Pentaketides relating to aspinonene and dihydroaspyrone from a marine-derived fungus, Aspergillus ostianus

Article abstract of DOI:10.1021/np070301n

Three new pentaketides, aspinotriols A (1) and B (3) and aspinonediol (5), were isolated together with two known compounds, aspinonene (7) and dihydroaspyrone (9), from the marine fungus Aspergillus ostianus strain 01F313, which was collected in Pohnpei and cultured with bromine-modified artificial seawater. The structures of the new compounds were determined by spectroscopic analyses including 1D and 2D NMR. Although 1 and 3 are diastereomers, they show nearly superimposable ¹H and ¹³C NMR spectra. The absolute configurations of compounds 1, 3, 5, and 9 were elucidated by the modified Mosher's method.

Full text of DOI:10.1021/np070301n

2022
J. Nat. Prod. 2007, 70, 2022–2025
Pentaketides Relating to Aspinonene and Dihydroaspyrone from a Marine-Derived Fungus,
Aspergillus ostianus
Keijiro Kito,† Ryuhei Ookura,† Sanae Yoshida,† Michio Namikoshi,‡ Takashi Ooi,† and Takenori Kusumi*,†
Faculty of Pharmaceutical Sciences, The UniVersity of Tokushima, Tokushima 770-8505, Japan, and Tohoku Pharmaceutical UniVersity,
Aoba-ku, Sendai 981-8558, Japan
ReceiVed June 22, 2007
Three new pentaketides, aspinotriols A (1) and B (3) and aspinonediol (5), were isolated together with two known
compounds, aspinonene (7) and dihydroaspyrone (9), from the marine fungus Aspergillus ostianus strain 01F313, which
was collected in Pohnpei and cultured with bromine-modified artificial seawater. The structures of the new compounds
were determined by spectroscopic analyses including 1D and 2D NMR. Although 1 and 3 are diastereomers, they show
nearly superimposable 1H and 13C NMR spectra. The absolute configurations of compounds 1, 3, 5, and 9 were elucidated
by the modified Mosher’s method.
A growing number of marine fungi have been reported to produce
novel and potentially useful bioactive secondary metabolites, and
the vast majority of compounds reported from marine fungi are
from strains that, based on morphological characteristics, are closely
related or identical to terrestrial species.1,2 We previously reported
three antibacterial chlorine-containing components of Aspergillus
ostianus strain 01F313 that had been isolated from an unidentified
marine sponge collected at Pohnpei.3 The chlorine must have
originated from the cultivation medium composed of natural
seawater. Expecting that bromine-containing compounds might be
obtained when a medium composed of a bromide solution in place
of seawater was used, we cultivated the same strain in a bromine-
modified 1/2 PD medium. Although we were unable to isolate
brominated compounds, we found that the metabolites were
considerably different from those obtained from the strain cultured
in the seawater medium and succeeded in isolating three new
compounds, named aspinotriols A (1) and B (3) and aspinonediol
(5), as well as two known compounds, aspinonene (7)4,5,6 and
dihydroaspyrone (9).6 This paper elucidates the structures of the
new compounds and the absolute configurations of all compounds.
Strain A. ostianus 01F313 was cultured in a 1/2 PD medium
containing bromine-modified artificial seawater (see the Experi-
mental Section). After the mycelial cake was removed by filtration,
the filtrate was subjected to HP-20 extraction. The extract was
separated by silica gel flash column chromatography (FCC)
followed by reversed-phase (ODS) HPLC to give compounds 1, 3,
5, 7, and 9 (Figure 1).
Figure 1. Structures of three new compounds, aspinotriols A (1)
and B (3) and aspinonediol (5), together with the known compounds
aspinonene (7) and dihydroaspyrone (9), and their derivatives.
Piv: pivaloyl (2,2-dimethypropanoyl), acetonide: isopropylidene,
TBDMS: tert-butyldimethylsilyl.
