Introduction

The genus Streptomyces is well-known as an excellent source of useful chemicals produced as secondary metabolites. Non-Streptomyces organisms can also be a good source of new compounds, as reported by Tiwari and Gupta [1]. Our group has discovered many important compounds, such as staurosporine [2], the clostomicins [3] and setamycin [4], from non-Streptomyces strains. Recently, the mangromicins (genus Lechevalieria) [5,6,7] and sagamilactam (genus Actinomadura) [8] were found from non-Streptomyces strains by physico-chemical screening by our group [9]. With respect to soil samples used to isolate actinomycetes, the genus Streptomyces is predominant, accounting for over 90% of actinomycetes found. We focused on plant roots as a source of actinomycetes producing new compounds as the frequency of isolating non-Streptomyces strains from plant root samples has been seen to be higher than that from soil samples, Streptomyces strains isolated from plant root samples estimated to constitute a mere 22.9% [10].

Endophytic actinomycete strains have a capacity to produce a wide variety of biological active compounds [10,11,12]. he spoxazomicins (genus Streptosporangium) [13], trehangelins (genus Polymorphospora) [14] and actinoallolides (genus Actinoallomurus) [15] were all found from endophytic actinomycetes by our group. This influenced our approach to search for new compounds from the cultured broths of endophytic non-Streptomyces strains.

Our screening program has discovered two new 22-membered macrolides, named hamuramicins A (1) and B (2), from a cultured broth of an endophytic non-Streptomyces strain, Allostreptomyces sp. K12-0794 (Fig. 1). The strain was isolated from a fern root gathered in Hamura, Tokyo. The strain, K12-0794, was identified as a member of the genus Allostreptomyces based on the morphology, chemical compositions and phylogenetic analysis. Allostreptomyces was proposed as new genus by Huang et al. in 2017 [16]. This is the first report of the isolation of new secondary metabolites from the cultured broth of the genus Allostreptomyces. In this paper, the isolation, physico-chemical properties, structure elucidation and some bioactivities of 1 and 2 are described.

Fig. 1
figure 1

Structures of hamuramicins A (1) and B (2)

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Results and discussion

Isolation of hamuramicins A (1) and B (2)

The procedure for isolation of 1 and 2 is summarized in Supplementary Scheme S1. A culture broth (200 L) was centrifuged at 12,000 rpm and the mycelial cake was mixed with acetone (21 L). After stirring for 3 h, the acetone extract was evaporated under reduced pressure to remove acetone. The aqueous solution was extracted twice by 7 L of ethyl acetate, and the organic layer was partitioned once by 7 L of H2O. The organic layer was concentrated to dryness in vacuo, and dissolved in a small amount of acetonitrile. The acetonitrile soluble fraction filtered by filter paper was concentrated to dryness in vacuo to yield a crude material (30.6 g). The material was dissolved in a small amount of CHCl3 and then applied to a silica gel column (50 i.d. × 120 mm; Fuji Silysia Chemical, Aichi, Japan), which was eluted stepwise with 1 L of a mixture of CHCl3-acetone (1:0, 4:1, 3:1, 2:1, 1:1 and 0:1) in this order, and the fractions were divided into two fractions (each 500 mL). Compounds 1 and 2 were included in four fractions (3:1–1 to 2:1–2). The CHCl3-acetone 3:1–2 and 2:1–1 fractions (total weight, 1.9 g), which contained 1 and 2 with high purity, were dissolved in a small amount of MeOH and applied to a preparative HPLC (Capcell pak C18 UG-120, 20 i.d. × 250 mm, Shiseido Co., Tokyo, Japan) with 80% of MeOH/H2O solvent (flow rate, 7.0 mL/minute; detection, UV 210 nm). The peaks with retention times of 16 and 20 min were collected and concentrated in vacuo in dark to dryness to yield hamuramicin A (1, 329.3 mg) and hamuramicin B (2, 397.9 mg), respectively.

