Difluoro Dipyridomethene Boron Complexes: Synthesis, Characterization, and Ab Initio Calculations.

Molecular engineering studies on the meso-cyano difluoro dipyridomethene boron complexes are presented and two series (a and b) of novel fluorophores are extensively studied. Halogenated derivatives were reacted under Suzuki-Miyaura or Sonogashira cross coupling reactions to introduce electron-donating or electron-withdrawing functional groups on positions 1 and 2 of the aromatic ligand. All derivatives were obtained in 14-90% yields and studied in detail by structural, photophysical, and computational analyses. Both series display excellent emissive properties in solution with blue to orange fluorescence emission upon blue light absorption and promising features as solid emitters. All the spectroscopic measurements are supported and confirmed by first-principles theoretical calculations combining TD-DFT and CC2. Series b, featuring an aryl substituent onto position 1 of the aromatic core, showed significantly large Stokes shifts values.

Urinary tract infections trigger synucleinopathy via the innate immune response.

Symptoms in the urogenital organs are common in multiple system atrophy (MSA), also in the years preceding the MSA diagnosis. It is unknown how MSA is triggered and these observations in prodromal MSA led us to hypothesize that synucleinopathy could be triggered by infection of the genitourinary tract causing ɑ-synuclein (ɑSyn) to aggregate in peripheral nerves innervating these organs. As a first proof that peripheral infections could act as a trigger in MSA, this study focused on lower urinary tract infections (UTIs), given the relevance and high frequency of UTIs in prodromal MSA, although other types of infection might also be important triggers of MSA. We performed an epidemiological nested-case control study in the Danish population showing that UTIs are associated with future diagnosis of MSA several years after infection and that it impacts risk in both men and women. Bacterial infection of the urinary bladder triggers synucleinopathy in mice and we propose a novel role of ɑSyn in the innate immune system response to bacteria. Urinary tract infection with uropathogenic E. coli results in the de novo aggregation of ɑSyn during neutrophil infiltration. During the infection, ɑSyn is released extracellularly from neutrophils as part of their extracellular traps. Injection of MSA aggregates into the urinary bladder leads to motor deficits and propagation of ɑSyn pathology to the central nervous system in mice overexpressing oligodendroglial ɑSyn. Repeated UTIs lead to progressive development of synucleinopathy with oligodendroglial involvement in vivo. Our results link bacterial infections with synucleinopathy and show that a host response to environmental triggers can result in ɑSyn pathology that bears semblance to MSA.

Lysosomes Targeting pH Activable Imaging-Guided Photodynamic Agents.

Photodynamic therapy (PDT) is a photochemistry-based medical treatment combining light at a specific wavelength and a photosensitizer (PS) in the presence of oxygen. Application of PDT as a conventional treatment is limited and clearly the approval in clinics of new PS is challenging. The selective accumulation of the PS in the targeted malignant cells is of paramount importance to reduce the side effects that are typical of the current worldwide approved PS. Here we report a new series of aniline- and iodine-substituted BODIPY derivatives (1-3) as promising lysosome-targeting and pH-responsive theranostic PS, which displayed a significant in vitro light-induced cytotoxicity, efficient imaging properties and low dark toxicity (for 2 and 3). These compounds were obtained in few reproducible synthetic steps and good yields. Spectroscopic and electrochemical measurements along with computational calculations confirmed the quenching of the emissive properties of the PS, while both fluorescence and 1 O2 emission were obtained only under acidic conditions inducing amine protonation. The pKa values and pH-dependent emissive properties of 1-3 being established, their cellular uptake and activation in the lysosomal vesicles (pH≈4-5) were confirmed by their co-localization with the commercial LysoTracker deep red and light-induced cytotoxicity (IC50 between 0.16 and 0.06 µM) against HeLa cancer cells.

Hetero-Substituted αß-Fused BODIPY.

