Article pubs.acs.org/jnp
Cite This: J. Nat. Prod. 2018, 81, 10−15
Alveolarides: Antifungal Peptides from Microascus alveolaris Active against Phytopathogenic Fungi Serge Fotso,*,† Paul Graupner,† Quanbo Xiong,† Jeffrey R. Gilbert,† Don Hahn,† Cruz Avila-Adame,† George Davis,† and Kengo Sumiyoshi‡ †
Discovery Research, Dow AgroSciences, 9330 Zionsville Road, Indianapolis, Indiana 46268, United States Bioscience Laboratories, Meiji Seika Pharma Co. Ltd., 788 Kayama, Odawara-shi, Kanagawa 250-0852, Japan
‡
S Supporting Information *
ABSTRACT: Three novel cyclodepsipeptides, alveolarides A (1), B (2), and C (3), each possessing the rare 2,3-dihydroxy4-methyltetradecanoic acid unit and a β-phenylalanine amino acid residue, along with the known peptide scopularide were isolated and identified from the culture broth of Microascus alveolaris strain PF1466. The pure compounds were evaluated for biological activity, and alveolaride A (1) provided strong in vitro activity against the plant pathogens Pyricularia oryzae, Zymoseptoria tritici, and Ustilago maydis. Moderate activity of alveolaride A was observed under in planta conditions against Z. tritici, Puccinia triticina, and Phakopsora pachyrhizi. Structures of 1, 2, and 3 were determined by detailed analysis of NMR (1D and 2D) and mass spectrometry data. The partial absolute configuration of alveolaride A (1) was established.
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INTRODUCTION Plant diseases continue to be an emerging threat to global food security. Many crops are destroyed each year by phytopathogens such as fungi, bacteria, and yeast, leading to economic losses to farmers.1 Direct yield losses caused by pathogens, animals, and weeds are together responsible for losses ranging between 20% and 40% of global agricultural productivity.2,3 The losses due to pests and pathogens are direct, as well as indirect; they have a number of facets, some with short- and others with long-term consequences.4 Some of the consequences contribute to food insecurity, which include food availability and food utilization.5 In order to feed the growing world population, agricultural research will have to deal with the growing resistance to known pesticides, find new solutions to protect crops from yield losses, and increase crop productivity. To address this challenging threat, Dow AgroSciences strives to search for natural pesticides from microorganisms with potent activity and an acceptable environmental profile. An evaluation of crude extracts from various sources led to the discovery of the extract from Microascus alveolaris strain PF1466, which initially showed promising antifungal activity against Puccinia triticina. Bioassay-guided fractionation using antifungal screens led to the isolation and identification of the known scopularide6 and three new cyclodepsipeptides named alveolarides A (1), B (2), and C (3). Normal phase and reverse phase HPLC of the crude acetone extract of M. alveolaris yielded the new compounds 1−3. Compound 1 was isolated as white powder and was highly soluble in MeOD-d4 and DMSO-d6. Based on HRMS its molecular formula was determined as C43H70N6O12 containing 12 unsaturations. The IR spectrum indicated absorbances at 3337.4, 1738.3, and 1650.2 cm−1, characteristic of the presence © 2017 American Chemical Society and American Society of Pharmacognosy
