Achikanath C. Bhasikuttan

Thioflavin T as an Efficient Inducer and Selective Fluorescent Sensor for the Human Telomeric G-Quadruplex DNA

The quest for a G-quadruplex specific fluorescent sensor among other DNA forms under physiological salt conditions has been addressed in this article. We demonstrate for the first time the application of a water-soluble fluorogenic dye, Thioflavin T (ThT), in a dual role of exclusively inducing quadruplex folding in the 22AG human telomeric DNA, both in the presence and absence of Tris buffer/salt, and sensing the same through its fluorescence light-up having emission enhancement of the order of 2100-fold in the visible region. Appropriate conditions allow an apparent switch over of the parallel quadruplex structure in 22AG-ThT (50 mM Tris, pH 7.2) solution to the antiparallel form just by the addition of K(+) ions in the range 10-50 mM. Moreover, addition of ThT cooperatively stabilizes the K(+) induced antiparallel quadruplexes by a ΔT(m) ∼11 °C. The distinction of ThT as a quadruplex inducer has been contrasted with the erstwhile used structurally related dye, Thiazole Orange (TO), which did not induce any quadruplex folding in the 22AG strand in the absence of salt. The striking fluorescence light-up in ThT on binding to the human telomeric G-quadruplex is shown to be highly specific compared to the less than 250-fold enhancement observed with other single/double strand DNA forms. This work has implication in designing new generation dyes based on the ThT scaffold, which are highly selective for telomeric DNA, for potential diagnostic, therapeutic, and ion-sensing applications.

Cooperative Metal Ion Binding to a Cucurbit[7]uril−Thioflavin T Complex: Demonstration of a Stimulus-Responsive Fluorescent Supramolecular Capsule

We report an intriguing noncovalent interaction of thioflavin T (ThT), a fibril diagnostic dye, with the versatile macrocyclic host molecule cucurbit[7]uril (CB7) in the presence of metal cations. ThT forms both 1:1 (CB7.ThT) and 2:1 [(CB7)(2).ThT] complexes with CB7 host, leading to specific structural arrangements. Addition of competitive guests like metal cations to the 1:1 stoichiometric complex displays expected competitive binding interactions with CB7, leading to decreased fluorescence intensity from ThT. However, addition of metal ions to the 2:1 complex leads to unusual enhancement in the fluorescence emission ( approximately 270-fold in the presence of Ca(2+) and approximately 160-fold in the presence of Na(+)). These contrasting observations on the fluorescence enhancement with change in the stoichiometric equilibrium have been investigated explicitly for a feasible binding model. Detailed photophysical characterization with supporting data from NMR and anisotropy measurements has led to the revelation of a novel stimulus-responsive cooperative metal ion binding to the stoichiometrically selected (CB7)(2).ThT complex, demonstrating a highly fluorescent supramolecular nanocapsule. The first example of a noncovalently packed fluorescent complex became feasible due to the structural arrangement of the host-guest complex in the 2:1 stoichiometry with two CB7 portals providing strong negative charge density for the metal ions to group and seal the complex, thus protecting the incorporated dye. To further strengthen the usefulness of the supramolecular capsule established here, rupture of the capsular complex has been demonstrated with a strong competitive guest, 1-amantadine hydrochloride, which helped in disrupting the capsule to release the dye. It is proposed here that by judicious design of the chromophore (guest) structure, such capsular assemblies can be explored for the binding and release of drug molecules, for fluorescence on-off systems, and as building blocks for molecular architectures displaying unique properties.

Noncovalent interaction of 5,10,15,20-tetrakis(4-N-methylpyridyl)porphyrin with Cucurbit[7]uril: a supramolecular architecture.

Noncovalent interaction of two water-soluble synthetic macromolecules, Cucurbit[7]uril (CB7) and 5,10,15,20-tetrakis(4- N-methylpyridyl)porphyrin (TMPyP), has been studied from the viewpoint of organizing them through supramolecular interactions and thereby modulating their functional activities. Steady-state and time-resolved fluorescence measurements along with NMR results establish that CB7 crowns the N-methylpyridyl group of the TMPyP in a 1:4 stoichiometry. The overall binding constant was evaluated to be approximately 4.5 x 10 (19) M (-4). The high binding affinity, promoting a stable and extendable molecular assembly in aqueous solution, could open new frontiers in the design and synthesis of higher-order supramolecular structures with photofunctional moieties and project their utility in therapeutic applications.

