Alternaramide, a cyclic depsipeptide from the marine-derived fungus Alternaria sp. SF-5016

Article abstract of DOI:10.1021/np900464p

Alternaramide (1), a cyclic pentadepsipeptide, has been isolated from an EtOAc extract of the marine-derived fungus Alternaria sp. SF-5016 by various chromatographic methods. The structure of 1 was mainly determined by analysis of the NMR spectroscopic da

Full text of DOI:10.1021/np900464p

J. Nat. Prod. 2009, 72, 2065–2068
2065
Alternaramide, a Cyclic Depsipeptide from the Marine-Derived Fungus Alternaria sp. SF-5016
Min-Young Kim,† Jae Hak Sohn,† Jong Seog Ahn,‡ and Hyuncheol Oh*,†
College of Medical and Life Sciences, Silla UniVersity, Busan 617-736, Korea, and Korea Research Institute of Bioscience and Biotechnology
(KRIBB), 52 Eoun-dong, Yuseong-gu, Daejeon 305-333, Republic of Korea
ReceiVed July 29, 2009
Alternaramide (1), a cyclic pentadepsipeptide, has been isolated from an EtOAc extract of the marine-derived fungus
Alternaria sp. SF-5016 by various chromatographic methods. The structure of 1 was mainly determined by analysis of
the NMR spectroscopic data, along with chemical methods such as Marfey’s and Mosher’s methods. Alternaramide (1)
showed weak antibiotic activity against Bacillus subtilis and Staphylococcus aureus.
Marine microorganisms are recognized as an important source
for structurally diverse bioactive secondary metabolites.1,2 As a part
of our ongoing studies on bioactive secondary metabolites from
marine microorganisms from Korea, we have investigated the
chemical constituents of the extracts obtained from cultures of the
marine-derived fungus Alternaria sp. SF-5016. This paper describes
the isolation, structure elucidation, and biological activity of the
metabolite, named alternaramide (1), encountered in this investigation.
Alternaramide (1) was isolated as a white powder and analyzed
for the molecular formula C33H40N4O6 by HRESIMS data (m/z
of a NOESY correlation of H-26 with H2-24, along with the
necessity that the compound must have an additional ring to fulfill
the unsaturations required by the molecular formula.
1
589.3024 [M + H]+), which was fully supported by the H and
13C NMR data (Table 1). The 1H NMR and DEPT spectra in CDCl3
revealed the presence of two methyl groups, 10 aromatic methines,
six sp3 methines (one oxygenated), and eight methylene units. In
addition to the signals corresponding to the above carbons, analysis
of the 13C NMR spectrum revealed the presence of five carbonyl
and two quaternary aromatic carbons. Furthermore, the presence
of characteristic signals for four R-amino carbons (δC/H 60.0/4.30,
52.2/4.99, 58.4/4.86, and 53.0/4.94) and five carbonyl/amide
carbonyl carbons (δC 172.3-168.4) in the NMR spectra of 1 was
indicative of a pentapeptide. The presence of one oxygen-bearing
methine carbon (δC/H 76.8/5.35) in the NMR spectra further
suggested that one of the carbonyl signals was from an ester moiety
and that the molecule contained a hydroxy acid moiety, thus
indicating a depsipeptidic nature of 1. Analysis of the 2D NMR
data (COSY, HMQC, and HMBC) allowed the identification of all
of the amino and hydroxy acid residues in the molecule, revealing
the presence of two phenylalanine (Phe), two proline (Pro), and
one R-hydroxyisovaleric acid (Hiv) residue. Because two aromatic
rings, two pyrrolidine rings, and five carbonyls accounted for 15
out of the 16 unsaturations in 1, alternaramide was proposed to be
a monocyclic depsipeptide. Interpretation of HMBC and NOESY
data readily led to the construction of the sequence of the five
residues in the molecule. Long-range correlations from H2-27 and
H-26 to C-25 revealed that this carbonyl carbon was positioned in
Phe-2. HMBC correlations of H-26 and H-2 with C-1 then
established the linkage of Phe-2 to Hiv. H-2 also showed a three
bond correlation to C-6, which was positioned in the Pro-1 unit on
the basis of HMBC correlations of H-7 and H-8 with C-6. Therefore,
the further extended sequence of Phe-2 f Hiv f Pro-1 was
unambiguously defined. A HMBC correlation of H-7 in the Pro-1
unit with C-11 and HMBC correlations of H2-13 and H-12 with
C-11 allowed the sequence of Pro-1 to Phe-1 to be established.
