Two new isoflavone 7-O-α-4″-anhydro-4″,5″-didehydroglucuronides from Streptomyces sp. LZ35ΔgdmAI

Two isoflavone 7-O-α-4″-anhydro-4″,5″-didehydroglucuronides, namely daidzein 7-O-α-4″-anhydro-4″,5″-didehydroglucuronide (1) and genistein 7-O-α-4″-anhydro-4″,5″-didehydroglucuronide (2), were isolated and identified from the mutant strain of Streptomyces sp. LZ35ΔgdmAI. Their structures were elucidated by the analysis of their high resolution mass spectrometry (HR–MS) and 1D, 2D Nuclear magnetic Resonance (NMR) spectroscopic data. They are new natural products and maybe the transformed products of the soybean meal by Streptomyces sp. LZ35ΔgdmAI.


Introduction
Streptomyces sp. LZ35 is a geldanamycin high-yield strain which was isolated from the intertidal soil collected at Jimei, Xiamen, P.R. China. So far, five different classes of natural products have been identified from strain LZ35, including geldanamycins (Shi et al. 2011), cuevaenes , hygrocins , 16,17dihydroxycyclooctatin (Zhao et al. 2013) and echosides (Deng, Lu, Li, Hao, et al. 2014). The analysis of genome sequence indicated that Streptomyces sp. LZ35 has the potential of producing different types of secondary metabolites (data not shown). In the course of our ongoing research activities towards the isolation of new natural products, the mutant strain Streptomyces sp. LZ35DgdmAI, which was constructed by disrupting the first PKS module of geldanamycin gene cluster (gdm), was fermented in soybean meal media.

