Flapping Peryleneimide as a Fluorogenic Dye with High Photostability and Strong Visible-Light Absorption

Flapping fluorophores (FLAP) with a flexible 8π ring are rapidly gaining attention as a versatile photofunctional system. Here we report a highly photostable “flapping peryleneimide” with an unprecedented fluorogenic mechanism based on a bent-to-planar conformational change in the S₁ excited state. The S₁ planarization induces an electronic configurational switch, almost quenching the inherent fluorescence (FL) of the peryleneimide moieties. However, the FL quantum yield is remarkably improved with a prolonged lifetime upon a slight environmental change. This fluorogenic function is realized by sensitive π-conjugation design, as a more π-expanded analogue does not show the planarization dynamics. With strong visible-light absorption, the FL lifetime response synchronized with the flexible flapping motion is useful for the latest optical techniques such as FL lifetime imaging microscopy (FLIM).     

A Double‐Clicking Bis‐Azide Fluorogenic Dye for Bioorthogonal Self‐Labeling Peptide Tags

Herein, we give the very first example for the development of a fluorogenic molecular probe that combines the two‐point binding specificity of biarsenical‐based dyes with the robustness of bioorthogonal click‐chemistry. This proof‐of‐principle study reports on the synthesis and fluorogenic characterization of a new, double‐quenched, bis‐azide fluorogenic probe suitable for bioorthogonal two‐point tagging of small peptide tags by double strain‐promoted azide–alkyne cycloaddition. The presented probe exhibits remarkable increase in fluorescence intensity when reacted with bis‐cyclooctynylated peptide sequences, which could also serve as possible self‐labeling small peptide tag motifs.     

New Red‐Emitting Tetrazine‐Phenoxazine Fluorogenic Labels for Live‐Cell Intracellular Bioorthogonal Labeling Schemes

The synthesis of a set of tetrazine‐bearing fluorogenic dyes suitable for intracellular labeling of proteins in live cells is presented. The red excitability and emission properties ensure minimal autofluorescence, while through‐bond energy‐transfer‐based fluorogenicity reduces nonspecific background fluorescence of unreacted dyes. The tetrazine motif efficiently quenches fluorescence of the phenoxazine core, which can be selectively turned on chemically upon bioorthogonal inverse‐electron‐demand Diels–Alder reaction with proteins modified genetically with strained trans‐cyclooctenes.     

Molecular Insight into Long‐Wavelength Fluorogenic Dye Design: Hydrogen Bond Induces Activation of a Dormant Acceptor

The detection of chemical or biological analytes in response to molecular changes relies increasingly on fluorescence methods. Therefore, there is a substantial need for the development of improved fluorogenic dyes. In this study, we demonstrated how an intramolecular hydrogen bond activates a dormant acceptor through a charge induction between phenolic hydrogen and a heteroaryl nitrogen moiety. As a result, a new fluorochrome is produced, and the molecule exhibits a strong fluorescent emission. When the strength of the hydrogen bonding was increased by conformational locking, the obtained dye emitted at longer wavelengths and fluoresced under physiological conditions. The dye was implemented in a turn‐ON system responsive to hydrogen peroxide. The molecular insight provided by this study should assist in the design of fluorescent dyes that are suitable for in vitro and in vivo applications.     

Benzo[1,2‐d:4,5‐d′]bisimidazoles as a Convenient Platform Towards Dyes that are Capable of Excited‐State Intramolecular Proton Transfer and of Two‐Photon Absorption

New, strongly fluorescent benzo[1,2‐d:4,5‐d′]bisimidazoles have been prepared by the reaction of Bandrowski′s base with various aldehydes. Their structures were carefully designed to achieve efficient excited‐state intramolecular proton transfer and good two‐photon‐absorption (2PA) cross‐sections. Functional dyes that possessed both high fluorescence quantum yields and large Stokes shifts were prepared. A π‐expanded D‐A‐D derivative that possessed Φ_fl=50 % and σ₂=230 GM in the spectroscopic area of interest for biological imaging is an excellent candidate as a fluorescent probe. Thanks to the presence of two reactive amino groups, such compounds can be easily transformed into probes for bioconjugation. All of these benzo[1,2‐d:4,5‐d′]bisimidazoles were also strongly fluorescent in the solid state.     

