Bioactive phenylalanine derivatives and cytochalasins from the soft coral-derived fungus, Aspergillus elegans

Article abstract of DOI:10.3390/md11062054

One new phenylalanine derivative 4′-OMe-asperphenamate (1), along with one known phenylalanine derivative (2) and two new cytochalasins, aspochalasin A1 (3) and cytochalasin Z24 (4), as well as eight known cytochalasin analogues (5-12) were isolated from the fermentation broth of Aspergillus elegans ZJ-2008010, a fungus obtained from a soft coral Sarcophyton sp. collected from the South China Sea. Their structures and the relative configurations were elucidated using comprehensive spectroscopic methods. The absolute configuration of 1 was determined by chemical synthesis and Marfey's method. All isolated metabolites (1-12) were evaluated for their antifouling and antibacterial activities. Cytochalasins 5, 6, 8 and 9 showed strong antifouling activity against the larval settlement of the barnacle Balanus amphitrite, with the EC₅₀ values ranging from 6.2 to 37 μM. This is the first report of antifouling activity for this class of metabolites. Additionally, 8 exhibited a broad spectrum of antibacterial activity, especially against four pathogenic bacteria Staphylococcus albus, S. aureus, Escherichia coli and Bacillus cereus.

Full text of DOI:10.3390/md11062054

Mar. Drugs 2013, 11, 2054-2068; doi:10.3390/md11062054
OPEN ACCESS
Marine Drugs
ISSN 1660-3397
www.mdpi.com/journal/marinedrugs
Article
Bioactive Phenylalanine Derivatives and Cytochalasins from the
Soft Coral-Derived Fungus, Aspergillus elegans
Cai-Juan Zheng 1,2, Chang-Lun Shao 1, Lu-Yong Wu 2, Min Chen 1, Kai-Ling Wang 1,
Dong-Lin Zhao 1, Xue-Ping Sun 3, Guang-Ying Chen 2,* and Chang-Yun Wang 1,*
1
Key Laboratory of Marine Drugs, Ministry of Education, School of Medicine and Pharmacy,
Ocean University of China, Qingdao 266003, China; E-Mails: caijuan2002@163.com (C.-J.Z.);
shaochanglun@163.com (C.-L.S.); dieying0719@163.com (M.C.); kailingw@163.com (K.-L.W.);
lanseyimi@sina.com (D.-L.Z.)
2
Key Laboratory of Tropical Medicinal Plant Chemistry of Ministry of Education, College of
Chemistry and Chemical Engineering, Hainan Normal University, Haikou 571158, China;
E-Mail: wuluyong@hainnu.edu.cn
3
Guangxi Key Laboratory of Marine Biotechnology, Guangxi Institute of Oceanology, Beihai
536000, China; E-Mail: pingty2006@163.com
* Authors to whom correspondence should be addressed; E-Mails: changyun@ouc.edu.cn (C.-Y.W.);
chgying123@163.com (G.-Y.C.); Tel.: +86-532-82031536 (C.-Y.W.);
Fax: +86-532-82031381 (C.-Y.W.); Tel./Fax: +86-898-65889422 (G.-Y.C.).
Received: 1 April 2013; in revised form: 10 May 2013 / Accepted: 14 May 2013 /
Published: 10 June 2013
Abstract: One new phenylalanine derivative 4′-OMe-asperphenamate (1), along with one
known phenylalanine derivative (2) and two new cytochalasins, aspochalasin A1 (3) and
cytochalasin Z24 (4), as well as eight known cytochalasin analogues (5–12) were isolated
from the fermentation broth of Aspergillus elegans ZJ-2008010, a fungus obtained from a
soft coral Sarcophyton sp. collected from the South China Sea. Their structures and the
relative configurations were elucidated using comprehensive spectroscopic methods. The
absolute configuration of 1 was determined by chemical synthesis and Marfey’s method.
All isolated metabolites (1–12) were evaluated for their antifouling and antibacterial
activities. Cytochalasins 5, 6, 8 and 9 showed strong antifouling activity against the larval
settlement of the barnacle Balanus amphitrite, with the EC50 values ranging from 6.2 to
37 μM. This is the first report of antifouling activity for this class of metabolites.
Additionally, 8 exhibited a broad spectrum of antibacterial activity, especially against four
pathogenic bacteria Staphylococcus albus, S. aureus, Escherichia coli and Bacillus cereus.