Structure elucidation of hamuramicin A (1)

Compound 1 was obtained as a pale yellow powder ([α]D23 −140: c 0.1, MeOH) and readily soluble in CHCl3, CH2Cl2, acetonitrile, acetone, MeOH and DMSO. It showed UV absorption (Supplementary Figure S1) maxima at 330 nm (ε 19850), 275 nm (ε 58210) and 265 nm (ε 54310) in MeOH. The IR absorption (Supplementary Figure S2) at 1720 and 3424 cm−1 indicated the presence of ketones and hydroxyl groups, respectively. The molecular formula of 1 was elucidated by HR-ESI-MS to be C35H50O7 (found m/z 605.3450 [M + Na]+, calcd. m/z 605.3454), requiring 11 degrees of unsaturation. The 1H and 13C NMR spectral data of 1 is listed in Table 1, Supplementary Figures S3 and S4. The 13C NMR and HSQC (Supplementary Figure S5) spectra indicated 35 carbons, which were classified into five sp2 fully substituted carbons including two carbonyl carbons, 13 sp2 methines, five oxygenated sp3 methines, two sp3 methines, four methylenes, and six methyls. The 1H–1H COSY (Fig. 2a and Supplementary Figure S6) of 1 indicated the assignments from H2-2 to H-3, from H-5 to H-6, from H-8 to H-10 and from H-13 to H3-30. The HMBC (Fig. 2a and Supplementary Figure S7) correlations from H2-2 (δH 2.99 and 3.15) to C-1 (δC 173.8) and C-4 (δC 141.5), from H-3 (δH 5.51) to C-1, from H-5 (δH 4.81) to C-4 and C-7 (δC 135.6), from H3-31 (δH 1.66) to C-3 (δC 118.0), C-4 and C-5 (δC 72.2), from H-8 (δH 6.69) to C-7 and C-32 (δC 12.5), from H3-32 (δH 1.88) to C-6 (δC 140.5), C-7 and C-8 (δC 148.0), from H-10 (δH 7.37) to C-12 (δC 200.6) and C-33 (δC 11.8), from H-13 (δH 5.40) to C-12, from H3-33 (δH 1.88) to C-10 (δC 146.3), C-11 (δC 131.4) and C-12, from H-14 (δH 5.34) to C-12 established the connectivity between C-1 to C-30, which was the presence of 5,13,21,23,27-pentahydroxyl-4,7,11,22,24-pentametheyl-12-oxotriaconta-3,6,8,10,14,16,18,28-octaenoic acid. Finally, the HMBC correlation from H-21 (δH 5.16) to C-1 and remaining unsaturation indicated the esterification between C-1 and C-21.

Table 1 1H and 13C NMR spectral data of hamuralides A (1) and B (2) in CD3OD
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Fig. 2
figure 2

Structure elucidation of hamuramicins A (1) and B (2). a gCOSY and key gHMBC correlations. b Key ROESY correlations and coupling constants

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The geometries of double bonds and relative configurations of 1 were established from coupling constants and the ROESY correlations (Fig. 2b and Supplementary Figure S8). The coupling constants were 15.0 Hz between H-8 (δH 6.69) and H-9, 14.5 Hz between H-14 and H-15(δH 6.63), 14.9 Hz between H-16 (δH 6.07) and H-17 (δH 6.28), 15.2 Hz between H-18 (δH 6.11) and H-19, respectively. The ROESY correlations were observed between H3-31 and H-5, between H-5 and H3-32, between H3-32 and H-9, between H-9 and H3-33, between H-6 (δH 5.66) and H-8, between H-8 and H-10, between H-10 and H-13, between H-13 and H-15, between H-15 and H-17, between H-14 and H-16, between H-16 and H-18, resulting in the geometry of double bonds, were all E-configuration in the 22-menbered macrolide. However, the relative configurations of C-5 and C-13 were not determined, although the coupling constants were 9.2 Hz between H-5 and H-6, 9.5 Hz between H-13 and H-14, respectively. The stereochemistry of the two hydroxyl groups at C-5 and C-13 was able to be both orientation, owing to the flexible 22-membered ring. A previous report of pulvomycin showed that the relative stereochemistry at C-5 and C-13 using nOe [17], plus the absolute configuration of pulvomycin, was confirmed by crystallographic analysis with an elongation factor (EF-) Tu (PDB entry 2C78) [18]. Likewise, the ROESY correlations were observed between H-18 and H2-20 (δH 2.33), between H2-20 (δH 2.62) and H-23 (δH 3.28), between H-19 (δH 5.61) and H-21, between H-22 (δH 1.82) and H3-35 (δH 0.82), between H-24 (δH 1.57) and H3-34 (δH 0.89), between H-27 (δH 4.41) and H3-30 (δH 1.68), between H-28 (δH 5.37) and H-29 (δH 5.53). The small coupling constants between H-20eq and H-21 (2.4 Hz), and between H-22 and H-23 (3.4 Hz) would support their syn-relationship, and the large coupling constants between H-19 and H-20ax (10.4 Hz), between H-20ax and H-21 (10.4 Hz) and between H-21 and H-22 (6.8 Hz) would support their anti-relationship. In addition, a coupling constant with 10.6 Hz between H-28 and H-29 suggested 28Z. We tried to elucidate the absolute configuration of C-27 using modified Mosher’s method but, unfortunately, the sample was decomposed. Thus, the geometries of double bonds and relative configurations of 1 was elucidated to be 3E, 6E, 8E, 10E, 14E, 16E, 18E, 21 R*, 22 S*, 23 R*, 24 R* and 28Z (Fig. 2b).

