Note pubs.acs.org/jnp
Phoslactomycins from Streptomyces sp. MLA1839 and Their Biological Activities Serge Fotso,* Paul Graupner, Quanbo Xiong, Don Hahn, Cruz Avila-Adame, and George Davis Discovery Research, Dow AgroSciences, 9330 Zionsville Road, Indianapolis, Indiana 46268, United States S Supporting Information *
ABSTRACT: Phoslactomycins H (1) and I (2), two new members of the phoslactomycin class of chemistry, were isolated from Streptomyces sp. MLA1839 on the basis of their antifungal activities. Their structures were elucidated using extensive NMR spectroscopy and mass spectrometry. Phoslactomycin H (1) featured a rare and unprecedented N,Ndimethylamine substitution at C-4 and existed as a hydroxy acid rather than the more common lactone. Herein, we report the structure of these compounds and their biological activities.
T
was confirmed by a 31P NMR spectrum in MeOH-d4, which indicated a signal from phosphate (δ 1.73). Searches using the molecular formula and the high-resolution mass in the Dictionary of Natural Products [Chapman and Hall] (DNP) and in SciFinder indicated no known chemistry hits, suggesting compound 1 is a novel natural product. The 13C NMR spectrum indicated 22 carbon atoms, which were attributed to eight sp2 methines, five sp3 methines, six methylenes, and one methyl group by an HSQC spectrum. One carbonyl at δC 168.5 and an aliphatic quaternary carbon at δC 76.0 were also visible. Interpretation of the H, H-COSY spectrum allowed the construction of three distinct spin systems: a proton signal appearing as a triplet at δH 6.18 (J = 9.5 Hz, H-3) coupled with protons at δH 6.35 (H-2) and 5.11 (H-4) ppm. In addition, proton H-4 indicated a coupling with a proton at δH 4.41 (H-5) bound to an oxygenated carbon; this first fragment was completed with the coupling of H-6 to protons at δH 6.03 (H7) and 4.41 (H-5). The second spin system was observed from H, H-COSY coupling between the methylene protons H-10 and the protons H-9 and H-11. This fragment was extended with the coupling of H-12 to H-11 and H-13 and by correlations from H-14 to H13 and H-15. Six methylene protons in the aliphatic region range of δH 1.20−1.80 appeared as multiplets. Among them, only one appeared in the first and second spin systems, suggesting that the last spin was probably a cyclohexyl ring based on the coupling of the methine proton at δH 2.50 (H-16) to two methylenes (C-17, C-21).
he phoslactomycins (PLMs, Figure 1), also known as phosphazomycins or phospholines, are antifungal polyketide-derived antibiotics belonging to a limited group of natural products structurally related to fostriecins and leustroducsins (LSN, Figure 1) produced by various Streptomyces spp. (S. pulveraceus, S. hygroscopicus, Streptomyces sp. HK803, S. nigrescens, etc.).1 Biological activities including antifungal, induction of colony-stimulating factors in bone marrow stromal cells,2 and cytotoxic activities have been reported. They act by inhibition of the serine/threonine protein phosphatase 2A (PP2A).3 Recently, the PP2A inhibition activity of PLMs has been shown to be responsible for the inhibition of tumor metastasis through the augmentation of natural killer cells.4 To date, phoslactomycins A−F have been isolated and reported.5 PLM G was produced enzymatically from Streptomyces sp. HK 803 by Reynolds et al.6 During our screening program on natural product extracts for the discovery of useful bioactive principles against plant and insect pathogens, the antifungal activities of the methanolic extract from Streptomyces sp. MLA1839 against Puccinia triticina (Bayer code PUCCRT) and Septoria tritici (SEPTTR) drew our attention. The bioactivity-guided investigation of this extract led to the isolation of two new metabolites, phoslactomycins H (1) and I (2). Herein, we report their structure elucidation and biological activities. The chromatogram profile of the methanolic extract indicated a major and a minor metabolite, which were isolated through bioassay-guided fractionation and identified using spectroscopy and spectrometry techniques as two new compounds belonging to the family of phoslactomycins. HRMS of 1 indicated a pseudomolecular ion at m/z 518.2530 [M + H]+ suggesting the molecular formula C24H41NO9P (requires 518.2519). The presence of phosphate © XXXX American Chemical Society and American Society of Pharmacognosy
Received: March 19, 2013
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Figure 1. Structures of known phoslactomycin (PLMs, 3) and leustroducins (LSN).
