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Aldehyde decarbonylation is a vital chemical transformation in the synthesis of natural products. Nature accomplishes this process through a family of decarbonylase enzymes, while in the laboratory, harsh transition metals and elevated temperatures are required. Herein, we report a mild aldehyde decarbonylation reaction that exhibits exclusive selectivity for ortho -aldehydes during a tandem nitrile boronic acid cross-coupling reaction. A wide substrate scope is displayed that includes regioselective removal of the ortho -aldehyde from phenyl boronic acids in the presence of meta - or para -aldehydes. A mechanistic investigation of the observed regioselectivity for ortho -aldehydes by density functional theory (DFT) calculations shows that the CO ligand extrusion is energetically more favorable for the ortho position as compared to the para position. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. Methionine plays an essential role in various biological and cell regulatory processes, making its chemoproteomic profiling necessary to understand its function. Here, the authors present Copper(I)-Nitrene Platform (CuNiP) for robust and selective labelling of methionine to generate highly stable sulfonyl sulfimide conjugates under physiological conditions. [ABSTRACT FROM AUTHOR]

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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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. Homogeneous engineering of proteins is promising to study biological functions and for the development of therapeutic conjugates. Here, the authors report a one-pot multicomponent Furan-Thiol-Amine reaction for diverse applications, including labelling of peptides, synthesis of macrocyclic and stapled peptides, selective modification of different proteins with varying payloads, and labelling of lysine and cysteine in a complex human proteome. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The capsid core of HIV-1 is a large macromolecular assembly that surrounds the viral genome and is an essential component of the infectious virus. In addition to its multiple roles throughout the viral life cycle, the capsid interacts with multiple host factors. Owing to its indispensable nature, the HIV-1 capsid has been the target of numerous antiretrovirals, though most capsid-targeting molecules have not had clinical success until recently. Lenacapavir, a long-acting drug that targets the HIV-1 capsid, is currently undergoing phase 2/3 clinical trials, making it the most successful capsid inhibitor to-date. In this review, we detail the role of the HIV-1 capsid protein in the virus life cycle, categorize antiviral compounds based on their targeting of five sites within the HIV-1 capsid, and discuss their molecular interactions and mechanisms of action. The diverse range of inhibition mechanisms provides insight into possible new strategies for designing novel HIV-1 drugs and furthers our understanding of HIV-1 biology. [ABSTRACT FROM AUTHOR]

Lysine monomethylation (Kme) is an impactful post‐translational modification (PTM) responsible for regulating biological processes and implicated in diseases, thus there is great interest in identifying these methylation marks globally. However, the progress in this area has been challenging because the addition of a small methyl group on lysine leads to negligible change in the bulk, charge, and hydrophobicity. Herein, we report an empowering chemical technology selective triazenation reaction, which we term "STaR", of secondary amines using arene diazonium salts to achieve highly selective, rapid, and robust tagging of Kme peptides from a complex mixture under biocompatible conditions. Although the resulting triazene‐linkage with Kme is stable, we highlight the efficient decoupling of the triazene‐conjugate to afford unmodified starting components under mild conditions when desired. Our work establishes a unique chemoselective, traceless bioconjugation strategy for the selective enrichment of Kme PTMs. [ABSTRACT FROM AUTHOR]

Lysine monomethylation (Kme) is an impactful post‐translational modification (PTM) responsible for regulating biological processes and implicated in diseases, thus there is great interest in identifying these methylation marks globally. However, the progress in this area has been challenging because the addition of a small methyl group on lysine leads to negligible change in the bulk, charge, and hydrophobicity. Herein, we report an empowering chemical technology selective triazenation reaction, which we term "STaR", of secondary amines using arene diazonium salts to achieve highly selective, rapid, and robust tagging of Kme peptides from a complex mixture under biocompatible conditions. Although the resulting triazene‐linkage with Kme is stable, we highlight the efficient decoupling of the triazene‐conjugate to afford unmodified starting components under mild conditions when desired. Our work establishes a unique chemoselective, traceless bioconjugation strategy for the selective enrichment of Kme PTMs. [ABSTRACT FROM AUTHOR]

The chemical modification of proteins in a site‐selective manner leads to many advances in various scientific fields. The major challenges with conventional N‐terminal bioconjugation techniques are the lack of universal sequence compatibility and poor mass‐detection sensitivity of the resulting bioconjugates. This approach efficiently analyzes proteolytic fragments and native proteins in a complex mixture. Multiple chemical steps are usually required for the site‐selective synthesis of bioconjugates with enhanced mass‐detection sensitivity. We present a single‐step, versatile strategy for the selective modification of protein N‐termini with mass boosters. The chemical tag enhances the peptide detection by multiple orders thus leading to the unambiguous analysis of the resulting bioconjugates. We demonstrate that tagging proteolytic fragments with mass sensitivity probes in a complex mixture improves the detection of resulting bioconjugates. [ABSTRACT FROM AUTHOR]

