Substrate specificity profiling of SARS-CoV-2 Mpro protease provides basis for anti-COVID-19 drug design

Wioletta Rut; Katarzyna Groborz; Linlin Zhang; Xinyuanyuan Sun; Mikolaj Zmudzinski; Rolf Hilgenfeld; Marcin Drag

Gene cloning, recombinant production of the SARS-CoV and SARS-CoV-2 M pro are described elsewhere. [7, 13] Combinatorial library synthesis Synthesis of H 2 N-ACC-resin. ACC synthesis was carried out according to Maly et al. [14] To a glass reaction vessel, 1 eq (9.62 mmol, 13 g) of Rink AM resin was added and stirred gently once per 10 min in DCM for 1 h, then filtered and washed 3 times with DMF. Fmoc-group deprotection was performed using 20% piperidine in DMF (three cycles: 5, 5, and 25 min), filtered and washed with DMF each time (six times). Next, 2.5 eq of Fmoc-ACC-OH (24.05 mmol, 10.64 g) was preactivated with 2.5 eq HOBt monohydrate (24.05 mmol, 3.61 g) and 2.5 eq DICI (24.05 mmol, 3.75 mL) in DMF and the slurry was added to the resin. The reaction was shaked gently for 24 hours at room temperature. After this time, the resin was washed four times with DMF and the reaction was repeated using 1.5 eq of above reagents in order to improve the yield of ACC coupling to the resin. After 24 hours, the resin was washed with DMF and the Fmoc protecting group was removed using 20% piperidine in DMF (5, 5, and 25 min), filtered and washed with DMF (six times).