Three new asperentin derivatives from the algicolous fungus aspergillus sp

Article abstract of DOI:10.3390/md12125993

Three new asperentin-type compounds, 6-O-α-D-ribosylasperentin (1) and 6-O-α- D-ribosyl-8-O-methylasperentin ( 2) and 5-hydroxyl-6-O-methylasperentin (3), along with asperentin (4) and its known analogues (5-9), were isolated from a halotolerant Aspergillus sp. strain F00785, an endotrophic fungus from marine alga. Their structures were determined using extensive NMR and HRESIMS spectroscopic analysis, including the X-ray crystallographic data for the assignment of the absolute configurations of compound 9. Compound 4 exhibited highly potent inhibitory activity against crop pathogens, Colletotrichum gleosporioides Penz. and Colletotrichum gleosporioides (Penz.) Sacc.

Full text of DOI:10.3390/md12125993

Mar. Drugs 2014, 12, 5993-6002; doi:10.3390/md12125993
OPEN ACCESS
marine drugs
ISSN 1660-3397
www.mdpi.com/journal/marinedrugs
Article
Three New Asperentin Derivatives from the Algicolous Fungus
Aspergillus sp. F00785
Qian Tang 1,2, Kai Guo 1,2, Xiao-Yang Li 1,2, Xiu-Ying Zheng 3, Xiang-Jian Kong 3,
Zhong-Hui Zheng 1,2, Qing-Yan Xu 1,2,* and Xianming Deng 1,2,
*
1
State Key Laboratory of Cellular Stress Biology, School of Life Sciences, Xiamen University,
Xiamen, Fujian 361102, China
2
3
State-Province Joint Engineering Laboratory of Targeted Drugs from Natural Products,
Xiamen University, Xiamen, Fujian 361102, China; E-Mails: 986713276@qq.com (Q.T.);
guokai1129@qq.com (K.G.); xiaoyang19901107@sina.com (X.-Y.L.); zhzheng@xmu.edu.cn (Z.-H.Z.)
State Key Laboratory for Physical Chemistry of Solid Surface, College of Chemistry and Chemical
Engineering, Xiamen University, Xiamen, Fujian 361005, China;
E-Mails: zhengxiuying37@163.com (X.-Y.Z.); xjkong@xmu.edu.cn (X.-J.K.)
* Authors to whom correspondence should be addressed; E-Mails: xuqingyan@xmu.edu.cn (Q.-Y.X.);
xmdeng@xmu.edu.cn (X.D.); Tel.: +86-592-2180328 (Q.-Y.X.); +86-592-2184180 (X.D.);
Fax: +86-592-2181722 (Q.-Y.X.); +86-592-2181722 (X.D.).
External Editor: Orazio Taglialatela-Scafati
Received: 21 October 2014; in revised form: 21 November 2014 / Accepted: 4 December 2014 /
Published: 15 December 2014
Abstract: Three new asperentin-type compounds, 6-O-α-D-ribosylasperentin (1) and
6-O-α-D-ribosyl-8-O-methylasperentin (2) and 5-hydroxyl-6-O-methylasperentin (3), along
with asperentin (4) and its known analogues (5–9), were isolated from a halotolerant
Aspergillus sp. strain F00785, an endotrophic fungus from marine alga. Their structures
were determined using extensive NMR and HRESIMS spectroscopic analysis, including
the X-ray crystallographic data for the assignment of the absolute configurations of
compound 9. Compound 4 exhibited highly potent inhibitory activity against crop pathogens,
Colletotrichum gleosporioides Penz. and Colletotrichum gleosporioides (Penz.) Sacc.
