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Excited-state intramolecular proton transfer (ESIPT) reactions are crucial in photoresponsive materials and fluorescent markers. The fluorescent compound 4-aminophthalimide (4-AP) has been reported to exhibit solvent-assisted ESIPT in protic solvents, such as methanol, wherein the solvent interacts with 4-AP to form a six-membered hydrogen-bonded ring that is strengthened upon excitation. Although the controversial observation of ESIPT in 4-AP has been extensively studied, the molecular mechanism has yet to be fully explored. In this study, femtosecond infrared spectroscopy was used to investigate the dynamics of 4-AP in methanol and acetonitrile after excitation at 350 and 300 nm, which promoted 4-AP to the S 1 and S 2 states, respectively. The excited 4-AP in the S 1 state relaxed to the ground state, while 4-AP in the S 2 state relaxed via the S 1 state without the occurrence of ESIPT. The enol form of 4-AP (Enol 4-AP) in the S 1 state was calculated to be ~10 kcal/mol higher in energy than the keto form in the S 1 state, indicating that keto-to-enol tautomerization was endergonic, ultimately resulting in no observable ESIPT for 4-AP in the S 1 state. Upon the excitation of 4-AP to the S 2 state, the transition to Enol-4-AP in the S 1 state was found to be exergonic; however, ESIPT must compete with an internal conversion from the S 2 to the S 1 state. The internal S 2 → S 1 conversion was significantly faster than the solvent-assisted ESIPT, resulting in a negligible ESIPT for the 4-AP excited to the S 2 state. The detailed excitation dynamics of 4-AP clearly reveal the molecular mechanism underlying its negligible ESIPT, despite the fact that it forms a favorable structure for solvent-assisted ESIPT.

Mesoporous silica nano-channel (MCM-41) based molecular switching of a biologically important anticancer drug, namely, ellipticine (EPT) has been utilized to probe its efficient loading onto MCM-41, and its subsequent release to intra-cellular biomolecules, like DNA. By exploiting various spectroscopic techniques (like, steady state fluorescence, time-resolved fluorescence and circular dichroism), it has been shown that EPT can be easily translocated from MCM-41 to DNA without using any external stimulant. Blue emission of EPT in a polar aprotic solvent, i.e., dichloromethane (DCM), completely switches to green upon loading inside MCM-41 due to the conversion from a neutral to a protonated form of the drug inside nano-pores. Powder X-ray diffraction (PXRD), N 2 gas adsorption and confocal fluorescence microscopy results confirm the adsorption of EPT inside the nano-pores of MCM-41. Here, the lysozyme (Lyz) protein has been utilized as a pore blocker of MCM-41 in order to prevent premature drug release. Interestingly, EPT is released to DNA even from the EPT-MCM-Lyz composite system, and results in intensification of green fluorescence. Electron microscopy results reveal the formation of a distinctive garland kind of morphology involving MCM-41 and DNA probably through non-covalent interactions, and this is believed to be responsible for the DNA assisted release of drug molecules from silica nano-pores. Confocal laser scanning microscopy (CLSM) imaging revealed that EPT-MCM is successfully internalized into the HeLa cervical cancer cells and localized into the nucleus. Cell viability assay results infer that EPT-MCM and EPT-MCM-Lyz showed much improved efficacy in HeLa cancer cells compared to free ellipticine.