Structure elucidation of hamuramicin B (2)

Compound 2 contained a lot of impurities. The NMR analysis revealed approximately a 2:1 mixture. The minor compound, which is very similar to structure of 2, could not be determined. Significant differences of 1H and 13C NMR data were observed at the midpoint of the 27-position (Supplementary Figure S9). We concluded that the minor compound may be a diastereomer of 2.

Compound 2 was obtained as a pale yellow powder ([α]D23 −166: c 0.1, MeOH) and readily soluble in CHCl3, CH2Cl2, acetonitrile, acetone, MeOH and DMSO. It showed UV absorption (Supplementary Figure S10) maxima at 330 nm (ε 16010), 275 nm (ε 47720) and 265 nm (ε 43870) in MeOH. The IR absorption (Supplementary Figure S11) at 1716 and 3432 cm−1 indicated the presence of ketones and hydroxyl groups, respectively. The molecular formula of 2 was elucidated by HR-ESI-MS to be C35H52O7 (found m/z 583.3620 [M − H]−, calcd. m/z 583.3635), requiring 10 degrees of unsaturation. The physico-chemical properties and NMR spectral data were similar to those of 1 (Table 1, Supplementary Figures S12, S13 and S14). The structure of 2 indicated that it was a reductant of 1, according to the absence of a double bond at the H-28 position in NMR spectra coupled with decreased degrees of unsaturation. The planar structure of 2 was further elucidated to be a reductant of 1 at the 28 position using COSY and HMBC (Fig. 2a and Supplementary Figures S15 and S16), and the geometries of double bonds and relative configurations of 2 was the same as 1 by data of ROESY (Supplementary Figure S17) correlation and coupling constants (Fig. 2b). We tried to elucidate the absolute configuration of the H-27 using modified Mosher’s method but the sample was decomposed.

Biological activity

Compounds 1 and 2 were evaluated for antimicrobial activity and cytotoxicity (Tables 2 and 3). Compounds 1 and 2 showed growth inhibition activity against Kocuria rhizophia ATCC9341 and Xanthomonas oryzae pv. oryzae KB88 but they showed no antimicrobial activities against six other bacteria at a concentration of 256 μg/mL using the agar dilution method [19]. However, both compounds broadly showed cytotoxicity against five adherent cell lines. The similar pulvomycin was evaluated for antimicrobial activity by Landini et al [20]. The report showed that MIC values of pulvomycin were 2 μg/mL and 8 μg/mL against Bacillus subtilis ATCC6633 and Staphylococcus aureus L165, respectively. However, 1 and 2 exhibited no antimicrobial activity against B. subtilis ATCC6633 and S. aureus ATCC6538P. It is suggested that the side chain is important for the expression of broad antibacterial activity.