HMBC correlations of H-12 to C-20 and of H-21 and H2-22 to the
same carbonyl carbon established the linkage of Phe-1 to Pro-2.
Finally, the sequence of Pro-2 to Phe-2 was established on the basis
The absolute configurations of the chiral centers in 1 were
determined by Marfey’s method3 and Mosher’s method.4 The
absolute configurations of the amino acid units were established
by acid hydrolysis (6 N HCl, 120 °C, 24 h) of 1, followed by
derivatization with Marfey’s reagent (1-fluoro-2,4-dinitrophenyl-
5-L-alanine amide)3 and subsequent HPLC analysis. By comparing
the retention times of authentic standards of L- and D-forms of Phe
and Pro, the hydrolysate was identified to contain two units each
of D-Phe and L-Pro. To establish the absolute configuration at C-2
of Hiv, alternaramide (1) was first converted to the linear peptidic
methyl ester (2) by treatment of 1 with NaOMe in MeOH. Next,
the S- and R-Mosher esters (3a and 3b) of the linear peptide 2
were prepared. Analysis of H NMR and COSY spectra allowed
the assignment of the proton chemical shifts for the two diastere-
omeric esters 3a and 3b in proximity of the esterified carbon (C-
2). The differences in chemical shift values (∆δ ) δS - δR) for
the two diastereomeric esters 3a and 3b were calculated in order
to assign the absolute configurations at C-2 (Figure 1). Calculations
for all of the relevant signals suggested the S absolute configuration
at C-2. Taken together, the overall absolute configuration of
alternaramide (1) could be assigned as 2S, 7S, 12R, 21S, and 26R.
Alternaramide (1) appears to have similar structural features to
those of several depsipeptides isolated from marine-derived fungi,
such as exumolides from Scytalidium sp.,5 sansalvamides from
4
1
* To whom correspondence should be addressed. Tel: +82-51-999-5026.
Fax: +82-51-999-5176. E-mail: hoh@silla.ac.kr.
† Silla University.
‡ Korea Research Institute of Bioscience and Biotechnology.
10.1021/np900464p CCC: $40.75  2009 American Chemical Society and American Society of Pharmacognosy
Published on Web 10/08/2009

2066 Journal of Natural Products, 2009, Vol. 72, No. 11
Notes
Table 1. NMR Spectroscopic Data for Alternaramide (1) in CDCl3
a
position
δC
δH, mult. (J in Hz)b
key NOESY
HMBC (HfC#)
L-Hiv
L-Pro-1
D-Phe-1
1
2
3
4
5
168.4
76.8
29.7
15.9
19.2
5.35, d (2.2)
2.53, m
0.71, d (7.0)
0.82, d (7.0)
H-5, H-26
1, 3, 4, 5, 6
H-24
2, 3, 5
2, 3, 4
6
7
8
9
170.3
60.0
29.7
24.9
47.0
4.30, dd (5.2, 8.8)
1.88, m 1.94, m
1.46, m 1.75, m
2.51, m 3.43, m
6, 8, 9, 10, 11
6, 7, 9, 10
7, 8, 10
H-12
H-12
10
8, 9
11
12
172.3
52.2
4.99, m
3.00, m
H-10
11, 13, 20
11, 12, 14, 15/19
13
40.5
14
136.1
129.4
128.5
126.6
15/19
16/18
17
7.18-7.33, m
7.18-7.33, m
7.18-7.33, m
L-Pro-2
20
21
22
23
24
170.0
58.4
24.55
24.57
46.0
4.86, d (7.3)
1.65, m 2.41, m
1.87, m 2.00, m
NH-20
H-26
20, 22, 24
20, 21, 23
24
2.90, dd (5.5, 13.2) 3.36, dd (9.5, 13.2)
25, 26, 28, 29
D-Phe-2
25
26
170.7
53.0
4.94, m
H-24
1, 25, 27, 28
27
37.0
2.90, dd (5.5, 13.2) 3.36, dd (9.5, 13.2)
25, 26, 28, 29/33
28
137.6
129.8
128.7
127.4
29/33
30/32
31
7.18-7.33, m
7.18-7.33, m
7.18-7.33, m
7.24c
NH-1
NH-20
1
20
8.13, d (9.5)
H-12, H-13, H-21
a Recorded at 100 MHz. b Recorded at 400 MHz. c Observed in the COSY data.
