 1), a neutral molecule belonging to the eminent chloride channel blocker family,21 with β-CD (and methyl β-CD) and CB7 (other CD and CB homologues are found to interact only nominally); the modulated excited state photophysics of the probe within the macrocyclic cavities has been exploited as the monitoring index to caste light towards the binding process. Since the aforementioned macrocycles are efficient drug delivery vehicles,8,9,22 the study is likely to furnish information related to the bio-distribution of the biologically potent probe.

EXPERIMENTAL Materials. The synthesis and purification process of 9-MA has been reported earlier.23 The molecular containers, viz. β-cyclodextrin (β-CD), methyl β-cyclodextrin (mβ-CD) and cucurbit[7]uril hydrate (CB7) were obtained from Sigma Chemical Co., USA and were used as received (vide Scheme 1 for the structures of the probe and the macrocycles). Triply distilled de-ionized Milli-Q water (Millipore) was used for spectral measurements. Purity of the solvents was further checked by a silent fluorescence spectrum in the wavelength range under consideration. In order to avoid aggregation and re-absorption effects, we kept the concentration of the probe in the range of ~10-6 M. Potassium Iodide (KI) was procured from E-Merck and was used without further purification. Instrumentation and Methods. Steady-State Spectral Measurements. The steady state absorption and emission spectral measurements were carried out respectively on a Hitachi UV-Vis U-3501 spectrophotometer and a Perkin-Elmer LS55 fluorimeter. The obtained spectra were


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properly rectified for background responses using a similar set of solutions without the probe, to avoid any spectral interference. All the experiments were performed at 298K, unless specified otherwise. Only freshly prepared solutions were subjected to spectroscopic measurements. Time-Resolved Fluorescence Measurements. Fluorescence lifetimes were obtained by the method of Time Correlated Single-Photon counting (TCSPC) on FluoroCube-01NL spectrometer (Horiba Jobin Yvon) equipped with a TC-125 peltier-controlled cuvette holder (Quantum Northwest). Laser-Diode of 375 nm was used as the excitation source and the fluorescence signals at the respective emission maxima were collected at the magic angle (54.7ο) to negate any significant involvement arising from fluorescence anisotropy decay.24 The characteristic time resolution of the setup was ~100 ps. The obtained decays were deconvoluted using DAS-6 decay analysis software and the suitability of the fits was evaluated by χ2 criteria and scrutiny of the residuals of the fitted functions. Mean (average) fluorescence lifetimes (〈〈 τf〉) were computed using the following equation:24

τ

α iτ i2 = ∑ α iτ i

(1)

∑

f

in which αi is the amplitude corresponding to the ith decay component, τi. To obtain the time-resolved ﬂuorescence anisotropy decays, the ﬂuorescence decays corresponding to parallel [IVV] and perpendicular [IVH] emission polarizations with respect to a fixed vertical excitation polarization were collected at the emission maxima of the fluorophore. The anisotropy decay function r(t) was constructed from these IVV and IVH decays using the following equation:24


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r (t ) =

(2)

I VV − G.I VH I VV + 2G.I VH

in which G is the correction factor for the detector sensitivity of the instrument.

RESULTS AND DISCUSSION In aqueous medium, 9-MA displays a broad and structured absorption spectrum with a maximum at ~ 360 nm, which is the impression of the π–π* type S1←S0 transition of the anthracene chromophore.23 Among all the cyclodextrins used in this study, only β-CD is found to impart visible changes in the spectrum (and hence after only the β- variety has been used in spectroscopic experiments). On addition of β-CD to the aqueous solution of 9-MA, the absorbance is found to decrease without any substantial shift of the band maximum (vide Figure S1a in the supplementary information), possibly indicating the formation of an inclusion complex which results in a reduced exposure of the probe towards the bulk. Similar results were obtained in the case of methyl β-CD (vide Figure S1b); and the decrement in absorbance is found to be similar with that in case of β-CD signifying that both the CDs provide commensurate protection to the probe. Owing to its comparable cavity dimension to that of β-CDs, we have extended this study to CB7 macrocycle. Akin to the observations reported above, with increasing concentration of CB7 in the solution, the absorbance of the probe is found to decrease, although to a lesser extent (with respect to the β-CDs, vide Figure S1c), possibly inferring that although the mode of complexation is identical in both the cases, the overall protection conveyed to the probe by CB7 is comparatively less.