Mar. Drugs 2013, 11
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Keywords: soft coral-derived fungus; Aspergillus elegans; phenylalanine derivative;
cytochalasin; antifouling
1. Introduction
Marine-derived fungi have proven to be a promising source of structurally novel and biologically
active secondary metabolites that have become interesting and significant resources for drug
discovery [1]. Especially, the genus Aspergillus has been known to be a major contributor to the
bioactive secondary metabolites of marine fungal origin, for example, antibacterial bisabolene-type
sesquiterpenoids from sponge-derived fungus, Aspergillus sp. [2], cytotoxic hetero-spirocyclic
γ-lactams from A. sydowii [3], 14-membered macrolides from A. ostianus [4], anthraquinone
derivatives with naphtho[1,2,3-de]chromene-2,7-dione skeleton from A. glaucus [5] and cytochalasins
from A. flavipes [6].
In our search for new antibacterial, cytotoxic and antifouling natural products from marine fungi in
the South China Sea, we have found several bioactive compounds, including sesquiterpenoids,
quinolinone alkaloids, azaphilone derivatives, resorcylic acid lactones and anthraquinone derivatives,
from marine fungi through the bioassay-guided isolation [2,7–12]. In our ongoing investigations on the
marine-derived fungi isolated from marine invertebrates, a fungus, Aspergillus elegans ZJ-2008010,
obtained from a soft coral, Sarcophyton sp., attracted our attention. The EtOAc extract of a
fermentation broth of the fungus exhibited antifouling activity against the larval settlement of the
barnacle, Balanus amphitrite. Bioassay-guided fractionation of the bioactive extract led to the isolation
of one new phenylalanine derivative, 4′-OMe-asperphenamate (1), together with one known
phenylalanine derivative asperphenamate (2) [13,14] and two new cytochalasins, aspochalasin A1 (3)
and cytochalasin Z24 (4), as well as eight known cytochalasins (5–12), determined as aspochalasins
I (5) and J (6) [15], aspochalasins B (7) and D (8) [16,17], aspochalasin H (9) [18], aspergillin PZ
(10) [19], zygosporin D (11) [20] and rosellichalasin (12) [21] (Figure 1). All isolated metabolites
(1–12) were evaluated for their antifouling and antibacterial activities. Herein, we report the isolation,
structure elucidation and biological activities of these compounds.

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Figure 1. Structures of compounds 1–12.
2. Results and Discussion
4′-OMe-asperphenamate (1) was obtained as a white powder. Its molecular formula was established
1
13
as C33H32N2O5 (19 degrees of unsaturation) from HRESIMS, combined with H and C NMR
spectroscopic data (Table 1). The 1H NMR spectrum of 1 showed the presence of 19 aromatic protons
in the downfield, including three monosubstituted phenyls at δH [7.38 (2H, m), 7.24 (1H, m) and 7.23
(2H, m); 7.65 (2H, m), 7.50 (1H, m) and 7.30 (2H, m); and 7.70 (2H, m), 7.42 (1H, m) and 7.30 (2H, m)]
and one disubstituted phenyl at δH [7.12 (1H, d, J = 8.4 Hz), 7.10 (1H, d, J = 8.4 Hz), 6.82 (1H, d,
J = 8.4 Hz) and 6.81 (1H, d, J = 8.4 Hz)]. In addition, two secondary amino protons at δH 6.71 (1H, d,
J = 8.4 Hz) and 6.59 (1H, d, J = 6.6 Hz) were also observed in the downfield. In the highfield, two
methine groups at δH 4.88 (1H, m) and 4.62 (1H, m), one oxygenated methylene group at δH [4.52 (1H,
dd, J = 10.8, 4.2 Hz) and 4.05 (1H, dd, J = 10.8, 3.0 Hz)], one methoxyl group at δH 3.74 (3H, s), two
methylene groups at δH [3.22 (1H, dd, J = 14.4, 6.6 Hz) and 3.16 (1H, dd, J = 14.4, 6.6 Hz)] and at δH
[3.00 (1H, dd, J = 13.2, 6.6 Hz) and 2.89 (1H, dd, J = 13.2, 8.4 Hz)] were observed. These
13
spectroscopic features combined with the C NMR spectrum suggested that 1 very closely resembled
asperphenamate (2) [13,14]. The only difference in the 1H NMR spectrum was the presence of a singlet
methyl signal at δH 3.74 (3H, s) in 1 instead of an aromatic proton signal at δH 7.30 (1H, m) in 2.
Furthermore, in the 13C NMR spectrum, the C-4′ signal moved downfield significantly [δC 158.9 (C) in
1 vs. 126.8 (CH) in 2], indicating a methoxy group was located at C-4′. The location of the methoxy
group at C-4′ was confirmed by the heteronuclear multiple bond correlation (HMBC) correlation from
4′-OCH3 at δH 3.74 (3H, s) to C-4′ at δC 158.9 (C). The planar structure of 1 was further confirmed by
the 1H-1H COSY and HMBC experiments (Figure 2).

