Fluorescent Molecular Rotors Research Articles

Thieno[3,2-b]thiophene for the Construction of Far-Red Molecular Rotor Hemicyanines as High-Affinity DNA Aptamer Fluorogenic Reporters.

The construction of far-red fluorescent molecular rotors (FMRs) is an imperative task for developing nucleic acid stains that have superior compatibility with cellular systems and complex matrices. A typical strategy relies on the methine extension of asymmetric cyanines, which unfortunately fails to produce sensitive rotor character. To break free from this paradigm, we have synthesized far-red hemicyanines using a dimethylamino thieno[3,2-b]thiophene donor. The resultant probes, designated as ATh2Ind and ATh2Btz, possess excitation maxima (λmax) of >600 nm and have been rigorously characterized by NMR, electrochemistry, and computational methods. The dyes possess alternating charge patterns like indodicarbocyanine (Cy5), but with twisted intramolecular charge transfer (TICT) rotational barriers at 60°, akin to the classical FMR thiazole orange (TO1). ATh2Btz also displays cyanine characteristics, enhancing its response upon binding to nucleic acids and allowing for efficient staining of cellular nuclei. When binding to the DNA aptamer for quinine (MN4), ATh2Btz exhibits a Kd of 17 nM, a 660-fold light-up response, brightness (Φfl x εmax) of ∼37,000 M-1cm-1, and λex/λem of 655/677 nm. The resulting far-red DNA-based MN4-ATh2Btz platform has been termed "pomegranate."

A near-infrared fluorescent molecular rotor for viscosity detection in biosystem and fluid beverages

Viscosity is closely associated with physiological and pathological processes, as well as food quality. Herein, a novel fluorescent molecular rotor, BMCY-V, was presented and applied for detection of viscosity. BMCY-V contained a benzoindole unit as electron donor and a malononitrile group as acceptor. In low-viscous solvents, the rotor can freely rotate, leading to dissipation of excited-state energy. In high-viscous media, however, the free rotation of the rotor is severely restricted, thus reducing non-radiative transition and resulting in significantly enhanced fluorescence intensity. BMCY-V is extremely sensitive to viscosity, showing about 3968 times increase of fluorescence intensity at 728 nm from water to 95 % glycerol. Due to the excellent photophysical property such as near-infrared emission, BMCY-V was successfully used to visualize viscosity in live cells and in liver tissues. In addition, BMCY-V can also evaluate the thickening effect of various thickeners and visualize the changes of viscosity during deterioration of fluid drinks.

Fluorescent Molecular Rotors Quantify an Adjuvant-Induced Softening of Plant Wax

Fluorescent Molecular Rotors Quantify an Adjuvant-Induced Softening of Plant Wax

A Red-Emission Fluorescent Probe with Large Stokes Shift for Detection of Viscosity in Living Cells and Tumor-Bearing Mice.

Abnormal viscosity is closely related to the occurrence of many diseases, such as cancer. Therefore, real-time detection of changes in viscosity in living cells is of great importance. Fluorescent molecular rotors play a critical role in detecting changes in cellular viscosity. Developing red emission viscosity probes with large Stokes shifts and high sensitivity and specificity remains an urgent and important topic. Herein, a novel viscosity-sensitive fluorescent probe (TCF-VIS1) with a large stokes shift and red emission was prepared based on the 2-dicyanomethylene-3-cyano-4,5,5-trimethyl-2,5-dihydrofuran (TCF) skeleton. Due to intramolecular rotation, the probe itself does not fluorescence at low viscosity. With the increase in viscosity, the rotation of TCF-VIS1 is limited, and its fluorescence is obviously enhanced. The probe has the advantages of simple preparation, large Stokes shift, good sensitivity and selectivity, and low cytotoxicity, which make it successfully used for viscosity detection in living cells. Moreover, TCF-VIS1 showed its potential for cancer diagnosis at the cell level and in tumor-bearing mice by detecting viscosity. Therefore, the probe is expected to enrich strategies for the detection of viscosity in biological systems and offer a potential tool for cancer diagnosis.

Excited State Rotational Freedom Impacts Viscosity Sensitivity in Arylcyanoamide Fluorescent Molecular Rotor Dyes.

The microviscosity of intracellular environments plays an important role in monitoring cellular function. Thus, the capability of detecting changes in viscosity can be utilized for the detection of different disease states. Viscosity-sensitive fluorescent molecular rotors are potentially excellent probes for these applications; however, the predictable relationships between chemical structural features and viscosity sensitivity are poorly understood. Here, we investigate a set of arylcyanoamide-based fluorescent probes and the effect of small aliphatic substituents on their viscosity sensitivity. We found that the location of the substituents and the type of π-network of the fluorophore can significantly affect the viscosity sensitivity of these fluorophores. Computational analysis supported the notion that the excited state rotational energy barrier plays a dominant role in the relative viscosity sensitivity of these fluorophores. These findings provide valuable insight into the design of molecular rotor-based fluorophores for viscosity measurement.

