A small-molecule probe to decipher stress-induced ER microenvironments and ER-Golgi communication.

Cellular stress is a crucial factor in regulating and maintaining both organismal and microenvironmental homeostasis. It induces a response that also affects the micropolarity of specific cellular compartments, which is essential for early disease diagnosis. In this contribution, we present a quantitative study of micropolarity changes inside the endoplasmic reticulum (ER) during the G1/S and G2/M phases, using a biocompatible small-molecule fluorophore called ER-Oct. This probe is selectively driven to the ER by its hydrophobicity, and it has the fastest diffusion properties among a series of analogous probes. We found that induced ER stress caused cell cycle arrests leading to an increase in ER micropolarity which is well supported by lambda scanning experiments and fluorescence lifetime imaging microscopy (FLIM) as well. ER-Oct is a versatile staining agent that could effectively stain the ER in various living/fixed mammalian cells, isolated ER, Caenorhabditis elegans, and mice tissues. Furthermore, we used this probe to visualize a well-known biological event, ER to Golgi transport, by live-cell fluorescence microscopy. Our exhaustive investigation of micropolarity using ER-staining dye provides a new way to study ER stress, which could provide a deeper understanding of proteostasis in model systems and even in fixed patient samples.

Mitochondria-Directing Fluorogenic Probe: An Efficient Amyloid Marker for Imaging Lipid Metabolite-Induced Protein Aggregation in Live Cells and Caenorhabditis elegans.

The design and development of optical probes for sensing neurotoxic amyloid fibrils are active and important areas of research and are undergoing continuous advancements. In this paper, we have synthesized a red emissive styryl chromone-based fluorophore (SC1) for fluorescence-based detection of amyloid fibrils. SC1 records exceptional modulation in its photophysical properties in the presence of amyloid fibrils, which has been attributed to the extreme sensitivity of its photophysical properties toward the immediate microenvironment of the probe in the fibrillar matrix. SC1 also shows very high selectivity toward the amyloid-aggregated form of the protein as compared to its native form. The probe is also able to monitor the kinetic progression of the fibrillation process, with comparable efficiency as that of the most popular amyloid probe, Thioflavin-T. Moreover, the performance of SC1 is least sensitive to the ionic strength of the medium, which is an advantage over Thioflavin-T. In addition, the molecular level interaction forces between the probe and the fibrillar matrix have been interrogated by molecular docking calculations which suggest the binding of the probe to the exterior channel of the fibrils. The probe has also been demonstrated to sense protein aggregates from the Aß-40 protein, which is known to be responsible for Alzheimer's disease. Moreover, SC1 exhibited excellent biocompatibility and exclusive accumulation at mitochondria which allowed us to successfully demonstrate the applicability of this probe to detect mitochondrial-aggregated protein induced by an oxidative stress indicator molecule 4-hydroxy-2-nonenal (4-HNE) in A549 cell lines as well as in a simple animal model like Caenorhabditis elegans. Overall, the styryl chromone-based probe presents a potentially exciting alternative for the sensing of neurotoxic protein aggregation species both in vitro as well as in vivo.

Construing the metaxin-2 mediated simultaneous localization between mitochondria and nucleolus using molecular viscometry.

Fluorescent probes for specific inter-organelle communication are of massive significance as such communication is essential for a diverse range of cellular events. Here, we present the microviscosity-sensitive fluorescence marker, Quinaldine Red (QR), and its dual organelle targeting light-up response in live cells. This biocompatible probe was able to localize in mitochondria and nucleolus simultaneously. While QR was able to sense the viscosity change inside these compartments under the induced effect of an ionophore and ROS-rich microenvironment, the probe's ability to stain mitochondria remained unperturbed even after protonophore-induced depolarization. Consequently, a systematic quantification was performed to understand the alteration of microviscosity. Similar behavior in two distinct organelles implied that QR binds to metaxin-2 protein, common to mitochondrial and nucleolar proteomes. We believe this is the first of its kind investigation that identifies the inter-organelle communications marker and opens up a new dimension in this field.

Intracellular Physical Properties with Small Organic Fluorescent Probes: Recent Advances and Future Perspectives.

