J. Phys. Chem. B 2009, 113, 1891–1898
1891
Photophysical Studies on the Noncovalent Interaction of Thioflavin T with Cucurbit[n]uril Macrocycles Sharmistha Dutta Choudhury, Jyotirmayee Mohanty,* Hari P. Upadhyaya, Achikanath C. Bhasikuttan,* and Haridas Pal Radiation & Photochemistry DiVision, Bhabha Atomic Research Centre, Mumbai 400085, India ReceiVed: September 29, 2008; ReVised Manuscript ReceiVed: December 16, 2008
Noncovalent interaction of Thioflavin T (ThT) with versatile macrocyclic host molecules, namely, cucurbit[7]uril (CB7) and cucurbit[5]uril (CB5), has been investigated in aqueous solutions by photophysical methods. Steadystate and time-resolved fluorescence studies illustrate significant enhancements/modifications in the ThT fluorescence yield, lifetime, and spectral features on interaction with the CBs and are assigned due to the formation of 1:1 and 2:1 complexes between the CBs and the ThT. The high binding constant values for the 1:1 complex (K1 ∼105 M-1) indicate the strong ion-dipole interaction between the host and guest molecules, whereas the 2:1 complex formation is mainly driven by weaker forces like hydrophobic interaction as evident from the lower binding constants (K2 ∼103 M-1). From the characteristic differences in the photophysical properties of the CB7-ThT and CB5-ThT complexes, it has been adjudged that ThT forms an inclusion complex with CB7 whereas with CB5, the interaction is through an exclusion complex formation. These contentions have been further verified by the rotational relaxation dynamics, NMR, and quantum chemical calculations on CB-ThT systems. The present results have also been compared with those reported for the dye in the presence of cyclodextrin hosts. Introduction Modulating the molecular characteristics of small guest molecules by introducing noncovalent interactions through macrocyclic receptors is well suited for applications like controlled uptake and release of potential drug molecules and exploiting their cooperative effect. Cucurbiturils (CBs), one of the interesting class of water-soluble macrocyclic receptors, composed of glycoluril units coupled in a cyclic manner by the pairs of methylene bridges, have attracted considerable attention in recent years owing to their excellent physicochemical properties.1-3 The pumpkin-shaped CBs have highly symmetrical cage structures with two identical portal ends flanked with highly polarizable carbonyl groups and an interior hydrophobic cavity, quite similar to that of cyclodextrins and calixarenes.2,4 Depending upon the number of glycoluril units, many homologues of cucurbiturils with different cavity and portal dimensions are known. They are proficient in binding small guest molecules like organic dyes into their rigid cavities through hydrophobic interaction,5 or metal cations6,7 and protonated alkyl and aryl amines via ion-dipole interaction involving their carbonyl portal ends,2,8,9 to form stable inclusion complexes in aqueous solution. Such supramolecular noncovalent interactions are aptly exploited to inflict variations in the molecular properties, especially to tune the radiative properties and the excited-state dynamics in the desired manner.9-13 Demonstrating this, a large number of host-guest complexation studies have been reported with a variety of dyes, using cucurbit[n]urils, mainly cucurbit[7]uril (CB7), with proper validation for specific application area.12-15 Noncovalent, extrinsic fluorescent probes find extensive usage as local reporters in many biological applications, especially in * To whom correspondence should be addressed. Tel.: (+91) 22 25593771. Fax: (+91) 22 2550 5151. E-mail:
[email protected] (J.M.);
[email protected] (A.C.B).
