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

To each well of the reaction apparatus, 1 eq of dry H 2 N-Gln(Trt)-ACC-resin (0.059 mmol, 80 mg) was added and stirred gently for 30 minutes in DCM, and then washed four times with DMF. In separate Eppendorf tubes, 2.5 eq (0.15 mmol) Fmoc-P2-OH was preactivated with 2.5 eq HOBt (0.15 mmol, 22.5 mg) and 2.5 eq DICI (0.15 mmol, 23.55 μL) in DMF. Next, preactivated amino acids were added to wells of the apparatus containing H 2 N-Gln(Trt)-ACCresin, followed by 3 h of agitation at room temperature. Then, the reaction mixture was filtered, washed with DMF (4 times), and the ninhydrin test was carried out in order to confirm P2-amino acid coupling. Subsequently, Fmoc protecting groups were removed with the use of 20% piperidine in DMF (5, 5, and 25 min). For P3 and P4 position coupling, an isokinetic mixture for 48 portions was prepared from 18 Fmoc-protected natural amino acids (omitting cysteine; plus norleucine mimicking methionine; 19 amino acids in total). Next, 5 eq of isokinetic mixture, 5 eq HOBt (14.16 mmol, 2.13 g), and 5 eq DICI (14.16 mmol, 2.22 mL) were diluted in DMF and preactivated for 3 min. The activated isokinetic mixture was added to each of 48 wells containing 1 eq of H 2 N-P2-Gln(Trt)-ACC-resin. After 3 h of gentle agitation, the slurry was filtered off and washed with DMF (4 times). A ninhydrin test was carried out and the Fmoc protecting group was removed using 20% piperidine in DMF (5, 5, and 25 min). The same procedure was applied for the remaining compounds. The isokinetic mixture was added to prepare the P4 position in the same manner as for the P3 position. In the last step of the synthesis, N-terminus acetylation was performed; to prepare the mixture for 48 compounds, 5 eq of AcOH (14.16 mmol, 807 µL), 5 eq HBTU (14.16 mmol, 5.37 g), and 5 eq DIPEA (14.16 mmol, 2.44 mL) in ~45 mL of DMF were added to a 50-mL falcon tube. After gentle stirring for 1 min, the mixture (~800 µL) was added to each well in the reaction apparatus, containing the H 2 N-Mix-Mix-P2-Gln(Trt)-ACC-resin, followed by gentle agitation for 30 min. Next, the resin was washed six times with DMF, three times with DCM, three times with MeOH, and dried over P 2 P4 sublibraries was performed in the same manner as described above; P3 and P4 sublibraries were synthesized by coupling fixed amino-acid residues to P3 (isokinetic mixture coupled to P2 and P4) and P4 position (isokinetic mixture coupled to P2 and P3).