phosphatase 1B (PTP1B),10 showing 49% inhibition at the level
of 150 µg/mL.
Experimental Section
General Experimental Procedures. Optical rotations were recorded
on a Perkin-Elmer 341 digital polarimeter. UV spectra were recorded
on a Biochrom 1300 UV/visible spectrophotometer. NMR spectra (1D
and 2D) were recorded in pyridine-d5 or CDCl3 using a JEOL JNM
1
ECP-400 spectrometer (400 MHz for H and 100 MHz for 13C), and
chemical shifts were referenced relative to tetramethylsilane (δH/δC )
1
0). HMQC and HMBC experiments were optimized for JCH ) 140
Figure 1. ∆δ values [∆δ (in ppm) ) δS - δR] obtained for the
(S)- and (R)-MTPA esters of the methanolysis product (3a and 3b,
respectively).
Hz and nJCH ) 8 Hz, respectively. ESIMS data were obtained using a
Q-TOF micro LC-MS/MS instrument (Waters, USA) at Korea Uni-
versity, Seoul, Korea. Solvents for extractions and flash column
chromatography were reagent grade and used without further purifica-
tion. Solvents used for HPLC were analytical grade. Flash column
chromatography was carried out using YMC octadecyl-functionalized
silica gel (C18). HPLC separations were performed on a Shiseido Capcell
Pak C18 column (10 × 250 mm; 5 µm particle size; 2 mL/min) or
Agilent prep-C18 column (21.2 × 150 mm; 5 µm particle size; 5 mL/
min). Compounds were detected by UV absorption at 210 nm.
Fungal Materials and Identification. Alternaria sp. SF-5016
(deposited at the College of Medical and Life Sciences fungal strain
repository, Silla University) was isolated from a shoreline sediment
sample collected from the shoreline in the Masan Bay area, Korea, in
January 2006. The sediment sample was stored in a sterile plastic bag
and transported to the laboratory, where it was kept frozen until
processed. The sample was diluted 10-fold using sterile seawater. One
milliliter of the diluted sample was processed utilizing the spread plate
method in PDA medium (24 g of potato dextrose agar, 1 L of seawater)
plates. The plate was incubated at 25 °C for 14 days. After purifying
Fusarium sp.,6,7 and zygosporamide from Zygosporium masonii.8
These peptides possess hydrophobic amino acid residues and an
ester linkage. In addition, the presence of D-amino acid residues in
this class of depsipeptide seems quite rare since zygosporamide is
the only example among the class. It is noteworthy that the
important role of a D-amino acid in the sansalvamide analogues
has been indicated in extensive SAR studies of their antitumor
activity.9
Alternaramide showed weak antimicrobial activity against Bacil-
lus subtilis (KCTC 1021) and Staphylococcus aureus (KCTC 1928),
affording zones of inhibition of 8 and 13 mm, respectively, at 400
µg/disk. However, 1 did not show antimicrobial activity against
Candida albicans (KCTC 7965), Proteus Vulgaris (KACC 12674),
or Filobasidiella neoformans (KCTC 7003) at the same level. In
addition, 1 weakly inhibited the activity of protein tyrosine

Notes
Journal of Natural Products, 2009, Vol. 72, No. 11 2067
1
1
the isolates several times, the final pure cultures were selected and
preserved at -70 °C.
