Cladosins A-E, hybrid polyketides from a deep-sea-derived fungus, Cladosporium sphaerospermum

Article abstract of DOI:10.1021/np400833x

Five new fungal hybrid polyketides, cladosins A-D (1-4), that contain a novel linear 6(3)-enamino-8,10-dihydroxy-tetraketide (1 and 2) or 6-enamino-7(8)-en-10-ol (3 and 4) moiety, as well as the biogenetically related cladosin E (5), were isolated from the deep-sea-derived fungus Cladosporium sphaerospermum 2005-01-E3. Their structures (1-5) were elucidated through a combination of spectroscopic data, chemical conversion, and both Mosher's and Marfey's methods for stereochemical assignment. A plausible biogenetic pathway to 1-5 is proposed. Cladosin C (3) possesses mild anti-influenza A H1N1 virus activity.

Full text of DOI:10.1021/np400833x

Article
pubs.acs.org/jnp
Cladosins A−E, Hybrid Polyketides from a Deep-Sea-Derived Fungus,
Cladosporium sphaerospermum
Guangwei Wu, Xinhua Sun, Guihong Yu, Wei Wang, Tianjiao Zhu, Qianqun Gu, and Dehai Li*
Key Laboratory of Marine Drugs, Chinese Ministry of Education, School of Medicine and Pharmacy, Ocean University of China,
Qingdao 266003, People’s Republic of China
S
* Supporting Information
ABSTRACT: Five new fungal hybrid polyketides, cladosins A−D (1−4),
that contain a novel linear 6(3)-enamino-8,10-dihydroxy-tetraketide (1 and
2) or 6-enamino-7(8)-en-10-ol (3 and 4) moiety, as well as the
biogenetically related cladosin E (5), were isolated from the deep-sea-
derived fungus Cladosporium sphaerospermum 2005-01-E3. Their structures
(1−5) were elucidated through a combination of spectroscopic data,
chemical conversion, and both Mosher’s and Marfey’s methods for
stereochemical assignment. A plausible biogenetic pathway to 1−5 is
proposed. Cladosin C (3) possesses mild anti-influenza A H1N1 virus
activity.
atural products containing the 2,4-pyrrolidinedione
(tetramic acid) core are well known as hybrids generally
N
constructed by fusion of amino acid and polyketide units.1 This
family of structures has attracted particular attention because of
the structurally diverse scaffolds,2 such as pyrrospirones A and
B,3 diaporthichalasin,3 cylindramide,4 cyclopiazonic acid,5 and
the penicillenols,6 as well as a range of biological effects
including antibiotic, antiviral, antitumor, anti-HIV, herbicidal,
and RNA polymerase inhibitory activities.7 This has led to
several studies of their total synthesis and biogenesis in recent
years.1,3,8 Although new members of this large family are being
discovered, there has been no report of congeners with a
polyketide component containing 1,3-diol, polyol, or enamino
moieties to date.
As part of our ongoing work exploring the bioactive
secondary metabolites from deep-sea-derived fungi,9 the strain
Cladosporium sphaerospermum 2005-01-E3, isolated from sedi-
ments collected in the Pacific Ocean, was selected for further
development due to its interesting HPLC-UV profiles and
bioactivity. Chemical investigation of this fungal extract finally
led to the isolation of four novel tetramic acid derivatives
containing either 6(3)-enamino-8,10-dihydroxy or 6(3)-enam-
ino-7(8)-en-10-ol side chains, named cladosins A−D (1−4).
Each compound exists as two tautomeric forms differing in
configuration of the enamine. We also isolated a new
biosynthetically related compound, cladosin E (5), from the
same culture. Details of the structure elucidation, bioactivity
screening, and a hypothetical biogenesis of 1−5 are reported
herein.
the HRESIMS adduct ion detected at m/z 305.1475 [M +
Na]+, the mixture (1a/1b) had the same molecular formula of
C14H22N2O4, requiring five degrees of unsaturation. The major
1D NMR resonances (1a) were categorized into four sp3
methyls (including a methoxy), two sp3 methylenes, two sp3
RESULTS AND DISCUSSION
■
Cladosin A (1) was isolated as an inseparable mixture of two
geometric isomers (1a:1b) present in a ratio of 5:3. Based on
Received: October 7, 2013
Published: February 5, 2014
© 2014 American Chemical Society and
American Society of Pharmacognosy
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Figure 1. Key 2D NMR correlations of 1−6.
