Reaction Of Amino Acids Research Articles

Top-down Proteomics for the Characterization and Quantification of Calreticulin Arginylation.

Arginylation installed by arginyltransferase 1 (ATE1) features an addition of arginine (Arg) to the reactive amino acids (e.g., Glu and Asp) at the protein N-terminus or side chain. Systemic removal of arginylation after ATE1 knockout (KO) in mouse models resulted in heart defects leading to embryonic lethality. The biological importance of arginylation has motivated the discovery of arginylation sites on proteins using bottom-up approaches. While bottom-up proteomics is powerful in localizing peptide arginylation, it lacks the ability to quantify proteoforms at the protein level. Here we developed a top-down proteomics workflow for characterizing and quantifying calreticulin (CALR) arginylation. To generate fully arginylated CALR (R-CALR), we have inserted an R residue after the signaling peptide (AA1-17). Upon overexpression in ATE1 KO cells, CALR and R-CALR were purified by affinity purification and analyzed by LCMS in positive mode. Both proteoforms showed charge states ranging from 27-68 with charge 58 as the most intense charge state. Their MS2 spectra from electron-activated dissociation (EAD) showed preferential fragmentation at the protein N-terminals which yielded sufficient c ions facilitating precise localization of the arginylation sites. The calcium-binding domain (CBD) gave minimum characteristic ions possibly due to the abundant presence of >100 D and E residues. Ultraviolet photodissociation (UVPD) compared with EAD and ETD significantly improved the sequence coverage of CBD. This method can identify and quantify CALR arginylation at absence, endogenous (low), and high levels. To our knowledge, our work is the first application of top-down proteomics in characterizing post-translational arginylation in vitro and in vivo.

Cysteine S-acetylation is a post-translational modification involved in metabolic regulation.

Cysteine is a reactive amino acid central to the catalytic activities of many enzymes. It is also a common target of post-translational modifications (PTMs), such as palmitoylation. This longchain acyl PTM can modify cysteine residues and induce changes in protein subcellular localization. We hypothesized that cysteine could also be modified by short-chain acyl groups, such as cysteine S-acetylation. To test this, we developed sample preparation and non-targeted mass spectrometry protocols to analyze the mouse liver proteome for cysteine acetylation. Our findings revealed hundreds of sites of cysteine acetylation across multiple tissue types, revealing a previously uncharacterized cysteine acetylome. Cysteine acetylation shows a marked cytoplasmic subcellular localization signature, with tissue-specific acetylome patterns and specific changes upon metabolic stress. This study uncovers a novel aspect of cysteine biochemistry, highlighting short-chain modifications alongside known long-chain acyl PTMs. These findings enrich our understanding of the landscape of acyl modifications and suggest new research directions in enzyme activity regulation and cellular signaling in metabolism.

Abstract 3315: Preclinical studies of TSN1611, a potent, selective, and orally bioavailable KRASG12D inhibitor

Abstract Background: KRAS mutations are the most frequently encountered driver oncogene, involved in ~25% of all human cancers [1,2]. KRASG12D is the predominant KRAS mutation isoform, detected in approximately 35% of pancreatic cancer, 13% of colorectal cancer, and 5% of NSCLC [3]. Compared to KRASG12C, targeting KRASG12D has proven to be more challenging since the target protein lacks a reactive amino acid residue for irreversible inhibitory modification by a ligand. Herein, we disclose TSN1611, a potent and selective KRASG12D inhibitor, which possesses favorable oral PK profiles and demonstrates significant in vitro and in vivo anti-tumor activity in various KRASG12D-mutant models. Method: Biochemical HTRF assay was used to measure the inhibition of TSN1611 to both GDP-bound and GTP-bound state of KRASG12D. Biophysical SPR method was used to directly measure the binding of TSN1611 to GDP-bound KRASG12D and KRASWT. Cell based activities were evaluated in a series of in vitro cell proliferation assay utilizing Ba/F3 cells engineered with KRASG12D or non-KRASG12D mutations and tumor cell lines harboring KRASG12D mutation. Human cancer cell-derived xenograft models of HPAC (pancreatic) and GP2D (colorectal) were used to evaluate its in vivo antitumor effect. in vitro and in vivo PK studies were performed in mouse, rat, and dog. Systematic nonclinical safety evaluations, including safety panel screen testing, safety pharmacology studies, and repeat-dose toxicity studies were carried out to assess its preliminary toxicity profile. Results: TSN1611 inhibited both active (GTP-bound) and inactive (GDP-bound) forms of KRASG12D protein at IC50 1.23 and 1.49 nM, respectively; the KD value of its direct binding to KRASG12D protein is 1.93 pM in SPR assay. TSN1611 demonstrated potent anti-proliferation activity against several tumor cell lines harboring KRASG12D mutation, and excellent selectivity over cells of NRAS, HRAS, and other KRAS isoforms. It also showed dose-dependent anti-tumor efficacy in GP2D and HPAC models. Mechanism of action studies concluded that the antitumor effect of TSN1611 is resulted from its effective inhibition of KRAS signaling pathway. Oral bioavailability and safety profile across multiple species supported its further development. Conclusion: TSN1611 is a selective KRASG12D inhibitor. It exhibited excellent selectivity and activity both in vitro and in vivo; it demonstrated favorable physicochemical properties, oral PK profiles, and brain penetration potential; it also showed acceptable margins of safety. The preclinical data supports further development. Pending regulatory submission and review, the phase I/II study is planned to start in H1 of 2024.