The partial relative stereochemistry of 1 was deduced from the NOESY spectrum, which indicated correlations between the protons H-4 and H-5; in addition, the methyl at position 8 indicated a correlation with H-9, and no correlation was observed between H-9 and H-11.
The spins were connected by interpretation of the HMBC spectrum. Spin systems II and III were connected based on the observed cross correlations between H-16 (δC 2.50) and the carbons at δC 123.1 (H-14) and 140.3 (H-15). In addition, HMBC correlations confirmed the fragment derived from the H, H-COSY couplings, with important cross-peaks visible between the proton at δH 4.96 (H-11) and carbons at δC 134.8 (C-12) and 124.8 (C-13). Some key HMBC correlations were also observed from the methyl singlet at δH 1.30 to the quaternary carbon at δC 76.0 (C-8) and to the oxygenated carbon C-9 (δC 79.4). The first and second spin systems were linked through the 4J correlation of the 8-CH3 to the carbon at δC 127.5 (C-6) and a 3 J correlation to the carbon at δC 141.6 (C-7). Determination of the location of the phosphate moiety was based on the splitting pattern (doublet) for the carbon C-9 resulting from coupling with phosphorus. A substructure search in the DNP was performed using spin system II, and the resulting hits indicated that this compound belongs to the group of compounds known as the phoslactomycins. Further HMBC cross-peaks indicated the correlations between H-2 and H-3 and the carbonyl at δC 168.5, leading to the substructure shown.
Figure 3. Structure of phoslactomycin H (1).
The decomposition of phoslactomycin B has been reported to occur at low or high pH, at different temperatures, and as a function of time, with the major decomposition product being the ring-opened lactone.7 The derivative of 1 possessing a lactone ring was not detected, and no methanolysis product was observed. In addition, LC analysis using neutral conditions indicated that the major metabolite was phoslactomycin H (1). These observations suggested that 1 was not an artifact formed during extraction or isolation. On the basis of the literature searches, this is the first example of a naturally occurring phoslactomycin containing a hydroxy acid rather than a lactone bearing an amino group at C-4.
Figure 2. Important HMBC (↷) correlations and spin systems (bold) derived from the COSY spectrum in the substructure of phoslactomycin H (1).
Figure 4. Structure of the methylated phoslactomycin H.
Accounting for this substructure, the molecular formula left an N(CH3)2 group to complete the structure of phoslactomycin H (1). The HMBC spectrum provided the location of the N,Ndimethylamino group through the cross-peak observed from the singlet of six protons at δH 2.87 to the carbon at δC 66.6 (C4). The broad signal due to the N(CH3)2 group at δC 40.0 in the 13C spectrum may be attributed to the zwitterionic nature of phoslactomycin H (1).
Methylation of phoslactomycin H (1) was performed, and the proton NMR spectrum of the obtained product indicated two methoxy signals; one appeared as a doublet and was assigned as connected to the phosphoric acid ester, while the second was the methyl ester of a carboxylic acid. The signal of the six-proton singlet due to the N(CH3)2 group that appeared at δH 2.87 (s) in compound 1 had shifted to δH 3.29 (s) in the methylated product and integrated for nine protons, indicating the presence of a trimethylammonium group. The HRESIMS B
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Figure 5. Structure of phoslactomycin I (left, 2) and fostriecin (right, 4).
