Gram scale enantioselective biocatalytic C-H fluorination. E. coli BL21(DE3) cells harbouring the recombinant plasmid encoding ACCOCHF were grown overnight at 37 ℃ and 230 rpm in 25 mL LB media supplemented with 0.05 mg/mL kanamycin (LBkan). Preculture (20 mL, 2% v/v) was used to inoculate 1 L TB media supplemented with 0.05 mg/mL kanamycin (TBkan) in a 4 L Erlenmeyer flask. The culture was incubated at 37 °C and 230 rpm for 2.5 h to reach an OD600 of approximately 1.5. The culture was then cooled on ice for 30 min and induced with 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG, final concentrations). Protein expression was performed at 20 ℃ and 200 rpm for 20 h. The cells were harvested by centrifugation (4000 rpm, 5 min, 4 °C) using a Thermo Scientific Sorvall Lynx 6000 superspeed centrifuge and resuspended in M9-N buffer (OD600 = 30). An aliquot of E. coli cell suspension (2 mL) was taken to determine protein concentration using the SDS-PAGE assay. Cell suspensions in M9-N buffer were kept on ice until use. (Note: leaving the cell suspension at room temperature for an extended period of time will lead to significantly reduced enzyme activity.) To a 1 L Erlenmeyer flask equipped with a screw cap was added a suspension of E. coli cells in M9-N buffer expressing the desired enzyme variant. The flask was transferred to an anaerobic chamber, where a solution of Mohr's salt (5 mL, 150 mM stock solution in degassed H2O) and the N-fluoroamide substrate (1.67 g, 7.5 mmol, 500 mM in MeCN, 15 mL for substrate 1a) were added. The flask was capped, sealed with parafilm, taken out of the anaerobic chamber and allowed to shake in an Eppendorf Innova 44R shaker at room temperature and 230 rpm for 24 h.