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Methionine plays a critical role in various biological and cell regulatory processes, making its chemoproteomic profiling indispensable for exploring its functions and potential in protein therapeutics. Building on the principle of rapid oxidation of methionine, we report Copper(I)-Nitrene Platform for robust, and selective labeling of methionine to generate stable sulfonyl sulfimide conjugates under physiological conditions. We demonstrate the versatility of this platform to label methionine in bioactive peptides, intact proteins (6.5-79.5 kDa), and proteins in complex cell lysate mixtures with varying payloads. We discover ligandable proteins and sites harboring hyperreactive methionine within the human proteome. Furthermore, this has been utilized to profile oxidation-sensitive methionine residues, which might increase our understanding of the protective role of methionine in diseases associated with elevated levels of reactive oxygen species. The Copper(I)-Nitrene Platform allows labeling methionine residues in live cancer cells, observing minimal cytotoxic effects and achieving dose-dependent labeling. Confocal imaging further reveals the spatial distribution of modified proteins within the cell membrane, cytoplasm, and nucleus, underscoring the platform's potential in profiling the cellular interactome.
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Cobre , Metionina , Proteómica , Humanos , Metionina/metabolismo , Metionina/química , Cobre/metabolismo , Cobre/química , Proteómica/métodos , Oxidación-Reducción , Proteoma/metabolismo , Línea Celular Tumoral , Péptidos/metabolismo , Péptidos/química , IminasRESUMEN
Lysine dimethylation (Kme2) is a crucial post-translational modification (PTM) that regulates biological processes and is implicated in diseases. There is significant interest in globally identifying these methylation marks. Unfortunately, this remains challenging due to the lack of robust technologies for selectively labeling Kme2. To address this, we present a chemical method named tertiary amine coupling by oxidation (TACO). This method selectively modifies Kme2 to aldehydes using Selectfluor and a base. The resulting aldehydes from Kme2 were then functionalized using reductive amination, thiolamine, and oxime chemistry. We successfully demonstrated the versatility of TACO in selectively labeling Kme2 peptides and proteins in complex cell lysate mixtures with varying payloads, including affinity tags and fluorophores. We further showed the application of TACO chemistry for the identification of Kme2 sites at a single-molecule level by fluorosequencing. We discovered novel 30 Kme2 sites, in addition to previously known 5 Kme2 sites, by proteomics analysis of TACO-modified nuclear extracts. Our work establishes a unique strategy for covalently modifying Kme2, facilitating the global identification of low-abundance Kme2-PTMs and their sites within complex cell lysate mixtures.
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Lisina , Procesamiento Proteico-Postraduccional , Lisina/química , Proteínas/química , Aminas , AldehídosRESUMEN
Aldehydes, pervasive in various environments, pose health risks at elevated levels due to their collective toxic effects via shared mechanisms. Monitoring total aldehyde content in living systems is crucial due to their cumulative impact. Current methods for detecting cellular aldehydes are limited to UV and visible ranges, restricting their analysis in living systems. This study introduces an innovative reaction-based trigger that leverages the exceptional selectivity of 2-aminothiophenol for aldehydes, leading to the production of dihydrobenzothiazole and activating a fluorescence response. Using this trigger, we developed a series of fluorescent probes for aldehydes by altering the fluorophore allowing for excitation and emission wavelengths across the visible to near-infrared spectral regions without compromising the reactivity of the bioorthogonal moiety. These probes exhibit remarkable aldehyde chemoselectivity, rapid kinetics, and high quantum yields, enabling the detection of diverse aldehyde types, both exogenous and endogenous, within complex biological contexts. Notably, we employed the most red-shifted near-infrared probe from this series to detect aldehydes in living systems, including biliary organoids and mouse organs. These probes provide valuable tools for exploring the multifaceted roles of aldehydes in biological functions and diseases within living systems, laying the groundwork for further investigations.
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Inspired by the enzyme lysyl oxidase, which selectively converts the side chain of lysine into allysine, an aldehyde-containing post-translational modification, we report herein the first chemical method for the synthesis of allysine by selective oxidation of dimethyl lysine. This approach is highly chemoselective for dimethyl lysine on proteins. We highlight the utility of this biomimetic approach for generating aldehydes in a variety of pharmaceutically active linear and cyclic peptides at a late stage for their diversification with various affinity and fluorescent tags. Notably, we utilized this approach for generating small-molecule aldehydes from the corresponding tertiary amines. We further demonstrated the potential of this approach in generating cellular models for studying allysine-associated diseases.
