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 (>300 000 samples). The compound exhibits strong cytotoxicity against human ovarian adenocarcinoma SKOV-3 cells (IC50=0.3 μM). 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 JH,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 RK741 was subjected to silica gel medium-pressure liquid chromatography (MPLC; Purif-Pack SI-30, Shoko Scientific Co., Yokohama, Japan), eluting with a CHCl3–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 CHCl3–MeOH (0, 2, 5, 10, 20, 30 and 100% MeOH). The 20% MeOH elute fraction (15.4 mg), containing methyl α-olivoside as a major component and methyl β-olivoside as a minor component, was purified by reversed phase-HPLC-MS (CAPCELL PAK C18 MGII column, 4.6 i.d. × 150 mm, Shiseido Co., Ltd., Tokyo, Japan) eluted with 5% MeOH (aq) containing 0.1% formic acid (isocratic; flow: 1.0 ml min−1; detected at m/z 185 [M+Na]+) to afford methyl α-D-olivoside (3.0 mg, Rt=11.7 min) as a colorless oil: +156 (c 0.23, acetone) lit.4 +149 (c 0.54, acetone); 1H NMR (600 MHz, acetone-d6) δ 4.64 (d, J=3.6 Hz, H-1), 3.71 (ddd, J=4.8, 9.6, 12.6 Hz, H-3), 3.50 (dq, J=6.0, 9.6 Hz, H-5), 3.24 (s, OMe-1), 2.92 (dd, J=9.6, 9.6 Hz, H-4), 1.97 (dd, J=4.8, 12.6 Hz, Heq-2), 1.53 (ddd, J=3.6, 12.6, 12.6 Hz, Hax-2), 1.18 (d, J=6.0 Hz, H3-6). 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 °C for 4 h; the resulting material was further fractionated by silica gel MPLC (Purif-Pack SI-30) using the same gradient CHCl3–MeOH elution conditions as described above. The 10% MeOH fraction (31.8 mg) containing α-quinovoside (major) and β-quinovoside (minor) was purified by preparative reversed phase-HPLC-MS using a CAPCELL PAK C18 MGII column (20 i.d. × 150 mm) eluted with 3% MeOH (aq) containing 0.1% formic acid (isocratic; flow: 10.0 ml min−1; detected at m/z 201 [M+Na]+) to give methyl α-D-quinovoside (2.3 mg, Rt=12.0 min) as a colorless oil: +122 (c 0.11, CHCl3) lit.5 +147 (c 1.00, CHCl3); 1H NMR (500 MHz, MeOH-d4) δ 4.59 (d, J=3.5 Hz, H-1), 3.58 (dq, J=6.0, 10.0 Hz, H-5), 3.55 (dd, J=10.0, 10.0 Hz, H-3), 3.39 (dd, J=3.5, 10.0 Hz, H-2) 3.37 (s, OMe-1), 2.97 (dd, J=10.0, 10.0 Hz, H-4), 1.24 (d, J=6.0 Hz, H3-6). In contrast to methyl α-D-olivoside and methyl α-D-quinovoside, the remaining sugar unit, methyl α-amicetoside could not be obtained by purification due to its volatile nature. Therefore, methyl α-D-amicetoside was prepared by organic synthesis according to the method reported previously.6 These sugars were employed as authentic samples for optical rotation measurements.

The absolute configurations of the sugar units of JBIR-129 were determined as described below. JBIR-129 (20.1 mg) was added to a solution of 5% HCl–MeOH (1 ml) and heated at 40 °C for 20 h. After cooling, the mixture was analyzed by HPLC using a YMC-Pack Hydrosphere C18 (4.6 i.d. × 250 mm, YMC Co., Ltd., Kyoto, Japan; eluent: MeOH/H2O 20:80, isocratic) in conjunction with a OR-2090 Plus chiral detector (JASCO Co., Tokyo, Japan). The sugar components of JBIR-129 were identified as D-olivoside, D-quinovoside and D-amicetoside by comparison with the retention times of authentic methyl α-D-olivoside, methyl α-D-quinovoside and methyl α-D-amicetoside (Rt=4.67, 6.19 and 9.06 min, respectively) and the signs (all positive) of optical rotation.

In a previous study, we have established the relative configuration of two fragments of JBIR-129, from C-7 to C-27 and from C-33 to C-39. Therefore, determination of the absolute configuration at C-13 and C-39 would firmly establish the absolute configuration of JBIR-129 itself. The absolute configurations of a β-quinovopyranoside (Qui1) and a β-amicetopyranoside (Ami1), which are substituents at the C-13 and C-39 positions, respectively, offered key insights into the determination of the macrolactone absolute configuration as follows: the ROESY spectrum exhibited a strong correlation between H-13 and H-Qui1-1; furthermore, ROESY correlations from methyl proton H3-42 to H-Qui1-1 and H-Qui1-2 were observed, whereas the correlations from H2-14 to H-Qui1-2 were not observed. These results suggested the spatial location of C-13 in MeOH-d4 as depicted in Figure 1; consequently, the absolute configuration at C-13 was deduced as S. In the same manner, the absolute configuration at C-39 was concluded to be S based on the presence of ROESY correlations from H-39 to H-Ami1-1 and from H-38 and H3-47 to Heq-Ami1-2, and the absence of a correlation from H-40b and H3-41 to Heq-Ami1-2. Hence, the absolute configuration of macrolactone moiety was determined as 7S, 9R, 12S, 13S, 15S, 16R, 17S, 19S, 20R, 21S, 23S, 24R, 25S, 27S, 33S, 34S, 35S, 36R, 37R, 38R and 39S (Figure 2).

Figure 1
figure 1

Configurational analysis of Qui1 and Ami1 glycosidic fragments of JBIR-129 (dashed arrow: ROESY correlation).

Figure 2
figure 2

Absolute configuration of JBIR-129.

JBIR-129 and its congener JBIR-139 showed strong cytotoxicities against human ovarian adenocarcinoma SKOV-3 cells with nearly identical IC50 values (0.3 and 0.4 μm, respectively). The structural difference between JBIR-129 and -139 is an additional N-acetylgalactosamine (GalNAc) moiety found at C-21 in JBIR-139. Therefore, the aglycone of JBR-129 is considered to make a significant contribution to the cytotoxicities of these compounds. As previously mentioned, the producing strain Streptomyces sp. RK74 produces many congeners of JBIR-129, making it difficult to study detailed biological activities specific to the compound such as antitumor activities in mice models. It should be possible to generate the aglycone of JBIR-129 by heterologous expression; in this regard, we have employed a BAC (Bacterial Artificial Chromosome) vector to clone the complete relevant biosynthetic gene cluster (126.5 kbp). Heterologous production using a deletion mutant of the glycosylation enzyme found in the biosynthetic gene cluster could provide the aglycone moiety of JBIR-129 solely. Attempts are currently underway to accomplish the heterologous production of JBIR-129 in a SUKA (Special Use of Kitasato Actinobacteria) host strain.7, 8