Browsing by Author "Pati, Avik K."

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BPS2025 - Dissecting the mechanism of aminoglycoside-induced miscoding with spatially multiplexed single-molecule FRET
(Elsevier, 2025-02) Pati, Avik K.
The kinetic mechanism of mRNA decoding has been established through ensemble kinetic investigations using purified translation components from Escherichia coli. Pre-steady state single-molecule fluorescence resonance energy transfer (smFRET) measurements have directly revealed insights into the movement of aminoacyl-tRNA (aa-tRNA) within the A site of the ribosome and their mRNA codon-dependencies. Here, we have interrogated the conformational dynamics of aa-tRNA on the ribosome during mRNA decoding using a multiplexed smFRET approach that facilitates side-by-side comparisons of multiple samples under identical experimental conditions. We demonstrate the utility of this approach by simultaneously examining the process of elongation factor-Tu (EF-Tu) facilitated Phe-tRNAPhe incorporation into 4 distinct initiation complexes bearing either cognate or near-cognate codons in the A site. As expected from prior ensemble investigations, our results show that the cognate Phe codons, UUU and UUC, exhibit distinctions in the rates of tRNA selection and those near-cognate codons (CUC and UCC) efficiently reject Phe-tRNAPhe during early steps of tRNA selection prior to GTP hydrolysis. We then examined the impacts of the miscoding antibiotic streptomycin on mRNA decoding. Prior ensemble investigations report that streptomycin slows cognate tRNA selection and accelerates near-cognate tRNA selection during intermediate steps of mRNA decoding to homogenize the rates of GTP hydrolysis and peptide bond formation. Our findings show that the primary impact of streptomycin is to accelerate the rates of mRNA decoding for both cognate and near-cognate ribosome complexes, while slowing the rates of aa-tRNA binding to the A site of the ribosome. These data are consistent with streptomycin favoring “closed” conformational states of the small subunit shoulder domain prior to aa-tRNA binding that inhibit early codon recognition steps where the tRNA anticodon is escorted by EF-Tu into the A site decoding region on the small ribosomal subunit.
Contrasting Solid-State Fluorescence of Diynes with Small and Large Aryl Substituents: Crystal Packing Dependence and Stimuli-Responsive Fluorescence Switching
(ACS, 2015-09) Pati, Avik K.
There has been a significant current interest in solid state luminescence of organic molecules and their stimuli responsive fluorescence switching behavior. Although small organic derivatives with olefinic, acetylenic, phenylenevinylenic, phenyleneethynylenic spacers are widely documented as solid state emitters in the literature, the solid state photophysics of organic derivatives with “butadiyne” spacer still remains unexplored. We provide detailed investigation on the solid state fluorescence properties of a series of butadiynyl fluorophores. Replacement of a phenyl ring, which is at periphery of the butadiyne bridge, with a large moiety such as pyrenyl group furnishes contrasting emissions in the solid state. While the butadiyne bridged phenyl derivatives show a blue shift of emission maxima in the solid powder with respect to monomer spectra in solution state, the butadiyne bridged pyrenyl derivatives exhibit a red shift in the solid state. The blue shift of the emission maxima of the butadiyne bridged phenyl derivatives in the solid powder is attributed to allowed excitonic transition in aggregates with nearly parallel transition dipoles. On the other hand, formation of pyrenyl excimer accounts for the red shift of the butadiyne bridged pyrenyl derivatives in the solid powder. In addition to that, the solid state fluorescence of the pyrenyl analogues is reversibly switched between two aggregate forms through external heating and rubbing stimuli.
Deciphering the Photophysical Role of Conjugated Diyne in Butadiynyl Fluorophores: Synthesis, Photophysical and Theoretical Study
(ACS, 2013-07) Pati, Avik K.
