Pyrene Functionalized Norbornadiene-Quadricyclane Fluorescent Photoswitches: Characterization of their Spectral Properties and Application in Imaging of Amyloid Beta Plaques.

This study presents the synthesis and characterization of two fluorescent norbornadiene (NBD) photoswitches, each incorporating two conjugated pyrene units. Expanding on the limited repertoire of reported photoswitchable fluorescent NBDs, we explore their properties with a focus on applications in bioimaging of amyloid beta (Aß) plaques. While the fluorescence emission of the NBD decreases upon photoisomerization, aligning with what has been previously reported, for the first time we observed luminescence after irradiation of the quadricyclane (QC) isomer. We deduce how the observed emission is induced by photoisomerization to the excited state of the parent isomer (NBD) which is then the emitting species. Thorough characterizations including NMR, UV-Vis, fluorescence, X-ray structural analysis and density functional theory (DFT) calculations provide a comprehensive understanding of these systems. Notably, one NBD-QC system exhibits exceptional durability. Additionally, these molecules serve as effective fluorescent stains targeting Aß plaques in situ, with observed NBD/QC switching within the plaques. Molecular docking simulations explore NBD interactions with amyloid, unveiling novel binding modes. These insights mark a crucial advancement in the comprehension and design of future photochromic NBDs for bioimaging applications and beyond, emphasizing their potential in studying and addressing protein aggregates.

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).

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.

NanoFN10: A High-Contrast Turn-On Fluorescence Nanoprobe for Multiphoton Singlet Oxygen Imaging.

An "off-on" fluorescent nanoprobe for near-infrared multiphoton imaging of singlet oxygen has been developed. The nanoprobe comprises a naphthoxazole fluorescent unit and a singlet-oxygen-sensitive furan derivative attached to the surface of mesoporous silica nanoparticles. In solution, the fluorescence of the nanoprobe increases upon reaction with singlet oxygen both under one- and multiphoton excitation, with fluorescence enhancements up to 180-fold. The nanoprobe can be readily internalized by macrophage cells and is capable of imaging intracellular singlet oxygen under multiphoton excitation.

E/Z Molecular Photoswitches Activated by Two-Photon Absorption: Comparison between Different Families.

Nonlinear optical techniques as two-photon absorption (TPA) have raised relevant interest within the last years due to the capability to excite chromophores with photons of wavelength equal to only half of the corresponding one-photon absorption energy. At the same time, its probability being proportional to the square of the light source intensity, it allows a better spatial control of the light-induced phenomenon. Although a consistent number of experimental studies focus on increasing the TPA cross section, very few of them are devoted to the study of photochemical phenomena induced by TPA. Here, we show a design strategy to find suitable E/Z photoswitches that can be activated by TPA. A theoretical approach is followed to predict the TPA cross sections related to different excited states of various photoswitches' families, finally concluding that protonated Schiff-bases (retinal)-like photoswitches outperform compared to the others. The donor-acceptor substitution effect is therefore rationalized for the successful TPA activatable photoswitch, in order to maximize its properties, finally also forecasting a possible application in optogenetics. Some experimental measurements are also carried out to support our conclusions.

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.

Identification of an antigenic determinant of clavulanic acid responsible for IgE-mediated reactions.

BACKGROUND: Selective reactions to clavulanic acid (CLV) account for around 30% of immediate reactions after administration of amoxicillin-CLV. Currently, no immunoassay is available for detecting specific IgE to CLV, and its specific recognition in patients with immediate reactions has only been demonstrated by basophil activation testing, however with suboptimal sensitivity. The lack of knowledge regarding the structure of the drug that remains bound to proteins (antigenic determinant) is hampering the development of in vitro diagnostics. We aimed to identify the antigenic determinants of CLV as well as to evaluate their specific IgE recognition and potential role for diagnosis. METHODS: Based on complex CLV degradation mechanisms, we hypothesized the formation of two antigenic determinants for CLV, AD-I (N-protein, 3-oxopropanamide) and AD-II (N-protein, 3-aminopropanamide), and designed different synthetic analogs to each one. IgE recognition of these structures was evaluated in basophils from patients with selective reactions to CLV and tolerant subjects. In parallel, the CLV fragments bound to proteins were identified by proteomic approaches. RESULTS: Two synthetic analogs of AD-I were found to activate basophils from allergic patients. This determinant was also detected bound to lysines 195 and 475 of CLV-treated human serum albumin. One of these analogs was able to activate basophils in 59% of patients whereas CLV only in 41%. Combining both results led to an increase in basophil activation in 69% of patients, and only in 12% of controls. CONCLUSION: We have identified AD-I as one CLV antigenic determinant, which is the drug fragment that remains protein-bound.