Table 1. 13C NMR (150 MHz) and 15N (90 MHz) Data of 1−4 in DMSO-d6 (δ ppm)
1
2
3
4
no.
a
b
a
b
a
b
a
b
2
3
4
5
6
7
8
9
168.6
97.2
171.4
96.0
169.0
97.3
171.5
96.0
168.3
96.9
169.7
95.6
166.5
103.8
183.8
129.8
166.9
120.4
144.1
31.0
168.0
103.7
186.4
130.6
165.0
120.4
144.7
31.0
186.5
130.3
169.6
36.4
183.5
131.0
169.6
36.7
186.4
130.3
170.0
39.5
183.7
131.0
170.2
39.8
187.3
130.3
161.2
123.6
142.1
43.4
183.8
130.9
161.5
123.3
142.1
43.4
a
a
a
a
a
a
77.8
77.4
68.2
68.0
a
a
a
a
a
a
43.6
43.5
47.0
46.8
a
a
10
11
12
13
14
15
NH
63.7
63.7
64.7
64.8
66.1
66.1
74.0
74.0
23.9
24.0
23.9
24.0
23.8
23.8
20.7
20.7
118.4
21.3
118.6
21.4
118.4
21.3
118.5
21.4
118.5
21.3
118.7
21.4
119.0
21.4
119.4
21.5
a
a
a
a
a
a
18.4
18.4
18.4
18.4
18.5
18.4
18.5
18.8
56.7
56.7
b
c
c
c
c
c
c
c
c
c
c
c
c
254.2
257.5
254.9
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
b
NH2
258.3
N.D.
a
b
c
1
Tautomers exhibited the same shifts for these carbons. Chemical shifts were recorded in the H−15N HSQC spectra. N.D.: not detected.
oxygenated methines, an amide carbonyl, an α/β-unsaturated
ketone carbon, and two tetrasubstituted double bonds,
indicating one additional ring existed in 1a to match the
degrees of unsaturation. The gross structure of 1a was
established by comprehensive 2D NMR analysis (Figure 1).
The sequential COSY correlations of H2-7/H-8/H2-9/H-
10(10-OH)/H3-11 unambiguously established the spin system
from C-7 to C-11, with the methyl ether (δC 56.7) and hydroxy
groups attached to C-8 and C-10, respectively, deduced by the
HMBC correlations between H3-15 (δH 3.24) and C-8 (δC
77.8), and the exchangeable proton (δH 4.51) with C-10. This
spin system was extended to include an enamine double bond
(δC 97.2, C-3; 169.6, C-6) by the corresponding HMBC
correlations from H-7 to C-3, with the amino group attached to
C-6 supported by the long-range 1H−15N HMBC from H-7 to
N-6 (Figure 1). The HMBC correlations from H3-13 and H3-14
to C-5 (δC 130.3) and C-12 enabled identification of an
isobutenyl moiety. Finally, a highly substituted 2,4-pyrrolidine-
dione (tetramic acid) scaffold10 was established based on the
HMBC correlations from the exchangeable amide proton NH-1
(δH 9.13) to alkene carbon C-3, α/β-unsaturated ketone carbon
C-4 (δC 186.5), isobutenyl carbon C-5, and the remaining
upfield carbonyl C-2 (δC 168.6). To confirm the 2,4-
pyrrolidinedione core, compound 1 was hydrogenated to
form the reduced diastereomers 6a/6b, which displayed the
relevant COSY correlation between NH-1/H-5 and the HMBC
correlation from the additional H-5 to C-3 (Figure 1 and Table
S1, Supporting Information). Thus, the planar structure of 1a
was assigned as a tetramic acid derivative with the new 6(3)-
enamino-8,10-dihydroxy system.
