Steric Course of the NIH Shift in the Enzymic Formation of Homogentisic Acid

1 4‐Hydroxyphenylpyruvate dioxygenase has been purified from beef liver to a specific activity of 0.021 U/mg at 25°C. 2 Samples of (3 R )‐ and (3 S )‐4′‐hydroxyphenyl[3‐ 2 H 1 ]pyruvate were prepared by taking advantage of the known stereospecificity of phenylpyruvate keto—enol‐isomerase (tautomerase). These were converted in situ by the dioxygenase into the corresponding deuterium‐labelled homogentisic acids, which were isolated as the crystalline 3′,5′‐dimethyl derivatives after treatment with dimethyl sulphate. 3 2′,5′‐Dimethoxy‐( E )‐cinnamate was deuterated by enoate reductase from Clostridium sp. La 1 to afford (2 S , 3 R )‐3‐(2′,5′‐dimethoxyphenyl)‐[2‐ 2 H 1 , 3‐ 2 H 1 ]propionate which was in turn chemically degraded to ( S )‐2,5‐dimethoxyphenyl[ 2 H 1 ]acetic acid. 4 After the chiroptical characterisation of the chiral 2,5‐dimethoxyphenyl[ 2 H 1 ]acetic acid had failed, the following samples of 2,5‐dimethoxyphenylacetic acids were converted into the (–)‐menthyl ester: (a) unlabelled, (b) statistically deuterated in position 2 (and in the aromatic positions), (c) (2 S )‐[2‐ 2 H 1 ] as reference, (d) produced by the dioxygenase from (3 R )‐4′‐hydroxyphenyl[3‐ 2 H 1 ]pyruvate, and (e) produced by the dioxygenase from (3 S )‐4′‐hydroxyphenyl[3‐ 2 H 1 ]pyruvate. 5 In the 500‐MHz 1 H‐NMR spectrum the 2‐methylene protons of unlabelled 2,5‐dimethoxyphenylacetic acid (–)‐menthyl ester appeared as a well resolved AB system. Using a triple resonance technique and the reference samples (b) and (c) the 2‐H Re atom could be correlated with the high‐field half and the 2‐H Si atom with the lowfield half of the AB system. 6 By comparison of the spectra of the enzymically obtained samples (d) and (e) with the reference spectra it was concluded that in the dioxygenase reaction the side‐chain migration occurred with retention of configuration at the methylene C atom.