Mar. Drugs 2013, 11
Table 1. NMR spectroscopic data (600/150 MHz, DMSO-d6) for compound 1.
2057
Position
1H (J in Hz)
-
13C
Position
1H (J in Hz)
13C
1
2
3
4
5
6
7
134.2, C
8′
9′
4.62, m
50.4, CH
7.23, m
129.4, CH
128.8, CH
127.2, CH
128.8, CH
129.4, CH
37.4, CH2
4.52, dd (10.8, 4.2) 65.4, CH2
4.05, dd (10.8, 3.0)
7.38, m
7.24, m
10′
1″
6.71, d (8.4)
-
-
7.23, m
133.4, C
7.38, m
2″
7.65, m
7.30, m
7.50, m
7.30, m
7.65, m
-
127.2, CH
128.5, CH
132.1, CH
128.5, CH
126.9, CH
167.5, C
3.22, dd (14.4, 6.6)
3.16, dd (14.4, 6.6)
4.88, m
3″
4″
8
9
55.2, CH
172.1, C
-
5″
-
6″
10
1′
2′
3′
4′
5′
6′
7′
6.59, d (6.6)
-
7″
137.3, C
130.3, CH
114.3, CH
158.9, C
114.3, CH
130.3, CH
36.7, CH2
1′″
-
137.2, C
7.10, d (8.4)
6.81, d (8.4)
-
2′″
7.70, m
7.30, m
7.42, m
7.30, m
7.70, m
-
127.1, CH
128.5, CH
131.5, CH
128.5, CH
127.1, CH
166.7, C
3′″
4′″
6.82, d (8.4)
7.12, d (8.4)
3.00, dd (13.2, 6.6)
2.89, dd (13.2, 8.4)
5′″
6′″
7′″
4′-OCH3
3.74, s
54.6, CH3
1
Figure 2. H-1H COSY correlations and key heteronuclear multiple bond correlation
(HMBC) correlations of compounds 1, 3 and 4.
The absolute configurations of C-8 and C-8′ in 1 were determined by chemical synthesis and
Marfey’s method [22]. The major acid hydrolysis products of 1 with 6 N HCl at 105 °C for 19 h were
Phe and 2-amino-3-(4-methoxyphenyl)-1-propanol. The hydrolysates of 1, as well as the standard
L-Phe and DL-Phe, were derivatized with Marfey’s reagent, 1-fluoro-2,4-dinitrophenyl-5-L-alanine
amide (FDAA). Analysis of the FDAA derivatives of the hydrolysates from 1 by HPLC, compared
with the FDAA derivatives from L-Phe and DL-Phe, revealed the presence of an L-Phe moiety in 1
(Figure S32). Therefore, the absolute configuration at C-8 was assigned as S.
To determine the absolute configuration at C-8′, (R/S)-2-amino-3-(4-methoxyphenyl)-1-propanol
(14a) and (S)-2-amino-3-(4-methoxyphenyl)-1-propanol (14b) were synthesized. Compounds 14a and
14b were prepared from DL-Tyr (13a) and L-Tyr (13b) (Figure 3), respectively, according to the
literature method [23]. Also, 14a and 14b were derivatized with Marfey’s reagent (FDAA). Analysis
of the FDAA derivatives of the hydrolysates from 1 by HPLC, compared with the FDAA derivatives

Mar. Drugs 2013, 11
2058
from 14a and 14b, revealed the presence of a 14b moiety in 1 (Figure S32). Accordingly, the absolute
configuration at C-8′ was assigned as S. The 8S8′S configurations in 1 were consistent with those in
asperphenamate (2), confirming that these two compounds share the same biogenetic path way.
Figure 3. The preparation of compounds 14a and 14b.
Aspochalasin A1 (3) was isolated as a white powder. Its molecular formula was established as
1
C24H35NO5 (eight degrees of unsaturation) by HRESIMS, combined with H and 13C NMR
1
spectroscopic data (Table 2). The H NMR spectrum recorded in CDCl3 intuitively revealed five
methyl groups, two olefinic protons [δH 6.00, 1H, d, J = 11.4 Hz, H-13; δH 5.37, 1H, br s, H-7], an
amide group (δH 5.93, 1H, br s, NH), six methine protons, five methylene units, along with one
hydroxy group (Table 2). These features characteristically revealed the structure of 3 as possessing a
(2-methylpropyl) isoindolone moiety, consistent with a cytochalasin skeleton [16]. These structural
features were also confirmed by the 13C NMR and DEPT spectra (Table 2). In the 13C NMR spectrum,
except for the carbon signals of the (2-methylpropyl) isoindolone moiety, there were also ten
resonance signals revealed that 3 possessed a 12-membered lactone macrocycle, containing a methyl
group (δC 15.9, CH3, C-25), a double bond [δC 125.6 (CH), C-13 and δC 136.2 (C), C-14] and two
carbonyl groups [δC 212.7 (C), C-17 and δC 171.8 (C), C-21]. The 1H and 13C NMR spectra of 3 were
closely related to those of aspochalasin M [24]. The obvious difference was the absence of the
macrocyclic ketone moiety as in aspochalasin M at δC 214.5 (C-21) and the presence of a lactone at δC
171.8 (C-21) in 3. The differences were also observed for the chemical shifts of H-18/C-18 [δH 5.00
(1H, ddd, J = 10.8, 3.6, 3.6 Hz) and δC 74.0 (CH) in 3 vs. δH 4.15 (1H, dd, J = 5.0, 5.0 Hz) and δC 77.4
(CH) in aspochalasin M] and the large coupling constant J = 10.8 Hz between H-18 (δH 5.00) and
H-19 (δH 1.53) in 3, instead of the small coupling constant J = 5.0 Hz between H-18 (δH 4.15) and
H-19 (δH 2.00) in aspochalasin M. These features established an axial H-18 in 3 in contrast to an
equatorial H-18 in aspochalasin M. This suggested that the OH at C-18 in 3 has a α-orientation
1
different from the β-orientation in aspochalasin M. The H-1H COSY and HMBC allowed the
deduction of the gross structure of 3 as shown in Figure 2.

