New α-glucosidase inhibitors from marine algae-derived Streptomyces sp. OUCMDZ-3434

Wailupemycins H (1) and I (2) with a new skeleton coupled two 6-(2-phenylnaphthalene-1-yl)pyrane-2-one nuclei to a –CH2– linkage were identified from the culture of Streptomyces sp. OUCMDZ-3434 associated with the marine algae, Enteromorpha prolifera. Compounds 1 and 2 are two new α-glucosidase inhibitors with the Ki/IC50 values of 16.8/19.7 and 6.0/8.3 μM, respectively. In addition, the absolute configurations of wailupemycins D (3) and E (4) are also resolved in this paper for the first time.

Key NOE correlations from both H-14a (δ H 3.63) and H-17 (δ H 7.48) to H-6 (δ H 4.71) and from H-17 (δ H 7.48) to H-14a (δ H 3.63) indicated the anti-orientation of the pyrone and phenyl moieties, suggesting the same relative configuration as 3 9 . The absolute configuration of 1 (6S, 15R) was determined by ECD calculations of the simplified model compounds I (6S, 15R) and ent-I (6R, 15S) using TD-DFT method at the B3LYP/6-31G(d) level 8 . The results showed that the measured CD curve of 1 is matched well with the calculated ECD for I and opposite to that of ent-I (Fig. 6).
The molecular formula of wailupemycin I (2) was also C 43 H 30 O 11 from the HRESIMS peak at m/z 723.1847 [M + H] + , indicating an isomer of 1. The similar UV, IR and 1D NMR spectra (Table 1)     However, no NOESY correlation between H-6 (δ H 4.28) and H-14a (δ H 2.92) indicated a 6-epimer. This deduction was confirmed by ECD calculations of the simplified model compounds II (6R, 15R) and ent-II (6S, 15S) using TD-DFT method at the B3LYP/6-31G(d) level 8 . The consistency of CD curve of 2 to the calculated ECD for II and the opposition to ECD of ent-II ( Fig. 7) indicated (6R, 15R)-configuration of 2.
Compounds 1 and 2 were postulated to be biosynthesized from compounds 3, 4 and 5 whose biosynthetic pathway had been elucidated to be from benzoyl-CoA and malonyl-CoA 9,10 . The aldol condensation took place between 3 or its enolate anion and formaldehyde to produce the key conjugated enone intermediate (a). The intermediate a further reacted with 5 or its enolate anion via a Michael addition to yield the keto-tautomer (b) of 1 that formed the more favorable enol-tautomer 1. By the same procedure, the bio-reactions between compounds 4 and 5 produced compound 2 (Fig. 8).
To elucidate the postulation and to further identify the structures of 1 and 2, a chemical transformation was performed using compounds 3, 4 and 5 as the materials. When reacted with 5 and HCHO in EtOH, compounds 3 and 4 formed 1 and 2 that were identified by ESIMS and co-HPLC experiments, respectively ( Figure S31).

Discussion
The aromatic dimers having a methylene linkage are rare in nature, especially originated from microorganism. Most of them were found in plant kingdom, such as italipyrone and homoarenol from Helichrysum stoechas 21 , two phloroglucinol derivatives from H. stoechas var. olonnense 22 , helipyrone and norhelipyrone from H. arenarium 23 , kunzeagin A (a dimeric flavonol glycoside) from Kunzea ambigua 24 , gerberinol from Diospyros kaki var. sylvestris 25 , and four acylphloroglucinol derivatives from Hypericum andinum 26 . As far as we know, very few of these natural dimers was identified in microbial kingdom, that is phaeochromycins F from Streptomyces sp. 27 , xyloketal F from Xylaria sp. 28 , and squarrosidine from Pholiota squarrosa 29 . This may be because there was less evidence on the biosynthesis of formaldehyde in microorganisms 30 . The isolation of wailupemycins H and I further reinforced the formaldehyde biosynthetic system could be occurred in microorganisms. And the good α -glucosidase inhibitory activity could also provide the alternative bioactivity screening for this kind of natural dimmers.

Methods
General experimental procedures. Optical rotations were measured with a JASCO P-1020 digital polarimeter. UV data were recorded with a Beckman DU 640 spectrophotometer, and CD data were collected using a JASCO J-815 spectropolarimeter. IR spectra were taken on a Nicolet NEXUS 470 spectrophotometer as KBr disks. 1 H NMR, 13 C NMR, DEPT, HMQC, HMBC, COSY, and NOESY spectra were recorded using Bruker Avance 500 spectrometer using TMS as an internal standard, and chemical shifts were recorded as δ values. Chemical shift values were referenced to residual solvent signals for DMSO (δ H /δ C , 2.50/ 39.5). HRESIMS data were recorded using a Q-TOF ULTIMA GLOBAL GAA076 LC mass spectrometer. HPLC and semi-preparative HPLC were performed using a Cholester column (COSMOSIL-pack, 4.6 × 250 mm, 5 μm, 1 mL/min) and an ODS column Actinobacterial material. The actinobacterial strain Streptomyces sp. OUCMDZ-3434 was isolated from E. prolifera collected from the Zhanqiao Beach (E 120°18′ 56.982″ , N 36°03′ 42.659″ , pH 6.0 in sea water), Qingdao, China in July 2012. The E. prolifera (1 g) were clipped and ground suspending in sterile distilled water. And then serially diluted to 1 mg/mL, 100 μL of which was deposited on a Gause's synthetic agar plate containing chloramphenicol (100 μg/mL) as a bacterial inhibitor and incubated at 28 °C for 8 days. A single colony was transferred onto another Gause's synthetic agar plate and was identified according to its morphological characteristics and 16S rRNA gene sequences (GenBank access No. KJ818249). A reference culture is maintained in our lab. at − 80 °C. Working stocks were prepared on Gause's synthetic agar slants and stored at 4 °C.
Fermentation and extraction. Spores were directly inoculated into 500 mL Erlenmeyer flasks containing 150 mL fermentation media consisted of glucose 20 g, beef extract 3 g, yeast extract 10 g, soluble starch 10 g, peptone 10 g, K 2 HPO 4 0.5 g, MgSO 4 0.5 g, CaCO 3 2 g, and 1 L of old sea water, pH nature). The flasks were incubated on a rotatory shaker at 180 rpm and 28 °C for 8 days. 45 L of whole broth was extracted with equal volumes of EtOAc for three times. The EtOAc extract was concentrated under reduced pressure to give a dark brown gum (28.0 g).