Hangtaimycin, a Peptide Secondary Metabolite Discovered from Streptomyces Spectabilis CPCC 200148 by Chemical Screening

The Journal of Antibiotics (2016) 69, 835–838 & 2016 Japan Antibiotics Research Association All rights reserved 0021-8820/16 www.nature.com/ja NOTE Hangtaimycin, a peptide secondary metabolite discovered from Streptomyces spectabilis CPCC 200148 by chemical screening Lijie Zuo, Bingya Jiang, Zhibo Jiang, Wei Zhao, Shufen Li, Hongyu Liu, Bin Hong, Liyan Yu, Limin Zuo and Linzhuan Wu The Journal of Antibiotics (2016) 69, 835–838; doi:10.1038/ja.2016.29; published online 9 March 2016 Streptomyces spectabilis is well known for the production of the revealed a major peak with UV-visible absorption profile different aminocyclitol antibiotic spectinomycin that is used clinically for from any of the above known secondary metabolites (Supplementary infections caused by Neisseria gonorrhoeae.1,2 Strains of S. spectabilis Figure S3), suggesting that it contained an unidentified secondary were also reported to produce streptovaricin (an ansamycin), metabolite. The hyphenated HRMS of the peak displayed a molecular metacycloprodigiosin (a tri-pyrrole), spectomycin (a tetrahydro- ion m/z 936.45150 ([M+H]+), which established the molecular + anthracene), bafilomycin (a macrolide), desertomycin (a macrolide), formula C50H61N7O11 (calculated at 936.45018 for [M+H] ) for the spectinabilin and SNF4435 (nitrophenyl pyrone), and TDD (trypto- unidentified secondary metabolite, with 24° of unsaturation and an phan–dehydrobutyrine diketopiperazine, a dipeptide).3–9 Therefore, odd number of nitrogen atoms (Supplementary Figure S4). It was S. spectabilis is capable of synthesizing secondary metabolites with believed to be a new compound after a formula search in SciFinder, structure diversity. which resulted in four compounds with their UV-absorption profiles S. spectabilis CPCC 200148 is a soil isolate from Hangzhou of different from the unidentified secondary metabolite. Zhejiang Province, China. It was identified as a producer of The new compound was then purified for structure elucidation. spectinomycin (actinospectacin) and streptovaricins in the year Fresh spores of S. spectabilis CPCC 200148 were inoculated into 350 1979.10,11 In recent times, we made a chemical screening of secondary ISP2 medium plates (Φ 9.0 cm, each containing 25 ml medium). They metabolites from S. spectabilis CPCC 200148, which revealed that it were incubated at 28 °C for 36 h, to develop into even layers of yellow was a talented strain with many secondary metabolites, including mycelial lawn, which produced comparatively high levels of the new metacycloprodigiosin, spectinabilin, SNF4435 and TDD. In particular, compound (Supplementary Figure S5). These agar cultures were cut we identified a new peptide secondary metabolite (designated as into small pieces, pooled and then extracted with an equal volume of hangtaimycin) with unusual structure moieties. Details of the EtOAc for three times. The EtOAc extract was concentrated and dried discovery, purification and structure determination of hangtaimycin (1.34 g) by rotary evaporation at room temperature, re-dissolved in a are presented below. small volume of EtOAc. It was loaded onto preparative silica gel TLC Fresh agar cultures of S. spectabilis CPCC 200148 from ISP2 for fractionation with a more polar mobile phase of EtOAc-n-hexane- medium (yeast extract 0.4%, malt extract 1.0%, glucose 0.4% and CH2Cl2-CH3OH, 5:5:2:2, v/v. The dark band at Rf 0.32 under UV agar 1.5%) plates incubated at 28 °C for 6 days were extracted with 254 nm (corresponding to the above band at Rf 0.21) was scraped off, ethyl acetate (EtOAc). The extract was analyzed by HPLC, TLC and extracted with EtOAc and dried (150 mg). It was re-dissolved in LC-MS. HPLC peaks with UV-visible absorption profiles identical or MeCN and then subjected to semi-preparative reverse-phase HPLC very similar to streptovaricins, prodigiosins, spectinabilin and for final purification. After vacuum evaporation, a white amorphous SNF4435 were found, and MS verified them to be members or powder of the pure new compound (1, 6.0 mg) was obtained. It 20 components of these antibiotics (Supplementary Figures S1 and S2). showed [α] D − 30.4 (c 0.10, MeOH) and UV (MeOH) λmax (log ε): Besides, an HPLC peak with UV absorption profile identical to TDD 219 (5.37), 266 (5.22) nm. was also observed (Supplementary Figure S3). Compound 1 was analyzed by NMR. The 1HNMRspectrumof1 In the silica gel TLC developed with EtOAc-n-hexane-CH2Cl2- showed distinctly 