Comparison of the NMR data of 1a and 1b indicated that
they had a similar planar core structure. The only significant
differences between 1a and 1b were the chemical shifts of
carbonyls C-2 and C-4 (Table 1), indicating 1 likely existed as a
pair of interconverting geometric isomers in solution.11 The
only reported example of an enaminotetramic acid core is
cyclopiazonic acid imine, which existed as a pair of tautomers
and was explained probably by the interconversion of keto-
enamine and enol-imine forms.12 However, the cladosin A
isomers 1a/1b were only found in the keto-enamine form, in
obvious difference from cyclopiazonic acid imine. The isomeric
behavior of 1 was readily explained through conversion via
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tautomerism of the 3-acyltetramic acids,11 with 1a (major) and
1b (minor) assigned separately as the exo-form A (Δ3(6): E)
and exo-form B (Δ3(6): Z). The forms of 1a and 1b were
assigned as exo-form A and exo-form B, respectively, based on
similar NMR evidence between 1 and 3-acyltetramic acids,
where the hydrogen-bonded carbonyl is shifted downfield when
compared to that of the corresponding free carbonyl.11,13
The molecular formula of tautomeric cladosin B (2) was
established as C13H20N2O4 from an adduct ion [M + Na]+ at
m/z 291.1316. The UV spectrum of 2 was almost identical to
that of 1, indicating a 6(3)-enaminotetramic acid system. The
1H and 13C NMR data of 2 showed similar resonances to those
of 1, except that the methyl ether of 1 was replaced by an
alcohol (δH 4.96) in 2. Similar to 1, 2 also existed as exo-A
(major) and exo-B (minor) geometric isomers in the same ratio
of 5:3. The relative configuration of the 1,3-diol unit in 2 was
determined from the 13C NMR analysis after derivatization to
acetonide 7 (Figure 2). The signals of the methyl carbons of
for the geminal protons H2-9 between the two compounds
(Table 2). The 10S configuration in 1 was assigned by the
modified Mosher’s method (Figure 3). Accordingly, the
absolute configuration of 1 was determined to be 8R, 10S, in
complete agreement with congener 2.
To assess whether 1 was an artifact of isolation, compound 2
was dissolved in MeOH and then stirred at room temperature
for a week. Monitoring by HPLC indicated no conversion of 1
to 2. It is also notable that when 2 was treated under harsher
conditions, such as elevated temperature (40 °C), the
compound degraded quickly.
Cladosins C and D (3 and 4) were assigned the same
molecular formula C13H18N2O3 from their adduct ions detected
under HRESIMS conditions, being 18 mass units less than that
of 2. Their UV absorption spectra suggested an extended 6(3)-
enaminotetramic acid chromophore. Analysis of the NMR data
of 3 revealed that the side chain differed from the 1,3-diol units
in 1 and 2, with COSY correlations between H-7/H-8/H2-9/H-
10/H3-11 establishing a homoallylic alcohol subunit, which was
further supported by relevant HMBC cross-peaks as shown in
Figure 1. The Δ7 moiety was unambiguously connected to C-6
based on the correlations from both H-7 and H-8 to enamine
C-6. The E geometry of Δ7 was deduced from the large vicinal
3
coupling constant JH‑7,H‑8 = 16.5 Hz. Moreover, the 10S-
configuration was assigned by the modified Mosher’s method.
1
The H and 13C NMR spectra of 3 and 4 were almost
Figure 2. NMR data of acetonide 7 and the consequential syn-1,3-diol
form.
identical, and analysis of 2D NMR spectra of 4 established the
same planar structure as 3. The only difference between 3 and 4
was the Z geometry of Δ7 deduced from the smaller vicinal
coupling constant (3JH‑7,H‑8 = 9.9 Hz) in 4. Due to lack of
material and slow decomposition under storage, the absolute
configuration at C-10 in 4 could not be established. However,
considering their common biogenetic origin, compound 4 is a
Z/E geometric isomer of compound 3 and is likely to have the
10S configuration. Unlike compounds 1−3, exo-form B of 4
dominated exo-form A in a ratio of 5:3. This suggests that
changes in the side chain of the 6(3)-enaminotetramic acid
affect the tautomeric equilibrium.
Cladosin E (5) was isolated as a white, amorphous powder
and had the molecular formula C13H17NO4 by HRESIMS. The
NMR resonances of 5 were attributed to a trisubstituted
benzene ring, two upfield carbonyl carbons, two sp3 methines,
two geminal methyls, an aryl methyl, a hydrogen-bonded
proton, and an exchangeable amide proton. The assembly of a
6-methylsalicylic acid moiety, which is one of the oldest fungal
polyketide skeletons known,17,18 was readily established from
the 1D NMR data. This was further confirmed from key COSY
and HMBC correlations in its 2D NMR spectra and in
particular identifying a valine residue, which accounted for the
remaining atoms required by the molecular formula (Figure 1).
