Isolation and Characterization of Antifungal Metabolites from the Melia azedarach-Associated Fungus Diaporthe eucalyptorum

Article abstract of DOI:10.1021/acs.jafc.9b07825

Two biosynthetically related new metabolites, eucalyptacid A (1) and eucalactam B (2), along with six known compounds (3-8), eugenitol (3), cytosporone C (4), 4-hydroxyphenethyl alcohol (5), 1-(4-hydroxyphenyl)ethane-1,2-diol (6), N-(2-hydroxy-2-phenyleth

Full text of DOI:10.1021/acs.jafc.9b07825

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Bioactive Constituents, Metabolites, and Functions
Isolation and Characterization of Antifungal Metabolites from the
Melia azedarach-Associated Fungus Diaporthe eucalyptorum
Yu-Qi Gao, Shuang Tian Du, Jian Xiao, Da Cheng Wang, Wen-Bo Han, Qiang Zhang, and Jin-Ming Gao
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b07825 • Publication Date (Web): 03 Feb 2020
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Isolation and Characterization of Antifungal Metabolites from
the Melia azedarach-Associated Fungus Diaporthe
eucalyptorum
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Yu-Qi Gao,§ Shuang-Tian Du,§ Jian Xiao, Da-Cheng Wang, Wen-Bo Han, Qiang Zhang,* and
Jin-Ming Gao*,
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Shaanxi Key Laboratory of Natural Products & Chemical Biology, College of Chemistry &
Pharmacy, Northwest A&F University, Yangling 712100, People’s Republic of China
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Corresponding Authors
*Telephone: + 86-29-87092335.
E-mail: jinminggao@nwsuaf.edu.cn (J.M.G); zhangq@nwsuaf.edu.cn (Q.Z.)
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ABSTRACT: Two biosynthetically related new metabolites, eucalyptacid A (1) and eucalactam B
(2), along with six known compounds (3–8), eugenitol (3), cytosporone C (4), 4-hydroxyphenethyl
alcohol (5), 1-(4-hydroxyphenyl)ethane-1,2-diol (6), N-(2-hydroxy-2-phenylethyl)acetamide (7)
and phomopene (8), were isolated from the solid rice cultures of the endophytic fungus Diaporthe
eucalyptorum KY-9 that had been isolated from Melia azedarach. Also, two further new derivatives
(2a, 2b) were prepared from 2. The structures were elucidated by exhaustive analysis of NMR and
ESIMS data, and chemical methods such as Marfey’s protocol. Compound 1 was identified as a rare
polyketide fatty acid, (8E)-3,5,11-trihydroxy-2,10,12-trimethyltetradecenoic acid, and 2 was
determined to be the first cyclic depsipeptide containing the same fatty acid unit as 1 and a
Gly-Gly-Thr tripeptide chain. Its N-terminal end is N-acylated by an 11-hydroxy fatty acid with a
branch alkyl chain of 14:1. The 11-hydroxyl group connects to the carboxylic group of the
C-terminal amino acid to form a 22-membered lactone ring. A hypothetical biosynthetic pathway
for the new polyketides is proposed. The isolated compounds were assayed for their inhibition
against four plant pathogenic fungi, Alternaria solani, Botrytis cinerea, Fusarium solani, and
Gibberella saubinettii. Compounds 1, 4, 6, and 7 exhibited antifungal activities against Alternaria
solani, with minimal inhibitory concentration (MIC) values from 6.25 to 50 μM. Thus, strain KY-9
represents an untapped source for the development of biological control agents to prevent the
infection of pathogenic fungus A. solani.
KEYWORDS: Diaporthe eucalyptorum; cyclic depsipeptides; antifungal activity; lipopeptides;
endophytic fungus; biopesticide
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43 INTRODUCTION
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Plant endophytes establish remarkable mutualistic associations with their hosts. They have attracted
considerable attention recently due to ecological and biotechnological potential.1–4 The symbiotic
fungi can provide favorable metabolites to stimulate plant growth or defense against plant pathogen
invasions.5,6 Thus, the endophytic fungi have been considerable sources of new chemical entities
with agricultural and pharmaceutical importance. For example, the endophytes from a medicinal
plant (Melia azedarach L.) generate numerous structurally unique and bioactive metabolites, such
as fusarimine, toxic fusariumin, insecticidal meroterpenes, allelopathic indole diketopiperazines,5
and antifungal alkaloids.6,7
Lipopeptides are a hybrid class of secondary metabolite connecting head to tail of hydroxyl fat
acid peptide. They have a broad bioresource, such as Streptomyces, Pseudomonas, and Bacillus,8 as
well as fungi, including Fusarium, Aspergillus, Pochonia,9 and Diaporthe.10 These compounds
exhibited a wide range of activities, such as cytotoxic, antimicrobial, immunosuppressant, and
surfactant properties.9
In our ongoing search for novel bioactive metabolites from endophytes, two new compounds,
eucalyptacid A (1) and eucalactam B (2), along with six known ones (3−8) (Figure 1), were isolated
from the solid culture extract of an endophyte Diaporthe eucalyptorum KY-9, associated with Melia
azedarach. These compounds were tested for their antifungal activities against four
phytopathogenic fungi. Herein, we deal with the isolation, structure analysis, and antifungal
activities of these compounds.
