Reagents for interrogating biological processes with bioorthogonal chemistry have been in high demand for their ability to enable dynamic imaging or mass spectrometric characterization of otherwise inaccessible biomolecules like sugars, lipids, and nucleic acids. Accordingly, much work has been done to create new reactions, increase the rate of these reactions, and create reagents with orthogonal reactivity that permit analysis of different processes in the same system. However, another dimension of bioorthogonal reactivity is ripe for development: controlling the time, cellular or subcellular location, or cellular metabolic state when/where the reactions occur. Here, we describe the development and optimization of chemical reactions that are specific, do not interfere with biology, and can be “turned-on” in the cell when desired. Essentially, we exploit the observation that cyclopropenes modified at the 3 position with bulky groups react more than 7000-times slower with tetrazines than those lacking bulky groups. We have recently developed several such cyclopropenes with N-substitution at the 3 position, which permits decoration with photo-, enzyme-, or small molecule-labile bulky groups that can be removed when and where desired by the experimenter. We describe strategies to accelerate the reaction between the uncaged cyclopropene and tetrazine by tuning the molecule’s electron withdrawing substituents. Further, we discuss strategies to control the activation of this reaction using exposure to light, native and non-native enzymes, and heightened levels of disease-associated metabolites. We explore additional classes of bioorthogonal reactions for the potential for modular control of reactivity. Finally, we evaluate these reactions in living cells to develop a strategy for cell-specific and subcellular targeting of bioorthogonal reactivity.

A modular turn-on bioorthogonal reaction

ACS Spring Meeting 2021

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