Molecular Rotors for In Situ Viscosity Mapping during Evaporation of Confined Fluid Mixtures.

Numerous formulation processes of materials involve a drying step, during which evaporation of a solvent from a multicomponent liquid mixture, often confined in a thin film or in a droplet, leads to concentration and assembly of nonvolatile compounds. While the basic phenomena ruling evaporation dynamics are known, precise modeling of practical situations is hindered by the lack of tools for local and time-resolved mapping of concentration fields in such confined systems. In this article, the use of fluorescence lifetime imaging microscopy and of fluorescent molecular rotors is introduced as a versatile, in situ, and quantitative method to map viscosity and concentration fields in confined, evaporating liquids. More precisely, the cases of drying of a suspended liquid film and of a sessile droplet of mixtures of fructose and water are investigated. Measured viscosity and concentration fields allow characterization of drying dynamics, in agreement with simple modeling of the evaporation process.

Visualization of the Sol-Gel Transition in Porous Networks Using Fluorescent Viscosity-Sensitive Probes.

The sol-gel transition involves the transformation of a colloidal suspension into a system-spanning, interconnected gel. This process is widely used to reinforce mechanically weakened porous artifacts, such as sculptures but the impact of the restricted geometry (porous network) on the gelation dynamics of the solution remains unclear. Here, using fluorescent viscosity-sensitive molecular rotors, confocal microscopy, and model pores, we visualize the local viscosity changes at the microscale that accompany the sol-gel transition of a methyltriethoxysilane solution into a gel network. We show that, with evaporation of the solvent, a viscosity gradient develops near the free surface, triggering the sol-gel transition inside small pores near the surface. In homogeneous porous media, this leads to skin formation, which reduces the evaporation rate. In heterogeneous porous media, a gradient in gel density develops toward the heart of the porous material, where the gel formation mainly occurs as capillary bridges within smaller pores.

Trimethylguanosine cap-fluorescent molecular rotor (TMG-FMR) conjugates are potent, specific snurportin1 ligands enabling visualization in living cells.

The trimethylguanosine (TMG) cap is a motif present inter alia at the 5' end of small nuclear RNAs, which are involved in RNA splicing. The TMG cap plays a crucial role in RNA processing and stability as it protects the RNA molecule from degradation by exonucleases and facilitates its export from the nucleus. Additionally, the TMG cap plays a role in the recognition of snRNA by snurportin, a protein that facilitates nuclear import. TMG cap analogs are used in biochemical experiments as molecular tools to substitute the natural TMG cap. To expand the range of available TMG-based tools, here we conjugated the TMG cap to Fluorescent Molecular Rotors (FMRs) to open the possibility of detecting protein-ligand interactions in vitro and, potentially, in vivo, particularly visualizing interactions with snurportin. Consequently, we report the synthesis of 34 differently modified TMG cap-FMR conjugates and their evaluation as molecular probes for snurportin. As FMRs we selected three GFP-like chromophores (derived from green fluorescent protein) and one julolidine derivative. The evaluation of binding affinities for snurportin showed unexpectedly a strong stabilizing effect for TMGpppG-derived dinucleotides containing the FMR at the 2'-O-position of guanosine. These newly discovered compounds are potent snurportin ligands with nanomolar KD (dissociation constant) values, which are two orders of magnitude lower than that of natural TMGpppG. The effect is diminished by ∼50-fold for the corresponding 3'-regioisomers. To deepen the understanding of the structure-activity relationship, we synthesized and tested FMR conjugates lacking the TMG cap moiety. These studies, supported by molecular docking, suggested that the enhanced affinity arises from additional hydrophobic contacts provided by the FMR moiety. The strongest snurportin ligand, which also gave the greatest fluorescence enhancement (Fm/F0) when saturated with the protein, were tested in living cells to detect interactions and visualize complexes by fluorescence lifetime monitoring. This approach has potential applications in the study of RNA processing and RNA-protein interactions.

Fluorogenic cell surface glycan labelling with fluorescence molecular rotor dyes and nucleic acid stains.

We show that covalent labelling of sialic acids on live cell surfaces or mucin increases the fluorescence of the fluorescence molecular rotors (FMRs) CCVJ, Cy3 and thioazole orange, enabling wash-free imaging of cell surfaces. Dual labelling with an FMR and an environmentally insensitive dye allows detection of changes that occur, for example, when cross-linking is altered.

