Speaker: Biotinylated Indoles as Probes for Enzymes of Auxin Biosynthesis

Recent evidence indicates that one of the biosynthetic pathways leading to the plant hormone (auxin) indole-3-acetic acid involves indole as an intermediate (1, 2). In an attempt to provide tools for the detection of the so far unknown enzymes, we prepared indole derivatives coupled to biotin via an oligopeptide spacer. With the indole moiety anchored to the active site of the enzyme, the biotin moiety would be detected by means of avidin coupled to a suitable indicator molecule. A hydrophilic spacer of sufficient length (chain of 12 or more C, N, or O) is essential because both indole and biotin are poorly water-soluble, and the biotin-binding pocket is situated deeply within the avidin molecule (3). As an example, we here report on constructs composed of 3-(2-aminoethyl)indole (tryptamine), oligo-(beta-alanine), and d-biotin. Two strategies were explored: assembling the molecules starting with tryptamine or with biotin. The latter approach involved intermediates for which no suitable solvent could be found and was thus abandoned. With tryptamine as the starting compound, the synthesis of compound 2 (tripeptide spacer) was initiated by condensation with the hydroxysuccinimide ester of N-tert-butyloxycarbonyl (beta-alanine)*3 . The product was, after N-deprotection, reacted with the hydroxysuccinimide ester of biotin (4). Compound 2 was, after purification on a column of Sephadex G-10, obtained as a colorless solid moderately soluble in water. Characterization by LC-MS-FAB (glycerol matrix) afforded the protonated molecular ion M+H at 600 amu and daughter ions containing a. the tryptamine moiety plus 1 to 3 beta-alanyl residues protonated at the terminal nitrogen (232, 303, 374 amu), b. the biotinyl moiety plus 0 to 3 beta-alanyl residues (227, 298, 369, 440 amu), c. S(3aS-(3aalpha, 4beta, 6aalpha))-hexahydro-2-oxo-1H-thieno(3, 4-d)imidazoleC+ at 143 amu, d. the M+H - 143C+ ion (457 amu). Further confirmation of the structure was obtained from C-13 and...