No abstract

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 η2-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.

Chemo-and regio-selective catalysis of the C(sp 3 )-H halogenation reaction is a formidable goal in chemical synthesis. 2-Oxoglutarate (2OG)-dependent non-heme iron halogenases catalyze selective chlorination/bromination of C−H bonds and exhibit high sequence and structural similarities with non-heme iron hydroxylases. How the secondary coordination sphere (SCS) of these two enzyme systems differentiate and determine their reactivity is not well understood. In this work, we show that specific positioning of redox-active tyrosine residues in the SCS of non-heme iron halogenases has a huge impact on their structure, function, and reactivity. We discover that a tyrosine residue (F121Y) rationally incorporated to hydrogen bond to iron's chloride ligand in SyrB2 halogenase undergoes post-translational oxidation to dihydroxyphenylalanine (DOPA) physiologically. A combination of spectroscopic, mass-spectrometric, and biochemical studies demonstrate that DOPA modification in SyrB2 renders the enzyme non-functional. Bioinformatic analysis suggests that SyrB2-like halogenases, unlike hydroxylases, have a conserved placement of phenylalanine at position 121 to preclude such unproductive oxidation. Furthermore, molecular dynamics simulations in tandem with experimental demonstration of DOPA incorporation exclusively at position 121 enables us to uniquely identify that an axial-chloro haloferryl isomer is operant in SyrB2. We also identify conserved redox-inactive residues in the SCS of other 2OG-dependent non-heme iron halogenases to avoid DOPA-like unproductive oxidations. Overall, this study demonstrates the importance of the SCS in controlling the structure and enzymatic activity of non-heme iron halogenases and will have significant implications toward the design of small-molecule and protein-based halogenation catalysts.

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.

We present classical molecular dynamics (MD), Born-Oppenheimer molecular dynamics (BOMD), and hybrid quantum mechanics/molecular mechanics (QM/MM) data. MD was performed using the GPU accelerated pmemd module of the AMBER14MD package. BOMD was performed using CP2K version 2.6. The reaction rates in BOMD were accelerated using the Metadynamics method. QM/MM was performed using ONIOM in the Gaussian09 suite of programs. Relevant input files for BOMD and QM/MM are available.