Catalysis driven by an amyloid-substrate complex.

Amine modification through nucleophilic attack of the amine functionality is a very common chemical transformation. Under biorelevant conditions using acidic-to-neutral pH buffer, however, the nucleophilic reaction of alkyl amines (pKa ≈ 10) is not facile due to the generation of ammonium ions lacking nucleophilicity. Here, we disclose a unique molecular transformation system, catalysis driven by amyloid-substrate complex (CASL), that promotes amine modifications in acidic buffer. Ammonium ions attached to molecules with amyloid-binding capability were activated through deprotonation due to the close proximity to the amyloid catalyst formed by Ac-Asn-Phe-Gly-Ala-Ile-Leu-NH2 (NL6), derived from islet amyloid polypeptide (IAPP). Under the CASL conditions, alkyl amines underwent various modifications, i.e., acylation, arylation, cyclization, and alkylation, in acidic buffer. Crystallographic analysis and chemical modification studies of the amyloid catalysts suggested that the carbonyl oxygen of the Phe-Gly amide bond of NL6 plays a key role in activating the substrate amine by forming a hydrogen bond. Using CASL, selective conversion of substrates possessing equivalently reactive amine functionalities was achieved in catalytic reactions using amyloids. CASL provides a unique method for applying nucleophilic conversion reactions of amines in diverse fields of chemistry and biology.

Modular synthetic strategy for N/C-terminal protected amyloidogenic peptides.

N/C-terminal protected amyloidogenic peptides are valuable biomaterials. Optimization of the protective structures at both termini is, however, synthetically laborious because a linear sequence of solid-phase peptide synthesis protocol (on-resin peptide assembly/peptide removal from resin/high-performance liquid chromatography purification) is required for the peptides each time the protective group is modified. In this study, we demonstrate a modular synthetic strategy for the purpose of rapidly deriving the N/C-terminal structures of amyloidogenic peptides. The precursor sequences that can be easily synthesized due to a non-amyloidogenic property were stocked as the synthetic intermediates. Condensation of the intermediates with N/C-terminal units in a liquid phase followed by high-performance liquid chromatography purification gave the desired peptides P1-P8. The amyloidogenic peptides that have various N/C-terminal protective structures were therefore synthesized in a labor-effective manner. This method is suggested to be useful for synthesizing amyloidogenic peptides possessing divergent protective structures at the N/C-terminus.

Knoevenagel Condensation between 2-Methyl-thiazolo[4,5-b]pyrazines and Aldehydes.

Knoevenagel condensation, an olefin-forming reaction from active methyl/methylene-containing compounds and aldehydes, is a fundamental and useful synthetic method. Benzothiazoles are, however, out of the scope of Knoevenagel condensation. Here, we report that Knoevenagel condensation between aldehydes and 2-methyl-thiazolo[4,5-b]pyrazines (MeTPy), a fused ring structure comprising pyrazine and thiazole, proceeded smoothly, despite minor structural differences from benzothiazoles. This finding will be useful for short synthesis of MeTPy-containing functional molecules, such as a tau probe analog 1.

Chemical catalyst-promoted photooxygenation of amyloid proteins.

Misfolded proteins produce aberrant fibrillar aggregates, called amyloids, which contain cross-ß-sheet higher order structures. The species generated in the aggregation process (i.e., oligomers, protofibrils, and fibrils) are cytotoxic and can cause various diseases. Interfering with the amyloid formation of proteins could be a drug development target for treating diseases caused by aberrant protein aggregation. In this review, we introduce a variety of chemical catalysts that oxygenate amyloid proteins under light irradiation using molecular oxygen as the oxygen atom donor (i.e., photooxygenation catalysts). Catalytic photooxygenation strongly inhibits the aggregation of amyloid proteins due to covalent installation of hydrophilic oxygen atoms and attenuates the neurotoxicity of the amyloid proteins. Recent in vivo studies in disease model animals using photooxygenation catalysts showed promising therapeutic effects, such as memory improvement and lifespan extension. Moreover, photooxygenation catalysts with new modes of action, including interference with the propagation of amyloid core seeds and enhancement in the metabolic clearance of amyloids in the brain, have begun to be identified. Manipulation of catalytic photooxygenation with secured amyloid selectivity is indispensable for minimizing the side effects in clinical application. Here we describe several strategies for designing catalysts that selectively photooxygenate amyloids without reacting with other non-amyloid biomolecules.

Nanoscale View of Amyloid Photodynamic Damage.

