Probing photoinduced electron transfer events in phenylferrocene-corrole dyad.

Herein, we investigated PhFC (10-phenylferrocenyl-5,15-diphenyl corrole), a corrole-based donor-acceptor (D-A) dyad, to understand the energy/electron transfer reaction dynamics. Phenylferrocene acts as the donor moiety when attached to the meso position of the corrole ring in the PhFC D-A system. The photophysical properties of the PhFC dyad and its parent molecule, TPC (5,10,15-triphenyl corrole), were studied by UV-vis spectroscopy, steady state fluorescence spectroscopy, TCSPC and optical microscopy techniques. A slight red shift and broadening of both the Q-band and Soret bands are observed in the absorption spectra of the PhFC dyad in comparison to TPC, representing the weak electronic interaction between the phenylferrocene moiety and corrole ring. The fluorescence emission spectrum of the PhFC dyad is significantly quenched (>80%) in comparison to TPC, attributed to the photoinduced electron transfer (PET) from phenylferrocene to the corrole ring. We observed that the electron transfer rate in the PhFC system is solvent dependent. Our theoretical investigation supported the experimental findings on the electron transfer mechanism. The HOMO and LUMO arrangements of these PhFC dyads demonstrate the electron density distribution and the ability of the donor moieties to transfer electrons to the corrole moiety. Fluorescence lifetime imaging microscopy (FLIM) was used to image the homogeneous lifetime distribution of the PhFC dyad and TPC.

Unraveling energy transfer and fluorescence quenching dynamics in biomolecular complexes: a comprehensive study of imiquimod-rifampicin interaction.

In nature, numerous biomolecules are implicated in charge transfer (CT) and energy transfer (ET) mechanisms crucial for fundamental processes such as photosynthesis. Unveiling these mechanisms is pertinent to multiple disciplines including chemistry, engineering and biochemistry. This article presents a detailed study involving two molecules forming a model system with efficient ET properties. Specifically, their complex exhibits dark quenching phenomena arising from fluorescence resonance energy transfer (FRET) from the donor (imiquimod) to the acceptor (rifampicin). In addition, the energy transfer properties were also elucidated by considering the two forms of rifampicin (RIF), non-ionic and zwitter-ionic in the solution. Supplemented by spectroscopic findings, molecular dynamics simulations and time dependent density functional theory (TD-DFT) calculations conclusively validate the ET properties from imiquimod (IMQ) to RIF forms. Interestingly, these ET processes were found to be associated with pi-pi and hydrogen bond interactions. Their contribution was observed to depend upon the non-ionic and zwitter-ionic form of RIF.

Identification of allosteric hotspots regulating the ribosomal RNA binding by antibiotic resistance-conferring Erm methyltransferases.

Antibiotic resistance via epigenetic methylation of ribosomal RNA is one of the most prevalent strategies adopted by multidrug resistant pathogens. The erythromycin-resistance methyltransferase (Erm) methylates rRNA at the conserved A2058 position and imparts resistance to macrolides such as erythromycin. However, the precise mechanism adopted by Erm methyltransferases for locating the target base within a complicated rRNA scaffold remains unclear. Here, we show that a conserved RNA architecture, including specific bulge sites, present more than 15 Å from the reaction center, is key to methylation at the pathogenic site. Using a set of RNA sequences site-specifically labeled by fluorescent nucleotide surrogates, we show that base flipping is a prerequisite for effective methylation and that distal bases assist in the recognition and flipping at the reaction center. The Erm-RNA complex model revealed that intrinsically flipped-out bases in the RNA serve as a putative anchor point for the Erm. Molecular dynamic simulation studies demonstrated the RNA undergoes a substantial change in conformation to facilitate an effective protein-rRNA handshake. This study highlights the importance of unique architectural features exploited by RNA to impart fidelity to RNA methyltransferases via enabling allosteric crosstalk. Moreover, the distal trigger sites identified here serve as attractive hotspots for the development of combination drug therapy aimed at reversing resistance.

What Makes Thienoguanosine an Outstanding Fluorescent DNA Probe?