analysis of the H NMR and COSY spectra: H NMR (pyridine-d5,
400 MHz) (integration, multiplicity, J in Hz, assignment): δ 5.55 (1H,
d, J ) 5.1, H-2), 2.43 (1H, m, H-3), 0.91 (3H, d, J ) 7.0 Hz, H3-4),
0.99 (3H, d, J ) 7.0 Hz, H3-5), 8.88 (1H, br s, NH-1), 5.30 (1H, m,
H-26), 3.35 (1H, m, H2-27), 3.22 (1H, m, H2-27).
This fungus was identified on the basis of the analysis of the rRNA
sequences. A GenBank search with the 28S rRNA gene of SF-5016
(Genbank accession number GQ865634) indicated Alternaria alternata
(DQ678082), Alternaria thalictrigena (EU040211), and Alternaria
malorum (AY251081) as the closest matches, showing sequence
identities of 99.51%, 99.51%, and 99.14%, respectively. Therefore, the
marine-derived fungal strain SF-5016 was characterized as Alternaria
sp.
Similarly, the reaction mixture from another sample of 2 (0.8 mg,
0.0013 mmol), (S)-MPTACl (7.5 mg, 0.029 mmol), and pyridine-d5
(0.5 mL) was processed as described above for 3a to afford 3b. H
1
NMR (pyridine-d5, 400 MHz) (integration, multiplicity, J in Hz,
assignment): δ 5.49 (1H, d, J ) 4.4, H-2), 2.45 (1H, m, H-3), 1.00
(3H, d, J ) 7.0 Hz, H3-4), 1.08 (3H, d, J ) 7.0 Hz, H3-5), 8.77 (1H,
NH-1), 5.28 (1H, m, H-26), 3.34 (1H, m, H2-27), 3.16 (1H, m, H2-27).
Purification of the (R)- and (S)-MTPA Ester Derivatives (3a and
3b). The reaction mixtures were recovered from the NMR tubes. After
removal of solvents under vacuum, the residues were purified by
reversed-phase HPLC (Agilent prep-C18; 21.2 × 150 mm; 5 µm particle
size; 5 mL/min) separately, using a gradient from 50% to 100% MeOH
in H2O (0.1% formic acid) over 35 min, then 100% MeOH for 20 min,
to yield pure 3a (1.2 mg; tR ) 41.6 min) or 3b (0.7 mg; tR ) 41.0
min), respectively. 1H NMR of 3a (CDCl3, 400 MHz, data were
Fermentation, Extraction, and Isolation of Alternaramide (1).
The fungal strain was cultured on 10 Petri-dish plates (90 mm), each
containing 20 mL of potato dextrose agar media [0.4% (w/v) potato
starch, 2% (w/v) dextrose, 3% (w/v) NaCl, 1.5% (w/v) agar]. Plates
were individually inoculated with 2 mL of seed cultures of the fungal
strain. Plate cultures were incubated at 25 °C for a period of 14 days.
Extraction of the agar media with EtOAc (2 × 500 mL) provided an
organic phase, which was then concentrated in Vacuo to yield 352.2
mg of an extract. The EtOAc extract was subjected to C18 flash column
chromatography (5 × 40 cm), eluting with a stepwise gradient of 20%,
40%, 60%, 80%, and 100% (v/v) MeOH in H2O (500 mL each). The
fractions eluted at 80% and 100% MeOH (49.6 mg) were combined
and purified by semipreparative reversed-phase HPLC eluting with a
gradient from 65% to 80% CH3CN in H2O (0.1% formic acid) over 35
min, then 100% CH3CN for 20 min, to yield 1 (6.1 mg, tR ) 35.0
min).
assigned on the basis of the correlations of H-1H COSY spectrum):
1
δ 5.09 (1H, d, J ) 4.4, H-2), 2.21 (1H, m, H-3), 0.87 (3H, d, J ) 7.0
Hz, H3-4), 0.85 (3H, d, J ) 7.0 Hz, H3-5), 7.20 (1H, NH-1), 4.87 (1H,
m, H-26), 3.08 (1H, m, H2-27), 2.98 (1H, m, H2-27), 7.40 (1H, br s,
NH-20), 4.82 (1H, m, H-12), 3.00 (1H, m, H-13), 2.97 (1H, m, H-13).
