Auxochrome Dimethyl-Dihydroacridine Improves Fluorophores for Prolonged Live-Cell Super-Resolution Imaging.

Superior photostability, minimal phototoxicity, red-shifted absorption/emission wavelengths, high brightness, and an enlarged Stokes shift are essential characteristics of top-tier organic fluorophores, particularly for long-lasting super-resolution imaging in live cells (e.g., via stimulated emission depletion (STED) nanoscopy). However, few existing fluorophores possess all of these properties. In this study, we demonstrate a general approach for simultaneously enhancing these parameters through the introduction of 9,9-dimethyl-9,10-dihydroacridine (DMA) as an electron-donating auxochrome. DMA not only induces red shifts in emission wavelengths but also suppresses photooxidative reactions and prevents the formation of triplet states in DMA-based fluorophores, greatly improving photostability and remarkably minimizing phototoxicity. Moreover, the DMA group enhances the fluorophores' brightness and enlarges the Stokes shift. Importantly, the "universal" benefits of attaching the DMA auxochrome have been exemplified in various fluorophores including rhodamines, difluoride-boron complexes, and coumarin derivatives. The resulting fluorophores successfully enabled the STED imaging of organelles and HaloTag-labeled membrane proteins.

Photoinduced Charge Centralization Quenches the Fluorescence of Conjugation-Fused Tetrazine Labels with Red-to-Near-Infrared Emissions.

Tetrazine-derived fluorogenic labels are extensively studied for their potential in biological and medical imaging. Nonetheless, the fluorescence quenching mechanism in numerous precursors continues to be debated, particularly as the wavelengths extend into the red and near-infrared (NIR) regions. This challenge poses obstacles to systematically optimizing their fluorogenicity, i.e., achieving red-shifted wavelengths and improved fluorescence turn-on signals through click reactions. This paper highlights the significance of photoinduced charge centralization (PCC), a quenching mechanism observed in tetrazine-fused fluorogenic labels with integrated π-conjugations. PCC is primarily responsible for the quenching effects observed in such labels emitting in the red-to-NIR spectrum. Drawing from structure-property relationships, this study proposes two molecular design strategies that incorporate the PCC mechanism and constitutional isomerization to develop high-performance tetrazine-based labels. These strategies facilitate multiplex fluorescence imaging following click reactions, promising significant advancements in bio-orthogonal imaging techniques.

Tetrazine-Isonitrile Bioorthogonal Fluorogenic Reactions Enable Multiplex Labeling and Wash-Free Bioimaging of Live Cells.

Developing fluorogenic probes for simultaneous live cell labeling of multiple targets is crucial for understanding complex cellular events. The emerging [4+1] cycloaddition between tetrazine and isonitriles holds promise as a bioorthogonal tool, yet existing tetrazine probes lack reactivity and fluorogenicity. Here, we present the development of a series of tetrazine-functionalized bioorthogonal probes. By incorporating pyrazole adducts into the fluorophore scaffolds, the post-reacted probes displayed remarkable fluorescence turn-on ratios, up to 3184-fold. Moreover, these modifications are generalizable to various fluorophores, enabling a broad emission range from 473 to 659 nm. Quantum chemical calculations further elucidate the turn-on mechanisms. These probes enable the simultaneous labeling of multiple targets in live cells, without the need for a washing step. Consequently, our findings pave the way for advanced multiplex imaging and detection techniques for cellular studies.

Force-Induced Near-Infrared Chromism of Mechanophore-Linked Polymers.

A near-infrared (NIR) mechanophore was developed and incorporated into a poly(methyl acrylate) chain to showcase the first force-induced NIR chromism in polymeric materials. This mechanophore, based on benzo[1,3]oxazine (OX) fused with a heptamethine cyanine moiety, exhibited NIR mechanochromism in solution, thin-film, and bulk states. The mechanochemical activity was validated using UV-vis-NIR absorption/fluorescence spectroscopies, gel permeation chromatography (GPC), NMR, and DFT simulations. Our work demonstrates that NIR mechanochromic polymers have considerable potential in mechanical force sensing, damage detection, bioimaging, and biomechanics.

Molecular-Dimension-Dependent ESIPT Break for Specific Reversible Response to GSH and Its Real-Time Bioimaging.