Here, we report the synthesis and properties of heterosubtituted αß-fused BODIPY fluorophores. The compounds were obtained in good yields by sequential and selective Stille cross-coupling reactions from 2,3,5,6-tetrahalo-BODIPY, allowing the introduction of different substituents at the 3,5 and 2,6 positions of the BODIPY ring. The final fused compounds were synthesized using oxidative cyclisation with ferrous chloride. The fully fused compounds show a strong bathochromically shifted emission along with a hyperchromic shift of the absorption maxima. The fluorescence quantum yields remain relatively large for compounds emitting in this wavelength range. Computational studies have been carried out to fully understand the photophysical behaviour of these dyes.

Rational affinity maturation of anti-amyloid antibodies with high conformational and sequence specificity.

The aggregation of amyloidogenic polypeptides is strongly linked to several neurodegenerative disorders, including Alzheimer's and Parkinson's diseases. Conformational antibodies that selectively recognize protein aggregates are leading therapeutic agents for selectively neutralizing toxic aggregates, diagnostic and imaging agents for detecting disease, and biomedical reagents for elucidating disease mechanisms. Despite their importance, it is challenging to generate high-quality conformational antibodies in a systematic and site-specific manner due to the properties of protein aggregates (hydrophobic, multivalent, and heterogeneous) and limitations of immunization (uncontrolled antigen presentation and immunodominant epitopes). Toward addressing these challenges, we have developed a systematic directed evolution procedure for affinity maturing antibodies against Alzheimer's Aß fibrils and selecting variants with strict conformational and sequence specificity. We first designed a library based on a lead conformational antibody by sampling combinations of amino acids in the antigen-binding site predicted to mediate high antibody specificity. Next, we displayed this library on the surface of yeast, sorted it against Aß42 aggregates, and identified promising clones using deep sequencing. The resulting antibodies displayed similar or higher affinities than clinical-stage Aß antibodies (aducanumab and crenezumab). Moreover, the affinity-matured antibodies retained high conformational specificity for Aß aggregates, as observed for aducanumab and unlike crenezumab. Notably, the affinity-maturated antibodies displayed extremely low levels of nonspecific interactions, as observed for crenezumab and unlike aducanumab. We expect that our systematic methods for generating antibodies with unique combinations of desirable properties will improve the generation of high-quality conformational antibodies specific for diverse types of aggregated conformers.

Shared and divergent phase separation and aggregation properties of brain-expressed ubiquilins.

The brain-expressed ubiquilins, UBQLNs 1, 2 and 4, are highly homologous proteins that participate in multiple aspects of protein homeostasis and are implicated in neurodegenerative diseases. Studies have established that UBQLN2 forms liquid-like condensates and accumulates in pathogenic aggregates, much like other proteins linked to neurodegenerative diseases. However, the relative condensate and aggregate formation of the three brain-expressed ubiquilins is unknown. Here we report that the three ubiquilins differ in aggregation propensity, revealed by in-vitro experiments, cellular models, and analysis of human brain tissue. UBQLN4 displays heightened aggregation propensity over the other ubiquilins and, like amyloids, UBQLN4 forms ThioflavinT-positive fibrils in vitro. Measuring fluorescence recovery after photobleaching (FRAP) of puncta in cells, we report that all three ubiquilins undergo liquid-liquid phase transition. UBQLN2 and 4 exhibit slower recovery than UBQLN1, suggesting the condensates formed by these brain-expressed ubiquilins have different compositions and undergo distinct internal rearrangements. We conclude that while all brain-expressed ubiquilins exhibit self-association behavior manifesting as condensates, they follow distinct courses of phase-separation and aggregation. We suggest that this variability among ubiquilins along the continuum from liquid-like to solid informs both the normal ubiquitin-linked functions of ubiquilins and their accumulation and potential contribution to toxicity in neurodegenerative diseases.

High-throughput screening yields several small-molecule inhibitors of repeat-associated non-AUG translation.