The present work focuses on the current interest in diyne bridged chromophores necessitating a clearer understanding of the photophysics of such molecules. The significance of the diyne moiety in the photophysics has been investigated by synthesizing simple substituted diphenyl butadiynyl derivatives following a quick and efficient microwave assisted Eglinton coupling of terminal alkynes. Emission of the fluorophores is observed from the usual locally excited (LE) state and intramolecular charge transfer (ICT) state. Separation of pure ICT emission from pure LE emission has been carried out by Gaussian/Lorentzian curve fitting. The vibronic coupling in the local transitions appears to be confined to the normal mode involving the C–C triple bond stretching of the diyne moiety. This implies that the LE transition involves the diyne moiety, a conclusion supported by quantum chemical calculations. The resolved ICT emission follows double linear dependence on ET(30) solvent polarity scale. The important role of the diyne moiety in the photophysics of this class of molecules is clearly discernible in this study.
Diverse fluorescent probe concepts for detection and monitoring of reactive oxygen species
(Wiley, 2025-02) Pati, Avik K.
World-wide research on reactive oxygen species (ROS) continues to reveal new information about the role and impact of ROS on human health and disease. ROS are generated in live cells as a byproduct of aerobic metabolism. Physiological concentrations of cellular ROS are important for signaling and homeostasis, but excessive generation of ROS causes apoptotic and necrotic cell death and various health disorders. Fluorescence technology is a powerful tool to detect, monitor, and image cellular ROS. The present review provides an overview of diverse organic dye-based fluorescent probe concepts that involve modifications of traditional fluorescent dyes utilizing basic principles of dye chemistry and photophysics. Fluorescence responses of the probes and their specificity towards ROS are discussed through analyses of their photophysical and photochemical parameters. We also provide an outlook on future directions of ROS-responsive fluorescent dyes, which could enable the design and development of advanced probes for gaining deeper insights into redox biology.
Fluorescent 1-Arylidene-1,3-dihydroisobenzofuran: Ligand-Free Palladium Nanoparticles, Catalyzed Domino Synthesis and Photophysical Studies
(Wiley, 2017-07) Pati, Avik K.
The present work describes synthesis and photophysical studies of a class of fluorophores containing biologically relevant 1,3-dihydroisobenzofuran scaffold. Ligand-free palladium nanoparticles have been utilized for domino synthesis of 1-arylidene-1,3-dihydroisobenzofurans from 2-iodobenzyl alcohol and terminal arylacetylenes under mild reaction conditions. Reaction of terminal alkylacetylenes with 2-iodobenzyl alcohol however, provided only the Sonogashira products (2-(alk-1-ynyl)phenyl)methanols. The 1-arylidene-1,3-dihydroisobenzofuran derivatives showed structured fluorescence spectra in non-polar cyclohexane, originated from a locally excited state and structureless emission in polar acetonitrile involving intramolecular charge transfer process. The vibronic structures in the locally excited emission were confined to the ring C=C stretching of the dyes. A detailed structure-property relationship of the 1-arylidene-1,3-dihydroisobenzofuran dyes is presented in this study.
Leveraging Baird aromaticity for advancement of bioimaging applications
(Wiley, 2022-10) Pati, Avik K.
In this perspective, we highlight the recent progress in utilizing Baird aromatic species to improve fluorophore performance in microscopy and imaging applications. We specifically focus on the origins of the use of Baird aromaticity in fluorescence applications, the development of “self-healing” fluorophores leveraging cyclooctatetraene’ Baird aromaticity, and where developments need to occur to optimize this technology.
Meta Effect of Absorption Energy in Donor–Acceptor Substituted Benzenoids: A Computational Study of Its Dependence on Acceptor Strength, Solvent Polarity, and Conjugation Length
(ACS, 2014-08) Pati, Avik K.
The present work focuses on theoretical understanding of electronic absorption energies of N,N-dimethylaniline with different ortho-, meta-, and para-substituted acceptor groups. The meta isomers exhibit the lowest absorption energy compared to the ortho and para derivatives. This unusual behavior of absorption energies of the meta isomers is related to the “meta effect” well-known in organic photochemical reactions. The meta effect of absorption energy of the derivatives is found to depend on the strength of acceptors, solvent polarity, and conjugation length. The meta derivatives with strong acceptor groups generally exhibit the lowest absorption energy over the other isomers irrespective of solvent polarity. However, the meta isomers with weak acceptor groups exhibit the meta effect only in highly polar solvents. The trend of the lowest absorption energies of the meta isomers is observed to change if the acceptor group is bridged through π conjugation unit (n) with the core moiety. The normal pattern of absorption energy that is the para isomer is of lowest energy is observed to occur for the derivatives where the repeated conjugation units (n) are between 2 and 4. The normal pattern of absorption energy is continued to observe from n > 4 for all the derivatives.