Keywords: asperentin; endophytic fungus; crop pathogen; α-D-ribofuranose

Mar. Drugs 2014, 12
5994
1. Introduction
Marine microorganisms inhabiting relatively uninvestigated and extreme environments have been
the focus of attractive sources for novel and bioactive secondary metabolites [1,2]. Marine natural
products with broad spectra of bioactivities such as anti-tumor, anti-microtubule, photoprotective,
antibiotic and anti-infective [3–5], are exceptionally interesting high value products for applications in
the pharmaceutical industry. At the moment there are eight Food and Drug Administration (FDA) or
European Medicines Agency (EMEA) approved drugs [6], for example trabectedin [7,8], an anti-tumor
agent, and brentuximab vedotin [9], an antibody-drug conjugate. Moreover, several compounds are in
different phases of the clinical trials [10,11]. Halotolerant microorganisms are microbes that are able to
grow well in varied saline conditions, such as marine, salt lake, salt field, etc. [12,13]. It is believed
that the high-salt environment might activate some silent genes and induce unique biosynthesis
pathway in these microbes [14,15]. In the course of our ongoing search for marine-originated
bioactive microbial metabolites, a halotolerant endogenic fungal Aspergillus sp. F00785 was isolated
from the marine alga collected in Jinjiang Saltern, Fujian province, China. A solvent partition
followed by repeated chromatographic purifications of the fermentation extracts afforded three new
asperentin derivatives, 6-O-α-D-ribosylasperentin (1), 6-O-α-D-ribosyl-8-O-methylasperentin (2) and
5-hydroxyl-6-O-methylasperentin (3), along with (−)-asperentin (4), and five asperentin derivatives,
5′-hydroxyasperentin
(5),
4′-hydroxyasperentin
(6),
asperentin-8-methyl
ether
(7),
5′-hydroxyasperentin-8-methyl ether (8) and 4′-hydroxyasperentin-6-methyl ether (9) (Figure 1). Here,
we report the isolation, structure determination and biological activity of asperentin and its derivatives
(1−9). The absolute configurations of stereocentres, C-2′, C-6′ and C-3 in 9, were determined for the
first time by X-ray crystallography. Compound 4 exhibited potent activity against Colletotrichum
gleosporioides Penz. (C. gleosporioides Penz.) and C. gleosporioides (Penz.) Sacc.
Figure 1. Structures of new asperentin analogs (1–3), (−)-asperentin (4) and its
derivatives (5–9).

Mar. Drugs 2014, 12
5995
2. Results and Discussion
2.1. Structure Elucidation
6-O-α-D-Ribosylasperentin (1) was isolated as pale yellow oil, which is soluble in acetone,
chloroform and methanol. The Rf value was 0.3 on TLC over silica gel developed with CHCl3/MeOH
(v/v, 10:1). The molecular formula of 1 was deduced as C21H28O9 based on the HRESIMS
(m/z 447.1632 [M + Na]+, calculated for C21H28O9Na, 447.1631). The IR absorptions at 3364 and 1667
1
13
cm−1 suggested the presence of hydroxyl and carbonyl groups. The H- and C-NMR spectra of 1 in
CDCl3 displayed signals for one methyl, six aliphatic methylenes, seven aliphatic methines, two
meta-coupled aromatic methines, four aromatic quaternary carbons, and one ester carbonyl carbon
(Table 1). The structure of fragment 1a was established on the basis of HMBC correlations from H-4
to C-1′, C-3, C-8a, C-4a and C-5, H-7 to C-6, C-8 and C-8a, H-3′ to C-4′, and H-4′ to C-5′; and 1H-1H
13
COSY correlations (H-1′/H-2′, H-2′/H-3′, H3-6′/H-6′, and H-6′/H-5′) (Figure 2). Its 1H and C NMR
data are compatible with those reported for asperentin [16]. Fragment 1b was further determined on
1
the basis of HMBC correlations from H-3″ to C-1″, H-1″ to C-3″ and C-4″, and the H-1HCOSY
correlations from H-5″ to H-4″, H-2″ to H-1″. Finally, fragments 1a and 1b were linked together via a
glycosidic bond indicated by the H-1″−C-6 three-bond correlation in the HMBC spectrum.
20
Acid hydrolysis of 1 afforded a pentofuranose and (−)-asperentin (4) ([α] = −23°, c = 0.83, EtOH) [17].
