Stereoselective Olefin Cyclopropanation under Aerobic Conditions with an Artificial Enzyme Incorporating an Iron-Chlorin e6 Cofactor

Abstract

Myoglobin has recently emerged as a promising biocatalyst for catalyzing carbene-mediated cyclopropanation, a synthetically valuable transformation not found in nature. Having naturally evolved for binding dioxygen, the carbene transferase activity of this metalloprotein is severely inhibited by it, imposing the need for strictly anaerobic conditions to conduct these reactions. In this report, we describe how substitution of the native heme cofactor with an iron-chlorin e6 complex enabled the development of a biocatalyst capable of promoting the cyclopropanation of vinylarenes with high catalytic efficiency (up to 6,970 TON), turnover rate (>2,000 turnovers/min), and stereoselectivity (up to 99% de and ee) in the presence of oxygen. The artificial metalloenzyme can be recombinantly expressed in bacterial cells, enabling its application also in the context of whole-cell biotransformations. This work makes available a robust and easy-to-use oxygen-tolerant biocatalyst for asymmetric cyclopropanations and demonstrates the value of porphyrin ligand substitution as a strategy for tuning and enhancing the catalytic properties of hemoproteins in the context of abiological reactions.

Iron‐porphyrin Catalyzed Carbene Transfer Reactions – an Evolution from Biomimetic Catalysis towards Chemistry‐inspired Non‐natural Reactivities of Enzymes

“…Fasan and co‐workers showed the incorporation of the Fe‐chlorin e6 complex into myoglobin leading to very high catalytic efficiencies in presence of oxygen and performed well in absence of a reducing agent (Table , Entry 5) . Arnold and coworkers achieved the independence of a reducing agent and oxygen tolerance by extensive rounds of directed evolution and thereby solely relying on natural amino acids and natural prosthetic groups (Table , Entry 6) .…”

An Artificial Heme Enzyme for Cyclopropanation Reactions

“…Engineered heme proteins such as cytochrome P450 enzymes, myoglobin, and cyctochrome C have emerged as excellent catalysts for new‐to‐nature reactions, including carbene‐transfer reactions such as cyclopropanations, olefinations, and N−H, Si−H, and B−H insertion reactions . These enzymes have proven amenable to optimization by both genetic methods and co‐factor replacement . A common feature of these (designed) heme enzymes is that they contain a large hydrophobic substrate binding pocket orthogonal to the plane of the heme moiety.…”

An Artificial Heme Enzyme for Cyclopropanation Reactions

“…These enzymes have proven amenable to optimization by both genetic methods and co‐factor replacement 14, 15, 16, 17, 18, 19, 20. A common feature of these (designed) heme enzymes is that they contain a large hydrophobic substrate binding pocket orthogonal to the plane of the heme moiety.…”

“…[13] These enzymes have proven amenable to optimization by both genetic methods and co-factor replacement. [14][15][16][17][18][19][20] Ac ommon feature of these (designed) heme enzymes is that they contain al arge hydrophobic substrate binding pocket orthogonal to the plane of the heme moiety. Alternatively,s ignificant effort has been devoted to the de novo design of heme proteins,particularly based on 4-helix bundles, [21][22][23][24][25] antibodies,o ro ther proteins, [26,27] yet none of these has found application in catalysis of new-to-nature reactions.Akey difference is that these artificial heme enzymes generally do not present ad efined binding site suitable for binding the often hydrophobic substrates.H ere, we report an ovel artificial heme enzyme based on the lactococcal multidrug resistance regulator (LmrR), which is capable of catalyzing abiological enantioselective cyclopropanation reactions.Moreover,wepropose that the structural dynamics of the artificial heme enzyme are key to its catalytic activity.…”