1H NMR of 3b (CDCl3, 400 MHz, data were assigned on the basis of
Alternaramide (1): white powder; [R]25D -6 (c 0.53, MeOH); UV
(MeOH) λmax (log ε) 208 (4.1); IR (neat) νmax 3298, 3008, 2930, 1742,
1674, 1634, 1525, 1450, 1236, 750 cm-1; 1H, 13C NMR data, Table 1;
HRESIMS m/z 589.3024 [M + H]+ (calcd for C33H41N4O6, 589.3026).
Preparation and Analysis of Marfey’s Derivatives. Alternaramide
(1, 0.6 mg) was hydrolyzed by heating in 6 N HCl (1 mL) at 110 °C
for 24 h. After cooling, the solution was evaporated to dryness and
redissolved in H2O (50 µL). To the mixture from acid hydrolysis was
added a 1% (w/v) solution (100 µL) of FDAA (Marfey’s reagent;
1-fluoro-2,4-dinitrophenyl-5- L-alanine amide) in acetone. After the
addition of NaHCO3 solution (1 M, 20 µL), the mixture was incubated
at 40 °C for 1 h. The reaction was stopped by the addition of HCl (2
N, 10 µL), the solvent was evaporated to dryness, and the resulting
residue was dissolved in MeOH/H2O (1:1; 1 mL). An aliquot of this
solution (20 µL for 1 and 10 µL for the standards) was analyzed by
HPLC [Capcell Pak C18 column, linear gradient from 30% to 60%
CH3CN in H2O (0.1% formic acid) over 200 min; 1 mL/min; 25 °C;
340 nm]. Separately, L-Phe, D-Phe, L-Pro, and D-Pro were derivatized
with FDAA in the same manner as that of 1. Retention times (min) of
the FDAA amino acid derivatives used as standards were as follows:
L-Phe (101.5), D-Phe (119.0), L-Pro (53.5), D-Pro (57.7). Retention times
(min) of the observed peaks in the HPLC trace of the FDAA-derivatized
hydrolysis product of 1 were 53.5 and 119.0.
Methanolysis of 1. Alternaramide (1, 3.0 mg) was dissolved in 0.5
N NaOMe solution (1.5 mL of MeOH) and stirred at ambient
temperature for 1 h. The mixture was neutralized by adding 1 N HCl
and dried in Vacuo. The residual material was partitioned with H2O
and EtOAc, and the resulting residue in EtOAc was purified by
reversed-phase HPLC, eluting with a gradient from 65% to 80% MeOH
in H2O (0.1% formic acid) over 35 min, then 100% MeOH for 20 min,
to yield the linear peptide 2 (2.1 mg, tR ) 35.0 min). The methanolysis
product was confirmed by ESIMS (m/z 643 [M + Na]+, molecular
formula; C34H44N4O7) and 1H NMR analysis. The linear peptide 2
showed the following 1H NMR spectroscopic properties. 1H NMR
(CDCl3, 400 MHz) δ (integration, multiplicity, J in Hz, assignment):
δ 3.91 (1H, br s, H-2), 2.10 (1H, m, H-3), 0.81 (3H, d, 7.0, H3-4), 0.99
(3H, d, 7.0, H3-5), 4.28 (1H, dd, 8.1, 3.7, H-7), 1.90 (1H, m, H2-8),
1.83 (1H, m, H2-8), 1.78 (1H, m, H2-9), 1.57 (1H, m, H2-9), 3.50 (1H,
m, H-10), 2.66 (1H, m, H2-10), 4.89 (1H, m, H-12), 3.10 (1H, m, H2-
13), 3.02 (1H, m, H2-13), 7.03-7.18 (10H, m, Ar-H), 4.48 (1H, br d,
5.8, H-21), 2.14 (1H, m, H2-22), 1.65 (1H, m, H2-22), 2.13 (1H, m,
H2-23), 1.84 (1H, m, H2-23), 3.56 (1H, m, H2-24), 2.74 (1H, m, H2-
24), 4.82 (1H, ddd, 13.9, 9.2, 5.5, H-26), 2.99 (1H, m, H2-27), 2.94
(1H, m, H2-27).