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MATERIALS AND METHODS
General experimental procedures
Optical rotations were measured on an Autopol III automatic polarimeter (Rudolph Research
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Analytical, USA). IR spectra were recorded on a Bruker Tensor 27 spectrophotometer with KBr
disks (Bruker Corp., German). ESI mass spectra (MS) were recorded on an LTQ Fleet instrument
(Thermo Fisher Scientific Inc., USA). HR-ESI-MS were recorded on an Agilent 6520
Accurate-Mass Q-TOF spectrometer (Agilent Technologies Ltd., USA). 1D and 2D NMR spectra
were recorded on an AVANCE III (500 MHz) instrument (Bruker Corp., Germany). Chemical
shifts were reported using solvent residual as the internal standard. HPLC analysis and isolation
were carried out on a Waters 1525 instrument (Waters Corp., USA). Column chromatography (CC)
was performed on silica gel (90−150 μm; Qingdao Marine Chemical Inc., China), Sephadex LH-20
(40−70 μm; Amersham Pharmacia Biotech AB, Sweden), and Chromatorex C18 gel (40−75 μm;
Fuji Silysia Chemical LTD., JPN). GF254 plates (Qingdao Marine Chemical Inc., China) were used
for thin-layer chromatography (TLC).
The standard amino acids (Glycine, L- and D-threonine) and other chemicals were bought from
Aladdin Industrial Corporation, Shanghai, China.
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Fungal Material
The endophytic strain of Diaporthe eucalyptorum was isolated from healthy leaves of Melia
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azedarach Linn collected in Yangling, Shaanxi Province, P. R. China. This strain was authenticated
by analysis of the ITS region of rDNA (GenBank Accession No. MK396569.1, Supporting
Information). The results indicate that KY-9 was closely related to D. eucalyptorum (KACC48653),
with the ITS sequence similarity of 99%. Combined with morphological characteristics, the fungus
was identified as D. eucalyptorum KY-9.
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The fungal strain was deposited in the culture collection of the College of Chemistry &
Pharmacy, Northwest A&F University. The fungi isolation and maintenance were carried out
according to the reported protocols.5
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Fermentation, Extraction, and Isolation
The strain was cultured on potato dextrose agar (PDA) at 28 °C for 5 days. One piece (7 mm2) of
mycelium was transferred into a sterile flask containing 30 mL of PDA liquid media. The seed
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liquid was incubated at 28 C for 3 days on a rotary shaker at 120 rpm. A suspension (100 μL) of
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the strain was inoculated aseptically to 500-mL Erlenmeyer flasks containing rice medium. The rice
medium contained 60 g rice and 90 mL distilled water, and was sterilized under 120 °C for 30 min
before use. The flask cultures were incubated under 28 °C for 3 weeks.
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The cultures were extracted four times with MeOH. The solvent was removed in vacuum to
afford a residue. The extract was dispersed in 90% MeOH/H2O (2 L) and extracted with petroleum
ether 2 times. The raffinate was adjusted to 50% MeOH/H2O, and then extracted four times with
EtOAc to yield a crude extract (8.0 g). The EtOAc extract was chromatographed on an RP-18
column eluted with MeOH and H2O to give three fractions Fr. 1~3. Fr.1 (2.1 g) was separated by
CC eluted by ether-ethyl acetate mixtures (v/v, 10:11:1) to give four subfractions (Fr.1a~Fr.1d).
Fr.1b was separated on a Sephadex LH-20 column with MeOH and by RP-HPLC purification to
afford compounds 6 (11 mg) and 7 (25 mg). Fr.1c was subjected to CC over ODS-A gel with
aqueous MeOH (25%85%) to afford 3 (80 mg). Fr.2 (1.3 g) was separated on a Sephadex LH-20
column (MeOH) followed by RP-HPLC to give 5 (18 mg) and 8 (15 mg). Fr.3 (950 mg) was
separated by CC over Sephadex LH-20 (MeOH) and ODS-A gel with MeOH-H2O (1:1) to give
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subfractions Fr.3a~Fr.3c. Semipreparative RP-HPLC of Fr.3b with MeOH-H2O (55:45) afforded 1
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(5 mg) and 4 (8 mg). Fr.3c was purified by CC over Sephadex LH-20 (MeOH) and semipreparative
RP-HPLC with MeOH-H2O (60:40) to afford 2 (32 mg) and 3 (5.2 mg).
Eucalyptacid A (1): colorless oil; [α]D21−16 (c 0.15, CHCl3); IR (KBr) vmax 3417, 2961, 2930,
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2875, 1713, 1458, 1379, 1030 cm–1; H NMR and C NMR data see Table 1; HRESIMS m/z
321.2017 [M−H2O +Na]+ (calcd. for C17H30O4Na+, 321.2042).
Eucalactam B (2): colorless, amorphous solids; [α]D27 −17 (c 0.23, CH3OH); IR (KBr) vmax 3489,
1
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3440, 3355, 3280, 2971, 2937, 1744, 1680, 1659, 1511, 1450, 1214 cm–1; H NMR and C NMR
data see Table 2; ESIMS m/z 512.02 [M–H]–, 547.93 [M+Cl]–; ESIMS m/z 536.19 [M+Na]+;
HRESIMS m/z 512.2982 ([M–H]–, calcd. for C25H42N3O8, 512.2972).
Acetylation of Compound 2. In a flame dried 20 mL reaction tube, compound 2 (5 mg) and
Ac2O (0.5 mL) were dissolved in 0.5 ml pyridine. After stirring for 12 hours at room temperature,
the reaction was quenched by adding water. The mixture was concentrated in vacuo to yield
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compound 2a (5.1 mg, 88%): colorless powder; [α]D + 70 (c 0.04, MeOH); H NMR and C
NMR data see Table 2; HRESIMS m/z 638.3331 ([M–H]–, Calcd. for C31H48N3O11, 638.3289).
Preparation of Acetonide 2b. To a flame-dried 50 mL reaction tube was added a stir bar,
2,2-dimethoxypropane (3.0 mL), CuSO4 (500 mg), compound 2 (20 mg) and acetone (10 mL).
After being stirred for 1 day at reflux temperature, the mixture was concentrated directly in vacuo.
