59th Annual Report on Research 2014

1. History of Assay Development. Inspiration for this assay came from the food chemistry literature. In 2001, Ou et al. described the use of fluorescein (FL, Fig. 1) in an assay of antioxidants.¹ In this assay, a standard oxidant degrades fluorescein, which is followed either by fluorescence or absorbance. The ability of an antioxidant to protect fluorescein is quantitated using intensities measured as a function of time.

2. Progress.

2.1. Dye Selection. Here the assay is adapted to measure oxidant potency. Initially we worked with fluorescein, for which some oxidative degradation pathways have been discussed.² However, quickly it became clear that FL is a poor substrate for this application: its solubility, ε and fluorescence in MeCN are limited. Most problematic, its absorption spectrum (λ_max 490 nm) overlaps significantly with those of many Fe catalysts. This means we cannot easily distinguish the degradation of the dye from changes in the metal complex as the reaction proceeds. This led to a search for other dye substrates. We searched for dyes that have an absorption maximum at 600 or more nm and also have some precedent for radical-based oxidation.³ This led us to malachite green (MG, Fig. 1).

2.2. Catalysts. This year we worked with two catalysts, the commercially available White-Chen catalyst (WC) and [Fe(dpa)₂]OTf₂ (Fig. 1). Fig. 2 illustrates the time-dependent degradation of MG in the presence of WC catalyst and H₂O₂. The loss of MG absorption is easily observed centered near 618 nm, and spectral overlap with WC is negligible.

2.3. Kinetic Results. We follow the loss of dye absorption peak at or near its maximum, tracking any overlap with the metal complex. We restrict the peroxides to those most commonly used in this area: H₂O₂ and tBuOOH. There are two modes of data collection: The first is spectral, in which we collect UV-vis spectra as a function of time. This allows us to better judge overlap and artifact issues, as well as provides inroads to mechanistic studies. The other mode is kinetic, observing at one or two wavelengths as a function of time. To stay the linear range of Beer’s Law, we are working at low uM MG concentrations. For the two metal complexes tested so far (WC and [Fe(dpa)₂]OTf₂), it is necessary for the metal complex concentration to be 10 to 50 times above that of the dye to get a convenient reaction rate (10-20 min at room temperature).

To normalize data obtained under different conditions for the purpose of comparison, we obtained first order rate constant for the reaction by collecting absorbance of the dye as a function of time at a range of metal complex concentration. The slope of this dependence is the first order rate constant with a unit of /time-metal complex concentration. Using this approach, we have already obtained preliminary 1^st order rate constants for the oxidation of MG as catalyzed by WC catalyst in the presence of H₂O₂ (3.2e-2 μM^-1min^-1) and tBuOOH (6.2e-4 μM^-1min^-1). Similar measurements for [Fe(dpa)₂]OTf₂ are in progress.

2.4. Mechanistic Investigations of Iron Oxidation Catalysis.  While performing control experiments during assay development, we observed spectra changes associated with the metal complex. By collecting spectra as a function of time, we determined rate constants for changes in peak intensities. As shown in Fig. 3, there are spectra changes on different timescales for [Fe(dpa)₂]OTf₂ and MG in the presence of H₂O₂. This suggests that there are changes in the metal complex that precede and possibly also accompany the degradation of the dye. Thus this assay provides a route to exploring the mechanism of the reaction.

2.5. Electrochemical Characterization. To better understand the spectral changes and possibly to correlate them to oxidation-reduction reactions, we have begun spectroelectrochemical measurements in collaboration with Dr. Mike Shaw at Southern Illinois University Edwardsville. We obtained preliminary CV and spectroelectrochemical data for WC and [Fe(dpa)₂]OTf₂ in MeCN. Fig. 4. The redox behavior of [Fe(dpa)₂]OTf₂ is reversible, and there are measurable spectral changes as a function of potential.

3. Summary and Outlook.

During the first year of the grant period, we worked, as outlined above, on the development of the assay. Some issues such as choosing the correct reporter dye needed to be resolved. This required the direct expertise of Co-PI Dr. Dupureur. While students were involved where possible, their experiments were limited during this time, leading to underspending on the budget. However, more recently research students are synthesizing iron complexes and collecting spectral data on a much larger scale. The remaining funds will be applied to support this work and advance the project.

A manuscript to be submitted for publication describing the assay is currently in preparation. During the first year of the grant, we collected enough data to submit a NSF grant proposal, which happened on September 29, 2014.

References

(1) Ou, B.; Hampsch-Woodill, M.; Flanagan, J.; Deemer, E. K.; Prior, R. L.; Huang, D. Journal of agricultural and food chemistry 2002, 50, 2772.

(2) Huang, D.; Ou, B.; Hampsch-Woodill, M.; Flanagan, J. A.; Deemer, E. K. Journal of agricultural and food chemistry 2002, 50, 1815.

(3) Chen, F.; Ma, W.; He, J.; Zhao, J. J. Phys. Chem. A 2002, 106, 9485.