Towards Mimicking Phosphoester Hydrolases in Aqueous Media
This document presents the research into the synthesis, characterization, and application of synthetic structural and functional model complexes of phosphoester hydrolases. Phosphoester hydrolases are a class of enzyme that specifically cleaves phosphoester bonds by hydrolytic insertion. While efforts have been put into understanding the mechanism for both the native enzymes as well as model complexes, many aspects of the mechanism remain unclear. This work focuses on new metal complexes utilizing a tetradentate ligand, H3camb, and heptadentate ligand, H5ccdp. These complexes serve as mimics of the active site structures and activity of phosphoester hydrolases. The catalytic activity of each complex was investigated using spectroscopic techniques including UV-vis in aqueous solutions. The ability of these complexes to hydrolyze phosphoester bonds was tested using 4-nitrophenyl phosphate, PNPP, and bis(4-nitrophenyl) phosphate, BNPP, as DNA model substrates for both monophosphoester and diphosphoester, respectively. The kinetic rates of hydrolysis for each metal complex were determined and compared. X-ray crystallographic and mass spectrometry information were utilized to propose mechanisms of action for the model complexes. The dicopper complex [NMe4]2[Cu2(ccdp)(μ-OAc)] performed twice as well with both PNPP and BNPP substrates as compared to the [NMe4]2[Zn2(ccdp)(μ-OAc)] complex, but both proved to be slower than the monometallic [Fe(camb)(H2O)2] and [Ga(camb)(H2O)2]. Additionally, the H5ccdp ligand also displayed a remarkable flexibility and a bound-substrate complex was isolated, crystallized, and examined for kinetic activity, gaining a new level of insight into the complex workings of the mechanism.