18 olefinic proton signals and 5 amino/amide proton CH3OH, 5:5:2:1, v/v, a dark band at Rf 0.21 under UV 254 nm aroused signals in the lower field region (Supplementary Figure S6). In our attention, as the following HPLC of this band’s EtOAc extract addition, the spectrum displayed seven methyl groups (including Key Laboratory of Biotechnology of Antibiotics of Ministry of Health, Institute of Medicinal Biotechnology, CAMS & PUMC, Beijing, China Correspondence: Professor L Wu or Dr B Jiang, Key Laboratory of Biotechnology of Antibiotics of Ministry of Health, Institute of Medicinal Biotechnology, CAMS & PUMC, Tiantan Xili, Beijing 100050, China. E-mail: [email protected] or [email protected] Received 2 December 2015; revised 30 January 2016; accepted 4 February 2016; published online 9 March 2016 Hangtaimycin from Streptomyces spectabilis LZuoet al 836 one oxygenated methyl group at δ 3.18 and one nitrogenated methyl The connectivity of fragments 1 and 2 via the methylene C-10 was group at δ 2.94), five methylene and six methine proton signals, clearly demonstrated by the HMBC correlations from H-10 to C-2, together with an overlapping singlet attributed to hydroxyl C-3, C-9 and C-16, and from H-11 to C-3 (Figure 1). 1 1 proton signal. The 13C NMR and DEPT spectra of 1 showed 50 The H- HCOSYcorrelationsofH3-22/H-21/H-20/NH-23 and carbon resonances corresponding to the above groups, as well as 15 H-29/H-30/H-31/H-32/H2-33/H2-34/H2-35, and HMBC correlations quaternary carbons (8 amide or ester carbonyls, 7 sp2 carbons) of H-20 to C-19, C-24, H-21 to -OCH3,H2-26 to C-24, NH-27 to (Supplementary Figures S7 and S8). C-24, C-26 and C-28, H-29 and H-30 to C-28 revealed the presence of The proton and hydrogen-bearing carbon resonances in the fragment 3 that contained a 3-O-methyl-threonine, a dehydroalanine 1 NMR spectra of 1 were assigned unambiguously by HSQC and a conjugated octanoic acid in (Figure 1). 1 1 spectroscopic data interpretation (Table 1 and Supplementary The H- H COSY correlations of H-52/H-53/H-54/H-55/H3-56 and H -37/H-38/H-42/NH-43, and HMBC correlations from H-52, H-53 Figure S9). 2 to C-51, H -46 to C-44, C-45 and C-47, H-47 to C-44, C-46, and In the 1H-1H gCOSY spectrum of 1 (Supplementary Figure S10), 3 H-43, H-46 and H-47 to C-44 demonstrated the presence of fragment the homonuclear coupling correlations NH-1/H-2, H-4/H-5/H-6/H-7 4 consisted of 1-amino-1-propanol, 2-methyl-2-ene-4-amino-penta- revealed the presence of structural units containing the vicinal 1 1 noic acid and 2,4-hexadienoic acid in (Figure 1). coupled protons (Figure 1, thick lines). In the HMBC spectrum of The linkage of fragments 3 and 4 by a δ-valerolactone ring was (Supplementary Figure S11), the following cross-peaks were observed: clearly demonstrated by the HMBC correlations from H-34, H-35, NH-1, H-2/C-3, C-8 and C-9; H-4, H-6/C-9; H-5/C-8, C-9; H-7/C-3, H-37 and H-38 to C-36, H-35, H-37 to C-41 and H-41 to C-40 C-8. These correlations, in combination with the shifts of these (Figure 1). proton and carbon resonances, demonstrated the presence of a C-3- The association of C-19 and the diketopiperazine moiety via a substituted indolyl moiety in 1 (Figure 1, fragment 1). nitrogen atom (N-15) was suggested by the chemical shift of C-19 1 1 The H- H gCOSY correlations (H-10/H-11 and H-17/H3-18) and (δ 167.0), the accurate assignment five out of the six exchangeable HMBC correlations (H3-18, H-17/C-13, C-14; H-11/C-13, C-16 and proton signals, together with 1’s molecular formula C50H61N7O11 with N-CH3; N-CH3/C-13, C-14), together with the chemical shift values of 24° of unsaturation. these carbons, indicated the presence of a cyclodipeptide moiety The geometrical configurations of four double bonds in compound (N-methylalanine-dehydrobutyrine; Figure 1, fragment 2) in 1. 1 were determined to be 29Z,31E,52Z and 54E by coupling constants Table 1 The NMR spectra data of hangtaimycin (compound 1) in DMSO-d6 Position δC δH (J in Hz) Position δC δH (J in Hz) 1 — 10.93 br. s 29 123.1 6.26 d (10.2) 2 125.3 6.94 d (2.4) 30 140.9 7.09 dd (15.0, 10.2) 3 107.3 31 129.0 6.22 (overlap) 4 111.1 7.29 d (8.4) 32 142.2 6.20 dt (15.0, 6.6) 5 121.0 7.02 t (7.2) 33 31.7 2.18 q (7.2) 6 118.6 6.92 t (7.2) 34 25.1 1.62 m 7 118.0 7.32 d (8.4) 35 35.3 2.29 t (7.2) 8 136.2 36 163.7 9 127.0 37 28.6 2.48 (overlap) 10 26.3 3.24 dd (14.4, 4.8) 38 77.2 4.37 m 3.35 dd (14.4, 4.2) 40 161.6 11 64.6 4.70 dd (4.8, 4.2) 41 114.6 5.74 s N-CH3 31.4 2.94 s 42 73.3 5.36 dd (13.2, 7.2) 13 162.3 42-OH 6.25 br.

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