Table 2 Biological activity of of hamuralides A (1) and B (2), The MIC values against eight bacteria
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Table 3 The IC50 values of cytotoxicity against five adherent cell lines
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This is the first report that new secondary metabolites from the cultured broth of the genus Allostreptomyces have been isolated. It reinforces the belief that there is significant possibility of discovering a significant number of useful compounds from culture broths of endophytic non-Streptomyces organisms.

Materials and methods

Fermentation

A loop of strain K12-0794 cells grown on an agar slant was inoculated into 10 mL of the seed medium consisting of 2.4% starch, 0.1% glucose, 0.3% peptone, 0.3% meat extract, 0.5% yeast extract, 0.4% CaCO3 and incubated until sufficient growth at 280 rpm at 27 °C. The cultured broth was stored at −28 °C as a frozen stock. Two hundred microliters of the frozen stock were inoculated into 10 mL of fresh seed medium with 5 pieces of glass beads (ϕ 6.3–7.8 mm) in test tubes for 3 days at 280 r.p.m. at 27 °C. Two milliliters of the seed culture were inoculated into 100 mL of the seed medium in 500-mL Erlenmeyer flasks and cultured for 4 days at 210 rpm at 27 °C. The seed (1.2 L) culture was inoculated into 20 L of fermentation medium consisting of 2.0% soluble starch, 1.0% defatted wheat germ, 0.3% meat extract, 0.4% CaCO3 and 0.08% Disform® as a defoaming agent in 30-L jar fermenter and incubated for 4 days (agitation, 200 rpm; temperature, 27 °C; aeration, 0.5 v/v/m).

General experiments

NMR spectra were measured by a Varian XL-400 spectrometer (Agilent Technologies Japan Ltd., CA, USA), with 1H NMR at 400 MHz and 13C NMR at 100 MHz in CD3OD. The chemical shifts are expressed in ppm and referred to CHD2OD (3.31 p.p.m.) in the 1H NMR spectra and to CD3OD (49.0 p.p.m.) in the 13C NMR spectra. ESI-MS spectra were measured with a JMS AX-505 HA mass spectrometer (JEOL Ltd., Tokyo, Japan). IR spectra (KBr) were taken on a FT-210 Fourier transform infrared spectrometer (Horiba Ltd., Kyoto, Japan). UV spectra were measured with a Hitachi U-2801 spectrophotometer (Hitachi Ltd., Tokyo, Japan). Optical rotation was measured with a JASCO P-2200 polarimeter (JASCO Co., Tokyo, Japan).

Antimicrobial activities

The agar dilution method was carried out according to the method recommended by CLSI19. Antimicrobial activities of 1 and 2 against eight microorganisms, B. subtilis ATCC6633, K. rhizophila ATCC9341, S. aureus ATCC6538P, Escherichia coli NIHJ, X. oryzae pv. oryzae KB88, Klebsiella pneumoniae ATCC10031, Proteus vulgaris NBRC3167 and Pseudomonas aeruginosa NBRC12582, were evaluated. The cultured broths of test microorganisms were diluted to log 6.0 CFU/mL and were spotted with Mueller Hinton II agar containing 1 or 2 (final concentration: 0.25–256 μg/mL). All microorganisms, except X. oryzae, were incubated at 37 °C for 24 h. X. oryzae was incubated at 27 °C for 48 h.

Cytotoxicity

Cytotoxic evaluation of 1 and 2 used five adherent human cell lines, HeLa S3 (human cervical cancer cell line), HT29 (human colorectal adenocarcinoma cell line), A549 (human adenocarcinoma cell line derived from lung cancer), H1299 (human non-small cell lung carcinoma cell line) and PANC-1 (human cell line derived from pancreatic cancer). All cell lines were incubated in DMEM medium (Wako Pure Chemical Industries, Osaka, Japan) supplemented with 10% FBS and 1% penicillin streptomycin at 37 °C under 5% CO2. The cultivated cell lines (5 × 104 cells per well) were seeded in 96-well plates. After overnight culture, MeOH solution of 1 or 2 was added into each well. After 2 days of incubation at 37 °C, WST-8 solution was added to each well and kept for 4 h at 37 °C. The absorbance of each well was measured using a Corona Grating Microplate Reader SH-9000 (Corona Electric Co. Ltd., Ibaraki, Japan) at 460 nm.