of hydroxy and amide functionalities. The molecular formula was supported by the 1H and 13C NMR data (Table 1), with 43 signals observed in the 13C NMR spectrum. The 1H NMR data for 1 in DMSO-d6 displayed features characteristic of a peptidic structure, illustrated by the signals of four amide NH protons (δH 8.55, 8.12, 7.54, 7.62) and four α-amino protons (δH 4.85, 4.47, 4.02, 3.89). In addition, signals of an NH2 (δH 7.29 and 6.78), an ester carbinol proton at δH 4.72, and two methyls appearing as doublets at δH 1.02 (d, J = 6.8), δH 0.70 (d, J = 6.8) and a methyl triplet at δH 0.87 (t, J = 6.9) were observed. The 13C NMR data revealed that compound 1 possessed seven amide or ester resonances [δC 174.1, 172.6, 172.3, 171.9, 171.6, 171.5, 170.1], and three sp3-oxygenated carbons were seen at δC 76.7, 72.7, and 71.2. By detailed evaluation of the COSY, HSQC, and HMBC data along with H2BC, HSQCTOCSY, and 1,1-ADEQ experiments, four amino residues were identified: serine (Ser), β-phenylalanine (Phe), and two glutamines (Gln). Since only one NH2 group was observed, further analysis of the 1H NMR and HMBC spectra indicated that the terminal amide of the second glutamine residue was alkylated. The nature of the alkyl fragment was determined by analysis of the COSY spectrum, which showed the methyl protons at δH 1.02 (d, J = 8.6 Hz, 20-CH3) coupling with the doublet at δH 7.47 (J = 8.6 Hz, 19-NH) and the latter coupled with an oxymethine proton at δH 3.38 (H-21); H-21 indicated further correlations with a hydroxy group (21-OH) and a methylene group at δH 1.56 (m)/1.37 (m, H2-22). The alkyl fragment was completed with the couplings of the methylene H2-23 to the oxygenated methylene (δH 3.39, H2-24) and to Received: April 18, 2017 Published: December 28, 2017 10
DOI: 10.1021/acs.jnatprod.7b00337 J. Nat. Prod. 2018, 81, 10−15
Journal of Natural Products
Article
Table 1. 1H and 13C NMR Data of Alveolarides A (1), B (2), and C (3) in DMSO-d6, δ (Multiplicity, J in Hz) 1 residue Phe
Gln-1
Gln-2
DHEXa
Serine
DHMTDAb
position
δH (m, J in Hz)
170.1, C 38.4, CH2
3 3-NH 4 5 6 7 8 9 10 11 11-NH 12 13 14 14-NH2 15 16 16-NH 17 18 19 19-NH 20 20-CH3 21 21-OH 22 23 24 24-OH 25 26 26-NH 27
49.8, CH 141.5, 126.3, 128.2, 126.7, 128.2, 126.3, 171.9, 54.74,
C CH CH CH CH CH C CH
27.1, CH2 32.7, CH2 174.1, C
3.21 (dd,15.7, 7.2); 2.88 (dd, 15.7, 4.2) 4.85 (td, 7.3, 4.2) 7.62 (d, 7.5) 7.26 7.26 7.17 7.26 7.26
(m) (m) (m) (m) (m)
4.02 8.12 1.98 2.14
(ddd, 9.7, 8.0, 5.5) (d, 8.0) (m) (m)
27.6, CH2 32.6, CH2 171.5, C 48.8, CH 17.4, CH3 72.7, CH 29.6, CH2 29.7, CH2 61.4, CH2
3.89 8.55 1.95 2.26
(dt, 9.4, 5.5) (d, 5.6) (m) (m)
7.47 3.81 1.02 3.38 4.64 1.42 1.56 3.39 4.38
(d, 8.6) (ddd, 8.6, 6.7, 3.4) (d, 6.8) (m) (d, 5.5) (m); 1.29 m m; 1.37 m (m) (s)