Aspinotriol A (1), [R]25D -10.1 (c 0.23, MeOH), has a molecular
formula of C9H16O3 deduced from HRTOFMS [(M + Na)+ m/z
195.1012, calc 195.0997]. The 1H NMR (400 MHz, CD3OD)
spectrum (Table 1) showed the presence of two secondary methyls
[δ 1.294 (d, J ) 6.4 Hz), 1.289 (d, J ) 6.4 Hz)], three olefinic
methines [δ 6.17 (d, J ) 15.9 Hz), 5.97 (dd, J ) 15.9, 6.4 Hz),
5.60 (d, J ) 8.6 Hz)], an oxymethylene [δ 4.330 (s)], and two
oxymethines [δ 4.76 (dq, J ) 8.6, 6.4 Hz), δ 4.325]. The two olefins
must be conjugated considering the absorption maximum at 231
nm in the UV spectrum, and one of them is a disubstituted E-olefin
olefinic carbon (δ 137.8), an oxymethylene carbon (δ 57.6), and
two oxymethine carbons (δ 69.4, 64.7). These data, together with
1
the H-1H COSY, HSQC, and HMBC spectra (Table 1), readily
revealed the planar structure of aspinotriol A (1). At this stage, the
stereochemistry of the two asymmetric carbons was unknown.
Aspinotriol B (3), C9H16O3 [(M + Na)+ m/z 195.1007, calc
195.0997], was obtained from the HPLC fraction, whose retention
time (tR ) 19 min) was largely different from the fraction giving
3
showing JHH of 15.9 Hz. The Z-configuration of the other olefin
was confirmed by the NOESY correlations of H-4/H-6 and H-7/
H-9. The 13C NMR (100 MHz, CD3OD) spectrum (Table 1)
confirmed the presence of two methyl carbons (δ 23.9, 23.7), three
olefinic methine carbons (δ 138.8, 135.0, 131.7), a quaternary
1
1 (tR ) 13 min). We were puzzled to find that the H (400 MHz,
CD3OD) and 13C NMR (100 MHz, CD3OD) spectra of 3 were
practically identical to those of 1 (Table 2). Changing the NMR
solvent to pyridine-d5 gave the same results (Table 3). Analyses of
the 1D and 2D NMR data of 3 (Table 2) led to the same planar
structure as 1, including the geometry of the diene group. Consider-
ing the chiroptical property of 3, [R]25D +6.1 (c 0.21, MeOH), we
* To whom correspondence should be addressed. Fax: +81-88-633-7288.
E-mail: tkusumi@ph.tokushima-u.ac.jp.
† The University of Tokushima.
‡ Tohoku Pharmaceutical University.
10.1021/np070301n CCC: $37.00
2007 American Chemical Society and American Society of Pharmacognosy
Published on Web 11/10/2007

Notes
Journal of Natural Products, 2007, Vol. 70, No. 12 2023
Table 1. NMR Data (100/400 MHz, CD3OD) of Aspinotriol
A (1)
only a single set of 1H NMR signals, implying that 7 was
enantiomerically pure. The ∆δ values obtained for 8 (Figure 2)
indicated a 2(S)-configuration of 7, which is consistent with the
reported absolute configuration.
position δC, mult.
δH (J in Hz)
23.9, CH3 1.29, d (6.4)
69.4, CH 4.33, m
COSY
HMBC
NOESY
3
1
2
3
4
5
6
7
8
9
2
2, 3
Compound 9 was identified as dihydroaspyrone by comparing
its spectroscopic data with those reported in the literature.6 The
relative and absolute stereochemistry of 9 has been presumed on
the basis of CD spectroscopy and biosynthetic reasoning.6 This
compound has two secondary hydroxy groups, one on the ring (C-
5) and another on the side chain (C-9). The hydroxy group at C-5
appears more hindered than the one at C-9 because the former is
neighboring a secondary methyl group. This supposition proved
correct: when 9 was treated with 1.2 equiv of (S)- and (R)-MTPA
chlorides, only the 9-OH was esterified, giving (R)- and (S)-MTPA
(10), respectively, which enabled us to assign the S-configuration
at C-9 (Figure 2). On the other hand, the more reactive hydroxy
group at C-9 of 9 was first protected by a tert-butyldimethylsilyl
(TBDMS) group, and the C-5 hydroxy group was esterified with
(R)- and (S)-MTPA chlorides. The ∆δ values obtained from the
MTPA esters (11) (Figure 2) indicated an S-configuration for C-5.
Since the trans relationship of H-5 and H-6 is obvious from the
NOESY correlation between H-5 and Me-7, and because JH-5,H-6
) 7.9 Hz observed for 9, the absolute configuration of dihydroaspy-
rone was confirmed, as shown in structure 9.
