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.

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.

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.

Photo-oxygenation by a biocompatible catalyst reduces amyloid-ß levels in Alzheimer's disease mice.

Amyloid formation and the deposition of the amyloid-ß peptide are hallmarks of Alzheimer's disease pathogenesis. Immunotherapies using anti-amyloid-ß antibodies have been highlighted as a promising approach for the prevention and treatment of Alzheimer's disease by enhancing microglial clearance of amyloid-ß peptide. However, the efficiency of antibody delivery into the brain is limited, and therefore an alternative strategy to facilitate the clearance of brain amyloid is needed. We previously developed an artificial photo-oxygenation system using a low molecular weight catalytic compound. The photocatalyst specifically attached oxygen atoms to amyloids upon irradiation with light, and successfully reduced the neurotoxicity of aggregated amyloid-ß via inhibition of amyloid formation. However, the therapeutic effect and mode of actions of the photo-oxygenation system in vivo remained unclear. In this study, we demonstrate that photo-oxygenation facilitates the clearance of aggregated amyloid-ß from the brains of living Alzheimer's disease model mice, and enhances the microglial degradation of amyloid-ß peptide. These results suggest that photo-oxygenation may represent a novel anti-amyloid-ß strategy in Alzheimer's disease, which is compatible with immunotherapy.

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.

Photo-Oxygenation: An Innovative New Therapeutic Approach Against Amyloidoses.

Many types of amyloidoses are pathologically characterized by the deposition of amyloid, which is comprised of fibrils formed by abnormally aggregated proteins, in various peripheral tissues and the central nervous system (CNS). Neurodegenerative disorders, such as Alzheimer disease (AD), Parkinson disease (PD), frontotemporal dementia (FTD), and amyotrophic lateral sclerosis (ALS), are well-known CNS amyloidoses that are characterized by amyloid deposition both inside and outside of cells. The amyloidogenic proteins of each disease have distinct primary sequences, and they normally function as soluble proteins. However, these proteins all aggregate and form amyloid with a common intermolecular tertiary structure, namely, a cross-ß-sheet structure, finally leading to the onset of each disease. Therefore, inhibition of the aggregation of amyloid proteins or efficient clearance of the already formed amyloids are thought to be promising therapeutic strategies against amyloidoses.

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.

Design, Synthesis, and Properties of a Chemically Tethered Amyloid-ß Segment Trimer Resistant to Intertrimer Mis-aggregation.

The oligomer species of amyloid-ß peptide (Aß) may be relevant to the development of Alzheimer's disease. Isolating specific oligomer species of Aß (i.e., dimers, trimers, tetramers, etc.), however, is difficult due to the transient, labile property of the oligomers. Here, we improved the resistance to intertrimer mis-aggregation of chemically tethered Aß25-35 trimers by introducing charged structures to the cyclic peptide tether that is covalently attached to the Aß chain. The resistance to aggregation of the chemically tethered trimers positively correlated with the number of negative charges at the tether. Thus, a chemical trimer possessing three malonic acids at the tether exhibited high resistance because of the attenuated self-association by anionic repulsion. In addition, the malonic acid trimer possessed amyloidogenic properties such as cross-ß-sheet structures, seeding activity, and cytotoxicity. This is the first study demonstrating that chemical modifications at the non-Aß component enhance the resistance to aggregation of chemically tethered Aß oligomers, by which the structural integrity of Aß is maintained. Biological/biophysical evaluations of the intertrimer aggregation-resistant trimer may offer new, useful insights into the pathological functions of Aß oligomers.

Design and properties of [Met35(O)]Aß42-lactam(Asp23/Lys28) possessing a lactam tether as a salt-bridge surrogate.

A characteristic feature of higher-order structures of amyloid ß peptide (Aß) aggregates observed in Alzheimer disease is the salt-bridge between the side-chains of Asp23 (carboxylate) and Lys28 (ammonium). We synthesized an [Met35(O)]Aß42 possessing a covalently bound lactam tether as an Asp23/Lys28 salt-bridge surrogate (compound 3). The lactam tether of 3 markedly promoted the formation of stable protofibril-like species that exhibited amyloidogenic properties such as a cross-ß-sheet structure and cytotoxicity. This finding is consistent with reports that the Asp23/Lys28 salt-bridge of Aß42 is transiently formed in aggregation intermediates.

Palladium-Catalyzed C-H Heteroarylation of 2,5-Disubstituted Imidazoles.

We developed a palladium-catalyzed C-H N-heteroarylation of N-protected-2,5-disubstituted imidazoles at the C4-position using N-heteroaryl halides as a coupling partner. Intensive reaction condition screening led us to identify fluorinated bathophenanthroline 7 as the optimum ligand for the palladium catalyst. This reaction will enhance lead optimization of drug candidates by facilitating the synthesis of heterobiaryl compounds containing an imidazole ring.

