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...

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Two general procedures were developed for the synthesis of chiral N mono , N,N´ di , N,N´N´´ tri , and N,N´,N´´,N´´´ tetraalkylglycolurils based on the reactions of 4,5 dihydroxy imidazolidin 2 ones or glyoxal with one or two moles of alkylureas, respectively, by acid catalysis. The reactions of N monoalkyl and N,N´ dialkylureas with glyoxal proceed regioselectively. The mechanism of these reactions was suggested and partly confirmed by quantum chemical calculations and experimental data. The enantiomeric separation of some chiral glycolurils by chiral phase HPLC was carried out for the first time.

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An efficient approach for the synthesis of monosubstituted aromatic compounds relying on a ring‐closing metathesis followed by spontaneous 1,2‐elimination is presented. The efficiency for late‐stage functionalization is highlighted in various solvents (up to 920 TON). This approach is compatible with strained cycles and other multiple bonds in the substrate.