Visible‐Light Photocatalytic Hydrogen Generation by Using Dye‐Sensitized Graphene Oxide as a Photocatalyst

Dye‐sensitized graphene oxide is able to generate hydrogen from water/methanol mixtures (80:20) by using visible or solar light. The most efficient photocatalyst tested contained a tris(2,2‐bipyridyl) ruthenium(II) complex incorporated in the interlayer spaces of a few layers of graphene oxide with a moderate degree of oxidation. The graphene oxide‐based photocatalyst does not contain noble metals and we have determined that it is two orders of magnitude more active than catalysts based on conventional titania.     

Sterically Shielded Heptamethine Cyanine Dyes for Bioconjugation and High Performance Near‐Infrared Fluorescence Imaging

The near‐infrared window of fluorescent heptamethine cyanine dyes greatly facilitates biological imaging because there is deep penetration of the light and negligible background fluorescence. However, dye instability, aggregation, and poor pharmacokinetics are current drawbacks that limit performance and the scope of possible applications. All these limitations are simultaneously overcome with a new molecular design strategy that produces a charge balanced and sterically shielded fluorochrome. The key design feature is a meso‐aryl group that simultaneously projects two shielding arms directly over each face of a linear heptamethine polyene. Cell and mouse imaging experiments compared a shielded heptamethine cyanine dye (and several peptide and antibody bioconjugates) to benchmark heptamethine dyes and found that the shielded systems possess an unsurpassed combination of photophysical, physiochemical, and biodistribution properties that greatly enhance bioimaging performance.     

Isolation and Characterization of Precise Dye/Dendrimer Ratios

Fluorescent dyes are commonly conjugated to nanomaterials for imaging applications using stochastic synthesis conditions that result in a Poisson distribution of dye/particle ratios and therefore a broad range of photophysical and biodistribution properties. We report the isolation and characterization of generation 5 poly(amidoamine) (G5 PAMAM) dendrimer samples containing 1, 2, 3, and 4 fluorescein (FC) or 6‐carboxytetramethylrhodamine succinimidyl ester (TAMRA) dyes per polymer particle. For the fluorescein case, this was achieved by stochastically functionalizing dendrimer with a cyclooctyne “click” ligand, separation into sample containing precisely defined “click” ligand/particle ratios using reverse‐phase high performance liquid chromatography (RP‐HPLC), followed by reaction with excess azide‐functionalized fluorescein dye. For the TAMRA samples, stochastically functionalized dendrimer was directly separated into precise dye/particle ratios using RP‐HPLC. These materials were characterized using ¹H and ¹⁹F NMR spectroscopy, RP‐HPLC, UV/Vis and fluorescence spectroscopy, lifetime measurements, and MALDI.     

Fluorescence Nanoscopy with Optical Sectioning by Two‐Photon Induced Molecular Switching using Continuous‐Wave Lasers

During the last decade far‐field fluorescence microscopy methods have evolved that have resolution far below the wavelength of light. To outperform the limiting role of diffraction, all these methods, in one way or another, switch the ability of a molecule to emit fluorescence. Here we present a novel rhodamine amide that can be photoswitched from a nonfluorescent to a fluorescent state by absorption of one or two photons from a continuous‐wave laser beam. This bright marker enables strict control of on/off switching and provides single‐molecule localization precision down to 15 nm in the focal plane. Two‐photon induced nonlinear photoswitching of this marker with continuous‐wave illumination offers optical sectioning with simple laser equipment. Future synthesis of similar compounds holds great promise for cost‐effective fluorescence nanoscopy with noninvasive optical sectioning.     

