Spatiotemporal and direct capturing global substrates of lysine-modifying enzymes in living cells.

Protein-modifying enzymes regulate the dynamics of myriad post-translational modification (PTM) substrates. Precise characterization of enzyme-substrate associations is essential for the molecular basis of cellular function and phenotype. Methods for direct capturing global substrates of protein-modifying enzymes in living cells are with many challenges, and yet largely unexplored.

Genetically Encoded Noncanonical Amino Acids in Proteins to Investigate Lysine Benzoylation.

Posttranslational modifications (PTMs) of lysine residues are major regulators of gene expression, protein-protein interactions, and protein localization and degradation. Histone lysine benzoylation is a recently identified epigenetic marker associated with active transcription, which has physiological relevance distinct from histone acetylation and can be regulated by debenzoylation of sirtuin 2 (SIRT2). Herein, we provide a protocol for the incorporation of benzoyllysine and fluorinated benzoyllysine into full-length histone proteins, which further serve as benzoylated histone probes with NMR or fluorescence signal for investigating the dynamics of SIRT2-mediated debenzoylation.

Spatiotemporal and global profiling of DNA-protein interactions enables discovery of low-affinity transcription factors.

Precise dissection of DNA-protein interactions is essential for elucidating the recognition basis, dynamics and gene regulation mechanism. However, global profiling of weak and dynamic DNA-protein interactions remains a long-standing challenge. Here, we establish the light-induced lysine (K) enabled crosslinking (LIKE-XL) strategy for spatiotemporal and global profiling of DNA-protein interactions.

Genetically Encoded Benzoyllysines Serve as Versatile Probes for Interrogating Histone Benzoylation and Interactions in Living Cells.

Histone posttranslational modifications (PTMs) are vital epigenetic regulators in many fundamental cell signaling pathways and diverse biological processes. Histone lysine benzoylation is a recently identified epigenetic mark associated with active transcription; however, it remains to be explored. Herein, we first report the genetic encoding of benzoyllysine and fluorinated benzoyllysines into full-length histone proteins in a site-specific manner in live cells, based on our rationally designed synthetase and fine-integrated fluorine element into benzoyllysines.

Light-induced efficient and residue-selective bioconjugation of native proteins indazolone formation.

Chemical modification of proteins has emerged as a powerful tool to realize enormous applications, such as development of novel biologics and functional studies of individual protein. We report a light-induced lysine-selective native protein conjugation approach indazolone formation, conferring reliable chemoselectivity, excellent efficiency, temporal control and biocompatibility under operationally simple and mild conditions, and in living systems. This straightforward protocol demonstrates the generality and accessibility for direct and rapid functionalization of diverse native proteins, which suggests a new avenue of great importance to bioconjugation, medicinal chemistry and chemical biology.

Light-induced primary amines and o-nitrobenzyl alcohols cyclization as a versatile photoclick reaction for modular conjugation.

The advent of click chemistry has had a profound impact on many fields and fueled a need for reliable reactions to expand the click chemistry toolkit. However, developing new systems to fulfill the click chemistry criteria remains highly desirable yet challenging. Here, we report the development of light-induced primary amines and o-nitrobenzyl alcohols cyclization (PANAC) as a photoclick reaction via primary amines as direct click handle, to rapid and modular functionalization of diverse small molecules and native biomolecules.

Rapid and halide compatible synthesis of 2--substituted indazolone derivatives photochemical cyclization in aqueous media.

Indazolone derivatives exhibit a wide range of biological and pharmaceutical properties. We report a rapid and efficient approach to provide structurally diverse 2--substituted indazolones photochemical cyclization in aqueous media at room temperature. This straightforward protocol is halide compatible for the synthesis of halogenated indazolones bearing a broad scope of substrates, which suggests a new avenue of great importance to medicinal chemistry.