27-OH 28 29 29-OH 30 31 31-CH3 32 33 34 35
29.2, CH2
1.25 (m)
36
29.45, CH2 29.49, CH2 29.6, CH2
1.25 (m)
31.8, CH2 22.6, CH2 14.4, CH3
1.25 (m) 1.25 (m) 0.87 (t, 6.9)
38 39 40 41 CO
δC
49.8, CH2 141.5, 126.3, 128.2, 126.7, 128.2, 126.3, 171.9, 54.77,
1.25 (m) 1.25 (m)
3
δH (m, J in Hz)
170.1, C 38.4, CH2
C CH CH CH CH CH C CH
27.1, CH2 32.7, CH2 174.1, C
7.29 (brs), 6.78 (d, 2.5) 172.6, C 55.6, CH
171.6, C 54.69, CH 4.47 (dt, 6.9, 5.0) 7.54 (d, 6.9) 62.7, CH2 3.75 (brd, 10.9); 3.56 (brd, 10.9) 5.15 (s) 172.3, C 71.2, CH 4.08 (d, 6.3) 6.02 (d, 6.8) 76.7, CH 4.72 (d, 8.0) 34.3, CH 1.83 (m) 15.7, CH3 0.70 (d, 6.8) 32.2, CH2 1.25 (m); 1.05 (m) 26.6, CH2 1.25 (m) 30.0, CH2 1.25 (m)
37
Trp
δC
1 2
2
3.21 (dd, 7.3, 15.9); 2.87 (dd, 15.9, 4.3) 4.83 (td, 7.3, 4.3), 7.62 (d, 7.5) 7.25 7.25 7.17 7.25 7.25
(m), (m), (m), (m), (m),
CH CH CH CH CH
4.00 8.14 1.97 2.12
(brdd, 8.0, 7.4) (brs) (m) (m)
δC
δH (m, J in Hz)
170.1, C 38.5, CH2
3.15 (m); 2.87 (dd, 15.7, 4.4)
49.9, CH2 141.5, C 126.4, CH 126.4, CH 126.8, CH 126.4, CH 126.4, CH 171.9, C 55.0, CH 27.1, CH2 32.2, CH2 174.2, C
7.31 (brs), 6.76 (brs) 172.6, C 55.6, CH 27.6, CH2 32.5, CH2 171.5, C 48.8, CH2 17.4, CH3 72.7, CH 29.6, CH2 29.7, CH2 61.4, CH2 171.6, C 54.71, CH 62.7, CH2
172.3, C 71.2, CH 76.7, CH 34.3, CH 15.7, CH3 32.2, CH2 26.5, CH2 29.97, CH2 29.61, CH2 29.54, CH2 29.48, CH2 29.91, CH2 131.8, CH 124.8, CH 18.2, CH3
11
3.88 8.64 1.93 2.27
4.82 (m) 7.61 (d, 7.4) 7.25 7.25 7.16 7.25 7.25
(m) (m) (m) (m) (m)
3.93 (ddd, 9.6, 7.6, 5.5) 8.20 (d, 7.6) 1.95; 1.83 (m) 2.04 (m) 7.24 (brs), 6.78 (brs)
dt (10.4, 5.5) (brs) (m) (m)
7.50 (brs) 3.79 ddd (8.6, 6.8, 3.6) 1.01(d, 6.8) 3.44 (m) 4.66 (brs) 1.43 (m); 1.29 (m) 1.54 (m); 1.37 (m) 3.44 (m) 4.38 (s) 4.45 (brq, 7.1) 7.54 (d, 7.1) 3.74 (dd, 10.9, 4.9); 3.55 (brdd, 10.9, 5.5) 5.20 (s) 4.10 6.08 4.70 1.83 0.69 1.25 1.25 1.25
(brs) (brs) (d, 8.0) (m) (d, 6.7) (m); 1.05 (m) (m) (m)
171.7, C 54.7, CH 62.6, CH2
4.47 (m) 7.55 (d, 6.9) 3.59 (dd, 11.0, 5.4); 3.46 (dd, 11.0, 5.4)
71.1, CH
4.06
76.8, 34.1, 15.7, 32.7, 22.5, 26.5,
4.71 1.83 0.68 1.26 1.25 1.25
CH CH CH3 CH2 CH2 CH2
(d, 8.2) (m), CH (d, 6.8) (m); 1.02 (m) (m) (m)
1.25 (m)
29.2, CH2
1.25 (m)
1.25 (m)
29.4, CH2
1.25 (m)
1.25 (m)
29.49, CH2 29.52, CH2 29.7, CH2 31.8, CH2 14.4, CH3 172.7, C
1.25 (m)
1.25 (m) 5.40 (m) 5.40 (m) 1.61 (d, 4.4)
1.25 (m) 1.25 (m) 1.25 (m) 0.86 (t, 7.1)
DOI: 10.1021/acs.jnatprod.7b00337 J. Nat. Prod. 2018, 81, 10−15
Journal of Natural Products
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Table 1. continued 1 residue
position
δC
δH (m, J in Hz)
2 δC
δH (m, J in Hz)
NH Hα Hβ NH 2 3 3a 4 5 6 7 8a a
3 δC
δH (m, J in Hz)
56.4, CH 27.3, CH2 124.1, 110.2, 127.6, 118.5, 118.8, 121.4, 111.8, 136.6,
CH C C CH CH CH CH C
8.62 (d, 6.0) 4.22 (d, 9.1, 5.5) 3.16 (m) 10.91 (d, 2.6) 7.27 (d, 2.6)
7.54 7.01 7.08 7.35
(brd, 7.7) (ddd, 7.7, 6.9, 1.0) (ddd, 8.2, 6.9, 1.2) (brd, 8.2)
DHEX: 1,4-dihydroxyhexyl. bDHMTDA: 2,3-dihydroxy-4-methyltetradecanoic acid.
Figure 1. Selected COSY (bold lines) and HMBC (↶) correlations in substructure I of 1.