The residue was chromatographed over silica gel CC eluted by CHCl3-MeOH (30:1) to afford 2b
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(12 mg, 56%): colorless powder; [α]D −10 (c 0.05, MeOH); H NMR (500 MHz, CDCl3) δ ppm
2.53 (dq, J = 7.3, 4.3 Hz, H-2), 3.91 (dt, J=11.7, 2.8 Hz, H-3), 1.53 (m, H2-4), 3.76 (m, H-5), 1.37
(m, H2-6), 1.83 (m, H-7a), 2.07 (m, H-7b), 5.40 (ddd, J=15.0, 8.2, 4.1 Hz, H-8), 5.11 (dd, J=15.0,
9.3 Hz, H-9), 2.31 (m, H-10), 4.79 (dd, J = 10.4, 1.6 Hz, H-11), 1.63 (m, H-12), 1.15 (m, H-12a),
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1.25 (m, H-12b), 0.88 (m, overlapped, H3-14, H3-17), 1.34 (d, J=7.6 Hz, H3-15), 0.91 (d, J = 6.9 Hz,
H3-16), 3.61 (m, H-19a), 4.36 (m, H-19b), 3.83 (dd, J = 16.9, 3.6 Hz, H-21a), 4.26 (dd, J = 17.0, 5.7
Hz, H-21b), 4.33 (m, H-23), 4.50 (quin, J = 6.6 Hz, H-24), 1.13 (d, J = 6.6 Hz, H3-25), 6.81 (d, J =
6.3 Hz, HN-19), 7.43 (t, J = 4.4 Hz, HN-21), 7.80 (d, J = 8.8 Hz, HN-23), 1.45 (s, H3-27), 1.40 (s,
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H3-18); C NMR (125 MHz, CDCl3) δ ppm 176.0 (s, C-1), 46.8 (d, C-2), 70.8 (d, C-3), 34.8 (t,
C-4), 69.6 (d, C-5), 35.8 (t, C-6), 28.3 (t, C-7), 131.1 (d, C-8), 132.2 (d, C-9), 40.1 (d, C-10), 80.3
(d, C-11), 35.0 (d, C-11), 27.2 (t, C-12), 12.1 (q,C-14), 16.5 (q, C-15), 16.7 (q, C-16), 11.9 (q,
C-17), 170.0 (s, C-18), 40.6 (t, C-19), 167.9 (s, C-20), 42.7 (t, C-21), 172.0 (s, C-22), 56.7 (d,
C-23), 65.7 (d, C-24), 18.8 (q, C-25), 99.2 (s, C-26), 30.0 (q, C-27), 19.4 (q, C-28); HRESIMS m/z
552.3304 ([M–H]–, calcd. for C28H46N3O8, 552.3284).
Advanced Marfey’s Analysis. The absolute configurations of amino acid residues were
determined by the advanced Marfey’s analysis protocol.11 To a flame-dried 50 mL reaction tube
was added a stir bar, 0.5 mL HCl (6 N) and compound 2 (0.5 mg). After stirring for 1 day at 100 oC,
the mixture was concentrated directly in vacuo. The residue was added with 50 μL H2O, 20 μL
NaHCO3 (1M) and 100 μL L-FDLA (25 mM in MeOH). After stirring for 1 h at 37 oC, the mixture
was added 20 μL HCl (1M) and 810 μL MeCN to afford solution A. Glycine, L- and D-threonine
were treated with L-FDLA to make into solutions B, C and D, respectively, using the same
procedure. All the solutions were analyzed on HPLC (Thermo Hypersil BDS 5 µm C18 250 × 4.6,
gradient 10-100% MeOH in 40 min). Solution A gave 2 peaks with tR at 17.03 and 18.76 min, while
solution B at 18.78 min, solution C at 17.00 min and solution C 18.71 min.
Hydrolysis of Compound 2. To a stirred solution of 2 (5.0 mg) in MeOH (1.0 mL) was added 1
mL of 1.2 N KOH in MeOH-H2O (1:1). The reaction mixture was stirred at 65 ℃ for 24 h and then
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treated with 1 N HCl. After being extracted with EtOAc, the crude product was purified by HPLC
(Synergic 5 μm RP-C18, 250 × 10 mm, 75% MeCN/H2O at 3 mL/min, detection at 210 nm) to give
pure compound 1 (2.4 mg, tR = 7.8 min), which was confirmed by 1 H NMR and ESIMS data.
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Antifungal Bioassay. Antifungal activities in vitro were assayed on four phytopathogenic fungi
(Alternaria solani, Botrytis cinerea, Fusarium solani, and Gibberella saubinettii). The bioassay was
performed on 96-well microtiter plates with potato dextrose (PD) medium.5 All the plant pathogens
were provided by the College of Plant Protection, Northwest A&F University, China. The fungicide,
hymexazol (Aladdin Chemistry Co. Ltd.), was employed as the positive control. DMSO was used
as the negative control. Each assay was performed in triplicate.
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Brine Shrimp Bioassay. The toxicity against brine shrimp (Artemia salina) were determined
according to the previously reported method.5 The final concentrations of the tested compounds
were 10, 50, and 100 μM. Toosendanin, an insecticidal triterpenoid derivative isolated from Melia
toosendan, and DMSO were used as the positive and negative controls. The lethality of each
concentration was recorded.