The chemical modification of proteins in a site‐selective manner leads to many advances in various scientific fields. The major challenges with conventional N‐terminal bioconjugation techniques are the lack of universal sequence compatibility and poor mass‐detection sensitivity of the resulting bioconjugates. This approach efficiently analyzes proteolytic fragments and native proteins in a complex mixture. Multiple chemical steps are usually required for the site‐selective synthesis of bioconjugates with enhanced mass‐detection sensitivity. We present a single‐step, versatile strategy for the selective modification of protein N‐termini with mass boosters. The chemical tag enhances the peptide detection by multiple orders thus leading to the unambiguous analysis of the resulting bioconjugates. We demonstrate that tagging proteolytic fragments with mass sensitivity probes in a complex mixture improves the detection of resulting bioconjugates. [ABSTRACT FROM AUTHOR]

The emergence of cyclic peptides as pharmaceuticals has led to an eruption of new methodologies for macrocyclization. However, the cyclization of peptides at high concentrations presents a challenge due to the production of side products like dimers and oligomers. This factor is more pronounced with the cyclization of peptides composed of fewer than seven amino acids, thus has created a need for a new synthetic strategy. Herein, we will elucidate a new chemoselective method termed 'CyClick' that works in an exclusively intramolecular fashion preventing the formation of commonly occurring side products such as dimers and oligomers, even at relatively high concentration. 1 Introduction 2 Known Methodologies 3 Novel CyClick Chemistry 4 Conclusion and Outlook [ABSTRACT FROM AUTHOR]

Nitroalkanes react specifically with aldehydes, providing rapid, stable, and chemoselective protein bioconjugation. These nitroalkylated proteins mimic key post‐translational modifications (PTMs) of proteins and can be used to understand the role of these PTMs in cellular processes. Demonstrated here is the substrate scope of this bioconjugation by attaching a variety of tags, such as NMR tags, fluorescent tags, affinity tags, and alkyne tags, to proteins. The structure and enzymatic activity of modified proteins remain conserved after labeling. Notably, the nitroalkane group leads to easy characterization of proteins by mass spectrometry because of its distinct fingerprint pattern. Importantly, the nitro‐alkylated peptides provide a new handle for site‐selective fluorination of peptides, thus installing a specific probe to study peptide–protein interactions by 19F NMR spectroscopy. Furthermore, nitroalkane reagents can be used for the late‐stage diversification of peptides and for the synthesis of peptide staples. [ABSTRACT FROM AUTHOR]

Nitroalkanes react specifically with aldehydes, providing rapid, stable, and chemoselective protein bioconjugation. These nitroalkylated proteins mimic key post‐translational modifications (PTMs) of proteins and can be used to understand the role of these PTMs in cellular processes. Demonstrated here is the substrate scope of this bioconjugation by attaching a variety of tags, such as NMR tags, fluorescent tags, affinity tags, and alkyne tags, to proteins. The structure and enzymatic activity of modified proteins remain conserved after labeling. Notably, the nitroalkane group leads to easy characterization of proteins by mass spectrometry because of its distinct fingerprint pattern. Importantly, the nitro‐alkylated peptides provide a new handle for site‐selective fluorination of peptides, thus installing a specific probe to study peptide–protein interactions by 19F NMR spectroscopy. Furthermore, nitroalkane reagents can be used for the late‐stage diversification of peptides and for the synthesis of peptide staples. [ABSTRACT FROM AUTHOR]

Here, we report a novel "CyClick" strategy for the macrocyclization of peptides that works in an exclusively intramolecular fashion thereby precluding the formation of dimers and oligomers via intermolecular reactions. The CyClick chemistry is highly chemoselective for the N‐terminus of the peptide with a C‐terminal aldehyde. In this protocol, the peptide conformation internally directs activation of the backbone amide bond and thereby facilitates formation of a stable 4‐imidazolidinone‐fused cyclic peptide with high diastereoselectivity (>99 %). This method is tolerant to a variety of peptide aldehydes and has been applied for the synthesis of 12‐ to 23‐membered rings with varying amino acid compositions in one pot under mild reaction conditions. The reaction generated peptide macrocycles featuring a 4‐imidazolidinone in their scaffolds, which acts as an endocyclic control element that promotes intramolecular hydrogen bonding and leads to macrocycles with conformationally rigid turn structures. [ABSTRACT FROM AUTHOR]

Here, we report a novel "CyClick" strategy for the macrocyclization of peptides that works in an exclusively intramolecular fashion thereby precluding the formation of dimers and oligomers via intermolecular reactions. The CyClick chemistry is highly chemoselective for the N‐terminus of the peptide with a C‐terminal aldehyde. In this protocol, the peptide conformation internally directs activation of the backbone amide bond and thereby facilitates formation of a stable 4‐imidazolidinone‐fused cyclic peptide with high diastereoselectivity (>99 %). This method is tolerant to a variety of peptide aldehydes and has been applied for the synthesis of 12‐ to 23‐membered rings with varying amino acid compositions in one pot under mild reaction conditions. The reaction generated peptide macrocycles featuring a 4‐imidazolidinone in their scaffolds, which acts as an endocyclic control element that promotes intramolecular hydrogen bonding and leads to macrocycles with conformationally rigid turn structures. [ABSTRACT FROM AUTHOR]

