Controllable multi-halogenation of a non-native substrate by SyrB2 iron halogenase.

Geminal, multi-halogenated functional groups are widespread in natural products and pharmaceuticals, yet no synthetic methodologies exist that enable selective multi-halogenation of unactivated C-H bonds. Biocatalysts are powerful tools for late-stage C-H functionalization, as they operate with high degrees of regio-, chemo-, and stereoselectivity. 2-oxoglutarate (2OG)-dependent non-heme iron halogenases chlorinate and brominate aliphatic C-H bonds offering a solution for achieving these challenging transformations. Here, we describe the ability of a non-heme iron halogenase, SyrB2, to controllably halogenate non-native substrate alpha-aminobutyric acid (Aba) to yield mono-chlorinated, di-chlorinated, and tri-chlorinated products. These chemoselective outcomes are achieved by controlling the loading of 2OG cofactor and SyrB2 biocatalyst. By using a ferredoxin-based biological reductant for electron transfer to the catalytic center of SyrB2, we demonstrate order-of-magnitude enhancement in the yield of tri-chlorinated product that were previously inaccessible using any single halogenase enzyme. We also apply these strategies to broaden SyrB2's reactivity scope to include multi-bromination and demonstrate chemoenzymatic conversion of the ethyl side chain in Aba to an ethylyne functional group. We show how steric hindrance induced by the successive addition of halogen atoms on Aba's C4 carbon dictates the degree of multi-halogenation by hampering C3-C4 bond rotation within SyrB2's catalytic pocket. Overall, our work showcases the synthetic potential of iron halogenases to facilitate multi-C-H functionalization chemistry.

Electrostatically regulated active site assembly governs reactivity in non-heme iron halogenases.

Non-heme iron halogenases (NHFe-Hals) catalyze the direct insertion of a chloride/bromide ion at an unactivated carbon position using a high-valent haloferryl intermediate. Despite more than a decade of structural and mechanistic characterization, how NHFe-Hals preferentially bind specific anions and substrates for C-H functionalization remains unknown. Herein, using lysine halogenating BesD and HalB enzymes as model systems, we demonstrate strong positive cooperativity between anion and substrate binding to the catalytic pocket. Detailed computational investigations indicate that a negatively charged glutamate hydrogen-bonded to iron's equatorial-aqua ligand acts as an electrostatic lock preventing both lysine and anion binding in the absence of the other. Using a combination of UV-Vis spectroscopy, binding affinity studies, stopped-flow kinetics investigations, and biochemical assays, we explore the implication of such active site assembly towards chlorination, bromination, and azidation reactivities. Overall, our work highlights previously unknown features regarding how anion-substrate pair binding govern reactivity of iron halogenases that are crucial for engineering next-generation C-H functionalization biocatalysts.

A conserved asparagine in a ubiquitin-conjugating enzyme positions the substrate for nucleophilic attack.

The mechanism used by the ubiquitin-conjugating enzyme, Ubc13, to catalyze ubiquitination is probed with three computational techniques: Born-Oppenheimer molecular dynamics, single point quantum mechanics/molecular mechanics energies, and classical molecular dynamics. These simulations support a long-held hypothesis and show that Ubc13-catalyzed ubiquitination uses a stepwise, nucleophilic attack mechanism. Furthermore, they show that the first step-the formation of a tetrahedral, zwitterionic intermediate-is rate limiting. However, these simulations contradict another popular hypothesis that supposes that the negative charge on the intermediate is stabilized by a highly conserved asparagine (Asn79 in Ubc13). Instead, calculated reaction profiles of the N79A mutant illustrate how charge stabilization actually increases the barrier to product formation. Finally, an alternate role for Asn79 is suggested by simulations of wild-type, N79A, N79D, and H77A Ubc13: it stabilizes the motion of the electrophile prior to the reaction, positioning it for nucleophilic attack. © 2019 Wiley Periodicals, Inc.

Carbodiimide Synthesis via Ti-Catalyzed Nitrene Transfer from Diazenes to Isocyanides.

Simple Ti imido halide complexes such as [Br2Ti(N t Bu)py2]2 are competent catalysts for the synthesis of unsymmetrical carbodiimides via Ti-catalyzed nitrene transfer from diazenes or azides to isocyanides. Both alkyl and aryl isocyanides are compatible with the reaction conditions, although product inhibition with sterically unencumbered substrates sometimes limits the yield when diazenes are employed as the oxidant. The reaction mechanism has been investigated both experimentally and computationally, wherein a key feature is that the product release is triggered by electron transfer from an η 2-carbodiimide to a Ti-bound azobenzene. This ligand-to-ligand redox buffering obviates the need for high-energy formally TiII intermediates and provides further evidence that substrate and product "redox noninnocence" can promote unusual Ti redox catalytic transformations.

Molecular dynamics simulations reveal a new role for a conserved active site asparagine in a ubiquitin-conjugating enzyme.

The role of a highly conserved active site asparagine (N79) in the ubiquitin conjugating enzyme, Ubc13, is probed using molecular dynamics simulations. Both wild type and mutant enzymes (N79A and N79D) are studied. Contrary to a popular hypothesis, we show that it is unlikely that N79 stabilizes a reaction intermediate, but instead preferentially hydrogen bonds to a loop near the active site. This keeps the sidechain carboxylate of an aspartate in the loop (D119) near the sidechain amine of the substrate lysine. Our simulations show that this distance increases in the mutants. D119 has been hypothesized to play a variety of roles in the enzyme, including deprotonating the substrate lysine, so changing this distance can have an effect on the enzyme's efficiency. Finally, we show that it is possible for the aspartate to deprotonate the substrate even across long distances if short water wires form that connect the proton donor and acceptor. Short water wires form with greater probability in the wild type than in mutant enzymes.