H2 -22, and the fragment was determined to be 1,4dihydroxyhexyl (Figure 1). The connection of this fragment to the glutamine C16−C19 fragment was defined by the HMBC cross-peaks observed from 19-NH, H-20, and H2-18 to the carbonyl C-19 (δc 171.5), leading to the substructure I. Eleven out of 12 unsaturations were accounted for by the amino acid residues; the new compound 1 was then deduced to be monocyclic. With the structures of these amino acid residues and the alkanol fragment established, 15 carbon signals remained unaccounted for one carbonyl (δC 172.3), two methyl (δC 15.7, 14.4), nine methylene (δC 32.2, 22.6, 26.6, 29.2, 29.45, 29.49, 29.6, 30.0, 31.8), and two oxygenated carbons (δC 76.7, 71.2). The 2D NMR data revealed that these carbons formed a 2,3-dihydroxy-4-methyltetradecanoic acid (DHMTDA) group or myristic acid derivative. The sequence of the amino acids and DHMTDA in 1 was established by analysis of the HMBC data using long-range correlations between the α-amino proton and/or the secondary amide proton and the carbonyl carbon resonances. Correlations of the α-amino proton of Phe (δH 4.85), H-2 (δH 3.21/2.88), and the oxymethine proton H-30 signals to the carbonyl carbon signal at δC 170.1 suggested that the oxygen at the β-position of the DHMTDA (C-30) was involved in the ester linkage. In addition, 3-NH (7.62, d, J = 7.5) and H-11 (δH 4.02) signals indicated correlations to the carbonyl C-10 (δC 171.9), suggesting the presence of a DHMTDA-Phe-Gln linkage. Additional correlations were observed between the 11-NH (δH 8.12) and the α-amino proton of the alkylated glutamine (H16) to the carbonyl C-15 (δC 172.6), leading to the substructure or sequence DHMTDA-Phe-Gln-Gln-C16. Furthermore, the 16-NH (δH 8.55), the α-amino proton of the serine (H-26), and the 26-NH (δH 7.54) showed long-range correlations to C-25 (δC 171.6). The basic structure of the molecule was completed with correlations from the 26-NH (δH 7.54) and the oxymethine H-29 (δH 4.08) signals to the carbonyl at C-28 (δC 172.3), leading unambiguously to the planar structure in Figure 2. Tetraacetate derivative 1a was formed and confirmed the presence of four hydroxy groups.
Figure 2. Selected COSY (bold lines and double-headed arrows) and HMBC (single-headed arrows) correlations in 1.
Figure 3. Selected NOESY correlations in the NMR spectrum of 1.
The planar structure and amino acid residue linkages were also confirmed by NOESY correlations (Figure 3), which were observed between the 16-NH and the protons H-16, H-17, H18, H-26, and H-27. Further cross-peaks were seen between 11NH and the protons H-11, H-12, and 3-NH. The proton 29OH indicated correlations with protons 27-OH and H-29, and also cross-peaks were observed between H-29 and H-30. The ester linkage in 1 was confirmed by hydrolysis using sodium methanolate to yield the ring-opened acid compound, instead of the methyl ester, which was confirmed by NMR data and high-resolution mass experiments, providing the molecular formula C43H72N6O13. The absolute configurations of the amino acid residues were determined by complete acid hydrolysis of compound 1 and HPLC-TOF-MS analysis of the Marfey’s derivatives.7 Derivatization of the hydrolysate of compound 1 (6 N HCl, 110 °C, overnight) with Nα-(4,6-dinitro-5-fluorophenyl)-L-alaninamide (FDAA) yielded Marfey’s derivatives. The obtained derivatives 12
DOI: 10.1021/acs.jnatprod.7b00337 J. Nat. Prod. 2018, 81, 10−15
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Figure 4. Structure of the hydrolysis product of 1.
Figure 5. Important HMBC correlations of compound 2.
were identified based on their retention times and mass spectra in comparison with the appropriate L- or D-amino acid standards derivatized with FDAA. The retention times for the amino acids in the 1 hydrolysate were as follows: βphenylalanine (7.44 min), serine (5.29 min), and glutamic acid (5.9 min), which were correlated to D-β-Phe and D-Ser and both Gln were L when compared to the standards. To determine the absolute configuration of the DHMTDA moiety, acid hydrolysis of 1 was performed, and the hexanesoluble 2,3-dihydroxy-4-methyltetradecanoic acid was isolated. The structure of the acid as well as that of the methyl ester derivative was confirmed by NMR. Unfortunately all attempts to prepare the Mosher esters failed, so the absolute stereochemistry of DHMTDA remained undetermined.