D
The latter was also known as (−)-cladosporin [18], its absolute configuration of (R)-3,4-dihydro-3-
(((2′R,6′S)tetrahydro-6′-methyl-2H-pyran-2-yl)-methyl)-6,8-dihydroxy-isochromen-1-one was confirmed
by comparing its NMR and physiochemical data with literature data [17,18]. The pentofuranose was
determined to be ribose on the basis of 1H- and 13C-NMR data [19]. The sugar moiety was determined
20
as D-ribose by comparison of the optical rotation of its tetra-acetate derivative ([α] = −17°, c = 0.68,
D
MeOH) with the reported data [20,21]. Additionally, the stereochemistry of the anomeric carbon of the
D-ribofuranose moiety was determined as α-configuration on the basis of the chemical shift and
coupling constant of C-1″ (δH 5.69 (d, J = 3.5 Hz), δC 100.1) that is consistent with the reported value [21].
The two hydrolysates of 1 further validated the structures of fragments 1a and 1b. With all the
obtained data, the structure of 6-O-α-D-ribosylasperentin (1) was deduced as (R)-3,4-dihydro-3-(((2′R,
6′S) tetrahydro-6′-methyl-2H-pyran-2′-yl)-methyl)-6-O-(α-D-ribofuranosyl)-8-hydroxyl-isochromen-1-one,
an asperentin-6-O-riboside.
6-O-α-D-Ribosyl-8-O-methylasperentin (2) was isolated as pale yellow oil, which is soluble in
acetone, chloroform and methanol. The Rf value was 0.35 eluting with the mixed solvent of
CHCl3/MeOH (v/v, 10:1). The molecular formula of 2 was deduced as C22H30O9 based on the
HRESIMS (m/z 439.1975 [M + H]+, calculated for C22H31O9, 439.1968). Analysis of the IR spectrum
indicated the presence of hydroxyl and carbonyl functionalities with IR absorption at 3445 and
1700 cm−1, respectively.

Mar. Drugs 2014, 12
5996
Table 1. 1H and 13CNMR data for 1–4 in CDCl3.
No.
1
2
3
4
b
b
b
b
δH a (J in Hz)
δC
δH a (J in Hz)
δC
δH a (J in Hz)
δC
δH a (J in Hz)
δC
1
3
4
169.6, C
76.5, CH
33.6, CH2
161.6, C
74.6, CH
35.4, CH2
170.0, C
76.3, CH
27.3, CH2
169.9, C
76.4, CH
4.72, m
2.87, m
4.60, m
4.63, m
4.72, m
2.86, m
2.92, dd, (16.2, 11.5)
2.83, dd, (16.2, 2.9)
3.10, dd, (16.8, 3.4)
2.62, dd, (16.8, 11.5)
33.7, CH2
4a
141.5, C
144.0, C
122.6, C
134.3, C
141.8, C
5
6.42, s
6.56, s
107.6, CH
6.56, d, (2.0)
106.8, CH
6.32, s
6.19, s
106.7, CH
OH-5
5.20, s
6
162.5, C
162.6, C
153.1, C
56.2, CH3
97.8, CH
157.8, C
162.9, C
OCH3-6
3.86, s
6.34, s
7
102.9, CH
163.8, C
6.61, d, (2.0)
3.94, s
100.1, CH
162.9, C
102.0, CH
164.3, C
8
OCH3-8
8a
56.3, CH3
106.8, C
103.2, C
39.2, CH2
66.4, CH
30.8, CH2
18.2, CH2
31.3, CH2
67.6, CH
19.1, CH3
100.1, CH
72.1, CH
70.1, CH
86.4, CH
62.3, CH2
100.7, C
39.5, CH2
66.3, CH
30.8, CH2
18.3, CH2
31.1, CH2
67.4, CH
19.2, CH3
101.7, C
39.4, CH2
66.5, CH
30.9, CH2
18.2, CH2
31.0, CH2
67.9, CH
18.9, CH3
1′
1.98, m; 1.82, m
4.10, m
1.92, m; 1.83, m
4.10, m
39.5, CH2
66.2, CH
30.9, CH2
18.3, CH2
31.0, CH2
67.7, CH
18.9, CH3
100.5, CH
72.2, CH
70.4, CH
86.5, CH
62.5, CH2
1.95, m; 1.78, m
4.05, m
1.99, m; 1.87, m
4.13, m
2′
3′
1.69, m; 1.35, m
1.70, m; 1.62, m
1.68, m; 1.32, m
3.94, m
1.36, m
1.64, m; 1.29, m
1.65, m; 1.56, m
1.63, m; 1.26, m
3.89, m
1.73, m; 1.38, m
1.74, m; 1.65, m
1.72, m; 1.36, m
4.02, m
4′
1.73, m; 1.64, m
1.72, m
5′
6′
3.96, m
CH3-6′
1″
1.21, d, (4.1)
5.69, d, (3.5)
4.29, brs
1.22, d, (6.5)
1.14, d, (6.5)
1.25, d, (6.5)
5.77, d, (4.5)
2″
4.34, m
3″
4.19, brs
4.24, dd, (6.2, 2.7)
4.28, dd, (6.2, 2.7)
3.89, dd, (12.1, 3.1); 3.81, dd, (12.1, 3.1)
4″
4.21, brs
5″
3.78, d, (11.6); 3.74, d, (11.6)
11.13, s
8-OH
10.86, s
11.10, s
a 600 MHz; b 150 MHz.

