Chemical biology research often requires precise covalent attachment of labels to the native proteins. Such methods are sought after to probe, design, and regulate the properties of proteins. At present, this demand is largely unmet due to the lack of empowering chemical technology. Here, we report a chemical platform that enables site-selective labeling of native proteins. Initially, a reversible intermolecular reaction places the "chemical linchpins" globally on all the accessible Lys residues. These linchpins have the capability to drive site-selective covalent labeling of proteins. The linchpin detaches within physiological conditions and capacitates the late-stage installation of various tags. The chemical platform is modular, and the reagent design regulates the site of modification. The linchpin is a multitasking group and facilitates purification of the labeled protein eliminating the requirement of additional chromatography tag. The methodology allows the labeling of a single protein in a mixture of proteins. The precise modification of an accessible residue in protein ensures that their structure remains unaltered. The enzymatic activity of myoglobin, cytochrome C, aldolase, and lysozyme C remains conserved after labeling. Also, the cellular uptake of modified insulin and its downstream signaling process remain unperturbed. The linchpin directed modification (LDM) provides a convenient route for the conjugation of a fluorophore and drug to a Fab and monoclonal antibody. It delivers trastuzumabdoxorubicin and trastuzumab-emtansine conjugates with selective antiproliferative activity toward Her-2 positive SKBR-3 breast cancer cells.

Labeling of native proteins invites interest from diverse segments of science. However, there remains the significant unmet challenge in precise labeling at a single site of a protein. Here, we report the site-specific labeling of natural or easy-to-engineer N-terminus Gly in proteins with remarkable efficiency and selectivity. The method generates a latent nucleophile from N-terminus imine that reacts with an aldehyde to deliver an aminoalcohol under physiological conditions. It differentiates N-Gly as a unique target amongst other proteinogenic amino acids. The method allows single-site labeling of proteins in isolated form and extends to lysed cells. It administers an orthogonal aldehyde group primed for late-stage tagging with an affinity tag, 19 F NMR probe, and a fluorophore. A user-friendly protocol delivers analytically pure tagged proteins. The mild reaction conditions do not alter the structure and function of the protein. The cellular uptake of fluorophore-tagged insulin and its ability to activate the insulin-receptor mediated signaling remains unperturbed.

This Communication reports the first general method for rapid, chemoselective, and modular functionalization of serine residues in native polypeptides, which uses a reagent platform based on the P­(V) oxidation state. This redox-economical approach can be used to append nearly any kind of cargo onto serine, generating a stable, benign, and hydrophilic phosphorothioate linkage. The method tolerates all other known nucleophilic functional groups of naturally occurring proteinogenic amino acids. A variety of applications can be envisaged by this expansion of the toolbox of site-selective bioconjugation methods.

The necessity for precision labeling of proteins emerged during the efforts to understand and regulate their structure and function. It demands selective attachment of tags such as affinity probes, fluorophores, and potent cytotoxins. Here, we report a method that enables single‐site labeling of a high‐frequency Lys residue in the native proteins. At first, the enabling reagent forms stabilized imines with multiple solvent‐accessible Lys residues chemoselectively. These linchpins create the opportunity to regulate the position of a second Lys‐selective electrophile connected by a spacer. Consequently, it enables the irreversible single‐site labeling of a Lys residue independent of its place in the reactivity order. The user‐friendly protocol involves a series of steps to deconvolute and address chemoselectivity, site‐selectivity, and modularity. Also, it delivers ordered immobilization and analytically pure probe‐tagged proteins. Besides, the methodology provides access to antibody‐drug conjugate (ADC), which exhibits highly selective anti‐proliferative activity towards HER‐2 expressing SKBR‐3 breast cancer cells.

Protein bioconjugation poses outstanding questions of selectivity to the organic transformations. Besides, the presence of a pool of functional groups in the structural outfit of a protein brings its own set of characteristics. In this minireview, we highlight the challenges faced by a chemical transformation to deliver selectivity in the modification of proteins. The examples of pre‐engineered proteins outline the attributes associated with chemoselectivity and chemical orthogonality. Building on this foundation, we discuss the complexity of site‐selectivity in the single‐site modification of native proteins. The gradual evolution of chemical methods while addressing the challenges associated with different amino acids are outlined. The modular methods for labeling a residue independent of its reactivity order closes the discussion.

The conservation of chemoselectivity becomes invalid for multiple electrophilic warheads during protein bioconjugation. Consequently, it leads to unpredictable heterogeneous labeling of proteins. Here, we report that a linchpin can create...

We report a chemoselective and site-selective formylation of ε-amine in native proteins. The aldehyde auto-oxidation re-routing, regulated generation of formate, and reversible N-terminus protection drive the transformation. It labels a single ε-amine in a pool of its copies, other nucleophilic residues, and α-amine. The extension of the methodology leads to site-selective acylation.

The cytoplasmic level of a messenger RNA, and hence protein, depends not only upon its rates of synthesis, processing, and transport, but its decay rate as well. mRNA decay rates are frequently not static, but vary in response to extracellular stimuli and viral infections. Sequence elements within an mRNA, together with the protein and/or small noncoding RNA factors that bind these elements, dictate its decay rate. Not surprisingly, genetic alterations in mRNA stability can lead to various diseases, including cancer, heart disease, and immune disorders. However, we now have the capacity to alter selective aspects of the mRNA decay machinery by design in order to tune expression of any given gene to desired levels as a means of achieving therapeutic results. Our intent in this review is to introduce the reader to the intricacies of regulated gene expression at the level of mRNA stability, describe the roles of mRNA stability in pathology and drug development, and discuss some recent developments in the field of computational biology that are providing novel tools for understanding specific protein-RNA interactions, which drive the mRNA degradation machinery.