Alveolaride B (2) contains a novel 2,3-dihydroxy-4methyltetradec-12-enoic acid that was not previously reported in any natural products. Compound 3 was isolated as a minor component and purified as an oil. The molecular weight was calculated from the molecular ion adduct, and the molecular formula was deduced as C43H60N6O9. Comparison of the 1H NMR data of 3 with those of 1 (Table 1) revealed similarities such as signals of the DHMTDA, four NH-amides, four α-amino acid protons, the βphenylalanine, and the serine, all of which suggested that the macrocyclic cyclic depsipeptide was intact. The major differences in the 1H NMR spectrum were the absence of the signals of the alkylated glutamine that were observed in compound 1; instead signals for five aromatic protons at δH 7.74, 7.27, 7.35, 7.08, and 7.02 and an exchangeable proton at δH 10.91 (d, J = 2.6 Hz) were present. These observations were confirmed by the presence of six carbonyl carbons for 3 instead of seven as in 1; in addition, eight aromatic carbon signals were observed. Interpretation of the H−H COSY, HSQC, and HMBC spectra revealed that the alkylated glutamine in 1 was replaced by a tryptophan residue in 3, leading to the new alveolaride C. Due to the limited amount of material available, the absolute configuration of 3 was not determined, but because three of the amino acids in compound 3 along with the DHMTDA chain were identical to those in 1 and 2, it was assumed that the stereochemistry of the amino acid residues β-phenylalanine, serine, and glutamine in 3 were identical to those in 1 and 2 based on the comparison of the 1H and 13C NMR data (Table 1).
Compound 1, named alveolaride A, is a new cyclodepsipeptide possessing a rare 2,3-dihydroxy-4-methyltetradecanoic acid fragment. A synthesis of 2,3-dihydroxytetradecanoic acid has been reported.8 Also, analysis of hydroxy fatty acid profiles from several Legionella species has revealed the presence of 2,3-dihydroxytetradecanoic acid.9 To our knowledge, this is the first report of this fragment in an isolated natural product. HRMS analysis of compound 2 revealed a molecular ion adduct [M + Na]+ in positive ion mode, corresponding to a molecular formula of C43H68O6N12, which requires 13 doublebond equivalents and suggested a dehydro analogue of 1. Careful examination and comparison of the 1H NMR data of 2 and 1 (Table 1) indicated the loss of the signal attributed to the methyl triplet H-14, which appeared in 1 at δH 0.87 (t, J = 6.9). Instead, signals of a methyl doublet and two sp2 protons were seen at δH 1.61 (d, J = 4.4) and δH 5.40 (m, 2H) in 2. Analysis of the COSY data indicated correlations between both sp2 protons and between the methyl at δH 1.61 (d, J = 4.4) and the sp2 proton at δH 5.40 (m), suggesting that the methyl was connected to the double bond. The location of the methyl was finally confirmed through HMBC correlations, which indicated cross-peaks from the proton at δ 1.61 (H-41) to the carbons at δ 124.8 (C-40) and δ 131.8 (C-39). The absolute configuration of 2 was not elucidated due to the scarcity of the material but is assumed to be the same as in 1 based on the comparison of the 1 H and 13C NMR shifts of most amino acids in 1, which indicated minimal differences (Table 1). The geometry of the double bond was also not determined, as the chemical shifts of both olefinic protons were not resolved and appeared as multiplets.