various fields of protein analysis, e.g., to characterize folding intermediates and detect aggregation or fibrillation.16-18 Here, the specific interaction with the protein environment introduces considerable change in the photophysical characteristics of the dye, projecting the details of its local microenvironment.19 One of such probes, Thioflavin T, 3,6-dimethyl-2-(4-dimethylaminophenyl)benzthiazolium cation (ThT), is a benzthiazolium dye that has been used quite extensively to detect the presence of amyloid fibrils.17,20-22 Binding of ThT with protein fibrils is shown to be quite specific and it accompanies with a significant enhancement in the fluorescence yield of the dye, thus acting as a sensor for detecting the protein fibrils.17,20 In recent years, the use of ThT as an extrinsic fluorescent dye has increased significantly, due to its versatility, sensitivity, and suitability for high-throughput screening in detecting amyloid fibrils in tissues.16 Though the application of ThT for the analysis of different aggregating systems is rapidly rising, the exact mechanism for ThT binding into the cavities of the amyloid fibrils causing dramatic changes in its fluorescence properties is still largely unknown. Apart from the restriction in the torsional motion between the benzthiazole and dimethylaminobenzene groups,23,24 excimer formation within the binding site is also proposed to cause further enhancement in the fluorescence for ThT on binding with amyloid fibrils.24-26 Addressing this issue, rationalizations have been put forward considering the void volumes in amyloid fibrils and its probable binding interaction, in comparison with synthetic water-soluble macrocylic cavities.27 Needless to say, several studies have been carried out on ThT with cyclodextrin (CD) hosts to explore the binding interactions in restricted environments and subsequent fluorescence changes.24,27,28 The truncated cone-shaped CDs are the well-known macrocyclic hosts studied for host-guest interaction, where the driving force for inclusion complex formation is mainly hydrophobic in nature.24 On the other hand, as discussed above, CBs possess a unique symmetrical structure,
10.1021/jp8103062 CCC: $40.75 2009 American Chemical Society Published on Web 01/28/2009
1892 J. Phys. Chem. B, Vol. 113, No. 7, 2009
Dutta Choudhury et al.
SCHEME 1
having highly polarizable carbonyl-laced portals with a hydrophobic interior cavity. These features of CBs have been exploited here to introduce strong and multiple binding with the cationic dye, Thioflavin T, where the CBs can engulf ThT from either ends of the dye. This can provide a large stabilization of the inclusion complex via ion-dipole interaction between the carbonyl portals and the cationic charge on the dye along with the hydrophobic interaction exerted by the cavity. In the present study, we have investigated the supramolecular interaction of ThT with two cucurbituril macrocyclic receptors of differing cavity dimensions, cucurbit[7]uril (CB7) and cucurbit[5]uril (CB5), by following the changes in the photophysical characteristics of the dye. Depending on the cavity sizes, inclusion and exclusion complexes have been detected, both with 1:1 and 2:1 host-guest stoichiometric ratio. Supporting the mechanisms proposed, quantum chemical calculations have also been carried out to obtain the geometry-optimized structures of the possible complexes using Gaussian computational methods. Experimental Section CB7 and CB5 were obtained from Sigma-Aldrich and were used as received. Thioflavin T (ThT), obtained from SigmaAldrich, was purified by column chromatography using silica gel column and mildly acidic methanol as eluent. Nanopure water (conductivity less than 0.06 µS cm-1), obtained from a Millipore Gradiant A10 system, was used to prepare the sample solutions. General chemical structures of the dye and the typical cucurbituril, CB7, are shown in Scheme 1. Absorption spectra were recorded with a Jasco V530 UV-vis spectrophotometer (Tokyo, Japan). Steady-state fluorescence spectra were recorded using a Hitachi F-4500 spectrofluorimeter (Tokyo, Japan). The time-resolved (TR) fluorescence measurements were carried out using a time-correlated single-photon-counting (TCSPC) spectrometer (IBH, UK), described elsewhere.11 In the present work, a 408 nm diode laser (∼100 ps, 1 MHz repetition rate) was used for excitation and a MCP PMT was used for fluorescence detection. From the measured decay traces, the time constants were evaluated following a reconvolution procedure.29 The fluorescence decays, I(t) were analyzed using a multiexponential function as
I(t) )
∑ Bi exp(-t/τi)
(1)
i
where Bi and τi are the pre-exponential factor and the fluorescence lifetime, respectively, for the ith component of the fluorescence decay.1H NMR spectra were recorded on a Bruker Avance WB 500 MHz spectrometer at TIFR, India. Computational studies were performed with Gaussian 92 suite of package.30
Figure 1. Absorption spectra of ThT (3 µM) in aqueous solution with CB7 (A) and CB5 (B). [CB7]/mM: (1) 0.0, (2) 0.001, (3) 0.002, (4) 0.004, (5) 0.1, and (6) 1.0. [CB5]/mM: (1) 0.0, (2) 0.01, (3) 0.05, (4) 0.1, and (5) 1.0.