Microsolvation-driven hours-long spectral dynamics in phenoxazine dyes
(ACS, 2024-12) Pati, Avik K.
The phenoxazine class of dyes has found widespread applications in chemistry and biology for more than a century, particularly for lipid membrane studies. Here, we report a general phenomenon on the ensemble spectral stability of traditional phenoxazine class of dyes (nile red, cresyl violet, and nile blue) that exhibit hours-long microstructural transitions reflected through systematic changes of electronic spectra over an hour. Mechanistic investigations reveal that such spectral dynamics of the dyes can be mitigated by tuning microenvironments, where microsolvation plays an underlying role. These microsolvation-induced microstructural changes in a single dye species tend to follow zeroth-order kinetics. The half-life values of such processes systematically vary with solvent hydrogen bonding strength and ionic radius of the dyes’ counteranions. In so doing, using a model lipid membrane, we demonstrate that the spectral response of a phenoxazine dye must be utilized appropriately for studying membrane properties. These findings of the phenoxazine class of dyes are of high significance for their careful applications in chemistry and biology
On the photophysics of butadiyne bridged pyrene–phenyl molecular conjugates: multiple emissive pathways through locally excited, intramolecular charge transfer and excimer states
(RSC, 2015-01) Pati, Avik K.
The present work describes the photophysical properties of a group of butadiyne bridged pyrene–phenyl molecular hybrids having different substitutions with varying donor and acceptor abilities. In addition to emission from the locally excited (LE) state originating from the pyrene moiety, intramolecular charge transfer (ICT) emissions were observed in molecules with donor–acceptor character. The positions of the ICT emission maxima varied over a wide range of wavelengths (475–600 nm). Pyrene behaved as a donor when a strong acceptor group (–CN) was attached to the phenyl ring and it behaved as an acceptor when the phenyl group contained a strong donor group (–NMe2). In mixed aqueous solvents at higher percentages of water (80–99%), the derivatives showed emissions from the aggregate state in addition to the LE and ICT states. Emissions from the aggregate states of the derivatives were centred in the range 510–560 nm. The aggregate state emissions were found to originate from static excimers involving pyrene moieties. A detailed structure–property relationship of the butadiynyl derivatives was revealed in this study.
Organic fluorophores for studying lipid membrane structures and dynamics
(Wiley, 2025-09) Pati, Avik K.
Biomembranes act as boundaries between cells and their outside environment and between intracellular compartments, playing integral roles in cellular signaling and communications. A vast body of research has shown that biomembranes are dynamic and heterogeneous, and that they largely vary in lipid compositions and their organizations, governing a plethora of membrane-associated biological processes. Fluorescent tools, including fluorescent contrast agents (fluorophores) and fluorescence imaging modalities, have been demonstrated to be powerful in studying lipid membrane structures and dynamics. Here, we review recent progress in lipid membrane probes based on organic fluorophore designs and their uses in studying lipid membrane properties in plasma membranes and various organelle membranes. In this context, we also highlight applications of these fluorescent membrane probes in diverse fluorescence-based imaging settings that advance lipid membrane research.
Photoinduced intramolecular charge transfer in a cross-conjugated push–pull enediyne: implications toward photoreaction
(RSC, 2018) Pati, Avik K.
Push–pull organic fluorophores are important owing to their interesting optoelectronical properties. Here we report the photophysics of a new cross-conjugated push–pull enediynyl dye which belongs to an unexplored class of π-conjugated donor–acceptor systems. Two N,N-dimethylaniline moieties serve as donors and one pyrene ring functions as an acceptor via a common Y-shaped ‘enediyne’ bridge which facilitates the cross-electronic communication. The dye exhibits dual emission from locally excited (LE) and intramolecular charge transfer (ICT) states. While the LE emission is dominant in non-polar solvents, the ICT emission predominates in polar solvents. Time-resolved fluorescence decay experiments reveal a relatively shorter lifetime component (∼0.5–0.9 ns) belonging to an ICT state and a relatively longer lifetime species (∼1.6–2.8 ns) corresponding to the LE state. The strong ICT behavior of the dye is manifested through the huge red-shift (4166 cm−1) of the emission spectra from non-polar cyclohexane to polar N,N-dimethylformamide. In contrast to many small push–pull organic dyes, the LE and ICT states of the push–pull enediynyl dye follow the same excitation pathway. The dominant red-shifted ICT emission (∼550 nm) intensity of the dye in polar solvent decreases with a concomitant appearance of the blue-shifted LE emission (∼385 nm) upon prolonged exposure to photons. This opens up a new photophysical strategy of achieving high contrast two fluorescence color conversion from yellow to blue.