Glutathione (GSH) plays many important roles in maintaining intracellular redox homeostasis, and determining its real-time levels in the biological system is essential for the diagnosis, treatment, and pathological research of related diseases. Fluorescence imaging has been regarded as a powerful tool for tracking biomarkers in vivo, for which specificity, reversibility, and fast response are the main issues to ensure the real-time effective detection of analytes. The determination of GSH is often interfered with by other active sulfur species. However, in addition to the common features of nucleophilic addition, GSH is unique in its large molecular scale. 2-(2-Hydroxyphenyl) benzothiazole (HBT) was often formed in the ESIPT process. In this study, HBT was installed with α,ß-unsaturated ketone conjugated coumarin derivates or nitrobenzene, which were used to adjust the reactivity of α,ß-unsaturated ketone. Experimental and theoretical calculations found ESIPT to be favorable in HBT-COU but not HBT-COU-NEt2 or HBT-BEN-NO2 due to the higher electronic energies in the keto form. Thus, for HBT-COU, in the presence of GSH, the hydrogen-bonding interaction between C═N of the HBT unit and carboxyl of GSH would inhibit the process, simultaneously promoting the Michel addition reaction between α,ß-unsaturated ketone and GSH. As a consequence, probe HBT-COU could exhibit a rapid reversible ratiometric response to GSH. Small structures of Hcy and Cys are passivated for such reactions. Cell imaging demonstrated the specific response of the probe to GSH, and the probe was successfully used to monitor fluctuations in GSH concentration during cells apoptosis in real-time.

Thermal equilibria between conformers enable highly reliable single-fluorophore ratiometric thermometers.

Fluorescent thermometers based on organic dyes play an important role in the visualization of dynamic temperature topography with high resolution. Many molecular thermometers contain two fluorophores with different temperature responses to achieve ratiometric temperature detection. However, the stability and reliability of such ratiometric thermometers are highly dependent on the susceptibility of the fluorophores towards photobleaching. Despite the use of single-fluorophore ratiometric thermometers in previous reports, the difficulty and complexity in their molecular design and synthesis severely hinder their widespread applicability. In this work, we have investigated the use of commercially available dyes (i.e., BD140 and LD688) with conformational isomers as ratiometric thermometers. Each of these dyes consists of a mixture of conformers with distinct UV-vis absorption and emission spectra. The thermal equilibrium of these conformers successfully enabled the development of ratiometric thermometers with good stability and reliability.

Restriction of Twisted Intramolecular Charge Transfer Enables the Aggregation-Induced Emission of 1-(N,N-Dialkylamino)-naphthalene Derivatives.

Understanding the mechanisms of aggregation-induced emission (AIE) is essential for the rational design and deployment of AIEgens toward various applications. Such a deep mechanistic understanding demands a thorough investigation of the excited-state behaviors of AIEgens. However, because of considerable complexity and rapid decay, these behaviors are often not experimentally accessible and the mechanistic comprehension of many AIEgens is lacking. Herein, utilizing detailed quantum chemical calculations, we provide insights toward the AIE mechanism of 1-(N,N-dialkylamino)-naphthalene (DAN) derivatives. Our theoretical analysis, corroborated by experimental observations, leads to the discovery that modulating the formation of the twisted intramolecular charge transfer (TICT) state (caused by the rotation of the amino groups) and managing the steric hindrance to minimize solid-state intermolecular interactions provides a plausible explanation for the AIE characteristics of DAN derivatives. These results will inspire the deployment of the TICT mechanism as a useful design strategy toward AIEgen development.

Modular assembly of arenes, ethylene and heteroarenes for the synthesis of 1,2-arylheteroaryl ethanes.

The 1,2-arylheteroaryl ethane motif stands as a privileged scaffold with promising implications in drug discovery. Conventional de novo syntheses of these molecules have relied heavily on pre-functionalized synthons, entailing harsh conditions and multi-step processes. Here, to address these limitations, we present a modular approach for the direct synthesis of 1,2-arylheteroaryl ethanes using feedstock chemicals, including ethylene, arenes and heteroarenes. We disclosed a photo triplet-energy-transfer-initiated radical cascade process, leveraging homolytic cleavage of C-S bonds in aryl sulfonium salts as the key step to access aryl radicals with excellent regioselectivity. This method allows for rapid structural diversification of bioactive molecules, showcasing excellent functional group tolerance and streamlining the synthesis of bioactive compounds and their derivatives. Furthermore, our approach can be extended to propylene, non-gaseous terminal alkenes and various other electrophilic radical precursors, including heteroaryl radicals, hydroxyl radicals, trifluoromethyl radicals and α-carbonyl alkyl radicals. This study highlights the significance of radical polarity matching in designing selective multi-component couplings.

1,2,4,5-Tetrazine-tethered probes for fluorogenically imaging superoxide in live cells with ultrahigh specificity.