Mar. Drugs 2014, 12
5997
Figure 2. The structures of fragments 1a and 1b of 6-O-α-D-ribosylasperentin (1) and
selected HMBCs (H→C) and 1H-1HCOSY correlations (bold line).
The structure of 2 was determined as 8-methoxyl analogue of 1 on the basis of the similar NMR
data of both compounds with the exception of the absence of a hydroxyl group and the presence of a
methoxyl at C-8 (δH-OMe 3.94, δc-OMe56.3) (Table 1). That the methoxyl substituent on C-8 was further
confirmed by HMBC correlation from OCH3 (δH 3.94) to C-8 (δC-8 162.9). Thus, 2 was
8-methoxyasperentin-6-O-riboside (Figure 1).
5-Hydroxyl-6-O-methylasperentin (3), another structurally similar metabolite, was isolated as white
needle, which is soluble in acetone, chloroform and methanol. The molecular formula of 3 was
deduced as C17H22O6 based on the HRESIMS (m/z 345.1308 [M + Na]+, calculated for C17H22O6Na,
345.1314). The IR absorptions at 3319 and 1657 cm−1 suggested the presence of hydroxyl and
carbonyl groups. The NMR spectra were closely related to those of fragment 1a, except that the signals
(δH-5 6.42, δC-5 107.6) of 1a was replaced with an aromatic oxygenated quaternary carbon (δc 134.3)
which indicated a hydroxyl-substitution at C-5 (Table 1). Additionally, HMBC correlations from
phenol hydrogen (δH5.20) at C-5 to C-4a (δC-4a 122.6), C-5 (δC-5 134.3) and C-6 (δC-6 153.1), and from
OCH3 (δH 3.86) to C-6 (δC-6 153.1) further confirmed that 3 was 5-hydroxyasperentin-6-methyl ether.
Compounds 4−9 were isolated along with 6-O-α-D-ribosylasperentin (1), 6-O-α-D-ribosyl-8-O-
methylasperentin (2) and 5-hydroxyl-6-O-methylasperentin (3). By analyses and comparisons of their
NMR spectra, MS data and specific rotations with those reported in the literatures [17,18,22],
compounds 4−9 were identified as (−)-asperentin (4) [17,18], 5′(S)-hydroxy-asperentin (5) [22],
4′(R)-hydroxyasperentin (6) [22], asperentin-8-methyl ether (7) [22], 5′(S)-hydroxy-asperentin-8-methyl
ether (8) [22] and 4′(R)-hydroxy-asperentin-6-methyl ether (9) [22], respectively. The absolute
configuration of stereocentres, C-2′, C-6′ and C-3 in 9, was determined as (R)-8-hydroxy-3-
(((2′R,4′R,6′S)-4-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)methyl)-6-methoxy-isochroman-1-one by the
results of X-ray single-crystal diffraction using Flack parameters (Figure 3 and please see
Supplementary Information). An intramolecular hydrogen bond was observed, which is consistent with
the NMR data of δ8-OH (11.20), downfield shift compared with the free phenol hydrogen.
2.2. Bioactivity Results
The antifungal activity of compounds 1–9 against three crop pathogens, C. gleosporioides Penz,
C. gleosporioides (Penz) Sacc. and Botrytis cinerea Pers, were evaluated by filter-paper disk method
using amphotericin B as positive control. The results showed that only (−)-asperentin (4) exhibited
strong inhibitory activity and no activity were observed for the other compounds. At a concentration of

Mar. Drugs 2014, 12
5998
5 mg/mL, the inhibition zone of 4 to C. gleosporioides Penz. was 19.7 ± 0.58 mm, while that of
amphotericin B was 15.7 ± 1.25 mm (Table 2).