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2059
Table 2. NMR spectroscopic data (600/150 MHz, CDCl3) for compounds 3 and 4.
3
4
Position
1H (J in Hz)
13C
1H (J in Hz)
-
13C
1
2
-
5.93, br s
3.00, m
2.49, dd (4.2, 3.6)
2.74, m
-
172.0, C
-
166.9, C
-
6.30, br s
3.68, m
3
52.6, CH
52.6, CH
35.1, CH
141.2, C
122.5, CH
42.1, CH
86.8, C
53.6, CH
49.2, CH
35.9, CH
57.2, C
60.8, CH
47.0, CH
85.2, C
45.1, CH2
4
3.04, br d (5.4)
2.22, m
5
6
-
7
5.37, br s
3.43, d (10.8)
-
2.74, d (5.4)
2.99, d (10.8, 5.4)
-
8
9
10
1.96, m
1.57, m
1.21, d (6.6)
1.78, br s
6.00, d (10.8)
-
46.4, CH2
2.86, dd (12.6, 8.4)
2.77, dd (12.6, 6.6)
0.93, d (6.6)
1.17, br s
11
12
13
14
15
14.3, CH3
20.3, CH3
125.6, CH
136.2, C
12.7, CH3
19.5, CH3
124.2, CH
138.8, CH
44.1, CH2
6.03, dd (13.2, 10.8)
5.21, ddd (13.2, 10.8, 3.6)
2.11, m
2.68, m
2.39, m
2.69, m
2.45, m
-
37.2, CH2
2.08, m
16
17
35.5, CH2
212.7, C
1.27, m
29.3, CH
53.8, CH2
1.76, dd (13.2, 1.8)
1.68, dd (13.2, 5.4)
-
18
19
5.00, ddd (10.8, 3.6, 3.6)
74.0, CH
29.1, CH2
72.8, C
2.10, m
7.11, d (15.6)
159.1, CH
1.53, m
20
2.65, m
31.4, CH2
5.69, d (15.6)
119.1, CH
2.44, m
21
22
-
171.8, C
-
172.8, C
27.1, CH3
22.0, CH3
-
1.19, m
25.6, CH
1.03, d (6.6)
23
0.93, d (6.6)
21.0, CH3
1.33, s
24
0.90, d (6.6)
23.9, CH3
-
25
1.80, br s
15.9, CH3
-
-
-
1′
-
-
-
-
-
137.0, C
129.4, CH
129.0, CH
127.1, CH
2′/6′
3′/5′
4′
-
7.15, br d (7.2)
7.30, m
7.25, m
-
-
18-OH
3.56, d (3.6)
The relative stereochemistry of 3 was determined by NOESY experiments and comparison of NMR
data with those of aspochalasin M [24]. In the NOESY experiments (Figure 4), the signal of H-3
showed correlations with H-10 and H-11 and H-4 with H-5 and H-8, indicating that the relative
configurations of the perhydroisoindol-1-one moiety in 3 were in accord with those of reported
cytochalasins. The modified Mosher’s method [25] was tried to determine the absolute configuration
of C-18 in 3; unfortunately, the reactions failed. A literature search revealed that the stereochemistry of