Abstract: A broadly applicable chemical cleavage methodology to facilitate MS/MS sequencing was developed for macrocyclic and lasso peptides, which hold promise as exciting new therapeutics. Existing methods such as Edman degradation, CNBr cleavage, and enzymatic digestion are either limited in scope or completely fail in cleavage of constrained nonribosomal peptides. Importantly, the new method was utilized for synthesizing a unique peptide‐based rotaxane (both cyclic and threaded) from the lasso peptide, benenodin‐1 ΔC5. [ABSTRACT FROM AUTHOR]

Abstract: A broadly applicable chemical cleavage methodology to facilitate MS/MS sequencing was developed for macrocyclic and lasso peptides, which hold promise as exciting new therapeutics. Existing methods such as Edman degradation, CNBr cleavage, and enzymatic digestion are either limited in scope or completely fail in cleavage of constrained nonribosomal peptides. Importantly, the new method was utilized for synthesizing a unique peptide‐based rotaxane (both cyclic and threaded) from the lasso peptide, benenodin‐1 ΔC5. [ABSTRACT FROM AUTHOR]

We describe a lasso peptide, albusnodin, that is post-translationally modified with an acetyl group, the first example of a lasso peptide with this modification. Using heterologous expression, we further show that the acetyltransferase colocalized with the albusnodin gene cluster is required for the biosynthesis of this lasso peptide. This type of lasso peptide is widespread in Actinobacteria with 44 examples found in currently sequenced genomes. [ABSTRACT FROM AUTHOR]

C-terminally modified peptides are of high significance due to the therapeutic properties that accompany various C-terminal functional groups and the ability to manipulate them for further applications. Thus, there is a great necessity for an effective solid phase technique for the synthesis of C-terminally modified peptides. Here, we report a universal solid phase strategy for the synthesis of various C-terminal modified peptides which is independent of the type of resins, linkers, and unnatural moieties typically needed for C-terminal modifications. The technique proceeds by the modification of C-terminal serine to a cyclic urethane moiety which results in the activation of the backbone amide chain for nucleophilic displacement by various nucleophiles to generate C-terminally modified acids, esters, N-aryl amides, and alcohols. This cyclic urethane technique (CUT) also provides a general strategy for synthesis of C-terminal protected peptides that can be used for convergent synthesis of large peptides. The C-terminal protecting groups are cleaved by facile hydrolysis to release the free peptide. [ABSTRACT FROM AUTHOR]

Site-selective cleavage of extremely unreactive peptide bonds is a very important chemical modification that provides invaluable information regarding protein sequence, and it acts as a modulator of protein structure and function for therapeutic applications. For controlled and selective cleavage, a daunting task, chemical reagents must selectively recognize or bind to one or more amino acid residues in the peptide chain and selectively cleave a peptide bond. Building on this principle, we have developed an approach that utilizes a chemical reagent to selectively modify the serine residue in a peptide chain and leads to the cleavage of a peptide backbone at the N-terminus of the serine residue. After cleavage, modified residues can be converted back to the original fragments. This method exhibits broad substrate scope and selectively cleaves various bioactive peptides with post-translational modifications (e.g. N-acetylation and -methylation) and mutations (d- and β-amino acids), which are a known cause of age related diseases. [ABSTRACT FROM AUTHOR]

Organocatalysts have emerged as a third major way of catalyzing a wide variety of reactions, besides metal catalysts and biocatalysts. They have gained tremendous importance because of their green chemistry perspective. The criteria for green chemistry would be largely fulfilled if the major component of the reaction mixture, i.e. the solvent, is water which is a suitable solvent in various biosynthetic reactions. In this feature article we have described reactions promoted by organocatalysts in a large excess of water, without any organic solvent or excess of any reactant. We have also explained the structural features required for organocatalysts to work well in aqueous media. [ABSTRACT FROM AUTHOR]

Organocatalysts have emerged as a third major way of catalyzing a wide variety of reactions, besides metal catalysts and biocatalysts. They have gained tremendous importance because of their green chemistry perspective. The criteria for green chemistry would be largely fulfilled if the major component of the reaction mixture, i.e.the solvent, is water which is a suitable solvent in various biosynthetic reactions. In this feature articlewe have described reactions promoted by organocatalysts in a large excess of water, without any organic solvent or excess of any reactant. We have also explained the structural features required for organocatalysts to work well in aqueous media. [ABSTRACT FROM AUTHOR]

Amide bonds are the most prevalent structures found in organic molecules and various biomolecules such as peptides, proteins, DNA, and RNA. The unique feature of amide bonds is their ability to form resonating structures, thus, they are highly stable and adopt particular three-dimensional structures, which, in turn, are responsible for their functions. The main focus of this review article is to report the methodologies for the activation of the unactivated amide bonds present in biomolecules, which includes the enzymatic approach, metal complexes, and non-metal based methods. This article also discusses some of the applications of amide bond activation approaches in the sequencing of proteins and the synthesis of peptide acids, esters, amides, and thioesters. [ABSTRACT FROM AUTHOR]