167 RESULTS AND DISCUSSION
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Structure Elucidation. Eucalyptacid A (1) was obtained as colorless oil. Its molecular formula
was determined to be C17H32O5 by HRESIMS, 13C NMR and DEPT data, indicating two degrees of
unsaturation. The IR spectrum of 1 displayed the presence of carbonyl (1713 cm–1) and hydroxyl
(3417 cm–1) groups. The 13C NMR spectrum of 1 (Table 1) showed 17 signals: four methyls [three
secondary ones (δC 12.3, 12.6, 17.2)], four methylenes (δC 27.0−35.9), eight methines (one double
bond at δC 130.5, 134.4, three oxygenated ones at δC 67.4, 75.3 and 77.2), and one carbonyl carbon
atom (δC 173.5). The 1H NMR spectrum of 1 (Table 1) showed four methyls [three secondary ones
at δH 0.85 (3H, d, J = 6.7 Hz, H-17), 0.94 (3H, d, J = 6.8 Hz, H-16), and 1.32 (3H, d, J = 7.2 Hz,
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H-15)], four methylenes (δH 1.28−2.20), and eight methines [one double bond at δH 5.50 (1H, dt, J =
15.3, 6.6 Hz, H-8), 5.36 (1H, dd, J = 15.3, 8.6 Hz, H-9), three oxygenated ones at δH 4.17 (1H, m,
H-3), 4.74 (1H, m, H-5) and 3.18 (1H, dd, J = 8.0, 3.4 Hz, H-11)]. These data suggest that this
metabolite may be a branched-chain fatty acid containing three secondary OH and three secondary
1
CH3 groups. The planar structure of 1 was established based on analysis of the HSQC, H−1H
COSY, and HMBC correlations (Figure 2). The 1H−1H COSY correlations disclosed a spin system
(−CH3CHCHCH2CHCH2CH2CHCHCH(CH3)CHCH(CH3)CH2CH3−), from H3-15 at δH 1.32 to
H3-14 at δH 0.90. The HMBC correlations of H-11/C-16, H3-17/C-11, H-9/C-16, H-11/C-16,
H2-7/C-5, H3-14/C-12, and H3-15/C-3 and C-1 confirmed the positions of the three OH at C-3, C-5
and C-11, and three CH3 groups at C-2, C-10 and C-12, respectively. In addition, the HMBC
correlations of H2-6/C-8, and H-9/C-16 indicated the olefin to be located at C-8 and C-9. The
trans-geometry at Δ8,9 was indicated by a large coupling constant (J = 15.3 Hz). The absolute
configuration determination of the hydroxyl groups at C-3, C-5 and C-11 by the modified Mosher’s
method could not be performed due to limited amounts. Thus, compound 1 was elucidated as
(8E)-3,5,11-trihydroxy-2,10,12-trimethyltetradec-8-enoic acid (THTMT) and was named
eucalyptacid A.
The molecular formula of eucalactam B (2) was established as C25H43N3O8, as deduced from
HRESIMS (m/z 512.2982 [M–H]–), indicating six degrees of unsaturation. The IR spectra indicated
the presence of both，ester (1744 cm−1 ) and amide (1680, 1659 cm−1) functionalities. An analysis
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of the C and H NMR spectra (Table 2) aided by phase-sensitive HSQC correlations identified
four carbonyl signals (δC 176.4, 172.1, 170.5, and 168.8) from δC 168 to 176, ten methine carbons
(five heteroatom-attached methine ones between δC 57 and δC 80, one olefinic group (δC 132.7,
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130.7), and three amide proton signals (δH 7.45, 7.64, 7.72). In addition, the presence of the four
carbonyls and one olefinic group accounted for five degrees of unsaturation, and the remaining was
attributed to the macrocycle, required by the molecular formula. Upon acylation, compound 2
afforded a triacetylated product 2a (Scheme 1), indicating the three free OH groups present in 2. In
addition, the hydrolysis of compound 2 in the presence of KOH in MeOH-H2O (1:1) gave 1, which
1
was confirmed by H NMR and ESIMS data. This result revealed that compound 2 was
biogenetically derived from 1.
The planar structure of 2 was elucidated on the basis of the HSQC, COSY and HMBC
correlations shown in Figure 2. Detailed analyses of the 1D and 2D NMR data including COSY,
HMBC, and HSQC (Table 2) clearly indicated the presence of two amino acids, glycine (Gly) and
threonine (Thr). Further COSY correlations disclosed the same chain substructure a as that of 1
(THTMT) (Figure 2). The sequence of Gly1-Gly2-Thr was established by the HMBC correlations
from H2-21 (Gly) at δH 3.99 to C-22 (Thr) at δC 172.1, and H2-19 (Gly1) at δH 3.62/4.33 to C-20
(Gly2) at δC 168.8. Additional HMBC cross-peaks (Table 2) from H-23 (Thr) at δH 4.42 to C-1
(THTMT) at δC 176.4 and H-11 (THTMT) at δH 4.79 to C-18 (Gly-1) at δC 170.5 indicated the
moiety a was connected to the Thr residue via an amide bond and to the Gly1 residue through an
ester linkage, respectively, to form a macrolide derivative (Figure 2), thus leading to establishment
of the structure of a cyclic depsipeptide. Further chemical information on structure 2 was obtained
from tandem ESI-MS analysis of the [M+Na]+ (see Supporting Information). The MS2 fragment at
m/z 479 (−57) generated from the MS1 peak at m/z 536 [M + Na]+ by simple cleavage, which
indicted the loss of amino acid residue via the cleavage of the ester bond (Figure 4).12 A double
hydrogen transfer occurred in the tandem MS signals of cyclic lipopeptides (Figure 5A).13 In
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particular, the ion peaks at m/z 518 (−18), 492 (−44), 461 (−75), and 404 (−132) resulting from m/z
536 (Figures 5B) were ascribable to the consecutive losses of two Gly residues from the C-terminal
end. This further confirmed that two sequential Gly-Gly residues connect to the 11-hydroxyl group
and are involved in the typical 22-membered lactone ring skeleton. The ion peak at m/z 312 was
considered as loss the fragment CH3CHCO-Thr-Gly-Gly-OHNa (Figure 5C), which indicated the
remaining Thr residue had to connect the N-terminal end.
The absolute configurations of the amino acid units in 2 were determined by Marfey’s
methodology. The acid hydrolysate of 2 was derivatized with Marfey’s reagent,
N-α-(2,4-dinitro-5-fluorophenyl)-L-leucinamide (L-FDLA)11 and analyzed by RP18 HPLC
comparison with FDLA-derivatized D- and L-amino acid standards (Table 3) (Supporting
Information). Compound 2 thus possesses the L-configuration for Thr unit.
