Abstract
The selective oxygenation of nonactivated carbon atoms is an ongoing synthetic challenge, and biocatalysts, particularly hemoprotein oxygenases, continue to be investigated for their potential, given both their sustainable chemistry credentials and also their superior selectivity. However, issues of stability, activity, and complex reaction requirements often render these biocatalytic oxygenations problematic with respect to scalable industrial processes. A continuing focus on Cytochromes P450 (P450s), which require a reduced nicotinamide cofactor and redox protein partners for electron transport, has now led to better catalysts and processes with a greater understanding of process requirements and limitations for both in vitro and whole-cell systems. However, the discovery and development of unspecific peroxygenases (UPOs) has also recently provided valuable complementary technology to P450-catalyzed reactions. UPOs need only hydrogen peroxide to effect oxygenations but are hampered by their sensitivity to peroxide and also by limited selectivity. In this Perspective, we survey recent developments in the engineering of proteins, cells, and processes for oxygenations by these two groups of hemoproteins and evaluate their potential and relative merits for scalable reactions.
Highlights
The selective oxygenation of nonactivated carbon centers remains a significant challenge in organic chemistry and has been addressed over several decades using either microorganisms or enzymes.[1,2] Biocatalyzed oxygenations have advantages over chemical reagents that accomplish similar, if less selective, transformations: They occur at ambient temperatures and pH and do not utilize harmful metals such as chromium
Unspecific peroxygenases (UPOs)[8,9] are a class of secreted fungal hemoproteins that catalyze the oxygenation of organic substrates at the expense of H2O2 only and have no requirement for NAD(P)H or electron transfer proteins, in part addressing the “oxygen dilemma” contextualized by Holtmann and Hollmann.[88]
In this Perspective, we have surveyed recent advances in biocatalytic oxygenations by hemoproteins, with an emphasis on reactions that result in isolatable yields from the tens of milligrams to, in one case,[45] kilogram quantities
Summary
The selective oxygenation of nonactivated carbon centers remains a significant challenge in organic chemistry and has been addressed over several decades using either microorganisms or enzymes.[1,2] Biocatalyzed oxygenations have advantages over chemical reagents that accomplish similar, if less selective, transformations: They occur at ambient temperatures and pH and do not utilize harmful metals such as chromium. Unspecific peroxygenases (UPOs)[8,9] are a class of secreted fungal hemoproteins that catalyze the oxygenation of organic substrates at the expense of H2O2 only and have no requirement for NAD(P)H or electron transfer proteins, in part addressing the “oxygen dilemma” (the dependence upon oxygen, balanced against the harmful ROS formation created during uncoupling processes) contextualized by Holtmann and Hollmann.[88] This simplicity, coupled with high activity and stability, makes UPOs attractive alternatives to P450s for scalable oxygenations if systems could be facilitated for their heterologous expression and their activities engineered Since their description by Hofrichter and co-workers,[20] researchers have suggested that there are two major groupings of UPOs: “short” enzymes of approximately 29 kDa molecular weight, typified by MroUPO from Marasmius rotula,[89] and “long” enzymes of 44 kDa, typified by the AaeUPO from Agrocybe aegerita,[20] the most studied UPO. Alcalde and co-workers showed that a variant of the PaDa AaeUPO, WamPa, which features nine additional mutations, displayed 23-fold greater stability in the presence of 30% acetonitrile.[132]
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