Concanamycin H from the soil actinomycete Streptomyces sp. R1706-8

The actinomycete strain R1706-8 is isolated from a soil sample collected from the nest of the horned-face bee (Osmia cornifrons) and identified as Streptomyces sp. based upon the results of 16SrRNA sequence analysis. Two concanamycin derivatives obtained from the solid fermentation have been determined by analysis of the infrared, high-resolution electrospray ionization mass spectrometry, 1D and 2D NMR spectra as well as by comparison with literature data. Of the two derivatives, one is a new compound, named concanamycin H, and the other is the known compound, concanamycin G. These compounds are assayed for antibacterial activity, with concanamycins H and G displaying inhibitory activity against Bacillus subtilis (minimum inhibitory concentration = 0.625 µg mL−1).


Introduction
To date, seven concanamycin derivatives (A-G) have been isolated from the mycelia of Streptomyces. 1-3 These compounds possess antimalarial, [3][4][5] antifungal, 6-8 antiviral, 9,10 anticancer, 11,12 and other biological activities. This family of compounds features an 18-membered macrolide ring 13 and a pyran ring, 14,15 but the side-chain structures are rare. Structure-activity relationship studies showed that molecules of the concanamycin family are responsible for potent inhibitory activity. Many researchers have studied the synthesis, 7 semisynthesis, 16 and biosynthesis 17 of these concanamycin derivatives.
In this paper, we report that a sample of the Streptomyces sp. R1706-8, which was isolated from the soil found in the front wall of the honeycomb chamber of the nest of the horned-face bee (Osmia cornifrons), located between Yantai and Weihai in Shandong Province, China, yielded one new compound, concanamycin H (1), and the known compound, concanamycin G (2) (Figure 1). These compounds were identified by analytical techniques, and their antibiotic activity has been investigated.

Results and discussion
Compound 1 was isolated as a colorless oil. The highresolution electrospray ionization mass spectrometry (HR-ESIMS) data showed a sodiated molecular ion at m/z (HR-ESIMS) 697.4279 [M + Na] + (calcd for C 39 H 62 O 9 Na: 697.4286) (see Figure S9 in the Supporting information), which in conjunction with 13 C NMR data (Table 1) indicated a molecular formula of C 39 H 62 O 9 , requiring nine degrees of unsaturation. The IR spectrum showed characteristic absorption bands attributed to hydroxy (3425 cm −1 ), carbonyl (1712 cm −1 ), and alkenyl (1456 cm −1 ) functionalities (see Figure S10 in the Supporting information). The 1 H, 13 C, and heteronuclear single quantum coherence (HSQC) NMR (Table 1) data demonstrated the presence of eleven methyl, two methylene, twenty-one methine, and five quaternary carbons, including six olefinic, two carbonyl, five oxymethine, and two methoxy groups. Compound 2 was isolated as a colorless oil. The HR-ESIMS data showed a sodiated molecular ion at m/z 683.4120 [M + Na] + (calcd for C 38 H 60 O 9 Na: 683.4129) (see Figure S19 in the Supporting information). The 1 H and 13 C NMR data of compound 2 closely resembled those of 1 except for the C-8 ethyl signal disappeared and replaced by a methyl group (δ C = 10.0; δ H = 0.94 (d, 7.0)) in the 13 C NMR of 2. Thus, compound 2 was assigned as the anhydroaglycone of concanamycin B 1 ( Figure 1) and named concanamycin G. 1 Concanamycin H (1) and concanamycin G (2) showed weak inhibitory activity against Bacillus subtilis with clear inhibition zones in disk diffusion assays ( Table 2). Accurate data on the inhibitory activity were obtained through determination of the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values (Table 3). Compounds 1 and 2 demonstrated minor inhibitory activity against Bacillus subtilis (MIC = 0.625 mg mL −1 and MBC = >1 mg mL −1 ).

Conclusion
Two concanamycin derivatives have been isolated from Streptomyces sp. R1706-8, including a new macrocycle named concanamycin H (1) along with the reported concanamycin G (2). The antibacterial activity compounds 1 and 2 have been evaluated by the disk diffusion method against four bacteria. Concanamycins H (1) and G (2) exhibited inhibitory activity against to Bacillus subtilis and displayed moderate activity (MIC = 0.625 μg mL −1 and MBC = >1 μg mL −1 ).

Media and culture conditions
The culture medium used Gauze's Synthetic Medium No. 1 that was prepared from soluble starch (20 g), KNO 3 (1 g), K 2 HPO 4 (0.5 g), MgSO 4 ·7H 2 O (0.5 g), NaCl (0.5 g), FeSO 4 ·7 H 2 O (0.01 g), agar (20 g), and pH = 7.0-8.0, After sterilization by steaming at 121 °C for 31 min and reducing the temperature to room temperature, actinomycete colonies were purified by streaking on the same agar medium. The solid medium was cultured at a constant temperature of 32 °C for around 10 days. Genomic DNA was extracted using a DNA isolation kit (Shanghai Sangon Biotech Co., China). Polymerase chain reaction (PCR) amplification of the 16S ribosomal RNA (rRNA) was performed using primers Fd2 (5'-GAGTTTGATCATGGCTCAG-3') and 16Sr (5'-TTGCGGGACTTAACCCAAC-AT-3') and sequenced by Shanghai Sangon Biotech Co., China. The nucleotide sequence was submitted to GenBank (accession numbers KX588720) and compared with reference 16SrRNA gene sequences in databases by BLAST searching. A phylogenetic tree was constructed by the neighborjoining method using MEGA 5.0 software (Figure 3). Streptomyces sp. R1706-8 was deposited at the Laboratory of Natural Products Chemistry, Shandong University, Weihai, P.R. of China.