Superoxide versus peroxide activation of dye decolorizing peroxidases for bioelectrocatalysis

Here we show that dye decolorizing peroxidases can be electrochemically activated for substrate oxidation without addition of exogenous H2O2, yielding activities similar or better than in their conventional operation. The method is based on the in situ generation of O2•−, •OH and H2O2. These species bind the heme to form catalytically competent oxoiron intermediates. Activation of the Pseudomonas putida enzyme is mainly mediated by O2•−, while those from Thermobifida fusca and Saccharomonospora viridis show preferential activation by H2O2. This differential reactivity is explained by the dynamics of a conserved distal aspartate residue. In contrast to previous reports on the Bacillus subtilis enzyme, none of the peroxidases studied here show significant •OH mediated activation, as this radical is quenched by reactive amino acids abundant in these proteins. These results pave the way for the development of bioelectrocatalytic reactors based on dye decolorizing peroxidases for water remediation and biomass processing.

Site-Selective Polyfluoroaryl Modification and Unsymmetric Stapling of Unprotected Peptides.

Peptide stapling is recognized as an effective strategy for improving the proteolytic stability and cell permeability of peptides. In this study, we present a novel approach for the site-selective unsymmetric perfluoroaryl stapling of Ser and Cys residues in unprotected peptides. The stapling reaction proceeds smoothly under very mild conditions, exhibiting a remarkably rapid reaction rate. It can furnish stapled products in both liquid and solid phases, and the presence of nucleophilic groups other than Cys thiol within the peptide does not impede the reaction, resulting in uniformly high yields. Importantly, the chemoselective activation of Ser β-C(sp3)-H enables the unreacted -OH to serve as a reactive handle for subsequent divergent modification of the staple moiety with various therapeutic functionalities, including a clickable azido group, a polar moiety, a lipid tag, and a fluorescent dye. In our study, we have also developed a visible-light-induced chemoselective C(sp3)-H polyfluoroarylation of the Ser β-position. This reaction avoids interference with the competitive reaction of Ser -OH, enabling the precise late-stage polyfluoroarylative modification of Ser residues in various unprotected peptides containing other highly reactive amino acid residues. The biological assay suggested that our peptide stapling strategy would potentially enhance the proteolytic stability and cellular permeability of peptides.

Hexafluoroisopropanol as a Bioconjugation Medium of Ultrafast, Tryptophan-Selective Catalysis.

The past decade has seen a remarkable growth in the number of bioconjugation techniques in chemistry, biology, material science, and biomedical fields. A core design element in bioconjugation technology is a chemical reaction that can form a covalent bond between the protein of interest and the labeling reagent. Achieving chemoselective protein bioconjugation in aqueous media is challenging, especially for generally less reactive amino acid residues, such as tryptophan. We present here the development of tryptophan-selective bioconjugation methods through ultrafast Lewis acid-catalyzed reactions in hexafluoroisopropanol (HFIP). Structure-reactivity relationship studies have revealed a combination of thiophene and ethanol moieties to give a suitable labeling reagent for this bioconjugation process, which enables modification of peptides and proteins in an extremely rapid reaction unencumbered by noticeable side reactions. The capability of the labeling method also facilitated radiofluorination application as well as antibody functionalization. Enhancement of an α-helix by HFIP leads to its compatibility with a certain protein, and this report also demonstrates a further stabilization strategy achieved by the addition of an ionic liquid to the HFIP medium. The nonaqueous bioconjugation approaches allow access to numerous chemical reactions that are unavailable in traditional aqueous processes and will further advance the chemistry of proteins.

