ABSTRACT
The direct formation of a C-N bond at the ortho-position of naphthalene monoimides (NMI) and perylene monoimides (PMI) is reported herein using dioxazolones as the amide source. This method affords direct access to ortho-amino NMI and PMI through an amidation and deprotection sequence. One-pot telescopic bay-bromination of ortho-amino PMIs was also achieved. The ortho-amidated NMIs and PMIs, accessed by the current methodology, show significant red shifts in their absorption and fluorescence spectra compared with the NMI and PMI alone. An improvement in the quantum yield and fluorescence lifetime was observed by the incorporation of pivalamide groups at the ortho-positions of NMI and PMI.
ABSTRACT
Intrinsically disordered proteins (IDPs) are a class of proteins that do not follow the unanimated perspective of the structure-function paradigm. IDPs enunciate the dynamics of motions which are often difficult to characterize by a particular experimental or theoretical approach. The chameleon nature of the IDPs is a result of an alteration or transition in their conformation upon binding with ligands. Experimental investigations via ensemble-average approaches to probe this randomness are often difficult to synchronize. Thus, to sense the substates of different conformational ensembles of IDPs, researchers have often targeted approaches based on single-molecule measurements. In this Perspective, we will discuss various single-molecule approaches to explore the conformational transitions of IDPs in different scenarios, the outcome, challenges, and future prospects.
Subject(s)
Intrinsically Disordered Proteins , Intrinsically Disordered Proteins/chemistry , Single Molecule Imaging , Protein Conformation , LigandsABSTRACT
Intercalated-motif (i-motif) DNA formed by cytosine (C)-rich sequences has been considered a novel target in anticancer research. Interestingly, this type of noncanonical DNA structure is highly dynamic and can display several conformational polymorphisms based on the immediate surrounding environment. However, studies regarding the folding pathway of i-motifs having disease-specific sequences under a confined environment at physiological pH are relatively scarce. This thereby warrants more explorations that will decipher their structural and functional properties inside constrained media. Herein, using the single-molecule Förster Resonance Energy Transfer (smFRET) studies, for the first time, we have illustrated the conformational dynamics of c-MYC promoter-based i-motif structures at physiological pH inside microemulsions of different dimensions. We concluded that the folding of such motifs under confined space is not a direct transition between the random coil and i-motif conformations; rather it occurs through a partially folded intermediate, depending on the confined dimension.
Subject(s)
DNA , Fluorescence Resonance Energy Transfer , Cytosine/chemistry , DNA/chemistry , Nucleic Acid Conformation , Promoter Regions, GeneticABSTRACT
Trypsin, the most abundant pancreatic protein, aids in protein digestion by hydrolysis and exhibits aggregation propensity in presence of alcohol, which can further lead to pancreatitis and eventually pancreatic cancer. Herein, by several experimental and theoretical approaches, we unearth the inhibition of alcohol-induced aggregation of Trypsin by macrocyclic cavitand, ß-cyclodextrin (ß-CD). ß-CD interacts with the native protein and shows inhibitory effect in a dose dependent manner. Moreover, the secondary structures and morphologies of Trypsin in presence of ß-CD also clearly emphasize the inhibition of fibril formation. From Fluorescence Correlation Spectroscopy, we observed an enhancement in diffusion time of Nile Red with â¼2.5â times increase in hydrodynamic radius, substantiating the presence of fibrillar structure. Trypsin also shows reduction in its functional activity due to alcohol-induced aggregation. Our simulation data reports the probable residues responsible for fibril formation, which was validated by molecular docking studies.
Subject(s)
Cyclodextrins , beta-Cyclodextrins , Cyclodextrins/chemistry , Ethanol/chemistry , Ethers, Cyclic , Molecular Docking Simulation , Resorcinols , Trypsin/chemistry , beta-Cyclodextrins/chemistry , beta-Cyclodextrins/pharmacologyABSTRACT
A one-step Rh-catalyzed site-selective ortho-C-H alkynylation of perylene as well as naphthalene mono- and diimides is reported. A single step regioselective access to ortho-C-H alkynylated derivatives of these ryleneimides not only increases the step economy of the ortho-functionalization on these dyes but also provides a quick access route towards highly functionalized dyes that have potential optoelectronic applications. Increased solubility of tetra(triisopropylsilyl)acetylenyl PDIs in organic solvents greatly enhances their utility for further derivatization.