Mar. Drugs 2013, 11
2060
the cyclohexane and isoindole moieties in all isolated cytochalasins, so far, are the same [26].
Therefore, based on the above data and the biogenesis consideration, 3 was determined as
aspochalasin A1.
Figure 4. Key NOESY correlations of compound 3.
Cytochalasin Z24 (4) was also obtained as a white power. Its molecular formula was established as
C28H35NO5 by HRESIMS. The general features of its NMR spectroscopic data (Table 2) were
markedly similar to those of cytochalasin Z22 [27]. Detailed comparison of NMR data of these two
compounds suggested that they had the same 10-phenyl-substituted 6,7-epoxyperhydroisoindol-1-one
1
skeleton. The only significant difference in the H NMR spectrum was the presence of a methylene
group at δH [1.76 (dd, J = 13.2, 1.8 Hz) and 1.68 (dd, J = 13.2, 5.4 Hz)] for H-17 in 4, and in the
13C-NMR spectrum, a methylene group at δC 53.8 (CH2) for C-17 was observed in 4, instead of a
carbonyl carbon at δC 212.3 (C) for C-17 in cytochalasin Z22. The gross structure of 4 was further
confirmed by 1H-1H COSY and HMBC spectra (Figure 2).
The configuration of the core skeleton, 10-phenyl-substituted 6,7-epoxyperhydroisoindol-1-one in 4
1
could be assigned based on biogenetic origin with those of cytochalasin Z22 [27]. In the H and
13C NMR spectra of 4, the chemical shifts and coupling constants of the H-atoms of C-3, C-4, C-5,
C-7, C-8, 11-Me and 12-Me and the chemical shifts of the C-atoms of C-3–C-8, 11-Me and 12-Me
were similar to those of cytochalasin Z22, suggesting that C-3–C-8 in 4 possess the same
configurations as in Z22. The configurations of C-16 and C-18 of the macrocyclic moiety in 4 were
assigned by comparison of the NMR data with those of [11]-cytochalasa-6(12),13,19-triene-1,
21-dione-7,18-dihydroxy-16,18-dimethyl-10-phenyl-(7S*,13E,16S*,18S*,19E) [28], [δH 1.27 (1H, m)
and δC 29.3 (CH) for C-16, δC 72.8 (C) for C-18 in 4] vs. [δH 1.33 (1H, m) and δC 28.8 (CH) for
C-16, δC 72.8 (C) for C-18 in [11]-cytochalasa-6(12),13,19-triene-1,21-dione-7,18-dihydroxy-16,
18-dimethyl-10-phenyl-(7S*,13E,16S*,18S*,19E)]. These results indicated that the two compounds
shared the same macrocyclic moiety, and these were also confirmed by other known cytochalasins
based on the shared biogenesis. The absolute configuration of 4 was then deduced as
3S,4S,5S,6R,7S,8S,9S,13E,16S,18S,19E. Therefore, the structure of 4 was established and named
cytochalasin Z24.

Mar. Drugs 2013, 11
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The structures of known compounds (2, 5–12) were identified by comparison of their spectroscopic
data with those in the literature [13–21]. Among them, asperphenamate (2), an uncommon
phenylalanine derivative, has already been isolated from some bioactive natural sources, such as fungi,
Aspergillus flavipes [29], Penicillium megasporum [30], P. brevicompactum [31] and P. canadense [32],
and plants, Anaphalis subumbellata [33], Artemisia anomala [34] and Croton hieronymi [13].
In the present paper, this is the first report of isolated asperphenamate from marine-derived fungus.
Cytochalasins are a large group of fungal alkaloids with a wide range of biological activities
targeting cytoskeletal processes [35]. Cytochalasin biosynthesis has been revealed by the formation of
an acetate- and methionine-derived octa- or nonaketide chain and the attachment of an amino acid. The
type of cytochalasins depends on the incorporated amino acids as structural subunits [36,37].
Compounds 3–9, 11 and 12 are a class of cytochalasins with 12-membered or 11-membered carbocyclic
(or oxygen-containing) rings connecting the C-8 and C-9 positions of a perhydroisoindol-1-one
moiety. The substituents at C-3 in compounds 3 and 5–9 is a 2-methylpropyl group; and in compounds
4, 11 and 12 is a phenyl group, belonging to the class of 10-phenyl-[11]-cytochalasin. Compound 10
belongs to an unusual type of aspochalasins with a pentacyclic system, of which only three examples
have been reported.
All the isolated compounds were evaluated for antifouling activity against the larval settlement of
the barnacle, Balanus amphitrite. Compounds 5, 6, 8 and 9 showed strong antifouling activity with the
EC50 values of 34, 14, 6.2 and 37 μM, respectively. Despite the slight structural differences in their
macrocycles, 8, bearing an α,β-unsaturated ketone moiety, was found to be considerably more active than
6, with an α,β-unsaturated lactone moiety, suggesting the importance of an electrophilic α,β-unsaturated
carbonyl moiety for the antifouling activity of these cytochalasins. Compound 8 displayed more
activity than 9, indicating that the double-bond at C-19 and C-20 might be essential for the antifouling
activity of cytochalasins. This is the first report of antifouling activity for this class of metabolites.
The antibacterial activity of all isolated compounds were also assessed against six terrestrial
pathogenic bacteria and two marine pathogenic bacteria. Compounds 1, 2, 5, 8 and 10 exhibited
selective antibacterial activity (Table 3). Compounds 1 and 2 showed selective antibacterial activity
against S. epidermidis, with the MIC values of 10 μM for each. Compound 5 showed moderate activity
against S. epidermidis and S. aureus with the MIC values of 20 and 10 μM, respectively, while 8 showed
a broad spectrum of antibacterial activity, especially towards four pathogenic bacteria, S. epidermidis,
S. aureus, E. coli and B. cereus.
Table 3. Antibacterial activity for compounds 1, 2, 5, 8 and 10.
MIC (μM)
Compound
S. epidermidis
S. aureus
>20
E. coli
>20
>20
>20
10
B. cereus
>20
1
10
10
2
>20
>20
5
20
10
>20
8
10
10
10
10
20
>20
>20
>20
Ciprofloxacin a
0.30
0.30
0.60
1.20
a Ciprofloxacin was used as a positive control.

