An Artificial Metalloenzyme Based on a Copper Heteroscorpionate Enables sp3 C-H Functionalization via Intramolecular Carbene Insertion.

The selective functionalization of sp3 C-H bonds is a versatile tool for the diversification of organic compounds. Combining attractive features of homogeneous and enzymatic catalysts, artificial metalloenzymes offer an ideal means to selectively modify these inert motifs. Herein, we report on a copper(I) heteroscorpionate complex embedded within streptavidin that catalyzes the intramolecular insertion of a carbene into sp3 C-H bonds. Target residues for genetic optimization of the artificial metalloenzyme were identified by quantum mechanics/molecular mechanics simulations. Double-saturation mutagenesis yielded detailed insight on the contribution of individual amino acids on the activity and the selectivity of the artificial metalloenzyme. Mutagenesis at a third position afforded a set of artificial metalloenzymes that catalyze the enantio- and regioselective formation of ß- and γ-lactams with high turnovers and promising enantioselectivities.

Directed evolution of artificial metalloenzymes for in vivo metathesis.

The field of biocatalysis has advanced from harnessing natural enzymes to using directed evolution to obtain new biocatalysts with tailor-made functions. Several tools have recently been developed to expand the natural enzymatic repertoire with abiotic reactions. For example, artificial metalloenzymes, which combine the versatile reaction scope of transition metals with the beneficial catalytic features of enzymes, offer an attractive means to engineer new reactions. Three complementary strategies exist: repurposing natural metalloenzymes for abiotic transformations; in silico metalloenzyme (re-)design; and incorporation of abiotic cofactors into proteins. The third strategy offers the opportunity to design a wide variety of artificial metalloenzymes for non-natural reactions. However, many metal cofactors are inhibited by cellular components and therefore require purification of the scaffold protein. This limits the throughput of genetic optimization schemes applied to artificial metalloenzymes and their applicability in vivo to expand natural metabolism. Here we report the compartmentalization and in vivo evolution of an artificial metalloenzyme for olefin metathesis, which represents an archetypal organometallic reaction without equivalent in nature. Building on previous work on an artificial metallohydrolase, we exploit the periplasm of Escherichia coli as a reaction compartment for the 'metathase' because it offers an auspicious environment for artificial metalloenzymes, mainly owing to low concentrations of inhibitors such as glutathione, which has recently been identified as a major inhibitor. This strategy facilitated the assembly of a functional metathase in vivo and its directed evolution with substantially increased throughput compared to conventional approaches that rely on purified protein variants. The evolved metathase compares favourably with commercial catalysts, shows activity for different metathesis substrates and can be further evolved in different directions by adjusting the workflow. Our results represent the systematic implementation and evolution of an artificial metalloenzyme that catalyses an abiotic reaction in vivo, with potential applications in, for example, non-natural metabolism.

Ultrahigh-Throughput Screening of an Artificial Metalloenzyme using Double Emulsions.

The potential for ultrahigh-throughput compartmentalization renders droplet microfluidics an attractive tool for the directed evolution of enzymes. Importantly, it ensures maintenance of the phenotype-genotype linkage, enabling reliable identification of improved mutants. Herein, we report an approach for ultrahigh-throughput screening of an artificial metalloenzyme in double emulsion droplets (DEs) using commercially available fluorescence-activated cell sorters (FACS). This protocol was validated by screening a 400 double-mutant streptavidin library for ruthenium-catalyzed deallylation of an alloc-protected aminocoumarin. The most active variants, identified by next-generation sequencing, were in good agreement with hits obtained using a 96-well plate procedure. These findings pave the way for the systematic implementation of FACS for the directed evolution of (artificial) enzymes and will significantly expand the accessibility of ultrahigh-throughput DE screening protocols.

Light-driven electron injection from a biotinylated triarylamine donor to [Ru(diimine)3](2+)-labeled streptavidin.