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Lisina , Péptidos , Péptidos/química , Péptidos/síntesis química , Péptidos/metabolismo , Lisina/química , Lisina/metabolismo , Humanos , Aldehídos/química , Oxidación-Reducción , Ácido 2-Aminoadípico/análogos & derivadosRESUMEN
In the realm of high-throughput screening (HTS), macrocyclic peptide libraries traditionally necessitate decoding tags, essential for both library synthesis and identifying hit peptide sequences post-screening. Our innovation introduces a tag-free technology platform for synthesizing cyclic peptide libraries in solution and facilitates screening against biological targets to identify peptide binders through unconventional intramolecular CyClick and DeClick chemistries (CCDC) discovered through our research. This combination allows for the synthesis of diverse cyclic peptide libraries, the incorporation of various amino acids, and facile linearization and decoding of cyclic peptide binder sequences. Our sensitivity-enhancing derivatization method, utilized in tandem with nano LC-MS/MS, enables the sequencing of peptides even at exceedingly low picomolar concentrations. Employing our technology platform, we have successfully unearthed novel cyclic peptide binders against a monoclonal antibody and the first cyclic peptide binder of HIV capsid protein responsible for viral infections as validated by microscale thermal shift assays (TSA), biolayer interferometry (BLI) and functional assays.
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Biblioteca de Péptidos , Péptidos Cíclicos , Péptidos Cíclicos/química , Péptidos Cíclicos/síntesis química , Ensayos Analíticos de Alto Rendimiento , Espectrometría de Masas en TándemRESUMEN
C-Terminal residues play a pivotal role in dictating the structure and functions of proteins. Herein, we report a mild, efficient, chemoselective, and site-selective chemical method that allows for precise chemical proteolysis at C-terminal arginine dictated by 9,10-phenanthrenequinone independent of the remaining sequence. This biomimetic approach also exhibits the potential to synthesize C-terminal methyl ester (-CO2Me) peptides.
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Arginina , Péptidos , Carboxipeptidasa B/metabolismo , Secuencia de Aminoácidos , Arginina/química , Péptidos/química , ProteínasRESUMEN
Aliphatic aldehydes are reactive electrophilic carbonyls that cross-link with DNA and proteins leading to cellular toxicity and disease pathogenesis. This toxicity is due to the cooperative effect of multiple aldehydes via a common mechanism. Therefore, live-cell imaging of total aliphatic aldehydes, small-to-long chain (C1-C10), is highly desired to decipher their physiological and pathological functions. However, sensors for imaging total cellular aliphatic aldehydes are currently lacking despite their high concentrations (â¼80 to >500 µM) inside cells. Herein, we report chemical sensors that generate a benzimidazole moiety upon reaction with aliphatic aldehydes of different chain lengths (C1-C10), resulting in turn-on fluorescence. These sensors exhibit high quantum yields, high dynamic range, and enable the quantification of changes in both the exogenous administration of aldehydes and endogenous real-time formation of aliphatic aldehydes in live mammalian cells. This tool has great potential to transform aldehyde research by illuminating cellular metabolites that have remained elusive in living systems.
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Macrocyclic peptides have become increasingly important in the pharmaceutical industry. We present a detailed computational investigation of the reaction mechanism of the recently developed "CyClick" chemistry to selectively form imidazolidinone cyclic peptides from linear peptide aldehydes, without using catalysts or directing groups (Angew. Chem. Int. Ed. 2019, 58, 19073-19080). We conducted computational mechanistic to investigate the effects of intramolecular hydrogen bonds (IMHBs) in promoting a kinetically facile zwitterionic mechanism in "CyClick" of pentapeptide aldehyde AFGPA. Our DFT calculations highlighted the importance of IMHB in pre-organization of the resting state, stabilization of the zwitterion intermediate, and the control of the product stereoselectivity. Furthermore, we have also identified that the low ring strain energy promotes the "CyClick" of hexapeptide aldehyde AAGPFA to form a thermodynamically more stable 15+5 imidazolidinone cyclic peptide product. In contrast, large ring strain energy suppresses "CyClick" reactivity of tetra peptide aldehyde AFPA from forming the 9+5 imidazolidinone cyclic peptide product.
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Péptidos Cíclicos , Péptidos , Enlace de Hidrógeno , Teoría Funcional de la DensidadRESUMEN
One-pot multicomponent coupling of different units in a chemoselective manner and their late-stage diversification has wide applicability in varying chemistry fields. Here, we report a simple multicomponent reaction inspired by enzymes that combines thiol and amine nucleophiles in one pot via a furan-based electrophile to generate stable pyrrole heterocycles independent of the diverse functionalities on furans, thiols and amines under physiological conditions. The resulting pyrrole provides a reactive handle to introduce diverse payloads. We demonstrate the application of Furan-Thiol-Amine (FuTine) reaction for the selective and irreversible labeling of peptides, synthesis of macrocyclic and stapled peptides, selective modification of twelve different proteins with varying payloads, homogeneous engineering of proteins, homogeneous stapling of proteins, dual modification of proteins with different fluorophores using the same chemistry and labeling of lysine and cysteine in a complex human proteome.
