Evidence of p-quinoid enamine formation during the oxidative desamination of p-hydroxy-d-phenylglycine catalyzed by d-amino acid oxidase

An enzymatic process for obtaining the corresponding α-keto acid of p-hydroxy-d-phenylglycine, a unnatural aromatic amino acid used in the synthesis of β-lactam antibiotics, was developed. For this, p-hydroxy-d-phenylglycine was oxidized by d-amino acid oxidase in the presence of catalase at pH 8.0. The gradual development of a 370 nm maximum was initially observed, which evolved to generate another maximum at 335 nm. The latter represented a product that was purified by anionic exchange chromatography and identified by NMR as the α-keto acid corresponding to p-hydroxy-d-phenylglycine. The intermediate with a maximum at 370 nm was taken as direct evidence that the enamine is formed as result of the chemical stabilization of the first imine form released by the enzyme. This imine–enamine equilibrium was demonstrated spectrophotometrically due to the particular structure of p-hydroxy-d-phenylglycine, which allows chemical stabilization via the formation of a p-quinone enamine. When the reaction was carried out with d-amino acid oxidase and catalase co-immobilized on Eupergit C, the enamine intermediate was separated from the enzyme, and its chemical decomposition constant was obtained (k = 0.16 min−1). However, this chemical evolution was not enough to explain the levels of enamine accumulated, when the enzyme concentration was increased. This observation indicates for the first time that the enzyme also contributes to the evolution of enamine into α-keto acid. A reaction scheme is proposed to explain the above-mentioned results.