Results and Discussion Absorption Spectral Characteristics of ThT in the Presence of Cucurbiturils. Figure 1 shows the characteristic absorption profile of ThT in aqueous solution exhibiting maximum at 412 nm. Spectral changes were observed on introducing the macrocyclic hosts, CB7 or CB5, to the ThT solution. Incremental addition of CB7 to the ThT solution resulted in a gradual decrease in the ThT absorbance with ∼11 nm bathochromic shift and the spectral changes displayed a distinct isosbestic point at 433 nm (Figure 1A), characteristic of a complexation equilibrium. Notably, on increasing the concentration of CB7 beyond 20 µM, the absorbance increased marginally, with disappearance of the isosbestic point indicating the presence of multiple complexation equilibria in the solution. In the presence of CB5 (Figure 1B), the spectral changes were only nominal with slight decrease in the absorbance, ∼2 nm bathochromic shift and an unclear isosbestic point around 442 nm. It is true that the nature and the extent of changes on the ThT absorption characteristics are indicative of a possible difference in the interaction mode of ThT with CB7 and CB5; however, they are not large enough to extract any definite information about the complexation interaction in detail. On the other hand, significant changes were observed in the fluorescence characteristics of ThT, distinctively with CB7 and CB5, and are discussed in the following sections. Steady-State Fluorescence Characteristics of ThT in the Presence of Cucurbiturils. The emission characteristics of ThT in aqueous solution were recorded by exciting the sample at 390 nm. As presented in Figure 2, the fluorescence spectrum displayed a broad profile with a maximum at ∼490 nm having quantum yield less than 10-3 (Φf ) 0.0003).31 The low fluorescence yield in solvents of low viscosity and high polarity was attributed to the formation of a nonfluorescent charge transfer state accompanied by a change in the angle between the benzthiazole and the dimethylaminobenzene rings of ThT from ∼37° to ∼90° in the excited state.32 Notably, significant changes in the fluorescence characteristics were seen on introducing noncovalent interactions through the complexation with the CBs. As shown in Figure 2A, with increasing the amount of CB7, the fluorescence intensity (If) monitored at 490 nm increased gradually. At the highest concentration of CB7 used (1 mM), the observed fluorescence enhancement was about ∼30 fold along with ∼12 nm hypsochromic shift. However, in the case of CB5, as presented in Figure 2B, the changes in the
Noncovalent Interaction of ThT with CB7 and CB5
J. Phys. Chem. B, Vol. 113, No. 7, 2009 1893 From the binding curve presented in Figure 3, the binding constant values for 1:1 and 2:1 stoichiometries were evaluated by considering the following complexation equilibria; K1
CB + ThT h CB · ThT
(2a)
K2
CB · ThT + CB h (CB)2 · ThT
Figure 2. Fluorescence spectra of ThT (3 µM) in aqueous solution with CB7 (A) and CB5 (B). [CB7]/mM: (1) 0.0, (2) 0.004, (3) 0.05, (4) 0.35, (5) 0.6, and (6) 1.0. [CB5]/mM: (1) 0.0, (2) 0.002, (3) 0.02 (4) 0.05, (5) 0.1, and (6) 1.0. The spectral feature marked r corresponds to the Raman scattering.
where K1 and K2 are the binding constants for the formation of the respective 1:1 and 2:1 complexes. At any stage, the observed fluorescence intensity If corresponds to the sum of the fluorescence intensities arising from the free ThT, CB · ThT, and (CB)2 · ThT and are directly proportional to their respective concentrations present in the solution. Therefore, one can write
If ) I0f
[ThT]eq [CB · ThT]eq + ICB · ThT + [ThT]0 [ThT]0 I(CB)2 · ThT
Figure 3. Fluorescence titration curves of ThT in the presence of CB7 (b) and CB5 (O). The solid lines represent the fitted curves as per eq 4. Insets show the changes in the fluorescence intensity for the lower concentration range,