Mar. Drugs 2013, 11
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3. Materials and Methods
3.1. General Experimental Procedures
Optical rotations were measured on a JASCO P-1020 digital polarimeter. IR spectra were recorded
1
on a Nicolet Nexus 470 spectrophotometer. H and 13C NMR spectra were recorded on a JEOL
1
13
Eclips-600 spectrometer at 600 MHz for H and 150 MHz for C in DMSO-d6 or CDCl3. Chemical
shifts δ are reported in ppm, using TMS as internal standard, and coupling constants (J) are in Hz.
ESIMS and HRESIMS spectra were measured on a Q-TOF Ultima Global GAA076 LC mass
spectrometer. HPLC separation was performed using a Hitachi L-2000 prep-HPLC system coupled
with a Hitachi L-2455 photodiode array detector. A Kromasil C18 preparative HPLC column
(10 × 250 mm, 5 μm and 4.6 × 250 mm, 5 μm) was used. Analysis of FDAA derivatives by HPLC was
performed using Waters 2695 prep-HPLC system coupled with a Waters 2489 UV detector. A Waters
C18 analytical HPLC column (4.6 × 250 mm, 5 μm) was used. Silica gel (Qing Dao Hai Yang
Chemical Group Co.; Qing Dao, China; 200–300 mesh), octadecylsilyl silica gel (Unicorn, Merck
KGaA, Darmstadt, Germany; 45–60 μm) and Sephadex LH-20 (Amersham Biosciences Inc.,
Piscataway, NJ, USA) were used for column chromatography (CC). Precoated silica gel plates
(Yan Tai Zi Fu Chemical Group Co.; Yan Tai, China; G60, F-254) were used for thin layer
chromatography (TLC).
3.2. Fungal Materials
The fungal strain, Aspergillus elegans ZJ-2008010, was isolated from a piece of tissue from the
inner part of the fresh soft coral, Sarcophyton sp. (GX-WZ-20080011), which was collected from the
Weizhou coral reef in the South China Sea in September, 2008. The strain was deposited in the Key
Laboratory of Marine Drugs, the Ministry of Education of China, School of Medicine and Pharmacy,
Ocean University of China, Qingdao, China, with the access code ZJ-2008010. The fungal strain was
cultivated in 50 L potato glucose liquid medium (15 g of glucose and 30 g of sea salt in 1 L of potato
infusion, in 1 L Erlenmeyer flasks, each containing 300 mL of culture broth) at 25 °C without shaking
for 4 weeks.
3.3. Identification of Fungus
The Fungus was identified according to its morphological characteristics and a molecular biological
protocol by 16S rRNA amplification and sequencing of the ITS region. The sequence data have been
submitted to GenBank, with an accession number JF694928, and the fungal strain was identified as
Aspergillus elegans.
3.4. Extraction and Isolation
The fungal cultures were filtered through cheesecloth, and the filtrate was extracted with EtOAc
(3 × 50 L, 12 h each). The organic extracts were concentrated in vacuo to yield an oily residue
(20.2 g), which was subjected to silica gel column chromatography (CC) (petroleum ether, EtOAc v/v,
gradient 100:0–0:100) to generate nine fractions (Fr. 1–Fr. 9). Fr. 2 was isolated by CC on silica gel

