Nicotinamide adenine dinucleotide (NAD+) is a biological redox mediator responsible for a large number of chemical reactions necessary for life. About 15% of documented enzymes rely on NAD+ or the related nicotinamide adenine dinucleotide phosphate (NADP+). Both cofactors have a redox-active nicotinamide site, which can undergo a net two-electron-one-proton reduction to form a high-energy 1,4-dihydropyridine, NAD(P)H. This dihydropyridine can reduce a substrate of interest through a net hydride transfer, regenerating NAD(P)+ in the process. As the chemical industry looks increasingly to bioderived compounds, the ability to control reactions involving NAD(P)H is a potential market opportunity, not only for their ability to transform biological feedstocks, but also for their safety (operating at or near room temperature) and their selectivity (reacting with high regio- and stereoselectivity). However, the cost of these cofactors – tens to hundreds of thousands of dollars per mole – is prohibitively high. Researchers have explored ways to regenerate NAD(P)H from NAD(P)+, which would make the cofactor more accessible for industry by enabling a catalytic amount to be used. An appealing way to regenerate NAD(P)H is by electrochemical reduction of NAD(P)+; however, the reduction is often intercepted after the first electron transfer to give an enzymatically-inactive (NAD(P)¬)2 dimer. The ability to design systems for regenerable NADH is hindered by a lack of understanding of which structural features correlate with dimerization, and which features correlate with reduction to NAD(P)H. Cofactor mimetics (mNAD+), which retain the redox active nicotinamide site but have variable molecular structures, have been explored as a platform for understanding the structure-function relationships governing the redox behavior of these cofactors. The purpose of the present thesis is to explore the electrochemistry of mNAD+, to understand which structural features correlate with dimerization, and how systems can be designed to favor reduction to mNADH over mNAD dimer. There are four chapters in this thesis. The first chapter is a literature review of the electrochemistry of NAD+ and mNAD+, with a special emphasis on methods of quantifying dimerization rates. The second chapter explores the effect of supporting electrolyte on the electrochemical reduction of mNAD+, and it is shown that sodium pyruvate favors the reduction of mNAD+ to a new product that has the same electrochemical oxidation signature as a sample of chemically-prepared mNADH. The third chapter explores the effect of both the molecular structure and the counterion of mNAD+ on the dimerization rate. It is shown that dimerization is faster at lower reduction potentials and, counterintuitively, when sterics at the 1-position are larger. This last trend suggests dimerization takes place preferentially at the 4-position, and that large 1-substituents favor arrangements in solution that place these redox-active sites close to each other. The electrochemistry data suggest that the reduction is more likely of mNAD+X- ion pairs than of lone mNAD+ ions, and NMR data reveal ionic interactions between mNAD+ and X- that are localized at the pyridinium 2- and 4-positions. The fourth chapter explores the mechanism of interaction between mNAD+ and pyruvate when they are reduced together. Evidence is provided in support of a mechanism whereby the intermediate mNAD radical mediates electron transfer to pyruvate, and that a pyruvate radical interacts with an mNAD radical to form mNADH in a non-catalytic way. Efforts to characterize the desired mNADH were unsuccessful, although NMR data show the appearance of a new CH2 unit with nonequivalent protons when mNAD+ and N,N-dimethylpyruvamide (DMP) are reduced together compared to when they are reduced separately. The major product of mNAD+ reduction without DMP is the 4,4’-dimer, consistent with the findings of Chapter 3. However, mNADH could not be conclusively identified by NMR. It is suggested that bulk electrolysis conditions mirror cyclic voltammetry conditions in every way possible to maximize mNADH formation.
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