Table 1. 1H (J in Hz) and 13C NMR of Phoslactomycins H (1) and I (2) in MeOH-d4
data support the structure of the methylated product by giving the empirical formula C27H46NO9P. Compound 2 was obtained as an oil with HRESIMS indicating the molecular formula C23H35O8P. The UV spectrum of 2 indicated a maximum absorption at 235 nm as in compound 1. Although, the proton and carbon NMR spectra of 2 showed similarities to those of compound 1, some differences were also observed, especially for the proton H-3, which appeared at δH 7.10 (δC 154.5) in compound 2 instead of δH 6.18 (δC 133.8) in compound 1. In addition, the H-4 in compound 2 was observed at δH 2.63 (δC 34.3) against δH 5.11 (δC 66.6) in compound 1. Furthermore, the 1H NMR spectrum of compound 2 indicated the lack of the singlet of the N(CH3)2 group, which appeared at δH 2.87 in compound 1; instead a doublet attributed to a methyl group was observed at δH 1.04. Additional evidence such as the downfield chemical shift of H-5 at δH 5.05 (δC 82.2) compared to δH 4.41 (δC 71.3) in compound 1 was observed, indicating that H-5 was connected to an oxygen. These differences suggested that a modification has occurred on the α,β-unsaturated carboxylic acid moiety of compound 2. Careful interpretation of the HMBC spectrum revealed the correlations of protons H-2, H-3, and H-5 to the carbonyl at δC 166.7 (C-1), suggesting the presence of a lactone ring. The methyl doublet at δH 1.04 indicated correlations to carbons at δC 154.5 (C-3), 82.2 (C-5), and 34.3 (C-4), leading to the structure of compound 2 as phoslactomycin I (Figure 5). The structure of phoslactomycin I (2) is a closely related to fostriecin (4), lacking the methyl at position C-4 and the cyclohexyl at position C-15. Compared to other PLMs, phoslactomycin I (2) lacks also the ethylamine substituent at C-8. Phoslactomycins H (1) and I (2) exhibited antifungal activity in vitro against multiple plant pathogenic fungi. Phoslactomycin I (2) was more active than phoslactomycin H (1), indicating that the lactone ring is important for the activity as previously reported for PLMs A−F.8 Both phoslactomycins showed potential in vitro antifungal activity against three plant pathogens (Table 2); however, this antifungal activity was not fully expressed when fungi were grown on the respective hosts. Pathogen specificity was observed with phoslactomycin I (2), which was active against the oomycete Phytophthora infestans, providing 87% control at 111 ppm, and against the basidiomycete P. triticina, providing 88% control at 200 ppm. Lack of disease control was observed against the imperfect fungi Alternaria solani, Septoria tritici, and Cochliobolus sativum.
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phoslactomycin H (1) 13
no.