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The emission spectrum of the probe is characterized by an amply Stokes shifted, unstructured, broad, polarity responsive band having a maximum at ~ 485 nm in aqueous medium.23 Gradual addition of β-CD, methyl β-CD and CB7 to the aqueous solution of 9MA lead to significant augmentations in emission intensity along with considerable blue shifts in emission wavelength (~ 15 nm, ~ 18 nm and ~ 28 nm for β-CD, methyl β-CD and CB7 respectively, vide Figure 1). Such shift in the emission wavelength can be interpreted in terms of inclusion of the probe molecules within the hydrophobic cavities of the concerned supramolecules, resulting reduced polarity in the neighborhood of the probe as compared to the aqueous phase. This inference is thoroughly supported via a scrutiny of the emission spectra of the probe in solvents having lower polarity.23 The reduced polarity within the core of the macrocycles amplifies the energy difference between the triplet/ground state and the emissive state of the probe leading to a reduction of the non-radiative decay channels and, hence, the emission yield improves.25 To acquire an insight of the polarity in the immediate vicinity the probe inside the cavity of the macrocycles, a calibration plot scrutinizing the energies associated with the emission maximum of 9-MA in different compositions of water–dioxane mixtures having accurately known polarity in terms of the standard ET(30) scale, a scale quantifying the ionizing power (loosely polarity) of a solvent, based on the maximum wavenumber of the longest wavelength electronic absorption band of 2,6-Diphenyl-4-(2,4,6-triphenyl-1pyridinio)phenolate in a given solvent (or mixture of solvents)26, has been used (vide Figure 2a; however, considering the possibilities of specific solvation of the probe in this solvent mixture as well as the gross approximation that these binary solvents mimic the real biomimicking environment, the ET(30) value thus obtained should be assessed as


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only a qualitative one for the sake of comparison). The thus determined micropolarity values of the cyclodextrin cavities are found to be 55.15, 52.72 and 44.18 for β-CD, methyl β-CD and CB7 respectively, which are appreciably less as compared to bulk water (ET(30)= 63.1). The aforesaid figure also reveals that methyl β-CD provides a more hydrophobic environment to the probe as compared to β-CD which can be connected to the presence of hydrophobic methyl groups.10 Interestingly, our result concerning the polarity of the CB7 cavity differ significantly from literature reports, which suggest that the polarity within CB7 cavity is comparable to that inside β-CD.3 The underlying reason has been discussed in the forthcoming section. To quantitatively determine the strength of the binding interactions of the probe with the studied supramolecules as well as the stoichiometry of the formed inclusion complex, the emission data have been analyzed using the following modified Benesi–Hildebrand equation,27

1 1 1 = + ( I − I 0 ) ( I1 − I 0 ) ( I 1− I 0 )K a [S ]n

(3)

in which I0, I and I1 are the respective emission intensities in the absence of, at intermediate and infinite concentration (indicating saturation of interaction) of the added supramolecule S, Ka is the association constant whereas n represents the stoichiometry of the formed complex (n = 1 or 2) with respect to the supramolecule. Thus, a plot of 1/[I – I0] vs. 1/[S]n should yield a straight line for the correct stoichiometry (n). In case of βCDs, the graphs of 1/[I – I0] vs. 1/[S] (n = 1) demonstrate linear variations (vide Figure 2b) with excellent correlation factors (R = 0.9961 and 0.9997 respectively for β-CD and methyl β-CD), confirming the formation of 1:1 inclusion complex between the probe and the β-CDs. However, in case of CB7, only a plot of 1/[I – I0] vs. 1/[S]2 is found to show a


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linear correlation (vide Figure 2c, the corresponding correlation factor being 0.9975, the plot of 1/[I – I0] vs. 1/[S] for CB7 shows an upward curvature, indicating higher order complex formation, vide Figure S2 in the supplementary information), inferring a 1:2 complexation between the guest and the hosts. For a quantitative idea of the strength of binding, the binding constants (Ka) have been determined using the ratio of the corresponding intercept and slope of the aforesaid plots and the calculated values are 65.62 M-1, 113.03 M-1 and 2.83×107 M-2 (± 5%) respectively for 9-MA/β-CD, 9MA/methyl β-CD and 9-MA/CB7 complexes, ensuring the binding strength to follow the order CB7 >> methyl β-CD > β-CD. However, since the strength of the binding process between the probe and the macrocycles is not proportionally related to the extent of protection awarded to the probe by the macrocycle, we refrain from drawing a comparison between the absorption and emission spectral data. It is now pertinent to discuss the results regarding the polarity inside the nanocavities of the molecular containers used in this study. It is a well known fact that the polarity in the interior of β-CD is comparable to that of tetrahydrofuran (THF, ET(30)= 37.4),28 but it should be emphasized that using a solvatochromic probe one can obtain a polarity analogous to THF within the β-CD cavity only if the probe is completely secluded inside the cavity (i.e. it is not exposed to the bulk water in any manner). Considering the dimensions of our probe (the axial length is ~ 9.5 Å and the equatorial lengths are ~ 7.8 Å and ~ 5 Å for the substituted and the unsubstituted rings respectively) and that of β-CD (the inner radius and the depth of the cavity being ~ 6 Å and ~ 8 Å respectively10), it can be easily concluded that an axial inclusion is not at all possible. As far as an equatorial insertion is concerned, it is logical to assume that only one unsubstituted aromatic ring


