1 Department of Chemical and Physical Sciences, University of Toronto Mississauga, Mississauga, Ontario, L5L 1C6, Canada. andrew.beharry@utoronto.ca.
2 Department of Chemistry and Biochemistry, Concordia University, 7141 Sherbrooke St W., Montréal, Québec, H4B 1R6, Canada.
3 Keenan Chair in Surgery, Division of Neurosurgery, St Michael's Hospital, University of Toronto, Ontario, M5B 1W8, Canada.

Fluorescent chemosensors offer a direct means of measuring enzyme activity for cancer diagnosis, predicting drug resistance, and aiding in the discovery of new anticancer drugs. O⁶-methylguanine DNA methyltransferase (MGMT) is a predictor of resistance towards anticancer alkylating agents such as temozolomide. Using the fluorescent molecular rotor, 9-(2-carboxy-2-cyanovinyl)julolidine (CCVJ), we synthesized, and evaluated a MGMT fluorescent chemosensor derived from a chloromethyl-triazole covalent inhibitor, AA-CW236, a non-pseudosubstrate of MGMT. Our fluorescence probe covalently labelled the MGMT active site C145, producing a 18-fold increase in fluorescence. Compared to previous fluorescent probes derived from a substrate-based inhibitor, our probe had improved binding and reaction rate. Overall, our chloromethyl triazole-based fluorescence MGMT probe is a promising tool for measuring MGMT activity to predict temozolomide resistance.