Post-Translational Modifications Control Phase Transitions of Tau.

The self-assembly of Tau(297-391) into filaments, which mirror the structures observed in Alzheimer's disease (AD) brains, raises questions about the role of AD-specific post-translational modifications (PTMs) in the formation of paired helical filaments (PHFs). To investigate this, we developed a synthetic approach to produce Tau(291-391) featuring N-acetyllysine, phosphoserine, phosphotyrosine, and N-glycosylation at positions commonly modified in post-mortem AD brains, thus facilitating the study of their roles in Tau pathology. Using transmission electron microscopy (TEM), cryo-electron microscopy (cryo-EM), and a range of optical microscopy techniques, we discovered that these modifications generally hinder the in vitro assembly of Tau into PHFs. Interestingly, while acetylation's effect on Tau assembly displayed variability, either promoting or inhibiting phase transitions in the context of cofactor free aggregation, heparin-induced aggregation, and RNA-mediated liquid-liquid phase separation (LLPS), phosphorylation uniformly mitigated these processes. Our observations suggest that PTMs, particularly those situated outside the fibril's rigid core are pivotal in the nucleation of PHFs. Moreover, in scenarios involving heparin-induced aggregation leading to the formation of heterogeneous aggregates, most AD-specific PTMs, except for K311, appeared to decelerate the aggregation process. The impact of acetylation on RNA-induced LLPS was notably site-dependent, exhibiting both facilitative and inhibitory effects, whereas phosphorylation consistently reduced LLPS across all proteoforms examined. These insights underscore the complex interplay between site-specific PTMs and environmental factors in modulating Tau aggregation kinetics, enhancing our understanding of the molecular underpinnings of Tau pathology in AD and highlighting the critical role of PTMs located outside the ordered filament core in driving the self-assembly of Tau into PHF structures.

Desulfurative Borylation of Small Molecules, Peptides, and Proteins.

We introduce a direct conversion of alkyl thiols into boronic acids, facilitated by a water-soluble phosphine, 1,3,5-triaza-7-phosphaadamantane (PTA), in conjunction with tetrahydroxydiboron (B2(OH)4), acting as both a radical initiator and a boron source. This desulfurative borylation reaction has been successfully applied to various substrates, including cysteine residues in oligopeptides and small proteins, primary alkyl thiols found in pharmaceutical compounds, disulfides, and selenocysteine. Optimization of reaction conditions was undertaken to reduce the formation of unwanted reactions, such as the reduction of alanyl or other primary radicals, and to prevent deleterious reactions between the phosphine and N-terminal amine that lead to methylene adducts by utilizing a buffer containing glycine-glycine (GG) dipeptide. The developed method is characterized by its operational simplicity and robustness. Moreover, its compatibility with various functional groups present in peptides and proteins makes it a promising tool for late-stage functionalization, extending its potential application across a broad spectrum of chemical and biological targets.

Chemical Synthesis of Microtubule-Associated Protein Tau.

Deposits of the microtubule-associated protein Tau (MAPT) serve as a hallmark of neurodegenerative diseases known as tauopathies. Numerous studies have demonstrated that in diseases such as Alzheimer's disease (AD), Tau undergoes extensive remodeling. The attachment of post-translational modifications distributed throughout the entire sequence of the protein correlates with clinical presentation. A systematic examination of these protein alterations can shed light on their roles in both healthy and diseased states. However, the ability to access these modifications in the entire protein chain is limited as Tau can only be produced recombinantly or through semisynthesis. In this article, we describe the first chemical synthesis of the longest 2N4R isoform of Tau, consisting of 441 amino acids. The 2N4R Tau was divided into 3 major segments and a total of 11 fragments, all of which were prepared via solid-phase peptide synthesis. The successful chemical strategy has relied on the strategic use of two cysteine sites (C291 and C322) for the native chemical ligations (NCLs). This was combined with modern preparative protein chemistries, such as mercaptothreonine ligation (T205), diselenide-selenoester ligation (D358), and mutations of mercaptoamino acids into native residues via homogeneous radical desulfurization (A40, A77, A119, A157, A246, and A390). The successful completion of the synthesis has established a robust and scalable route to the native protein in multimilligram quantities and high purity. In broader terms, the presented strategy can be applied to the preparation of other shorter isoforms of Tau as well as to introduce all post-translational modifications that are characteristic of tauopathies such as AD.

TMEM106B Fibrils from FTLD Patients and Healthy Controls.

Recent studies involving four research teams have revealed that amyloid fibrils in FTLD-TDP patients and cognitively healthy individuals primarily consist of TMEM106B, a protein previously identified as a risk factor for FTLD-TDP. Through cryogenic electron microscopy, the studies identified various protofilament structures of TMEM106B fibrils from individuals with several neurodegenerative diseases. These findings raise new questions and opportunities for future research, as they suggest that TMEM106B plays a central role in FTLD pathology. These discoveries also prompt the need for the development of specific antibodies for fibrillar TMEM106B and necessitate further investigation of the potential mechanistic link between TMEM106B and other filamentous aggregates. The power of cryo-EM techniques is underscored in these unexpected findings and may be a vital tool for gaining further molecular insights into neurodegenerative diseases characterized by amyloid deposits.