To determine the relative configuration of HO-groups at C-3 and C-5, a 1,3-diol acetonide
derivative of 2 was prepared (Scheme 1). Generally, the difference in chemical shifts between the
two methyl groups on the 1,3-diol acetonide moiety is the most reliable indicator of
stereochemistry, with the syn isomers showing a difference of > 9 ppm, and anti isomers showing a
difference of < 5 ppm. The typical chemical shift of acetal carbon can also ascertain the
stereochemistry in 1,3-diol acetonides. In general, syn stereochemistry will afford δC below 99.5,
while anti stereochemistry afford δC above 100.5.14 In the current product 2b, the δC of the two
methyls are 19.4 and 30.0 ppm with a difference of 10.6 ppm, and the δC of the acetal carbon is
99.2. Thus, compound 2 contained syn 1,3-diol moiety at C-3 and C-5. This conclusion is supported
by the NOESY correlations between H-3and H-5/H3-28 and NH(Thr)/H3-27 (Figure 3), which also
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241
confirmed the relative configuration of the OH groups at C-3 and C-5 of 1. Based on the above
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evidence, 2 was elucidated as shown and named eucalactam B. Interestingly, 2 belongs to a family
of fungal cyclic lipopeptides that are different from bacterial depsipeptides that contain a fatty acid
residue with a characteristic triple-, or double-bond and involves PKS and an NRPS
biogenetically.8,15
However, unfortunately, attempts to use X-ray crystallography and the crystalline sponge
methods to establish the absolute stereochemistry of 1 or 2 have not yet been successful,16 the likely
cause being the flexible appendix on the depsipeptide ring. It should be noted that the relative and
absolute configurations of 1 and 2 remain undetermined partially. To the best of our knowledge,
compound 1 was a rare polyketide fatty acid,17 and compound 2 represents the first lipopeptide with
a subunit which is the same as compound 1.
Biosynthetically, eucalactam B (2) is a polyketide synthase−non-ribosomal peptide synthetase
(PKS−NRPS) hybrid cyclic lipopeptide, constructed by two fragments, namely
3,5,11-trihydroxy-2,10,12-trimethyltetradec-8-enoic acid (1) and threonyl-glycyl-glycine through
intermolecular addition (Scheme 2). Compound 1 is a highly branched new fatty acid, which could
originate from one molecule of acetyl-CoA and six molecules of malonyl-CoA. Alternatively,
threonyl-glycyl-glycine is a linear tripeptide that was assembled by one Thr residue and two Gly
residues.
The known metabolites 3–8, by comparison of their NMR data with those reported in the
literature, were determined as eugenitol (3),18 cytosporone C (4),19 4-hdroxyphenethyl alcohol (5),20
1-(4-hydroxyphenyl)ethane-1,2-diol (6),21 N-(2-hydroxy-2-phenylethyl)acetamide (7),22 and
phomopene (8).23
Evaluation of Antifungal Activities. All isolated compounds (except 8) and semi-synthesized
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derivatives 2a and 2b were tested in vitro for their antifungal activity against the four plant
pathogenic fungi A. solani, B. cinerea, F. solani, and G. saubinettii. As shown in Table 4, the
metabolites 1, 4, 6, and 7 were found to inhibit the growth of A. solani, with minimal inhibitory
concentration (MIC) values ranging from 6.25 to 50 μM, and 6 and 7 had same antifungal activity
against A. solani to the positive control hymexazol (MIC 6.25 μM), which revealed that A. solani
was more sensitive than other fungi to the test compounds. In contrast, 2 and its derivatives 2a and
2b did not display any antifungal activity. The results suggest that the free 11-OH and -COOH
groups of 1 are essential for the activity. In addition, 1 suppressed the growth of the four fungi, with
MIC values from 12.5 to 50 μM, and the remaining substances displayed weak growth suppression.
Brine Shrimp Bioassay. Brine shrimp can be emplyed as a convenient model for insecticidal
bioassay.5 Compounds 1−7 were evaluated for toxicity, and brine shrimp lethality. Compounds 6
and 8 showed the activity with the lethality of ca. 36% at 100 μM, whereas the other compounds
were inactive.
In conclusion, two new metabolites, eucalyptacid A (1) and eucalactam B (2), as well as the
known ones (3–8), have been identified from the endophytic fungus D. eucalyptorum KY-9.
Compound 1 represents an unusual fatty acid, while 2 was believed as the first naturally occurring
cyclic depsipeptide containing the same fatty acid unit as 1. Compounds 1, 4, 6, and 7 displayed the
growth inhibition of pathogenic fungus Alternaria solani. Thus, strain KY-9 could provide an
untapped source for the development of biological control agents to prevent the infection of
pathogenic fungus A. solani. These findings expand the chemical diversity of polyketides derived
from the genus Diaporthe, and also provide a basis for further development and utilization of
polyketide derivatives as a source of crops-protecting agents.
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ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the ACS Publications website at DOI:
1D NMR, 2D NMR, and HRESIMS spectra of 1 and 2 (PDF).
AUTHOR INFORMATION
Corresponding Author
*Telephone: + 86-29-87092335. E-mail: jinminggao@nwsuaf.edu.cn (J.M.G.); zerkang@126.com
(Q.Z.)
ORCID
Jin-Ming Gao: 0000-0003-4801-6514
Author Contributions
§These authors contributed equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
This research was financially supported by the National Natural Science Foundation of China
(21702169, 31800281), the Fundamental Research Funds for the Central Universities
(2452019185), the Natural Science Basic Research Plan in Shaanxi Province of China (2018JQ2009
and 2014JZ2-001), and the Program of Unified Planning Innovation Engineering of Science &
Technology in Shaanxi Province (2015KTCQ02-14).
REFERENCES
(1) Huang, R.; Jiang, B. G.; Li, X. N.; Wang, Y. T.; Liu, S. S.; Zheng, K. X.; He, J.; Wu, S. H.
