Chemical Investigation of the Secondary Metabolites of Streptomyces sp. 4205

Abstract

Chemical investigation of the secondary metabolites of Streptomyces sp. 4205 resulted in the isolation of 1 new compound, named as 3,8-dimethyl-9-hydroxy-cyclodeca-3,5-diene-1,2-diketone (1), along with 3 known compounds. Their structures were elucidated on the basis of extensive spectroscopic analysis. All the compounds were tested for their inhibitory activity against Candida albicans ATCC 90028, Escherichia coli ATCC 35218, Pseudomonas aeruginosa ATCC 28753, Crytococcus neoformans ATCC 90113, Mycobacterium intracellulare ATCC 23068, Aspergillus fumigatus ATCC 90906, and methicillin-resistant Staphylococcus aureus ATCC 43300. Compound 1 showed activity against C. albicans ATCC 90028, with minimum inhibitory concentration and half-maximal inhibitory concentration values of 10.0 and 1.0 mg/mL, respectively.

Streptomyces is a group of Gram-positive actinomycetes, which are mainly distributed in soils, ocean, lakes, and rivers. ^{1 -4} Members of the genus produce plenty of bioactive secondary metabolites, ³ which could be divided into several types with antibacterial, antifungal, antiviral, and antitumor activities based on their biological functions. Thus, the genus Streptomyces is one of the most important sources for finding new leading compounds, especially antibiotic medicines. The most famous antibiotics, including streptomycin, oxytetracycline, bleomycin, and validamycin, were produced by Streptomyces species. ^{3 -6} Approximately 60% of antibiotics used in modern medicine are synthesized by Streptomyces spp. according to the statistics. ^{7 -9} Hence, the secondary metabolites produced by Streptomyces attract more and more attention of scientists.

In the present paper, we report the isolation from Streptomyces sp. 4205, which was isolated from paddy soil, and structure elucidation of 1 new (1) and 3 known compounds, identified as 3-hydroxybenzoic acid (2), ¹⁰ 2-hydroxybenzoic acid (3), ¹¹ and 2-ethyl-3-hydroxyl-1-hexanol (4), ¹² by comparison of their spectroscopic data with literature values (Figure 1). The antimicrobial activity was evaluated of all the isolates against Candida albicans ATCC 90028, Escherichia coli ATCC 35218, Pseudomonas aeruginosa ATCC 28753, Crytococcus neoformans ATCC 90113, Mycobacterium intracellulare ATCC 23068, Aspergillus fumigatus ATCC 90906, and methicillin-resistant Staphylococcus aureus ATCC 43300.

Figure 1

The structures of compounds 1 to 4.

Compound 1 has a molecular formula of C₁₂H₁₆O₃, on the basis of the positive high-resolution electrospray ionization mass spectrometry (HRESIMS) ion at m/z 231.0995 [M]⁺, and ¹³C nuclear magnetic resonance (NMR) (Figure S2) and distortionless enhancement by polarization transfer (DEPT) spectroscopic data, corresponding to 5 degrees of unsaturation. Its IR absorption bands (Figure S6) at 3 440, 1 753, and 1 720 cm^-1 indicated the presence of hydroxyl and carbonyl groups. The ¹H NMR spectrum (Figure S1) showed the presence of 3 olefinic protons (δ_H 6.84 [1H, d, J = 11.1 Hz], 6.36 [1H, t, J = 12.5 Hz], 5.98 [1H, m]), 1 singlet methyl (δ_H 1.75, s), and 1 double methyl (δ_H 0.77, d, J = 6.7 Hz). The ¹³C NMR and DEPT spectra exhibited 12 carbon resonances attributed to 2 methyls, 2 methylenes, 5 methines with 3 olefinic (δ_C 139.5, 131.1, 143.8) and 1 oxygenated (δ_C 76.4), and 3 quaternary carbons including 1 olefinic (δ_C 133.1) and 2 carbonyls (δ_C 183.0, 180.4). The above evidence showed 2 carbonyls and 2 double bonds in compound 1 corresponding to 4 degrees of unsaturation. That left 1 degree of unsaturation, which indicated that there was a ring in 1. The ¹H-¹H correlation and heteronuclear single quantum coherence spectra established the fragment structure of C4-C5-C6-C7-C8-C9-C10 (Figure S3 and S5). Furthermore, heteronuclear multiple bond correlations (HMBCs) (Figure S4) from H-4 (δ_H 6.84, d, J = 11.1 Hz) and H-5 (δ_H 6.36, t, J = 12.5 Hz) to δ_C 133.1 assigned the signal at δ_C 133.1 to C-3. HMBC correlations from H-4 (δ_H 6.84, d, J = 11.1 Hz) to δ_C 180.4, and from H-10 (δ_H 2.18, 2.37) to δ_C 183.0 assigned these 2 carbons to C-2 and C-1, respectively. Therefore, there was a 10-member ring in compound 1. Me-11 and Me-12 were located at C-3 and C-8 according to HMBC correlations of Me-11 (δ_H 1.75, s) with C-3, and Me-12 (δ_H 0.77, d, J = 6.7 Hz) with C-8. Thus, compound 1 was established as 3,8-dimethyl-9-hydroxy-cyclodeca-3,5-diene-1,2-diketone.

