A chemo-enzymatic oxidation cascade to activate C-H bonds with in situ generated H2O2

Abstract

Continuous low-level supply or in situ generation of hydrogen peroxide (H₂O₂) is essential for the stability of unspecific peroxygenases, which are deemed ideal biocatalysts for the selective activation of C-H bonds. To envisage potential large scale applications of combined catalytic systems the reactions need to be simple, efficient and produce minimal by-products. We show that gold-palladium nanoparticles supported on TiO₂ or carbon have sufficient activity at ambient temperature and pressure to generate H₂O₂ from H₂ and O₂ and supply the oxidant to the engineered unspecific heme-thiolate peroxygenase PaDa-I. This tandem catalyst combination facilitates efficient oxidation of a range of C-H bonds to hydroxylated products in one reaction vessel with only water as a by-product under conditions that could be easily scaled.

Fig. 1

Proposed reaction system. A combination of heterogeneous catalysis and biocatalysis can be used to oxidize cyclohexane using in situ produced H₂O₂

Fig. 2

Feasibility of Coupling Catalytic Systems. a Cyclohexanol production in control reactions to determine the effect of the metal catalyst on unspecific peroxygenase activity when H₂O₂ is generated using GOX. b H₂O₂ synthesis with various catalyst concentrations under flowing (30 ml min⁻¹) 80% H₂ in air. Reaction Conditions: Solvent (H₂O) 50 ml, ambient temperature and pressure, gas flow 30 ml min⁻¹, 500 rpm stirring. Errors associated in the measurement of H₂O₂ by titration were ± 3 ppm. c Cyclohexane conversion to cyclohexanol using PaDa-I (15 U ml_RM⁻¹) in potassium phosphate buffer (100 mM, pH 6) and H₂O₂ generated in situ by metal catalysts. Efficiency of different AuPd catalysts (0.5 mg ml⁻¹) in the presence of 77% H₂ in air. d Time course results for cyclohexanol formation by PaDa-I (15 U ml_RM⁻¹) was coupled with in situ H₂O₂ generation by 2.5% Au-2.5% Pd/TiO₂ (0.1 mg ml_RM⁻¹) using a gas mixture of 77% H₂ in air (solid diamonds) or by GOX (0.2 U ml_RM⁻¹) using 200 mM glucose and air (circles). The GOX reactions were carried out using the same experimental setup as the metal catalyst reactions. Typically analysis of products was ± 0.2 mM as determined by GC (on duplicate experiments)

Fig. 3

Hydroxylation reactions using tandem catalysis system. Time on line hydroxylation reactions of a H₂O₂ production (black squares) (in the absence of enzyme) and cyclohexane (10 mM) oxidation to cyclohexanol and cyclohexanone b Ethylbenzene (10 mM) oxidation to 1-phenylethanol c Isophorone (30 mM) oxidation to 4 and 7-hydroxyisophorone when PaDa-I (15 U ml_RM⁻¹) was coupled with in situ H₂O₂ generation by 0.5% Au-0.5% Pd/TiO₂ (0.1 mg ml_RM⁻¹) using a gas mixture of 80% H₂ in air in a sealed system under 2 bar total pressure