Figure 3. X-ray crystal structure of 9.
Table 2. Antimicrobial activity of (−) asperentin (4).
Inhibition Zone (mm)
Compounds
C. gleosporioides Penz C. gleosporioides (Penz) Sacc. B. cinerea Pers
(−)-asperentin (4)
19.7 ± 0.58
15.7 ± 1.25
13.3 ± 3.40
16.0 ±1.41
1.67 ± 1.11
11.0 ± 0.82
amphotericin B
3. Experimental Section
3.1. General Experimental Procedures
Optical rotations were measured using a Perkin-Elmer 341 polarimeter (PerkinElmer Inc., Waltham,
MA, USA). UV spectra were recorded on Jasco V-530 spectrophotometer (JASCO International Co.,
Tokyo, Japan). IR spectra were obtained on Perkin-Elmer 552 spectrophotometer. NMR spectra were
recorded on a Bruker Avance-600 spectrometer (600 MHz) (Bruker Co., Bremen, Germany) using
TMS as the internal standard. ESI-MS was measured on a Thermo-Finnigan LCQ Advantage mass
spectrometer (Thermo Fisher Scientific Inc, San Jose, CA, USA). HR-ESI-MS was obtained on a
Bruker LC-QTOF mass spectrometer. Semi-preparative high pressure liquid chromatography (HPLC)
was performed on Agilent 1200 using XDB C18 column (10 × 250 mm, 5 μm, flow = 2 mL/min)
(Agilent Technologies Inc., Santa Clara, CA, USA). TLC detection was carried out using precoated
silica gel GF254 plates (10–40 μm, Qingdao Marine Chemical Plant, Qingdao, China). Column
chromatography was performed with silica gel (200–300 mesh, Qingdao Marine Chemical Plant,
Qingdao, China), reverse phase RP-18 (40–63 μm, Merck, Darmstadt, Germany), and Sephadex
LH-20 (Amersham Biosciences, Sweden). All solvents were of analytical grade.
3.2. Fungi Materials
The marine-derived endophytic fungus Aspergillums sp. strain F00785 was identified by
morphological characteristics. It was isolated from marine alga, Enteromorpha prolifera, collected in

Mar. Drugs 2014, 12
5999
Jinjiang Saltern, Fujian province, China, in 2006, and preserved in liquid paraffin at 4 °C until its use
for fermentation.
3.3. Extraction and Purification
The marine-derived fungal strain F00785 was statically cultured on potato-dextrose-agar (PDA)
medium at 28 °C for 18 days. The fermented material was diced and extracted with the mixed solvent
of EtOAc/MeOH/AcOH (v/v/v, 80/15/5) (3 × 5 L). The organic solution was combined and
concentrated in vacuum at 40 °C to yield crude syrup (49.2 g). The crude syrup was suspended in
EtOAc and washed with H2O, then the EtOAc layer was concentrated and resuspended in MeOH and
petroleum ether. The MeOH layer was concentrated to give the crude extract.
