In this study, a remarkably simple and direct strategy has been successfully developed to selectively label target cysteine residues in fully unprotected peptides and proteins. The strategy is based on the reaction between allenamides and the cysteine thiol, and proceeds swiftly in aqueous medium with excellent selectivity and quantitative conversion, thus forming a stable and irreversible conjugate. The combined simplicity and mildness of the process project allenamide as robust and versatile handles to target cysteines and has potential use in biological systems. Additionally, fluorescent-labeling studies demonstrated that the installation of a C-terminal allenamide moiety onto various molecules of interest may supply a new methodology towards the site-specific labeling of cysteine-containing proteins. Such a new labeling strategy may thus open a window for its application in the field of life sciences.

In this study, a remarkably simple and direct strategy has been successfully developed to selectively label target cysteine residues in fully unprotected peptides and proteins. The strategy is based on the reaction between allenamides and the cysteine thiol, and proceeds swiftly in aqueous medium with excellent selectivity and quantitative conversion, thus forming a stable and irreversible conjugate. The combined simplicity and mildness of the process project allenamide as robust and versatile handles to target cysteines and has potential use in biological systems. Additionally, fluorescentlabeling studies demonstrated that the installation of a Cterminal allenamide moiety onto various molecules of interest may supply a new methodology towards the site-specific labeling of cysteine-containing proteins. Such a new labeling strategy may thus open a window for its application in the field of life sciences.Selective chemical modification of protein structure is of significant importance to directly visualize protein dynamics, clearly understand their interaction mechanisms, and precisely mediate protein-protein interactions under single-cell and molecule resolution. Among the various remarkable labeling strategies, the sulfhydryl group in peptides and proteins has remained an attractive target for site-selective modification [1] owing mainly to its higher nucleophilicity and relatively lower natural abundance. [2] Briefly, the direct modification of cysteine can be mainly represented by two typical chemical pathways, that is, the nucleophilic substitution of a leaving group through the thiol of cysteine, as is the case for a-halocarbonyl derivatives (e.g. 2-iodoacetic acid and related variants), and the Michael addition of a thiol group to Michael acceptors like a,b-unsaturated systems. Another less common strategy is the metal-catalyzed cystein modification where most notable works involve cross-metathesis of allyl sulphides using the ruthenium catalyst reported by the group of Davis, [3] and thiol-allene coupling using a gold catalyst reported by the group of Che. [4] In recent years, some notable efforts to successfully target cysteine residues can be summed up as cross-metathesis of allyl sulfides, [3] and conjugation of thiol groups with allyl selenosulfate salts, [5] electron-deficient alkynes, [6] or bromomaleimides, [7] dithiomaleimide [8] derivatives, and PEGylation of native disulfide bond using a double cysteine alkylating reagent. [9] However, most strategies suffer from one or more drawbacks and the search for a versatile methodology remains elusive. While the cross-reactivity of classical reagents, including a-halo acyl analogues and maleimides, with other amino acids such as histidine and lysine residues potentially affects the selectivity, their reversibility/irreversibility is a matter of interest in newer findings. For example, bromomaleimides, dithiomaleimides, and electron-deficient alkynes form a bond with cysteine thiols, and can be reversed with excess glutathione, DTT, and other reactants. Thi...

Adenosine triphosphate-binding cassette (ABC) transporters, such as multidrug resistance protein 1 (MRP1), protect against cellular toxicity by exporting xenobiotic compounds across the plasma membrane. However, constitutive MRP1 function hinders drug delivery across the blood–brain barrier, and MRP1 overexpression in certain cancers leads to acquired multidrug resistance and chemotherapy failure. Small-molecule inhibitors have the potential to block substrate transport, but few show specificity for MRP1. Here we identify a macrocyclic peptide, named CPI1, which inhibits MRP1 with nanomolar potency but shows minimal inhibition of a related multidrug transporter P-glycoprotein. A cryoelectron microscopy (cryo-EM) structure at 3.27 Å resolution shows that CPI1 binds MRP1 at the same location as the physiological substrate leukotriene C4 (LTC 4 ). Residues that interact with both ligands contain large, flexible sidechains that can form a variety of interactions, revealing how MRP1 recognizes multiple structurally unrelated molecules. CPI1 binding prevents the conformational changes necessary for adenosine triphosphate (ATP) hydrolysis and substrate transport, suggesting it may have potential as a therapeutic candidate.

Rapid surface functionalization of nanomaterials using covalent linkage following "green chemistry" remains challenging, and the quest for developing simple protocols is persisting. We report a nanomechanical microfluidic approach for the coupling of allenamide functionalized organic derivatives on the surface of thiol-modified silica nanoparticles using allenamide-thiol chemistry. The coupling principle involves the use of a microfluidic surface acoustic wave device that generates acoustic streaming-based chaotic fluid micromixing that enables mixing of laterally flowing fluids containing active components. This approach was used to demonstrate the direct surface labeling of thiol-modified silica nanoparticles using a selected group of modified fluorescent tags containing allenamide handles and achieved a total labeling efficiency of 83−90%. This green approach enabled a highly efficient surface functionalization under aqueous conditions, with tunable control over the conjugation process via the applied field. The dye-labeled silica particles were characterized using various analytical techniques and found to be biocompatible with potential in live cell bioimaging. It is envisaged that this bioconjugation strategy will find numerous applications in the field of bioimaging and drug delivery.

Display technology platforms offer their own unique set of challenges for chemical transformations, at the heart of which lies peptide macrocyclization. The amenable reactions for peptide macrocyclization on this platform need to meet a number of criteria like high reactivity, selectivity, mild conditions, irreversibility, and in many cases, a unique requirement to be assimilated into the translation machinery. Skillful utilization of these reactions has led to the formation of huge macrocyclic peptide libraries with varied linkages and topographies which have in turn led to the discovery of a number of hits for purposes such as drug discovery and others. Herein, we review those reactions which have mainly been applied in mRNA and phage display and discuss their technical characteristics and significance.