The 6-methylsalicylic acid moiety and valine residue were
linked via an amide bond that was confirmed by the key HMBC
correlation from H-2′ to C-1 and from the presence of an
amide proton (δH 7.46). The absolute configuration of the
valine residue was established as L by application of Marfey’s
method.19
acetonide 7 were distinct at δC 19.5 and 29.0 ppm, requiring a
syn 1,3-diol system in 2.14 Furthermore, the coupling pattern
for the C-9 methylene protons of 7 provided further evidence
for a syn 1,3-diol.15 The modified Mosher’s method has been
applied to ascertain the absolute configuration of the simple
acyclic syn 1,3-diols from their di-MTPA esters.16 The
systematically distributed Δδ values of positive (ΔδL), negative
(ΔδC), and negative (ΔδR) corroborated the 8R,10S config-
urations in 2 (Figure 3). Biogenetically, compound 1 may be
the methylation product of 2, which suggested the same syn 1,3-
diol unit as 2 and was supported by the similar splitting pattern
Biosynthetically, all of the isolated compounds (cladosins A−
E) are composed of a tetraketide unit and a valine residue,
involving both PKS and amino acid pathways (Figure 4). In the
putative biogenesis of cladosins A−D, the linear tetraketide
initially condenses with an activated valine to form the
intermediate ii, a 1,3-dione-5,7-diol conjugate.1,20 The con-
densed hybrid molecule ii is released as a tetramic acid core
Figure 3. ΔδS−R values for the Mosher’s ester derivatives of 1−3.
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1
Table 2. H NMR (600 MHz) Data of 1−4 in DMSO-d6 (δ ppm, J in Hz)
1
2
3
4
no.
a
b
a
b
a
b
a
b
NH-1
7
9.13, s
9.31, s
9.10, s
9.25, s
9.22, s
9.40, s
9.80, s
9.65, s
2.96, m;
3.01, m
3.64, m
1.71, m;
1.36, m
3.77, m
1.04, d (6.0)
1.75, s
2.96, m;
3.01, m
3.62, m
1.71, m;
1.36, m
3.77, m
1.04, d (6.0)
1.74, s
3.08, m;
2.65, m
3.93, brs
1.56, m;
1.39, m
3.80, brs
1.03, d (5.5)
1.73, s
3.08, m;
2.65, m
3.93, brs
1.56, m;
1.39, m
3.80, brs
1.03, d (5.5)
1.72, s
7.43, d (16.5)
7.43, d (16.5)
7.72, dd
(9.9, 1.6)
6.94, m
2.38, m;
2.57, m
4.42, m
1.40, d (6.6)
1.75, s
7.68, dd
(9.9, 1.6)
6.97, m
2.35, m;
2.60, m
4.42, m
1.40, d (6.6)
1.75, s
8
9
6.92, m
2.34, m
6.92, m
2.34, m
10
3.77, m
3.77, m
11
1.11, d (6.4)
1.75, s
1.11, d (6.4)
1.74, s
13
14
2.16, s
2.13, s
2.14, s
2.11, s
2.16, s
2.13, s
2.11, s
2.12, s
15
3.24, s
3.26, s
8-OH
10-OH
6-NH2
4.96, s
4.94, s
4.51, d (4.4)
10.06, brs;
8.85, brs
4.49, d (4.4)
9.46, brs;
4.59, s
4.59, s
4.71, d (5.0)
9.89, brs;
4.71, d (5.0)
9.26, brs;
10.05, brs;
8.77, brs
9.45, brs;
8.65, brs
8.73, brs
8.68, brs
8.58, brs
Figure 4. Plausible biogenetic origin of 1−5.
through a catalyzed Dieckmann cyclization and a series of
subsequent transformations, including hydroxylation, dehydra-
tion, transamination,21 and O-methylation that lead to the
production of cladosins A−D.1,20 However, the linear
intermediate i could undergo cyclization, dehydration, and
aromatization to produce a 6-methylsalicylic acid core and
finally release cladosin E (5).