Extended Alkenyl and Alkynyl Benzotriazoles with Enhanced Two-Photon Absorption Properties as a Promising Alternative to Benzothiadiazoles.

A series of donor-π-acceptor-π-donor (D-π-A-π-D) benzoazole dyes with 2H-benzo[d][1,2,3]triazole or BTD cores have been prepared and their photophysical properties characterized. The properties of these compounds display remarkable differences, mainly as a result of the electron-donor substituent. Dyes with the best properties have visible-light absorption over λ=400 nm, large Stokes shifts in the range of about 3500-6400 cm-1 , and good fluorescence emission with quantum yields of up to 0.78. The two-photon absorption properties were also studied to establish the relationship between structure and properties in the different compounds synthesized. These results provided cross sections of up to 1500 GM, with a predominance of S2 ←S0 transitions and a high charge-transfer character. Time-dependent DFT calculations supported the experimental results.

Synthesis of Amino Terminal Clicked Dendrimers. Approaches to the Application as a Biomarker.

Herein, we present an easy and efficient synthesis of amino terminal dendrons, combining protection/deprotection reactions with copper-catalyzed azide alkyne cycloaddition in a convergent way. This new approach affords dendrons in gram scale with excellent yields and easy purification. By choosing the appropriate azido-functionalized core, these dendrons lead to a more efficient and controlled convergent synthesis of dendrimers with different sizes and shapes and multivalence. The amino terminal dendrimers were analyzed by diffusion-ordered spectroscopy experiments. The observed dendrimer size is in excellent correlation with the expected size and shape by molecular dynamic simulations. The construction of these kinds of nanostructures, in a simple and efficient way, opens new opportunities for biomedical applications. Moreover, by choosing the appropriate core, these versatile macromolecules become an excellent fluorescent biomarker.

Bis(dioxaborine) Dyes with Variable π-Bridges: Towards Two-Photon Absorbing Fluorophores with Very High Brightness.

Bis(dioxaborine) dyes of the A-π-A format (A: acceptor, π: conjugated bridge) were prepared and photophysically characterized. The best performing dyes feature (a) visible-light absorption (>400 nm), (b) high molar absorption coefficients (up to 70000 m-1  cm-1 ), (c) Stokes shifts in the range of ca. 2500-5800 cm-1 , and (d) strong fluorescence emission with quantum yields of up to 0.74. This yields very bright-emitting dyes for one-photon excitation. However, the most intriguing feature of the dyes is their strong two-photon absorption. This was achieved by means of increased π-conjugation in the phenylene or phenylene-thiophene bridges through the variation of the conjugation length and rigidity. This provided two-photon absorption cross sections of up to 2800 GM (1 Goeppert-Mayer (GM)=10-50  cm4 s photon-1 ). Considering the mentioned high fluorescence quantum yields, exceptionally bright-emitting A-π-A two-photon absorbing dyes with low molecular mass are obtained. Time-dependent density-functional theory calculations corroborated the experimental results.

Dendrimeric Antigens for Drug Allergy Diagnosis: A New Approach for Basophil Activation Tests.

Dendrimeric Antigens (DeAns) consist of dendrimers decorated with multiple units of drug antigenic determinants. These conjugates have been shown to be a powerful tool for diagnosing penicillin allergy using in vitro immunoassays, in which they are recognized by specific IgE from allergic patients. Here we propose a new diagnostic approach using DeAns in cellular tests, in which recognition occurs through IgE bound to the basophil surface. Both IgE molecular recognition and subsequent cell activation may be influenced by the tridimensional architecture and size of the immunogens. Structural features of benzylpenicilloyl-DeAn and amoxicilloyl-DeAn (G2 and G4 PAMAM) were studied by diffusion Nuclear Magnetic Resonance (NMR) experiments and are discussed in relation to molecular dynamics simulation (MDS) observations. IgE recognition was clinically evaluated using the basophil activation test (BAT) for allergic patients and tolerant subjects. Diffusion NMR experiments, MDS and cellular studies provide evidence that the size of the DeAn, its antigen composition and tridimensional distribution play key roles in IgE-antigen recognition at the effector cell surface. These results indicate that the fourth generation DeAns induce a higher level of basophil activation in allergic patients. This approach can be considered as a potential complementary diagnostic method for evaluating penicillin allergy.