Hydrophilicity Control of Visible‐Light Hydrogen Evolution and Dynamics of the Charge‐Separated State in Dye/TiO2/Pt Hybrid Systems

Visible‐light‐driven H₂ evolution based on Dye/TiO₂/Pt hybrid photocatalysts was investigated for a series of (E)‐3‐(5′‐{4‐[bis(4‐R¹‐phenyl)amino]phenyl}‐4,4′‐(R²)₂‐2,2′‐bithiophen‐5‐yl)‐2‐cyanoacrylic acid dyes. Efficiencies of hydrogen evolution from aqueous suspensions in the presence of ethylenediaminetetraacetic acid as electron donor under illumination at λ>420 nm were found to considerably depend on the hydrophilic character of R¹, varying in the order MOD (R¹=CH₃OCH₂, R²=H)≈ MO4D (R¹=R²=CH₃OCH₂)> HD (R¹=R²=H)> PD (R¹=C₃H₇, R²=H). In the case of MOD /TiO₂/Pt, the apparent quantum yield for photocatalyzed H₂ generation at 436 nm was 0.27±0.03. Transient absorption measurements for MOD ‐ or PD ‐grafted transparent films of TiO₂ nanoparticles dipped into water at pH 3 commonly revealed ultrafast formation (<100 fs) of the dye radical cation (Dye^.+) followed by multicomponent decays, which involve minor fast decays (<5 ps) almost independent of R¹ and major slower decays with significant differences between the two samples: 1) the early decay of the major components for MOD is about 2.5 times slower than that for PD and 2) a redshift of the spectrum occurred for MOD with a time constant of 17 ps, but not for PD . The substituent effects on H₂ generation as well as on transient behavior have been discussed in terms substituent‐dependent charge recombination (CR) of Dye^.+ with electrons in bulk, inner‐trap, and/or interstitial‐trap states, arising from different solvent reorganization.     

Asymmetric Distyrylpyridinium Dyes as Red‐Emitting Fluorescent Probes for Quadruplex DNA

The interactions of three cationic distyryl dyes, namely 2,4‐bis(4‐dimethylaminostyryl)‐1‐methylpyridinium ( 1 a ), its derivative with a quaternary aminoalkyl chain ( 1 b ), and the symmetric 2,6‐bis(4‐dimethylaminostyryl)‐1‐methylpyridinium ( 2 a ), with several quadruplex and duplex nucleic acids were studied with the aim to establish the influence of the geometry of the dyes on their DNA‐binding and DNA‐probing properties. The results from spectrofluorimetric titrations and thermal denaturation experiments provide evidence that asymmetric (2,4‐disubstituted) dyes 1 a and 1 b bind to quadruplex DNA structures with a near‐micromolar affinity and a fair selectivity with respect to double‐stranded (ds) DNA [K_a(G4)/K_a(ds)=2.5–8.4]. At the same time, the fluorescence of both dyes is selectively increased in the presence of quadruplex DNAs (more than 80–100‐fold in the case of human telomeric quadruplex), even in the presence of an excess of competing double‐stranded DNA. This optical selectivity allows these dyes to be used as quadruplex‐DNA‐selective probes in solution and stains in polyacrylamide gels. In contrast, the symmetric analogue 2 a displays a strong binding preference for double‐stranded DNA [K_a(ds)/K_a(G4)=40–100), presumably due to binding in the minor groove. In addition, 2 a is not able to discriminate between quadruplex and duplex DNA, as its fluorescence is increased equally well (20–50‐fold) in the presence of both structures. This study emphasizes and rationalizes the strong impact of subtle structural variations on both DNA‐recognition properties and fluorimetric response of organic dyes.     

Iron(III)‐Selective Chelation‐Enhanced Fluorescence Sensing for In Vivo Imaging Applications

A “turn‐on” pattern Fe³⁺‐selective fluorescent sensor was synthesized and characterized that showed high fluorescence discrimination of Fe³⁺ over Fe²⁺ and other tested ions. With a 62‐fold fluorescence enhancement towards Fe³⁺, the probe was employed to detect Fe³⁺ in vivo in HeLa cells and Caenorhabditis elegans, and it was also successfully used to elucidate Fe³⁺ enrichment and exchange infected by innexin3 (Inx3) in hemichannel‐closed Sf9 cells.     

