Studies in Peroxidase-like Activity of Dinuclear Metal Complexes: Application to Molecular Recognition and Catalysis

Abstract

Recently, dinuclear complexes with two metal ions in close proximity have been extensively investigated as a promising strategy for mimicking the function of natural enzymes because of the possibility of the cooperative effect of the two metal ions in enzymatic reactions. In particular, the mimicking of peroxidase which catalyzes the oxidation of various compounds in the presence of hydrogen peroxide (H2O2), have received significant attention because of their applicability in various fields such as the chemical, pharmaceutical, and in biosensing systems. In this dissertation, (1) the application of peroxidase activity of dinuclear complex to biological important target detection through catalytic signal amplification process and (2) the derivation of novel dinuclear complex which has superior peroxidase activity through high-throughput approach was dealt. In the field of molecular recognition, selective detection of adenosine monophosphate (AMP) which has a lower charge than adenosine triphosphate (ATP) and pyrophosphate is very challenging because of its relatively low binding affinity for phosphate receptors. In chapter 1, colorimetric discrimination of nucleoside phosphates was achieved based on catalytic signal amplification through the activation of Mn2(bpmp) complex which have peroxidase activity. This method showed high selectivity for AMP over ATP and pyrophosphate, unlike previous phosphate sensors that use Zn2+-dipicolylamine-based receptors. High selectivity of the suggested method allowed discrimination of AMP in aqueous solution by the naked eye, and the detection limit was estimated to be 0.5 μM. Mechanism analysis revealed AMP acted as activators in the peroxidation cycle of the Mn2(bpmp)/ABTS/H2O2 system despite having relatively low binding affinity. Additionally, high selectivity and quantitative signal amplification allowed for the development of colorimetric phosphodiesterases assay method. The newly proposed assay method showed significant advantages over other assay methods by: (1) being label- and antibody free; (2) detecting the phosphate derivative directly, without conversion; (3) providing both end-point and real-time assays, enabling quantitative analysis of enzyme activities; and (4) providing a screen for inhibitors and determination of IC50 values. In nature, some enzymes, which have the bimetallic systems, utilize two different metal ions in their catalytic system. Also, some synthetic catalysts that have heterodinuclear metal system show better synergistic effects than homodinuclear metal system. In chapter 2, inspired by these points, we attempted to derive novel heterodinuclear complex with superior peroxidase activity based on the high-throughput approach. For parallel analysis of peroxidase activity of metal complexes, we used ABTS as colorimetric substrate because its peroxidation catalysed by metal complexes induce the change colorless ABTS to the deep green ABTS radical cation (ABTS•+) and it allow colorimetric high-throughput screening of peroxidase activity. From the in-situ generated metal complex library, which composed of combination of six dinucleating unsymmetrical ligands, eight 1st row transition metal ions, and three pH, we derived one “hit” metal complex, L3-(Ni2+, Co2+) complex which have superior peroxidase activity. Steady-state kinetics revealed the kcat/Km ratio, the catalytic efficiency, of L3-(Ni2+, Co2+) complex increased than Mn2(bpmp) complex about 3~4 times in both the oxidation and reduction process, respectively, in peroxidation reaction cycle. Also, spin trap experiment based on the EPR was revealed that the generation of hydroperoxyl radical (•OOH) as key intermediates by the reaction between L3-(Ni2+, Co2+) complex and H2O2. Furthermore, unlike previous peroxidase mimic, L3-(Ni2+, Co2+) complex was not catalyze the over-oxidation of ABTS, as a result, the colorimetric response originated from ABTS•+ was maintained more than 3 hours. For this reason, it is expected L3-(Ni2+, Co2+) complex could show great advantages when applied to colorimetric cascade assays in analytical method as alternative of horseradish peroxidase (HRP) or their mimic.