1
C
H
phoslactomycin I (2) 13
C
1 2 3 4 4N(CH3)2 4-CH3 5
168.6 132.7 133.8 66.6 40.0 (br)
6
127.5
7 8 8-CH3 9
141.6 76. 0 24.8 79.4
1.30, s 4.25, br t (10.2)
139.4 76.0 24.3 79.3
10 11 12 13 14 15 16 17 18 19 20 21
40.3 64.8 134.8 124.8 123.1 140.3 37.8 34.5 27.0 27.2 27.0 34.5
1.50; 1.68 4.96, m 5.45, m 6.28, m 6.28, m 5.35, m 2.50, m 1.60−1.71, m 1.75, m; 1.36, m 1.68, m; 1.24, m 1.68, m; 1.24, m 1.11, m
40.8 64.6 134.5 124.5 122.9 140.1 37.6 34.5 26.9 27.1 26.9 34.4
71. 30
6.35, 6.18, 5.11, 2.87,
brd t (9.5) brt (9.5) s
4. 41, t (7.2) 5.69, dd (15.7, 7.2) 6.03, d (15.7)
166.7 120.1 154.5 34.3
12.6 82.2 125.5
1
H
5.96, dd (9.7, 1.1) 7.10, dd (9.7, 5.6) 2.63, m
1.04, d (7.2) 5.05, ddd (6.6, 3.9, 1.2) 5.92, dd (15.6, 6.6) 6.01, dd (15.6, 1.2) 1.37, s 4.27, ddd (2.5, 10.7, 10.4) 1.69, m; 1.52, m 4.96, m 5.45, m 6.26, m 6.27, m 5.34, m 2.50, m 1.70−1.20, m 1.70−1.20, m 1.70−1.20, m 1.70−1.20, m 1.63, m; 1.09, m
Table 2. Concentration (μM) to Achieve 50% Growth Inhibition (ED50) of Three Fungal Plant Pathogens Growing on Phoslactomycin-Amended Liquid Mediaa pathogen
phoslactomycin H (1)
phoslactomycin I (2)
Pyricularia oryzae Septoria tritici Ustilago maydis
16.6 7.2 11.4
2.6 2.0 9.2
The commercial fungicide azoxystrobin was used at 5 μM as the positive control. At this concentration azoxystrobin provided >70% inhibition of all three pathogens.
a
preparative system. IR was recorded on a Nicolet 6700 FT-IR (Thermo Scientific). Taxonomy of the Producing Strain. The strain MLA1839 was isolated from a soil sample collected in Singapore by Merlion Pharmaceuticals, Inc. The isolate was identified on the basis of morphology and a partial 16S rRNA sequence. The 16S rRNA sequence of strain MLA1839 showed high similarity to the 16S rRNA gene sequences of several species in the Streptomyces hygroscopicus clade including Streptomyces sioyeansis NRRL B-5408 (DQ026654), Streptomyces tuberciticus DSM 40261 (AJ621612), and Streptomyces nigrescens NRRL B-12176 (DQ442530). The partial 16S rRNA gene
EXPERIMENTAL SECTION
General Experimental Procedures. High-resolution mass spectra were acquired on an Agilent 6220 time-of-flight mass spectrometer (LC-TOF) introduced by HPLC in both positive electrospray (+ESI) and negative electrospray (−ESI) modes. All NMR experiments were acquired on a Bruker DRX600 spectrometer operating at 600.13 MHz for proton (1H) and 150.62 MHz for carbon (13C). HPLC purification was performed using an Agilent 1100 C
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Tomato was inoculated with either A. solani using approximately 5 × 104 conidia mL−1 or P. infestans using around 5 × 104 sporangia per mL. Conidia and sporangia of these two pathogens were suspended in water (no Tween-20 was required). After inoculation, plants inoculated with A. solani were incubated at 20−22 °C and 100% RH for 48 h, then moved to a growth chamber at 22 °C until disease evaluation. Tomato inoculated with P. infestans was incubated at 22 °C and 100% RH, then transferred to a growth chamber at 20 °C until final evaluation. Percent disease severity was visually estimated using a scale ranging from 0 to 100, where 0 indicated no visible disease and 100 corresponded to 100% disease severity. Percent disease control was calculated by subtracting from 1 the value obtained from dividing the disease severity observed in the treatments by the disease severity observed in the untreated control and multiplying the result by 100. Untreated