Development of epoxide probes for protein labeling, and, Development of fluorescent reporter substrate probes for oxidoreductases

Development of epoxide probes for protein labeling. The research presented herein describes the development of new affinity probes for protein labeling based on an epoxide reactive group. Systematic screening revealed that an epoxide functionality possesses the special combination of stability and reactivity which renders it stable toward proteins in solution but reactive on the protein surface outside the active site (proximity-induced reactivity). Highly efficient and selective labeling of purified HCA II (human carbonic anhydrase II) was achieved. Importantly, the epoxide probe selectively labeled HCA II both in simple protein mixtures and in cellular extracts. The utility of our modular probe design was confirmed by an epoxide probe containing an adamantyl ligand instead of a benzenesulfonamide, which was constructed and shown to label P450cam efficiently and selectively in a proteomic context. In addition to being a promising platform for mild, selective in vitro protein labeling, this system has the potential to be an interesting and useful tool for proteomic analysis and exploration.;Development of fluorescent reporter substrates probes for oxidoreductases . Based on the rational design of fluorescent switches and exploration of enzymes promiscuity, the research presented herein describes the development of fluorogenic small molecule reporters for dehydrogenases and monoamine oxidases. Rational design of fluorescence switch based on PET and TICT quenching mechanism was exploited, novel oxidative deamination coupled with pyrrole-formation cascade reporting pathway was utilized. Systematic exploration of amino- and pyrrole-coumarin motifs was undertaken which led to the development of two systems with desired optical properties (fluorogenic probes). Ultimately, the ability of these compounds to serve as reporter substrate was demonstrated, most notably by the development of selective probe for MAO B. Moreover, the effectiveness of the diamine probes was demonstrated in context of intact mitochondria. These probes represent exciting leads for novel imaging probes that can be used in vivo.