The intracellular physical parameters i. e., polarity, viscosity, fluidity, tension, potential, and temperature of a live cell are the hallmark of cellular health and have garnered immense interest over the past decade. In this context, small molecule organic fluorophores exhibit prominent useful properties including easy functionalizability, environmental sensitivity, biocompatibility, and fast yet efficient cellular uptakability which has made them a popular tool to understand intra-cellular micro-environmental properties. Throughout this discussion, we have outlined the basic design strategies of small molecules for specific organelle targeting and quantification of physical properties. The values of these parameters are indicative of cellular homeostasis and subtle alteration may be considered as the onset of disease. We believe this comprehensive review will facilitate the development of potential future probes for superior insight into the physical parameters that are yet to be quantified.

Red to NIR-emissive anthracene-conjugated PMI dyes with dual functions: singlet-oxygen response and lipid-droplet imaging.

The rich chemistry of solution-processable red and near-infrared (NIR) organic emitters has emerged as an attractive and progressive research field because of their particular applications in organic optoelectronics and bioimaging. Also, one can see that the research area of perylene monoimide-based red and NIR-emissive fluorophores is underexplored, which prompted us to design and synthesize three anthracene-conjugated PMI dyes exhibiting strong emission in the red and NIR window in solution. Three PMI-based fluorophores were synthesized via conjoining anthracene and donor moieties (-Ph, -N,N-PhNMe2) with a PMI core via an acetylene linkage at the peri-position, which helped to attain extensive electronic conjugation, which was reflected in red and NIR-emission in solution. The key molecular features to be highlighted here are: all three dyes are strongly emissive in solution, as unveiled by the excellent absolute fluorescence QYs; and they possess tuneable emission properties, guided by the donor strength and a profound Stokes shift (100-200 nm). The three fluorescent dyes demonstrated appreciable singlet-oxygen (1O2) sensitivity when photoirradiated with methylene blue (MB) in solution, showing a substantial blue-shift in emission in a ratiometric manner. Further, the treatment of dye-MB solution with α-tocopherol (1O2 scavenger) validated the presence of 1O2 as the only oxidizing species generated by MB in solution. Computational investigations gave insight into the twisting of donor moieties in their ground-state optimized geometries, the modulation of the FMO energy gap, and the thermodynamic feasibility of the 1O2 reaction. Finally, via taking advantage of the red and NIR-emission, we successfully utilized one of the fluorophores as a lipid-droplet marker for bioimaging in HepG2 cells.

Interplay of Anion-π+ and π+ -π+ Interactions in Novel Pyrido[2,1-a]isoquinolinium-Based AIEgens - Substituent- and Counterion-Dependent Fluorescence Modulation and Applications in Live Cell Mitochondrial Imaging.

Recently, the concept of anion-π+ interactions has witnessed unique applications in the field of AIEgen development. In this contribution, we disclose a consolidated study of a library of N-doped ionic AIEgens accessed through silver-mediated cyclization of pyridino-alkynes. A thorough photophysical, computational and crystallographic study has been conducted to rationalize the observed substituent- and counterion-dependent fluorescence properties of these luminogens. We further elucidate the prominent role of anion-π+ interactions, π+ -π+ interactions and other non-covalent interactions, in inhibiting the undesired ACQ effect. Finally, we have also demonstrated the application of selected AIEgens for imaging of mitochondria in live cells.

Janus -faced oxidant and antioxidant profiles of organo diselenides.