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(or one unsubstituted ring along with some portion of the substituted middle ring) can be inserted within the aforesaid molecular container. In that case a significant portion of the probe will still be exposed to bulk water; thereby the average polarity experienced by the probe would increase. However, in case of CB7, the stoichiometry of the inclusion complex is 1:2, i.e. two cucurbit units are shielding the probe from external exposure. Assuming the cavity radius of CB7 and β-CD to be comparable; we propose that two CB7 units encapsulate two terminal unsubstituted rings along with some portion of the substituted ring in such a fashion that only the –COOMe group and a minor segment of the substituted ring remain exposed to the bulk water, which drastically reduces the polarity experienced by the probe, resulting an ET(30) value (44.18) close to that of THF. The aforementioned binding trend, obtained from Benesi-Hildebrand analysis, is further supported by the steady state anisotropy data (vide Figure 3a), which is known to serve as an indicator of the extent of motional restriction enforced on the probe and thereby permitting us to compare of the extent of stringency exerted by different supramolecular hosts.24 As is seen from the figure, with increasing concentration of the supramolecules, the anisotropy increases from that in aqueous solution, and finally reaches a plateau signifying the saturation of interaction, concluding that the probe experiences substantial motional restriction being encapsulated within the host cavities compared to the aqueous medium. An increased value of the anisotropy parameter (r) at the saturation concentration of CB7 (~ 0.29) compared to that in methyl β-CD (~ 0.24), which in turn is higher compared to that in β-CD (~ 0.16), indicates that CB7 provides a stiffer environment to the probe molecule than the β-CDs ( as well as methyl β-CD imparts more motional restriction on the guest probe than β-CD), again confirming the


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strength of binding to follow the order CB7 > methyl β-CD > β-CD (though the substantial difference in the binding constants between the 9-MA/CB7 and 9-MA/β-CDs, as has been observed from steady state emission measurements is not manifested here). A closer scrutiny of the anisotropy profiles however reveals an interesting observation. In case of β-CDs, the anisotropy profile exhibits a sigmoidal shape, i.e. at the initial stage, with increasing concentration of the β-CDs, the anisotropy parameter (r) shows of an approximately exponential growth; then, with further increase in concentration, the growth slows, and finally saturates at a limiting value, whereas in case of CB7, the shape of the profile is analogous to an exponential growth towards a limiting value (with increasing concentration of CB7, the anisotropy parameter increases very rapidly at first, and then level off to become asymptotic). Thus, it is evident that the rate of increase in emission anisotropy (with respect to the added host) is also greater in the case of CB7 than in the β-CDs. This observation can be rationalized invoking the dimension of the formed host-guest complexes, which is one of the most prominent contributing factors in emission anisotropy; the larger the dimension of the complex, the larger would be the time corresponding to its overall tumbling motion, thereby contributing more to the emission anisotropy.29 Since, in the present case, the stoichiometry of the host-guest complexes between 9-MA and the supramolecules are 1:1 and 1:2 respectively for β-CDs and CB7; it is expected that in the latter case the dimension of the formed complex would be larger (since the molecular dimension of β-CD and CB7 are comparable), leading to an enhanced fluorescence anisotropy. To further assess the probable locations of the probe within the concerned supramolecular assemblies, iodide induced emission quenching study has been


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undertaken. An ionic quencher has been chosen due to the fact that quenching anion is expected to be preferentially present in the bulk solvent instead of the hydrophobic interior of the concerned macrocycles. Hence, the deeper the fluorophore would be embedded within the macrocyclic cavity, the lesser will be the extent of quenching suggesting a greater efficacy of the host system to shield the fluorophore from external influence thereby furnishing a comparative account of the encapsulation efficiency of the macrocycles towards the guest.24 The quenching phenomenon has been followed using the typical Stern-Volmer relation: 24 I0 =1+ K I

SV

[Q ]

(4)

in which I0 is the emission intensity of the fluorophore (9-MA) in the absence of the quencher Q (here iodide); I is the quenched intensity of the fluorophore in the presence of the quencher; and KSV is the Stern-Volmer quenching constant. A higher magnitude of KSV indicates proficient quenching, ascertaining that the probe molecule is more accessible to the quencher. Figure 3b displays the Stern-Volmer plots for iodide–induced quenching of 9-MA in various experimental conditions, a glance at which at once infers that the extent of fluorescence quenching of 9-MA in all the concerned supramolecular assemblies is appreciably lower than that in the aqueous medium (KSV = 1343.65 ± 11.54 M-1) indicating that the probe is encapsulated within the hydrophobic interiors of the concerned supramolecules (although to different extents) thereby enjoying ample protection from external influences. But surprisingly, the aforesaid figure reveals that the KSV value in CB7 (889.64 ± 7.79 M-1) is considerably greater than that in β-CD (653.04 ± 5.83 M-1) which in turn is greater than that in methyl β-CD (412.97 ± 8.72 M-1), which demonstrates the extent of protection the probe molecule (by the quencher) within the