Thienoguanosine (thG) is an isomorphic guanosine (G) surrogate that almost perfectly mimics G in nucleic acids. To exploit its full potential and lay the foundation for future applications, 20 DNA duplexes, where the bases facing and neighboring thG were systematically varied, were thoroughly studied using fluorescence spectroscopy, molecular dynamics simulations, and mixed quantum mechanical/molecular mechanics calculations, yielding a comprehensive understanding of its photophysics in DNA. In matched duplexes, thG's hypochromism was larger for flanking G/C residues but its fluorescence quantum yield (QY) and lifetime values were almost independent of the flanking bases. This was attributed to high duplex stability, which maintains a steady orientation and distance between nucleobases, so that a similar charge transfer (CT) mechanism governs the photophysics of thG independently of its flanking nucleobases. thG can therefore replace any G residue in matched duplexes, while always maintaining similar photophysical features. In contrast, the local destabilization induced by a mismatch or an abasic site restores a strong dependence of thG's QY and lifetime values on its environmental context, depending on the CT route efficiency and solvent exposure of thG. Due to this exquisite sensitivity, thG appears ideal for monitoring local structural changes and single nucleotide polymorphism. Moreover, thG's dominant fluorescence lifetime in DNA is unusually long (9-29 ns), facilitating its selective measurement in complex media using a lifetime-based or a time-gated detection scheme. Taken together, our data highlight thG as an outstanding emissive substitute for G with good QY, long fluorescence lifetimes, and exquisite sensitivity to local structural changes.

Excited-State Dynamics of Thienoguanosine, an Isomorphic Highly Fluorescent Analogue of Guanosine.

Thienoguanosine (th G) is an isomorphic analogue of guanosine with promising potentialities as fluorescent DNA label. As a free probe in protic solvents, th G exists in two tautomeric forms, identified as the H1, being the only one observed in nonprotic solvents, and H3 keto-amino tautomers. We herein investigate the photophysics of th G in solvents of different polarity, from water to dioxane, by combining time-resolved fluorescence with PCM/TD-DFT and CASSCF calculations. Fluorescence lifetimes of 14.5-20.5 and 7-13 ns were observed for the H1 and H3 tautomers, respectively, in the tested solvents. In methanol and ethanol, an additional fluorescent decay lifetime (≈3 ns) at the blue emission side (λ≈430 nm) as well as a 0.5 ns component with negative amplitude at the red edge of the spectrum, typical of an excited-state reaction, were observed. Our computational analysis explains the solvent effects observed on the tautomeric equilibrium. The main radiative and nonradiative deactivation routes have been mapped by PCM/TD-DFT calculations in solution and CASSCF in the gas phase. The most easily accessible conical intersection, involving an out-of plane motion of the sulfur atom in the five-membered ring of th G, is separated by a sizeable energy barrier (≥0.4 eV) from the minimum of the spectroscopic state, which explains the large experimental fluorescence quantum yield.

A Molecular Tool Targeting the Base-Flipping Activity of Human UHRF1.

During DNA replication, ubiquitin-like, containing PHD and RING fingers domains 1 (UHRF1) plays key roles in the inheritance of methylation patterns to daughter strands by recognizing through its SET and RING-associated domain (SRA) the methylated CpGs and recruiting DNA methyltransferase 1 (DNMT1). Herein, our goal is to identify UHRF1 inhibitors targeting the 5'-methylcytosine (5mC) binding pocket of the SRA domain to prevent the recognition and flipping of 5mC and determine the molecular and cellular consequences of this inhibition. For this, we used a multidisciplinary strategy combining virtual screening and molecular modeling with biophysical assays in solution and cells. We identified an anthraquinone compound able to bind to the 5mC binding pocket and inhibit the base-flipping process in the low micromolar range. We also showed in cells that this hit impaired the UHRF1/DNMT1 interaction and decreased the overall methylation of DNA, highlighting the critical role of base flipping for DNMT1 recruitment and providing the first proof of concept of the druggability of the 5mC binding pocket. The selected anthraquinone appears thus as a key tool to investigate the role of UHRF1 in the inheritance of methylation patterns, as well as a starting point for hit-to-lead optimizations.

Dynamics of Methylated Cytosine Flipping by UHRF1.

DNA methylation patterns, which are critical for gene expression, are replicated by DNA methyltransferase 1 (DNMT1) and ubiquitin-like containing PHD and RING finger domains 1 (UHRF1) proteins. This replication is initiated by the recognition of hemimethylated CpG sites and further flipping of methylated cytosines (mC) by the Set and Ring Associated (SRA) domain of UHRF1. Although crystallography has shed light on the mechanism of mC flipping by SRA, tools are required to monitor in real time how SRA reads DNA and flips the modified nucleobase. To accomplish this aim, we have utilized two distinct fluorescent nucleobase surrogates, 2-thienyl-3-hydroxychromone nucleoside (3HCnt) and thienoguanosine (thG), incorporated at different positions into hemimethylated (HM) and nonmethylated (NM) DNA duplexes. Large fluorescence changes were associated with mC flipping in HM duplexes, showing the outstanding sensitivity of both nucleobase surrogates to the small structural changes accompanying base flipping. Importantly, the nucleobase surrogates marginally affected the structure of the duplex and its affinity for SRA at positions where they were responsive to base flipping, illustrating their promise as nonperturbing probes for monitoring such events. Stopped-flow studies using these two distinct tools revealed the fast kinetics of SRA binding and sliding to NM duplexes, consistent with its reader role. In contrast, the kinetics of mC flipping was found to be much slower in HM duplexes, substantially increasing the lifetime of CpG-bound UHRF1, and thus the probability of recruiting DNMT1 to faithfully duplicate the DNA methylation profile. The fluorescence-based approach using these two different fluorescent nucleoside surrogates advances the mechanistic understanding of the UHRF1/DNMT1 tandem and the development of assays for the identification of base flipping inhibitors.