To date, organoseleniums are pre-eminent for peroxide decomposition and radical quenching antioxidant activities. On the contrary, here, a series of Janus-faced aminophenolic diselenides have been prepared from substituted 2-iodoaniline and selenium powder using copper-catalyzed methodology. Subsequently, condensation with substituted salicylaldehyde afforded the Schiff base, which on reduction, yielded the desired substituted aminophenolic diselenides in 72%-88% yields. The generation of reactive oxygen species (ROS) from oxygen gas by the synthesized aminophenolic diselenides was studied by analyzing the oxidation of dichlorofluorescein diacetate (DCFDA) dye and para-nitro-thiophenol by fluorescence and UV-Visible spectroscopic methods. Furthermore, density functional theory calculations and crystal structure analysis revealed the role of functional amine and hydroxyl sites present in the Janus-faced organoselenium catalyst for the activation of molecular oxygen, where NH and phenolic groups bring the oxygen molecule close to the catalyst by N-H⋯O and O-H⋯O intermolecular interactions. Additionally, these functionalities stabilize the selenium-centered radical in the formed transition states. Antioxidant activities of the synthesized diselenides have been explored as the catalyst for the decomposition of hydrogen peroxide using benzenethiol sacrificial co-reductant by a well-established thiol assay. Radical quenching antioxidant activity was studied by the quenching of DPPH radicals at 516 nm by UV-Visible spectroscopy. The structure activity correlation suggests that the electron-rich phenol and electron-rich and sterically hindered selenium center enhance the oxidizing property of the aminophenolic diselenides. Janus-faced diselenides were also evaluated for their cytotoxic effect on HeLa cancer cells via MTT assay, which suggests that the compounds are effective at 15-18 µM concentration against cancer cells. Moreover, the combination with therapeutic anticancer drugs Erlotinib and Doxorubicin showed promising cytotoxicity at the nanomolar concentration (8-28 nM), which is sufficient to suppress the growth of the cancer cells.

Strategic engineering of alkyl spacer length for a pH-tolerant lysosome marker and dual organelle localization.

Long-term visualization of lysosomal properties is extremely crucial to evaluate diseases related to their dysfunction. However, many of the reported lysotrackers are less conducive to imaging lysosomes precisely because they suffer from fluorescence quenching and other inherent drawbacks such as pH-sensitivity, polarity insensitivity, water insolubility, slow diffusibility, and poor photostability. To overcome these limitations, we have utilized an alkyl chain length engineering strategy and synthesized a series of lysosome targeting fluorescent derivatives namely NIMCs by attaching a morpholine moiety at the peri position of the 1,8-naphthalimide (NI) ring through varying alkyl spacers between morpholine and 1,8-naphthalimide. The structural and optical properties of the synthesized NIMCs were explored by 1H-NMR, single-crystal X-ray diffraction, UV-Vis, and fluorescence spectroscopy. Afterward, optical spectroscopic measurements were carefully performed to identify a pH-tolerant, polarity sensitive, and highly photostable fluoroprobes for further live-cell imaging applications. NIMC6 displayed excellent pH-tolerant and polarity-sensitive properties. Consequently, all NIMCs were employed in kidney fibroblast cells (BHK-21) to investigate their applicability for lysosome targeting and probing lysosomal micropolarity. Interestingly, a switching of localization from lysosomes to the endoplasmic reticulum (ER) was also achieved by controlling the linker length and this phenomenon was subsequently applied in determining ER micropolarity. Additionally, the selected probe NIMC6 was also employed in BHK-21 cells for 3-D spheroid imaging and in Caenorhabditis elegans (C. elegans) for in vivo imaging, to evaluate its efficacy for imaging animal models.

An Enumerated Outlook of Intracellular Micropolarity Using Solvatochromic Organic Fluorescent Probes.

The spatiotemporal distribution of intracellular physical parameters of a live cell is heterogeneous and complex. Measuring physical properties inside given cellular compartments (organelles) is challenging and important for therapy and diagnostics. The tiny volume of a single cell and even tinier organelles are not accessible by classical measuring devices. The microenvironment inside an organelle vastly controls the outcome of any biochemical and biophysical processes taking place inside it, which is crucial for the overall cellular health. Therefore, it is very important to understand the microenvironmental physical properties inside cellular organelles. Moreover, specific alterations of such microenvironmental properties were observed in the disease condition, making them a diagnostic hallmark. With this high demand, small-molecule organic fluorophores are emerging as the most successful tool due to their small relative size, bioavailability, and ease of functionalization. In this mini-review, the development of micropolarity-sensitive small organic fluorophore with the capability of targeting a specific cellular organelle has been discussed. Here, we have highlighted the strategies of targeting a specific organelle, the micropolarity, and the challenges and prospects of the field.