Members of the class of cyclodepsipeptide natural products have previously been shown to be cytotoxic,10 antifungal,11 insecticidal,12 and histone deacetylase13 and elastase inhibitors.14 Compounds 1, 2, and 3 were tested for fungicidal activity. Alveolaride A (1) showed strong in vitro fungicidal activity against the plant pathogens Pyricularia oryzae, Ustilago maydis, and Zymoseptoria tritici (Table 2). Alveolaride A (1) was evaluated under in planta conditions at 200 and 50 ppm, and its average fungicidal inhibition at 200 ppm was 60.5% against Z. tritici, 49.5% against Phakopsora pachyrhizi, and up to 94% against Puccinia triticina. No activity was observed at 50 ppm. 13
DOI: 10.1021/acs.jnatprod.7b00337 J. Nat. Prod. 2018, 81, 10−15
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Table 2. Antifungal Activity as Percent of Growth Inhibition by Alveolarides 1, 1a, 2, and 3
a
1
1a
2
3
tebuconazole
dose (μg/mL)
dose (μg/mL)
dose (μg/mL)
dose (μg/mL)
dose (μg/mL)
targeta
10
2
0.4
10
10
2
0.4
2
0.4
10
2
0.4
10
2
0.4
PYRIOR USTIMA SEPTTR
100 95 95
100 100 100
55 100 95
10 100 40
0 60 0
0 60 0
0 0 0
0 90 20
0 10 0
30 100 90
10 60 20
20 0 10
95 100 90
90 100 85
63 95 80
PYRIOR = Pyricularia oryzae; USTIMA = Ustilago maydis; SEPTTR = Zymoseptoria tritici. 10 mg L−1. Final solvent concentration was 1%, and it did not affect fungal growth. For in vivo evaluation against Z. tritici and Puccinia triticina, alveolarides A (1), B (2), and C (3) were dissolved in methanol (10% final concentration) and formulated in 0.01% Triton X-100 (Fisher Scientific, Fair Lawn, NJ, USA) in distilled water. For evaluation against P. pachyrhizi, alveolaride A (1) was dissolved in acetone (10% final concentration) and formulated in 0.01% Tween-20 (Acros, Morris Plains, NJ, USA). In Vitro Test. In vitro evaluation was performed using fresh fungal spores as a source of inoculum. Spores of Z. tritici were collected from 3-day-old cultures grown on potato dextrose agar; spores of Ustilago maydis were produced in cultures grown in potato dextrose broth for 24 h; Pyricularia oryzae spores were collected from 10- to 14-day-old cultures grown on rice agar. Inocula of P. oryzae and Z. tritici were 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. Inocula were dispensed in a 96-well microtiter plate using 200 μL per well. Percentage inhibition was estimated measuring light scattering in a Nephelometer plate reader. In Vivo Evaluation. Ten- to 13-day-old soybean and 8- to 10-dayold winter wheat plants were treated with alveolarides at 50 and 200 mg·L−1 24 h before inoculation. They were sprayed to runoff and airdried afterward for 24 h at room temperature. Wheat used for Z. tritici evaluation was inoculated with a conidial suspension containing 0.05% Tween-20 (Acros) 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 Puccinia 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. Soybean plants were inoculated with Phakopsora pachyrhizi using approximately 1 × 105 urediniospores mL−1. Urediniospores were suspended in 0.02% Tween-20. After inoculation, plants were incubated at 22 °C and 100% RH for 24 h, then moved to a growth chamber at 23 °C until disease 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 included in experiments to evaluate the effect against Z. tritici and P. triticina were sprayed with water containing 0.01% Triton X-100 and 10% methanol. Untreated controls included in experiments using soybean plants were sprayed with 0.01% Tween-20 and 10% acetone. Acid Hydrolysis and Marfey’s Analysis. Compound 1 (2.0 mg) was dissolved in 2 mL of 6 N HCl, and 200 μL was taken and heated at 110 °C, overnight. The hydrolysate was evaporated to dryness, resuspended in 1 M NaHCO3 (400 μL), and treated with Nα-(4,6dinitro-5-fluorophenyl)-L-alaninamide (50 μL, 2.8 mg/mL solution in acetone). The mixture was heated at 80 °C for 30 min followed by cooling at room temperature and diluted with 2 N HCl (200 μL). The mixture was evaporated to dryness and dissolved in 500 μL of aqueous DMSO, and the Marfey’s derivatives were analyzed by LC-TOF using a Hypersil Gold C18 column (2.1 mm × 50 mm, 1.9 μm, Thermo). The mobile phase at 0.4 mL/min consisted of 0.1% formic acid in
Likewise, alveolaride B (2) was active under in vitro conditions against U. maydis and Z. tritici, and the tetraacetate derivative 1a and alveolaride C (3) were only active against U. maydis (Table 2).
