Artificial metalloenzymes (ArMs) catalyzing new-to-nature reactions could play an important role in transitioning toward a sustainable economy. While ArMs have been created for various transformations, attempts at their genetic optimization have been case specific and resulted mostly in modest improvements. To realize their full potential, methods to rapidly discover active ArM variants for ideally any reaction of interest are required. Here, we introduce a reaction-independent, automation-compatible platform, which relies on periplasmic compartmentalization in Escherichia coli to rapidly and reliably engineer ArMs based on the biotin-streptavidin technology. We systematically assess 400 ArM mutants for five bioorthogonal transformations involving different metals, reaction mechanisms, and reactants, which include novel ArMs for gold-catalyzed hydroamination and hydroarylation. Activity enhancements up to 15-fold highlight the potential of the systematic approach. Furthermore, we suggest smart screening strategies and build machine learning models that accurately predict ArM activity from sequence, which has crucial implications for future ArM development.

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Artificial metalloenzymes (ArMs) result from anchoring an organometallic catalyst within an evolvable protein scaffold. Thanks to its dimer of dimers quaternary structure, streptavidin allows the precise positioning of two metal cofactors to activate a single substrate, thus expanding the reaction scope accessible to ArMs. To validate this concept, we report herein on our efforts to engineer and evolve an artificial hydroaminase based on dual-gold activation of alkynes. Guided by modelling, we designed a chimeric streptavidin equipped with a hydrophobic lid shielding its active site which enforces the advantageous positioning of two synergistic biotinylated gold cofactors. Three rounds of directed evolution using E. coli cellfree extracts led to the identification of mutants favouring either the anti-Markovnikov product (an indole carboxamide with 96% regioselectivity, 51 TONs) resulting from a dual gold activation of an ethynylphenylurea substrate or the Markovnikov product (a phenyldihydroquinazolinone with 99% regioselectivity, 333 TONs) resulting from the -activation of the alkyne by gold. genetic means. Thus far, more than 40 reactions can be catalysed by ArMs. 25 Current challenges in the field include; protein-accelerated catalysis, whereby a pre-catalyst is activated upon incorporation within the host protein, 26 dual catalysis 27,28 and compatibility of the ArM with a cytosolic environment. 29 Privileged scaffolds for ArMs include: carbonic anhydrase, 30 hemoproteins, 31,32 prolyl oligopeptidase, 33 lactococcal multiresistance regulator, 23 four helix-bundles, 34,35 nitrobindin, 36 human serum albumin, 37 and (strept)avidin. 20,[38][39][40] The work presented herein capitalizes on the unique topology of Sav enabling the localization of two close-lying biotinylated probes within a hydrophobic environment. This enabled the engineering and evolution of a biocompatible artificial hydroaminase (HAMase hereafter) based on either single-or dual-gold activation of an alkyne, Figure 1. Results Design of the artificial hydroaminaseAs reported by Asensio 5,41 and van der Vlugt 42 , the regioselectivity for the hydroamination of ethynylurea 1 is by-and-large governed by the mode of activation of the alkyne by gold: the canonical -activation favours the quinazolinone 3 (Markovnikov, 6-exo-dig addition product), while the dual -gold activation affords preferentially the indole 2 (anti-Markovnikov, 5endo-dig addition product) 5,42,43 Upon -coordination of the alkyne to gold, the pKa of the terminal C-H bond is lowered, thus favouring its deprotonation and coordination by a second gold to afford the -activation mode. 41 Accordingly, the spatial arrangement of the two gold species is critical in determining the regioselectivity of the reaction. We thus selected the gold-catalyzed cyclization of the ethynylurea 1 to engineer and evolve a dual-gold catalysed hydroaminase (HAMase) based on the biotin-streptavidin technology.Thanks to its dimer of dimers quaternary structure, which places the valeric acid side ch...

A mild and practical Barbier-Negishi coupling of secondary alkyl bromides with aryl and alkenyl triflates and nonaflates has been developed. This challenging reaction was enabled by the use of a very bulky imidazole-based phosphine ligand, which resulted in good yields as well as good chemo- and site selectivities for a broad range of substrates at room temperature and under non-aqueous conditions. This reaction was extended to primary alkyl bromides by using an analogous pyrazole-based ligand.

Artificial metalloenzymes (ArMs) catalyze new-to-nature reactions under mild conditions and could therefore play an important role in the transition to a sustainable, circular economy. While ArMs have been created for a variety of reactions, their activity for most biorthogonal transformations has remained modest and attempts at optimizing them by means of enzyme engineering have been case-specific and unsystematic. To realize the great potential of ArMs for biocatalysis and synthetic biology, there is a need for methods that enable the rapid discovery of highly active ArM variants for any reaction of interest. Here, we present a broadly applicable, automation-compatible ArM engineering platform. It relies on periplasmic compartmentalization of the ArM in Escherichia coli to rapidly and reliably identify improved variants based on the biotin-streptavidin technology. We assess 400 sequence-verified ArM mutants for five bio-orthogonal transformations involving different metal cofactors, reaction mechanisms and substrate-product pairs, including novel ArMs for gold-catalyzed hydroamination and hydroarylation. The achieved activity enhancements of six-to fifteen-fold highlight the potential of the proposed systematic approach to ArM engineering. We further capitalize on the sequence-activity data to suggest and validate smart strategies for future screening campaigns. This systematic, multi-reaction study has important implications for the development of highly active ArMs for novel applications in biocatalysis and synthetic biology.