Peptide-Based Covalent Inhibitors Bearing Mild Electrophiles to Target a Conserved His Residue of the Bacterial Sliding Clamp.

Peptide-based covalent inhibitors targeted to nucleophilic protein residues have recently emerged as new modalities to target protein-protein interactions (PPIs) as they may provide some benefits over more classic competitive inhibitors. Covalent inhibitors are generally targeted to cysteine, the most intrinsically reactive amino acid residue, and to lysine, which is more abundant at the surface of proteins but much less frequently to histidine. Herein, we report the structure-guided design of targeted covalent inhibitors (TCIs) able to bind covalently and selectively to the bacterial sliding clamp (SC), by reacting with a well-conserved histidine residue located on the edge of the peptide-binding pocket. SC is an essential component of the bacterial DNA replication machinery, identified as a promising target for the development of new antibacterial compounds. Thermodynamic and kinetic analyses of ligands bearing different mild electrophilic warheads confirmed the higher efficiency of the chloroacetamide compared to Michael acceptors. Two high-resolution X-ray structures of covalent inhibitor-SC adducts were obtained, revealing the canonical orientation of the ligand and details of covalent bond formation with histidine. Proteomic studies were consistent with a selective SC engagement by the chloroacetamide-based TCI. Finally, the TCI of SC was substantially more active than the parent noncovalent inhibitor in an in vitro SC-dependent DNA synthesis assay, validating the potential of the approach to design covalent inhibitors of protein-protein interactions targeted to histidine.

Unusual cysteine modifications in natural product biosynthesis.

l-Cysteine is a highly reactive amino acid that is modified into a variety of chemical structures, including cysteine sulfinic acid in human metabolic pathways, and sulfur-containing scaffolds of amino acids, alkaloids, and peptides in natural product biosynthesis. Among the modification enzymes responsible for these cysteine-derived compounds, metalloenzymes constitute an important family of enzymes that catalyze a wide variety of reactions. Therefore, understanding their reaction mechanisms is important for the biosynthetic production of cysteine-derived natural products. This review mainly summarizes recent mechanistic investigations of metalloenzymes, with a particular focus on recently discovered mononuclear non-heme iron (NHI) enzymes, dinuclear NHI enzymes, and radical-SAM enzymes involved in unusual cysteine modifications in natural product biosynthesis.

Protein painting for structural and binding site analysis via intracellular lysine reactivity profiling with o-phthalaldehyde.

The three-dimensional structure and the molecular interaction of proteins determine their roles in many cellular processes. Chemical protein painting with protein mass spectrometry can identify changes in structural conformations and molecular interactions of proteins including their binding sites. Nevertheless, most current protein painting techniques identify protein targets and binding sites of drugs in vitro using a cell lysate or purified protein. Here, we tested 11 membrane-permeable lysine-reactive chemical probes for intracellular covalent labeling of endogenous proteins, which reveals ortho-phthalaldehyde (OPA) as the most reactive probe in the intracellular environment. An MS workflow and a new data analysis strategy termed RAPID (Reactive Amino acid Profiling by Inverse Detection) was developed to enhance detection sensitivity. RAPID with OPA successfully identified structural changes induced by the allosteric drug TEPP-46 on its target protein PKM2 and was applied to profile the conformation change of the proteome occurring in cells during thermal denaturation. The application of RAPID-OPA on cells treated with geldanamycin, selumetinib, and staurosporine successfully revealed their binding sites on target proteins. Thus, RAPID-OPA for cellular protein painting enables the identification of ligand-binding sites and detection of protein structural changes occurring in cells.

The Molecular Frequency, Conservation and Role of Reactive Cysteines in Plant Lipid Metabolism.

