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

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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. s 14 125.3 43 - 8.21 d (8.4) 16 172.1 44 168.3 17 129.6 5.53 q (7.2) 45 130.5 18 13.4 0.61 d (7.2) 46 12.8 1.83 s 19 167.0 47 137.4 6.13 d (9.0) 20 55.7 5.72 dd (8.4, 3.6) 48 42.4 4.64 m 21 75.8 3.66 m 49 20.3 1.15 d (6.6)

21-OCH3 56.4 3.18 s 50 - 8.13 d (7.2) 22 15.5 0.98 d (6.0) 51 164.6 23 - 8.01 d (8.4) 52 119.1 5.57 d (11.4) 24 164.2 53 140.1 6.36 t (11.4) 25 135.8 54 128.6 7.49 dd (14.4, 12.0) 26 105.2 5.50 s, 6.10 s 55 136.8 5.94 m 27 - 9.27 s 56 18.3 1.78 d (6.6) 28 164.7

1Hand13C NMR spectral data (δ) were obtained at 600 and 150 MHz, respectively, on a VNS-600 spectrometer or Bruker 600 spectrometer at room temperature.

The Journal of Antibiotics Hangtaimycin from Streptomyces spectabilis LZuoet al 837

6' 5' 7' 4' O 7'a 3'a HN 6a HN HN H 1' 3' H O OCH3 2' 6 O N C C N 1N 5 NH HN O 2 NH O O O O 4 O OH O 3 HN O 3a 3b

Figure 2 Chemical structure of TDD (tryptophan–dehydrobutyrine diketopi- 56 55 perazine) from S. spectabilis CPCC 200148. fragment 1 53 54 6 5 fragment 4 O 7 4 52 CPCC 200148 (7.0 l, with an incubation period 36 h at 28 °C) 8 9 δ-valerolactone HN 49 was first fractionated by a silica flash column (Santai Technologies HN ring 50 3 10 22 1 46 Inc., Changzhou, China; 80 g) eluted with EtOAc-methanol (49:1, 2 16 O OCH3 47 − 1 21 30 min; 33:1, 60 min; 25 ml min ). Fractions containing the putative N C 12 23 44 TDD (Supplementary Figures S12 and S13) were pooled, concentrated N 19 NH H HN O O 14 26 42 and then loaded onto preparative silica gel TLC for refined fractiona- O 25 O O 17 O H 39 OH tion with a mobile phase of CH2Cl2-CH3OH, 10:1, v/v.Theputative 18 41 HN O 37 TDD band (Rf 0.50, Supplementary Figure S14) was scrapped off and fragment 2 28 31 33 extracted with EtOAc. The extract revealed a molecular ion at m/z 29 35 284.13928 ([M+H]+, Supplementary Figure S15) by HRMS, which fragment 3 30 32 34 established a molecular formula C16H17O2N3 (calculated at 284.13935 Figure 1 The planar structure of hangtaimycin (compound 1, upper), and for [M+H]+) identical to TDD.12 The extract was then subjected to the 1H-1H COSY (thick bonds, lower) and main HMBC correlations (arrows, reverse-phase HPLC, which yielded 5 mg pure putative TDD as white fi lower). A full color version of this gure is available at The Journal of amorphous powder after evaporation. Its 1H-NMR and 13C-NMR Antibiotics journal online. spectra (Supplementary Figures S16 and S17) were found to be the same as the chemosynthesis Z-isomer of TDD (δH:5.41(q,1H, J29–30 = 10.2 Hz, J31–32 = 15.0 Hz, J52–53 = 11.4 Hz and J54–55 = 14.4 Hz J = 7.4 Hz, H-3a); 0.84 (d,3H,J = 7.4 Hz, H-3b)), not the chemo- 1 (Table 1). Accordingly, the planar structure of , designated as synthesis E-isomer of TDD (δH:4.51(q,1H,J = 7.4 Hz, H-3a); 1.66 hangtaimycin hereafter, was determined as depicted in Figure 1. (d,3H,J = 7.4 Hz, H-3b)), disclosed in the reference.12 Thus, the TDD Hangtaimycin is a peptide secondary metabolite. It has a linear from S. spectabilis CPCC 200148 was identified as its Z-isomer chain as the terminal unit comprises 2,4-hexadienoic acid and (Figure 2 and Supplementary Table S1). 