Superoxide (O2·-) is the primary reactive oxygen species in mammal cells. Detecting superoxide is crucial for understanding redox signaling but remains challenging. Herein, we introduce a class of activity-based sensing probes. The probes utilize 1,2,4,5-tetrazine as a superoxide-responsive trigger, which can be modularly tethered to various fluorophores to tune probe sensitivity and emission color. These probes afford ultra-specific and ultra-fluorogenic responses towards superoxide, and enable multiplexed imaging of various cellular superoxide levels in an organelle-resolved way. Notably, the probes reveal the aberrant superoxide generation in the pathology of myocardial ischemia/reperfusion injury, and facilitate the establishment of a high-content screening pipeline for mediators of superoxide homeostasis. One such identified mediator, coprostanone, is shown to effectively ameliorating oxidative stress-induced injury in mice with myocardial ischemia/reperfusion injury. Collectively, these results showcase the potential of 1,2,4,5-tetrazine-tethered probes as versatile tools to monitor superoxide in a range of pathophysiological settings.

Unique assembly of carbonylpyridinium and chromene reveals mitochondrial thiol starvation under ferroptosis and novel ferroptosis inducer.

To reveal the delicate function of mitochondria, spatiotemporally precise detection tools remain highly desirable. However, current probes with positively charged warheads for targeting mitochondria diffuse out of the mitochondria as the potential of the mitochondrial membrane changes, which directly influences the accuracy of the detection. Herein, we assembled carbonylpyridinium and chromene to afford the probe CM-Mit. Following the ultrafast response to thiol and the dissociation of carbonylpyridinium, the formation of o-quinone methide from CM-Mit was proposed to label proteins, thus avoiding diffusion out of the mitochondria. Therefore, the accurate spatiotemporal detection of thiol in mitochondria was realized. With this excellent probe, ferroptosis inducers were proved to stimulate thiol starvation in mitochondria for the first time in cancer cells. Moreover, CM-Mit was used to screen a compound library developed in-house and the stemona alkaloid analog SA-11 was shown to induce ferroptosis in various cancer cell lines, including a drug-resistant one.

High-fidelity imaging of amyloid-beta deposits with an ultrasensitive fluorescent probe facilitates the early diagnosis and treatment of Alzheimer's Disease.

Background: Imaging amyloid-beta (Aß) deposits with high fidelity in naturally aging brains is crucial for the early diagnosis of Alzheimer's disease (AD). However, this is impeded by the lack of highly sensitive probes. Methods: By conducting computational modelling to quantitatively fine-tune the twisted intramolecular charge transfer (TICT) tendency of Thioflavin T (ThT) analogues, we developed an ultrasensitive probe AH-2. AH-2 retained the binding affinity and binding mode of ThT towards Aß deposits, and exhibited ca 10-fold less background fluorescence and 5-10 folds of improved signal-to-background contrast upon binding Aß deposits. These desirable features endowed AH-2 the sensitivity to detect Aß deposition in naturally aging wild-type mice. Results: AH-2 imaging revealed that Aß puncta signals appeared near the nuclei in young mice and spread through the intracellular and extracellular compartments in older mice. Moreover, Aß deposits were observed to emerge earlier in mice cerebral cortex than in the hippocampus region. Given this desirable sensitivity and good spatiotemporal resolution, AH-2 was successfully applied in the preclinical evaluation of Aß-targeted treatment by melatonin. Conclusions: We expect that AH-2 is promising for early diagnosis of AD and will serve as a sensitive tool for studying Aß-related AD pathology.

Methine-Quinoidal Fragment Induces Significant Bathochromic Shifts in Organic Dyes.

Bathochromical shifts in the UV-vis absorption (and emission) spectra and reduced molecular sizes are two desirable features of organic dyes in many applications, i.e., live-cell fluorescence imaging and solar cells. Yet, these two features are often viewed as contradictory requirements as expanding the π-conjugation is one of the most widely used methods for prompting this redshift. Interestingly, it has been reported that linking a methine-quinoidal unit can result in a significant redshift in the UV-vis absorption spectra in comparison with the methine-benzenoidal constitutional isomers (by 61 nm in dioxane). Herein, using comprehensive quantum chemical calculations with various functionals and solvent formalisms in 10 solvents with different polarities, we demonstrated that the formation of the quinoidal moiety plays a decisive role in this redshift. We further showed that the inclusion of a quinoidal unit without increasing the molecular size represents a general design strategy for the bathochromic shifts of many organic dyes.