Cladosins A−D (1−4) represent a novel class of tetramic
acid congeners characterized by previously unknown 6(3)-
enamino-8,10-dihydroxy- or 6(3)-enamino-7(8)-en-10-ol side
chains. To date, among the numerous known natural products
containing the tetramic acid core, only xyrrolin22 and
preaspyridone20 contain a C8 polyketide unit. Due to the
different side chains in 1−4 in relation to those previously
reported in other tetramic acids, the formation of the C8
polyketide chains in cladosins A−D may be encoded through
a PKS gene with a rare aminotransferase domain. An extensive
literature survey has also indicated that only cyclopiazonic acid
imine contains a similar enamine motif in the polyketide
chain,12b and only three examples, 3-acetyl-5-isopropytetramic
acid,23 dysidin,24 and L-tenuazonic acid25 incorporate a valine
residue within a 2,4-pyrrolidinedione core.
In addition, all the isolated compounds were evaluated for
antiviral activity against the influenza A H1N1 virus, as well as
antitumor, antitubercular, and NF-κB inhibitory activities. Only
compound 3 exhibited activity with an IC50 = 276 μM against
the influenza A H1N1 virus (ribavirin as positive control, IC50
131 μM).
EXPERIMENTAL SECTION
■
General Experimental Procedures. Specific rotations were
obtained on a JASCO P-1020 digital polarimeter. UV spectra were
recorded on a Beckman DU 640 spectrophotometer. NMR spectra
were recorded on a JEOL JNM-ECP 600 spectrometer using TMS as
internal standard. 1H−15N HSQC spectra were recorded on an Agilent
500 MHz DD2 spectrometer. ESIMS spectra were acquired on a
Thermo Scientific LTQ Orbitrap XL mass spectrometer or a
Micromass Q-TOF ULTIMA GLOBAL GAA076 LC mass spec-
trometer. Semipreparative HPLC was performed using an ODS
column [HPLC (YMC-Pack ODS-A, 10 × 250 mm, 5 μm, 4 mL/
min)]. Medium-pressure preparation liquid chromatography (MPLC)
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was performed on a Bona-Agela CHEETAH HP100 (Beijing Agela
Technologies Co., Ltd.). Column chromatography (CC) was
performed with silica gel (200−300 mesh, Qingdao Marine Chemical
Inc.) and Sephadex LH-20 (Amersham Biosciences), respectively.
Fungal Material. The fungal strain C. sphaerospermum 2005-01-E3
was isolated from deep-sea sludge in the Pacific Ocean (W 102°22′0,
N 00°15′9; depth over 1000 m). The isolate was identified as C.
sphaerospermum 2005-01-E3 according to morphological traits and ITS
rDNA sequence analysis. The sequence data have been submitted to
GenBank, accession number JQ670942.1. 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, PR China. Working stocks were prepared on potato dextrose
agar slants stored at 4 °C.
HPLC column to give an isomeric mixture of 6 (2.2 mg). For 1D and
2D NMR data, see Table S1 (Supporting Information); HRESIMS m/
z 285.1807 [M + H]+ (calcd for C14H25N2O4, 285.1809); 307.1626 [M
+ Na]+ (calcd for C14H24N2O4Na, 307.1628)
Preparation of Acetonide 7. To a solution of 2 (4 mg), in dry
MeOH (1 mL) was added 2,2-dimethoxypropane (1 mL) and p-
TsOH (5 mg) with stirring at rt under a nitrogen atmosphere for 12 h.