Photoinduced solid state keto–enol tautomerization of 2-(2-(3-nitrophenyl)-4,5-diphenyl-1H-imidazol-1-yloxy)-1-phenylethanone
(RSC, 2014) Pati, Avik K.
Excited state intramolecular proton transfer (ESIPT) plays an important role in biological systems and has also recently found applications in electronic devices such as transducers, switches etc. In this paper we report the synthesis and solid state photochromic behavior of 2-(2-(3-nitrophenyl)-4,5-diphenyl-1H-imidazol-1-yloxy)-1-phenylethanone (II) due to ESIPT. Compound II exhibits yellow color in dark and red color in light, with the yellow form attributed to the keto derivative and the red form assigned to its enol derivative The color change in the presence of light is thus attributed to the keto–enol tautomerism through ESIPT. The color change from yellow to red is a photochemical process which thermally decays to the yellow form in the dark. The solid state stability of the enol form upon phototautomerization of the keto form is a noteworthy phenomenon, and its stability has been substantiated by our experimental findings. In the solution state, the yellow form (keto) is stable in chloroform while the red form (enol) is stable in DMSO. Theoretical calculations have been performed to understand the geometries and electronic transitions of the keto and enol forms. In addition, ground and excited state equilibrium constants for the keto–enol tautomerism were calculated.
Photophysical Impact of Diacetylenic Conjugation on Classical Donor–Acceptor Electronic Energy Pair
(ACS, 2018-12) Pati, Avik K.
Organic fluorophores with extended π-conjugation are important for their widespread applications. The present work provides photophysical insights into a diacetylene bridged classical donor–acceptor electronic energy pair, naphthalene–pyrene, in comparison with its constituents’ molecular structures, naphthyl and pyrenyl acetylenes, as well as parent naphthalene and pyrene chromophores. The diacetylenic dye loses the individual spectral identities of the donor and acceptor fluorophores exhibiting a locally excited (LE) emission (∼411 nm) from the overall molecular entity with high fluorescence quantum yields (0.55–0.84) in nonaqueous media. In contrast to the parent pyrene, the hybrid derivative shows a strongly allowed S0 → S1 transition. In mixed-aqueous media, the dye forms aggregates displaying a new red-shifted absorption (∼425 nm) as well as emission (∼510 nm) band. Unlike the hybrid dye, the naphthyl and pyrenyl acetylenes do not form aggregates. In the aggregate state of the hybrid fluorophore, electronic energy transfer takes place from the naphthyl moiety to pyrenyl ring. The excited-state photophysical properties of the dye are exploited in vapor sensing in the solid state.
Photophysics and peripheral ring size dependent aggregate emission of cross-conjugated enediynes: applications to white light emission and vapor sensing
(RSC, 2018) Pati, Avik K.