14
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310
311
312
313
314
315
316
317
318
319
320
321
322
323
324
325
326
327
328
329
Polyoxygenated cyclohexenoids with promising α-glycosidase inhibitory activity produced by
Phomopsis sp. YE3250, an endophytic fungus derived from Paeonia delavayi. J. Agric. Food
Chem. 2018, 66, 1140−1146.
(2)
Zhang, Q.; Li, H. Q.; Zong, S. C.; Gao, J. M.; Zhang, A. L. Chemical and
bioactive diversities of the genus Chaetomium secondary metabolites, Mini-Rev. Med. Chem.
2012, 12, 127–148.
(3)
a source of novel biologically active secondary metabolites. Mycol. Res. 2002, 106, 996–1004.
(4) Yuan, Y.; Tian, J. M.; Xiao, J.; Shao, Q.; Gao, J.M. Bioactive metabolites isolated
Schulz, B.; Boyle, C.; Draeger, S.; Römmert, A.-K.; Krohn, K. Endophytic fungi:
from Penicillium sp. YY-20, the endophytic fungus from Ginkgo biloba, Nat. Prod. Res. 2014,
28, 278 –281.
(5)
Xiao, J.; Zhang, Q.; Gao, Y.-Q.; Tang, J.-J.; Zhang, A.-L.; Gao, J.-M. Secondary
metabolites from the endophytic Botryosphaeria dothidea of Melia azedarach and their
antifungal, antibacterial, antioxidant, and cytotoxic activities, J. Agric. Food Chem. 2014, 62,
3584−3590.
(6)
Li, H.; Wei, J.; Pan, S. Y.; Gao, J. M.; Tian, J. M. Antifungal, phytotoxic and
toxic metabolites produced by Penicillium purpurogenum, Nat. Prod. Res. 2014, 28,
2358−2361.
(7)
Xiao, J.; Zhang, Q.; Gao, Y.-Q.; Shi, X.-W.; Gao, J.-M. Antifungal and
antibacterial metabolites from an endophytic Aspergillus sp. associated with Melia azedarach,
Nat. Prod. Res. 2014, 28, 1388–1392.
(8)
Raaijmakers, J.; de Bruijn, I.; Nybroe, O.; Ongena, M. Natural functions of
15
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Page 16 of 30
330
331
332
333
334
335
336
337
338
339
340
341
342
343
344
345
346
347
348
349
350
351
lipopeptides from Bacillus and Pseudomonas: more than surfactants and antibiotics. FEMS
Microbiol. Rev. 2010, 34, 1037−1062.
(9)
Tan, K. C.; Wakimoto, T.; Abe, I. Lipodiscamides A−C, new cytotoxic
lipopeptides from Discodermia kiiensis. Org. Lett. 2014, 16, 3256−3259.
(10) Chepkirui, C.; Stadler, M. The genus Diaporthe: a rich source of diverse and
bioactive metabolites. Mycol. Progress 2017, 16, 477–494.
(11) Gao, Y.-Q.; Guo, C.-J.; Zhang, Q.; Zhou, W.-M.; Wang, C. C. C.; Gao, J.-M.
Asperaculanes A and B, two sesquiterpenoids from the fungus Aspergillus aculeatus,
Molecules 2015, 20, 325–334.
(12) Liu, X. Y.; Yang, S. Z.; Mu, B. Z. Isolation and characterization of a C12-lipopeptide
produced by Bacillus subtilis HSO 121. J. Pept. Sci. 2008, 14, 864–875.
(13) Yang, S. Z.; Wei, D. Z.; Mu, B. Z. J. Determination of the amino acid sequence in a
cyclic lipopeptide using MS with DHT mechanism. Biochem. Biophys. Methods 2006, 68, 69–
74.
13
(14) Rychnovsky, S. D.; Rogers, B.; Yang, G. Analysis of two C NMR correlations
for determining the stereochemistry of 1,3-diol acetonides. J. Org. Chem. 1993, 58, 3511–
3515.
(15) Sivanathan, S.; Scherkenbeck, J. Cyclodepsipeptides: a rich source of biologically
active compounds for drug research. Molecules 2014, 19, 12368–12420.
(16) Inokuma, Y.; Yoshioka, S.; Ariyoshi, J.; Arai, T.; Hitora, Y.; Takada, K.;
Matsunaga, S.; Rissanen, K.; Fujita, M. X-Ray analysis on the nanogram to microgram scale
using porous complexes. Nature 2013, 495, 461–466.
16
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Page 17 of 30
Journal of Agricultural and Food Chemistry
352
353
354
355
356
357
358
359
360
361
362
363
364
365
366
367
368
369
370
371
372
373
(17) Ishidoh, K.; Kinoshita, H.; Igarashi, Y.; Ihara, F.; Nihira, T. Cyclic lipodepsipeptides
verlamelin A and B, isolated from entomopathogenic fungus Lecanicillium sp. J. Antibiot.
2014, 67, 459–463.
(18) Li, L. M.; Zou, Q.; Li, G. Y. Chromones from an ascomycete, Chaetomium
aureus, Chin. Chem. Lett. 2010, 21, 1203–1205.
(19) Xu, J.; Kjer, J.; Sendker, J.; Wray, V.; Guan, H.; Edrada, R.; Müller, W. E. G.;
Bayer, M.; Lin, W.; Wu, J.; Proksch, P. Cytosporones, coumarins, and an alkaloid from the
endophytic fungus Pestalotiopsis sp. isolated from the Chinese mangrove plant Rhizophora
mucronata. Bioorg. Med. Chem. 2009, 17, 7362–7367.
(20) Capasso, R.; Cristinzio, G.; Evidente, A.; Scognamiglio, F. Isolation, spectroscopy
and selective phytotoxic effects of polyphenols from vegetable waste waters. Phytochemistry
1992, 31, 4125–4128.