The reaction mixture was poured into cold saturated aqueous
NaHCO3 and subsequently extracted with EtOAc. The organic layer
was washed with H2O and evaporated to dryness, finally yielding a
1
mixture of 7 and 2. H NMR (CD3OD 600 MHz) exo-form A [exo-
form B] δ 4.29 (1H, m, H-10) [4.29 (1H, m, H-10)]; 4.03 (1H, m, H-
8) [4.10 (1H, m, H-8)]; 3.25 (1H, m, H-7) [3.25 (1H, m, H-7)]; 2.71
(1H, m, H-7) [2.71 (1H, m, H-7)]; 2.22 (3H, s, H-14) [2.21 (3H, s,
H-14)]; 1.82 (3H, s, H-13) [1.82 (3H, s, H-13)]; 1.65 (1H, m, H-9)
[1.65 (1H, m, H-9)]; 1.40 (3H, s, (O)2C(CH3)2) [1.40 (3H, s,
(O)2C(CH3)2]; 1.34 (3H, s, (O)2C(CH3)2) [1.34 (3H, s, (O)2C-
(CH3)2]; 1.28 (3H, s, H-11) [1.28 (3H, s, H-11)]; 1.15 (1H, m, H-9)
[1.15 (1H, m, H-9)]; 13C NMR data of 7 (CD3OD 150 MHz) exo-
form A (exo-form B) δ 186.5 (184.2), 171.6 (170.6), 169.0 (169.5),
129.6 (129.6), 120.6 (120.9), 98.7 (98.7), 96.8 (97.0), 67.9 (67.6),
65.0 (65.6), 38.0 (38.0), 37.8 (38.3), 29.0 (29.3), 22.0 (22.8), 20.9
(22.3), 19.5 (19.7), 17.1 (17.2); ESIMS m/z 309.2 [M + H]+.
Preparation of MTPA Esters of 1−3. To a mixture of 1 (1 mg)
and DMAP (cat.) in pyridine (800 μL) was added (R)-MTPACl (10
μL) at rt under a nitrogen atmosphere for 2 h. The organic phase was
evaporated to dryness and separated by semipreparative HPLC to
afford the (S)-MTPA ester 1. In a similar manner, the (R)-MTPA ester
1 was prepared from 1 using (S)-MTPACl and DMAP. The same
procedure was used for the preparation of the (R)- or (S)-MTPA
diesters 2 and (R)- or (S)-MTPA esters 3 obtained from 2 and 3,
respectively.
Fermentation and Extraction. The fungus C. sphaerospermum
2005-01-E3 was cultured under static conditions at 28 °C in 1 L
Erlenmeyer flasks containing rice media (rice 80 g, artificial seawater
120 mL). After 30 days of cultivation, the fermentation from 50 flasks
was extracted with MeOH. The MeOH extract was evaporated under
reduced pressure to afford an aqueous solution that was extracted with
EtOAc. The EtOAc extract was redissolved in MeOH containing 20%
H2O and was extracted by hexane (three times). The MeOH layer was
concentrated under reduced pressure to give a defatted extract (5.0 g).
Isolation. The extract (5.0 g) was applied on a C-18 ODS column
using a stepped gradient elution of MeOH−H2O, yielding six
subfractions (A1−A6). Fraction A2, that eluted with 30:70 MeOH−
H2O, was separated by MPLC (C-18 ODS) using a stepped gradient
elution of MeOH−H2O (5:95 to 50:50) to furnish five subfractions
(A2.1−A2.5). A2.3 was chromatographed on SephadexLH-20
(MeOH) and further purified by semipreparative HPLC (20:80
MeCN−H2O, 4 mL/min) to afford compounds 1 (30.0 mg) and 2
(12.0 mg). A3 was fractionated on SephadexLH-20 (MeOH) and
further purified by semipreparative HPLC (60:40 MeOH−H2O, 4
mL/min) to afford compounds 3 (5.0 mg) and 4 (1.3 mg). A1 was
applied to a SephadexLH-20 column (MeOH) and further purified by
semipreparative HPLC (30:70 MeOH−H2O) to afford 5 (25.0 mg).
1
(S)-MTPA ester 1: H NMR (DMSO-d6) exo-form A (exo-form B)
δ 10.00 (9.40), 9.11 (9.32), 8.87 (8.79), 7.57−7.30 (7.57−7.30), 5.22
(5.22), 3.67 (3.61), 3.46 (3.45), 3.24 (3.23), 3.09 (3.04), 2.93 (2.91),
2.15 (2.10), 1.93 (1.93), 1.75 (1.72), 1.65 (1.65), 1.21(1.21);
HRESIMS m/z 499.2048 [M + H]+ (calcd for C24H30F3N2O6,
499.2050), 521.1862 [M + Na]+ (calcd for C24H29F3N2O6Na,
521.1870).
Cladosin A (1): pale yellow oil; [α]24 +17.7 (c 0.10, MeOH); UV
D
1
(MeOH) λmax (log ε) 278 (4.07) nm; H and 13C NMR data, see
Tables 1 and 2, respectively; HRESIMS m/z 305.1475 [M + Na]+
1
(calcd for C14H22N2O4Na, 305.1472).