Ultrathin g‐C3N4 Nanosheets Coupled with AgIO3 as Highly Efficient Heterostructured Photocatalysts for Enhanced Visible‐Light Photocatalytic Activity

The photocatalytic activity of graphite‐like carbon nitride (g‐C₃N₄) could be enhanced by heterojunction strategies through increasing the charge‐separation efficiency. As a surface‐based process, the heterogeneous photocatalytic process would become more efficient if a larger contact region existed in the heterojunction interface. In this work, ultrathin g‐C₃N₄ nanosheets (g‐C₃N₄‐NS) with much larger specific surface areas are employed instead of bulk g‐C₃N₄ (g‐C₃N₄‐B) to prepare AgIO₃/g‐C₃N₄‐NS nanocomposite photocatalysts. By taking advantage of this feature, the as‐prepared composites exhibit remarkable performances for photocatalytic wastewater treatment under visible‐light irradiation. Notably, the optimum photocatalytic activity of AgIO₃/g‐C₃N₄‐NS composites is almost 80.59 and 55.09 times higher than that of pure g‐C₃N₄‐B towards the degradation of rhodamine B and methyl orange pollutants, respectively. Finally, the stability and possible photocatalytic mechanism of the AgIO₃/g‐C₃N₄‐NS system are also investigated.     

Development of Photoactivated Fluorescent N‐Hydroxyoxindoles and Their Application for Cell‐Selective Imaging

Photoactivatable fluorophores are essential tools for studying the dynamic molecular interactions within important biological systems with high spatiotemporal resolution. However, currently developed photoactivatable fluorophores based on conventional dyes have several limitations including reduced photoactivation efficiency, cytotoxicity, large molecular size, and complicated organic synthesis. To overcome these challenges, we herein report a class of photoactivatable fluorescent N‐hydroxyoxindoles formed through the intramolecular photocyclization of substituted o‐nitrophenyl ethanol (ONPE). These oxindole fluorophores afford excellent photoactivation efficiency with ultra‐high fluorescence enhancement (up to 800‐fold) and are small in size. Furthermore, the oxindole derivatives show exceptional biocompatibility by generating water as the only photolytic side product. Moreover, structure–activity relationship analysis clearly revealed the strong correlation between the fluorescent properties and the substituent groups, which can serve as a guideline for the further development of ONPE‐based fluorescent probes with desired photophysical and biological properties. As a proof‐of‐concept, we demonstrated the capability of a new substituted ONPE that has an uncaging wavelength of 365–405 nm and an excitation/emission at 515 and 620 nm, for the selective imaging of a cancer cell line (Hela cells) and a human neural stem cell line (hNSCs).     

Pyrrolopyrrole Cyanine Dyes: A New Class of Near‐Infrared Dyes and Fluorophores

NIRer there : Pyrrolopyrrole cyanine (PPCys) dyes, a new class of near‐infrared (NIR) fluorophores, are obtained by condensation of heteroarylacetonitrile and diketopyrrolopyrrole compounds (see picture). Complexation with BF₂ or BPh₂ yields strongly fluorescent, photostable NIR dyes that show high absorption cross‐sections and fluorescence quantum yields. Furthermore, alteration of the heterocycle can tune the main absorption between λ = 684 and 864 nm.

