Azaborahelicene fluorophores derived from four-coordinate N,C-boron chelates: synthesis, photophysical and chiroptical properties.

A series of six azaborahelicenes with varying electron-donor substitution at the 4-position of the aryl residue (i.e., naphthyl) or with variable π-extension of the aryl residue (thianthrenyl, anthryl, pyrenyl) was prepared with an efficient and flexible synthetic protocol. These different types of functionalization afforded notably pronounced intramolecular charge-transfer (ICT) character for the dyes with the strongest electron donor substitution (NMe2) or easiest to oxidize aryl residues, as evidenced by photophysical investigations. These effects also impact the corresponding chiroptical properties of the separated M- and P-enantiomers, which notably display circularly polarized luminescence (CPL) with dissymmetry factors in the order of magnitude of 10-4 to 10-3. Theoretical calculations confirm the optical spectroscopy data and are in agreement with the proposed involvement of ICT processes.

BASHY Dyes Are Highly Efficient Lipid Droplet-Targeting Photosensitizers that Induce Ferroptosis through Lipid Peroxidation.

Ferroptosis is an iron-dependent lipid-peroxidation-driven mechanism of cell death and a promising therapeutic target to eradicate cancer cells. In this study, we discovered that boronic acid-derived salicylidenehydrazone (BASHY) dyes are highly efficient singlet-oxygen photosensitizers (PSs; ΦΔ up to 0.8) that induce ferroptosis triggered by photodynamic therapy. The best-performing BASHY dye displayed a high phototoxicity against the human glioblastoma multiform U87 cell line, with an IC50 value in the low nanomolar range (4.40 nM) and a remarkable phototoxicity index (PI > 22,700). Importantly, BASHY dyes were shown to accumulate in lipid droplets (LDs) and this intracellular partition was found to be essential for the enhanced phototoxicity and the induction of ferroptosis through lipid peroxidation. The safety and phototoxicity of this platform were validated using an in vivo zebrafish model (Danio rerio).

Photofunctional Scope of Fluorescent Dithienylethene Conjugates with Aza-Heteroaromatic Cations.

A series of dithienylethene (DTE) photoswitches with aza-heteroaromatic cationic moieties is synthesized. The switches are characterized regarding their photochemical and photophysical properties in acetonitrile and in water. The efficiency of the switching and the photostationary state composition depend on the degree of π-conjugation of the heteroaromatic systems. Thus, DTEs with acridinium-derived moieties have very low quantum yields for the ring-closing process, which is in contrast to switches with pyridinium and quinolinium moieties. All switches emit fluorescence in their open forms. The involved electronic transitions are traced back to an integrative picture including the DTE core and the cationic arms. The emission can be fine-tuned by the π-conjugation of the heteroaromatic cations, reaching the red spectral region for DTEs with acridinium moieties. On ring-closing of the DTEs the fluorescence is not observable anymore. Theoretical calculations point to rather low-lying energy levels of the highly conjugated ring-closed DTEs, which would originate near-infrared emission (> 1200 nm). The latter is predicted to be very weak due to the concurrent non-radiative deactivation, according to the energy-gap law. In essence, an ON-OFF fluorescence switching as the result of the electrocyclic ring-closing reaction is observed.

Phototransduction in a supramolecular cascade: a mimic for essential features of the vision process.

The tailored design of a light-triggered supramolecular cascade results in an artificial machinery that assimilates the transduction of photons into chemical communication and the final release of a neurotransmitter. This is reminiscent of key steps in the natural vision process.

Engineering the BASHY Dye Platform toward Architectures with Responsive Fluorescence.

A set of nine boronic-acid-derived salicylidenehydrazone (BASHY) complexes has been synthesized in good to very good chemical yields in a versatile three-component reaction. In an extension to previous reports on this dye platform, the focus was put on the electronic modification of the "vertical" positions of the salicylidenehydrazone backbone. This enabled the observation of fluorescence quenching by photoinduced electron transfer (PeT), which can be reverted by the addition of acid in organic solvent (OFF-ON fluorescence switching). The resulting emission is observed in the green-to-orange spectral region (maxima at 520-590 nm). In contrast, under physiological pH conditions in water, the PeT process is inherently decativated, thereby enabling the observation of fluorescence in the red-to-NIR region (maxima at 650-680 nm) with appreciable quantum yields and lifetimes. The latter characteristic supported the application of the dyes in fluorescence lifetime imaging (FLIM) of live A549 cells.