ABSTRACT
This work delineates an integrative approach combining spectroscopic and computational studies to decipher the association-induced fluorescence properties of a fluorescent molecular rotor, viz., auramine O (AuO), after interacting with 20-mer duplex DNA having diverse well-matched base pairs. While exploring the scarcely explored sequence-dependent interaction mechanism of AuO and DNA, we observed that DNA could act as a conducive scaffold to the formation of AuO dimer through noncovalent interactions at lower molecular density. The photophysical properties of AuO depend on the nucleotide compositions as described from sequence-dependent shifting in the emission and absorption maxima. Furthermore, we explored such DNA base pair-dependent fluorescence spectral characteristics of AuO toward discriminating the thermodynamically most stable single nucleotide mismatch in a 20-mer sequence. Our results are interesting and could be useful in developing analogues with further enhanced emission properties toward mismatched DNA sequences.
Subject(s)
Benzophenoneidum , DNA , Benzophenoneidum/chemistry , DNA/chemistry , Fluorescent Dyes , Nucleotides , Staining and LabelingABSTRACT
Understanding the fundamental interactions between plasmonic metal nanoparticles (MNPs) and small molecules is of utmost importance in various applications such as catalysis, sensing, drug delivery, optoelectronics, and surface-enhanced Raman spectroscopy. Herein, we have investigated the early stage of the aggregation pathway of citrate-stabilized Au NPs with surfactants and explored their catalytic efficacy. Our findings reveal that (17 ± 2)-nm-sized citrate-stabilized Au NPs undergo concentration and time-dependent aggregation with positively charged cetyltrimethylammonium bromide (CTAB). Kinetic analyses revealed the presence of two distinct kinds of aggregates, namely, smaller clusters and a larger branched network of Au nanochains. At longer times and in the presence of higher concentrations of CTAB, these branched networks of Au nanochains transform into dense compact globular aggregates. The catalytic efficacy of Au NPs, branched Au nanochains, and dense compact aggregates has been explored with respect to the reductive hydrogenation of 4-nitophenol in the presence of excess NaBH4. Our study revealed that the catalytic rate decreases in the order of Au NPs > branched Au nanochains > compact aggregates. Interestingly, pre-equilibrating different Au NP samples with excess NaBH4 prior to the onset of the reaction results in similar catalytic activity irrespective of the aggregation state of Au NPs. This observation has been explained by considering efficient surface restructuring via ligand exchange with H- ions and the subsequent disruption of CTAB-induced aggregates of Au NPs. Moreover, the aggregated Au NPs can be recycled over several consecutive cycles for the reductive hydrogenation of 4-NP upon ligand exchange with H- ions. Taken together, our present study highlights the early-stage aggregation kinetics of Au NPs with CTAB surfactants and demonstrates the importance of the surface restructuring of Au NPs on their catalytic efficacy.
Subject(s)
Gold , Metal Nanoparticles , Catalysis , Cetrimonium , Citric Acid , Gold/chemistry , Ions , Kinetics , Ligands , Metal Nanoparticles/chemistry , Surface-Active AgentsABSTRACT
Targeting mismatched base pairs containing DNA using small molecules and exploring the underlying mechanism involved during the binding interactions is one of the fundamental aspects of drug design. These molecules in turn are used in nucleic acid targeted therapeutics and cancer diagnosis. In this work, we systematically delineate the binding of the anticancer drug, epirubicin hydrochloride (EPR) with 20-mer duplex DNA, having both natural nucleobase pairing and thermodynamically least stable non-Watson-Crick base pairing. From the thermal denaturation studies, we observed that EPR can remarkably enhance the thermal stability of cytosine-cytosine (CC) and cytosine-thymine (CT) mismatched (MM) DNA over other 20-mer duplex DNA. From steady-state fluorescence spectroscopy and isothermal titration calorimetry studies, we concluded that EPR binds strongly with the mismatched duplex DNA through the intercalation binding mode. The interaction of EPR and duplex DNA has also been monitored at a single molecular resolution using fluorescence correlation spectroscopy (FCS). Dynamic quantitates such as diffusion coefficients and hydrodynamic radii obtained from an FCS study along with association and dissociation rate constants estimated from intensity time trace analyses further substantiate the stronger binding affinity of EPR to the thermally less stable mismatched DNA, formed by the most discriminating nucleobase (viz. cytosine). Additionally, we have shown that EPR can be sequestered from nucleic acids using a mixed micellar system of an anionic surfactant and a triblock copolymer. From thermal denaturation studies and circular dichroism spectroscopy, we found that the extent of drug sequestration depends on the binding affinity of EPR to the duplex DNA, and this mixed micellar system can be employed for the removal of excess drug in the case of a drug overdose.
