JBIR-129, isolated from the culture of Streptomyces sp. RK74, was discovered by the screening program first implemented for the search for bioactive substances in our extensive natural product extract library (4300 000 samples). The compound exhibits strong cytotoxicity against human ovarian adenocarcinoma SKOV-3 cells (IC 50 ¼ 0.3 mM). Its planar structure, consisting of a 34-membered polyol macrolide skeleton with five deoxysugars, has been elucidated by MS and NMR spectroscopic data analyses. 1 The relative configurations of the deoxysugar units have been elucidated based on J H,H values and ROESY data. 1 In addition, two partial relative configurations of the aglycone moieties have been independently determined by using J-based configuration analysis. 2 Herein, we report the determination of the absolute configuration of JBIR-129 by further investigating the stereochemistry of the deoxysugar moieties. 3 The absolute configurations of the sugar moieties of JBIR-129 were established by HPLC analysis using an optical rotation detector. As the JBIR-129-producing microorganism also produces many congeners consisting of the common aglycone and a variety of sugar units, the purification of JBIR-129 proved to be challenging. 1 According to previous analyses, the main sugar units of these congeners were identical to those of JBIR-129. Therefore, we attempted to obtain the main sugar moieties from a semi-purified fraction of JBIR-129.A 1.0 g portion of the n-BuOH extract obtained from the fermentation broth of RK74 1 was subjected to silica gel mediumpressure liquid chromatography (MPLC; Purif-Pack SI-30, Shoko Scientific Co., Yokohama, Japan), eluting with a CHCl 3 -MeOH gradient increasing in strength in 10% stepwise increments of MeOH to give a crude material containing the glycosidic macrolide (100% MeOH eluate, 239.5 mg). The material was added to a 5% HCl-MeOH solution (1 ml) and stirred (10 min, rt); the resulting material was applied to a silica gel MPLC column (Purif-Pack SI-30) and developed with a stepwise gradient system of CHCl 3 -MeOH (0, 2, 5, 10, 20, 30 and 100% MeOH). The 20% MeOH elute fraction (15.4 mg), containing methyl a-olivoside as a major component and methyl b-olivoside as a minor component, was purified by reversed phase-HPLC-MS (CAPCELL PAK C 18 MGII column, 4.6 i.d. Â 150 mm, Shiseido Co., Ltd., Tokyo, Japan) eluted with 5% MeOH (aq) containing 0.1% formic acid (isocratic; flow: 1.0 ml min À1 ; detected at m/z 185 [M þ Na] þ ) to afford methyl a-D-olivoside (3.0 mg, Rt ¼ 11.7 min) as a colorless oil: ½a 26 D þ 156 (c 0.23, acetone) lit. 4 ½a 23 D þ 149 (c 0.54, acetone); 1 H NMR (600 MHz, acetone-d 6 ) d 4.64 (d, J ¼ 3.6 Hz, H-1), 3.71 (ddd, J ¼ 4. 8, 9.6, 12.6 Hz, H-3), 3.50 (dq, J ¼ 6.0, 9.6 Hz, H-5), 3.24 (s, OMe-1), 2.92 (dd, J ¼ 9.6, 9.6 Hz, H-4), 1.97 (dd, J ¼ 4.8, 12.6 Hz, H eq -2), 1.53 (ddd, J ¼ 3.6, 12.6, 12.6 Hz, H ax -2), 1.18 (d, J ¼ 6.0 Hz, H 3 -6). The combined fraction (183.5 mg) of the 30 and 100% MeOH eluates was then subjected to acid hydrolysis with 5% HCl-MeOH at 50 1C...