Nature has evolutionarily integrated metal centers in some of its most crucial/efficient functional proteins, most of which successfully mediate catalytic chemical transformations utilizing non-precious metal catalysts under entirely environmentally benign reaction conditions. The pivotal nature of these metalloenzyme biochemistries often implicate them in human disease and diagnostics, whereupon a clear comprehension of their mechanistic details may lead to effective therapeutics against some of the most challenging pathological conditions humans face in present day, such as cancer, rheumatoid arthritis, Alzheimer’s or Huntington’s. The efficiency in which nature utilizes these greener, cheaper metal systems in enzymatic catalytic turnovers also offers invaluable chemistry lessons to humans on how to move away from toxic, expensive metal catalysts that are currently in use for industrial-scale bulk transformations.

Wijeratne Research Laboratory utilizes synthetic organic and inorganic chemical tools to generate inorganic model complexes that resemble metalloprotein active sites, and then studies their reactivity profiles with small molecule biological substrates such as dioxygen (O2) and/or nitrogen oxides (NOx’s; e.g., NO, N2O, NO­2, NO3) under inert laboratory conditions (i.e., utilizing glovebox/Schlenk techniques). One of the primary goals of this work is to identify biologically relevant intermediates/active species using synthetic model systems. Such reaction intermediates often display impaired thermal/chemical stability, and thus require specialized cryogenic techniques for unequivocal characterization.

An array of separation, purification, and spectroscopic and structural characterization strategies will be applied, such as chromatography, electronic absorption (UV-Vis), electronic paramagnetic resonance (EPR), nuclear magnetic resonance (NMR), Infrared (IR), resonance Raman (rR), and X-ray absorption (XANES & EXAFS) spectroscopies, mass spectrometry, and X-ray diffraction (XRD) along with electrochemical analysis (e.g., cyclic voltammetry (CV) and differential pulse voltammetry (DPV)).  Careful thermodynamic and kinetic analyses (i.e., Eyring-, Arrhenius-, Polanyi-, and Hamett-type, kinetic isotope effects (KIE), and bond dissociation (free) energy (BD(F)E) calculations) of substrate and/or self reactivities of these species will lead into crucial insights that relate to key unknowns pertaining to the corresponding metalloprotein systems. Complementary Density Functional Theory (DFT) computations will also be employed as warranted. Comprehensive understanding of the bio-related chemistries will pave the way into novel, highly effective therapeutics, as well as greener (nature-inspired) methodologies for industrial scale catalytic applications. The undergraduate, graduate and postdoctoral researchers engaged in these research attempts will progress to adepts in synthetic, spectroscopic and structural approaches for tackling mechanistic ambiguities, with sound knowledge of biological, inorganic, organic and physical chemistries, and their potential roles in industrial applications. Wijeratne Research Group actively collaborates with multiple on-campus, local and international research groups and national labs/facilities, including the UAB Centers for Free Radical Biology (CFRB) and Nanoscale Materials and Biointegration (CNMB).

Reactivity Landscapes involving Heme-superoxo/peroxo/oxo Intermediates

Heme-oxygen intermediates are key species in all dioxygen-activating heme enzymes, and heme-superoxide or heme-oxy species are the first adducts that form upon O2 binding to the heme center. These heme-oxy reaction intermediates may get further reduced to heme-peroxo compounds, eventually leading to O–O bond cleavage (typically with the involvement of protons) either generating Cmpd-II or Cmpd-I species. Therefore, heme-oxy/heme-superoxo species are central to a variety of heme enzymes, and mediate a diverse array of reaction pathways depending on the local environment (i.e., distal and proximal) about the active site.

Adapted from: Adam, S. A., Wijeratne, G. B. et al. Chem. Rev. 2018, 118, 10840–11022.

Biological heme-superoxide intermediates are proposed to mediate a number of pivotal, experimentally-demanding transformations, however, modeling such reactivities using synthetic analogues has remained a massive challenge. We strive to overcome this challenge, and synthesize/utilize bio-inspired small-molecule heme-superoxo models to effectively mimic their bio-relevant reactivity pathways, which could lead to the observation and characterization of critical mechanistic intermediates/events. One heme enzyme catalyzed transformation that has gained significant biochemical and biomedical research interest in the recent past is tryptophan dioxygenation by tryptophan 2,3-dioxygenase and indoleamine 2,3-dioxygenase (TDO/IDO).

Tryptophan Dioxygenation to N-formylkynurenine using Dioxygen in Biology

Adapted from: Mondal, P. et al. J. Am. Chem. Soc. 2020, 142, 1846-1856.

TDO/IDO are unique heme enzymes that utilize a heme superoxide adduct (O2 derived) in mediating the 2,3-oxidative ring-opening cleavage of the indole moiety in tryptophan, and only require a single reduction event during complete enzymatic turnover. This process is the first and rate-limiting step of the kynurenine pathway, and has been linked to an array of critically important human pathogenic conditions including Huntington’s disease, depression, rheumatoid arthritis, type-I diabetes, and cancer. Accordingly, TDO/IDO inhibitors are rapidly emerging (anti-cancer) drug targets. Nevertheless, the rational design of efficient inhibitors and/or the comprehension of aforementioned disease progression pathways is significantly impeded by the lack of understanding of the precise mechanistic details pertaining to TDO/IDO functionality.

A “base-catalyzed” TDO/IDO mechanism had been heavily reproduced in the literature (pathway A below), although recent key findings have warranted severe revisions of this proposal. Two current mechanistic proposals exist; in those, the initial indole activation step is either an electrophilic (pathway B below) or a radical (pathway C below) addition step. Both pathways converge at a common event; formation of the ferryl (FeIV=O) species and the indole epoxide upon O-O bond cleavage.

For the first time in model chemistry, our work has shown that the synthetic heme superoxide mimics can indeed mimic the indole dioxygenation chemistry of biological heme-oxy systems, where a series of electronically divergent heme-superoxo models were reported to demonstrate indole dioxygenation across an array of indoles (J. Am. Chem. Soc. 2020, 142, 1846-1856). This work also sheds light on key reaction intermediates involved (i.e., the ferryl intermediate that forms during the reaction), while providing evidence in support of the electrophilic mechanism proposed for TDO/IDO-dependent tryptophan dioxygenation (pathway B above). However, key unknowns still exist, which will be the focus of future studies.

Adapted from: Mondal, P. et al. J. Am. Chem. Soc. 2020, 142, 1846-1856.