Tertiary Amine Coupling by Oxidation for Selective Labeling of Dimethyl Lysine Post-Translational Modifications.

Lysine dimethylation (Kme2) is a crucial post-translational modification (PTM) that regulates biological processes and is implicated in diseases. There is significant interest in globally identifying these methylation marks. Unfortunately, this remains challenging due to the lack of robust technologies for selectively labeling Kme2. To address this, we present a chemical method named tertiary amine coupling by oxidation (TACO). This method selectively modifies Kme2 to aldehydes using Selectfluor and a base. The resulting aldehydes from Kme2 were then functionalized using reductive amination, thiolamine, and oxime chemistry. We successfully demonstrated the versatility of TACO in selectively labeling Kme2 peptides and proteins in complex cell lysate mixtures with varying payloads, including affinity tags and fluorophores. We further showed the application of TACO chemistry for the identification of Kme2 sites at a single-molecule level by fluorosequencing. We discovered novel 30 Kme2 sites, in addition to previously known 5 Kme2 sites, by proteomics analysis of TACO-modified nuclear extracts. Our work establishes a unique strategy for covalently modifying Kme2, facilitating the global identification of low-abundance Kme2-PTMs and their sites within complex cell lysate mixtures.

A Tag-Free Platform for Synthesis and Screening of Cyclic Peptide Libraries.

In the realm of high-throughput screening (HTS), macrocyclic peptide libraries traditionally necessitate decoding tags, essential for both library synthesis and identifying hit peptide sequences post-screening. Our innovation introduces a tag-free technology platform for synthesizing cyclic peptide libraries in solution and facilitates screening against biological targets to identify peptide binders through unconventional intramolecular CyClick and DeClick chemistries (CCDC) discovered through our research. This combination allows for the synthesis of diverse cyclic peptide libraries, the incorporation of various amino acids, and facile linearization and decoding of cyclic peptide binder sequences. Our sensitivity-enhancing derivatization method, utilized in tandem with nano LC-MS/MS, enables the sequencing of peptides even at exceedingly low picomolar concentrations. Employing our technology platform, we have successfully unearthed novel cyclic peptide binders against a monoclonal antibody and the first cyclic peptide binder of HIV capsid protein responsible for viral infections as validated by microscale thermal shift assays (TSA), biolayer interferometry (BLI) and functional assays.

Bioinspired one-pot furan-thiol-amine multicomponent reaction for making heterocycles and its applications.

One-pot multicomponent coupling of different units in a chemoselective manner and their late-stage diversification has wide applicability in varying chemistry fields. Here, we report a simple multicomponent reaction inspired by enzymes that combines thiol and amine nucleophiles in one pot via a furan-based electrophile to generate stable pyrrole heterocycles independent of the diverse functionalities on furans, thiols and amines under physiological conditions. The resulting pyrrole provides a reactive handle to introduce diverse payloads. We demonstrate the application of Furan-Thiol-Amine (FuTine) reaction for the selective and irreversible labeling of peptides, synthesis of macrocyclic and stapled peptides, selective modification of twelve different proteins with varying payloads, homogeneous engineering of proteins, homogeneous stapling of proteins, dual modification of proteins with different fluorophores using the same chemistry and labeling of lysine and cysteine in a complex human proteome.

Chemical sensors for imaging total cellular aliphatic aldehydes in live cells.

Aliphatic aldehydes are reactive electrophilic carbonyls that cross-link with DNA and proteins leading to cellular toxicity and disease pathogenesis. This toxicity is due to the cooperative effect of multiple aldehydes via a common mechanism. Therefore, live-cell imaging of total aliphatic aldehydes, small-to-long chain (C1-C10), is highly desired to decipher their physiological and pathological functions. However, sensors for imaging total cellular aliphatic aldehydes are currently lacking despite their high concentrations (∼80 to >500 µM) inside cells. Herein, we report chemical sensors that generate a benzimidazole moiety upon reaction with aliphatic aldehydes of different chain lengths (C1-C10), resulting in turn-on fluorescence. These sensors exhibit high quantum yields, high dynamic range, and enable the quantification of changes in both the exogenous administration of aldehydes and endogenous real-time formation of aliphatic aldehydes in live mammalian cells. This tool has great potential to transform aldehyde research by illuminating cellular metabolites that have remained elusive in living systems.