Repeat-associated non-AUG (RAN) translation is a noncanonical translation initiation event that occurs at nucleotide-repeat expansion mutations that are associated with several neurodegenerative diseases, including fragile X-associated tremor ataxia syndrome (FXTAS), ALS, and frontotemporal dementia (FTD). Translation of expanded repeats produces toxic proteins that accumulate in human brains and contribute to disease pathogenesis. Consequently, RAN translation constitutes a potentially important therapeutic target for managing multiple neurodegenerative disorders. Here, we adapted a previously developed RAN translation assay to a high-throughput format to screen 3,253 bioactive compounds for inhibition of RAN translation of expanded CGG repeats associated with FXTAS. We identified five diverse small molecules that dose-dependently inhibited CGG RAN translation, while relatively sparing canonical translation. All five compounds also inhibited RAN translation of expanded GGGGCC repeats associated with ALS and FTD. Using CD and native gel analyses, we found evidence that three of these compounds, BIX01294, CP-31398, and propidium iodide, bind directly to the repeat RNAs. These findings provide proof-of-principle supporting the development of selective small-molecule RAN translation inhibitors that act across multiple disease-causing repeats.

Mechanistic insights into the protective roles of polyphosphate against amyloid cytotoxicity.

The universally abundant polyphosphate (polyP) accelerates fibril formation of disease-related amyloids and protects against amyloid cytotoxicity. To gain insights into the mechanism(s) by which polyP exerts these effects, we focused on α-synuclein, a well-studied amyloid protein, which constitutes the major component of Lewy bodies found in Parkinson's disease. Here, we demonstrate that polyP is unable to accelerate the rate-limiting step of α-synuclein fibril formation but effectively nucleates fibril assembly once α-synuclein oligomers are formed. Binding of polyP to α-synuclein either during fibril formation or upon fibril maturation substantially alters fibril morphology and effectively reduces the ability of α-synuclein fibrils to interact with cell membranes. The effect of polyP appears to be α-synuclein fibril specific and successfully prevents the uptake of fibrils into neuronal cells. These results suggest that altering the polyP levels in the extracellular space might be a potential therapeutic strategy to prevent the spreading of the disease.

Functional panchromatic BODIPY dyes with near-infrared absorption: design, synthesis, characterization and use in dye-sensitized solar cells.

We report two novel functional dyes based on a boron-dipyrromethene (BODIPY) core displaying a panchromatic absorption with an extension to the near-infrared (NIR) range. An innovative synthetic approach for preparing the 2,3,5,6-tetramethyl-BODIPY unit is disclosed, and a versatile way to further functionalize this unit has been developed. The optoelectronic properties of the two dyes were computed by density functional theory modelling (DFT) and characterized through UV-vis spectroscopy and cyclic voltammetry (CV) measurements. Finally, we report preliminary results obtained using these functional dyes as photosensitizers in dye-sensitized solar cells (DSSCs).

Tau's Three-Repeat Domain and EFhd2 Co-incubation Leads to Increased Thioflavin Signal.

Aggregation of the protein tau is a pathological hallmark of Alzheimer's disease (AD) and related disorders. However, the molecular mechanisms that lead to tau protein aggregation are still unclear. Previously, we showed that EFhd2 protein is associated with pathological aggregated forms of tau in AD brain. Further, immuno-gold analyses of purified tau aggregates showed that EFhd2 co-localized with filamentous tau structures. We demonstrated that EFhd2's coiled-coil domain is required for its association with tau proteins. However, it is unknown the role that EFhd2 plays in tau aggregation. Here, we show that incubation of K19-tau with substoichiometric amount of EFhd2 promote the formation of amyloid structures in vitro. The result suggests that EFhd2 may play a role in the biogenesis of aggregated tau.

Fluorescent pH-Responsive Probes Based on Water-Soluble Boron-Dipyrromethene (BODIPY) Derivatives, Featuring Long-Wavelength Emission.