Herein, we explored the photophysical properties of the antimalarial, anticancer drug cryptolepine (CRYP) in the presence of the macrocyclic host cucurbit[7]uril (CB7) and DNA with the help of steady-state and time-resolved fluorescence techniques. Ground-state and excited-state calculations based on density functional theory were also performed to obtain insight into the shape, electron density distribution, and energetics of the molecular orbitals of CRYP. CRYP exists in two forms depending on the pH of the medium, namely, a cationic (charge transfer) form and a neutral form, which emit at λ=540 and 420 nm, respectively. In a buffer solution of pH 7, the drug exists in the cationic form, and upon encapsulation with CB7, it exhibits a huge enhancement in fluorescence intensity due to a decrement in nonradiative decay pathways of the emitting cryptolepine species. Furthermore, docking and quantum chemical calculations were employed to decipher the molecular orientation of the drug in the inclusion complex. Studies with natural DNA indicate that CRYP molecules intercalate into DNA, which leads to a huge quenching of the fluorescence of CRYP. Keeping this in mind, we studied the DNA-assisted release of CRYP molecules from the nanocavity of CB7. Strikingly, DNA alone could not remove the drug from the nanocavity of CB7. However, an external stimulus such as acetylcholine chloride was able to displace CRYP from the nanocavity, and subsequently, the displaced drug could bind to DNA., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Reverse hexagonal (HII) liquid crystalline material based on glycerol monooleate (GMO) is considered as a potential carrier for drugs and other important biomolecules due to its thermotropic phase change and excellent morphology. In this work, the dynamics of encapsulated water, which plays important role in stabilization and formation of reverse hexagonal mesophase, has been investigated by time dependent Stokes shift method using Coumarin-343 as a solvation probe. The formation of the reverse hexagonal mesophase (HII) and transformation to the L2 phase have been monitored using small-angle X-ray scattering and polarized light microscopy experiments. REES studies suggest the existence of different polar regions in both HII and L2 systems. The solvation dynamics study inside the reverse hexagonal (HII) phase reveals the existence of two different types of water molecules exhibiting dynamics on a 120-900 ps time scale. The estimated diffusion coefficients of both types of water molecules obtained from the observed dynamics are in good agreement with the measured diffusion coefficient collected from the NMR study. The calculated activation energy is found to be 2.05 kcal/mol, which is associated with coupled rotational-translational water relaxation dynamics upon the transition from "bound" to "quasi-free" state. The observed ∼2 ns faster dynamics of the L2 phase compared to the HII phase may be associated with both the phase transformation as well as thermotropic effect on the relaxation process. Microviscosities calculated from time-resolved anisotropy studies infer that the interface is almost ∼22 times higher viscous than the central part of the cylinder. Overall, our results reveal the unique dynamical features of water inside the cylinder of reverse hexagonal and inverse micellar phases.

The excited state proton transfer (ESPT) dynamics of a potentially important anticancer drug, Topotecan (TPT), has been explored in aqueous reverse micelle (RM) using steady-state and time-resolved fluorescence measurements. Both the time-resolved emission spectrum and time-resolved area normalized emission spectrum infer the generation of excited state zwitterionic form of TPT from the excited state cationic form of TPT, as a result of ESPT process from the -OH group of TPT to the nearby water molecule. The ESPT dynamics were found to be severely retarded inside the nanocavities of RMs, yielding time constants of 250 ps to 1.0 ns, which is significantly slower than the dynamics obtained in bulk water (32 ps). The observed slow ESPT dynamics in RM compared to bulk water is mainly attributed to the sluggish hydrogen-bonded network dynamics of water molecules inside the nanocavity of RM and the screening of the sodium ions present at the interface.

The role of Mg(2+) ion in flavin (flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN)) recognition by RNA aptamer has been explored through steady state and time-resolved fluorescence, circular dichroism (CD), thermal melting (TM) and isothermal titration calorimetry (ITC) studies. A strong quenching of flavin emission is detected due to stacking interaction with the nucleobases in the mismatched region of aptamer, and it enhances manifold with increasing Mg(2+) concentrations. A comparatively lower binding affinity toward FAD compared to FMN is attributed to the presence of intramolecular 'stack' conformer of FAD, which cannot participate in the intermolecular stacking interactions with the nucleobases. CD and TM studies predict that flavin detection causes structural reformation of RNA aptamer. ITC results indicate that flavin detection is thermodynamically feasible and highly enthalpy driven., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The G-quadruplex (GQ-DNA), an alternative structure motif of DNA, has emerged as a novel and exciting target for anticancer drug discovery. GQ-DNA formed in the presence of monovalent cations (Na(+)/K(+)) by human telomeric DNA is a point of interest due to their direct relevance for cellular aging and abnormal cell growths. Small molecules that selectively target and stabilize G-quadruplex structures are considered to be potential therapeutic anticancer agents. Herein, we probe G-quadruplex and proflavine (a well-known DNA intercalator, hence acting as an anticarcinogen) association through steady state and time-resolved fluorescence spectroscopy to explore the effect of stabilization of GQ-DNA by this well-known DNA intercalator. The structural modifications of G-quadruplex upon binding are highlighted through circular dichroism (CD) spectra. Moreover, a detailed insight into the thermodynamics of this interaction has been provided though isothermal titration calorimetry (ITC) studies. The thermodynamic parameters obtained from ITC help to gain knowledge about the nature as well as the driving forces of binding. This present study shows that proflavine (PF) can act as a stabilizer of telomeric GQ-DNA through an entropically as well as enthalpically feasible process with high binding affinity and thereby can be considered as a potential telomerase inhibitor.