A combination of time-resolved optical spectroscopy and nanoscale imaging has been used to study the complex binding to amyloids of a photocatalyst that selectively photo-oxygenates pathogenic aggregates, as well as the consequences of its irradiation. Correlative atomic force microscopy (AFM) and fluorescence microscopy reveals topography-dependent binding of the dye to model ß-lactoglobulin fibers, which may also explain the observed difference in their response to photodegradation. We provide direct evidence of the photosensitization of singlet oxygen by the photocatalyst bound to amyloid fibers by direct detection of its NIR phosphorescence. The effect of singlet oxygen at the molecular level brings about nanoscale morphological changes that can be observed with AFM at the single-fiber level. We also find differential response of two α-synuclein mutants to photodamage, which can be rationalized by the presence of amino acids susceptible to photo-oxygenation. Overall, our results help to unravel some of the complexity associated with highly heterogeneous amyloid populations and contribute to the development of improved phototherapeutic strategies for amyloid-related disorders.

Quantitative Assays for Catalytic Photo-Oxygenation of Alzheimer Disease-Related Tau Proteins.

Catalytic photo-oxygenation of tau amyloid is a potential therapeutic approach to tauopathies, including Alzheimer disease (AD). However, tau is a complex target containing great molecular size and heterogeneous isoforms/proteoforms. Although catalytic photo-oxygenation has been confirmed when using catalyst 1 and recombinant tau pretreated with heparin, its effects on tau from human patients have not yet been clarified. In this study, focusing on the histidine residues being oxygenated, we have constructed two assay systems capable of quantitatively evaluating the catalytic activity when used on human patient tau: (1) fluorescence labeling at oxygenated histidine sites and (2) LC-MS/MS analysis of histidine-containing fragments. Using these assays, we identified 2 as a promising catalyst for oxygenation of human tau. In addition, our results suggest that aggregated tau induced by heparin is different from actual AD patient tau in developing effective photo-oxygenation catalysts.

Photophysical properties and application in live cell imaging of B,B-fluoro-perfluoroalkyl BODIPYs.

The photophysical properties of newly identified B,B-fluoro-perfluoroalkyl BODIPYs (2 and 3), which possess a fluoro group and a trifluoromethyl or pentafluoroethyl group at the boron center, were investigated. B,B-Fluoro-perfluoroalkyl BODIPYs 2 and 3 exhibited better photophysical/chemical properties than B,B-difluoro-BODIPY 1, as follows: (1) higher photostability both in methanol solvent and in a live cell environment, (2) higher stability against acid degradation, and (3) improved fluorescence signal-to-noise ratios in a cell system. These favorable properties of B,B-fluoro-perfluoroalkyl BODIPYs are likely due to the highly electron-withdrawing nature of the perfluoroalkyl groups on the boron atom, which reduces the reactivity to 1O2 and strengthens the complexation of the dipyrromethene ligands to the boron atom. Thus, B,B-fluoro perfluoroalkyl BODIPYs may be useful functional molecules for various applications.

Photo-oxygenation inhibits tau amyloid formation.

Tau amyloid formation and deposition are responsible for the onset of Alzheimer's disease. In particular, the seeding activity of the tau protein plays an important role in the spreading of tau pathology via its propagation in the human brain. Here we demonstrate that catalytic photo-oxygenation markedly suppresses tau seeding activity, resulting in the inhibition of amyloid formation, both in vitro and in cultured cells.

Convergent and Functional-Group-Tolerant Synthesis of B-Organo BODIPYs.

Boron-dipyrromethenes (BODIPYs) are one of the most important fluorescent materials. Despite their potential unique properties, however, B,B-fluoro-organo BODIPYs (BFR-BODIPYs) possessing an organo group (R) on the boron center have not been studied in detail, due in part to challenges related to their synthesis. In this paper, a convergent synthesis of BFR-BODIPYs operative under mild conditions is reported. Conversions of the thus-synthesized functionalized BFR-BODIPYs by cross-coupling, condensation, and SN2 reactions at the R group are also demonstrated.

Hydroxy Group Directed Catalytic Hydrosilylation of Amides.

Chemo- and site-selective hydrosilylation of α- or ß-hydroxy amides using organocatalyst B(C6F5)3 and commercially available hydrosilanes is described. This transformation is operative under mild conditions and tolerates a wide range of functional groups. The reaction was applied for selective reduction of a specific amide group of the therapeutically important cyclic peptide cyclosporin A, demonstrating the potential usefulness of this catalytic method in late-stage structural transformations of drug lead molecules.