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concerned supramolecular assemblies to follow the order methyl β-CD > β-CD > CB7. Since, in the preceding section we have concluded that the binding strength of the probe with the macromolecules follows the order CB7 > β-CD > methyl β-CD; the only pertinent logic to substantiate this anomaly is to consider the fraction of probe molecules encapsulated within the hosts. If the fraction of unbound probe considerably outweighs that of the bound probe, prominent quenching will be observed, irrespective of the extent of encapsulation of the bound fraction, which might be the scenario here and this logic is also in line with our absorption spectral data. Time Resolved Emission Study. Time Resolved Fluorescence Emission Decay. To monitor how the fluorescence lifetime of 9-MA is perturbed from the aqueous medium to the supramolecular assemblies; we have collected its fluorescence decays in all the concerned microheterogeneous environments (vide Figure 4). In aqueous medium, 9-MA demonstrates a bi-exponential decay comprising of a fast component (~ 310 ps) with principal contribution (99.4%) and another comparatively slower component (3.14 ns) having only a meager amplitude (0.6%), ascribed respectively to the unbound (free) fluorophore and its solvated (hydrated) cluster.23 The lifetime decay curves of 9-MA in all the concerned supramolecular assemblies can be satisfactorily deconvoluted using a simple bi-exponential fitting function. The obtained bi-exponential decays are distinguished by a faster component τ1 which bear a resemblance with that of the unbound fluorophore, and a comparatively slower decay component τ2, having a significantly higher magnitude than that of the unbound (free) fluorophore in solution immediately implying that the species corresponds to a binding phenomenon (vide Table


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1). Thus the faster and the slower component in the fluorescence decays have been assigned to the free and the supramolecule-encapsulated probe, respectively. When the probe molecules are enveloped within the hydrophobic nanocavities of the molecular containers, lesser accessibility of water leads to a suppression of the non-radiative decay channels, thereby producing an enhancement of the lifetime of the encapsulated guest.30 Moreover, our preceding conclusion that CB7 provides a superior protection to the encapsulated guest from the neighboring water molecules than the β-CDs is further confirmed via a comparison of the magnitudes of the slower lifetime components τ2 in the supramolecular assemblies (vide Table 1). However, with increasing concentration of the hosts, the lifetime component τ1 (corresponding to the free probe) also demonstrates a perceptible increase (the order of increment being methyl β-CD > β-CD > CB7; being proportionate to the concentrations of the hosts being used), which has been attributed to the increased bulk viscosity of the solution due to the presence of the hosts. Moreover, the amplitude associated with the slower lifetime component (α2), which signifies the supramolecule-encapsulated fraction of the guests, shows a progressive increase with the concentration of the hosts along with a concerted decrement in the amplitude of the unbound probe (vide Table 1). It is however, interesting to note that the relative amplitude of the slower lifetime component (α2) at the point of saturation of interaction, is found to follow the order, methyl β-CD > β-CD > CB7, indicating the highest degree of encapsulation of the probe within methyl β-CD and least within the cavity of CB7, substantiating our conjecture regarding the fluorescence quenching experiment. It is now pertinent to discuss the reason behind the apparent anomaly between steady state and time resolved emission spectral results. Two factors are to be considered here.


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First, our earlier studies involving 9-MA in homogeneous solvents differing in polarity23 indicates that with increase in polarity, the emission yield of the compound reduces to a noteworthy extent, but this decrease is not linear for the solvents capable of forming hydrogen bond, e.g. alcohols, due to a stabilizing hydrogen bonding interaction between the oxygen atoms present in the probe and the solvents, which leads to a more prominent drop in the emission yield, which is believed to be caused via the operation of additional non-radiative channels (which are assisted by the formed hydrogen bonds). Thus, the increase in emission yield from polar aprotic acetonitrile (ET(30) = 46) to non-polar carbon tetrachloride (ET(30) = 32.5) is much more rapid than that from polar protic water (ET(30) = 63.1) to acetonitrile, viz. from water to ethanol (ET(30) = 51.9, analogous to that encountered by 9-MA within the cavities of the β-CDs) the increment in emission yield is only four fold (from ~ 0.01 in water to ~ 0.04 in ethanol), whereas in DMF (ET(30) = 43.8, comparable to that encountered by 9-MA within the CB7 cavity) the increase in emission yield is twelve fold (~ 0.12). Since, in general emission yield is proportional to emission intensity and for a probe in a medium containing a macrocycle (as is the case here); the obtained emission profile is a convolution of emissions originating from the encapsulated as well as the free form, therefore, if we consider two scenarios, (i) only a small fraction of the probe is encapsulated within the macrocycle (the remaining fraction is free), but the encapsulation renders exceptional hydrophobicity and motional restriction on the bound probe and (ii) a substantial fraction of the probe is encapsulated but the hydrophobicity and restriction imparted on the probe is much less than in the previous case, then the intensity of the emission spectral profile in the former situation could be greater than in the latter one. Thus, steady state emission spectral