2-methyl-2-ene-4-amino-pentanoic acid linked by amide bond, which To determine the absolute configuration of the only chiral carbon is hardly seen in microbial secondary metabolites. It contains an (C-6) in TDD from S. spectabilis CPCC 200148, we assayed its specific δ 20 -valerolactone ring embedded in a conjugated dodecanoic acid, a rotation and CD spectrum. The TDD showed an [α] D − 12.67 structural moiety rather rare in natural products. In addition, (c 0.03, EtOH), which is different from that of the chemically 21 12 hangtaimycin has a dipeptide unit of 3-O-methylthreonine-dehydroa- synthetic TDD with [α] D+13 (c 0.03, EtOH). Thus, the TDD lanine, which connects the terminal TDD moiety of the molecule. from S. spectabilis CPCC 200148 should possess an R configuration It is worthy to note that hangtaimycin had no structure similar (Figure 2). The experimental CD spectrum of TDD from S. spectabilis molecules in SciFinder with Tanimoto coefficient X 70%. CPCC 200148 showed Cotton effects at 218(− ), 255(+) and 293 Hangtaimycin may bear a hybrid structural skeleton assembled by ( − ) nm, which was very similar to the electronic circular dichroism PKS-NRPS (polyketide synthase and nonribosomal peptide synthase) (ECD) spectrum generated by semi-empirical ZINDO of the R for its biosynthesis in S. spectabilis CPCC 200148. The 2,4-hexadienoic configuration TDD13 (Supplementary Figures S18 and S19), which acid unit, the conjugated dodecanoic acid unit and the δ-valerolactone further supported the R configuration assignment to the only chiral ring were most probably formed by type I PKS, while the whole C-6 of TDD from S. spectabilis CPCC 200148. molecule was assembled by NRPS. Hangtaimycin should possess the same absolute configuration for Hangtaimycin contains six chiral carbons in its molecule, that is, its chiral C-11 as the corresponding chiral C-6 in the TDD identified C-11, 20, 21, 38, 42 and 48. To determine the absolute configurations above, as hangtaimycin must share the same set of biosynthetic of these chiral carbons, an initial attempt to crystallize hangtaimycin machinery for its TDD moiety with the TDD molecule that appeared was made but was not successful, possibly because it is a long linear in S. spectabilis CPCC 200148 synchronously with hangtaimycin. molecule that may be difficult to crystallize. Therefore, the chiral C-11 in hangtaimycin should take an R As hangtaimycin contains a TDD moiety, and that a putative TDD configuration. However, chiral C-20, 21, 38, 42 and 48 in hang- peak was detected in the previous HPLC analysis, we purified the taimycin remain unclear concerning their absolute configurations. putative TDD and then characterized its structure for comparison with Some natural or synthetic compounds with 2,5-diketopiperazine hangtaimycin. The EtOAc extract of the ISP2 culture of S. spectabilis moiety exhibited antibacterial or other biological activities.14,15 A

The Journal of Antibiotics Hangtaimycin from Streptomyces spectabilis LZuoet al 838