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Aminas , Compuestos de Sulfhidrilo , Humanos , Furanos , PirrolesRESUMEN
Protein α-N-terminal dimethylation (Nme2) is an underexplored posttranslational modification (PTM) despite the increasing implications of α-N-terminal dimethylation in vital physiological and pathological processes across diverse species; thus, it is imperative to identify the sites of α-N-terminal dimethylation in the proteome. So far, only â¼300 α-N-terminal methylation sites have been discovered including mono-, di-, and tri-methylation, due to the lack of a pan-selective method for detecting α-N-terminal dimethylation. Herein, we introduce the three-component coupling reaction, oxidative nitrile thiazolidination (OxNiTha) for chemoselective modification of α-Nme2 to thiazolidine ring in the presence of selectfluor, sodium cyanide, and 1,2 aminothiols. One of the major challenges in developing a pan-specific method for the selective modification of α-Nme2 PTM is the competing reaction with dimethyl lysine (Kme2) PTM of a similar structure. We tackle this challenge by trapping nitrile-modified Nme2 with aminothiols, leading to the conversion of Nme2 to a five-membered thiazolidine ring. Surprisingly, the 1,2 aminothiol reaction with nitrile-modified Kme2 led to de-nitrilation along with the de-methylation to generate monomethyl lysine (Kme1). We demonstrated the application of OxNiTha reaction in pan-selective and robust modification of α-Nme2 in peptides and proteins to thiazolidine functionalized with varying fluorescent and affinity tags under physiological conditions. Further study with cell lysate enabled the enrichment of Nme2 PTM containing proteins.
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Lisina , Nitrilos , Lisina/química , Tiazolidinas , Procesamiento Proteico-Postraduccional , Proteoma/metabolismo , Estrés OxidativoRESUMEN
OBJECTIVE: Snake bite is an emergency in tropical and subtropical countries. It is a neglected disease and is most commonly seen in rural setups, where people are ignorant about the venomous snake bites. It results in increased mortality and morbidity because precious time is wasted, either in consulting traditional healers or waiting for the development of signs and symptoms of envenomation. Then only the patient is shifted to a health center. Here we studied the clinical profile, management, and outcome of snake bite patients. MATERIALS AND METHODS: This study was done by retrieving the records of patients with snake bites admitted to the Department of Medicine, Indira Gandhi Medical College & Hospital, Shimla, from 1st January 2017 through December 2019. The recorded data was entered in a precoded performa, and analysis was done with respect to various variables. RESULT: We evaluated the records of 190 patients. The incidence of the bite was higher among females, 62.1% (n = 118). The commonest age group involved was 21-50 years, 70.1% (n = 34). In 55.8% (n = 106), the site of the bite was the upper limb. The daytime bite was present in 54.7% (n = 106). The maximum incidence of snake bites was found during the rainy season, 81.5% (n = 155). 28.4% (n = 54) of patients presented within 6 hours of the bite. Coagulopathy [whole blood clotting test (WBCT) of >20 minutes] and neurotoxicity were seen in 77.9 and 7.9% of patients, respectively. Anti-snake venom (ASV) was given to 87.8% (n = 167) of patients. In 80% (n = 152) of the cases, hospital stay was up to 3 days. Mortality was seen in only two (1.05%) cases. CONCLUSION: There is a need to create awareness among the community, particularly in rural areas, about snake bite envenomation and early transportation of victims to the nearest health center. Training of health professionals is also needed to manage cases of snake bites efficiently and judiciously, thereby reducing morbidity and morbidity.
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Mordeduras de Serpientes , Adulto , Femenino , Humanos , Persona de Mediana Edad , Adulto Joven , Antivenenos/uso terapéutico , Mordeduras de Serpientes/epidemiología , Mordeduras de Serpientes/terapia , Mordeduras de Serpientes/diagnóstico , Venenos de Serpiente , Centros de Atención Terciaria , Atención Terciaria de SaludRESUMEN
The chemo- and site-selective reaction at the particular C-N amide bond among a sea of other amides is a significant and long-standing challenge. Although the use of twisted amides has been demonstrated for modifications of inert C-N amide bonds, none of these methods can selectively activate a particular amide bond for C-C bond formation in the presence of similar amides. Using density functional theory as a guide, we report the first site-selective C-C bond modification of a particular C-N amide bond in polyamides with a low twist angle by combining ground-state steric distortion with electronic activation.