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profile is not a reliable source to extract information about the fraction of probe encapsulated within a supramolecule (similar argument is valid for steady state anisotropy analysis as it solely depends on steady state emission intensity). Second, it is to be emphasized that the binding constant is a measure of the stability of the formed complex; it is not at all related to the kinetic aspect of the reaction, i.e. to the activation barrier of the involved process.31 We thus recommend that the reaction between the probe and CB7 involves a much greater activation barrier owing to an entropic disadvantage associated with a 1:2 complexation process (than the reactions involving β-CDs, where 1:1 complexes are formed); therefore, though the exceptional stability of the formed complex might lead to anticipate a greater fraction of the said species to exist, only a minute fraction of the reactants (i.e. 9-MA and CB7) are capable of surmounting the energy barrier at ambient temperature, eventually furnishing the opposite observation. To experimentally support our assumption, we have collected time resolved emission decays of the probe-CB7 system at different temperatures (within the range 288K–323K, Figure 5). A linear increment of the relative amplitude corresponding to the longer lifetime component α2, representing the abundance of the probe–CB7 complex, with increasing temperature (which in turn induces an increment in the average lifetime, vide Table 2) convincingly suggests that with increasing temperature the formation of the inclusion complex is favored. It is readily comprehensible that with the involvement of external thermal energy, the fraction of reactants able to surpass the activation barrier is increased, which is manifested by an improved concentration of the reaction product/s, thereby thoroughly corroborating our argument. It is also stimulating to note that the variation of the magnitude of the longer lifetime component τ2 with temperature gives rise to a bell-


17


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Page 18 of 40

shaped curve (vide Figure S3 in the supplementary information); i.e. with an increase in temperature, the lifetime component τ2 gradually attains a maximum and then falls off with further increase in temperature. While the initial increase can be attributed to the stability of the formed complex; an elevated temperature is likely to enhance nonradiative decay rates, leading to a decrease in the lifetime component. Time-Resolved

Fluorescence

Anisotropy

Decay.

Time-resolved

fluorescence

anisotropy decay is associated with the time-dependent reorientation of the emission dipole of the concerned fluorophore and the corresponding correlation time (τr); which usually is slower in case of the fluorophore being enveloped within a microheterogeneous assembly as compared to while revolving freely in solution, therefore serves as an actuating tool to acquire information regarding the motional restriction exerted on the probe.24 In aqueous medium, the fluorophore demonstrates single exponential anisotropy decay with a rotational correlation time of ~ 100 ps.33 In the presence of the β-CDs, the decays exhibit bi-exponential characteristics which comprise of two rotational components, a faster one (τ1r) having magnitude akin to that of the free probe in aqueous medium and a relatively slower one (τ2r); confirming the encapsulation of the probe within the β-CD cavities, leading to severe rotational restriction to the probe (vide the inset of Figure 6a and Table 3). To compare the degree of motional stringency suffered by the fluorophore within the β-CD cavities, the magnitude of the slower component as well as the average rotational correlation time (〈τr〉) have been used which clearly suggest that the rotational dynamics of the probe in methyl β-CD is slower than that in β-CD, corroborating well to our preceding arguments. To deduce the origin of the relaxation time constants within the macromolecules, three possible relaxation pathways has been


18

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considered: (i) the unbound or free fluorophore rotates within the solution, (ii) the entrapped fluorophore rotates within the macrocycle; and (iii) the rotation of the entrapped fluorophore itself is not possible, but the macrocycle carrying the probe itself rotates eventually leading to a depolarization of fluorescence.32,33 Bearing the above possibilities in mind, the relaxation component τ1r having comparable amplitudes in both β-CD and methyl β-CD (vide Table 3), has been assigned to be originating from of the rotation of the free probe in solution. To confirm which one of the remaining two scenarios leads to the origin of the second component τ2r, the rotational relaxation times of the macromolecules as a whole (τL) have been theoretically calculated using the renowned Stokes–Einstein-Debye (SED) relation:34