The crude extract (12.87 g) was fractionated by RP-18 column (160 g) using MeOH–H2O gradient
(30%, 40%, 50%, and 100%) as the eluent to yield four fractions (Fraction I-IV). Fraction I (840.0 mg)
was sequentially subjected to a Sephadex LH-20 (160 g) using MeOH as the mobile phase and a
Sephadex LH-20 (160 g) using acetone as the eluent to yield two portions, part A (32.5 mg) and part B
(78.9 mg). Part A was subjected to preparative TLC to yield 6 (4.0 mg). Part B was purified on silica
column chromatography using PE (petroleum ether)/EtOAc (v/v, 3/1) as the eluent to obtain 8
(3.5 mg). Fraction II (339.0 mg) was subjected to Sephadex LH-20 (160 g) eluting with CH2Cl2/MeOH
(v/v, 1/2) and then silica gel column eluting with PE/EtOAc (v/v, 1/2) to afford 2 (7.0 mg). Fraction IV
(1.56 g) was sequentially subjected to a RP-18 column (80 g) eluting with a MeOH–H2O gradient
(30%, 40%, 50%, and 100%), Sephadex LH-20 (160 g) eluting with CH2Cl2/MeOH (v/v, 1/2),
Sephadex LH-20 (80 g) eluting with acetone, and preparative TLC to afford 1 (28.9 mg), 4 (10.0 mg),
5 (8.0 mg),7 (38.0 mg), and 9 (4.5 mg). Compound 3 (2 mg) was obtained by further purification using
semi-preparative HPLC (XDB C18 column, 10 × 250 mm, 5 μm, flow = 2 mL/min). The Rf values of
compounds 4–9 on TLC over silica gel developed with CHCl3/MeOH (v/v, 10:1) are 0.85, 0.40, 0.40,
0.65, 0.35, and 0.35, respectively.
20
6-O-α-D-Ribosylasperentin (1). Pale yellow oil; [α] = +122 (c = 0.7, MeOH), UV (MeOH) λmax
D
1
13
265.9 and 302.0 nm; IR (KBr) νmax 3364 and 1667 cm−1; H and C NMR, see Table 1; HR-ESI-MS
m/z 447.1632 [M + Na]+ (calcd for C21H28O9Na, 447.1631).
20
6-O-α-D-Ribosyl-8-O-methylasperentin (2). Pale yellow oil; [α] = +96 (c = 0.44, MeOH), UV
D
(MeOH) λmax 260.0 and 302.0 nm; IR (KBr) νmax 3445 and 1700 cm−1; 1H and 13C NMR, see Table 1;
HR-ESI-MS m/z 439.1975 [M + H]+ (calcd for C22H31O9, 439.1968).
20
5-Hydroxyl-6-O-methylasperentin (3). White needle; [α] = −12 (c = 0.18, MeOH), m.p. 125.7 °C,
D
UV (MeOH) λmax 268.9 and 333.0 nm; IR (KBr) νmax 3319 and 1657 cm−1; 1H and 13C NMR, see Table 1;
HR-ESI-MS m/z 345.1308 [M + Na]+, (calcd for C17H22O6Na, 345.1314).
3.4. Acid Hydrolysis and Stereochemistry Determination of the Ribofuranose of 6-O-α-D-Ribosyl
Asperentin (1)
A solution of 1 (90.0 mg) in 3 N HCl (60 mL) was stirred at 80 °C for 1 h. After the hydrolysis
reaction was completed, the reaction mixture was concentrated under vacuum. The resulting residue
was suspended in H2O (40 mL) and extracted with CH2Cl2 (3 × 40 mL). Then the aqueous layer was

Mar. Drugs 2014, 12
6000
concentrated and dried under vacuum. The resulting residue was acetylated with Ac2O/Py at room
temperature to yield an acetate product that was purified by Sephadex LH-20 (80 g) eluting with
acetone/MeOH (v/v, 4/1) to afford a tetra-acetate of ribofuranose (4.1 mg). The specific rotation of this
20
tetra-acetate product, [α] = −17 (c = 0.68, MeOH), is consistent with the reported value of D-ribose [20].
D
1H-NMR (600 MHz, CDCl3) δ 6.06 (1H, d, J = 4.9 Hz, H-1), 5.06 (1H, d, J = 3.6 Hz, H-2), 5.51 (1H,
brs, H-3), 5.18 (1H, d, J = 3.2 Hz, H-4), 3.94 (1H, dd, J = 12.5, 5.9 Hz, Ha-5), 4.05 (1H, dd,
J = 12.5, 3.0 Hz, Hb-5), 2.16 (3H, s, Ac), 2.15 (3H, s, Ac), 2.13 (3H, s, Ac), 2.12 (3H, s, Ac).
13C-NMR (150 MHz, CDCl3) δ 90.9 (C-1), 67.3 (C-2), 66.2 (C-3), 66.3 (C-4), 62.7 (C-5), 169.9
(CH3CO), 169.8 (CH3CO), 169.5 (CH3CO), 168.8 (CH3CO), 20.9 (CH3CO), 20.8 (CH3CO), 20.7
(CH3CO), 20.7 (CH3CO).
