Toolkit for Fast, Multipurpose and Inducible Bioorthogonal Chemistry

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Project Summary The goal of this proposal will be to develop a diverse set of new chemical tools centered on tetrazine ligation– the fastest known bioorthogonal reaction. This proposal describes a continued effort to develop new bioorthogonal reagents. Methods will be developed for the safe synthesis and direct coupling of ‘minimalist’ tetrazines to any molecule of interest, including chemical probes and fluorescent reporters. We also propose to develop tetrazines that can serve as “affinity bioorthogonal chemistry” tags that can serve a dual role in enabling protein purification followed by subsequent site-selective bioorthogonal chemistry. Applications to on-resin protein-protein and protein-biomolecule assembly are proposed. We propose to develop efficient catalytic methods for ‘turning on’ rapid bioorthogonal chemistry in cellular context, providing tool molecules with high stability in the cellular environment in their ‘off’ state, and the fastest bioorthogonal reactions to date in their ‘on’ state. Efficient photocatalysts have been developed that can target bioorthogonal turn-on in vivo and at the subcellular level. Near-IR photocatalysts as well as thermal catalysts will be developed as will new enabling technologies for studying protein dynamics and for proteomic target identification. Finally, we will also develop bioorthogonal N-acylmuramic acid (NAM) and N- acylglucosamine (NAG) molecules for use in probe applications in microbiology and immunology research.