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

ACC-labeled substrates were synthesized on the solid support according to the solid phase peptide synthesis method described elsewhere. [15] In brief, Fmoc-ACC-OH (2.5 eq) was attached to a Rink-amide resin using HOBt (2.5 eq) and DICI (2.5 eq) in DMF as coupling reagents. Then, the Fmoc protecting group was removed using 20% piperidine in DMF (three cycles: 5, 5, and 25 min). Fmoc-Gln(Trt)-OH (2.5 eq) was coupled to the H 2 N-ACC-resin using HATU (2.5 eq) and 2,4,6-collidine (2.5 eq) in DMF. After Fmoc group removal, Fmoc-P2-OH (2.5 eq) amino acid was attached (HOBt and DICI (2.5 eq) in DMF). Amino acids in P3 and P4 positions were coupled in the same manner. The free N-terminus was acetylated using HBTU, AcOH and DIPEA in DMF (5 eq of each reagent). Then, the resin was washed five times with DMF, three times with DCM and three times with MeOH, and dried over The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.03.07.981928 doi: bioRxiv preprint