the correlations of H-1H COSY spectrum): δ 5.22 (1H, br s, H-2),
1
2.30 (1H, m, H-3), 0.96 (3H, d, J ) 6.6 Hz, H3-4), 0.92 (3H, d, J )
6.6 Hz, H3-5), 6.67 (1H, br s, NH-1), 4.66 (1H, m, H-26), 2.87 (1H,
m, H2-27), 2.75 (1H, m, H2-27), 7.30 (1H, br s, NH-20), 4.82 (1H, m,
H-12), 2.97 (1H, m, H-13), 2.90 (1H, m, H-13).
Antimicrobial Assay Procedures. The bacterial strains were grown
on nutrient agar [0.3% (w/v) beef extract, 0.5% (w/v) peptone, 1.5%
(w/v) agar], and the yeast Candida albicans (KCTC 7965) and
Filobasidiella neoformans (KCTC 7003) were grown on yeast mold
agar [0.3% (w/v) yeast extract, 0.3% (w/v) malt extract, 0.5% (w/v)
peptone, 1% (w/v) dextrose, 2% (w/v) agar] and malt extract agar
[1.275% (w/v) maltose, 0.275% (w/v) dextrin, 0.235% (w/v) glycerol,
0.078% (w/v) peptone, 1.5% (w/v) agar], respectively. Test compound
was absorbed onto individual disks (6 mm diameter) at 400 µg/disk
and placed on the surface of the agar. The assay plates were incubated
at 25 °C for 48 h and examined for the presence of a zone of inhibition.
Gentamicin was employed as the positive control and displayed 13
mm (at 50 µg/disk) and 16 mm (at 5 µg/disk) zones of inhibition against
Staphylococcus aureus (KCTC 1928) and Bacillus subtilis (KCTC
1021), respectively.
PTP1B Assay Procedures. PTP1B (human, recombinant) was
purchased from BIOMOL Research Laboratories, Inc. The enzyme
activity was measured in a reaction mixture containing 2 mM
p-nitrophenyl phosphate (pNPP) in 50 mM citrate, pH 6.0, 0.1 M NaCl,
1 mM EDTA, and 1 mM dithiothreitol (DTT). The reaction mixture
was placed in a 30 °C incubator for 30 min, and the reaction was
terminated by the addition of 10 N NaOH. The amount of produced
p-nitrophenol was estimated by measuring the increase in absorbance
at 405 nm. The nonenzymatic hydrolysis of 2 mM pNPP was corrected
by measuring the increase in absorbance at 405 nm obtained in the
absence of PTP1B enzyme.
Acknowledgment. This research was supported in part by the grants
from Global Partnership Program (No. M60602000001-06E0200-
00100) of Korea Foundation for International Cooperation of Science
& Technology (KICOS) through the grant provided by the Korean
Ministry of Education, Science & Technology (MEST), and KRIBB
Research Initiation Program. We gratefully acknowledge S.-Y. Kim
for her technical support of this project.
Supporting Information Available: 1D- and 2D-NMR spectra for
compounds 1,2, 3a, and 3b. This material is available free of charge
via the Internet at http://pubs.acs.org.
Preparation of Mosher Esters of the Linear Peptide (2). A sample
of 2 (0.9 mg, 0.0014 mmol), (R)-MTPACl (7.5 mg, 0.029 mmol), and
pyridine-d5 (0.5 mL) were allowed to react in an NMR tube at ambient
temperature for 24 h. The 1H NMR data of the (S)-MTPA ester
derivative (3a) were obtained directly on the reaction mixture by
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NP900464P