Synthesis of [13C]-isotopomers of indole and tryptophan for use in the analysis of indole-3-acetic acid biosynthesis

Abstract

The direct conversion of indole to indole-3-acetic acid without tryptophan as an intermediate has previously been shown to occur in vivo, as well as in vitro, with seedlings of plants. In order to facilitate the purification of the enzymes that carry out the enzymatic synthesis of indole-3-acetic acid from labeled indole, it was necessary to develop an assay that had both high sensitivity and analytical precision. To obtain the required analytical resolution and to allow definitive product identification, [¹³C₆]indole was synthesized for use in GC-MS assays of the enzymatic conversion. Plants have been shown to be able to synthesize indole-3-acetic acid either directly from indole as well as by degradation of tryptophan. Thus, in order to allow the biochemical discrimination between these processes, the synthesized [¹³C₆]indole was used as a starting material for a novel enzymatic synthesis of [¹³C]isotopomers of L-tryptophan labeled at specific positions. Together, these isotope labeled indolic compounds offer powerful new approaches to understanding and differentiating routes of indole-3-acetic acid biosynthesis in vitro and in vivo. Copyright © 2004 John Wiley & Sons, Ltd.