We describe here the synthesis of water-soluble red/NIR-emissive, boron-dipyrromethene (BODIPY) derivatives displaying optical (absorption and emission) responses in pH range of 4-8. Substitution close to the tertiary aniline or the phenol subunits selected as the proton-sensitive sites allowed us to finely tune the pH ranges. Furthermore, the introduction of sulfobetaine functions at the boron centre of these pH-responsive BODIPYs afforded valuable fluorescent dyes in the red/NIR region in aqueous media, for which the steric hindrance and electrostatic repulsions prevent their non-emissive aggregation. All the absorption and emission studies, as well as the protonation properties were investigated in aqueous, ethanolic and saline solutions (mimicking physiological conditions). Interestingly, the systems present a fluorescent ratiometric protonation response in EtOH, but the non-protonated form is almost a non-fluorescent species under quasi-physiological conditions (saline aqueous solutions) due to the fading of the emissive character of the low-lying charge-transfer transition in the presence of a supporting electrolyte.

A bifurcated molecular pentad capable of sequential electronic energy transfer and intramolecular charge transfer.

An extended molecular array, comprising three distinct types of chromophores and two additional redox-active subunits, that harvests photons over most of the visible spectral range has been synthesized and characterised. The array exhibits a rich variety of electrochemical waves when examined by cyclic voltammetry but assignment can be made on the basis of control compounds and molecular orbital calculations. Stepwise electronic energy transfer occurs along the molecular axis, corresponding to a gradient of excitation energies, to populate the lowest-energy excited state of the ultimate acceptor. The latter species, which absorbs and emits in the far-red region, enters into light-induced charge transfer with a terminal amine group. The array is relatively stable under illumination with white light but degrades slowly via a series of well-defined steps, the first of which is autocatalytic. One of the main attributes of this system is the capability to harvest an unusually high fraction of sunlight while providing protection against exposure to UV light.

Stepwise photoconversion of an artificial light-harvesting array built from extended BODIPY units.

A molecular dyad, comprising two disparate extended boron dipyrromethene (BODIPY) units, has been identified as a potential component of artificial light-harvesting arrays. Highly efficient, intramolecular electronic energy transfer takes place under illumination but there is some competition from light-induced electron transfer along the molecular axis. The primary energy acceptor has a somewhat shortened excited-state lifetime and reduced emission quantum yield due to charge transfer from a terminal amine residue, the latter being required for the molecular system to operate in organic solar cells. Under continuous illumination with simulated solar light, the dyad undergoes very slow decomposition. In a protic solvent, both BODIPY units degrade at the same rate via an autocatalytic process. The products, one of which is a protonated analogue of the donor, degrade further by independent routes. In aprotic solvents or thin plastic films, the acceptor BODIPY dye absorbing at lowest energy undergoes photochemical degradation as above but the donor is much more stable under these conditions. At each stage of degradation, the molecule retains the ability to sensitize an amorphous silicon solar cell and the overall turnover number with respect to absorbed photons exceeds 10 million. The optical properties of the target compound nicely complement those of the solar cell and sensitization helps to avoid Staebler-Wronski photo-degradation.

Unsymmetrical p-carborane backbone as a linker for donor-acceptor dyads.

Fluorescent nanorods: Donor-acceptor dyads based on novel unsymmetrically disubstituted closo-1,12-dicarbadecaboranes have been prepared in a completely controlled manner by using a three-step procedure. Dyads with different donor-acceptor spacing were thereby obtained. Efficient energy transfer from the donor to the acceptor was determined in fluid solution at room temperature.

Carborane-Bodipy scaffolds for through space energy transfer.

Use of a closo-1,12-dicarbadecaborane backbone to link two different organic dyes separated by ca. 37 Å promotes exclusive through space electronic energy transfer with an efficiency of 81 to 87% with Stoke shifts between 4750 and 2800 cm(-1) depending on the pH.