τ

L

=

4 η π R h3 × 3 k BT

(5)

where η represents the bulk viscosity of the solution, Rh is the hydrodynamic radius of the concerned macromolecules,8,9 kB is the Boltzmann constant and T is the experimental temperature. The calculated rotational relaxation times of the whole macrocycles (β-CDs) are found to be significantly higher than the corresponding fluorescence depolarization times (vide Table S1 in the supplementary information); confirming that the component τ2r originates from the rotation of the host entrapped fluorophore, rather than from the rotation of the guest-bound macrocycle as a whole. However, in the presence of CB7, the probe demonstrates a “dip-and-rise” decay pattern (vide Figure 6a) which is regarded as a characteristic for the co-existence of at least two populations having significantly different values of both fluorescence lifetime and rotational correlation time.24,35,36 Such kind of time-resolved anisotropy behavior can be elucidated using an associated exponential model proposed by Ludescher et. al,35 in


19


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Page 20 of 40

which the fluorescence lifetime and amplitude of the total intensity decay components are explicitly linked with individual anisotropy parameters via the following relation: r ( t ) = r (0 )∑ n

i =1

 − t f i ( t ) exp   θi

  

(6)

where, fi (t ) =

α i exp( − t τ i ) I T (t )

(7)

in which fi(t) is the time-dependent weighing factor, IT(t) is the total emission intensity decay, θi is the ith rotational correlation time, αi, τi are the ith fluorescence amplitude and lifetime, respectively, and r(0) is the initial pre-rotational anisotropy. Now, it is comprehensible that the aforementioned dip-and-rise type anisotropy profile is best understood for systems having a reasonably low lifetime in unbound condition (i.e. in aqueous medium, where internal motions favor rapid non-radiative deactivation of the emissive state) and an amplified enough lifetime in the bound condition (in this case, the macromolecule-encapsulated condition where the internal motions are severely restricted). We thus have attributed the faster motion to the solvent exposed (free) probe and the slower motion to the bound counterpart (rotation of the drug along with a part or the whole of the macromolecule, the fitted parameters are shown in Table 3), resulting the dip-and-rise pattern in the anisotropy decay profile. It is also stimulating to observe that the prominence of the dip-and-rise pattern is progressively improved with the increase in temperature (vide Figure 6b), further reinforcing our conjecture.

CONCLUSION


20

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The present work demonstrates the study of interaction of a promising chloride channel blocker 9-MA with two different classes of molecular containers, viz. βcyclodextrins and cucurbit[7]uril, having comparable cavity dimension. An inspection of the spectral data reveals that CB7 encapsulates the probe more effectively in a 1:2 fashion whereas the β-CDs can only encapsulate some portion of the probe via forming a 1:1 complex. Interestingly, even though the probe–CB7 complexation is thermodynamically much more favorable than the probe–β-CD complexations, only a small fraction of the former complex is formed, whereas the fractions of the latter complexes are significantly high. This apparent anomaly has been answered on the basis of the proposition that since the formation of a 1:2 complex is entropically challenged, the activation barrier of the corresponding reaction is reasonably high, thereby only a small fraction of the reactants are able to surpass the energy barrier to form the products. This proposition has been thoroughly corroborated using fluorescence lifetime measurements at different temperatures. Therefore, the present results provide the detail pharmacokinetic behavior of the drug 9-MA and we are optimistic to extend this work to other biomimetics and bioenvironments for a thorough understanding of the biological implications of the drug.

Acknowledgements AG and SG gratefully acknowledge Senior Research Fellowships respectively from CSIR and UGC, New Delhi, Govt. of India. NG likes to acknowledge UPE and CRNN, CU and DST, India for financial assistance.

Associated Content


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Page 22 of 40

Supporting Information: Absorption spectra of 9-MA in the presence of increasing concentrations of the molecular containers ( β-CD, mβ-CD and CB7), Benesi-Hildebrand plot of corresponding to the 1:1 association between 9-MA and CB7, Variation of the longer lifetime component (τ2) as obtained from time-resolved fluorescence decay profiles of 9-MA in CB7 with increasing temperature, and comparison of the second rotational relaxation components τ2r with the theoretical rotational relaxation times (τL) of the macrocycles as a whole using the SED relation.