Contrasting guest binding interaction of cucurbit[7-8]urils with neutral red dye: controlled exchange of multiple guests

Interactions among macrocyclic hosts and dyes/drugs have been explored extensively for their direct usage in controlled uptake and release of large number of potential drug molecules. In this paper we report the non-covalent interaction of cucurbit[8]uril macrocycle (CB8) with a biologically important dye, neutral red, by absorption and fluorescence spectroscopy. A comparative analysis with the complexation behaviour of the dye with CB7, the lower homologue of CB8, indicates contrasting guest binding behaviour with significant changes in the photophysical characteristics of the dye. While CB7 interaction leads to a 1 ratio 1 stoichiometry resulting in approximately 6 fold enhancement in the fluorescence emission of the dye, CB8 displays signatures for a 1 ratio 2 host-guest stoichiometry with drastic reduction in the fluorescence emission. Apart from the evaluation of approximately 2 unit shift in the protolytic equilibrium on complexation (pK(a) shift), the measurements with tryptophan established a selective guest exchange to favour a co-localized dimer inside the CB8 cavity. In a protein medium (BSA), the 1 ratio 2 complex was converted to a 1 ratio 1 ratio 1 CB8-NRH(+)-BSA complex. The finding that NRH(+) can be transferred from CB8 to BSA, even though the binding constant for NRH(+)-CB8 is much higher than NRH(+)-BSA, is projected for a controlled slow release of NRH(+) towards BSA. Since the release and activity of drugs can be controlled by regulating the protolytic equilibrium, the macromolecular encapsulation and release of NRH(+) demonstrated here provide information relevant to host-guest based drug delivery systems and its applications.

Photophysical studies on the noncovalent interaction of thioflavin T with cucurbit[n]uril macrocycles.

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. Steady-state 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 (K(1) approximately 10(5) 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 (K(2) approximately 10(3) 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.

Control of the supramolecular excimer formation of thioflavin T within a cucurbit[8]uril host: a fluorescence on/off mechanism.

On or off? A new excimer band at lambda = 570 nm was visualized during the noncovalent host-guest interaction between thioflavin T (ThT) and cucurbit[8]uril (CB8). Controlled dissociation of this assembly in the presence of Ca(2+) was demonstrated as an on/off fluorescence switch (see picture).

Stimulus-responsive supramolecular pKa tuning of cucurbit[7]uril encapsulated coumarin 6 dye.

This article reports an efficient host-assisted guest protonation mechanism in coumarin 6 (C6) dye, upon its interaction with cucurbit[7]uril (CB7) macrocycle. C6 uptakes the CB7 macrocycle both in 1:1 and 2:1 (CB7/C6) stoichiometries, which brings out a large upward pK(a) shift (from ~0.8 to 6), and facilitates the protonation of C6 at normal pH conditions, having significant photochemical implications. Controlled dissociation of the assemblies has been achieved through their response to stimuli like temperature change or metal ions. By this approach, a specific form of the guest can be activated and could provide a simple stimulus for the controlled drug/dye delivery. Furthermore, the host-assisted guest protonation improves the stability and aqueous solubility of C6 and is a promising candidate for aqueous-based supramolecular dye laser system. Such simple protocol leading to photoswitchable systems having aqueous solubility and biocompatibility can in principle be evolved into a general strategy to deliver and operate potential functional molecular components under various trigger control.

Contrasting solvent polarity effect on the photophysical properties of two newly synthesized aminostyryl dyes in the lower and in the higher solvent polarity regions.