Subject(s)
Micelles , Nucleotides , Base Pairing , DNA , Epirubicin , Nucleic Acid Conformation , ThermodynamicsABSTRACT
Herein we report the binding interactions between lysozyme (Lyz) and an anthracycline drug, epirubicin hydrochloride (EPR), through an extensive spectroscopic approach at both ensemble average and single molecular resolution. Our steady-state and time-resolved fluorescence spectroscopy reveals that the drug-induced fluorescence quenching of the protein proceeds through a static quenching mechanism. Isothermal titration calorimetry (ITC) and steady-state experiments reveal almost similar thermodynamic signatures of the drug-protein interactions. The underlying force that plays pivotal roles in the said interaction is hydrophobic in nature, which is enhanced in the presence of a strong electrolyte (NaCl). Circular dichroism (CD) spectra indicate that there is a marginal increase in the secondary structure of the native protein (α-helical content increases from 26.9 to 31.4% in the presence of 100 µM EPR) upon binding with the drug. Fluorescence correlation spectroscopy (FCS) was used to monitor the changes in structure and conformational dynamics of Lyz upon interaction with EPR. The individual association (Kass = 0.33 × 106 ms-1 M-1) and dissociation (Kdiss = 1.79 ms-1) rate constants and the binding constant (Kb = 1.84 × 105 M-1) values, obtained from fluctuations of fluorescence intensity of the EPR-bound protein, have also been estimated. AutoDock results demonstrate that the drug molecule is encapsulated within the hydrophobic pocket of the protein (in close proximity to both Trp62 and Trp108) and resides â¼20 Å apart from the covalently labelled CPM dye. Förster resonance energy transfer (FRET) studies proved that the distance between the donor (CPM) and the acceptor (EPR) is â¼22 Å, which is very similar to that obtained from molecular docking analysis (â¼20 Å). The system also shows temperature-dependent reversible FRET, which may be used as a thermal sensor for the temperature-sensitive biological systems.
Subject(s)
Muramidase , Binding Sites , Circular Dichroism , Epirubicin , Molecular Docking Simulation , Muramidase/metabolism , Protein Binding , Spectrometry, Fluorescence , ThermodynamicsABSTRACT
Monitoring the DNA dynamics in solution has great potential to develop new nucleic acid-based sensors and devices. With spectroscopic approaches, both at the ensemble average and single-molecule resolution, this study is directed to differentiate a single nucleotide mismatch (SNM) via a metal ion-stabilized mismatched base-pairing (C-Ag+-C/C-Cu2+-T) (C = cytosine, T = thymine) and site-selective extrinsic fluorophore, specifically, Thioflavin T (ThT). This is the first approach of its kind where dynamic quantities like molecular diffusion coefficients and diffusion times have been utilized to distinguish the least-stable SNM (CC & CT) formed by the most discriminating nucleobase, specifically, cytosine in a 20-mer duplex DNA. Additionally, this work also quantifies metal ions (Ag+ and Cu2+) at lower concentrations using fluorescence correlation spectroscopy. Our results can provide greater molecular-level insights into the mismatch-dependent metal-DNA interactions and also illuminate ThT as a new fluorophore to monitor the dynamics involved in DNA-metal composites.
Subject(s)
Benzothiazoles/chemistry , Copper/chemistry , DNA/chemistry , Silver/chemistry , Base Pair Mismatch , Base Pairing , Ions/chemistry , Spectrometry, FluorescenceABSTRACT
We report the unfolding of the globular protein, Bovine Serum Albumin (BSA) induced by anionic surfactant sodium dodecyl sulfate (SDS) and subsequently monitored the refolding of this denatured BSA using triblock copolymers F127 and P123 through the formation of mixed micelles. Our study exclusively represents the reversibility of this unfolding-refolding process using pluronic triblock copolymers F127/P123 as refolding agents. We confirm the recovery of its native state from its denatured state estimating the α-helical structure of the denatured protein from the CD data which support our steady state fluorescence spectra monitoring the fluorescence of the intrinsic Trp molecules present in BSA. Time resolved study also corroborates the stepwise recovery of the denatured BSA as well as the reversibility of the processes. Isothermal Titration Calorimetry (ITC) data explain the negligible interactions between the triblock copolymers and the native state of BSA. The high binding constant of SDS and triblock copolymers probably play the crucial role in the stepwise recovery of the unfolded BSA followed by reversibility of the refolding processes through the formation of the mixed micelles. The mechanism of mixed-micelle formation has been substantiated by the fact that the Guanidine Hydrochloride denatured BSA does not react with F127/P123 whereby no recovery of the protein was observed.