The new compound (1) showed antimicrobial activity against C. albicans ATCC 90028, with minimum inhibitory concentration (MIC) and half-maximal inhibitory concentration (IC₅₀) values of 10.0 and 1.0 mg/mL, respectively. The MIC and IC₅₀ values of the positive control were 0.5 and 0.1 mg/mL, respectively.

Experimental

General

NMR, Bruker DRX-500 spectrometer; IR, Bruker Tensor 27 spectrometer; HRESIMS, Thermo high-resolution Q exactive focus mass spectrometer; CC, silica gel (200, 300 mesh) (Qingdao Marine Chemical and Industrial Factory, China), MCI-gel CHP20P (75, 100 µm). RP-18 gel (40, 60 µm), Sephadex LH-20, and Toyopearl HW-40C.

Biological Materials

Streptomyces strain 4205 was isolated from paddy soil collected in Anfeng Town, Anxiang County, Hunan Province, People’s Republic of China. C. albicans ATCC 90028, E. coli ATCC 35218, P. aeruginosa ATCC 28753, C. neoformans ATCC 90113, M. intracellulare ATCC 23068, A. fumigatus ATCC 90906, and methicillin-resistant S. aureus ATCC 43300 were provided by Microbiology Laboratory in the College of Agriculture, South China Agriculture University.

Fermentation and Extraction

Strain Streptomyces sp. 4205 was cultivated in a medium with starch (2%), saccharose (2%), soybean extract (4%), maize extract (8%), ammonium chloride (0.115%), sodium chloride (0.05%), ironvitriol (0.001%), magnesium sulfate (0.03%), K₂HPO₄·3H₂O (0.05%), and CaCO₃ (0.2%), at pH 7.2-7.4. The cultures were grown on a rotary shaker (180 rpm) at 30°C for 6 days. After the fermentation, the culture broth was extracted with n-butyl alcohol and the extract evaporated to dryness under reduced pressure in a rotatory evaporator.

Isolation and Purification

The dried n-butyl alcohol extract (140 g) was dissolved in water and extracted with ethyl acetate. After evaporation to dryness, the extract was subjected to a Sephadex LH-20 column, eluting with MeOH-H₂O (10:90 to 100:0), to afford 3 fractions, Fr. A-C. Fr. B (4 g) was applied to silica gel (CHCl₃:MeOH, 15:1 to 0:1), to afford 5 subfractions (Frs. B1-B5). Fr. B2 (506 mg) was applied to RP-18, eluting with MeOH-H₂O (10:90 to 90:10), then purified by MCI-gelCHP20P (MeOH-H₂O, 30:70 to 90:10) to give compounds 2 (103 mg) and 3 (96 mg). Fr. B3 (312 mg) was subjected to RP-18, eluting with MeOH-H₂O (30:70 to 100:0), and purified on an MCI-gel CHP20P column, eluting with MeOH-H₂O (80:20), to yield compound 1 (39 mg). Fr. B4 (207 mg) was subjected to MCI-gel CHP20P, eluting with MeOH-H₂O (30:70 to 90:10), and purified on a Toyopearl HW-40C column, eluting with MeOH-H₂O (80:20), to give compound 4 (76 mg).

Compound (1)

White Amorphous Powder.

IR (KBr) ν _max: 3441(-OH), 1 551, 1411 cm⁻¹.

UV (MeOH) λ_max nm: (log ε) 260.0 (3.91), 334.4 (2.21).

¹H and ¹³C NMR: Table 1

Table 1

¹H (500 MHz) and ¹³C (125 MHz) NMR Data for 1 in CD₃OD (Δ in ppm).

No.	1
	δC	δH(mult., J in Hz)
1	183
2	180.4
3	133.1
4	139.5	6.84, d (11.1)
5	131.1	6.36, t (12.5)
6	143.8	5.98, m
7	38.9	1.91, 2.28, m
8	41.8	1.61, m
9	76.4	3.78, m
10	44.5	2.18, 2.37, m
11	16.3	1.75 s
12	18.1	0.77 d (6.7)

HRESIMS: m/z 231.0995 [M + Na]⁺ (calculated for C₁₂H₁₆O₃Na, 231.0997).

In Vitro Antimicrobial Assay

The antimicrobial activity was examined using the disk inhibition zone method against C. albicans ATCC 90028, E. coli ATCC 35218, P. aeruginosa ATCC 28753, C. neoformans ATCC 90113, M. intracellulare ATCC 23068, A. fumigatus ATCC 90906, and methicillin-resistant S. aureus ATCC 43300. Ciprofloxacin was used as positive control.

Supplemental Material

Supplementary material - Supplemental material for Chemical Investigation of the Secondary Metabolites of Streptomyces sp. 4205

Supplemental material, Supplementary material, for Chemical Investigation of the Secondary Metabolites of Streptomyces sp. 4205 by Cheng-Zhen Gu, Jing Lv, Sheng-Hao Yuan, Yuan-Yuan Song, Chong-Ren Yang, Ying-Jun Zhang, and Ren-Sen Zeng in Natural Product Communications