(R)-MTPA ester 1: H NMR (DMSO-d6) exo-form A (exo-form B)
CladosinB (2): pale yellow oil; [α]24 +20.4 (c 0.10, MeOH); UV
δ 9.97 (9.36), 9.14 (9.36), 8.80 (8.73), 7.49−7.36 (7.49−7.36), 5.20
(5.26), 3.51 (3.49), overlap with H2O (3.38), 3.08 (3.01), 2.96 (2.95),
2.93 (2.84), 2.16 (2.13), 1.87 (1.87), 1.76 (1.74), 1.57 (1.57), 1.30
(1.31); HRESIMS m/z 499.2042 [M + H]+ (calcd for C24H30F3N2O6,
499.2050).
D
1
(MeOH) λmax (log ε) 279 (3.95) nm; H and 13C NMR data, see
Tables 1 and 2, respectively; HRESIMS m/z 291.1316 [M + Na]+
(calcd for C13H20N2O4Na, 291.1315).
Cladosin C (3): pale yellow oil; [α]24 +10.5 (c 0.10, MeOH); UV
D
1
1
(MeOH) λmax (log ε) 278 (3.40), 322 (3.98) nm; H and 13C NMR
(S)-MTPA diesters 2: H NMR (DMSO-d6) exo-form A (exo-form
data, see Tables 1 and 2, respectively; HRESIMS m/z 273.1213 [M +
B) δ 9.78 (9.19), 9.13 (9.34), 8.81 (8.68), 7.51−7.32 (7.51−7.32),
5.70 (5.60), 5.16 (5.21), 3.49 (3.51), 3.42 (overlap with H2O), 3.10
(3.10), 3.03 (2.95), 2.17 (2.04), 2.09 (2.09), 1.94 (1.94), 1.75 (1.72),
1.41 (1.42); HRESIMS m/z 701.2292 [M + H]+ (calcd for
C33H35F6N2O8, 701.2292).
Na]+ (calcd for C13H18N2O3Na, 273.1210).
Cladosin D (4): pale yellow oil; [α]24 +8.3 (c 0.10, MeOH); UV
D
1
(MeOH) λmax (log ε) 278 (3.45), 322 (4.05) nm; H and 13C NMR
data, see Tables 1 and 2, respectively; HRESIMS m/z 251.1389 [M +
H]+ (calcd for C13H19N2O3, 251.1390).
(R)-MTPA diesters 2: H NMR (DMSO-d6) exo-form A (exo-form
1
Cladosin E (5): colorless powder; [α]24 +15.2 (c 0.10, MeOH);
B) δ 9.98 (9.40), 9.18 (9.37), 9.05 (8.96), 7.53−7.37 (7.53−7.37),
5.71 (5.64), 5.03 (5.12), 3.24 (overlap with H2O), 3.15 (2.98), 2.17
(2.08), 2.01 (2.01), 1.91 (1.91), 1.75 (1.72), 1.24 (1.23); HRESIMS
m/z 701.2291 [M + H]+ (calcd for C33H35F6N2O8, 701.2292).
D
UV (MeOH) λmax (log ε) 225 (3.76), 268 (1.07); 1H NMR (DMSO-
d6 600 MHz) δ 6.60 (1H, d, J = 8.0 Hz, H-4); 7.02 (1H, t, J = 8.0, 7.7
Hz, H-5); 6.56 (1H, d, J = 7.7 Hz, H-6); 2.19 (3H, s, H-8); 4.02 (1H,
d, J = 4.4 Hz, H-2′); 2.23 (1H, m, H-3′); 0.81 (3H, d, J = 7.1 Hz, H-
4′); 0.91 (3H, d, J = 6.6 Hz, H-5′); 7.46 (1H, brs, −NH); 11.99 (1H,
brs, 3-OH); 13C NMR data (DMSO-d6 150 MHz) δ 168.8(C, C-1),
126.2 (C, C-2), 156.4 (C, C-3), 114.8 (CH, C-4), 129.6 (CH, C-5),
119.9 (C, C-6), 136.0 (C, C-7), 19.8 (CH3, C-8), 174.6 (C, C-1′), 60.9
(CH, C-2′), 29.7 (CH, C-3′), 18.5 (CH3, C-4′), 20.7 (CH3, C-5′);
HRESIMS m/z 274.1050 [M + Na]+ (calcd for C13H17NO4Na,
274.1050); 252.1228 [M + H]+ (calcd for C13H18NO4, 252.1230).