Solvent polarity effect on the photophysical properties of two newly synthesized aminostyryl-thiazoloquinoxaline dyes, one with a flexible diphenylamino group, namely, N,N-diphenyl-4-[2-(thiazolo[4,5-b]quinoxalin-2-yl)vinyl]aniline (TQ1), and the other with a rigid julolidinylamino group, namely, (9-[2-(thiazolo[4,5-b]quinoxalin-2-yl)vinyl]julolidine) (TQ2), have been investigated in different aprotic solvents and solvent mixtures. From the polarity dependent changes in the absorption and fluorescence spectral properties, it is indicated that the fluorescent states of the dyes are of intramolecular charge transfer (ICT) character. For both the dyes, the photophysical properties like fluorescence quantum yields (Phi(f)), fluorescence lifetimes (tau(f)), radiative rate constants (k(f) = Phi(f)/tau(f)), and nonradiative rate constants (k(nr) = 1/tau(f) - Phi(f)/tau(f)) show clearly contrasting solvent polarity effects in the lower and in the higher solvent polarity region, causing an interesting reversal in the properties below and above an intermediate solvent polarity. It is inferred that the domination of the cis-trans isomerization in the lower solvent polarity region and that of the twisted intramolecular charge transfer (TICT) state formation in the higher solvent polarity region are responsible for the observed contrasting solvent polarity effects on the photophysical properties of the two dyes. As both isomerization and TICT state formation causes an enhancement in the nonradiative decay rate of the excited dyes and both the processes become less significant at the intermediate solvent polarity region, the two dyes show their largest Phi(f) and tau(f) values at intermediate solvent polarities. Suitable mechanistic schemes have been proposed and qualitative potential energy diagrams have been presented to explain the observed results with the changes in the polarity of the solvents used.

Recognition-Mediated Light-Up of Thiazole Orange with Cucurbit[8]uril: Exchange and Release by Chemical Stimuli

This article reports a convenient supramolecular strategy to construct fluorescent photoswitchable molecular assemblies between a macrocyclic host, cucurbit[8]uril (CB8), and a fluorogenic dye, thiazole orange (TO). The interaction mechanism and the stable stoichiometric host-guest arrangements have been claimed on the basis of the optical absorption, steady-state and time-resolved fluorescence lifetime and anisotropy measurements, and also the geometry optimization studies. The CB8 recognized TO in its 2:2 stoichiometry exhibited spectacular fluorescence enhancement of the order of 1700 fold, which is the largest directly determined value so far reported for a dye in an organic macrocyclic system. This prospective 2CB8:2TO assembly responded to selected chemical stimuli such as metal ions, adamantylamine, and tryptophan, providing different dissociation mechanisms and demonstrating a controlled exchange and release action desired with such noncovalently linked assemblies. Positively, considering the aqueous solubility and biocompatibility of the host-guest constituents, this methodology can evolve into a general approach to deliver and operate intracellularly functional molecular components under chemical/thermal/optical trigger control, especially for therapeutic applications.

Supramolecular assembly of hoechst-33258 with cucurbit[7]uril macrocycle

Molecular assemblies of potential guest molecules through non-covalent host-guest interactions have found immense use in many applied areas. In this study supramolecular interaction of a biologically important dye Hoechst-33258 (H33258) has been investigated in aqueous solutions at different pHs, in the presence of a macrocyclic host, namely, cucurbit[7]uril (CB7). The pH dependent emission behaviour of H33258 is inherently connected with its protolytic equilibria which allow the dye in different geometrical conformations. This pH dependent structural orientation is greatly affected by the complexation with CB7. The significant structural changes in the monocationic H33258 brought out by CB7 at pH 7 have been documented in the fluorescence emission and lifetime data, which are comparatively less affected in case of the dicationic form, which is prominent in dye solutions at pH 4.5. The strong ion-dipole interactions provided by the carbonyl portals of the CB7 host adequately stabilize the CB7-H33258 complex, both in 1:1 and 2:1 stoichiometries at both the pH conditions. The Jobs plot method, fluorescence anisotropy, NMR measurements and geometry optimization calculations confirm the stoichiometric arrangement and are found to be tunable with the addition of metal ions. The non-covalently stabilized assembly brings out large enhancement in the fluorescence emission due to the unique structural orientation attained by H33258, which reduces the non-radiative relaxation pathways. Comparison of the spectral data of the dye at different pH conditions in the absence and presence of CB7 proposes a large upward pK(a) shift due to CB7 encapsulation, thus providing a handy tool to modulate the photophysical characteristics of the guest molecules.