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The non‐heme iron ergothioneine synthase (EgtB) is a sulfoxide synthase that catalyzes oxidative C−S bond formation in the synthesis of ergothioneine, which plays roles against oxidative stress in cells. Despite extensive experimental and computational studies of the catalytic mechanisms of EgtB, the root causes for the selective C−S bond formation remain elusive. Using quantum mechanics/molecular mechanics (QM/MM) calculations, we show herein that a coordination switch of the sulfoxide intermediate is involved in the catalysis of the non‐heme iron EgtB. This coordination switch from the S to the O atom is driven by the S/π electrostatic interactions, which efficiently promotes the observed stereoselective C−S bond formation while bypassing cysteine dioxygenation. The present mechanism is in agreement with all available experimental data, including regioselectivity, stereoselectivity and KIE results. This match underscores the critical role of coordination switching in the catalysis of non‐heme enzymes. [ABSTRACT FROM AUTHOR]

Artificial metalloenzymes (ArMs), created by introducing synthetic cofactors into protein scaffolds, are an emerging class of catalyst for non‐natural reactions. Although many classes of ArMs are known, in vitro reconstitution of cofactors and proteins has been a limiting step in the high‐throughput screening and directed evolution of ArMs because purification of individual host proteins is time‐consuming. We describe the application of a platform to combine mutants of the P450 enzyme CYP119 and the cofactor Ir(Me)MPIX in vivo, by coexpression of the CYP119 mutants with the heme transporter encoded by the hug operon, to the directed evolution of ArMs containing Ir(Me)MPIX in whole cells. We applied this platform to the development an ArMs catalyzing the insertion of the acyclic carbene from α‐diazopropanoate esters (Me‐EDA) into the N−H bonds of N‐alkyl anilines, a combination of carbene and amine classes for which mutant enzymes of natural hemoproteins previously reacted with low enantioselectivity. The mutants of the artificial metalloenzyme Ir(Me)CYP119 identified by an evolution campaign involving more than 4000 mutants are shown to catalyze the reaction of Me‐EDA with N‐methyl anilines to form chiral chiral amino esters with high TON and good enantioselectivity, thereby demonstrating that the directed evolution of ArMs can rival that of natural enzymes in vivo. [ABSTRACT FROM AUTHOR]

The selective functionalization of one C−H bond over others in nearly identical steric and electronic environments can facilitate the construction of complex molecules. We report site‐selective functionalizations of C−H bonds, differentiated solely by remote substituents, catalyzed by artificial metalloenzymes (ArMs) that are generated from the combination of an evolvable P450 scaffold and an iridium‐porphyrin cofactor. The generated systems catalyze the insertion of carbenes into the C−H bonds of a range of phthalan derivatives containing substituents that render the two methylene positions in each phthalan inequivalent. These reactions occur with site‐selectivity ratios of up to 17.8:1 and, in most cases, with pairs of enzyme mutants that preferentially form each of the two constitutional isomers. This study demonstrates the potential of abiotic reactions catalyzed by metalloenzymes to functionalize C−H bonds with site selectivity that is difficult to achieve with small‐molecule catalysts. [ABSTRACT FROM AUTHOR]

An efficient copper-promoted difluoromethylthiolation of aryl and heteroaryl diazonium salts is described. The reaction is conducted under mild reaction conditions and various functional groups were compatible. In addition, reactions of heteroaryl diazonium salts such as pyridyl, quinolinyl, benzothiazolyl, thiophenyl, carbazolyl, and pyrazolyl diazonium salts occurred smoothly to afford the medicinally important difluoromethylthiolated heteroarenes. Furthermore, a more practical one-pot direct diazotization and difluoromethylthiolation protocol was developed, and it converts the aniline derivatives into difluoromethylthiolated arenes. The utility of the method is demonstrated by difluoromethylthiolation of a number of natural products and drug molecules. [ABSTRACT FROM AUTHOR]