22

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Thomas, S. S.; Bohne, C. Determination of the kinetics underlying the pKa

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Page 28 of 40

FIGURE CAPTIONS: Figure 1: Representative emission spectra of 9-MA (λex = 360 nm) in the presence of increasing concentration (a) β-CD (Curves i → ix correspond to [β-CD] =0, 1, 2, 3, 4, 5, 6, 8, 10 mM respectively) (b) methyl β-CD (Curves i → ix correspond to [mβ-CD] = 0, 1, 3, 5, 7, 9, 11, 15, 20 mM respectively) and (c) CB7 (Curves i → ix correspond to [CB7] = 0, 30, 50, 75, 100, 150, 200, 300, 400 µM respectively). [9-MA] was kept fixed at ca. 2 µM in all the measurements. Figure 2: (a) The plot of the variation of emission maxima of 9-MA in dioxane-water mixture against ET(30) values indicating the micropolarity of the binding sites within the macrocycles. (b) Benesi-Hildebrand plot of 1/[I -I0] vs 1/[CD] (mM-1) for binding of 9MA with the β-CDs as indicated in the figure legends. (c) Benesi-Hildebrand plot of 1/[I I0] vs 1/[CB7]2 (µM-2) for binding of 9-MA with CB7. In both the B-H plots, each data point is an average of 5 individual measurements. The error bars are within the marker symbols if not apparent. [9-MA] was kept fixed at ca. 2 µM in all the measurements. Figure 3: (a) Variation of steady-state fluorescence anisotropy (r) of the probe as a function of macrocycle concentration as indicated in the figure legends. Each data point in the diagrams showing anisotropy variation is an average of 10 individual measurements. The error bars are within the marker symbols if not apparent. (b) SternVolmer plots for fluorescence quenching of 9-MA (λex = 360 nm) by iodide in various conditions as indicated in the figure legends. Concentrations of β-CD, methyl β-CD and CB7 were fixed at 10 mM, 20 mM and 400 µM respectively. Each data point is an average of 5 individual measurements. The error bars are within the marker symbols if not apparent. [9-MA] was kept fixed at ca. 2 µM in all the measurements.


28

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 4: Representative time-resolved fluorescence decay profiles (λex = 375 nm, λmonitored = λmax(em) ) of the probe 9-MA (a) in the presence of increasing β-CD concentration. Curves i → v correspond to [β-CD] = 0, 1, 2, 5 and 10 mM respectively. (b) in the presence of increasing methyl β-CD concentration. Curves i → v correspond to [mβ-CD] = 0, 2, 5, 10 and 15 mM respectively. (c) in the presence of increasing CB7 concentration. Curves i → vi correspond to [CB7] = 0, 30, 50, 100, 200 and 400 µM respectively. The sharp black profile on the extreme left in all the plots represents the instrument response function (IRF). [9-MA] was kept fixed at ca. 2 µM in all the measurements. Figure 5: Representative time-resolved fluorescence decay profiles of 9-MA (λex = 375 nm, λmonitored = λmax(em) ) with increasing temperature in a solution containing 400 µM CB7. Curves i → v correspond to T (temperature) = 288, 293, 298, 308 and 323 K respectively. The sharp black profile on the extreme left in all the plots represents the instrument response function (IRF). [9-MA] was kept fixed at ca. 2 µM in all the measurements. Figure 6: Fluorescence depolarization profile of the probe 9-MA in (a) different supramolecular environments (concentrations of β-CD, methyl β-CD and CB7 were fixed at 10 mM, 20 mM and 400 µM respectively) as mentioned in the figure legends and (b) 400 µM CB7 at two different temperatures (vide figure legends). [9-MA] was kept fixed at ca. 2 µM in all the measurements.


29


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Page 30 of 40

Table 1: Time-Resolved Fluorescence Decay Parameters of 9-MA with Increasing Concentrations of the Macrocycles α2 τ1 τ2 χ2 β-CD Conc. α1 〈τf〉 (mM) (ns) (%) (%) ( ps) (ns)

m-β-CD

CB7

0

99.35

0.66

310

3.14

0.51

0.98

1

97.49

2.51

313

3.48

1.04

1.02

2

94.39

5.61

318

4.04

1.92

1.05

4

88.56

11.44

359

4.39

2.83

1.09

7

85.05

14.95

404

4.71

3.30

1.11

10

81.38

18.63

472

4.96

3.64

1.13

0

99.35

0.66

310

3.14

0.51

0.98

2

90.44

9.56

417

4.26

2.32

1.13

5

87.47

12.53

453

4.63

2.93

1.12

8

85.96

14.04

562

5.05

3.23

1.09

12

73.65

26.35

675

5.80

4.54

1.07

15

64.57

35.43

758

6.27

5.27

1.02

Conc. (µM) 0

99.35

0.66

310

3.14

0.51

0.98

30

99.13

0.87

312

5.73

1.06

0.97

50

98.94

1.06

318

7.03

1.60

1.01

100

98.70

1.30

331

9.09

2.66

1.09

200

98.30

1.70

340

9.33

3.24

1.12

400

96.46

3.54

371

10.56

5.58

1.19

Conc. (mM)


30

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Table 2: Time-Resolved Fluorescence Decay Parameters of 9-MA in CB7 as a Function of Temperature α2 τ1 τ2 χ2 [CB7] Temp. α1 〈τf〉 (K) (%) (%) ( ps) (ns) (ns)