Excited-State Dynamics in Borylated Arylisoquinoline Complexes in Solution and in cellulo.

Two four-coordinate organoboron N,C-chelate complexes with different functional terminals on the PEG chains are studied with respect to their photophysical properties within human MCF-7 cells. Their excited-state properties are characterized by time-resolved pump-probe spectroscopy and fluorescence lifetime microscopy. The excited-state relaxation dynamics of the two complexes are similar when studied in DMSO. Aggregation of the complexes with the carboxylate terminal group is observed in water. When studying the light-driven excited-state dynamics of both complexes in cellulo, i. e., after being taken up into human MCF-7 cells, both complexes show different features depending on the nature of the anchoring PEG chains. The lifetime of a characteristic intramolecular charge-transfer state is significantly shorter when studied in cellulo (360±170 ps) as compared to in DMSO (∼960 ps) at 600 nm for the complexes with an amino group. However, the kinetics of the complexes with the carboxylate group are in line with those recorded in DMSO. On the other hand, the lifetimes of the fluorescent state are almost identical for both complexes in cellulo. These findings underline the importance to evaluate the excited-state properties of fluorophores in a complex biological environment in order to fully account for intra- and intermolecular effects governing the light-induced processes in functional dyes.

Metal-Mediated Organocatalysis in Water: Serendipitous Discovery of Aldol Reaction Catalyzed by the [Ru(bpy)2(nornicotine)2]2+ Complex.

The [Ru(bpy)2(Nor)2]2+ complex (Nor = nornicotine) is an efficient catalyst for the aldol reaction of acetone with activated benzaldehydes in a buffered aqueous solution. The metal plays the role of an activator for the nornicotine organocatalyst ligands. The resulting catalytic activity is potentiated by a factor of about 4.5 as compared to free nornicotine. Similar rate enhancements can be achieved by using Zn(II) cations as the activator. The observations are rationalized with the reduced basicity of the pyrrolidine N in nornicotine due to the enhanced electron withdrawal of the metal-complexed pyridyl moiety.

The BASHY Platform Enables the Assembly of a Fluorescent Bortezomib-GV1001 Conjugate.

In this study, we show that fluorescent boronic-acid derived salicylidenehydrazone complexes (BASHY) can function as fluorescent linkers for bioconjugates that were used to monitor the delivery of the proteasome inhibitor bortezomib (Btz) to HT-29 cancer cells. BASHY complexes were structurally optimized to improve the stability of the complex in buffered conditions (ammonium acetate, pH 7 up to t 1/2 = 40 h), photophysically characterized regarding their fluorescence properties and used in confocal microscopy colocalization studies that revealed their intracellular sequestration by lipid droplets. The accumulation in these hydrophobic organelles limited the hydrolysis of the complex and consequently the drug release, a problem that was circumvented by the conjugation of the BASHY-Btz complex with a cell-penetrating peptide GV1001-C. The conjugate exhibited an improved cytoplasmic availability as confirmed by confocal fluorescence microscopy studies and an improved potency against HT-29 cancer cells (IC50 = 100 nM) as compared to the nontargeted complex (IC50 = 450 nM).

BASHY Dye Platform Enables the Fluorescence Bioimaging of Myelin Debris Phagocytosis by Microglia during Demyelination.

Multiple sclerosis (MS) is a demyelinating disease of the central nervous system that is characterized by the presence of demyelinated regions with accumulated myelin lipid debris. Importantly, to allow effective remyelination, such debris must be cleared by microglia. Therefore, the study of microglial activity with sensitive tools is of great interest to better monitor the MS clinical course. Using a boronic acid-based (BASHY) fluorophore, specific for nonpolar lipid aggregates, we aimed to address BASHY's ability to label nonpolar myelin debris and image myelin clearance in the context of demyelination. Demyelinated ex vivo organotypic cultures (OCSCs) and primary microglia cells were immunostained to evaluate BASHY's co-localization with myelin debris and also to evaluate BASHY's specificity for phagocytosing cells. Additionally, mice induced with experimental autoimmune encephalomyelitis (EAE) were injected with BASHY and posteriorly analyzed to evaluate BASHY+ microglia within demyelinated lesions. Indeed, in our in vitro and ex vivo studies, we showed a significant increase in BASHY labeling in demyelinated OCSCs, mostly co-localized with Iba1-expressing amoeboid/phagocytic microglia. Most importantly, BASHY's presence was also found within demyelinated areas of EAE mice, essentially co-localizing with lesion-associated Iba1+ cells, evidencing BASHY's potential for the in vivo bioimaging of myelin clearance and myelin-carrying microglia in regions of active demyelination.