1, 3, 4
1
138.8, CH 5.97, dd (15.9, 6.4) 2, 4
131.7, CH 6.17, d (15.9)
137.8, C
135.0, CH 5.60, d (8.6)
64.7, CH 4.76, dq (8.6, 6.4) 6, 8
1, 2, 4, 5
1, 2
2, 6
2, 3, 6 2, 3, 5, 6, 9
4, 7, 9 4, 5, 8, 9
4, 8
9
6
5, 8
6, 7
4, 5, 6
23.7, CH3 1.29, d (6.4)
57.6, CH2 4.33 (s)
7
6
3, 7
Table 2. NMR Data (100/400 MHz, CD3OD) of Aspinotriol
B (3)
position δC, mult.
δH (J in Hz)
23.9, CH3 1.29, d (6.4)
69.4, CH 4.32, m
138.8, CH 5.97, dd (15.9, 6.4) 2, 4
COSY
HMBC
NOESY
3
1
2
3
4
5
6
7
8
9
2
2, 3
1, 3, 4 1, 3
1, 2, 4, 5, 6
1, 2
2, 6
131.7, CH 6.17, d (15.9)
137.8, C
135.0, CH 5.60, d (8.6)
64.7, CH 4.75, dq (8.6,6.2)
23.7, CH3 1.29, d (6.2)
57.6, CH2 4.33 (s)
2, 3, 6 2, 3, 5, 6, 9
4, 7, 9 4, 5, 7, 8, 9
6, 8
7
4, 8
9
6
5, 6, 8
6, 7
4, 5, 6
6
3, 7
The biosynthesis of aspinonene (7) and its analogues has been
exhaustively studied by Zeeck et al.5,6 They experimentally
confirmed that 7 is a pentaketide produced by a polyketide synthesis
(PKS) via rearrangement of I to II (Figure 3) and decarboxylation
followed by dehydration to afford 2(S),3(S),6(S),7(S)-diepoxide
(12a). The 7(S)-configurations of 1, 3, 5, and 7 and the 9(S)-
configuration of 9 reinforce the proposed biosynthetic precursor
12a.
We tested 1, 3, 5, 7, and 9 for inhibitory activity against MRSA
(methicillin-resistant Staphylococcus aureus), as well as 1, 7, and
9 for cytotoxic activity against L-1210. No anti-MRSA activity was
found at a concentration of 100 µg/disk for the tested samples.
Compounds 7 and 10 showed toxicity against mouse lymphocytic
leukemia cells at 25 ppm (27% and 25%, respectively), although 1
was inactive.
even wondered if 3 was the enantiomer of 1, although this was of
course impossible since the HPLC column (ODS) used for
separation was achiral. We therefore undertook to elucidate the
absolute configurations of the secondary alcohols of 1 and 3 (Figures
1 and 2) using the modified Mosher’s method.7,8
The primary alcohol of 1 was protected with a pivaloyl (2,2-
dimethylpropanoyl) moiety, and the resulting monopivaloyl ester
was converted to di-(R)- and di-(S)-MTPA esters (2). The ∆δ values
[δ(S)-MTPA – δ(R)-MTPA] were calculated, revealing 2(R)- and 7(S)-
configurations (Figure 2). Similar experiments performed on 3
through di-MTPA esters (4) gave 2(S)- and 7(S)-configurations of
3. Thus, it was established that 1 and 3 were in an epimeric
relationship at C-2. To our knowledge, it is very rare for such
distinct diastereomers to show practically the same 1H and 13C NMR
spectra.
Experimental Section
Aspinonediol (5), [R]24D +2.4 (c 0.63, MeOH), had a molecular
formula of C9H14O3 deduced from HRTOFMS [(M + Na)+ m/z
General Experimental Procedures. Optical rotation values were
determined on a JASCO P-1010 polarimeter. High-resolution MS
spectra were obtained with a Waters LCT-Premier 2695 mass spec-
trometer. 1D and 2D NMR were recorded on Bruker ARX-400, JEOL-
JNM-AL400, and JEOL-GSX-400 spectrometers. Chemical shifts (δ)
are expressed in parts per million (ppm) with reference to the solvent
signals. [1H NMR: CD3OD (δ 3.35), CDCl3 (δ 7.25), C5D5N (δ 7.55
for H-3), acetone-d6 (δ 2.05). 13C NMR: CD3OD (δ 49.0), CDCl3 (δ
77.0), C5D5N (δ 135.5 for C-3).] Multiplicities of 13C signals were
determined by DEPT and HSQC spectra. Flash column chromatography
(FCC) was carried out on silica gel (40–63 µm, Merck Co.). Thin-
layer chromatography was carried out on silica gel 60 F254 plates (Merck
Co.). Cultivation of the titled fungus was performed in a Sanyo grows
cabinet NLR-350H. HPLC was done using a Tosoh CCPS. Recycle-
HPLC was done using a Japan Analytical Industry Co., Ltd. LC-908.
