Scheme 14. Hyphenation of halogenation and cross-coupling to achieve C–H activation, (A) general concept in which the halogenase operates first, and then the cross-coupling is carried out on the halogenated material as a component of the crude extract. This is carried out stepwise in (B–F), enabling access to a broad series of compounds, and synchronously in G where the halogenation and cross-coupling events are spatially separated through application of a membrane. (B) Bromination of l-tryptophan by Thal, followed by Suzuki–Miyaura cross-coupling of the brominated product as a component of the crude lysate.215 The product of cross-coupling (λex 300 nm/λem 430 nm) is used in screening the directed evolution of RebH, resulting in a 2.5-fold increase in enzymatic activity. Cross-coupling conditions: 3-aminophenylboronic acid (10 equiv.), Na2PdCl4 (50 mol%), SSphos (150 mol%), K3PO4 (15 equiv.), 95 °C.215 (C) One-pot bromination of l-tryptophan by RebH-CLEA, followed by Mizoroki–Heck cross-coupling, in a stepwise fashion to afford C-7 substituted 7-(4-carboxystyryl)tryptophan with a λem of 485 nm (λex 360 nm). Cross-coupling conditions: 4-carboxystyrene (5 equiv.), PdOAc2 (0.1–0.2 equiv.), TPPTS (0.3–0.6 equiv.), K2CO3 (5 equiv.), water, degassed under argon, 100 °C.189 (D–F) Sequential enzymatic bromination by evolved RebH variants, followed by (D) Suzuki–Miyaura, (E) Buchwald–Hartwig, (F) alkoxylation of tryptophan as a component of a crude extract. Conditions D: ArB(OH)2 (1.5 equiv.), PdOAc2 (0.05 equiv.), SSPhos (0.05 equiv.), iPrOH: phosphate buffer (170 mM, pH 8.5) 1 : 1, 90 °C. (E) ArNH2 (3 equiv.), PdOAc2 (0.03 equiv.), BrettPhos (0.03 equiv.), NaOt-Bu (6 equiv.), dioxane, 100 °C. (F) CF3CH2OH (2 equiv.), [(allyl)PdCl]2(0.005 equiv.), RockPhos (0.015 equiv.), Cs2CO3 (2 equiv.), toluene, 90 °C.189 (G) One-pot, synchronous halogenation and Suzuki–Miyaura cross-coupling by PDMS membrane separated RebH-CLEA and Pd catalyst. Cross-coupling conditions: PdOAc2 (10 mol%), 2-(dimethylamino)-pyrimidine-4,6-diol (20 mol%), aryl boronic acid (5 equiv.) and CsF (10 equiv.), r.t. overnight then 80 °C.211.