     

Red‐Emitting Rhodamine Dyes for Fluorescence Microscopy and Nanoscopy

Fluorescent markers emitting in the red are extremely valuable in biological microscopy since they minimize cellular autofluorescence and increase flexibility in multicolor experiments. Novel rhodamine dyes excitable with 630 nm laser light and emitting at around 660 nm have been developed. The new rhodamines are very photostable and have high fluorescence quantum yields of up to 80 %, long excited state lifetimes of 3.4 ns, and comparatively low intersystem‐crossing rates. They perform very well both in conventional and in subdiffraction‐resolution microscopy such as STED (stimulated emission depletion) and GSDIM (ground‐state depletion with individual molecular return), as well as in single‐molecule‐based experiments such as fluorescence correlation spectroscopy (FCS). Syntheses of lipophilic and hydrophilic derivatives starting from the same chromophore‐containing scaffold are described. Introduction of two sulfo groups provides high solubility in water and a considerable rise in fluorescence quantum yield. The attachment of amino or thiol reactive groups allows the dyes to be used as fluorescent markers in biology. Dyes deuterated at certain positions have narrow and symmetrical molecular mass distribution patterns, and are proposed as new tags in MS or LC‐MS for identification and quantification of various substance classes (e.g., amines and thiols) in complex mixtures. High‐resolution GSDIM images and live‐cell STED‐FCS experiments on labeled microtubules and lipids prove the versatility of the novel probes for modern fluorescence microscopy and nanoscopy.     

o‐Fluorination of Aromatic Azides Yields Improved Azido‐Based Fluorescent Probes for Hydrogen Sulfide: Synthesis,Spectra, and Bioimaging

Hydrogen sulfide (H₂S) is an endogenously produced gaseous signaling molecule with multiple biological functions. To visualize the endogenous in situ production of H₂S in real time, new coumarin‐ and boron‐dipyrromethene‐based fluorescent turn‐on probes were developed for fast sensing of H₂S in aqueous buffer and in living cells. Introduction of a fluoro group in the ortho position of the aromatic azide can lead to a greater than twofold increase in the rate of reaction with H₂S. On the basis of o‐fluorinated aromatic azides, fluorescent probes with high sensitivity and selectivity toward H₂S over other biologically relevant species were designed and synthesized. The probes can be used to in situ to visualize exogenous H₂S and D ‐cysteine‐dependent endogenously produced H₂S in living cells, which makes them promising tools for potential applications in H₂S biology.     

Dihydronaphthalene‐Fused Boron–Dipyrromethene (BODIPY) Dyes: Insight into the Electronic and Conformational Tuning Modes of BODIPY Fluorophores

A new series of boron–dipyrromethene (BDP, BODIPY) dyes with dihydronaphthalene units fused to the β‐pyrrole positions ( 1 a – d , 2 ) has been synthesised and spectroscopically investigated. All the dyes, except pH‐responsive 1 d in polar solvents, display intense emission between 550–700 nm. Compounds 1 a and 1 b with a hydrogen atom and a methyl group in the meso position of the BODIPY core show spectroscopic properties that are similar to those of rhodamine 101, thus rendering them potent alternatives to the positively charged rhodamine dyes as stains and labels for less polar environments or for the dyeing of latex beads. Compound 1 d , which carries an electron‐donating 4‐(dimethylamino)phenyl group in the meso position, shows dual fluorescence in solvents more polar than dibutyl ether and can act as a pH‐responsive “light‐up” probe for acidic pH. Correlation of the pK_a data of 1 d and several other meso‐(4‐dimethylanilino)‐substituted BODIPY derivatives allowed us to draw conclusions on the influence of steric crowding at the meso position on the acidity of the aniline nitrogen atom. Preparation and investigation of 2 , which carries a nitrogen instead of a carbon as the meso‐bridgehead atom, suggests that the rules of colour tuning of BODIPYs as established so far have to be reassessed; for all the reported couples of meso‐C‐ and meso‐N‐substituted BODIPYs, the exchange leads to pronounced redshifts of the spectra and reduced fluorescence quantum yields. For 2 , when compared with 1 a , the opposite is found: negligible spectral shifts and enhanced fluorescence. Additional X‐ray crystallographic analysis of 1 a and quantum chemical modelling of the title and related compounds employing density functional theory granted further insight into the features of such sterically crowded chromophores.