Unimolecular Chemiexcited Oxygenation of Pathogenic Amyloids.

Pathogenic protein aggregates, called amyloids, are etiologically relevant to various diseases, including neurodegenerative Alzheimer disease. Catalytic photooxygenation of amyloids, such as amyloid-ß (Aß), reduces their toxicity; however, the requirement for light irradiation may limit its utility in large animals, including humans, due to the low tissue permeability of light. Here, we report that Cypridina luciferin analogs, dmCLA-Cl and dmCLA-Br, promoted selective oxygenation of amyloids through chemiexcitation without external light irradiation. Further structural optimization of dmCLA-Cl led to the identification of a derivative with a polar carboxylate functional group and low cellular toxicity: dmCLA-Cl-acid. dmCLA-Cl-acid promoted oxygenation of Aß amyloid and reduced its cellular toxicity without photoirradiation. The chemiexcited oxygenation developed in this study may be an effective approach to neutralizing the toxicity of amyloids, which can accumulate deep inside the body, and treating amyloidosis.

Leuco Ethyl Violet as Self-Activating Prodrug Photocatalyst for In Vivo Amyloid-Selective Oxygenation.

Aberrant aggregates of amyloid-ß (Aß) and tau protein (tau), called amyloid, are related to the etiology of Alzheimer disease (AD). Reducing amyloid levels in AD patients is a potentially effective approach to the treatment of AD. The selective degradation of amyloids via small molecule-catalyzed photooxygenation in vivo is a leading approach; however, moderate catalyst activity and the side effects of scalp injury are problematic in prior studies using AD model mice. Here, leuco ethyl violet (LEV) is identified as a highly active, amyloid-selective, and blood-brain barrier (BBB)-permeable photooxygenation catalyst that circumvents all of these problems. LEV is a redox-sensitive, self-activating prodrug catalyst; self-oxidation of LEV through a hydrogen atom transfer process under photoirradiation produces catalytically active ethyl violet (EV) in the presence of amyloid. LEV effectively oxygenates human Aß and tau, suggesting the feasibility for applications in humans. Furthermore, a concept of using a hydrogen atom as a caging group of a reactive catalyst functional in vivo is postulated. The minimal size of the hydrogen caging group is especially useful for catalyst delivery to the brain through BBB.

Redesign of a thioflavin-T-binding protein with a flat ß-sheet to evaluate a thioflavin-T-derived photocatalyst with enhanced affinity.

Amyloids, proteinous aggregates with ß-sheet-rich fibrils, are involved in several neurodegenerative diseases such as Alzheimer's disease; thus, their detection is critically important. The most common fluorescent dye for amyloid detection is thioflavin-T (ThT), which shows on/off fluorescence upon amyloid binding. We previously reported that an engineered globular protein with a flat ß-sheet, peptide self-assembly mimic (PSAM), can be used as an amyloid binding model. In this study, we further explored the residue-specific properties of ThT-binding to the flat ß-sheet by introducing systematic mutations. We found that site-specific mutations at the ThT-binding channel enhanced affinity. We also evaluated the binding of a ThT-based photocatalyst, which showed the photooxygenation activity on the amyloid fibril upon light radiation. Upon binding of the photocatalyst to the PSAM variant, singlet oxygen-generating activity was observed. The results of this study expand our understanding of the detailed binding mechanism of amyloid-specific molecules.

Designer Adaptor Proteins for Functional Conversion of Peptides to Small-Molecule Ligands toward In-Cell Catalytic Protein Modification.

Peptides are privileged ligands for diverse biomacromolecules, including proteins; however, their utility is often limited due to low membrane permeability and in-cell instability. Here, we report peptide ligand-inserted eDHFR (PLIED) fusion protein as a universal adaptor for targeting proteins of interest (POI) with cell-permeable and stable synthetic functional small molecules (SFSM). PLIED binds to POI through the peptide moiety, properly orienting its eDHFR moiety, which then recruits trimethoprim (TMP)-conjugated SFSM to POI. Using a lysine-acylating BAHA catalyst as SFSM, we demonstrate that POI (MDM2 and chromatin histone) are post-translationally and synthetically acetylated at specific lysine residues. The residue-selectivity is predictable in an atomic resolution from molecular dynamics simulations of the POI/PLIED/TMP-BAHA (MTX was used as a TMP model) ternary complex. This designer adaptor approach universally enables functional conversion of impermeable peptide ligands to permeable small-molecule ligands, thus expanding the in-cell toolbox of chemical biology.

Photo-oxygenation of histidine residue inhibits α-synuclein aggregation.