A chloromethyl-triazole fluorescent chemosensor for O6-methylguanine DNA methyltransferase.

Fluorescent chemosensors offer a direct means of measuring enzyme activity for cancer diagnosis, predicting drug resistance, and aiding in the discovery of new anticancer drugs. O6-methylguanine DNA methyltransferase (MGMT) is a predictor of resistance towards anticancer alkylating agents such as temozolomide. Using the fluorescent molecular rotor, 9-(2-carboxy-2-cyanovinyl)julolidine (CCVJ), we synthesized, and evaluated a MGMT fluorescent chemosensor derived from a chloromethyl-triazole covalent inhibitor, AA-CW236, a non-pseudosubstrate of MGMT. Our fluorescence probe covalently labelled the MGMT active site C145, producing a 18-fold increase in fluorescence. Compared to previous fluorescent probes derived from a substrate-based inhibitor, our probe had improved binding and reaction rate. Overall, our chloromethyl triazole-based fluorescence MGMT probe is a promising tool for measuring MGMT activity to predict temozolomide resistance.

Julolidine-based fluorescent molecular rotor: a versatile tool for sensing and diagnosis

Fluorescent molecular rotors incorporating julolidine have found diverse applications in various research fields.

Fluorescent molecular rotors detect O6-methylguanine dynamics and repair in duplex DNA.

Alkylation at the O6 position of guanine is a common and highly mutagenic form of DNA damage. Direct repair of O6-alkylguanines by the "suicide" enzyme O6-methylguanine DNA methyltransferase (MGMT, AGT, AGAT) maintains genome stability and inhibits carcinogenesis. In this study, a fluorescent analogue of thymidine containing trans-stilbene (tsT) is quenched by O6-methylguanine residues in the opposite strand of DNA by molecular dynamics that propagate through the duplex with as much as ∼9 Å of separation. Increased fluorescence of tsT or the cytosine analogue tsC resulting from MGMT-mediated DNA repair were distinguishable from non-covalent DNA-protein binding following protease digest. To our knowledge, this is the first study utilizing molecular rotor base analogues to detect DNA damage and repair activities in duplex DNA.

Dual role far red fluorescent molecular rotor for decoding the plasma membrane and mitochondrial viscosity.

The dysfunctions in the mitochondria are associated with various pathological conditions like neurodegeneration, metabolic disorder, and cancer, leading to dysregulated cell death. Here, we have designed and synthesized a julolidine-based molecular rotor (JMT) to target mitochondria with far-red emission accounting for mitochondrial dysfunction. JMT showed viscosity sensitivity with 160-fold enhancement in fluorescence intensity. The origin of the dark state in a lower viscous environment was investigated through density functional calculations. We have employed JMT to monitor mitochondrial dysfunction induced by nystatin using confocal and fluorescence lifetime imaging microscopy. Further, we investigated mitochondrial abnormalities under inflammatory conditions triggered by lipopolysaccharide in live HeLa cells. The cellular uptake mechanisms of JMT were studied using various endocytosis inhibitors. Moreover, we reported tracking small fluorescent molecule switching from mitochondria to the plasma membrane upon introducing mitochondrial depolarizer in cells. On treating the mitochondria potential uncoupler, JMT relocates to the cell membrane and can be utilized for understanding the interplay between mitochondria and cell membranes. Moreover, JMT was applied to stain the RBC plasma membrane isolated from human blood.

Fluorescent molecular rotors as versatile in situ sensors for protein quantitation

Accurate protein quantitation is essential for many cellular mechanistic studies. Existing technology relies on extrinsic sample evaluation that requires significant volumes of sample as well as addition of assay-specific reagents and importantly, is a terminal analysis. This study exploits the unique chemical features of a fluorescent molecular rotor that fluctuates between twisted-to-untwisted states, with a subsequent intensity increase in fluorescence depending on environmental conditions (e.g., viscosity). Here we report the development of a rapid, sensitive in situ protein quantitation method using ARCAM-1, a representative fluorescent molecular rotor that can be employed in both non-terminal and terminal assays.

Computational chemistry‐assisted design of hydrazine‐based fluorescent molecular rotor for viscosity sensors

AbstractDeep understanding of the fluorescence quenching mechanisms of probes plays a crucial role in developing their practical applications. The fluorescence quenching mechanism of hydrazine‐based fluorescence probes needs to be clarified up to the present. Herein, we designed and synthesized a new hydrazine‐based fluorescence probe (HA‐Na) based on the naphthalimide skeleton. We clarified the molecular origin of the non‐fluorescence of this probe with the aid of computational chemistry and spectroscopic analysis. We showed that the significant rotation of the hydrazine group in the excited state potential energy surface, which caused the complete charge separation, was responsible for the fluorescence quenching of the probe in an organic solvent. Once the rotation was prevented in an aggregative state or high‐viscosity solution, the fluorescence of the probe recovered. In other words, the fluorescence quenching mechanism of hydrazine‐based fluorescence probes was attributed to the formation of a twisted intramolecular charge transfer (TICT) state. More importantly, we demonstrated that this fluorescence molecular rotor could be used to monitor the autophagy process in living cells by detecting lysosomal viscosity changes during starvation. Altogether, this work provides an essential theoretical basis for the developing potential hydrazine‐based fluorescence molecular rotors.