A chemically engineered, stable oligomer mimic of amyloid ß42 containing an oxime switch for fibril formation.

Toxic aggregation of monomeric amyloid ß (Aß) into oligomers followed by the formation of fibrils is a causative process in the pathogenesis of Alzheimer's disease. The mechanism for furnishing the toxicity of Aß aggregates is elusive, however, mainly due to the transient, unstable properties of the oligomer states. Oligomer mimics stabilized by chemical protein engineering are potentially useful tools for elucidating the pathogenicity of Aß aggregates. Here we report a stable Aß oligomer mimic that is transformed into fibrils by a chemical stimulus, i.e., an oxime exchange reaction. A derivative of Aß42[Met35(O)], compound 2, containing an oxime tether between residues 23 and 28 (a salt-bridge surrogate between Asp23 and Lys28 of the Aß42 oligomer), rapidly and homogeneously formed stable, relatively large oligomers with preserved amyloid-like properties, such as the propensity to form ß-sheets and toxicity. Chemical cleavage of the tether via an oxime exchange reaction induced transformation of the oligomers into the fibril state. These results demonstrate that the oxime bond formation/cleavage can switch the aggregation state of the mimic by functionally surrogating the salt-bridge of Aß42. This novel system temporally dissects the dynamic process of Aß aggregation, and thus might offer a unique molecular tool for exploring the properties of Aß oligomers and fibrils.

Transition Metal-Free Tryptophan-Selective Bioconjugation of Proteins.

Chemical modifications of native proteins can facilitate production of supernatural protein functions that are not easily accessible by complementary methods relying on genetic manipulations. However, accomplishing precise control over selectivity while maintaining structural integrity and homogeneity still represents a formidable challenge. Herein, we report a transition metal-free method for tryptophan-selective bioconjugation of proteins that is based on an organoradical and operates under ambient conditions. This method exhibits low levels of cross-reactivity and leaves higher-order structures of the protein and various functional groups therein unaffected. The strategy to target less abundant amino acids contributes to the formation of structurally homogeneous conjugates, which may even be suitable for protein crystallography. The absence of toxic metals and biochemically incompatible conditions allows a rapid functional modulation of native proteins such as antibodies and pathogenic aggregative proteins, and this method may thus easily find therapeutic applications.

Medicinal Chemistry Focusing on Aggregation of Amyloid-ß.

The aggregation of peptides/proteins is intimately related to a number of human diseases. More than 20 have been identified which aggregate into fibrils containing extensive ß-sheet structures, and species generated in the aggregation processes (i.e., oligomers and fibrils) contribute to disease development. Amyloid-ß peptide (designated Aß), related to Alzheimer's disease (AD), is the representative example. The intensive aggregation property of Aß also leads to difficulty in its synthesis. To improve the synthetic problem, we developed an O-acyl isopeptide of Aß1-42, in which the N-acyl linkage (amide bond) of Ser(26) was replaced with an O-acyl linkage (ester bond) at the side chain. The O-acyl isopeptide demonstrated markedly higher water-solubility than that of Aß1-42, while it quickly converted to intact monomer Aß1-42 via an O-to-N acyl rearrangement under physiological conditions. Inhibition of the pathogenic aggregation of Aß1-42 might be a therapeutic strategy for curing AD. We succeeded in the rational design and identification of a small molecule aggregation inhibitor based on a pharmacophore motif obtained from cyclo[-Lys-Leu-Val-Phe-Phe-]. Moreover, the inhibition of Aß aggregation was achieved via oxygenation (i.e., incorporation of oxygen atoms to Aß) using an artificial catalyst. We identified a selective, cell-compatible photo-oxygenation catalyst of Aß, a flavin catalyst attached to an Aß-binding peptide, which markedly decreased the aggregation potency and neurotoxicity of Aß.

Synthesis of chemically-tethered amyloid-ß segment trimer possessing amyloidogenic properties.

As amyloid-ß (Aß) undergoes dynamic aggregation, it is impossible to isolate ('hook') the transient Aß oligomer in an assembly state-pure form (e.g., sole Aß dimer, trimer, tetramer, etc.). Obtaining such a pure Aß oligomer would allow us to establish an in vitro system to perform a more detailed investigation of the pathogenic properties of the oligomer. A chemically-tethered Aß oligomer, constructed only by covalent bonds, could satisfy this demand. Here we designed a chemically-tethered trimer of a pathogenic Aß fragment (Aß25-35) (1) and successfully generated it in situ from its precursor (4), a water-soluble and non-aggregative O-acyl isopeptide of 1, in neutral aqueous media. Chemically-tethered 1 possessed stronger amyloidogenic properties, that is, potential for ß-sheet structure, fibril formation, and cytotoxicity, than the corresponding monomer Aß25-35 (6). Trimerization of Aß25-35 sequence might affect both the aggregative properties and cytotoxicity, based on the present results. This work opens the door for chemical synthesis of oligomers bigger than trimers in an assembly state-pure form, allowing for identification of the most toxic Aß oligomer.