Toward Light-Controlled Supramolecular Peptide Dimerization.

The selective photodeprotection of the NVoc-modified FGG tripeptide yields the transformation of its 1:1 receptor-ligand complex with cucurbit[8]uril into a homoternary FGG2@CB8 assembly. The resulting light-induced dimerization of the model peptide provides a tool for the implementation of stimuli-responsive supramolecular chemistry in biologically relevant contexts.

Toward Two-Photon Absorbing Dyes with Unusually Potentiated Nonlinear Fluorescence Response.

The combination of two two-photon-induced processes in a Förster resonance energy transfer (FRET)-operated photochromic fluorene-dithienylethene dyad lays the foundation for the observation of a quartic dependence of the fluorescence signal on the excitation light intensity. While this photophysical behavior is predicted for a four-photon absorbing dye, the herein proposed approach opens the way to use two-photon absorbing dyes, reaching the same performance. Hence, the spatial resolution limit, being a critical parameter for applications in fluorescence imaging or data storage with common two-photon absorbing dyes, is dramatically improved.

Visible Light-Gated Organocatalysis Using a RuII -Photocage.

The light-gated organocatalysis via the release of 4-N,N-dimethylaminopyridine (DMAP) by irradiation of the [Ru(bpy)2 (DMAP)2 ]2+ complex with visible light was investigated. As model reaction the acetylation of benzyl alcohols with acetic anhydride was chosen. The pre-catalyst releases one DMAP molecule on irradiation at wavelengths longer than 455 nm. The photochemical process was characterized by steady-state irradiation and ultrafast transient absorption spectroscopy. The latter enabled the observation of the 3 MLCT state and the spectral features of the penta-coordinated intermediate [Ru(bpy)2 (DMAP)]2+ . The released DMAP catalyzes the acetylation of a wide range of benzyl alcohols with chemical yields of up to 99 %. Control experiments revealed unequivocally that it is the released DMAP which takes the role of the catalyst.

Cyanine-Like Boronic Acid-Derived Salicylidenehydrazone Complexes (Cy-BASHY) for Bioimaging Applications.

Boronic acid-derived salicylidenehydrazone complex (BASHY) dyes with a polymethine backbone were designed to yield efficient red-emitting and two-photon absorbing fluorophores that can be used as markers for astrocytes. The dyes are chemically stable in aqueous solution and do not undergo photodecomposition. Their photophysical properties can be electronically fine-tuned and thereby adapted to potentially different imaging situations and requirements.

Chemical signal cascading in a supramolecular network.

A chemically-triggered signalling cascade between cucurbituril host-guest complexes by means of multi-step competitive displacement is demonstrated. The inter-complex communication of chemical information yields the release of bio-relevant cargo, reminiscent of cellular signalling pathways.

Optical Supramolecular Sensing of Creatinine.

Calix[4]pyrrole phosphonate-cavitands were used as receptors for the design of supramolecular sensors for creatinine and its lipophilic derivative hexylcreatinine. The sensing principle is based on indicator displacement assays of an inherently fluorescent guest dye or a black-hole quencher from the receptor's cavity by means of competition with the creatinine analytes. The systems were thermodynamically and kinetically characterized regarding their 1:1 binding properties by means of nuclear magnetic resonance spectroscopy (1H and 31P NMR), isothermal titration calorimetry, and optical spectroscopies (UV/vis absorption and fluorescence). For the use of the black-hole indicator dye, the calix[4]pyrrole was modified with a dansyl chromophore as a signaling unit that engages in Förster resonance energy transfer with the indicator dye. The 1:1 binding constants of the indicator dyes are in the range of 107 M-1, while hexylcreatinine showed values around (2-4) × 105 M-1. The competitive displacement of the indicators by hexylcreatinine produced supramolecular fluorescence turn-on sensors that work at micromolar analyte concentrations that are compatible with those observed for healthy as well as sick patients. The limit of detection for one of the systems reached submicromolar ranges (110 nM).

Arylisoquinoline-derived organoboron dyes with a triaryl skeleton show dual fluorescence.