Aggregation of α-synuclein (α-syn) into amyloid is the pathological hallmark of several neurodegenerative disorders, including Parkinson disease, dementia with Lewy bodies, and multiple system atrophy. It is widely accepted that α-syn aggregation is associated with neurodegeneration, although the mechanisms are not yet fully understood. Therefore, the inhibition of α-syn aggregation is a potential therapeutic approach against these diseases. This study used the photocatalyst for α-syn photo-oxygenation, which selectively adds oxygen atoms to fibrils. Our findings demonstrate that photo-oxygenation using this photocatalyst successfully inhibits α-syn aggregation, particularly by reducing its seeding ability. Notably, we also discovered that photo-oxygenation of the histidine at the 50th residue in α-syn aggregates is responsible for the inhibitory effect. These findings indicate that photo-oxygenation of the histidine residue in α-syn is a potential therapeutic strategy for synucleinopathies.

Practical N-to-C peptide synthesis with minimal protecting groups.

Accessible drug modalities have continued to increase in number in recent years. Peptides play a central role as pharmaceuticals and biomaterials in these new drug modalities. Although traditional peptide synthesis using chain-elongation from C- to N-terminus is reliable, it produces large quantities of chemical waste derived from protecting groups and condensation reagents, which place a heavy burden on the environment. Here we report an alternative N-to-C elongation strategy utilizing catalytic peptide thioacid formation and oxidative peptide bond formation with main chain-unprotected amino acids under aerobic conditions. This method is applicable to both iterative peptide couplings and convergent fragment couplings without requiring elaborate condensation reagents and protecting group manipulations. A recyclable N-hydroxy pyridone additive effectively suppresses epimerization at the elongating chain. We demonstrate the practicality of this method by showcasing a straightforward synthesis of the nonapeptide DSIP. This method further opens the door to clean and atom-efficient peptide synthesis.

A chemical catalyst enabling histone acylation with endogenous acyl-CoA.

Life emerges from a network of biomolecules and chemical reactions catalyzed by enzymes. As enzyme abnormalities are often connected to various diseases, a chemical catalyst promoting physiologically important intracellular reactions in place of malfunctional endogenous enzymes would have great utility in understanding and treating diseases. However, research into such small-molecule chemical enzyme surrogates remains limited, due to difficulties in developing a reactive catalyst capable of activating inert cellular metabolites present at low concentrations. Herein, we report a small-molecule catalyst, mBnA, as a surrogate for a histone acetyltransferase. A hydroxamic acid moiety of suitable electronic characteristics at the catalytic site, paired with a thiol-thioester exchange process, enables mBnA to activate endogenous acyl-CoAs present in low concentrations and promote histone lysine acylations in living cells without the addition of exogenous acyl donors. An enzyme surrogate utilizing cellular metabolites will be a unique tool for elucidation of and synthetic intervention in the chemistry of life and disease.

Histidine Photooxygenation Chemistry: Mechanistic Evidence and Elucidation.

Histidine photooxygenation has been the subject of extensive investigation for many years. The intricate nature of histidine distinguishes it from other amino acids, as its side chain readily undergoes changes in charge state and tautomerization in response to pH, and the polarity of the imidazole ring inverts upon oxidation. This complexity gives rise to a diverse range of oxidation products and mechanisms, posing challenges in their interpretation. This review aims to provide a thorough overview of the chemistry involved in histidine photooxygenation, encompassing a comprehensive analysis of resulting products, mechanisms engaged in their formation, and analytical techniques that have contributed to their identification. Additionally, it explores a wide range of applications stemming from this transformation, offering valuable insights into its practical implications in fields such as materials science, biomedical research, and drug development. By bridging the existing gap in literature, this review serves as a resource for understanding the intricacies of histidine photooxygenation and its diverse ramifications.

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.

Catalytic Approaches to Chemo- and Site-Selective Transformation of Carbohydrates.

Carbohydrates are a fundamental unit playing pivotal roles in all the biological processes. It is thus essential to develop methods for synthesizing, functionalizing, and manipulating carbohydrates for further understanding of their functions and the creation of sugar-based functional materials. It is, however, not trivial to develop such methods, since carbohydrates are densely decorated with polar and similarly reactive hydroxy groups in a stereodefined manner. New approaches to chemo- and site-selective transformations of carbohydrates are, therefore, of great significance for revolutionizing sugar chemistry to enable easier access to sugars of interest. This review begins with a brief overview of the innate reactivity of hydroxy groups of carbohydrates. It is followed by discussions about catalytic approaches to enhance, override, or be orthogonal to the innate reactivity for the transformation of carbohydrates. This review avoids making a list of chemo- and site-selective reactions, but rather focuses on summarizing the concept behind each reported transformation. The literature references were sorted into sections based on the underlying ideas of the catalytic approaches, which we hope will help readers have a better sense of the current state of chemistry and develop innovative ideas for the field.

