Tunable nucleofugality in carbamoyl-bearing covalent cholinesterase inhibitors

A handful of carbamate warheads is utilised in chemical biology to target serine hydrolases. Here, we report an in-depth profiling of diverse halogen, chalcogen, and nitrogen-based leaving groups to identify the optimal leaving groups (nucleofuges) to tune the reactivity of the electrophilic carbamoyl group. Even though this is a case study on cholinesterases, it is a first comprehensive structure-<i>reactivity</i> exploration of the carbamoyl warhead, rather than one-target-oriented structure-<i>activity</i> study. With computational tools we correlated the experimentally observed reactivities with steric and electronic factors of the investigated warheads. Multiscale QM/MM simulations taking into account the enzymatic environment explained how substitution of carbon for nitrogen in the leaving groups of compounds <b>26</b> and <b>28</b> withdrawal through resonance stabilization, inductive bond polarization, and acidity amplification lowered the reaction barrier and increased the reaction rate &gt;360 million times, making compound <b>28 </b>a covalent inhibitor. Our findings underline the complexity of covalent inhibition and demonstrate that multiple complementary methods are required to interpret and predict covalent behaviour. Additionally, even though carbamates typically act as slow substrates, we were able to slow down decarbamoylation to a point where inhibition became <i>de facto</i> irreversible. The most interesting <i>O</i>-isoxazol-3-yl carbamate-decorated probe was further profiled against the wider human proteome and showed a surprising selectivity, making it useful in further drug discovery.Files: PCA.prism - Statistical analysis of molecular descriptorsbiotin pull-down_rev2.xlsx - Proteomic profilingKarbamati kinetika FFA.pzfx - Source data of initial velocity measurements and fittingChE TD-curves.pzfx - Time-dependency curves