John Steven Anderson - US grants

The objective of this project is to study how the enzymes of the cytoplasmic membrane of Micrococcus luteus, a gram positive bacterium, effect the biosynthesis of teichuronic acid which is covalently linked to the peptidoglycan. A series of intermediates containing undecaprenyl phosphate as carrier lipid effect teichuronic acid biosynthesis by sequential elongation of the carbohydrate chain of teichuronic acid. Modified cell preparations will be used to determine if some of the intermediates can transfer the putative teichuronic acid chain to peptidoglycan. Concomitant peptidoglycan synthesis may also be required. Another objective is to isolate and chemically characterize the unique linkage region oligosaccharide which joins teichuronic acid to peptidoglycan. An enzymatic and chemical degradation scheme based on the presumed structure of the linkage region will be followed by purification. Methylation analysis, mass spectrometry and nuclear magnetic resonance spectroscopy will be used for characterization. The glycosyltransferase which is involved in teichuronic acid chain elongation will be purified so that the mechanism can be determined by which glucosyl residues are incorporated into the polymer with retention of anomeric configuration while the alternate residues of the polymer are incorporated with inversion of configuration. Teichuronidase, an enzyme which degrades teichuronic acid, will be isolated from an organism which can utilize teichuronic acid as growth substrate. Characterization of the enzyme will follow. Immunoelectron microscopy utilizing antibodies directed against teichuronic acid will be used to evaluate the location of teichuronic acid in the cell wall and sites of synthesis in the cytoplasmic membrane. Proteus myxofaciens, a gram negative bacterium, produces an extracellular gel which will be isolated and characterized by methylation analysis, mass spectrometry and nuclear magnetic resonance spectroscopy.

Project Abstract This proposed research involves the investigation of distal anion effects on the properties of transition metal oxo complexes. Transition metal oxo species are invoked as central intermediates in a wide variety of enzymatic oxidations. This centrality has motivated substantial efforts at understanding their structure and function. Molecular model complexes have provided significant insights into oxo complexes by providing systems where hypotheses can be rationally and systematically studied. Nevertheless, it is becoming increasingly apparent that classic systems used to model oxo intermediates, which typically feature strongly donating anionic ligand sets, do not mimic the electronic structures or reactivities of some of the most interesting enzymatic active sites. Against this backdrop, recent results have underscored the importance of secondary coordination sphere effects in the function of oxo species. These studies have primarily focused on hydrogen bonding interactions. In this research program, we aim to investigate an alternative secondary coordination sphere influence, namely that of distal anionic charges. Enzymatic active sites can be highly charged and this effect can strongly influence the reactivity of different oxo species. However, the effect of the incorporation of distal charges has not been systematically investigated. We aim to rationally incorporate distal anions onto model oxo complexes in order to study the effect of anionic charge on the reactivity and properties of transition metal oxo species. By incorporating distal anion charges that are not in conjugation with transition metal centers we will be able to tune the redox potential of transition metal oxo complexes independent of the electronic structure of the M-O unit. This will allow us to modulate parameters such as O-H BDE's which should directly influence C-H abstraction capabilities. Furthermore, we anticipate that the incorporation of distal anions will enable the isolation and study of high-valent oxo complexes with comparatively weak ligand fields. These weak ligand fields should enable unusual electronic structures, such as high spin states, that have been proposed as important for crucial intermediates but have little to no synthetic precedent. As a final point, we will also target oxo complexes that have no structural precedent, particularly Cu oxo species. This broad strategy will enable fundamental insights into processes such as C-H activation/hydroxylation and oxygen evolution. The importance of charge on enzymatic function is well-established, but has not been investigated in the context of oxo intermediates. As such, our approach offers a great deal of promise in understanding the function of oxo complexes and rationally controlling their properties and reactivity.

Project Abstract This is a request for a diversity supplement to the existing grant R35GM133470 to hire Jorge Martinez as a post-doctoral scholar. Jorge Martinez is an outstanding Ph.D. candidate in the laboratory of Jeremy Smith. When he joins our group, Jorge will work on studying the fundamental effects of anionic charges on coordination complexes, particularly oxo complexes. This proposed research involves the investigation of distal anion effects on the properties of transition metal oxo complexes. Transition metal oxo species are invoked as central intermediates in a wide variety of enzymatic oxidations. This centrality has motivated substantial efforts at understanding their structure and function. Molecular model complexes have provided significant insights into oxo complexes by providing systems where hypotheses can be rationally and systematically studied. Nevertheless, it is becoming increasingly apparent that classic systems used to model oxo intermediates, which typically feature strongly donating anionic ligand sets, do not mimic the electronic structures or reactivities of some of the most interesting enzymatic active sites. Against this backdrop, recent results have underscored the importance of secondary coordination sphere effects in the function of oxo species. These studies have primarily focused on hydrogen bonding interactions. In this research program, we aim to investigate an alternative secondary coordination sphere influence, namely that of distal anionic charges. Enzymatic active sites can be highly charged and this effect can strongly influence the reactivity of different oxo species. However, the effect of the incorporation of distal charges has not been systematically investigated. We aim to rationally incorporate distal anions onto model oxo complexes in order to study the effect of anionic charge on the reactivity and properties of transition metal oxo species. Jorge's project will involve a fac-chelating ligand where the number and position of negative charges can be systematically controlled. Furthermore, the system that Jorge will synthesize will enable tuning of the dipole or electric field of systems while also changing the overall charge of the system. Jorge will investigate how these variable affect fundamental properties such as redox-potential, backbonding, basicity, as well as more exotic phenomena such as O-centered radical character. Jorge will then be able to link these properties and the effects of anionic charges to important reactions such as C?H activation and oxygen evolution. In parallel with this research effort, there will be a comprehensive and curated training program to prepare Jorge for a career as a PI at a major research institution. This will involve training in communication, both written and verbal, as well as new instrumental and analytical techniques. Furthermore, there is a multi-component plan aimed at networking interactions for Jorge with the community more broadly and with other faculty at UChicago and other institutions in particular.