Photophysical understanding of organic fluorophores with π-conjugated scaffolds is crucial as such dyes are central to optoelectronic applications. This work presents a detailed photophysical investigation of a class of cross-conjugated homo- and hetero-enediynes (Y-shaped) peripherally attached to common aromatic moieties such as benzene, naphthalene, and anthracene. The cross-communicated electronic communication among the three aromatic units located at the tri-poles of the Y-shaped enediynes results in a broad S0 → S1 absorption band and locally excited (LE) emission signals. In addition to the LE emission band, a red-shifted aggregate emission is observed for some of the dyes in non-aqueous solvents where a clear size dependence of the peripheral aromatic rings is noted for the appearance of the aggregate fluorescence. The aggregates are static in nature as is evident from ground-state absorption spectral changes and the absence of rise-time in the time-resolved fluorescence decay studies, which are substantiated further through nuclear magnetic resonance spectroscopy and single-crystal X-ray diffraction experiments. Molecular orbital calculations support the local nature of the dominant electronic transition. The optimized ground state geometries of the dyes from partially to fully propeller shaped structures confirm the ring-size dependence of the aggregates. The LE and aggregate state emissions are judiciously exploited to generate single-component white light emission in binary solvent mixtures. The excited state photophysics are further applied toward polar aprotic vapor sensing in the solid state
Photophysics and Photoreactivity of Cross-conjugated Enediynyl Aggregates: Applications to Multi-parametric Sensing of Microheterogeneity and Reversible Fluorescence Switching
(Elsevier, 2020-01) Pati, Avik K.
Fluorescent aggregates find wide-spread applications in modern optoelectronics. Here, we study the photophysics of organic fluorophores composed of benzene, pyrene, naphthalene, and anthracene moieties connected via a Y-shaped enediyne π-spacer in water-acetonitrile solvent mixtures and solid forms. The fluorophores show a predominant aggregate emission band, which is ca. 4000 cm−1 red-shifted compared to their locally excited (LE) emission energies in water-acetonitrile. The aggregate emission is however blue-shifted compared to an intramolecular charge transfer (ICT) emission band. The interplay of the LE, ICT, and aggregate emissive states depends on peripheral aromatic units, electronic substituents and microenvironments. The LE to aggregate emission spectral shift is exploited to sensitively probe micro-heterogeneity in bile salts, which are complemented by fluorescence intensity and fluorescence anisotropy changes. The solution phase aggregate emissions closely resemble the solid state aggregate emission bands, which are attributed to excimer states. The aggregate fluorescence states in the solids are altered by external stimuli and reversibly switched multiple of cycles. The aggregates undergo photoreactions in mixed-aqueous solvents, but they remain unreactive in solid powder forms.
Photophysics of Diphenylbutadiynes in Water, Acetonitrile–Water, and Acetonitrile Solvent Systems: Application to Single Component White Light Emission
(ACS, 2016-07) Pati, Avik K.
Diacetylenes have been the subject of current research because of their interesting optoelectronic properties. Herein, we report that substituted diphenylbutadiynes exhibit locally excited (LE) and excimer emissions in water and multiple emissions from the LE, excimer, and intramolecular charge transfer (ICT) states in acetonitrile–water solvent systems. The LE, excimer, and ICT emissions are clearly distinguishable for a diphenylbutadiynyl derivative with push (−NMe2)–pull (−CN) substituents and those are closely overlapped for non-push–pull analogues. In neat acetonitrile, the excimer emission disappears and the LE and ICT emissions predominate. In the case of the push (−NMe2)–pull (−CN) diphenylbutadiyne, the intensity of the ICT emission increases with increasing the fluorophore concentration. This suggests that the ICT emission accompanies with intermolecular CT emission which is of exciplex type. As the LE and exciplex emissions of the push–pull diphenylbutadiyne together cover the visible region (400–700 nm) in acetonitrile, a control of the fluorophore concentration makes the relative intensities of the LE and exciplex emissions such that pure white light emission is achieved. The white light emission is not observed in those diphenylbutadiynyl analogues in which the peripheral substituents of the phenyl rings do not possess strong push–pull character.
Quantitative comparison between sub-millisecond time resolution single-molecule FRET measurements and 10-second molecular simulations of a biosensor protein
(PLOS Biology, 2020-11) Pati, Avik K.