Cysteines (Cys) are chemically reactive amino acids containing sulfur that play diverse roles in plant biology. Recent proteomics investigations in Arabidopsis thaliana have revealed the presence of thiol post-translational modifications (PTMs) in several Cys residues. These PTMs are presumed to impact protein structure and function, yet mechanistic data regarding the specific Cys susceptible to modification and their biochemical relevance remain limited. To help address these limitations, we have conducted a wide-ranging analysis by integrating published datasets encompassing PTM proteomics (comparing S-sulfenylation, persulfidation, S-nitrosylation and S-acylation), genomics and protein structures, with a specific focus on proteins involved in plant lipid metabolism. The prevalence and distribution of modified Cys residues across all analyzed proteins is diverse and multifaceted. Nevertheless, by combining an evaluation of sequence conservation across 100+ plant genomes with AlphaFold-generated protein structures and physicochemical predictions, we have unveiled structural propensities associated with Cys modifications. Furthermore, we have identified discernible patterns in lipid biochemical pathways enriched with Cys PTMs, notably involving beta-oxidation, jasmonic acid biosynthesis, fatty acid biosynthesis and wax biosynthesis. These collective findings provide valuable insights for future investigations targeting the mechanistic foundations of Cys modifications and the regulation of modified proteins in lipid metabolism and other metabolic pathways.

Two-Step Covalent Docking with Attracting Cavities.

Due to their various advantages, interest in the development of covalent drugs has been renewed in the past few years. It is therefore important to accurately describe and predict their interactions with biological targets by computer-aided drug design tools such as docking algorithms. Here, we report a covalent docking procedure for our in-house docking code Attracting Cavities (AC), which mimics the two-step mechanism of covalent ligand binding. Ligand binding to the protein cavity is driven by nonbonded interactions, followed by the formation of a covalent bond between the ligand and the protein through a chemical reaction. To test the performance of this method, we developed a diverse, high-quality, openly accessible re-docking benchmark set of 95 covalent complexes bound by 8 chemical reactions to 5 different reactive amino acids. Combination with structures from previous studies resulted in a set of 304 complexes, on which AC obtained a success rate (rmsd ≤ 2 Å) of 78%, outperforming two state-of-the-art covalent docking codes, genetic optimization for ligand docking (GOLD (66%)) and AutoDock (AD (35%)). Using a more stringent success criterion (rmsd ≤ 1.5 Å), AC reached a success rate of 71 vs 55% for GOLD and 26% for AD. We additionally assessed the cross-docking performance of AC on a set of 76 covalent complexes of the SARS-CoV-2 main protease. On this challenging test set of mainly small and highly solvent-exposed ligands, AC yielded success rates of 58 and 28% for re-docking and cross-docking, respectively, compared to 45 and 17% for GOLD.

Antibody fragments functionalized with non-canonical amino acids preserving structure and functionality - A door opener for new biological and therapeutic applications

Functionalization of proteins by incorporating reactive non-canonical amino acids (ncAAs) has been widely applied for numerous biological and therapeutic applications. The requirement not to lose the intrinsic properties of these proteins is often underestimated and not considered. Main purpose of this study was to answer the question whether functionalization via residue-specific incorporation of the ncAA N6-[(2-Azidoethoxy) carbonyl]-l-lysine (Azk) influences the properties of the anti-tumor-necrosis-factor-α-Fab (FTN2). Therefore, FTN2Azk variants with different Azk incorporation sites were designed and amber codon suppression was used for production. The functionalized FTN2Azk variants were efficiently produced in fed-batch like μ-bioreactor cultivations in the periplasm of E. coli displaying correct structure and antigen binding affinities comparable to those of wild-type FTN2. Our FTN2Azk variants with reactive handles for diverse conjugates enable tracking of recombinant protein in the production cell, pharmacological studies and translation into new pharmaceutical applications.

Tyrosinase inhibitory peptides: Structure-activity relationship study on peptide chemical properties, terminal preferences and intracellular regulation of melanogenesis signaling pathways

