Diels-Alder cycloaddition for fluorophore targeting to specific proteins inside living cells

The inverse-electron-demand Diels-Alder cycloaddition between trans-cyclooctenes and tetrazines is biocompatible and exceptionally fast. We utilized this chemistry for site-specific fluorescence labeling of proteins on the cell surface and inside living mammalian cells by a two-step protocol. Escherichia coli lipoic acid ligase site-specifically ligates a trans-cyclooctene derivative onto a protein of interest in the first step, followed by chemoselective derivatization with a tetrazine-fluorophore conjugate in the second step. On the cell surface, this labeling was fluorogenic and highly sensitive. Inside the cell, we achieved specific labeling of cytoskeletal proteins with green and red fluorophores. By incorporating the Diels-Alder cycloaddition, we have broadened the panel of fluorophores that can be targeted by lipoic acid ligase.     

Evaluation of TILI-2 as an Anti-Tyrosinase,Anti-Oxidative Agent and Its Role in Preventing Melanogenesis Using a Proteomics Approach

There is a desire to develop new molecules that can combat hyperpigmentation. To this end, the N-terminal cysteine-containing heptapeptide TILI-2 has shown promising preliminary results. In this work, the mechanism by which it works was evaluated using a series of biochemical assays focusing on known biochemical pathways, followed by LC-MS/MS proteomics to discover pathways that have not been considered before. We demonstrate that TILI-2 is a competitive inhibitor of tyrosinase’s monophenolase activity and it could potentially scavenge ABTS and DPPH radicals. It has a very low cytotoxicity up to 1400 µM against human fibroblast NFDH cells and macrophage-like RAW 264.7 cells. Our proteomics study revealed that another putative mechanism by which TILI-2 may reduce melanin production involves the disruption of the TGF-β signaling pathway in mouse B16F1 cells. This result suggests that TILI-2 has potential scope to be used as a depigmenting agent.