As the most abundant intracellular antioxidant, glutathione (GSH) has been extensively studied for its roles in hydrogen peroxide reduction and other enzymatic pathways, but it has not been well studied for its metal binding properties. GSH has many potential sites for metal binding: the carboxylic acid groups on both the glycine and the glutamate ends of the molecule, the glutamate amine, and the sulfur on the central cysteine. GSH also has a γ-glutamyl bond rather than the α-glutamyl typical of most peptides, and the function of this unusual linkage has not been studied in light of its antioxidant or metal binding properties. To explore the structural and metal binding properties of GSH, two target GSH analogs were synthesized. The first, synthesized through typical peptide strategies, has the same amino acid sequence but with an α-glutamyl bond rather than the γ-glutamyl bond of GSH. The second target analog is a tri-peptoid, consisting of the same amino acid R groups as GSH but bound to the nitrogen atoms rather than the α-carbon. The γ-glutamyl bond was maintained in this analog, and synthesis of this bond was achieved using HOBt/carbodiimide coupling strategies that are common in the synthesis of γ-peptides. These analogs were then studied in DNA damage gel electrophoresis experiments to determine their ability to prevent copper- and iron-mediated oxidative DNA damage compared to glutathione. Electron paramagnetic resonance (EPR) studies with Cu<sup>2+</sup> experiments were used to determine the metal binding sites of each molecule to determine the importance of specifically the carboxylic acid and amine groups on the glutamate for the metal binding properties of glutathione. This work establishes the link between the antioxidant and metal binding properties of glutathione, an understanding that will allow for more targeted glutathione analog design.

Strategic synthesis of glutathione analogs for determining structural features required for antioxidant and metal binding properties

ACS Fall 2021

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