Photophysics and proton transfer dynamics of an eminent anticancer drug, ellipticine (EPT), have been investigated inside a biocompatible octyl-β-D-glucoside (OBG) micellar medium using steady state and time-resolved fluorescence spectroscopic techniques. EPT exists as protonated form in aqueous solution of pH 7. When EPT molecules are encapsulated in OBG micelles, protonated form is converted to neutral form in the ground state due to the hydrophobic effect of the micellar environment. Interestingly, steady state fluorescence results indicate the existence of both neutral and protonated forms of EPT in the excited state, even though neutral molecules are selectively excited, and it is attributed to the conversion of neutral to protonated form of EPT by the excited state proton transfer (ESPT) process. A clear isoemissive point in the time-resolved area normalized emission spectra (TRANES) further supports the excited state conversion of neutral to protonated form of EPT. Notably, this kind of proton transfer dynamics is not observed in other conventional micelles, such as, SDS, Triton-X and CTAB. Therefore, the observed ESPT dynamics is believed to be an outcome of combined effects of the local dielectric constant felt by EPT and the local proton concentration at the OBG micellar surface.

Graphene oxide based molecular switching of ellipticine (E) has been utilized to probe its efficient loading onto graphene oxide (GO) and subsequent release to intra-cellular biomolecules like DNA/RNA. The green fluorescence of E switches to blue in GO and switches back to green with polynucleotides. The intensified blue emission of the ellipticine-GO (E-GO) complex with human serum albumin (HSA), switches to a bluish green upon addition of dsDNA. Electron microscopy reveals the formation of distinctive 3D assemblies involving GO and biomolecule(s) probably through non-covalent interactions and this is primarily responsible for the biomolcule(s) assisted fluorescence-switching of E. To our knowledge, such morphological patterning of a GO-DNA complex is very unusual, reported here the first time and could find applications in the fabrication of biomedical devices. Moreover, our approach of direct optical detection of drug loading and releasing is very cheap, appealing and will be useful for clinical trial experiments once the cytotoxicity of GO is duly taken care.

A visible fluorescence switch of an eminent anti-carcinogen, ellipticine has been used to probe non-specific protein-DNA interaction. The unique pattern of protein-DNA complexation is depicted for the first time through field emission scanning electron microscopy (FE-SEM) images and spectroscopic techniques.

Host-guest interactions between cucurbit[7]uril (CB7) and a cardiotonic drug, milrinone, have been explored using steady state and pico-second time-resolved techniques. A novel fluorescence switch from ultraviolet (UV) to visible (cyan) is observed as a consequence of upward pKa shift of the drug inside the nano-cavity of cucurbit[7]uril.

Here, we investigate the effect of urea in the unfolding dynamics of flavin adenine dinucleotide (FAD), an important enzymatic cofactor, through steady state, time-resolved fluorescence spectroscopic and molecular dynamics (MD) simulation studies. Steady state results indicate the possibility of urea induced unfolding of FAD, inferred from increasing emission intensity of FAD with urea. The TCSPC and up-conversion results suggest that the stack-unstack dynamics of FAD severely gets affected in the presence of urea and leads to an increase in the unstack conformation population from 15% in pure water to 40% in 12 M urea. Molecular dynamics simulation was employed to understand the nature of the interaction between FAD and urea at the molecular level. Results depict that urea molecules replace many of the water molecules around adenine and isoalloxazine rings of FAD. However, the major driving force for the stability of this unstack conformations arises from the favorable stacking interaction of a significant fraction of the urea molecules with adenine and isoalloxazine rings of FAD, which overcomes the intramolecular stacking interaction between themselves observed in pure water.