A dual-functional probe that allows cascade response to hydrogen peroxide oxidative stress-induced protein aggregation in live cells

The misfolding and aggregation of specific proteins induced by endogenous oxidation are closely related to some incurable diseases including cancers, diabetes and neurodegenerative diseases. Reactive oxygen species (ROS), as a common oxidizing substance in vivo would induce the oxidation of endogenous biomacromolecules like proteins. However, it remains a challenge to detect protein aggregation induced by specific ROS. Herein, we demonstrate that the developed probe (P2) with a dual function that can simultaneously detect hydrogen peroxide (H2O2) levels and protein aggregates formed by oxidative stress. In this probe, the boronic acid pinacol ester group was conjugated to fluorescent molecular rotor 4-hydroxybenzylidene-imidazolinone (HBI) and served as the H2O2 detection group. Experimental results show that the fluorescence of P2 can be activated by H2O2 and exhibited a positive correlation with the degree of protein aggregation. Moreover, combined with AggTag method P2 was successfully applied to dual-channel imaging of protein aggregation that is stressed by H2O2 in cellular. In general, this work not only provides a potential method to study protein aggregation that associates with oxidation in live cells but also can be generally applied to study other biological processes.

Programming Positive Mechanofluorescence in Liquid Crystalline Elastomers

Programming Positive Mechanofluorescence in Liquid Crystalline Elastomers

Effects of Paclitaxel on Plasma Membrane Microviscosity and Lipid Composition in Cancer Cells.

The cell membrane is an important regulator for the cytotoxicity of chemotherapeutic agents. However, the biochemical and biophysical effects that occur in the membrane under the action of chemotherapy drugs are not fully described. In the present study, changes in the microviscosity of membranes of living HeLa-Kyoto tumor cells were studied during chemotherapy with paclitaxel, a widely used antimicrotubule agent. To visualize the microviscosity of the membranes, fluorescence lifetime imaging microscopy (FLIM) with a BODIPY 2 fluorescent molecular rotor was used. The lipid profile of the membranes was assessed using time-of-flight secondary ion mass spectrometry ToF-SIMS. A significant, steady-state decrease in the microviscosity of membranes, both in cell monolayers and in tumor spheroids, was revealed after the treatment. Mass spectrometry showed an increase in the unsaturated fatty acid content in treated cell membranes, which may explain, at least partially, their low microviscosity. These results indicate the involvement of membrane microviscosity in the response of tumor cells to paclitaxel treatment.

Cover Image

The cover image is based on the Review Boron difluoride hydrazone (BODIHY) complexes: A new class of fluorescent molecular rotors by Aisha N. Bismillah and Ivan Aprahamian, https://doi.org/10.1002/poc.4485.

Fluorescent Molecular Rotors Based on Hinged Anthracene Carboxyimides.

Temperature and viscosity are essential parameters in medicine, environmental science, smart materials, and biology. However, few fluorescent sensor publications mention the direct relationship between temperature and viscosity. Three anthracene carboxyimide-based fluorescent molecular rotors, 1DiAC∙Cl, 2DiAC∙Cl, and 9DiAC∙Cl, were designed and synthesized. Their photophysical properties were studied in various solvents, such as N, N-dimethylacetamide, N, N-dimethylformamide, 1-propanol, ethanol, dimethyl sulfoxide, methanol, and water. Solvent polarizability resulted in a solvatochromism effect for all three rotors and their absorption and emission spectra were analyzed via the Lippert-Mataga equation and multilinear analysis using Kamlet-Taft and Catalán parameters. The rotors exhibited red-shifted absorption and emission bands in solution on account of differences in their torsion angle. The three rotors demonstrated strong fluorescence in a high-viscosity environment due to restricted intramolecular rotation. Investigations carried out under varying ratios of water to glycerol were explored to probe the viscosity-based changes in their optical properties. A good linear correlation between the logarithms of fluorescence intensity and solution viscosity for two rotors, namely 2DiAC∙Cl and 9DiAC∙Cl, was observed as the percentage of glycerol increased. Excellent exponential regression between the viscosity-related temperature and emission intensity was observed for all three investigated rotors.