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EXPERIMENTAL SECTION
General Experimental Procedures. High-resolution mass spectra were acquired on an Agilent 6220; TOF-MS was introduced by HPLC in positive electrospray (+)-ESI mode. 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 preparative system. IR was recorded on a Nicolet 6700 FT-IR (Thermo Scientific). Fungus Isolation and Identification. The fungal strain PF1466 was isolated from soil collected in Okinawa Prefecture, Japan. This organism grew on various culture media, attaining 6−40 mm in diameter for 2 weeks at 25 °C, and it formed brownish-gray to olive, velvety colonies. It was identified as Microascus alveolaris based on morphological characteristics of ascomata (perithecial, superficial or immersed, globose to pyriform, lageniform, dark brown to black, with an ostiolar neck), conidia (annellidic, ellipsoidal, with truncate base, hyaline to light brown, unicellular, smooth-walled, arranged in chains), asci (eight-spored, globose to subglobose, evanescent), and ascospores (one-celled, triangular to ovariform, hyaline to light brown). Fermentation and Extraction. The strain PF1466 was inoculated in 500 mL Erlenmeyer flasks, each containing 100 mL of a seed medium (2.0% soluble starch, 1.0% glucose, 0.5% polypeptone, 0.6% wheat germ, 0.3% yeast extract, 0.2% soybean meal, pH 7.0 with NaOH). The flasks were incubated at 25 °C for 3 days while shaking at 220 rpm. Aliquots of 3 mL of this seed cultured broth were transferred to 500 mL Erlenmeyer flasks containing 2 g of oatmeal and 80 g of water-absorbed brown rice as a solid production medium. The flasks were incubated statically at 25 °C for 14 days. A total of 5 kg of solid fermentation of PF1466 was extracted with 10 L of 67% aqueous acetone by shaking for 2 h at room temperature. The extract was filtered through diatomaceous earth, the filtrate was concentrated under vacuum to remove acetone, and 2.0 g of extract was obtained. Isolation of Alveolarides A−C (1−3). The extract (2.0 g) was fractionated on a Combiflash apparatus using silica gel and dichloromethane with increasing methanol as the solvent system. Eight fractions were collected (fractions 1−8), and bioassay screens indicated that fractions F3 (21.3 mg), F4 (515.4 mg), and F6 (20.8 mg) were active against SEPTTR. Trituration of F4 in MeOH delivered compound 1 (468.0 mg). Preparative HPLC (Hypersil Gold RP18, 50 × 21.2 mm, 5 μm using a gradient of solvent, 0−5 min; 60− 100% aqueous MeCN, 5−7 min; 100% MeCN, 7−10 min; aqueous MeCN) of the mother liquor yielded compound 2 (8.7 min, 7.1 mg). Bioassay-guided fractionation of F3 on Sephadex-LH20 (MeOH) followed by preparative HPLC (0−9 min: 50−100% 0.1% formic acid aqueous + MeCN, 9−10 min: 100% MeCN, 10.01−16 min: 50% 0.1% formic acid aqueous + MeCN, Phenomenex C8, 150 × 21.2 mm, 10 μm) yielded 3 (10.7 min, 5.0 mg). The known scopularide (100 mg) was filtered as a white solid from the non-SEPTTR-active F2. 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 0.4, 2, and 14
DOI: 10.1021/acs.jnatprod.7b00337 J. Nat. Prod. 2018, 81, 10−15
Journal of Natural Products
Article
(2) Teng, P. S., Ed. Crop Loss Assessment and Pest Management; APS Press: St Paul, 1987. (3) (a) Oerke, E. C. J. Agric. Sci. 2006, 144, 31−43. (b) Oerke, E. C.; Dehne, H. W.; Schönbeck, F.; Weber, A.Crop Production and Crop Protection. Estimated Losses in Major Food and Cash Crops; Elsevier: Amsterdam, 1994. (4) Zadoks, J. C.Types of losses caused by plant diseases. In FAO Papers Presented at the Symposium on Crop Losses; Chiarappa, L., Ed.; FAO: Rome, 1967; pp 149−158. (5) Savary, S.; Ficke, A.; Aubertot, N. J.; Hollier, C. Food security 2012, 4, 519−537. (6) Yu, Z.; Lang, G.; Kajahn, I.; Schmaljohann, R.; Imhoff, J. F. J. Nat. Prod. 2008, 71, 1052−1054. (7) Blushan, R.; Bruckner, H. Amino Acids 2004, 27, 231−247. (8) Breusch, F. L.; Tulus Rasim. Rev. Faculte Sci. Univ. Istanbul 1947, 12A, 289−293. (9) Jantzen, E.; Sonesson, A.; Tangen, T. J. Clin. Microbiol. 1993, 31, 1413−1419. (10) Feng, Y.; Blunt, J. W.; Cole, A. L. J.; Cannon, J. F.; Robinson, W. T.; Munro, M. H. G. J. Org. Chem. 2003, 68, 2002−2005. (11) Tajima, H.; Wakimoto, T.; Takada, K.; Ise, Y.; Abe, I. J. Nat. Prod. 2014, 77, 154−158. (12) Langenfeld, A.; Blond, A.; Gueye, S.; Herson, P.; Nay, B.; Dupont, J.; Prado, S. J. Nat. Prod. 2011, 74, 825−830. (13) Vervoort, H. C.; Drašković, M.; Crews, P. Org. Lett. 2011, 13, 410−413. (14) Kanchan, T.; Matthew, S.; Rocca, J. R.; Paul, V. J.; Luesch, H. J. Nat. Prod. 2007, 70, 1593−1600.