Spectroscopy and Dynamics of Cryptolepine in the Nanocavity of Cucurbit[7]uril and DNA.

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.

Design and Development of a Two-Color Emissive FRET Pair Based on a Photostable Fluorescent Deoxyuridine Donor Presenting a Mega-Stokes Shift.

We report the synthesis and site-specific incorporation in oligodeoxynucleotides (ODNs) of an emissive deoxyuridine analog electronically conjugated on its C5-position with a 3-methoxychromone moiety acting as a fluorophore. When incorporated in ODNs, this fluorescent deoxyuridine analog exhibits remarkable photostability and good quantum yields. This deoxyuridine analog also displays a mega-Stokes shift, which allows for its use as an efficient donor for FRET-based studies when paired with the yellow emissive indocarbocyanine Cy3 acceptor.

Fluorescence switching of sanguinarine in micellar environments.

Sanguinarine (SANG), a key member of the benzylisoquinoline alkaloid family, is well-known for its various therapeutic applications such as antimicrobial, antitumor, anticancer, antifungal and anti-inflammatory etc. Depending on the medium pH, SANG exists in the iminium or alkanolamine form, which emits at 580 nm and 420 nm, respectively. Nucleophilic attack on the C6 carbon atom converts the iminium form to the alkanolamine form of SANG, and these two forms are equally important for the medicinal activities of SANG. To improve its potency as a drug, it is essential to get a physical insight into this conversion process. In this study, we have deployed steady sate and time-resolved spectroscopic techniques to probe this conversion process inside different micellar environments. We have observed that the conversion from the iminium to alkanolamine form takes place in neutral OBG (octyl-ß-d-glucopyranoside) and positively charged CTAB micelles, whereas the iminium form exclusively exists in negatively charged SDS micelles. This conversion from the iminium to alkanolamine form in the case of OBG and CTAB micelles may be attributed to the reduced pKa of this conversion process owing to the enhanced hydrophobicity experienced by the iminium form in between the surfactant head groups. On the other hand, the electrostatic attraction between positively charged iminium and negatively charged surfactant head groups stabilizes the iminium form in the stern layer of the SDS micelle. We believe that our observations are useful for selective transportation of any particular form of the drug into the active site. Moreover, loading of any particular form of drug can be easily monitored with the help of fluorescence color switch from orange (iminium) to violet (alkanolamine) without pursuing any sophisticated or complex technique.

Fluorescence Up-Conversion Studies of [2,2'-Bipyridyl]-3,3'-diol in Octyl-ß-d-glucoside and Other Micellar Aggregates.

In this present work, excited state double proton transfer dynamics (ESIDPT) of 2,2'-bipyridyl-3,3'-diol (BP(OH)2) molecules has been probed in a nontoxic, biocompatible sugar surfactant assembly, namely, octyl-ß-d-glucoside (OBG) micelle with the help of steady state and fluorescence up-conversion techniques. Moreover, the ultrafast double proton transfer dynamics in conventional micelles (SDS, CTAB) and bile salts aggregates have been probed and compared. Interestingly, in all these supramolecular aggregates, the ESIDPT dynamics is found to follow sequential pathway; however, the time-scale of proton transfer dynamics varies from 11 to 30 ps. This difference in proton transfer time scale in different supramolecular aggregates has been explained in terms of accessibility of water molecules in the vicinity of probe.

Femtosecond to nanosecond dynamics of 2,2'-bipyridine-3,3'-diol inside the nano-cavities of molecular containers.

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 ß-cyclodextrin (ß-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 ß-CD (having similar cavity size as that of CB7), inferring the presence of a two-step sequential process of PT inside the ß-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 ß-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.

Cucurbit[7]uril assisted ultraviolet to visible fluorescence switch of a heart medicine.

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.

Excited state proton transfer dynamics of an eminent anticancer drug, ellipticine, in octyl glucoside micelle.

Photophysics and proton transfer dynamics of an eminent anticancer drug, ellipticine (EPT), have been investigated inside a biocompatible octyl-ß-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.

An anticancer drug to probe non-specific protein-DNA interactions.

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.

Modulation of photophysics and pKa shift of the anti-cancer drug camptothecin in the nanocavities of supramolecular hosts.