Mar. Drugs 2013, 11
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eluted with petroleum ether-EtOAc (4:1) and then subjected to Sephadex LH-20 CC eluting with
mixtures of petroleum ether-CHCl3-MeOH (2:1:1) to obtain compounds 1 (3.0 mg) and 2 (20.0 mg).
Fr. 3 was subjected to repeated Sephadex LH-20 CC (CHCl3/MeOH, v/v, 1:1) and further purified by
using HPLC on an ODS semi-preparative column (Kromasil C18, 10 × 250 mm, 5 μm, 2 mL/min)
eluted with 85% MeOH/H2O to obtain compounds 3 (3.6 mg), 4 (4.0 mg), 7 (10.0 mg), 11 (7.0 mg)
and 12 (5.0 mg). Fr. 4 was subjected to repeated Sephadex LH-20 CC (CHCl3/MeOH, v/v, 1:1) and
further purified on HPLC (82% MeOH/H2O) to afford compounds 5 (10.8 mg), 6 (8.2 mg) and 8
(11.0 mg). Fr. 5 was subjected to repeated Sephadex LH-20 CC (MeOH) and further purified on HPLC
(60% MeOH/H2O) to afford compounds 9 (9.0 mg) and 10 (12.0 mg).
4′-OMe-asperphenamate (1): White powder; [α]24D −38.0 (c 0.20, MeOH); UV (MeOH) λmax (log ε)
1
13
214(2.90), 280(2.50), 314(1.20) nm; IR (KBr) νmax 3448, 1712, 1624 cm−1; H and C NMR: see
Table 1; HRESIMS m/z 537.2215 [M + H]+ (calcd. for C33H33N2O5, 537.2209).
Aspochalasin A1 (3): White powder; [α]24 +12.0 (c 0.15, CHCl3); UV (MeOH) λmax (log ε)
D
1
13
214(2.90); IR (KBr) νmax 3462, 1700, 1448 cm−1; H and C NMR: see Table 2; HRESIMS m/z
418.2582 [M + H]+ (calcd. for C24H36NO5, 418.2593).
Cytochalasin Z24 (4): White powder; [α]24 −25.0 (c 0.12, CHCl3); UV (MeOH) λmax (log ε)
D
1
13
214(4.1), 232(3.1), 265(3.0), 289(2.9); IR (KBr) νmax 3310, 1710, 1446 cm−1; H and C NMR: see
Table 2; HRESIMS m/z 466.2407 [M + H]+ (calcd. for C28H36NO5, 466.2410).
3.5. Synthesis of Compounds 14a and 14b
(S/R)-3-(4-Hydroxyphenyl)-2-[(ethoxycarbonyl)amino]
propionic
acid
(13a-1)
or
(S)-3-(4-hydroxyphenyl)-2-[(ethoxycarbonyl)amino] propionic acid (13b-1): Ethyl chloroformate
(255 μL, 3.3 mmol) was added to a solution of DL-Tyr or L-Tyr (13a or 13b, 543 mg, 3 mmol,
respectively) and NaHCO3 (750 mg, 9 mmol, respectively) in a mixture of H2O/THF (15 mL/15 mL,
respectively). After stirring at room temperature (rt) overnight, the mixture was diluted with H2O and
washed with Et2O, successively. The aqueous layer was acidified and extracted with EtOAc (3 × 18 mL).
The combined extracts were washed (H2O), dried (Na2SO4) and concentrated to afford 500 mg (92%)
of 13a-1 or 13b-1, respectively, colorless oil.
Methyl (S/R)-2-[(ethoxycarbonyl)amino]-3-(4-methoxyphenyl) propanoate (13a-2) or methyl
(S)-2-[(ethoxycarbonyl)amino]-3-(4-methoxyphenyl) propanoate (13b-2): CH3I (370 μL, 6 mmol)
was added to a solution of 13a-1 or 13b-1 (500 mg, 1.97 mmol, respectively) in dry acetone (5 mL,
respectively) containing suspended K2CO3 (822 mg, 6 mmol, respectively). The mixture was refluxed
at rt overnight, then diluted with H2O and extracted with EtOAc (3 × 50 mL, respectively). The
combined extracts were washed (H2O), dried (Na2SO4) and concentrated, afforded 410 mg (82%) of
13a-2 or 13b-2, respectively, colorless oil.
(R/S)-2-[(Ethoxycarbonyl)amino]-3-(4-methoxyphenyl) propan-1-ol (13a-3) and (S)-2-[(ethoxy
carbonyl)amino]-3-(4-methoxyphenyl) propan-1-ol (13b-3): A solution of 13a-2 or 13b-2 (200 mg,
0.71 mmol, respectively) in THF (10 mL, respectively) was added dropwise over 2 h to a cold (0 °C)
solution of LiAlH4 (48 mg, 1.26 mmol, respectively) in THF (10 mL, respectively) maintained under