BackgroundBioactive peptides have gained attention as potential alternatives to chemical-based skin lightening agents. Based on literature search, the reported articles focused mainly on the sources and preparation methods of tyrosinase inhibitory peptides and there is lacking information regarding the structure-activity relationship (SAR) between peptide property and tyrosinase inhibition. It was hypothesized that peptide properties such as hydrophobicity/hydrophilicity and the amino acid type and position/arrangement at the terminal positions could affect peptide mode of binding hence result in various degrees of tyrosinase inhibition. MethodsIn this study, the sequences of 128 tyrosinase inhibitory peptides were collected from peer-reviewed articles. The hydrophobicity/hydrophilicity property and the amino acid profile of peptides at the N- and C-terminals were analyzed using bioinformatics tools. Molecular docking analysis was employed to further elucidate the roles of reactive amino acids in tyrosinase-peptide binding interactions. The peptide-regulated intracellular melanogenesis pathways were also compiled and discussed. ResultsIt was found that hydrophobic and/or polar neutral properties may facilitate or stabilize peptide binding with tyrosinase. Moreover, short peptides featuring a cysteine and tyrosine at the N- and C- terminal ultimate positions tend to bind to the active site of tyrosinase whereas positively charged amino acid such as arginine at the N-terminal does not favor peptide binding to tyrosinase. ConclusionsThese findings provide detailed explanation on how peptide/amino acid structures are related and what function they play in tyrosinase inhibition. It could also inspire researchers to account for tyrosinase-peptide SAR and the underlying anti-melanogenesis mechanisms in formulating peptide-based treatments or strategies against skin hyperpigmentation.

Between eustress and distress: UVB induced changes in carotenoid accumulation in halophytic Salicornia europaea

Halophytes are potential future crops with a valuable nutritional profile. Produced in indoor farming, they are considered to contribute to sustainable and resilient food systems. Indoor farms operate using artificial light. In this context narrowband and low dose UVB radiation can be used to increase plant secondary metabolites, such as carotenoids, and provide an improved nutritional profile for a human diet. UVB radiation can cause eustress or distress in the plant depending on the lighting situation. The aim of this study was to identify the doses of UVB that lead to either eustress or distress and to analyze these responses in Salicornia europaea. Therefore, S. europaea plants were exposed to different UVB radiation levels, low, medium and high, and analyzed for reactive oxygen species (ROS), plant hormones, amino acids, and photosynthetic pigments. High UVB treatment was found to affect phenotype and growth, and the metabolite profile was affected in a UVB dose-dependent manner. Specifically, medium UVB radiation resulted in an increase in carotenoids, whereas high UVB resulted in a decrease. We also observed an altered oxidative stress status and increased SA and decreased ABA contents in response to UVB treatment. This was supported by the results of menadione treatment that induces oxidative stress in plants, which also indicated an altered oxidative stress status in combination with altered carotenoid content. Thus, we show that a moderate dose of UVB can increase the carotenoid content of S. europaea. Furthermore, the UVB stress-dependent response led to a better understanding of carotenoid accumulation upon UVB exposure, which can be used to improve lighting systems and in turn the nutritional profile of future crops in indoor farming.

Physalis Mottle Virus-Like Nanocarriers with Expanded Internal Loading Capacity.

An ongoing challenge in precision medicine is the efficient delivery of therapeutics to tissues/organs of interest. Nanoparticle delivery systems have the potential to overcome traditional limitations of drug and gene delivery through improved pharmacokinetics, tissue targeting, and stability of encapsulated cargo. Physalis mottle virus (PhMV)-like nanoparticles are a promising nanocarrier platform which can be chemically targeted on the exterior and interior surfaces through reactive amino acids. Cargo-loading to the internal cavity is achieved with thiol-reactive small molecules. However, the internal loading capacity of these nanoparticles is limited by the presence of a single reactive cysteine (C75) per coat protein with low inherent reactivity. Here, we use structure-based design to engineer cysteine-added mutants of PhMV VLPs that display increased reactivity toward thiol-reactive small molecules. Specifically, the A31C and S137C mutants show a greater than 10-fold increased rate of reactivity towards thiol-reactive small molecules, and PhMV Cys1 (A31C), PhMV Cys2 (S137C), and PhMV Cys1+2 (double mutant) VLPs display up to three-fold increased internal loading of the small molecule chemotherapeutics aldoxorubicin and vcMMAE and up to four-fold increased internal loading of the MRI imaging reagent DOTA(Gd). These results further improve upon a promising plant virus-based nanocarrier system for use in targeted delivery of small-molecule drugs and imaging reagents in vivo.

BODIPY Based OFF-ON Fluorescent Probe for Endogenous Carbon Monoxide Imaging in Living Cells.

Carbon monoxide (CO) is one of the signaling molecules that are ubiquitous in humans, which involves in the regulation of human physiology and pathology. In this work, the probe PEC was designed and synthesized based on BODIPY fluorophore that can selectively detect CO through reducing the nitro group to amino group, resulting in a "turn-on" fluorescence response with a simultaneous increase in the concentration of CO. The response is selective over a variety of relevant reactive free radicals, ions, and amino acid species. PEC has the advantages of good stability, good water solubility, and obvious changes in fluorescence signals. In addition, PEC can be used to detect and track endogenous CO in living cells.

