DOI: 10.1002/cctc.201200671 Artificial Copper Enzymes for Asymmetric Diels–Alder Reactions Peter J. Deuss, Gina Popa, Alexandra M. Z. Slawin, Wouter Laan,* and Paul C. J. Kamer* [a] Introduction Natural enzymes have evolved over millions of years to cata- lyze biologically relevant chemical reactions under mild reac- tion conditions with excellent rates and enantio-, regio-, and chemoselectivity. These properties render enzymes attractive synthetic tools as biocatalysts for the chemical industry. [1] Moreover, the powerful methods of directed evolution allow for significant improvements of the useful properties of en- zymes. [2] However, for many synthetically important, transition- metal-catalyzed transformations, for example, hydrogenation, allylic substitution and hydroformylation, enzymes do not exist. Therefore, these reactions are still confined to the realm of homogeneous catalysis, relying on man-made organo- metallic catalysts. Hybrid catalysts, consisting of a protein scaffold functional- ized with a synthetic transition metal catalyst, have emerged as a potential solution to this limitation. This approach aims to combine the catalytic activity of transition-metal complexes with the significant potential for selectivity-inducing interac- tions between proteins and substrates. Inspired by the pio- neering work of the groups of Kaiser [3] and Whitesides, [4] sever- al groups have developed artificial metalloenzymes, either by non-covalent or covalent incorporation of catalytic moieties within a macromolecular host. A particularly successful exam- ple of the non-covalent approach is the work of Whitesides, Chan, and particularly Ward, based on the well-developed biotin-avidin technology. Chemical modification of biotin with phosphorous ligands allows the introduction of transition metal catalysts in (strept)avidin. Subsequent tuning of both the transition-metal–ligand structure and the protein sequence has resulted in highly selective catalysts for asymmetric hydro- genation of amino acid precursors [5] and an impressive wide range of other catalytic transformations. [6] Moreover, Reetz sub- jected this system to directed evolution, demonstrating that this method can also be applied to improve the performance of hybrid catalysts. [7] Other examples that demonstrate the power of these hybrid catalysts are raised antibodies against transition-metal complexes and subsequent use in catalytic transformation [8] and the reconstitution of apomyoglobin with Cr III Schiff base complexes. [9] The group of Reetz has developed enantioselective artificial metalloenzymes based on commercially available copper phthalocyanine in combination with various serum albumins for copper-catalysed Diels–Alder reactions. [10] These systems proved surprisingly effective showing good conversions, high endo selectivities, and high enantioselectivities. Recently, Reetz et al. reported the introduction of a copper-binding site in the synthase subunit of imidazole glycerol phosphate synthase from Thermotoga maritime. [11] This protein was specifically se- lected for its thermo-stability which allows efficient purification by a simple heat treatment to separate this protein directly from the cell extract. The binding site consists of a geometrical- ly appropriately introduced histidine–histidine–asparagine motif, which was inspired by natural copper binding sites in proteins. These mutations were introduced using step-wise site-directed mutagenesis. Copper coordination to this motif was confirmed by using electron paramagnetic resonance spectroscopy. The obtained artificial metalloenzyme showed The development of artificial copper enzymes from sterol carri- er protein type 2 like domain (SCP-2L) for the use in asymmet- ric catalysis was explored. For this purpose, proteins were modified with various nitrogen donor ligands. Maleimide-con- taining ligands were found most suitable for selective cysteine bio-conjugation. Fluorescence spectroscopy was used to con- firm copper binding to an introduced phenanthroline ligand, which was introduced in two unique cysteine containing SCP- 2L mutants. Copper adducts of several modified SCP-2L tem- plates were applied in asymmetric Diels–Alder reactions. A clear influence of both the protein environment and the intro- duced ligand was found in the asymmetric Diels–Alder reaction between azachalcone and cyclopentadiene. A promising enan- tioselectivity of 25 % ee was obtained by using SCP-2LV83C modified with phenanthroline–maleimide ligand. Good endo selectivity was observed for SCP-2L modified with the dipicolyl- amine-based nitrogen donor ligand. These artificial metalloen- zymes provide a suitable starting point for the implementation of various available techniques to optimise the performance of this system. [a] Dr. P. J. Deuss, Dr. G. Popa, Prof. Dr. A. M. Z. Slawin, Dr. W. Laan, Prof. Dr. P. C. J. Kamer EaStCHEM, School of Chemistry University of St. Andrews North Haugh, St. Andrews (UK) E-mail : wwl1@st-andrews.ac.uk pcjk@st-andrews.ac.uk Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cctc.201200671.  2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ChemCatChem 2013, 5, 1184 – 1191 1184 CHEMCATCHEM FULL PAPERS