water containing 5% acetonitrile and 5 mM NH4OAc (A) and acetonitrile (B). The chromatography held the initial mobile phase composition of 0% B constant for 1 min, followed by a linear gradient to 100% B up to 15 min, and stayed at 100% B for 2 min. The gradient was then immediately dropped to 0% B over 0.1 min and held to reequilibrate for 3 min before the next injection. The obtained derivatives were identified based on their retention times and mass spectra in comparison with the appropriate L- or D-amino acid standards derivatized with FDAA. Amino acids in the hydrolysates of 1 had the following retention times for the FDAA derivatives: βphenylalanine (7.44 min), serine (5.29 min), and glutamic acid (5.49 min), which were correlated to D-β-Phe and D-Ser, and both Gln were L, instead of L-β-Phe (8.08 min), L-Ser (5.04 min), and D-Gln (5.90 min), by comparison with the standards. Methanolysis of Alveolaride A (1). Compound 1 (35 mg) was added to 4 mL of 0.5 N NaOMe, and the mixture was stirred at room temperature. After 2 h the reaction was stopped by adding 2 mL of 2 N HCl. Extraction of the reaction mixture with ethyl acetate and evaporation of the organic solvent yielded 31.3 mg of the ring-opened acid derivative of 1. Alveolaride A (1): white powder, C43H70N6O12, [α]25D −13.3 (c 0.045, CH3OH); IR (neat) νmax 3337.4, 2922.6, 2852.4, 1738.3, 1650.2, 1525.1, 1451.7, 1407.6, 1384.0, 1232.7, 1189.5, 1173.3, 1132.6, 1111.0, 1028.7, 983 cm−1; 1H NMR (600 MHz, DMSO-d6) and 13C NMR (150 MHz, DMSO-d6), see Table 1; (+)-ESI-HRMS m/z 863.5144 [M + H]+ (863.5124 calcd for C43H71N6O12+), 885.4969 [M + Na]+ (885.4944 calcd for C43H70N6O12Na+). Alveolaride B (2): C43H68N6O12, [α]25D −10.9 (c 0.11, CH3OH); IR (neat) νmax 3285.8, 2923.2, 2852.5, 1737.5, 1665.8, 1635.0, 1618.3, 1727.7, 1449.4, 1413.9, 1382.2, 1277.2, 1236.3, 1192.5, 1171.9, 1136.1, 1056.2, 1024.1, 1002.5, 964.4 cm−1; 1H NMR (600 MHz, DMSO-d6) and 13C NMR (150 MHz, DMSO-d6), see Table 1 (+)-ESI-HRMS m/ z 861.4981 [M + H]+ (861.4968 calcd for C43H69N6O12+), 883.4803 [M + Na]+ (884.4793 calcd for C43H68N6O12Na+). Alveolaride C (3): white powder, C43H60N6O9, [α]25D −10.2 (c 0.12, CH3OH); IR (neat) νmax 3259.9, 2922.5, 2852.6, 2253.2, 1727.4, 1655.8, 1527.2, 1456.3, 1342.0, 1327.3, 1193.1, 1171.5, 1048.8, 1023.1, 1003.3 cm−1; 1H NMR (600 MHz, DMSO-d6) and 13C NMR (150 MHz, DMSO-d6), see Table 1; (+)-ESI-HRMS m/z 805.4503[M + H]+ (805.4495 calcd for C43H61N6O9+), 827.4320 [M + Na]+ (827.4314 calcd for C43H61N6O9Na), 1631.8763 [2M + Na]+ (1631.8736 calcd for C86H120N12O18Na+).
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ASSOCIATED CONTENT
* Supporting Information S
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.7b00337. 1 H and 13C NMR, COSY, HSQC, and HMBC spectra of compound 1 (PDF)
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AUTHOR INFORMATION
Corresponding Author
*Tel (S. Fotso): +1-317-337-5155. Fax: +1-317-337-3546. Email:
[email protected]. ORCID
Serge Fotso: 0000-0003-3421-4480 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank Dr. P. Lewer for his corrections and suggestions. REFERENCES
(1) Khem, R. M.; Shamsher, S. K. BioMed Res. Int. 2015, 2015, 1−9. 15
DOI: 10.1021/acs.jnatprod.7b00337 J. Nat. Prod. 2018, 81, 10−15