This article reports the pK(a) shift of an anti-cancer drug, 20(S)-camptothecin (CPT), upon encapsulation into the nanocavity of a cucurbit[7]uril (CB7) macrocycle. Steady-state, time-resolved fluorescence and electrospray ionisation mass spectrometry (ESI-MS) studies provide evidence for the formation of both 1:1 and 2:1 (CB7⋅CPT) stoichiometries. Astonishingly, we have found that protonation of CPT takes place at a higher concentration of macrocycle (≥50 µM) when the 2:1 stoichiometric complex develops. However, we did not find any proof for protonation of CPT when it is encased by a ß-cyclodextrin cavity, which has a cavity size almost the same as that of CB7. Hence, we conclude that electron-rich carbonyl portals of CB7 have an important role in protonation of the drug in the 2:1 inclusion complex. Docking and semi-empirical quantum chemical calculations have been employed to gain an insight into the molecular picture of orientation of CPT in the inclusion complexes. It is clearly seen from the optimised structure of the 2:1 (CB7⋅CPT) inclusion complex that the quinoline nitrogen of CPT does not reside within either of the CB7 cavities, rather it is almost sandwiched between two CB7 rings, and therefore, it experiences huge electron density exerted by both carbonyl portals of the macrocycles. As a result, the pK(a) of CPT shifts from 1.2 to 6.2. Finally, controlled release of the drug has been achieved through the introduction of NaCl, which is rich in cells, as an external stimulus. We hope this recognition-mediated binding and release mechanism can be useful for activation of the drug and controlled release of the drug in therapeutic uses.

Supramolecular host-inhibited excited-state proton transfer and fluorescence switching of the anti-cancer drug, topotecan.

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.

Modulation of excimer formation of 9-(dicyano-vinyl)julolidine by the macrocyclic hosts.

This article reports the alteration of the excited state photophysics of a molecular rotor, namely 9-(dicyano-vinyl)julolidine (DCVJ), which has been extensively used to report protein aggregation and protein conformational changes, by the various cavity sizes of cyclodextrin (CD) macrocyclic hosts, with the help of steady state, time-resolved fluorescence techniques. It is observed that, in the presence of α-CD, the characteristic features of both the monomer and excimer emissions of DCVJ are almost unperturbed. However, in the presence of ß-CD, the excited photophysics of the molecule is significantly perturbed, and it is noted that ß-CD inhibits the excimer formation drift of DCVJ by incorporation of a DCVJ monomer inside its cavity. The most striking findings are observed in the case of γ-CD. Initially, the excimer peak intensity drops and the monomer intensity increases, due to the 1 : 1 DCVJ/γ-CD inclusion complex formation. Above a certain concentration, another DCVJ molecule is accommodated inside the γ-CD cavity and forms an excimer, which is reflected in the intensification of the excimer peak. At higher γ-CD concentration the fluorescence intensity of the excimer shoots up, due to the formation of 2 : 2 host-guest complex, in which an additional γ-CD molecule provides extra stabilization to the excimer. Insight on the molecular picture of this host-guest interaction has been provided by docking studies followed by quantum chemical calculations.

Spectroscopy and dynamics of beta-lactoglobulin complexed with rifampicin.

In this paper, we report the binding interaction of milk protein, beta-lactoglobulin (BLG), with an antibiotic against tuberculosis, rifampicin (RIF). BLG intrinsic fluorescence from tryptophan (Trp) amino acids was monitored to understand protein-drug interactions. Binding parameters and stoichiometry were estimated with the help of fluorescence spectral changes. Synchronous fluorescence spectroscopy was employed to exclusively monitor the Trp and Tyrosine (Tyr) environment in the presence of RIF. With the help of steady state fluorescence at different temperatures supported by time-resolved fluorescence, we confirmed that the protein forms a static complex with RIF. Thermodynamic parameters, ΔH and ΔS values, showed the involvement of hydrophobic forces between the RIF and BLG. Competitive displacement assay with ANS confirmed the BLG calyx as the binding site for RIF. Energy transfer mechanism from Trp to RIF was attributed to the fluorescence changes in protein upon complexation. The Förster resonance energy transfer (FRET) was used to find distance, energy transfer efficiency and rate of energy transfer between donor (BLG) and acceptor (RIF). Fourier-transform infrared (FTIR) spectroscopy was utilized for estimating changes in the secondary structure of BLG induced by RIF. Molecular docking was used to visualise the binding location of RIF on BLG. Molecular dynamics (MD) simulation studies showed a consistent binding interactions between BLG and RIF during the 100 ns simulation period and this well supported the increased beta sheet content in FTIR. Overall our results establish the potential of intrinsic fluorescence of BLG in combination with biophysical tools to rationalize drug-protein interactions.Communicated by Ramaswamy H. Sarma.