PREPARATION, C-13 NMR CHARACTERIZATION, AND ENZYMATIC TRANSFORMATION OF [U-C-13]CHORISMATE TO [U-C-13]PREPHENATE AND [U-C-13]HYDROXYPHENYL PYRUVATE

Chorismate is the common precursor of the aromatic amino acids and a variety of plant and microbial natural products. [U-13C]Chorismate has been prepared from[U-13C]glucose using Klebsiella pneumoniae 62-1. With respect to previously published procedures, our yield represents a threefold improvement. [U-13C]Chorismate was purified directly from the growth medium in a single step by preparative HPLC. The 13C NMR spectrum of [U-13C]chorismic acid was obtained at both 75 MHz and 150 MHz (7.05 and 14.1 T). Near complete spectral assignment was achieved on the basis of chemical shifts, JCC, and 13C-1H coupling patterns. Assignment of C2 and C5 was accomplished via spectral simulation (program courtesy of Dr. Milton Johnston, USF) and verified using homonuclear 13C decoupling. Inspection of the 150-MHz spectrum allowed assignment of additional long range JCC. Partially purified Escherichia coli chorismate mutase/prephenate dehydrogenase was used to convert [U-13C]chorismate to [U-13C]prephenate, which in turn was converted to [U-13C] hydroxyphenyl pyruvate by the addition of NAD. 13C NMR of [U-13]prephenate and [U-13C] hydroxyphenyl pyruvate have been assigned based on chemical shifts, JCC, spectral simulation, and homonuclear 13C decoupling. © 1992.