Catalytic Amide Activation with Thermally Stable Molybdenum(VI) Dioxide Complexes.

Oxazolines and thiazolines are important constituents of bioactive natural products and pharmaceuticals. Here, we report the development of an effective and practical method of oxazoline and thiazoline formation, which can facilitate the synthesis of natural products, chiral ligands, and pharmaceutical intermediates. This method capitalized on a Mo(VI) dioxide catalyst stabilized by substituted picolinic acid ligands, which is tolerant to many functional groups that would otherwise be sensitive to highly electrophilic alternative reagents.

Umpolung AlaB Reagents for the Synthesis of Non-Proteogenic Amino Acids, Peptides and Proteins.

Non-proteogenic amino acids and functionalized peptides are important motifs in modern drug discovery. Here we report that AlaB can serve as universal building blocks in the synthesis of a diverse collection of modified amino acids, peptides, and proteins. First, we develop the synthesis of AlaB from redox-active esters of aspartic acid resulting in a series of ß-boronoalanine derivatives. Next, we show that AlaB can be integrated into automated oligopeptide solid-phase synthesis. AlaB is compatible with common transformations used in preparative peptide chemistry such as native chemical ligation and radical desulfurization as showcased by total synthesis of AlaB -containing ubiquitin. Furthermore, AlaB reagents participate in Pd-catalyzed reactions, including C-C cross-couplings and macrocyclizations. Taken together, AlaB synthons are practical reagents to access modified peptides, proteins, and in the synthesis of cyclic/stapled peptides.

Site-Selective Amide Functionalization by Catalytic Azoline Engrafting.

Direct peptide and protein activation is a challenging transformation because of the stabilizing effect of the amide group. While enzymes can be considered as prototypical systems that have evolved to achieve high selectivity and specificity, small-molecule catalysts that functionalize the amide group may accommodate a much larger selection of substrates but currently remain scarce. Here, by combining the desired features from both catalytic regimes we designed an artificial cyclodehydratase, a catalytic system for the site-selective modification of peptides and natural products by engrafting heterocycles into their scaffolds. The catalytic system features a molybdenum(VI) center that was decorated with a sterically congested tripod ligand. The optimized catalyst can introduce azolines into small molecules, natural products, and oligopeptides with high efficiency and minimal waste. We further demonstrate the utility of the new protocol in the direct functionalization of a single amide group in the presence of up to seven other chemically similar positions and in the direct conversion of these groups into amines and thioamides. This new mechanistic paradigm may address an unmet need for a general method for the selective and sustainable functionalization of peptides and natural products.

Organometallic AlaM Reagents for Umpolung Peptide Diversification.

Selective modifications of peptides and proteins have emerged as a promising strategy to develop novel mechanistic probes and prepare compounds with translational potentials. Here, we report alanine carbastannatranes AlaSn as a universal synthon in various C-C and C-heteroatom bond-forming reactions. These reagents are compatible with peptide manipulation techniques and can undergo chemoselective conjugation in minutes when promoted by Pd(0). Despite their increased nucleophilicity and propensity to transfer the alkyl group, C(sp3)-C(sp2) coupling with AlaSn can be accomplished at room temperature under buffered conditions (pH 6.5-8.5). We also show that AlaSn can be easily transformed into several canonical L- and D-amino acids in arylation, acylation, and etherification reactions. Furthermore, AlaSn can partake in macrocyclizations exemplified by the synthesis of medium size cyclic peptides with various topologies. Taken together, metalated alanine AlaSn demonstrates unparalleled scope and represents a new type of umpolung reagents suitable for structure-activity relationship studies and peptide diversification.

Total synthesis of micrococcin P1 and thiocillin I enabled by Mo(vi) catalyst.

Thiopeptides are a class of potent antibiotics with promising therapeutic potential. We developed a novel Mo(vi)-oxide/picolinic acid catalyst for the cyclodehydration of cysteine peptides to form thiazoline heterocycles. With this powerful tool in hand, we completed the total syntheses of two representative thiopeptide antibiotics: micrococcin P1 and thiocillin I. These two concise syntheses (15 steps, longest linear sequence) feature a C-H activation strategy to install the trisubstituted pyridine core and thiazole groups. The synthetic material displays promising antimicrobial properties measured against a series of Gram-positive bacteria.

Asymmetric Synthesis of Chiral 1,2-Amino Alcohols and Morpholin-2-ones from Arylglyoxals.

Chiral 1,2-amino alcohols are privileged scaffolds with important applications as drug candidates and chiral ligands. Although various methods for the preparation of this structural motif have been reported, these methods are limited because of the use of precious metals and ligands. Here, we report a practical and high yielding synthesis of chiral 1,2-amino alcohols using arylglyoxals and pseudoephedrine auxiliary. This reaction is catalyzed by a Brønsted acid and provides morpholinone products in high yields and selectivities. The morpholine ring was converted into 1,2-amino alcohols in a two-step protocol.