Figure 2. Chemo-enzymatic fluorination of organic molecules.

(a) Screening of P450 library in 96-well format. Reactions were carried out in the presence of the substrate, P450 enzyme from cell lysate, and a glucose-6-phosphate dehydrogenase-based NADPH regeneration system. TON: turnover number. Standard error is within 15%. WT = wild-type P450_BM3. (b) Selective fluorination of cyclopentenone derivatives. Reagents and conditions: i) 1, 0.04 mol% var-H3, 88%; ii) DAST (1.2 equiv), CH₂Cl₂, −78°C, 12 h, 90% (20% ee) ; iii) 1, 0.04 mol% var-G6, 45%; iv) DAST (1.2 equiv), CH₂Cl₂, −78°C, 12 h, 85%; v) 2, 0.05 mol% var-H3, 85%; vi) DAST (1.3 equiv), CH₂Cl₂, −78°C, 12 h, 92% (78% ee); vii) 2, 0.05 mol% var-G4, 42%; viii) DAST (1.5 equiv), CH₂Cl₂, −78°C, 12 h, 89%; ix) 3, 0.05 mol% var-D10, 69%; x) DAST (1.2 equiv), CH₂Cl₂, −78°C, 3 h, 88% (dr 1:8.5, major: 5% ee, minor: 71% ee); xi) 3, 0.05 mol% var-G4, 62%; xii) DAST (1.2 equiv), CH₂Cl₂, −78°C, 5 h, 92% (dr 4:96, major: 0% ee, minor: 57% ee); xiii) 3, 0.07 mol% var-G5, 32%; xiv) DAST (1.2 equiv), CH₂Cl₂, −78°C, 5 h, 90% (dr not measurable). (c) Selective mono- and difluorination of prodrug ibuprofen methyl ester. Reagents and conditions: i) 0.1 mol% var-B4, 72%; ii) DAST (1.4 equiv), CH₂Cl₂, −78°C, 12 h, 86% (dr 1:3.2, major: 19% ee, minor: 44% ee); iii) 0.05 mol% var-G4, 88%; iv) DAST (1.4 equiv), CH₂Cl₂, −78°C, 12 h, 95%; v) 0.06 mol% var-B2, 93%; vi) DAST (1.2 equiv), CH₂Cl₂, −78°C, 12 h, 98% (dr 1:3.7, major: 9% ee, minor: 9% ee). All experimental procedures are described in detail in Supplementary Methods. The sequences of the P450 variants are described in Supplementary Table 2. Yields refer to the isolated products. Enantiomeric excess values were determined by chiral GC analysis.