Human Behavior-Inspired Linchpin-Directed Catalysis for Traceless Precision Labeling of Lysine in Native Proteins.

The complex social ecosystem regulates the spectrum of human behavior. However, it becomes relatively easier to understand if we disintegrate the contributing factors, such as locality and interacting partners. Interestingly, it draws remarkable similarity with the behavior of a residue placed in a social setup of functional groups in a protein. Can it inspire principles for creating a unique environment for the precision engineering of proteins? We demonstrate that localization-regulated interacting partner(s) could render precise and traceless single-site modification of structurally diverse native proteins. The method targets a combination of high-frequency Lys residues through an array of reversible and irreversible reactions. However, excellent simultaneous control over chemoselectivity, site selectivity, and modularity ensures that the user-friendly protocol renders acyl group installation, including post-translational modifications (PTMs), on a single Lys. Besides, it offers a chemically orthogonal handle for the installation of probes. Also, a purification protocol integration delivers analytically pure single-site tagged protein bioconjugates. The precise labeling of a surface Lys residue ensures that the structure and enzymatic activities remain conserved post-bioconjugation. For example, the precise modification of insulin does not affect its uptake and downstream signaling pathway. Further, the method enables the synthesis of homogeneous antibody-fluorophore and antibody-drug conjugates (AFC and ADC; K183 and K249 labeling). The trastuzumab-rhodamine B conjugate displays excellent serum stability along with antigen-specific cellular imaging. Further, the trastuzumab-emtansine conjugate offers highly specific antiproliferative activity toward HER-2 positive SKBR-3 breast cancer cells. This work validates that disintegrate theory can create a comprehensive platform to enrich the chemical toolbox to meet the technological demands at the chemistry, biology, and medicine interface.

Serine-Selective Bioconjugation.

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.

Chemoselective and Site-Selective Lysine-Directed Lysine Modification Enables Single-Site Labeling of Native Proteins.

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.

Single-Site Labeling of Native Proteins Enabled by a Chemoselective and Site-Selective Chemical Technology.

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 trastuzumab-doxorubicin and trastuzumab-emtansine conjugates with selective antiproliferative activity toward Her-2 positive SKBR-3 breast cancer cells.