Reetz, M.T.

Angew. Chem. Int. Ed. 2010, 49, 5151-5155, 10.1002/anie.201002106

Guided by nature: A designed binding site comprising the His/His/Asp motif for CuII complexation has been constructed in a robust protein by site‐specific mutagenesis (see picture). The artificial metalloenzyme catalyzes an enantioselective Diels–Alder reaction.

Reetz, M.T.

Chem. Rec. 2012, 12, 391-406, 10.1002/tcr.201100043

Numerous enzymes are useful catalysts in synthetic organic chemistry, but they cannot catalyze the myriad transition‐metal‐mediated transformations customary in daily chemical work. For this reason the concept of directed evolution of hybrid catalysts was proposed some time ago. A synthetic ligand/transition‐metal moiety is anchored covalently or non‐covalently to a host protein, thereby generating a single artificial metalloenzyme which can then be optimized by molecular biological methods. In the quest to construct an appropriate experimental platform for asymmetric Diels–Alder reactions amenable to this Darwinian approach to catalysis, specifically those not currently possible using traditional chiral transition‐metal catalysts, two strategies have been developed which are reviewed here. One concerns the supramolecular anchoring of a Cu(II)‐phthalocyanine complex to serum albumins; the other is based on the design of a Cu(II)‐specific binding site in a thermostable protein host (tHisF), leading to 46–98% ee in a model Diels–Alder reaction. This sets the stage for genetic fine‐tuning using the methods of directed evolution.

Reetz, M.T.

Angew. Chem. Int. Ed. 2006, 45, 2416-2419, 10.1002/anie.200504561

Chirality from blood: Serum albumins form strong complexes with CuII–phthalocyanines, leading to protein conjugates. These hybrid catalysts promote enantioselective Diels–Alder reactions, such as that of azachalcones 1 with cyclopentadiene (2) to give products 3 with 85–98 % ee.

Reetz, M.T.

Isr. J. Chem. 2015, 55, 51-60, 10.1002/ijch.201400087

Transition metal catalysis in asymmetric transformations plays a pivotal role in modern synthetic organic chemistry, with these catalysts being tuned by systematic variation of the chiral ligand. More than three decades ago it was recognized that an alternative approach is possible, namely the anchoring of an achiral ligand/metal entity in an appropriate protein host, with formation of an artificial metalloenzyme (hybrid catalyst). However, this procedure delivers a single transition metal catalyst, with high enantioselectivity being a matter of chance. In view of this restriction, we proposed in 2001/2002 the concept of directed evolution of such hybrid catalysts. The most intensively studied system involves biotinylated phosphine/metal entities which are non‐covalently anchored to streptavidin. The present review summarizes progress in this intriguing area of research. It includes the assessment of the requirements of a given Darwinian system to be successful, and offers hints on how to achieve success in future studies.

Reetz, M.T.

Acc. Chem. Res. 2019, 52, 336-344, 10.1021/acs.accounts.8b00582

Transition metal catalysts mediate a wide variety of chemo-, stereo-, and regioselective transformations, and therefore play a pivotal role in modern synthetic organic chemistry. Steric and electronic effects of ligands provide organic chemists with an exceedingly useful tool. More than four decades ago, chemists began to think about a different approach, namely, embedding achiral ligand/metal moieties covalently or noncovalently in protein hosts with formation of artificial metalloenzymes. While structurally fascinating, this approach led in each case only to a single (bio)catalyst, with its selectivity and activity being a matter of chance. In order to solve this fundamental problem, my group proposed in 2000−2002 the idea of directed evolution of artificial metalloenzymes. In earlier studies, we had already demonstrated that directed evolution of enzymes constitutes a viable method for enhancing and inverting the stereoselectivity of enzymes as catalysts inorganic chemistry. We speculated that it should also be possible to manipulate selectivity and activity of artificial metalloenzymes, which would provide organic chemists with a tool for optimizing essentially any transition metal catalyzed reaction type. In order to put this vision into practice, we first turned to the Whitesides system for artificial metalloenzyme formation, comprising a biotinylated diphosphine/Rh moiety, which is anchored noncovalently to avidin or streptavidin. Following intensive optimization, proof of principle was finally demonstrated in 2006, which opened the door to a new research area. This personal Account critically assesses these early studies as well as subsequent efforts from my group focusing on different protein scaffolds, and includes briefly some of the most important current contributions of other groups. Two primary messages emerge: First, since organic chemists continue to be extremely good at designing and implementing man-made transition metal catalysts, often on a large scale, those scientists that are active in the equally intriguing field of directed evolution of artificial metalloenzymes should be moderate when generalizing claims. All factors required for a truly viable catalytic system need to beconsidered, especially activity and ease of upscaling. Second, the most exciting and thus far very rare cases of directed evolution of artificial metalloenzymes are those that focus on selective transformations that are not readily possible using state of the art transition metal catalysts.

Reetz, M.T.

Chem. Commun. 2006, 4318, 10.1039/b610461d

The concept of utilizing the methods of directed evolution for tuning the enantioselectivity of synthetic achiral metal–ligand centers anchored to proteins has been implemented experimentally for the first time.

Reetz, M.T.

Top. Organomet. Chem. 2009, 10.1007/3418_2008_12

Whereas the directed evolution of stereoselective enzymes provides a useful tool in asymmetric catalysis, generality cannot be claimed because enzymes as catalysts are restricted to a limited set of reaction types. Therefore, a new concept has been proposed, namely directed evolution of hybrid catalysts in which proteins serve as hosts for anchoring ligand/transition metal entities. Accordingly, appropriate genetic mutagenesis methods are applied to the gene of a given protein host, providing after expression a library of mutant proteins. These are purified and a ligand/transition metal anchored site-specifically. Following en masse ee-screening, the best hit is identified, and the corresponding mutant gene is used as a template for another round of mutagenesis, expression, purification, bioconjugation, and screening. This allows for a Darwinian optimization of transition metal catalysts.

Reetz, M.T.

Chimia 2002, 56, 721-723, 10.2533/000942902777679920

The first step in applying the recently proposed concept concerning the application of directed evolution to the creation of selective hybrid catalysts is described, specifically the covalent attachment of Mn-salen moieties and of Cu-, Pd-, and Rh-complexes of dipyridine derivatives as well as the implantation of a diphosphine moiety in a protein, future steps being cycles of mutagenesis/screening.

Clark, D.S.; Hartwig, J.F.; Keasling, J.D.; Mukhopadhyay, A.

Nat. Chem. 2021, 13, 1186-1191, 10.1038/s41557-021-00801-3

Synthetic biology enables microbial hosts to produce complex molecules from organisms that are rare or difficult to cultivate, but the structures of these molecules are limited to those formed by reactions of natural enzymes. The integration of artificial metalloenzymes (ArMs) that catalyse unnatural reactions into metabolic networks could broaden the cache of molecules produced biosynthetically. Here we report an engineered microbial cell expressing a heterologous biosynthetic pathway, containing both natural enzymes and ArMs, that produces an unnatural product with high diastereoselectivity. We engineered Escherichia coli with a heterologous terpene biosynthetic pathway and an ArM containing an iridium–porphyrin complex that was transported into the cell with a heterologous transport system. We improved the diastereoselectivity and product titre of the unnatural product by evolving the ArM and selecting the appropriate gene induction and cultivation conditions. This work shows that synthetic biology and synthetic chemistry can produce, by combining natural and artificial enzymes in whole cells, molecules that were previously inaccessible to nature.