400 µM

288

97.90

2.10

358

10.05

4.00

1.09

293

97.37

2.63

362

10.19

4.61

1.11

298

96.46

3.54

371

10.56

5.58

1.19

303

95.42

4.58

365

11.10

6.74

1.16

308

95.05

4.95

367

10.95

6.80

1.19

313

94.46

5.54

374

10.78

6.91

1.15

318

94.03

5.97

376

10.61

6.95

1.18

323

92.96

7.04

382

10.39

7.12

1.20


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Page 32 of 40

Table 3: Time-Resolved Fluorescence Anisotropy Decay Parameters of 9-MA in Different Macrocycles α2r Environment α1r τ1r 〈τr〉 τ2r χ2 (ps) (ps) (ps) β-CD

0.775

0.225

132

658

443

1.06

mβ-CD

0.641

0.359

243

865

658

1.04

Environment

f1r

f2r

θ1r

θ2r

〈θr〉

χ2

(ps)

(ns)

(ns)

CB7 (298K)

0.925

0.075

110

2.18

1.39

1.09

CB7 (323K)

0.891

0.109

145

2.42

1.67

1.12


32

Page 33 of 40

480

(a)

Em. intensity (a.u.)

360

ix

300 240

[β-CD]

180

i

120 60 0

(b)

ix

400 320

[mβ-CD]

240

i

160 80 0

400

450

500

550

600

400

Wavelength (nm)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


600

450

500

550

Wavelength (nm)

(c)

ix

500 400

[CB7]

300

i

200 100 0 385

440

495

550

605

Wavelength (nm)

Figure 1


33

600


0.060

22400 β-CD mβ-CD

22000

0.045

1/(I-I0)

21600 21200

0.030

β-CD mβ-CD

0.015

20800

(a)

20400 35

40

45

50

55

-1

60

0.000 0.0

65

0.060

0.2

0.4

0.6

0.8

1/[CD] in mM

ET(30) kcal mol

1/(I-I0)

em

(b)

CB7

-1

Εmax(cm )

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 34 of 40

(c)

0.045 0.030 0.015 0.000 0.0000

0.0003

0.0006

0.0009

0.0012

2

1/[CB7] in µM

Figure 2


34

1.0

Page 35 of 40

(a)

0.25

β -CD mβ -CD

0.24

0.20 0.15 0.10 0.05

0.20 0.16

0.08

16 8

2

4

6

8

10 12 14

0.00 150

(b)

[CD] in mM 0

75

24

0.12

0.04

0

9-MA in water 9-MA + CB7 9-MA + β-CD 9-MA + mβ-CD

32

CB7

I0/I - 1

0.30

40

Anisotropy (r)

Anisotropy (r)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


225

300

375

450

0 0

[CB7] in µM

5

10

15

20

[KI] in mM

Figure 3


35

25

30


(a)

(b)

v log (Counts)

1000

[β-CD] 100

i

v

1000

10

[mβ-CD] 100

i 10

0

10

20

30

0

40

10

20

30

Time (ns)

Time (ns)

(c) log (Counts)

log (Counts)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 36 of 40

vi

1000

[CB7] 100

i 10 0

15

30

45

60

75

Time (ns)

Figure 4


36

40

Page 37 of 40

v log (Counts)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1000

T (K) 100

i 10 0

15

30

45

60

75

Time (ns)

Figure 5


37


0.2

(a)

β -CD mβ -CD

0.3

r(t)

(b)

298K 323K

0.2

0.2 0.1 0.0

Time (ns)

-0.1

0.1

r(t)

r(t)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 38 of 40

0

2

4

6

8

10

0.1

CB7

0.0

0.0 0

2

4

6

8

10

0

2

4

6

8

Time (ns)

Time (ns) Figure 6


38

10

Page 39 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Scheme 1: Structures of (a) β-cyclodextrins (R= –H and –CH3 corresponds to β-

cyclodextrin and methyl β-cyclodextrin respectively), (b) Cucurbit[7]uril (CB7) and (c) 9methyl anthroate (9-MA); the molecules used in this study.

O

OR OR O RO

RO

O

O OR OR

O

RO O

O RO

RO

N

OR OR

N

N N

N N

N N

N

OO

NN

N N

NN

N N

N N

N

N N

N

O

O OR RO

RO O

N

N

O RO

OO

O O

O OR O

OR RO O

OR

O

N

N

N

O O

O O

O O

OR

O RO

(b)

(a) O

OMe C

(c)


39


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 40 of 40

TOC Graphic

MeOOC

β-CD

CB7

9-MA 1:1

MeOOC

1:2


40

Inclusion of an Anthracene-based Fluorophore within Molecular

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