3.5. Anti-Crop Pathogens Test
Anti-crop pathogen activity against C. gleosporioides Penz, C. gleosporioides (Penz) Sacc., and
B. cinerea Pers was evaluated using a filter-paper disk method. The tested strains were cultivated on
PDA plates at 28 °C. Compounds 1–9 were tested at a concentration of 5 mg/mL in MeOH using
amphotericin B (5 mg/mL in DMSO) as the positive control. The tested compound solutions (5 μL)
were transferred onto filtering paper disks (Φ = 5 mm) placed in the center of assay plates. After 48 h
incubation at 28 °C, the diameters of inhibition zones were measured to evaluate the activity, a bigger
size of the diameter indicated a stronger inhibition. All experiments were repeated in triplicate.
4. Conclusion
Three new compounds, 6-O-α-D-ribosylasperentin (1), 6-O-α-D-ribosyl-8-O-methylasperentin (2)
and 5-hydroxyl-6-O-methylasperentin (3), along with six known asperentin derivatives (4–9), isolated
and identified from the fermentation products of a halotolerant fungus Aspergillums sp. strain F00785
from the inner tissue of the marine alga. 1 and 2, were found to be the first members with
D-ribofuranose via α-glycosidic linkage. To the best of our knowledge, the α-D-ribofuranosyl moiety is
unusual in metabolites from fungi, with only four natural products containing α-D-ribofuranosyl
moiety reported up to now [19,21,23,24]; their functions in microorganisms need to be further
investigated. Among the nine compounds, compound 4 exhibited strong inhibitory activity against
three crop pathogens. Preliminary study of the structure activity relationships (SARs) of these nine
compounds indicated that free hydroxyls at C-6 and C-8 are critical to the antifungal activity, while the
hydroxyl substitution at the 4′ or 5′ position of the pyranoid ring is not favorable. In summary,
discovery of these new secondary metabolites suggest that chemical investigations of marine
microorganisms in relatively uninvestigated environments may provide significant natural chemical
diversity for drug discovery.
Acknowledgments
This study was supported by grants from the National Natural Science Foundation of China
(81422045 and U1405223), the Science and Technology Plan Project of Xiamen (3502Z20133010),
and the Fundamental Research Funds for the Central Universities of China (2011121030, and

Mar. Drugs 2014, 12
6001
2013121032) to X. Deng, and the Science and Technology Plan Project of Xiamen (3502Z20123010),
the Fundamental Research Funds for the Central Universities of China (2010121092) to Q.-Y. Xu. We
thank ChemAxon for giving academic license to access free software products.
Author Contributions
Q. Tang carried out most of the experiments and wrote the manuscript. X.-Y. Li performed the
bioassay. K.Guo contributed to the structure determination. Z.-H. Zheng identified the fungus. X.-Y.
Zheng and X.-J. Kong contributed to the X-ray crystal structure study. Q.-Y. Xu and X. Deng
conceived and designed the experiments and wrote the manuscript. All authors read and revised
the manuscript.
Conflicts of Interest
The authors declare no conflict of interest.
References
1. Pettit, R.K. Culturability and secondary metabolite diversity of extreme microbes: Expanding
contribution of deep sea and deep-sea vent microbes to natural product discovery. Mar.
Biotechnol. 2011, 13, 1–11.
2. Fenical, W. Chemical studies of marine bacteria: Developing a new resource. Chem. Rev. 1993,
93, 1673–1683.
3. Schumacher, M.; Kelkel, M.; Dicato, M.; Diederich, M. Gold from the sea: marine compounds as
inhibitors of the hallmarks of cancer. Biotechnol. Adv. 2011, 29, 531–547.
4. Molinski, T.F.; Dalisay, D.S.; Lievens, S.L.; Saludes, J.P. Drug development from marine natural
products. Nat. Rev. Drug Discov. 2009, 8, 69–85.
5. Berdy, J. Bioactive microbial metabolites. J. Antibiot. 2005, 58, 1–26.
6. Martins, A.; Vieira, H.; Gaspar, H.; Santos, S. Marketed marine natural products in the
pharmaceutical and cosmeceutical industries: Tips for success. Mar. Drugs 2014, 12, 1066–1101.