controls were sprayed with water containing 0.01% Triton X-100 and 10% methanol. ED50 values were estimated using JMPPro 9.0.3 from SAS Institute Inc., Cary, NC, USA. Phoslactomycin H (1): C24H40NO9P; white powder; UV (from HPLC) 240 nm; IR (νmax, cm−1) 3364.5, 2928.8, 2851.6, 1713.3, 1448.3, 13.71.2, 1162.1, 1030.6, 977.9, 736.2; 1H and 13C NMR see Table 1; (+)-ESI HRMS m/z 518.2530 [M + H]+ (calcd for C24H41NO9P 518.2519), 1035.4942 [2M + H]+; HRESIMS m/z 516.2399 [M − H]−, 1033.4786 [2M − H]−. Phoslactomycin H methyl ester: C27H46NO9P; oil; UV (from HPLC) 240 nm; IR (νmax, cm−1) 3362, 2925, 2849, 2808.9, 1602.9, 1356.1, 1211.1, 1067.1; 1H NMR (400 MHz, MeOH-d4) δ 6.30 (m, 5H), 5.97 (1H, d, J = 15.4 Hz), 5.65 (1H, dd, J = 15.5, 7.9 Hz), 5.45 (1H, brt, J = 8.3 Hz), 5.37 (m, 2H), 4.58 (1H, t, J = 8.2 Hz), 4.27 (1H, brt, J = 8.6 Hz), 3.81 (3H, s), 3.66 (3H, d, J = 10.8 Hz), 3.29 (9H, s), 2.50 (1H, m), 1.56−1.80 (7H, m), 1.31−1.54 (4H, m), 1.30 (3H, s), 1.00−1.25 (4H, m); HRESIMS m/z 560.3008 [M + H]+ (calcd for C27H47NO9P 560.2988), 1119.5935 [2M + H]+; HRESIMS m/z 558.2839 [M − H]−. Phoslactomycin I (2): C23H35O8P; oil; IR (νmax, cm−1) 3364.5, 2928.8, 2851.6, 1713.3, 1448.3, 13.71.2, 1162.1, 1030.6, 977.9, 736.2; 1 H and 13C NMR see Table 1; (+)-ESIHRMS m/z 453.2057 [M − H2O + H]+, 529.2695 [M + MeCN + NH4]+; (−)-ESIHRMS m/z 469.2006 [M − H]− (calcd for C23H34O8P 469.1997), 939.4122 [2M − H]−.
sequence of strain MLA1839 was deposited in GenBank with the accession number JX312318. Fermentation and Isolation. Strain MLA1839 was cultivated in a 250 mL Erlenmeyer flask containing 50 mL of seed medium composed of 1.5% glucose, 1.5% glycerol, 1.5% soya peptone (Oxoid), and 0.1% CaCO3 (pH 7.0, adjusted before sterilization). The flask was shaken on an orbital shaker (200 rpm) at 28 °C for 3 days. Aliquots (2.5 mL) of the broth were then transferred to 250 mL Erlenmeyer flasks containing 50 mL of a production medium composed of 1.5% glycerol, 3.0% oatmeal, 0.5% yeast extract (Becton, Dickinson and Company), 0.5% KH2PO4, 0.5% Na2HPO4·12H2O, and 0.1% MgCl2·6H2O. The samples (1 L) were cultured on a shaker (200 rpm) at 28 °C for 6 days. The culture was then freeze-dried and extracted with methanol. Isolation. The methanolic extract obtained from the 1 L culture broth was processed as followed: fractionation on preparative flash HPLC (XTerra 19 × 30 mm, 5 μm RP18, Waters) using 0.1% formic acid in a water and acetonitrile gradient for 12 min at 9 mL/min, from 0% to 80% acetonitrile in 8 and 9 min, and 0% acetonitrile in 10−12 min. Ten fractions were collected and tested in vitro against SEPTTR. The most active fractions, 5 (380.0 mg) and 7 (35.1 mg), were further analyzed on an XBridge column (250 × 21.2 mm, 10 μm, Waters). NMR spectra of these fractions indicated that fraction 5 was the pure phoslactomycin H (1), and further purification of fraction 7 on HPLC using an XBridge column (250 × 21.2 mm, 10 μm, Waters, 12 mL/ min flow rate, using 0.1% formic acid in water and acetonitrile in a gradient as follows: 5−100% acetonitrile in 10 min, 100% acetonitrile from 10 to 15 min, back to 5% acteonitrile from 16 to 24 min) delivered phoslactomycin I (25.1 mg, 2). The methylation of phoslactomycin H (1) was performed as follows: 3 mg of the phoslactomycin H (1) was dissolved in 5 mL of 50% dichlomethane/methanol, the mixture was kept in a dry ice, 5 mL of trimethylsilyldiazomethane was added, and the mixture was maintained