Molecular Dynamics (MD) simulations seek to provide atomic-level insights into conformationally dynamic biological systems at experimentally relevant time resolutions, such as those afforded by single-molecule fluorescence measurements. However, limitations in the time scales of MD simulations and the time resolution of single-molecule measurements have challenged efforts to obtain overlapping temporal regimes required for close quantitative comparisons. Achieving such overlap has the potential to provide novel theories, hypotheses, and interpretations that can inform idealized experimental designs that maximize the detection of the desired reaction coordinate. Here, we report MD simulations at time scales overlapping with in vitro single-molecule Förster (fluorescence) resonance energy transfer (smFRET) measurements of the amino acid binding protein LIV-BPSS at sub-millisecond resolution. Computationally efficient all-atom structure-based simulations, calibrated against explicit solvent simulations, were employed for sampling multiple cycles of LIV-BPSS clamshell-like conformational changes on the time scale of seconds, examining the relationship between these events and those observed by smFRET. The MD simulations agree with the smFRET measurements and provide valuable information on local dynamics of fluorophores at their sites of attachment on LIV-BPSS and the correlations between fluorophore motions and large-scale conformational changes between LIV-BPSS domains. We further utilize the MD simulations to inform the interpretation of smFRET data, including Förster radius (R0) and fluorophore orientation factor (κ2) determinations. The approach we describe can be readily extended to distinct biochemical systems, allowing for the interpretation of any FRET system conjugated to protein or ribonucleoprotein complexes, including those with more conformational processes, as well as those implementing multi-color smFRET.
Recovering true FRET efficiencies from smFRET investigations requires triplet state mitigation
(Springer Nature, 2024-06) Pati, Avik K.
Single-molecule fluorescence resonance energy transfer (smFRET) methods employed to quantify time-dependent compositional and conformational changes within biomolecules require elevated illumination intensities to recover robust photon emission streams from individual fluorophores. Here we show that outside the weak-excitation limit, and in regimes where fluorophores must undergo many rapid cycles of excitation and relaxation, non-fluorescing, excitation-induced triplet states with lifetimes orders of magnitude longer lived than photon-emitting singlet states degrade photon emission streams from both donor and acceptor fluorophores resulting in illumination-intensity-dependent changes in FRET efficiency. These changes are not commonly taken into consideration; therefore, robust strategies to suppress excited state accumulations are required to recover accurate and precise FRET efficiency, and thus distance, estimates. We propose both robust triplet state suppression and data correction strategies that enable the recovery of FRET efficiencies more closely approximating true values, thereby extending the spatial and temporal resolution of smFRET.
Resolving fluorescence signatures of a photoconvertible fluorophore by fluorescence spectroscopy and MCR-ALS-based combinatorial approach
(Elsevier, 2022-03) Pati, Avik K.
Photoconvertible fluorophores are important for a myriad of applications in chemistry and biology. Here, we spectrally resolve and quantify individual photophysical information of a dual-emitting photoconvertible fluorophore by fluorescence spectroscopy and multivariate curve resolution-alternate least square techniques. We found that the reactant fluorophore, which shows a weak locally excited (LE) emission and a dominant intramolecular charge transfer (ICT) emission, also exhibits an intermolecular charge transfer emission. The ICT emission bands of both the reactant and product fluorophores are originated from their respective LE states. The reactant fluorophore is a mixture of its different ground state conformers. Higher yields of photoconversion of the yellow-emitting reactant fluorophore are achieved via a visible light photoreaction, leading to formation of pure white light at an intermediate photoreaction time. These findings together help us to glean new photophysical and photochemical insights into the photoreaction of a dual-emitting photoconvertible fluorophore.
Search for improved triplet-state quenchers for fluorescence imaging: a computational framework incorporating excited-state Baird-aromaticity
(RSC, 2025-03) Pati, Avik K.
Fluorescence imaging is crucial for studying biology. Triplet state quenchers (TSQs), especially cyclooctatetraene (COT), can dramatically improve fluorophore performance, particularly when linked intramolecularly so as to enable “self-healing”. Leveraging knowledge revealed through investigations of the self-healing mechanism enabled by COT, we computationally screened for cyclic 8π-electron species, and their annulated derivatives, with efficient triplet–triplet energy transfer potential, high photostability, and strong spin–orbit coupling (SOC) between the lowest triplet state to the singlet ground state. Here, we report theory-based analyses of a broad array of candidates that demonstrate various extents of triplet state Baird-aromaticity, indicating self-healing potential. We identify specific candidates with 7-membered ring structures predicted to exhibit favorable enhancements in fluorophore performance spanning the visible spectrum, with several possessing estimated intersystem crossing (ISC) rates up to 4 × 106 times faster than that of COT, the current benchmark for the self-healing strategy.