Attenuation of α-synuclein aggregation by catalytic photo-oxygenation.

We developed catalyst 11 to promote selective photo-oxygenation of α-synuclein amyloid and attenuate its aggregation. Catalyst 11 effectively oxygenated both small and large aggregates. The oxygenated α-synuclein exhibited lower seeding activity than intact α-synuclein. This study corroborates the feasibility of catalytic photo-oxygenation as an anti-synucleinopathy strategy.

Actin filaments accumulated in the nucleus remain in the vicinity of condensing chromosomes in the zebrafish early embryo.

In the cytoplasm, filamentous actin (F-actin) plays a critical role in cell regulation, including cell migration, stress fiber formation, and cytokinesis. Recent studies have shown that actin filaments that form in the nucleus are associated with diverse functions. Here, using live imaging of an F-actin-specific probe, superfolder GFP-tagged utrophin (UtrCH-sfGFP), we demonstrated the dynamics of nuclear actin in zebrafish (Danio rerio) embryos. In early zebrafish embryos up to around the high stage, UtrCH-sfGFP increasingly accumulated in nuclei during the interphase and reached a peak during the prophase. After nuclear envelope breakdown (NEBD), patches of UtrCH-sfGFP remained in the vicinity of condensing chromosomes during the prometaphase to metaphase. When zygotic transcription was inhibited by injecting α-amanitin, the nuclear accumulation of UtrCH-sfGFP was still observed at the sphere and dome stages, suggesting that zygotic transcription may induce a decrease in nuclear F-actin. The accumulation of F-actin in nuclei may contribute to proper mitotic progression of large cells with rapid cell cycles in zebrafish early embryos, by assisting in NEBD, chromosome congression, and/or spindle assembly.

Bioconjugation of Au25 Nanocluster to Monoclonal Antibody at Tryptophan.

We report the first bioconjugation of Au25 nanocluster to a monoclonal antibody at scarcely exposed tryptophan (Trp) residues toward the development of high-resolution probes for cryogenic electron microscopy (cryo-EM) and tomography (cryo-ET). To achieve this, we improved the Trp-selective bioconjugation using hydroxylamine (ABNOH) reagents instead of previously developed N-oxyl radicals (ABNO). This new protocol allowed for the application of Trp-selective bioconjugation to acid-sensitive proteins such as antibodies. We found that a two-step procedure utilizing first Trp-selective bioconjugation for the introduction of azide groups to the protein and then strain-promoted azide-alkyne cycloaddition (SPAAC) to attach a bicyclononyne (BCN)-presenting redox-sensitive Au25 nanocluster was essential for a scalable procedure. Covalent labeling of the antibody with gold nanoclusters was confirmed by various analytical methods, including cryo-EM analysis of the Au25 nanocluster conjugates.

Boron-Catalyzed α-Functionalizations of Carboxylic Acids.

Catalytic, chemoselective, and asymmetric α-functionalizations of carboxylic acids promise up-grading simple feedstock materials to value-added functional molecules, as well as late-stage structural diversifications of multifunctional molecules, such as drugs and their leads. In this personal account, we describe boron-catalyzed α-functionalizations of carboxylic acids developed in our group (five reaction types). The reversible boron carboxylate formation is key to the acidification of the α-protons and enolization using mild organic bases, allowing for chemoselective and asymmetric bond formations of carboxylic acids. The ligand effects on reactivity and stereoselectivity, substrate scopes, and mechanistic insights are summarized.

A Catalytic Alkylation of Ketones via sp3 C-H Bond Activation.

We identified a ternary hybrid catalyst system composed of an acridinium photoredox catalyst, a thiophosphoric imide (TPI) catalyst, and a titanium complex catalyst that promoted an intermolecular addition reaction of organic molecules with various ketones through sp3 C-H bond activation. The thiyl radical generated via single-electron oxidation of TPI by the excited photoredox catalyst abstracted a hydrogen atom from organic molecules such as toluene, benzyl alcohol, alkenes, aldehydes, and THF. The thus-generated carbon-centered radical species underwent addition to ketones and aldehydes. This intrinsically unfavorable step was promoted by single-electron reduction of the intermediate alkoxy radical by catalytically generated titanium(III) species. This reaction provided an efficient and straightforward route to a broad range of tertiary alcohols and was successfully applied to late-stage functionalization of drugs or their derivatives. The proposed mechanism was supported by both experimental and theoretical studies.