Hydrogenation of Compound 1. To a solution of 1 (4 mg) in
MeOH (1 mL) was added 10% palladium on activated charcoal (5
mg). The flask was degassed and filled with hydrogen. After stirring for
12 h at room temperature (rt), the reaction mixture was filtered
through a microporous filter (0.22 μm) and the filtrate was evaporated
to dryness. The residue was purified by a semipreparative RP-18
1
(S)-MTPA ester 3: H NMR (DMSO-d6) exo-form A (exo-form B)
δ 6.89 (6.89), 5.27 (5.27), 2.67 (2.67), 2.62 (2.62), 2.16 (2.12), 1.76
(1.74), 1.29 (1.29); HRESIMS m/z 467.1782 [M + H]+ (calcd for
C23H26F3N2O5, 467.1788).
1
(R)-MTPA ester 3: H NMR (DMSO-d6) exo-form A (exo-form B)
δ 6.79 (6.79), 5.28 (5.43), 2.62 (2.54), 2.17 (2.11), 1.75 (1.74), 1.38
(1.38); HRESIMS m/z 467.1781 [M + H]+ (calcd for C23H26F3N2O5,
467.1788).
Absolute Configuration of Valine. Compound 5 (1 mg) was
dissolved in 0.5 mL of 6 N HCl and heated at 95 °C for 24 h. After
removing the solvent under reduced pressure, the reaction mixture
(0.25 μmol) was redissolved in 50 μL of H2O, and then 0.25 μmol of
L-FDAA in 100 μL of acetone was added, followed by 25 μL of 1 N
NaHCO3. The mixture was heated for 1 h at 43 °C. After cooling to rt,
274
dx.doi.org/10.1021/np400833x | J. Nat. Prod. 2014, 77, 270−275

Journal of Natural Products
Article
the reaction was quenched by the addition of 25 μL of 2 N HCl.
Finally the resulting solution was filtered through a small 4.5 μm filter
and stored in a freezer until HPLC analysis. The standard L- and D-
valine were also derivatized with FDAA in the above-described
manner. The resulting mixture containing the L-FDAA derivative was
analyzed by reversed-phase HPLC under the following conditions: a 5
mm × 250 mm YMC C18 column, 5 μm, with a linear gradient of (A)
CH3CN and (B) 0.05% aqueous TFA from 10% to 50% (A) over 60
min at a flow rate of 1 mL/min, UV detection at 320 nm. The
chromatographic peaks were identified by comparing the retention
times for the L-FDAA derivatives of the L- and D-valine and FDAA
standards. The standards gave the following retention times: 32.70 min
for FDAA; 35.91 min for L-Val; 39.52 min for D-Val. The L-FDAA-
derivatized hydrolysate mixture showed a peak at 35.91 min. The
result demonstrated the valine in compound 5 was the L-form.
Biological Assay. Biological evaluation was carried out as
previously reported.9a,b,26
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* Supporting Information
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AUTHOR INFORMATION
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Corresponding Author
*Tel: 0086-532-82031619. Fax: 0086-532-82033054. E-mail:
dehaili@ouc.edu.cn.
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Notes
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The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
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(17) Mazzini, F.; Galli, F.; Salvadori, P. Eur. J. Org. Chem. 2006, 24,
5588−5593.
This work was financially supported by the National Natural
Science Fundation of China (Nos. 41176120 and 21372208),
the Promotive Research Fund for Excellent Young and Middle-
aged Scientists of Shandong Province (No. BS2010HZ027), the
National High Technology Research and Development
Program of China (No. 2013AA092901), the Program for
New Century Excellent Talents in University (No. NCET-12-
0499), the Public Projects of State Oceanic Administration
(No. 2010418022-3), and the Program for Changjiang Scholars
and Innovative Research Team in University (No. IRT0944).
We thank Dr. Z. Lin (University of Utah, USA) for the
suggestion of the plausible biosynthetic pathway and Dr. R. A.
Keyzers (Victoria University of Wellington, New Zealand) for
helping with the manuscript preparation.
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