Red-Emitting Tetracoordinate Organoboron Chelates: Synthesis, Photophysical Properties, and Fluorescence Microscopy.

Seven tetracoordinate organoboron fluorophores with heterobiaryl N,O- or N,N-chelate ligands were prepared and photophysically characterized (in toluene). The electronic variation of the heteroaromatic moiety provided a means for the fine-tuning of the UV/vis absorption and emission spectra. In the most interesting cases, the spectra were red-shifted to maximum absorbance at wavelengths longer than 500 nm and emission maxima between 620 and 660 nm. The pronounced intramolecular charge-transfer character of the dyes yielded large Stokes shifts (3500-5100 cm-1), while maintaining appreciable fluorescence quantum yields of up to 0.2 for emission maxima longer than 600 nm. The lipophilic character of the dyes enabled their application as stains of vesicle substructures in confocal fluorescence microscopy imaging.

Strongly Emissive and Photostable Four-Coordinate Organoboron N,C Chelates and Their Use in Fluorescence Microscopy.

Six strongly fluorescent four-coordinate organoboron N,C chelates containing an aryl isoquinoline skeleton were prepared. Remarkably, the fluorescence quantum yields reach values of up to 0.74 in oxygen-free toluene. The strong B-N interaction was corroborated by the single-crystal X-ray analysis of two dyes. The intramolecular charge-transfer character of the fluorophores was evidenced by solvatochromism studies and time-dependent DFT calculations at the PCM(toluene)/CAM-B3LYP/6-311++G(2d,p)//PCM(toluene)/B3LYP/6-311G(2d,p) level of theory. The compounds combine high chemical stability with high photostability, especially when equipped with electron-donating substituents. The strong fluorescence and the large Stokes shifts predestine these compounds for use in confocal fluorescence microscopy. This was demonstrated for the imaging of the N13 mouse microglial cell line. Moreover, significant two-photon absorption cross sections (up to 61 GM) allow the use of excitation wavelengths in the near-infrared region (>800 nm).

Recognition of multiepitope dendrimeric antigens by human immunoglobulin E.

In vitro drug allergy tests have limited sensitivity, partly due to a poor understanding of the immunological recognition of in vitro drug-protein conjugates. We have designed and synthesized multivalent mono- and bi-epitope dendrimeric antigen (DeAn) conjugates and studied their chemical and tridimensional structures. We describe differences in the spatial distribution and conformation of these conjugated epitopes for the first time: a partially hidden benzylpenicilloyl and a more exposed amoxicilloyl. Our data suggest that DeAn conjugates provide a useful model for studying IgE recognition in patients who suffer from an allergic reaction to benzylpenicillin and/or amoxicillin. 1D and 2D NMR, MDS and immunochemical studies provide evidence that both antigen composition and tridimensional distribution play key roles in IgE-antigen recognition. Bi-epitope DeAn conjugates could potentially allow the diagnosis of patients allergic to any of these two drugs with a single test and represent the basis for a broadly-applicable in vitro assay. From the clinical editor: The prevalence of allergic drug reactions is rising and there is an imperative need to identify patients at risk. In this interesting and important article, the authors developed a novel method for detecting drug specific IgE antibodies, responsible for allergic reactions, by using multivalent mono- and bi-epitope Dendrimeric Antigen (DeAn) conjugates. The continued success of this research may pave way of eventual development of a simple diagnostic test.

Effects of maize cultivation on nitrogen and phosphorus loadings to drainage channels in Central Chile.

There are concerns about the impact of maize cultivation with high applications of nitrogen (N) and phosphorus (P) on water quality in surface waters in Mediterranean Central Chile. This study estimated the contribution of N and P from maize fields to nearby drainage channels and evaluated the effects in water quality. An N and P budget was drawn up for three fields managed with a maize-fallow system, El Maitén (20.7 ha), El Naranjal (14.9 ha) and El Caleuche (4.2 ha), and water quality variables (pH, EC, dissolved oxygen, total solids, turbidity, NO3-N, NH4-N, PO4(3-), COD, total N, total P and sulphate) were monitored in nearby drainage channels. The N and P balances for the three fields indicated a high risk of N and P non-point source pollution, with fertiliser management, soil texture and climate factors determining the temporal variations in water quality parameters. Elevated levels of NH4-N and PO4(3-) in the drainage channels were usually observed during the winter period, while NO3- concentrations did not show a clear tendency. The results suggest that excessive slurry application during winter represents a very high risk of N and P runoff to drainage channels. Overall, great emphasis must be placed on good agronomic management of fields neighbouring drainage channels, including accurately calculating N and P fertiliser rates and establishing mitigation measures.