preliminary inspection of the antibacterial activity of hangtaimycin was 3 Spasova, D., Vesselinova, N. & Gesheva, R. Comparative investigation of a conducted, as it contains a 2,5-diketopiperazine moiety in the streptovaricin-producing strain of Streptomyces spectabilis and its selectant. Folia. Microbiol. 42,35–38 (1997). molecule. Hangtaimycin generated a small growth inhibition zone 4 Vesselinova, N., Gesheva, R. & Ivanova, V. Streptomyces species producing the on yeast extract peptone dextrose medium (YPD) plate inoculated streptovaricin complex. Folia. Microbiol. 36,538–541 (1991). 5 Isaka, M., Jaturapat, A., Kramyu, J., Tanticharoen, M. & Thebtaranonth, Y. Potent with Bacillus subtilis CMCC 63501 or Candida albicans ATCC 10231 in vitro antimalarial activity of metacycloprodigiosin isolated from Streptomyces when an amount of 100–800 μg hangtaimycin was applied on a spectabilis BCC 4785. Antimicrob. Agents. Chemother. 46, 1112–1113 (2002). Φ 5.0-mm ﬁlter paper disk (Supplementary Figure S20).16 Thus, it 6Benallaoua,S.et al. The mode of action of a nonpolyenic antifungal (desertomycin) produced by a strain of Streptomyces spectabilis. Can. J. Microbiol. 36, displayed very weak antimicrobial activities. Hangtaimycin produced 609–616 (1990). − 1 ~50% growth inhibition at 250 μM (233.75 μgml ) to human 7 Kurosawa, K., Takahashi, K. & Tsuda, E. SNF4435C and D, novel immunosuppressants produced by a strain of Streptomyces spectabilis. I. Taxonomy, fermentation, isolation pancreatic cancer cell lines Hup-T3 and MIA PaCa-2, indicating that and biological activities. J. Antibiot. 54,541–547 (2001). it had also very weak cytotoxicity against some tumor cells.17 8 Staley, A. L. & Rinehart, K. L. Spectomycins, new antibacterial compounds produced by Streptomyces spectabilis: isolation, structures, and biosynthesis. J. Antibiot. 47, 1425–1433 (1994). ACKNOWLEDGEMENTS 9 Kakinuma, K. & Rinehart, K. L. Jr. Tryptophan-dehydrobutyrine diketopiperazine, a This work was supported by the National Natural Science Foundation of China metabolite of Streptomyces spectabilis. J. Antibiot. 27,733–737 (1974). (81573328 and 81321004), National Mega-project for Innovative Drugs 10 Yu, Q. & Fan, C. [Taxonomy of 1043 strain producing actinospectacin]. Wei Sheng Wu Xue Bao 34,160–163 (1994). (2012ZX09301002-001-016 and 2012ZX09301002-003), Fundamental Research 11Yu,Q.W.et al. [Streptomyces strain 1043 and actinospectacin produced by it]. Wei Funds for the Central Universities (2012N09), Peking Union medical college Sheng Wu Xue Bao 19,81–87 (1979). graduate student Innovation Fund (10023-1007-1010) and National 12 Santamaria, A., Cabezas, N. & Avendano, C. Synthesis of Tryptophan-dehydrobutyrine diketopiperazines and analogues. Tetrahedron. Lett. 55, 1173–1186 (1999). Infrastructure of Microbial Resources (No. NIMR-2015-3). 13 Frisch, M. J. et al. Gaussian 09 Revision A.01, Gaussian, Inc., Wallingford, CT, (2009). 14 de Carvalho, M. P. & Abraham, W. R. Antimicrobial and bioﬁlm inhibiting diketopiperazines. Curr. Med. Chem. 19,3564–3577 (2012). 15 Kanoh, K. et al. Antitumor activity of phenylahistin in vitro and in vivo. Biosci. Biotechnol. Biochem. 63,1130–1133 (1999). 1 Mason, D. J., Dietz, A. & Smith, R. M. Actinospectacin, a new antibiotic. I. Discovery 16 Bauer, A. W., Kirby, W. M., Sherris, J. C. & Turck, M. Antibiotic susceptibility testing by and biological properties. Antibiot. Chemother. 11,118–122 (1961). a standardized single disk method. Am. J. Clin. Pathol. 45,493–496 (1966). 2 Hoeksema, H. & Knight, J. C. The production of dihydrospectinomycin by Streptomyces 17 Skehan, P. et al. New colorimetric cytotoxicity assay for anticancer-drug screening. spectabilis. J. Antibiot. 28,240–241 (1975). J. Natl Cancer. Inst. 82,1107–1112 (1990).

Supplementary Information accompanies the paper on The Journal of Antibiotics website (http://www.nature.com/ja)

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