Femtosecond fluorescence upconversion measurements are employed to elucidate the mechanism of ultrafast double proton transfer dynamics of BP(OH)2 inside molecular containers (cucurbit[7]uril (CB7) and β-cyclodextrin (β-CD)). Femtosecond up-converted signals of BP(OH)2 in water consist of growth followed by a long decay component (~650 ps). The appearance of the growth component (~35 ps) in the up-converted signal indicates the presence of a two-step sequential proton transfer process of BP(OH)2 in water. Surprisingly, the up-converted signal of BP(OH)2 inside the CB7 nano-cavity does not exhibit any growth component characteristic of a two-step sequential process. Interestingly, the growth component exists inside the nano-cavity of β-CD (having similar cavity size as that of CB7), inferring the presence of a two-step sequential process of PT inside the β-CD nano-cavity. The different features of PT dynamics of BP(OH)2 in the above mentioned two macrocyclic hosts may be attributed to the presence and absence of water solvation network surrounding the BP(OH)2 inside the nano-cavities of β-CD and CB7, respectively. Finally, docking and DFT calculations have been employed in deciphering the molecular pictures of the interactions between BP(OH)2 and the macrocyclic host.

Host-guest interactions between an anticancer drug, ellipticine (EPT), and molecular containers (cucurbitruils (CBn) and cyclodextrins (CD)) are investigated with the help of steady state and time-resolved fluorescence measurements. Our experimental results confirm the formation of 1:1 inclusion complexes with CB7 and CB8. The protonated form of EPT predominantly prevails in the inclusion complexes due to the stabilization achieved through ion-dipole interaction between host and positively charged drug. Drug does not form an inclusion complex with CB6, which is smaller in cavity size compared to either CB7 or CB8. In the case of cyclodextrins, α-CD does not form an inclusion complex, whereas β-CD forms a 1:1 inclusion complex with the protonated form of the drug, and the binding affinity of EPT with β-CD is less compared to CB7/CB8. Interestingly, in the case of γ-CD, drug exists in different forms depending on the concentration of the host. At lower concentration of γ-CD, 1:1 inclusion complex formation takes place and EPT exists in protonated form due to accessibility of water by the drug in the inclusion complex, whereas, at higher concentration, a 2:1 inclusion complex (γ-CD:EPT) is observed, in which EPT is completely buried inside the hydrophobic cavity of the capsule formed by two γ-CD molecules, and we believe the hydrophobic environment inside the capsule stabilizes the neutral form of the drug in the 2:1 inclusion complex. Deep insight into the molecular picture of these host-guest interactions has been provided by the docking studies followed by quantum chemical calculations.

The effect of cucurbit[7]uril (CB[7]) nano-caging on the photophysical properties, particularly excited-state proton transfer (ESPT) reaction, of an eminent anti-cancer drug, topotecan (TPT), is demonstrated through steady-state and time-resolved fluorescence measurements. TPT in water (pH 6) exists exclusively as the cationic form (C) in the ground state. However, the drug emission mainly comes from the excited-state zwitterionic form (Z*) of TPT, and is attributed to water-assisted ESPT between the 10-hydroxyl group and water, which leads to the transformation of C* to Z* of TPT. In the presence of CB[7], it is found that selective encapsulation of the C form of TPT results in the formation of a 1:1 inclusion complex (CB[7]:TPT), and the ESPT process is inhibited by this encapsulation process. As a result, C* becomes the dominant emitting species in the presence of CB[7] rather than Z*, and fluorescence switching takes place from green to blue. Time-resolved studies also support the existence of CB[7]-encapsulated cationic species as the major emitting species in the presence of the macrocyclic host. Semi-empirical quantum chemical calculations are employed to gain insight into the molecular picture of orientation of TPT in the inclusion complex. It is clearly seen from the optimised structure of 1:1 CB[7]:TPT inclusion complex that both 10-hydroxyl and 9-dimethylaminomethylene groups of TPT lie partly inside the cavity, and thereby inhibit the excited-state transformation of C* to Z* by the ESPT process. Finally, controlled release of the drug is achieved by means of fluorescence switching by introducing NaCl, which is rich in cells, as an external stimulus., (Copyright © 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim.)