Synthesis of deuterium-labeled (24R)-methyl brassinosteroids Vladimir A. Khripach, Vladimir N. Zhabinskii, Ã Olga V. Gulyakevich, Yurii V. Ermolovich, and Olga V. Konstantinova Synthesis of labeled brassinosteroids (24-epibrassinolide and its biosynthetic precursors) containing three deuterium atoms in the terminal part of the side chain is reported. Labeling was achieved by three-step reductive transformation of carbethoxy group into methyl using lithium aluminium deuteride. The proposed method ensured high isotopic purity of (24R)-methyl brassinosteroids containing a label in a position which is not subjected to its loss. Keywords: brassinosteroids; epibrassinolide; epicastasterone; episecasterone; episecasterol Introduction 24-Epibrassinolide holds a special place among natural brassino- steroids (BS). 1 It belongs to the most active representatives of this group of phytohormones, and at the same time it can be relatively easily prepared from available sterols. 2–4 As a result, now it is the most practically useful compound of this group; for a number of years preparations with 24-epibrassinolide (Figure 1) as an active ingredient have been used in agriculture. 5,6 Research in the recent years has revealed its potential usefulness in medicine also. 7–9 Further studies, both in agricultural and medicinal directions, require the availability of labeled 24-epibrassinolide. Recently, we have published synthesis of [7- 2 H 2 ]epibrassinolide. 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, 11–15 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 brassi- nosteroids for biochemical and other biological applications and deals with the preparation of [26- 2 H 3 ]epibrassinolide and its biosynthetic precursors. Experimental Melting points were recorded on a Boetius micro-melting point apparatus and are uncorrected. 1 H and 13 C NMR spectra were obtained using a Bruker AVANCE 500 (Bruker Biospin, Rhein- stetten, Germany) spectrometer in CDCl 3 operating at 500 MHz for 1 H and 125 MHz for 13 C. Assignment of 1 H and 13 C resonances was performed by the combined use of 1D and 2D experiments, including COSY, HSQC, HMBC, TOCSY, and NOESY methods using standard pulse sequences supplied in the instrument manufacturer’s software package. Mass spectra were performed on a LCQ Fleet mass spectrometer (Thermo Electron Corporation, USA) with an APCI source. Spectra were collected in the positive ion mode and analyzed by the Xcalibur software. Chemicals were purchased from Aldrich and Fluka and used as received. Reactions were monitored by TLC using aluminum or plastic sheets, silica gel 60 F 254 precoated (Merck Art. 5715). Column chromatography was carried out on Kieselgel 60 (Merck Art. 7734). [26 2 H 3 ](22E,24R)-6b-Methoxy-3a,5-cyclo-24-methyl-5a- cholest-22-ene (4) To a solution of (22E,24R)-6b-methoxy-3a,5-cyclo-24-methyl-5a- cholest-22-en-26-oic acid ethyl ester (3) 17 (1.22 g, 2.58 mmol) in THF-diethyl ether (7:1, 80 ml), LiAlD 4 (217 mg, 5.16 mmol) was added portionwise. The reaction was stirred at an ambient temperature for 1.5 h. Then water (0.22 ml), 15% NaOH (0.22 ml) and again water (0.65 ml) were consecutively added to the mixture. The precipitate was filtered off and the filtrate was evaporated to dryness. Purification of the residue by SiO 2 332 Figure 1. The Structure of 24-epibrassinolide. Institute of Bioorganic Chemistry, National Academy of Sciences of Belarus, Kuprevich str., 5/2, 220141 Minsk, Belarus *Correspondence to: Vladimir N. Zhabinskii, Institute of Bioorganic Chemistry, Kuprevich str., 5/2, 220141 Minsk, Belarus. E-mail: vz@iboch.bas-net.by Research Article Received 1 November 2010, Revised 28 December 2010, Accepted 9 January 2011 Published online 17 February 2011 in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/jlcr.1874 J. Label Compd. Radiopharm 2011, 54 332–336 Copyright r 2011 John Wiley & Sons, Ltd.