7. D’Incalci, M.; Galmarini, C.M. A review of trabectedin (ET-743): A unique mechanism of action.
Mol. Cancer Ther. 2010, 9, 2157–2163.
8. Monk, B.J.; Dalton, H.; Benjamin, I.; Tanovic, A. Trabectedin as a new chemotherapy option in
the treatment of relapsed platinum sensitive ovarian cancer. Curr. Pharm. Des. 2012, 18, 3754–3769.
9. Bauer, A.; Bronstrup, M. Industrial natural product chemistry for drug discovery and
development. Nat. Prod. Rep. 2014, 31, 35–60.
10. Mayer, A.M.; Glaser, K.B.; Cuevas, C.; Jacobs, R.S.; Kem, W.; Little, R.D.; McIntosh, J.M.;
Newman, D.J.; Potts, B.C.; Shuster, D.E. The odyssey of marine pharmaceuticals: A current
pipeline perspective. Trends Pharmacol. Sci. 2010, 31, 255–265.
11. Mayer, A.M. Marine Pharmaceuticals: The Clinical Pipeline. Available online:
http://marinepharmacology.midwestern.edu/clinPipeline.htm (accessed on 16 October 2014).
12. Aguilar, A.; Ingemansson, T.; Magnien, E. Extremophile microorganisms as cell factories:
Support from the European Union. Extremophiles 1998, 2, 367–373.

Mar. Drugs 2014, 12
6002
13. Kamekura, M. Diversity of extremely halophilic bacteria. Extremophiles 1998, 2, 289–295.
14. Koch, A.L. Genetic Response of Microbes to Extreme Challenges. J. Theor. Biol. 1993, 160, 1–21.
15. Mejanelle, L.; Lopez, J.F.; Gunde-Cimerman, N.; Grimalt, J.O. Ergosterol biosynthesis in novel
melanized fungi from hypersaline environments. J. Lipid Res. 2001, 42, 352–358.
16. Springer, J.P.; Cutler, H.G.; Crumley, F.G.; Cox, R.H.; Davis, E.E.; Thean, J.E. Plant-growth
regulatory effects and stereochemistry of cladosporin. J. Agric. Food Chem. 1981, 29, 853–855.
17. Grove, J.F. New metabolic products of Aspergillus flavus. I. Asperentin, its methyl ethers, and
5′-hydroxyasperentin. J. Chem. Soc. Perkin Trans. 1 1972, 19, 2400–2406.
18. Scott, P.M.; Van Walbeek, W.; MacLean, W.M. Cladosporin, a new antifungal metabolite from
Cladosporium cladosporioides. J. Antibiot. 1971, 24, 747–755.
19. Li, Y.; Li, X.; Lee, U.; Kang, J.S.; Choi, H.D.; Son, B.W. A new radical scavenging anthracene
glycoside, asperflavinribofuranoside, and polyketides from a marine isolate of the fungus
Microsporum. Chem. Pharm. Bull. 2006, 54, 882–883.
20. Zinner, H. Die Acetate der D-Ribose. Chem. Ber. 1953, 86, 817–824.
21. Pretsch, E.B.P.; Affolter, C. Structure Determination of Organic Compounds, Tables of Spectral
Data, 3rd ed.; Springer-Verlag Berlin: Heidelberg, Germany, 2000; p. 152.
22. Grove, J.F. New metabolic products of Aspergillus flavus. IV. 4′-Hydroxyasperentin and
5′-hydroxyasperentin 8-methyl ether. J. Chem. Soc. Perkin. 1 1973, 22, 2704–2706.
23. Liu, R.; Zhou, Z.Y.; Jiang, M.Y.; Wang, F.; Liu, J.K. A new isoprenyl phenyl ether riboside from
the culture of basidiomycete Laccaria amethystea. J. Asian Nat. Prod. Res. 2010, 12, 723–726.
24. Li, D.L.; Li, X.M.; Wang, B.G. Natural anthraquinone derivatives from a marine mangrove
plant-derived endophytic fungus Eurotium rubrum: Structural elucidation and DPPH radical
scavenging activity. J. Microbiol. Biotechnol. 2009, 19, 675–680.
© 2014 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/4.0/).