(21) Hisamoto, M.; Kikuzaki, H.; Nakatani, N. Constituents of the leaves of
Peucedanum japonicum Thunb. and their biological activity, J. Agric. Food Chem. 2004, 52,
445–450.
(22) Veum, L.; Pereira, S. R. M.; van der Waal, J. C.; Hanefeld, U. Catalytic
hydrogenation of cyanohydrin esters as a novel approach to N-acylated β-amino alcohols–
reaction optimisation by a design of experiment approach. Eur. J. Org. Chem. 2006, 2006,
1664–1671.
(23) Li, Y. Y.; Wang, M. Z.; Huang, Y.J.; Shen, Y.-M. Secondary metabolites from
Phomopsis sp. A123. Mycology. 2010, 1, 254–261.
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392
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394
Table 1. 1H (500 MHz) NMR and 13C (125 MHz) NMR Spectroscopic Data for 1 in CDCl3
Table 2. 1H (500 MHz) NMR and 13C (125 MHz) NMR Spectroscopic Data for 2 and 2a in CDCl3
Table 3. HPLC Analysis of L-FDLA Derivatives of Acid Hydrolysate from 2 and Amino Acid
Standards
Table 4. Antifungal Activities of Compounds 1−7, 2a and 2b against Phytopathogenic Fungi
Figure 1. Structures of compounds 1−8 isolated from Phomopsis sp.
Figure 2. Key COSY and HMBC Correlations of Compounds 1 and 2.
Figure 3. NOE Correlations of 2b
Figure 4. (A) Simple cleavage of compound 2 in ESI-MS. (B) Set of fragmentation ions obtained in
tandem ESI-MS of 2.
Figure 5. (A) Double hydrogen transfer cleavage of 2 in tandem ESI-MS. (B) Molecular and
fragmentation ions obtained in tandem ESI-MS of 2. The difference between the fragments
produced after double hydrogen transfer mechanism is 18 units higher than that after simple
cleavage. (C) Set of fragmentation ions obtained in tandem ESI-MS of 2.
Scheme 1. Chemical Transformation of 2
Scheme 2. Biosynthetic Routes for both 1 and 2
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Table 1. 1H (500 MHz) NMR and 13C (125 MHz) NMR Spectroscopic Data for 1 in CDCl3
no. δH (mult, J in Hz)
δC
1
173.5 (s)
2
3
4
2.49 (dq, 7.2, 3.3)
4.17 (m)
41.4 (d)
67.4 (d)
35.9 (t)
2.10 (dt, 17.4, 5.0)
1.74 (dt, 17.4, 1.8)
4.74 (m)
5
6
7
8
9
75.3 (d)
35.1 (t)
28.0 (t)
130.5 (d)
134.4 (d)
40.9 (d)
77.2 (d)
36.2 (d)
27.0 (t)
11.8 (q)
12.6 (q)
17.2 (q)
12.3 (q)
1.84 (m); 1.68 (m)
2.20 (m)
5.50 (dt, 15.3, 6.6)
5.36 (dd, 15.3, 8.6)
10 2.25 (m)
11 3.18 (dd, 8.0, 3.4)
12 1.49 (m)
13 1.41 (m); 1.28 (m)
14 0.90 (t, 7.4)
15 1.32 (d, 7.2)
16 0.94 (d, 6.8)
17 0.85 (d, 6.7)
399
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401
402
Table 2. 1H (500 MHz) NMR and 13C (125 MHz) NMR Spectroscopic Data for 2 and 2a in CDCl3
2a
δH (mult, J in Hz) b
2
no.
1
δH (mult, J in Hz)b
δC
δC
Fatty acid
176.4 (s)
48.1 (d)
74.2 (d)
41.5 (t)
174.1 (s)
45.4 (d)
72.3 (d)
36.1 (t)
2
2.46 (dq, 7.3, 2.7)
3.99 (m, overlapped)
1.65 (m, overlapped)
1.77 (dt, 14.7, 10.6)
3.73 (m)
2.83 (dq, 7.3, 4.3)
5.11 (m)
3
4
1.90 (m)
5
73.5 (d)
37.7 (t)
28.5 (t)
130.7 (d)
132.7 (d)
39.6 (d)
80.6 (d)
35.3 (d)
27.1 (t)
12.2 (q)
16.3 (q)
16.8 (q)
11.9 (q)
170.5 (s)
4.90 (m)
71.4 (d)
32.8 (t)
27.2 (t)
129.7 (d)
133.1 (d)
39.6 (d)
80.4 (d)
35.4 (d)
27.0 (t)
12.2 (q)
14.8 (q)
17.3 (q)
11.8 (q)
169.6 (s)
6
1.46 (m), 1.56 (m)
1.98 (m); 2.14 (m)
5.44 (m)
1.58 (m); 1.64 (m)
1.96 (dt, 14.1, 7.2)
5.41(dt, 15.3, 6.1)
5.17 (dd, 15.3, 9.0)
2.34 (m)
7
8
9
5.18 (dd, 15.3, 9.1)
2.35 (m)
10
11
12
13
14
15
16
17
18
HN
19
4.79 (dd, 10.4, 1.6)
1.65 (m, overlapped)
1.13 (m)
4.79 (dd, 10.1, 2.0)
1.63 (m, overlapped)
1.16 (m)
0.87 (t, 6.9)
0.88 (m, overlapped)
1.20 (d, 7.3)
1.39 (d, 7.6)
0.94 (d, 6.6)
0.93 (d, 6.8)
0.89 (d, 6.0)
0.88 (m, overlapped)
Gly1
Gly2
Thr
7.45 (dd, 7.2, 3.6)
3.62 (dd, 18.3, 3.6)
4.33 (dd, 18.3, 7.2)
7.16 (t, 4.7)
40.9 (t)
3.86 (dd, 18.3, 4.9)
4.08 (dd,18.3, 6.5)
40.9 (t)
20
168.8 (s)