Mar. Drugs 2013, 11
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Ar. The mixture was stirred overnight at rt; then, it was cooled to 0 °C and extracted with EtOAc. The
combined extracts were washed (H2O), dried (Na2SO4) and concentrated. The crude product over silica
gel (petroleum ether/EtOAc: 4/1) afforded 160 mg (80%) of 13a-3 or 13b-3, respectively, colorless oil.
(S/R)-2-Amino-3-(4-methoxyphenyl)-1-propanol (14a) and (S)-2-amino-3-(4-methoxyphenyl)
-1-propanol (14b): A solution of 13a-3 or 13b-3 (160 mg, 0.63 mmol) and KOH (900 mg, 16.3 mmol,
respectively) in CH3OH/H2O (2:1, 7 mL, respectively) was stirred at 80 °C 4 h. The mixture was then
extracted with EtOAc (3 × 50 mL). The combined extracts were washed (H2O), dried (Na2SO4) and
concentrated. The crude product was over silica gel (petroleum ether/EtOAc: 3/1) to yield 120 g (75%)
of 14a or 14b, respectively, white solid.
3.6. Absolute Configuration Determination of 1 by Marfey’s Method [22]
A solution of 1 (1.5 mg) in 6 M HCl (1 mL) was heated to 105 °C for 19 h. The solution was then
evaporated to dryness and the residue redissolved in H2O (250 μL). A 50 μL portion of the acid
hydrolysate solution was then placed in a 1 mL reaction vial and treated with a 1% solution of FDAA
(200 μL) in acetone followed by 1.0 M NaHCO3 (40 μL). The reaction mixture was heated at 45 °C for
1 h, cooled to room temperature and then acidified with 2.0 M HCl (20 μL). In a similar fashion, 14a
and 14b and the standard DL- and L-Phe were derivatized with FDAA separately. The FDAA
derivatives of the hydrolysates, 14a and 14b and standard amino acids were subjected to HPLC
analysis (Waters C18 column; 5 μm, 4.6 × 250 mm; 1.0 mL/min) at 30 °C using the following gradient
program: solvent A, H2O + 0.1% TFA; solvent B, MeCN; linear gradient: 0 min, 25% B, 40 min,
60% B, 45 min, 100% B; UV detection at 340 nm. The retention times for the FDAA derivatives of
hydrolysates of 1 were 22.3 and 23.6 min, respectively; standard L-Phe, D-Phe, (S)-2-amino-
3-(4-methoxyphenyl)-1-propanol (14b) and (R)-2-amino-3-(4-methoxyphenyl)-1-propanol were 23.6,
25.7, 22.3 and 24.1 min, respectively (Figure S28, Supporting Information).
3.7. Biological Assays
Antifouling activity against the larval-attachment was determined using cyprids of the barnacle,
B. amphitrite Darwin, according to literature procedures [38]. Adults of B. amphitrite exposed to air
for more than 6 h were collected from the intertidal zone in Hong Kong and then were placed in a
container filled with 0.22 μm of filtered seawater (FSW) to release nauplii. The collected nauplii were
reared to the cyprid stage according to the method described by Thiyagarajan et al. [38]. When kept at
26–28 °C and fed with Chaetoceros gracilis, larvae developed to cyprids on the fourth day. Fresh
cyprids were used in the tests. Larval settlement assays were performed using 24-well polystyrene
plates (Becton Dickinson 353047 [39]).
Antibacterial activity was determined against six terrestrial pathogenic bacteria, including
Staphylococcus epidermidis, S. aureus, Escherichia coli, Bacillus subtilis, B. cereus and
Micrococcus luteus, and two marine pathogenic bacteria, Vibrio parahaemolyticus and
Listonella anguillarum, by the microplate assay method [40].

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4. Conclusions
Twelve secondary metabolites, including two phenylalanine derivatives (1,2) and ten cytochalasins
(3–12), have been isolated from the fermentation broth of a soft coral-derived fungus,
Aspergillus elegans ZJ-2008010. Compound 1 is a new phenylalanine derivative, and 3 and 4 are new
cytochalasin analogues. Their structures and the relative configurations were elucidated using
comprehensive spectroscopic methods. The absolute configuration of 1 was determined by chemical
synthesis and Marfey’s method. Asperphenamate (2) is the first report of isolated asperphenamate from
marine-derived fungus. Compound 10 belongs to an unusual type of aspochalasins with a pentacyclic
system, of which only three examples have been reported. Cytochalasins showed strong antifouling
activity against the larval settlement of the barnacle, B. amphitrite. This is the first report of antifouling
activity for this class of metabolites.
Acknowledgements
We appreciate Pei-Yuan Qian and his coworkers from Hong Kong University of Science and
Technology for evaluation of the antifouling activity. This work was supported by the National Natural
Science Foundation of China (No. 41130858; 41176121), the National High Technology Research and
Development Program of China (863 Program) (No. 2013AA093001), the Natural Science Foundation
of Shandong Province (ZR2011HM085), the Program for New Century Excellent Talents in
University, Ministry of Education of China (No. NCET-11-0472) and the Natural Science Foundation
of Hainan Province (213021).
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