Results and discussion
The MeOH extract from a 6-L agar plate fermentation of the strain LZ35DgdmAI was fractionated by a combination of various column chromatographic methods, resulting in the isolation of two new isoflavone 7-O-a-4 00 -anhydro-4 00 ,5 00 -didehydroglucuronides (1 and 2).
The molecular formula of compound 1 was determined to be C 21 H 16 O 9 by HR-ESI-MS (m/z 413.3543 [M þ H] þ , calcd for 413.3537). The UV absorptions at l max 275 and 320 nm attributable to the bands I and II of the benzoyl and cinnamoyl chromophores of an isoflavone (Farag et al. 2001).
The 1 H NMR spectrum displayed 12 resonances for 9 unsaturated protons (d H 6.17-8.21), 3 oxymethine protons (d H 5.82, 4.26, 3.98). A characteristic singlet resonance for H-2 of isoflavone was observed at d H 8.15 (Horie et al. 1998). This assignment was confirmed by its long-range connectivity to the quaternary carbons at d C 178.1 (C-4), 159.1 (C-9), 126.2 (C-3) and 124.0 (C-1 0 ) in the HMBC spectrum. The presence of a tri-substituted benzene ring was indicated by two doublets and a singlet with the equal J values of 8.8 Hz at d H 7. 29, 8.15 and 7.40 in the 1H NMR, and HMBC correlations from H-8 to C-6, C-7 and C-10, and H-6 to C-7, C-9 (w) and C-10, and H-5 to C-7, C-4 and C-9. The remaining aromatic proton resonances comprised two doublets at d H 7.39 and 6.85, representing a p-substituted ring B, which was confirmed by the HMBC correlations from the proton at d H 7.39 (H-2 0 , H-6 0 ) to C-3, and C-4 0 (d C 158.8), and the proton at d H 6.85 (H-3 0 , H-5 0 ) to C-1 0 , C-4 0 and C-5 0 . According to the down field shifts of C-4 0 (d H 158.8) and C-7 (d 162.7), two oxy carbons were deduced at C-4 0 and C-7. Therefore, the aglycone of compound 1 was determined as daidzein (Li et al. 2009;Yang et al. 2013).
Compound 2 was obtained as white powder, and the molecular formula was determined to be C 21 H 16 O 10 by HR-ESI-MS (m/z 429.3537 [M þ H] þ , calcd for 429.3531). The NMR data of 2 were very similar to that of 1. A tetra-substituted ring A was deduced by two singlet protons at d H 6.83 (H-8) and 6.60 (H-6) and a p-substituted ring B was confirmed by two aromatic doublets protons at d H 7.39 and 6.85 with the J values of 7.5 and 8.8 Hz. The down field shifts of C-4 0 (d C 158.9), C-5 (d C 163.9) and C-7 (d C 163.6) revealed the presence of oxy carbons. Therefore, the aglycone moiety of 2 was determined as genistein (Wang et al. 1999;Kozerski et al. 2003). Additionally, the a-4-anhydro-4,5-didehydroglucuronate moiety was determined by NMR comparison with compound 1. Therefore, the structure of 2 was determined as genistein 7-O-a-4 00 -anhydro-4 00 ,5 00 -didehydroglucuronide.
It has been demonstrated that isoflavones isolated from Streptomyces cultivated in media containing plant-derived nutrients such as soybean are not of microbial biosynthetic origin (Anyanwutaku et al. 1992). Moreover, Streptomyces is well-known for its ability of biotransformation of isoflavones, such as converting genistein (5,7,4 0 -trihydroxyisoflavone), the major isoflavone of soybeans, into 8-methyl genistein (Hosny & Rosazza 1999), and daidzein into 1 00 -O-methyl-8-hydroxymethyl daidzein (Yang et al. 2013). Therefore, the two new isoflavone glucuronides (1 and 2) might be transformed from daidzein and genistein of the culture media by glucuronidation (Maatooq & Rosazza 2005). Indeed, glucuronidation is broadly involved in xenobiotic metabolism of substances such as drugs (Guillemette 2003;Sugatani 2013), and elimination of endobiotics such as biosynthetic intermediates . Previously, five p-terphenyl O-b-glucuronides, namely echosides A -E, were isolated from the strain Streptomyces sp. LZ35DgdmAI (Deng, Lu, Li, Hao, et al. 2014), indicating the presence of UDP-glucuronosyltransferases in this strain and further supporting the biotransformation origin of 1 and 2. Accordingly, we proposed that the production of 1 and 2 was derived from daidzein and genistein by the glucuronidation at 7-hydroxyl group and the dehydration of 5-hydroxyl group of glucuronate moiety, respectively (Figure 2).
The isolation of compounds 1 and 2 in this study indicated the readiness of glucuronidation of daidzein and genistein by Streptomyces species, which was consistent to the results of previous research. However, the dehydration of 5-hydroxyl group in the glucuronate moiety was novel among isoflavone glucuronides. Moreover, soybean isoflavones have been reported to possess numerous physiological properties, such as antitumour, anti-menopausal (female) osteoporosis and antiageing, antioxidant, anti-inflammation. They have also been reported to improve learning and memory skills in menopausal women and to aid in the prevention and treatment of heart disease, diabetes and Kawasaki disease (KD) . Therefore, the bioactivities and biosynthesis of compounds 1 and 2 are worth further investigation (Al-Maharik & Botting 2006;Kgomotso et al. 2008). Orbitrap XL (Thermo-Finnigan, San Jose, CA, USA). Column chromatography (CC) was performed with silica gel (200 -300 mesh, Qingdao Marine Chemical, Inc., Qingdao, P.R. China), Sephadex LH-20 (40 -70 mm, Amersham Pharmacia Biotech AB, Uppsala, Sweden) and 281 Lichroprep reversed phase RP-18 silica gel (40 -63 mm, Merck, Darmstadt, Germany). TLC (1.0 -1.5 mm) was conducted with glass precoated silica gel GF254 (Qingdao Marine Chemical, Inc.). Semipreparative high performance liquid chromatography (HPLC) were performed on an Agilent 1260 equipped with a ZORBAX XDB-C 18 column (9.4 £ 250 mm, 5 mm). All solvents used were of analytical grade. Compounds were visualised under UV light and/or by spraying with H 2 SO 4 /EtOH (1:9, v/v) followed by heating.

Supplementary material
Supplementary material including NMR spectra of compounds 1 and 2 are available online.