42.5 (t)
168.4 (s)
42.9 (t)
HN
21
7.64 (t, 4.7)
6.55 (t, 5.5)
3.99 (m, overlapped)
3.79 (dd, 16.9, 4.0)
4.22 (dd, 16.9, 6.2)
22
172.1 (s)
169.2 (s)
HN
23
7.72 (d, 6.3)
4.42 (br.d, 9.1)
4.54 (qd, 6.3, 0.9)
1.18 (d, 6.3)
-
6.92 (d, 8.4)
57.3 (d)
66.1 (d)
19.3 (q)
-
4.50 (dd, 8.4, 5.3)
5.54 (dq, 6.3, 5.3)
1.28 (d, 6.3)
56.9 (d)
24
69.0 (d)
25
17.0 (q)
3×Ac
2.03 (s), 2.04 (s)
2.16 (s)
21.0 (q), 21.0 (q),
21.2 (q), 170.2 (s),
170.9 (s), 170.0(s)
403
404
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406
407
Table 3. HPLC Analysis of L-FDLA Derivatives of Acid Hydrolysate from 2 and
Amino Acid Standards
Sample
Gly−L-FDLA
tR (min)
18.78
L-Thr−L-FDLA
D-Thr−L-FDLA
acid hydrolysate from 2
17.00
18.71
17.03, 18.76
408
409
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411
Table 4. Antifungal Activities of 1−7, 2a and 2b against Phytopathogenic Fungi
Minimal Inhibitory Concentration (MIC, μM)
Compounds
Botrytis
cinerea
Gibberella
saubinettii
Alternaria solani
Fusarium solani
25.0
1
12.5
NAa
NA
50.0
NA
50.0
NA
2
NA
2a
NA
NA
NA
2b
NA
NA
NA
NA
3
50.0
12.5
50.0
6.25
6.25
6.25
100.0
50.0
100.0
50.0
100.0
50.0
100.0
50.0
100.0
50.0
100.0
50.0
50.0
25.0
4
5
100.0
50.0
6
7
50.0
Hymexazol
a NA: not active.
50.0
412
413
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OH
OH OH
O
NH OH
COOH
NH
(R)
(S)
O
HN
HO
HO
O
1
O
O
OH
O
O
O
O
O
2
4
3
OH
HO
OH
O
H
N
O
HO
HO
OH
OH
OH
8
6
7
5
414
415
416
417
Figure 1. Chemical structures of 1−8 isolated from Phomopsis sp.
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OH
11
12
17
OH
5
OH
1
3
COOH
10
16
14
2
8
15
1
21
O
Gly-2
O
NH
OH
24
20
HMBC
COSY
L-Thr
22
NH
Gly-1
25
19
18
HN
O
O
O
1
2
16
HO
12
17
11
3
14
15
10
HO
5
8
a
2
418
419
420
Figure 2. Key COSY and HMBC correlations of compounds 1 and 2.
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422
Gly-2
20
L-Thr
24
23
22
19
Gly-1
18
1
17
11
27
26
16
2
12
15
10
3
28
14
8
5
423
424
425
Figure 3. NOE correlations of compound 2b.
426
427
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O
O
O
NH OH
NH
OH
NH OH
A
NH
NH
NH
O
O
O
Na ionized
ESIMS
.
HN
HO
HO
O
HN
HO
HO
O
.
HN
HO
HO
O
.
O
O
O
O
O
NaO
m/z 536 [M+Na]+
m/z 479
B
OH
ONa
OH OH
O
O
H
O
N
N
N
H
.
H
O
-57 (Gly)
428
429
430
Figure 4. (A) Simple cleavage of compound 2 in ESI-MS. (B) Set of fragmentation ions obtained in
tandem ESI-MS of 2.
431
432
O
O
O
NH OH
NH OH
NH OH
A
NH
NH
NH
O
O
O
+
+
HN
HO
HO
O
HN
HO
HO
O
HN
HO
HO
O
ESIMS
H2O
Na
H2O
Na ionized
O
O
O
O
H
.
2H transfer
H
m/z 536 [M+Na]+
m/z 461 m/z 492 m/z 518
m/z 404
B
C
OH OH
O
O
HO
H
Na
O
OH
N
N
H
N
H
O
+
2
-75 (Gly-H2O)
-132 (Gly-Gly-H2O)
m/z 312
HO
OH
OH
O
O
H
O
N
N
H
N
H
O
OHNa
+
312 (CH3CHCO-Thr-Gly-Gly-OHNa)
433
434
435
436
437
438
439
Figure 5. (A) Double hydrogen transfer cleavage of 2 in tandem ESI-MS. (B) Molecular and
fragmentation ions obtained in tandem ESI-MS of 2. The difference between the fragments produced after
double hydrogen transfer mechanism is 18 units higher than that after simple cleavage. (C) Set of
fragmentation ions obtained in tandem ESI-MS of 2.
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O
NH OH
OH
OH OH
1.2 M KOH, MeOH-H2O 1:1
NH
COOH
O
65 oC, 24 h
HN
HO
HO
O
O
O
1
O
NH OAc
Ac2O
NH
O
pyridine, r.t. 12 h
2
HN
O
O
O
AcO
O
O
CuSO4
acetone
65 oC
AcO
O
NH OH
12 h
NH
2a
O
HN
O
O
O
O
3
O
syn 1,3-diol
5
2b
441
442
443
Scheme 1. Chemical transformation of 2
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O
O
PKS
HO
HOOC
+
6 X
1 X
SCoA
SCoA
O
O
OH
OH
OH
1
L-Methionine
NRPS
2
OH
NH2
O
H
OH
N
OH
+
1 X
2 X H2N
OH
N
H
O
NH2
OH
O
O
N-end
C- end
445
446
447
448
449
Scheme 2. Biosynthetic routes for both 1 and 2.
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