Supramolecular Encapsulation of a Neurotransmitter Serotonin by Cucurbit[7]uril.

pH-dependent host-guest complexation of a monoamine neurotransmitter, Serotonin, with cucurbit[7]uril has been thoroughly investigated. The binding phenomena were explored using steady-state and time-resolved fluorescence spectroscopy at different pH values. At lower pH, i.e., protonated Serotonin, the binding affinity with cucurbit[7]uril was significantly higher compared to higher pH. Furthermore, detailed NMR titration experiments depicted the solution structure of the host-guest complex through the complexation induced chemical shift values. A competitive binding assay with cesium ions at pD 2.8 was subsequently performed for the further manifestation of the binding. Finally, the molecular docking studies provided well-documented proof of the 1:1 inclusion complex and the geometry of the complex. We believe that understanding from such studies can be important for pH-controlled delivery of serotonin for biological applications.

Biocompatible Fluorescent Probe for Selective Detection of Amyloid Fibrils.

The misfolding and aggregation of proteins leading to amyloid formation has been linked to numerous diseases, necessitating the development of tools to monitor the fibrillation process. Here, we report an intramolecular charge transfer (ICT) dye, DMNDC, as an alternative to thioflavin-T (ThT), most commonly used for monitoring amyloid fibrils. Using insulin as a model protein, we show that DMNDC efficiently reports on the kinetics of fibril formation. An approximately 70 nm hypsochromic shift along with a large enhancement in emission intensity was observed upon binding of DMNDC to protein fibrils. The aggregation kinetics of insulin were not significantly affected in the presence of DMNDC, suggesting that DMNDC does not inhibit insulin aggregation. Additionally, the efficient cellular internalization and low toxicity of DMNDC make it highly suited for sensing and imaging of amyloid fibrils in the complex biological milieu.

Cellular metabolic activity marker via selective turn-ON detection of transporter protein using nitrobenzoxadiazole-based fluorescent reporter.

A nitrobenzoxadiazole-based fluoroprobe (NBD-Bu) is designed to probe cellular metabolic activity in cancer and normal cells. NBD-Bu shows a significant fluorescence enhancement upon selective binding to the transport protein serum albumin in PBS buffer at ambient conditions. Encouraged by this finding, the site- specificity of NBD-Bu has been explored through a competitive displacement assay in the presence of site-specific markers such as warfarin and ibuprofen. Notably, even at micromolar concentrations, the probe possesses the ability to displace the site marker drug ibuprofen, efficiently. Subsequently, high-resolution fluorescence imaging results consolidated the potential of NBD-Bu for detection of abnormal cellular metabolic activity.

Orchestrated catalytic double rollover annulation: rapid access to N-enriched cationic and neutral PAHs.

Disclosed herein is a rhodium(iii)-catalyzed novel one-step back-to-back double rollover annulation on pyridine and pyrazine backbones leading to a structurally and optoelectronically diverse class of nicely decorated multi-ring-fused, extensively π-conjugated, N-enriched PAH molecules by virtue of orchestrated quadruple C-H activation events. Selected N-PAHs have been utilized as potential mitochondria and lysosome markers.

A rapid, naked-eye detection of hypochlorite and bisulfite using a robust and highly-photostable indicator dye Quinaldine Red in aqueous medium.

A "naked-eye" detection of health hazardous bisulfite (HSO3-) and hypochlorite (ClO-) using an indicator dye (Quinaldine Red, QR) in a wide range of pH is demonstrated. The molecule contains a quinoline moiety linked to an N,N-dimethylaniline moiety with a conjugated double bond. Treatment of QR with HSO3- and ClO-, in aqueous solution at near-neutral pH, resulted in a colorless product with high selectivity and sensitivity. The detection limit was 47.8µM and 0.2µM for HSO3- and ClO- respectively. However, ClO- was 50 times more sensitive and with 2 times faster response compared to HSO3-. The detail characterization and related analysis demonstrate the potential of QR for a rapid, robust and highly efficient colorimetric sensor for the practical applications to detect hypochlorite in water samples.