Azido Auxins: Synthesis and Biological Activity of Fluorescent Photoaffinity Labeling Agents

Abstract

Three auxin analogs, 4-, 5-, and 6-azido-3-indoleacetic acid (4N3-IAA, 5-N3-IAA, and 6-NA-IAA) have been synthesized for use as fluorescent photoaffinity labeling agents. The pKa values of these compounds (4-N3-IAA, 4.67; 5-Ns-IAA, 4.65; 6-N3-IAA, 4.66; all ± 0.04) are experimentally indisfinguishable from the pK. of 3-indoleacetic acid (IAA, 4.69 ± 0.04). The auxin activity of these IAA derivatives has been determined in several systems. In soybean, pea, and corn straight growth assays, all three analogs induce growth comparable to that caused by IAA. In the tobacco pith assay, anl three analogs elicit a maximum increase in fresh weight at least 40 to 50% of that caused by IAA. Optimal growth is attained in the tobacco pith assay at slightly higher concentrations of 4Ns-IAA and 6-N3-IAA (30 micromolar) than required for IAA (10 micromolar); however, maximal growth is achieved at a slightly lower concentration of 5-N3-IAA (3 micromolar). The NA-IAAs, like IAA, are transported basipetally through tobacco pith tissue.Auxins, the longest known class of plant hormones, modulate a wide variety of cell functions, the most significant being cell elongation (21,31,39,42,54,55). Despite extensive investigation, the mechanism of auxin action is still unknown, although it is now generally recognized that the response to auxins occurs in two phases (58,60,61). Within the past decade, reversible binding between auxins and several cell components has been demonstrated, establishing tentative locations of auxin receptors within the cell (17, 19, 23, 27, 43, 45, 50-52, 59, 61-66) (41).The decline in the rate of appearance of reports on auxin binding in the past 2 years compared to the rate during the 6 years immediately following the initial demonstration of auxin binding (23) and recent attempts to predict the properties of the auxin receptor from empirical structure-activity correlations (29,33,49) signal the need for a fresh approach to the problem of isolating an auxin receptor.To this end, we have synthesized three auxin analogs, 4-, 5-, and 6-azido-3-indoleacetic acid (4-N3-IAA, 5-N3-IAA, and 6-N3-IAA)7 for use as fluorescent photoaffinity labeling agents. The technique of photoaffinity labeling, which has been used to study animal hormones and to characterize the active sites of enzymes, has been thoroughly reviewed (6, 14-16, 30, 32

Azido Auxins

“…Electronic absorption and fluorescence emission spectra of IAA and the N3IAAs in buffer are summarized in Table I. Spectra of all four compounds agree with those previously obtained in water or in 95% ethanol (3,7,14). The N3IAAs are nonfluorescent, as expected.…”

“…The purpose of this loading step was to allow IAA or N3IAA to permeate the tissue and to occupy auxin-sensitive sites. At this point, the (14). The low growth observed under these conditions in sucrosebuffer implies that endogenous auxins were not being regenerated in significant amounts during the course of the experiments.…”

“…High quality, white, crystalline IAA (melting point > 167-169°C) was purchased from Sigma and stored in a brown vial at -5°C. The preparation of 4-, 5-, and 6-N3IAA has been described (14). These compounds were stored in brown, foilwrapped vials at 3C.…”

“…Earlier, we reported the synthesis of 4-, 5-, and 6-azido-3-indoleacetic acid and showed that, in the dark, these compounds are physiologically indistinguishable from IAA in every system tested (14). As a continuation ofthe assessment oftheir usefulness as fluorescent photoaffinity labeling agents, we now describe the photolysis of these compounds on TLC plates, in solution, and in vivo.…”

Auxin action and auxin‐binding proteins

“…Much use has been made of the photoactive auxin tritiated 5-azido-IAA (Melhado et al, 1981). Two comprehensive surveys and discussions of this work have been made Venis & Napier, 1995) and only a few summary remarks will be added here.…”