Live Cell Synthetic Histone Acetylation by Chemical Catalyst.

Posttranslational modifications (PTMs) of histones, such as lysine acetylation and ubiquitination, regulate chromatin structure and gene expression. In living organisms, histone PTMs are catalyzed by histone-modifying enzymes. Here, we describe an entirely chemical method to introduce histone modifications in living cells without genetic manipulation. The chemical catalyst PEG-LANA-DSSMe activates a thioester acetyl donor, N,S-diacetylcysteamine (NAC-Ac), and promotes regioselective, synthetic histone acetylation at H2BK120 in living cells.

Lewis Acid-Conjugated Pyrene Photoredox Catalyst Promoting the Addition Reaction of α-Silyl Amines with Benzalmalononitriles.

We developed the addition reaction of α-silyl amines with benzalmalononitriles catalyzed by a Mg2+-conjugated pyrene catalyst under visible light irradiation. The catalytic activity of this complex was higher than pyrene alone, a Mg2+ Lewis acid alone, and the sum of these two independent catalytic elements. The observed enhancement in catalytic activity was likely due to electrostatic interactions of the Mg2+ Lewis acid with the pyrene radical anion, which was generated through photoinduced single electron transfer from α-silyl amines to the catalyst's pyrene moiety.

Promotion in the Clearance of Aggregated Aß In Vivo Using Amyloid Selective Photo-Oxygenation Technology.

Alzheimer's disease (AD) is characterized by the aggregation and deposition of 2 amyloid proteins: amyloid ß peptide (Aß) and tau protein. Immunotherapies using anti-Aß antibodies to promote the clearance of aggregated Aß have recently been highlighted as a promising disease-modifying approach against AD. However, immunotherapy has still some problems, such as low efficiency of delivery into the brain and high costs. We have developed the "amyloid selective photo-oxygenation technology" as a comparable to immunotherapy for amyloids. The photo-oxygenation can artificially attach the oxygen atoms to specific amino acids in amyloid proteins using photocatalyst and light irradiation. We revealed that in vivo photo-oxygenation for living AD model mice reduced the aggregated Aß in the brain. Moreover, we also showed that microglia were responsible for this promoted clearance of photo-oxygenated Aß from the brain. These results indicated that our photo-oxygenation technology has the potential as a disease-modifying therapy against AD to promote the degradation of amyloids, resulting in being comparable to immunotherapy. Here, we introduce our technology and its effects in vivo that we showed previously in Ozawa et al., Brain, 2021, as well as a further improvement towards non-invasive in vivo photo-oxygenation described in another publication Nagashima et al., Sci. Adv., 2021, as expanded discussion.

Pathological complete remission of relapsed tumor by photo-activating antibody-mimetic drug conjugate treatment.

Antibody-mimetic drug conjugate is a novel noncovalent conjugate consisting of an antibody-mimetic recognizing a target molecule on the cancer cell surface and low-molecular-weight payloads that kill the cancer cells. In this study, the efficacy of a photo-activating antibody-mimetic drug conjugate targeting HER2-expressing tumors was evaluated in mice, by using the affibody that recognize HER2 (ZHER2:342 ) as a target molecule and an axially substituted silicon phthalocyanine (a novel potent photo-activating compound) as a payload. The first treatment with the photo-activating antibody-mimetic drug conjugates reduced the size of all HER2-expressing KPL-4 xenograft tumors macroscopically. However, during the observation period, relapsed tumors gradually appeared in approximately 50% of the animals. To evaluate the efficacy of repeated antibody-mimetic drug conjugate treatment, animals with relapsed tumors were treated again with the same regimen. After the second observation period, the mouse tissues were examined histopathologically. Unexpectedly, all relapsed tumors were eradicated, and all animals were diagnosed with pathological complete remission. After the second treatment, skin wounds healed rapidly, and no significant side effects were observed in other organs, except for occasional microscopic granulomatous tissues beneath the serosa of the liver in a few mice. Repeated treatments seemed to be well tolerated. These results indicate the promising efficacy of the repeated photo-activating antibody-mimetic drug conjugate treatment against HER2-expressing tumors.

Site-Selective α-Alkylation of 1,3-Butanediol Using a Thiophosphoric Acid Hydrogen Atom Transfer Catalyst.

Herein, we developed secondary-alcohol-selective C-H alkylation of 1,3-butane diol by combining an acridinium photoredox catalyst and a thiophosphoric acid hydrogen atom transfer (HAT) catalyst. The use of non-coordinating solvent such as dichloromethane (DCM) improved secondary α-alkoxy C-H selectivity by lowering bond dissociation energy (BDE) through intramolecular hydrogen bonding.