Inversion of supramolecular helicity in oligo-p-phenylene-based supramolecular polymers: influence of molecular atropisomerism.

The helical organization of oligo-p-phenylene-based organogelators has been investigated by atomic force microscopy, circular and vibrational circular dichroism, and Raman techniques. Whilst OPPs with more than two phenyl rings in the core self-assemble into left-handed helices, that with a biphenyl core shows an inversion of the supramolecular helicity depending on the formation conditions through the atropisomerism of the biphenyl central unit. The results presented herein outline a new example of kinetically controlled modulation of supramolecular helicity.

Investigating New Applications of a Photoswitchable Fluorescent Norbornadiene as a Multifunctional Probe for Delineation of Amyloid Plaque Polymorphism.

Amyloid beta (Aß) plaques are a major pathological hallmark of Alzheimer's disease (AD) and constitute of structurally heterogenic entities (polymorphs) that have been implicated in the phenotypic heterogeneity of AD pathology and pathogenesis. Understanding amyloid aggregation has been a critical limiting factor to gain understanding of AD pathogenesis, ultimately reflected in that the underlying mechanism remains elusive. We identified a fluorescent probe in the form of a turn-off photoswitchable norbornadiene derivative (NBD1) with several microenvironment-sensitive properties that make it relevant for applications within advanced fluorescence imaging, for example, multifunctional imaging. We explored the application of NBD1 for in situ delineation of structurally heterogenic Aß plaques in transgenic AD mouse models. NBD1 plaque imaging shows characteristic broader emission bands in the periphery and more narrow emission bands in the dense cores of mature cored plaques. Further, we demonstrate in situ photoisomerization of NBD1 to quadricyclane and thermal recovery in single plaques, which is relevant for applications within both functional and super-resolution imaging. This is the first time a norbornadiene photoswitch has been used as a probe for fluorescence imaging of Aß plaque pathology in situ and that its spectroscopic and switching properties have been studied within the specific environment of senile Aß plaques. These findings open the way toward new applications of NBD-based photoswitchable fluorescent probes for super-resolution or dual-color imaging and multifunctional microscopy of amyloid plaque heterogeneity. This could allow to visualize Aß plaques with resolution beyond the diffraction limit, label different plaque types, and gain insights into their physicochemical composition.

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.

Manganese-Oxidizing Antarctic Bacteria (Mn-Oxb) Release Reactive Oxygen Species (ROS) as Secondary Mn(II) Oxidation Mechanisms to Avoid Toxicity.

Manganese (Mn) oxidation is performed through oxidative Mn-oxidizing bacteria (MnOxb) as the main bio-weathering mechanism for Mn(III/IV) deposits during soil formation. However, with an increase in temperature, the respiration rate also increases, producing Reactive Oxygen Species (ROS) as by-products, which are harmful to microbial cells. We hypothesize that bacterial ROS oxidize Mn(II) to Mn(III/IV) as a secondary non-enzymatic temperature-dependent mechanism for cell protection. Fourteen MnOxb were isolated from Antarctic soils under the global warming effect, and peroxidase (PO) activity, ROS, and Mn(III/IV) production were evaluated for 120 h of incubation at 4 °C, 15 °C, and 30 °C. ROS contributions to Mn oxidation were evaluated in Arthrobacter oxydans under antioxidant (Trolox) and ROS-stimulated (menadione) conditions. The Mn(III/IV) concentration increased with temperature and positively correlated with ROS production. ROS scavenging with Trolox depleted the Mn oxidation, and ROS-stimulant increased the Mn precipitation in A. oxydans. Increasing the Mn(II) concentration caused a reduction in the membrane potential and bacterial viability, which resulted in Mn precipitation on the bacteria surface. In conclusion, bacterial ROS production serves as a complementary non-enzymatic temperature-dependent mechanism for Mn(II) oxidation as a response in warming environments.

Spontaneous macrocyclization through multiple dynamic cyclic aminal formation.

The use of aminals in dynamic covalent chemistry is slightly underexplored, probably due to their inherent instability. Here we report the spontaneous [2+2] macrocyclization of tetrakis(aminals). Their unexpected stability and structural modularity, the dynamic nature of the connections and their water tolerance make them appealing systems for future applications as stimulus-responsive materials.