Covalent modifier-type aggregation inhibitor of amyloid-ß based on a cyclo-KLVFF motif.

Inhibition of amyloid-ß (Aß) aggregation could be a drug development target for treating Alzheimer disease. Insufficient activity to inhibit aggregation, however, remains a key issue. Here, we report a covalent modifier-type aggregation inhibitor of Aß, diazirine-equipped cyclo-KLVF(ß-Ph)F (2). Due to the affinity of the cyclo-KLVFF motif for Aß, 2 selectively reacted with Aß1-42 under UV-light irradiation to form an irreversible covalent bond. The Tyr-10 residue of Aß1-42 was identified as the covalent modification site with 2. The extent of cross-ß-sheet structure, characteristics of amyloid aggregation, and toxicity of Aß1-42 were strongly attenuated by this chemical modification.

A cyclic KLVFF-derived peptide aggregation inhibitor induces the formation of less-toxic off-pathway amyloid-ß oligomers.

Inhibition of amyloid-ß (Aß) aggregation could be a target of drug development for the treatment of currently incurable Alzheimer's disease. We previously reported that a head-to-tail cyclic peptide of KLVFF (cyclic-KLVFF), a pentapeptide fragment corresponding to the Aß16-20 region (which plays a critical role in the generating Aß fibrils), possesses potent inhibitory activity against Aß aggregation. Here we found that the inhibitory activity of cyclic-KLVFF was significantly improved by incorporating an additional phenyl group at the ß-position of the Phe4 side chain (inhibitor 3). Biophysical and biochemical analyses revealed the rapid formation of 3-embedded oligomer species when Aß1-42 was mixed with 3. The oligomer species is an "off-pathway" species with low affinity for cross-ß-sheet-specific dye thioflavin T and oligomer-specific A11 antibodies. The oligomer species had a sub-nanometer height and little capability of aggregation to amyloid fibrils. Importantly, the toxicity of the oligomer species was significantly lower than that of native Aß oligomers. These insights will be useful for further refinement of cyclic-KLVFF-based aggregation inhibitors.

Non-pretreated O-acyl isopeptide of amyloid ß peptide 1-42 is monomeric with a random coil structure but starts to aggregate in a concentration-dependent manner.

An isopeptide of amyloid ß peptide 1-42 (isoAß42) was considered as a non-aggregative precursor molecule for the highly aggregative Aß42. It has been applied to biological studies after several pretreatments. Here we report that isoAß42 is monomeric with a random coil structure at 40 µM without any pretreatment. But we also found that isoAß42 retains a slight aggregative nature, which is significantly weaker than that of the native Aß42.

Synthesis of an O-acyl isopeptide by using native chemical ligation in an aqueous solvent system.

O-Acyl isopeptides, in which the N-acyl linkage on the hydroxyamino acid residue (e.g. Ser and Thr) is replaced by an O-acyl linkage, generally suppress unfavorable aggregation properties derived from the corresponding parent peptides. Here, we report the synthesis of an O-acyl isopeptide of 34-mer pyroGlu-ADan (2), a component of amyloid deposits in hereditary familial Danish dementia, by using native chemical ligation. Native chemical ligation of pyroGlu(1) -ADan(1-21)-SCH2 CH2 SO3 (-) Na(+) (3) and Cys(22) -O-acyl isopeptide (4), in which the amino group of the Ser(29) residue at the isopeptide moiety was protected by an allyloxycarbonyl group, proceeded well in an aqueous solvent to yield a ligated O-acyl isopeptide (5). Subsequent disulfide bond formation and deprotection of the allyloxycarbonyl group followed by HPLC purification gave 2 with a reasonable overall yield. 2 was converted to the parent peptide 1 via an O-to-N acyl migration reaction. The sequential method, namely (i) native chemical ligation of the O-acyl isopeptide, (ii) HPLC purification as the O-acyl isopeptide form, and (iii) O-to-N acyl migration into the desired polypeptide, would be helpful to solve problems with HPLC purification of hydrophobic polypeptides in the process of chemical protein synthesis.

O-acyl isopeptide method: development of an O-acyl isodipeptide unit for Boc SPPS and its application to the synthesis of Aß1-42 isopeptide.

The O-acyl isopeptide method was developed for the efficient preparation of difficult sequence-containing peptide. Furthermore, development of the O-acyl isodipeptide unit for Fmoc chemistry simplified its synthetic procedure by solid-phase peptide synthesis. Here, we report a novel isodipeptide unit for Boc chemistry, and the unit was successfully applied to the synthesis of amyloid ß peptide. Combination of Boc chemistry and the isodipeptide unit would be an effective method for the synthesis of many difficult peptides. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.