Electron transfer from a biotinylated electron donor to photochemically generated Ru(iii) complexes covalently anchored to streptavidin is demonstrated by means of time-resolved laser spectroscopy. Through site-selective mutagenesis, a single cysteine residue was engineered at four different positions on streptavidin, and a Ru(ii) tris-diimine complex was then bioconjugated to the exposed cysteines. A biotinylated triarylamine electron donor was added to the Ru(ii)-modified streptavidins to afford dyads localized within a streptavidin host. The resulting systems were subjected to electron transfer studies. In some of the explored mutants, the phototriggered electron transfer between triarylamine and Ru(iii) is complete within 10 ns, thus highlighting the potential of such artificial metalloenzymes to perform photoredox catalysis.

Improving the Catalytic Performance of an Artificial Metalloenzyme by Computational Design.

Artifical metalloenzymes combine the reactivity of small molecule catalysts with the selectivity of enzymes, and new methods are required to tune the catalytic properties of these systems for an application of interest. Structure-based computational design could help to identify amino acid mutations leading to improved catalytic activity and enantioselectivity. Here we describe the application of Rosetta Design for the genetic optimization of an artificial transfer hydrogenase (ATHase hereafter), [(η(5)-Cp*)Ir(pico)Cl] ⊂ WT hCA II (Cp* = Me5C5(-)), for the asymmetric reduction of a cyclic imine, the precursor of salsolsidine. Based on a crystal structure of the ATHase, computational design afforded four hCAII variants with protein backbone-stabilizing and hydrophobic cofactor-embedding mutations. In dansylamide-competition assays, these designs showed 46-64-fold improved affinity for the iridium pianostool complex [(η(5)-Cp*)Ir(pico)Cl]. Gratifyingly, the new designs yielded a significant improvement in both activity and enantioselectivity (from 70% ee (WT hCA II) to up to 92% ee and a 4-fold increase in total turnover number) for the production of (S)-salsolidine. Introducing additional hydrophobicity in the Cp*-moiety of the Ir-catalyst provided by adding a propyl substituent on the Cp* moiety yields the most (S)-selective (96% ee) ATHase reported to date. X-ray structural data indicate that the high enantioselectivity results from embedding the piano stool moiety within the protein, consistent with the computational model.

Anion-π catalysis: bicyclic products with four contiguous stereogenic centers from otherwise elusive diastereospecific domino reactions on π-acidic surfaces.

Anion-π interactions have been introduced recently to catalysis. The idea of stabilizing anionic intermediates and transition states on π-acidic surfaces is a new fundamental concept. By now, examples exist for asymmetric enolate, enamine, iminium and transamination chemistry, and the first anion-π enzyme has been created. Delocalized over large aromatic planes, anion-π interactions appear particularly attractive to stabilize extensive long-distance charge displacements during domino processes. Moving on from the formation of cyclohexane rings with five stereogenic centers in one step on a π-acidic surface, we here focus on asymmetric anion-π catalysis of domino reactions that afford bicyclic products with quaternary stereogenic centers. Catalyst screening includes a newly synthesized, better performing anion-π version of classical organocatalysts from cinchona alkaloids, and anion-π enzymes. We find stereoselectivities that are clearly better than the best ones reported with conventional catalysts, culminating in unprecedented diastereospecificity. Moreover, we describe achiral salts as supramolecular chirality enhancers and report the first artificial enzyme that operates in neutral water with anion-π interactions, i.e., interactions that are essentially new to enzymes. Evidence in support of contributions of anion-π interactions to asymmetric catalysis include increasing diastereo- and enantioselectivity with increasing rates, i.e., asymmetric transition-state stabilization in the presence of π-acidic surfaces and inhibition with the anion selectivity sequence NO3- > Br- > BF4- > PF6-.

An enantioselective artificial Suzukiase based on the biotin-streptavidin technology.

Introduction of a biotinylated monophosphine palladium complex within streptavidin affords an enantioselective artificial Suzukiase. Site-directed mutagenesis allowed the optimization of the activity and the enantioselectivity of this artificial metalloenzyme. A variety of atropisomeric biaryls were produced in good yields and up to 90% ee. The hybrid catalyst described herein shows comparable TOF to the previous aqueous-asymmetric Suzuki catalysts, and excellent stability under the reaction conditions to realize higher TON through longer reaction time.