Four new dyes that derive from borylated arylisoquinolines were prepared, containing a third aryl residue (naphthyl, 4-methoxynaphthyl, pyrenyl or anthryl) that is linked via an additional stereogenic axis. The triaryl cores were synthesized by Suzuki couplings and then transformed into boronic acid esters by employing an Ir(I)-catalyzed reaction. The chromophores show dual emission behavior, where the long-wavelength emission band can reach maxima close to 600 nm in polar solvents. The fluorescence quantum yields of the dyes are generally in the range of 0.2-0.4, reaching in some cases values as high as 0.5-0.6. Laser-flash photolysis provided evidence for the existence of excited triplet states. The dyes form fluoroboronate complexes with fluoride anions, leading to the observation of the quenching of the long-wavelength emission band and ratiometric response by the build-up of a hypsochromically shifted emission signal.

π-Extended Four-Coordinate Organoboron N,C-Chelates as Two-Photon Absorbing Chromophores.

Four-coordinate N,C-chelate organoboron dyes with alkynyl spacers were synthesized by Heck alkynylation. These dyes are π-extended analogues of the recently reported class of four-coordinate borylated arylisoquinolines (BAI). Depending on the electron-donor substitution, they feature an intramolecular charge-transfer (ICT) character in the excited state. This translates into pronounced apparent Stokes shifts (up to 8500 cm-1) and a solvatofluorochromic behavior. In general, the observed emission quantum yields are high in nonpolar media (ΦF ca. 0.5-0.6). For the dye with the most pronounced ICT rather high emission quantum yields (ΦF ca. 0.4) are observed for emissions with maxima longer than 600 nm in solvents of moderate polarity. The π-extended dyes show interesting two-photon absorption (TPA) properties, maintaining high cross sections (up to 60 GM) in the near-infrared wavelength window (>900 nm). One of the dyes was designed as dimeric chromophore, integrating the acceptor-π-acceptor (A-π-A) format. This alternative design showed no ICT behavior but led to the observation of high two-photon-absorption (TPA) cross sections (ca. 220 GM at 700 nm). All investigated dyes show pronounced photostability, providing added value to this structural and photofunctional extension of the BAI dye platform.

Binding of Flavylium Ions to Sulfonatocalix[4]arene and Implication in the Photorelease of Biologically Relevant Guests in Water.

The formation of host-guest complexes between seven flavylium cations and water-soluble p-sulfonatocalix[4]arene (SC4) was investigated by UV/vis absorption, fluorescence, and NMR spectroscopies. The results show the cationic guests form complexes with affinities in the submillimolar range. A representative chalcone/flavylium photoswitch was investigated in more detail regarding its pH- and light-triggered interconversion between the two forms. The dramatic affinity differentiation of the SC4 binding of the two switchable species (40 M-1 for the trans-chalcone versus 3.5 × 104 M-1 for the flavylium cation) enables the pH-gated photocontrol of the complexation process. These responsive properties were explored to demonstrate the competitive and selective release of biologically relevant guests from their supramolecular complexes with p-sulfonatocalix[4]arene (SC4), following the principle of AND logic. The guest release can be reverted by the thermally activated reaction of the flavylium ion back to the trans-chalcone.

Light-driven control of the composition of a supramolecular network.

The composition of a supramolecular network, constituted by several cucurbituril receptors and guests, can be controlled by the reversible and all-photonic switching of a dithienylethene guest.

Precise supramolecular control of surface coverage densities on polymer micro- and nanoparticles.

We report herein the controlled surface functionalization of micro- and nanoparticles by supramolecular host-guest interactions. Our idea is to exploit the competition of two high-affinity guests for binding to the surface-bound supramolecular host cucurbit[7]uril (CB7). To establish our strategy, surface azide groups were introduced to hard-sphere (poly)methylmethacrylate particles with a grafted layer of poly(acrylic acid), and a propargyl derivative of CB7 was coupled to the surface by click chemistry. The amount of surface-bound CB7 was quantified with the high-affinity guest aminomethyladamantane (AMADA), which revealed CB7 surface coverage densities around 0.3 nmol cm-2 indicative of a 3D layer of CB7 binding sites on the surface. The potential for surface functionalization was demonstrated with an aminoadamantane-labeled rhodamine (Ada-Rho) as a second high-affinity guest. Simultaneous incubation of CB7-functionalized particles with both high-affinity guests, AMADA and Ada-Rho, revealed a simple linear relationship between the resulting surface coverage densities of the model fluorescent dye and the mole fraction of Ada-Rho in the incubation mixture. This suggests a highly modular supramolecular strategy for the stable immobilization of application-relevant molecules on particle surfaces and a precise control of their surface coverage densities.