Table 2. Activity and Selectivity of Representative Engineered CYP119 Variants for the Intermolecular C–H Functionalization of N-Phenylpyrrolidine with EDAa.
| entry | enzyme variant | yield (%)b | TONc | e.r. (6a:7a) |
|---|---|---|---|---|
| 1 | CYP119 (WT) | 3 | 76 | nd |
| 2 | CYP 119 (C317S) | 5 | 110 | nd |
| 3 | CYP119 (T213A) | 6 | 135 | nd |
| 4 | CYP119 (T213A, C317S) | 18 | 812 | 71:29 |
| 5 | CYP119 (T213A, V254A, C317S)(CHI-g1) | 99 | 4,400 | 79:21 |
| 6 | CYP119 (F153G, T213A, V254A, C317S) (CHI-EDA) | 89 (64)d | 8,920 | 87:13 |
| 7e | CYP119 (F153G, T213A, V254A, C317S) (CHI-EDA) | 99 | 500 | 87:13 |
| 8f | CYP119 (F153G, T213A, V254A, C317S) (CHI-EDA) | 87 | 8,710 | 87:13 |
| 9f,g | CYP119 (F153G, T213A, V254A, C317S) (CHI-EDA) | 51 | 5,090 | 87:13 |
| 10 | CYP119 (A209W, T213G, V254A, C317S) | 71 | 6,750 | 19:81 |
a
Standard reaction conditions: CYP119-expressing C41 (DE3) E. coli cells, OD600 = 40, 10 mM 1a, 20 mM EDA, in KPi buffer (50 mM, pH 7), room temperature, 16 h, in an anaerobic chamber.
b
Assay yields as determined by GC using calibration curves generated with the isolated product.
c
TON as calculated based on the protein concentration measured from cell lysate.
d
Isolated yield.
e
Using 20 μM purified protein and 10 mM Na2S2O4.
f
Reaction time: 2 h.
g
Under aerobic conditions. nd = not determined.