for 3 min. The mixture was finally evaporated, and 5 mg of the methylated phoslactomycin H was obtained. Bioassay Sample Preparation. For in vitro evaluation, the active compounds were dissolved in dimethyl sulfoxide and tested in a 96well microtiter plate. Final compound concentrations were 0.08, 0.4, 2, and 10 mg L−1. Final solvent concentration was 1%, and it did not affect fungal growth. For in vivo evaluation, phoslactomycins H (1) and I (2) were dissolved in methanol (10% final concentration) and formulated in 0.01% Triton X-100 (Fisher Scientific, Fair Lawn, NJ, USA) in distilled water. In Vitro Test. In vitro evaluation was performed using fresh fungal spores as a source of inoculum. Spores of S. tritici were collected from 3-day-old cultures grown on potato dextrose agar; a 24-h-old culture of Ustilago maydis was produced in potato dextrose broth; Pyricularia oryzae spores were collected from 10- to 14-day-old cultures grown on rice agar. An inoculum of P. oryzae and S. tritici was adjusted to 4 × 104 and 1 × 105 spores mL−1, respectively, using yeast minimal phosphate media. U. maydis cultures were prepared at 1:500 dilution of a cell suspension previously adjusted to an OD450 of 0.2. The inoculum was dispensed in a 96-well microtiter plate at 200 μL per well. Percentage inhibition was estimated using an initial and a final absorbance reading as indicators of fungal growth. In Vivo Evaluation. Two-week-old tomato plants, 8-day-old winter wheat plants, and 7-day-old barley plants were treated with phoslactomycin at 6.9, 27.8 50, 111, and 200 mg·L−1 24 h before inoculation. They were sprayed to runoff and air-dried afterward for 24 h at room temperature. Wheat used for S. tritici evaluation was inoculated with a conidial suspension containing 0.05% Tween-20 (ACROS, NJ, USA) and 1 × 106 spores per mL, then incubated at 100% RH for 3 days at 20−22 °C. Plants were then incubated in a greenhouse at 20 °C, where they remained until the disease was fully expressed on untreated plants. Wheat infected with P. triticina was inoculated with 1 × 106 urediniospores per mL suspended in water with 0.05% Tween-20. Plants were incubated for 24 h at 22 °C and 100% RH, then transferred to a greenhouse at 25 °C. Barley was inoculated with C. sativum using a suspension of 5 × 104 conidia per mL in water with 0.05% Tween-20. Incubation and disease development were similar to conditions described for P. triticina.
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ASSOCIATED CONTENT
S Supporting Information *
1
H and 13C NMR and HSQC spectra of compounds 1 and 2 are available free of charge via the Internet at http://pubs.acs. org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Tel: (+1)317-337-5155. Fax: (+1) 317-337-3546. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank Merlion Pharmaceuticals, Singapore, who isolated and fermented strain MLA1839. The authors also thank N. Palaniappan for his helpful comments.
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REFERENCES
(1) Shibata, T.; Kurihara, S.; Oikawa, T.; Ohkawa, N.; Shimazaki, N.; Sasagawa, K.; Kobayashi, T.; Kohama, T.; Asai, F.; Shiraishi, A.; Sugimura, Y. J. Antibiot. 1995, 48, 1518−1520. (2) (a) Kohama, T.; Enokita, R.; Okazaki, T.; Miyaoka, H.; Torikata, A.; Inukai, M.; Kaneko, I.; Kagasaki, T.; Sakaida, Y.; Satoh, A.; Shiraishi, A. J. Antibiot. 1993, 46, 1503−1511. (b) Kohama, T.;
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