Monday, February 10, 2025
2/10/2025
Development and Applications of Bioorthogonal Chemistry ABSTRACT This MIRA renewal application combines three ongoing research projects in the PI’s lab to further develop bioorthogonal covalent chemistry to tackle significant biophysical, biochemical, and in vivo imaging problems in biomedical research. We have a long-standing interest in developing reactivity-based chemical tools to address biomedical problems that are difficult to solve using conventional molecular biology techniques alone. In the last five years, we continued our progress in tool development and applications of these tools to study class B G protein-coupled receptors (GPCRs) in live cells and small domain antibodies such as monobodies. Specifically, we optimized the tetrazole-based photoclick chemistry by designing new genetically encoded hydrophilic azaspiroalkene reporters, the sterically shielded tetrazoles for fast bioorthogonal ligation reactions with strained alkenes and alkyne on live cell surface, the tetrazole-based fluorescent reporters of hydrogen peroxide in mammalian cells, a new class of reagents called hydrazonyl sultones (HS) that release NI through tautomerization and display a balanced stability and cycloaddition reactivity, and biocompatible genetically encoded crosslinkers based on 2-arboxy-4-aryltriazoles and -lactam for efficient inter- and intramolecular crosslinking. In conjunction with genetic code expansion, we showed that these chemical tools can be used to install an organic fluorophore into class B GPCRs for FRET studies as well as endowing cell penetration to small domain antibodies through orthogonal crosslinking. In this application, we plan to leverage our most powerful tools developed recently, namely, hydrazonyl sultones (HS) and -lactam-lysine (BeLaK), and prepare modified membrane receptors, enzymes, and nanobodies with new capabilities using bioorthogonal chemistry. Specifically, in Project 1, we will design hydrazonyl sultone (HS)-based turn-on fluorophores for constructing FRET-based biosensors to probe class B GPCR activation and signal transduction dynamics in live cells. The organic fluorophore will be installed at the intracellular loop 3 of class B GPCRs via bioorthogonal HS−BCN ligation reaction to permit FRET-based single-cell kinetic analysis of receptor activation and recruitment of G protein and -arrestin. In Project 2, we will design β-lactam-encoded enzyme traps for covalent capture and subsequent identification of protein lysine methyltransferase substrates in living cells. In Project 3, we will develop covalent nanobody-based immunoPET radiotracers with enhanced performance. Both BeLaK-mediated, proximity-driven crosslinking chemistry and HS-mediated, environment-dependent bioorthogonal ligation reactions will be exploited to enhance tumor uptake of the nanobody-based PET tracers for greater sensitivity without unwanted nephrotoxicity. We expect these studies will offer new capabilities of covalent chemistry to biomedical research.
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