Reversible Conversion In The Brassinosteroid Quartet Castasterone, Brassinolide And Their 3β-Epimers

Abstract

The metabolism of deuterated brassinosteroids has been studied in excised leaves of Secale cereale, and in vitro in seedlings of Arabidopsis thaliana and cell suspension cultures of Lycopersicon esculentum. In addition to the known biosynthetic conversion of castasterone to brassinolide and epimerization to 3-epicastasterone, inversion of the 3α-configured hydroxyl group of brassinolide to a 3β -configured one in 3-epibrassinolide has been observed using liquid chromatography (HPLC) with electrospray ionization (ESI) and selected ion-monitoring mass-spectrometry (SIM-MS). Administration of deuterated 3-epicastasterone and 3-epibrassinolide to Arabidopsis and Secale seedlings resulted in the formation of castasterone and brassinolide, respectively, indicating conversion of configuration at C-3 of brassinosteroids is reversible.

Labeled brassinosteroids for biochemical studies

“…A new biosynthetic subpathway from typhasterol/teasterone via 2,3‐epoxybrassinosteroids to castasterone was found in seedlings of S. cereale 29. A reversible conversion of the 3α‐configured hydroxyl group of brassinolide and castasterone into a 3β‐configured one of 3‐epibrassinolide and 3‐epicastasterone was detected in seedlings of A. thaliana and S. cereale 30…”

“…This work was realized through the joint efforts of the scientists from our laboratory listed in the references10,11,17,18,25,27–30 and of Dr. Bernd Schneider from the Max‐Planck Institute for Chemical Ecology (Jena, Germany).…”

Synthesis of deuterium‐labeled (24R)‐methyl brassinosteroids

“…The residue was purified by SiO 2 chromatography using CHCl 3 /MeOH (100:1) to give tetraacetate 14 (90 mg, 97%) as an oil. (22R,23R,24R)-24-Methy-B-homo-7-oxa-5a-cholestan-6-on-2a,3a,22,23-tetraol tetraacetate (15) A solution of trifluoroperbenzoic acid was prepared by adding trifluoroacetic anhydride (3.5 ml) to a stirred emulsion of H 2 O 2 (30%, 0.4 ml) in CH 2 Cl 2 (8.4 ml) at 01C. An aliquot of the obtained trifluoroacetic acid solution (5.87 ml) was added to a stirred solution of tetraacetate 14 (80 mg, 0.126 mmol) in CH 2 Cl 2 (4 ml) at 01C.…”

“…10 This compound proved to be useful for biochemical and physiological investigations, but because of its relatively low isotopic purity (82%) it could not be used in some experiments. Based on the experience we have gained in brassinolide biosynthetic studies, [11][12][13][14][15] labeling the terminal part of the steroidal side chain was considered as an appropriate solution of the problems connected with low isotopic purity. The present work is an extension of our studies 16 toward synthesis of labeled brassinosteroids for biochemical and other biological applications and deals with the preparation of [26-2 H 3 ]epibrassinolide and its biosynthetic precursors.…”

An improved synthesis of [26‐2H3]castasterone

“…Deuterated analogs of steroids are widely used as internal standards for mass spectrometry‐based analyses of various compounds belonging to animal and human hormones, including corticosteroids, androgens, estrogens, neurosteroids, and so on . The same methodology proved to be successful also in analyzing steroidal plant hormones (brassinosteroids, BS) . Apart from high chemical and isotopic purity, an important prerequisite for a successful implementation of deuterium‐labeled BS as internal standards is the presence of at least three non‐exchangeable deuterium atoms in their molecules .…”