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Amidas , Nylons , Amidas/química , ElectrónicaRESUMEN
Gong Chen, Monika Raj and Andrei Yudin introduce the RSC Chemical Biology themed collection on the 'Synthesis and chemical biology of macrocycles'.
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Nature increases the functional diversity of the proteome through posttranslational modifications (PTMs); a process that involves the proteolytic processing or catalytic attachment of diverse functional groups onto proteins. These modifications modulate a host of biological activities and responses. Consequently, anomalous PTMs often correlate to a host of diseases, hence there is a need to detect these transformations, both qualitatively and quantitatively. One technique that has gained traction is the use of robust chemical strategies to label different PTMs. By utilizing the intrinsic chemical reactivity of the different chemical groups on the target amino acid residues, this strategy can facilitate the delineation of the overarching and inclusionary roles of these different modifications. Herein, we will discuss the current state of the art in post-translational modification analysis, with a direct focus on covalent chemical methods used for detecting them.
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Here, we report a novel rapid arene triazene strategy for the macrocyclization of peptides that generates an inbuilt chromophoric triazene moiety at the site of cyclization within a minute. The rapid arene triazene chemistry is chemoselective for secondary amines and p-amino phenylalanine. Importantly, the resulting triazene cyclic peptide is highly stable at neutral pH and under harsh conditions but rapidly responds to various external stimuli such as UV radiations and acidic conditions, resulting in the ring opening to generate the linear peptides in an unchanged form, which further cyclizes under neutral pH conditions. This method works with completely unprotected peptides and has been applied for the synthesis of 18- to 66-membered monocycles and bicycles with various amino acid compositions in one pot under neutral pH conditions. Due to the high stability of triazene cyclic peptides, the postcyclization modification was carried out with various functional groups. This rapid, macrocyclization strategy featuring a triazene scaffold, amenable to late-stage diversification and responsive to external stimuli, should find application in various fields of chemical biology, selective drug delivery, and identification of cyclic peptide hits after library screening.
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Péptidos Cíclicos , Péptidos , Aminas , Ciclización , Péptidos/química , Fenilalanina/químicaRESUMEN
Here we report a new series of hydrolytically stable chemotype heteroaromatic azoline thioethers (HATs) to achieve highly selective, rapid, and efficient covalent labeling of cysteine under physiological conditions. Although the resulting cysteine-azoline conjugate is stable, we highlight traceless decoupling of the conjugate to afford unmodified starting components in response to reducing conditions. We demonstrated that HAT probes reverse the reactivity of nucleophilic cysteine to electrophilic dehydroalanine (Dha) under mild basic conditions. We demonstrated the umpolung capability of HAT probes for the modification of cysteine on peptides and proteins with various nucleophiles. We demonstrated that HAT probes increase the mass sensitivity of the modified peptides and proteins by 100 fold as compared to the classical methods. Finally, we extended the application of HAT probes for specific modification of cysteines in a complex cell lysate mixture.
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Proteome profiling by activated esters identified >9000 ligandable lysines but they are limited as covalent inhibitors due to poor hydrolytic stability. Here we report our efforts to design and discover a new series of tunable amine-reactive electrophiles (TAREs) for selective and robust labeling of lysine. The major challenges in developing selective probes for lysine are the high nucleophilicity of cysteines and poor hydrolytic stability. Our work circumvents these challenges by a unique design of the TAREs that form stable adducts with lysine and on reaction with cysteine generate another reactive electrophiles for lysine. We highlight that TAREs exhibit substantially high hydrolytic stability as compared to the activated esters and are non-cytotoxic thus have the potential to act as covalent ligands. We applied these alternative TAREs for the intracellular labeling of proteins in different cell lines, and for the selective identification of lysines in the human proteome on a global scale.
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LisinaRESUMEN
Cyclic tetrapeptides exhibit high cellular permeability and a wide range of biological properties and thus have gained great interest in the field of medicinal chemistry. We synthesized highly strained 12-membered head to tail cyclic peptides with varying reactive amino acids, without oligomerization using the exclusively intramolecular CyClick chemistry. This occurs by a two-step process involving the low-energy formation of a 15 atom-containing cyclic imine, followed by a chemoselective ring contraction of the peptide backbone generating a highly strained 12 atom-containing cyclic tetrapeptide. This reaction exhibited high substrate scope and generated head to tail cyclic tetrapeptides with varying amino acids at the N-terminus, showing chemoselectivity without the need for side group protection.