Ordered immobilization of serine proteases enabled by a linchpin directed modification platform.

We report a chemoselective and site-selective precision engineering of lysine in proteases. The mild and physiological reaction conditions keep their auto-degradation under control. Furthermore, it enables single-site ordered immobilization, enhancing protein digestion and peptide mapping efficiency.

Chemical technology principles for selective bioconjugation of proteins and antibodies.

Proteins are multifunctional large organic compounds that constitute an essential component of a living system. Hence, control over their bioconjugation impacts science at the chemistry-biology-medicine interface. A chemical toolbox for their precision engineering can boost healthcare and open a gateway for directed or precision therapeutics. Such a chemical toolbox remained elusive for a long time due to the complexity presented by the large pool of functional groups. The precise single-site modification of a protein requires a method to address a combination of selectivity attributes. This review focuses on guiding principles that can segregate them to simplify the task for a chemical method. Such a disintegration systematically employs a multi-step chemical transformation to deconvolute the selectivity challenges. It constitutes a disintegrate (DIN) theory that offers additional control parameters for tuning precision in protein bioconjugation. This review outlines the selectivity hurdles faced by chemical methods. It elaborates on the developments in the perspective of DIN theory to demonstrate simultaneous regulation of reactivity, chemoselectivity, site-selectivity, modularity, residue specificity, and protein specificity. It discusses the progress of such methods to construct protein and antibody conjugates for biologics, including antibody-fluorophore and antibody-drug conjugates (AFCs and ADCs). It also briefs how this knowledge can assist in developing small molecule-based covalent inhibitors. In the process, it highlights an opportunity for hypothesis-driven routes to accelerate discoveries of selective methods and establish new targetome in the precision engineering of proteins and antibodies.

Disintegrate (DIN) Theory Enabling Precision Engineering of Proteins.

The chemical toolbox for the selective modification of proteins has witnessed immense interest in the past few years. The rapid growth of biologics and the need for precision therapeutics have fuelled this growth further. However, the broad spectrum of selectivity parameters creates a roadblock to the field's growth. Additionally, bond formation and dissociation are significantly redefined during the translation from small molecules to proteins. Understanding these principles and developing theories to deconvolute the multidimensional attributes could accelerate the area. This outlook presents a disintegrate (DIN) theory for systematically disintegrating the selectivity challenges through reversible chemical reactions. An irreversible step concludes the reaction sequence to render an integrated solution for precise protein bioconjugation. In this perspective, we highlight the key advancements, unsolved challenges, and potential opportunities.

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.