Impact of water content and bloom index on gelatin glycation

The increasing number of people around the world suffering from chewing disorders necessitates the design of foods adapted to their needs. Glycation could be considered as one of the possible texturing methods. This chemical reaction alters the texture of food products by changing their macromolecular structure. However, there is no consensus on its effects on food texture. In this study, gelatin is used as a model medium to study the effect of glycation on its texture by considering two different Bloom indices (gel strength) and two different initial water contents.Our results suggest that the initial water content of gelatin, as well as its Bloom index, have an impact on the glycation reaction and on the texture of gelatin gels. The effect of these two factors influences the binding of d-ribose to reactive amino acids, in particular due to a variable amount of gelatin triple helix aggregates.This study shows the importance of controlling the water content and the Bloom index of gelatin to regulate the glycation reaction when used for food texturing.

A supramolecular metalloenzyme possessing robust oxidase-mimetic catalytic function

Enzymes fold into unique three-dimensional structures to distribute their reactive amino acid residues, but environmental changes can disrupt their essential folding and lead to irreversible activity loss. The de novo synthesis of enzyme-like active sites is challenging due to the difficulty of replicating the spatial arrangement of functional groups. Here, we present a supramolecular mimetic enzyme formed by self-assembling nucleotides with fluorenylmethyloxycarbonyl (Fmoc)-modified amino acids and copper. This catalyst exhibits catalytic functions akin those of copper cluster-dependent oxidases, and catalytic performance surpasses to date-reported artificial complexes. Our experimental and theoretical results reveal the crucial role of periodic arrangement of amino acid components, enabled by fluorenyl stacking, in forming oxidase-mimetic copper clusters. Nucleotides provide coordination atoms that enhance copper activity by facilitating the formation of a copper-peroxide intermediate. The catalyst shows thermophilic behavior, remaining active up to 95 °C in an aqueous environment. These findings may aid the design of advanced biomimetic catalysts and offer insights into primordial redox enzymes.

Proteome-wide structural analysis identifies warhead- and coverage-specific biases in cysteine-focused chemoproteomics

Covalent drug discovery has undergone a resurgence over the past two decades and reactive cysteine profiling has emerged in parallel as a platform for ligand discovery through on- and off-target profiling; however, the scope of this approach has not been fully explored at the whole-proteome level. We combined AlphaFold2-predicted side-chain accessibilities for >95% of the human proteome with a meta-analysis of eighteen public cysteine profiling datasets, totaling 44,187 unique cysteine residues, revealing accessibility biases in sampled cysteines primarily dictated by warhead chemistry. Analysis of >3.5 million cysteine-fragment interactions further showed that hit elaboration and optimization drives increased bias against buried cysteine residues. Based on these data, we suggest that current profiling approaches cover a small proportion of potential ligandable cysteine residues and propose future directions for increasing coverage, focusing on high-priority residues and depth. All analysis and produced resources are freely available and extendable to other reactive amino acids.

Site-Selective Arylation of Carboxamides from Unprotected Peptides.

The amidated peptides are an important class of biologically active compounds due to their unique biological properties and wide applications as potential peptide drugs and biomarkers. Despite the abundance of free amide motifs (Asn, Gln, and C-terminal amide) in native peptides, late-stage modification of the amide unit in naturally occurring peptides remains very rare because of the intrinsically weak nucleophilicity of amides and the interference of multiple competing nucleophilic residues, which generally lead to undesired side reactions. Herein, chemoselective arylation of amides in unprotected polypeptides has been developed under an air atmosphere to afford the N-aryl amide peptides bearing various functional motifs. Its success relies on the combination of gold catalysis and silver salt to differentiate the relative inert amide among a collection of reactive nucleophilic amino acid residues (e.g., -NH2, -OH, and -COOH), favoring the C-N bond coupling toward amides over other more nucleophilic groups. Experimental and DFT studies reveal a crucial role of the silver cation, which serves as a transient coordination mask of the more reactive reaction sites, overcoming the inherently low reactivity of amides. The excellent biocompatibility of this strategy has been applied to functionalize a wide range of peptide drugs and complex peptides. The application could be further extended to peptide labeling and peptide stapling.