Fungal Isolation and Identification. The fungus, designated as
strain 01F313, was isolated from an unidentified marine sponge
collected in Pohnpei in 2001. Isolation and identification of the strain
are described in the literature.3 The fungus is maintained on 1/10 YSA
medium at the Marine Natural Products Laboratory, Faculty of
Pharmaceutical Sciences, the University of Tokushima, Japan.
1
193.0869, calc 193.0841]. The H NMR (CD3OD) spectrum of 5
was similar to those of 1 and 3, although one of the signals due to
the oxymethines was missing, the signals of the disubstituted olefin
appeared as lower field AB doublets at δ 7.24 (J 16.2 Hz) and
6.45 (J 16.2 Hz), and another olefin proton resonated at δ 6.09 (d,
J 8.5 Hz). The 13C NMR signal at δ 201.5 suggested the presence
of a carbonyl carbon, and the UV maximum at 269 nm showed
the existence of an R,ꢀ,γ,δ-unsaturated enone system (λmaxcalc 263
nm). Other NMR properties, including the 2D NMR data (Table
4), gave the planar structure 5 of aspinonediol. The S-configuration
1
at C-7 was deduced by comparing the H chemical shifts of (R)-
and (S)-MTPA esters (6) (Figure 2). This finding suggested that 5
was the precursor of aspinotriols A (1) and B (3), whose 2-hydroxy
groups are formed by enantiomerically nonspecific reduction of the
carbonyl of 5.
The spectroscopic data of 7, [R]24D -7.3 (c 0.38, MeOH), were
identical to those reported for aspinonene4 except for the reported
optical rotation value, [R]24 -78 (c 0.3, MeOH). The absolute
D
Cultivation of Fungus and Isolation of Metabolites. The fungus
was cultured in 67 500-mL Erlenmeyer flasks each containing 150 mL
of a 1/2 PD medium using 90% BrSW [NaBr (52.78 g), KBr (1.27 g),
CaBr2 (2.76 g), and MgSO4 (6.60 g) dissolved in distilled H2O (1 L);
acidity of the medium was adjusted by 1 M HBr and 1 M NaOH to
pH 8.3] for five weeks at 20 °C. The broth was filtered, and the filtrate
was passed through a column packed with HP-20 (205 g). After washing
with 1 L of distilled H2O, MeOH (6 L) was passed through the column
stereochemistry of aspinonene has already been established by X-ray
analysis. The marked difference in the specific rotation values made
us suspect that 7 might be enantiomerically impure. To confirm
the purity, the following experiments were performed. The primary
and tertiary OH groups of 7 were protected by treatment with 2,2-
dimethoxypropane, and the resulting acetonide4 was converted to
(R)- and (S)-MTPA esters (8). Each of the MTPA esters exhibited

2024 Journal of Natural Products, 2007, Vol. 70, No. 12
Notes
Table 3. 1H NMR Data (100/400 MHz, C5D5N) of Aspinotriol A (1) and Aspinotriol B (3)
1
3
position
δC, mult.
δH (J in Hz)
1.42, d (6.3)
4.63, dq (6.3, 5.6)
6.47, dd (15.9, 5.6)
6.57, d (15.9)
COSY
δC, mult.
δH (J in Hz)
COSY
1
2
3
4
5
6
7
8
9
24.4, CH3
68.1, CH
135.5, CH
130.9, CH
137.7, C
138.9, CH
63.7, CH
24.7, CH3
57.5, CH2
2
24.4, CH3
68.2, CH
135.6, CH
130.9, CH
137.8, C
138.9, CH
63.8, CH
24.8, CH3
57.6, CH2
1.43, d (6.3)
2
1, 3, 4
2, 4
2, 3, 6
4.64, dq (6.3, 5.6)
6.49, dd (15.9, 5.6)
6.58, d (15.9)
1, 3, 4
2, 4
2, 3, 6
5.97, d (8.2)
5.19, dq (8.2, 6.3)
1.45, d (6.3)
4, 7
6, 8
7
5.99, d (8.4)
5.19, dq (8.4,6.3)
1.46, d (6.3)
4, 7
6, 8
7
4.73/4.68, d (12.1)
4.74/4.69, d (12.2)
and the MeOH solution was concentrated. The brown extract (2.04 g)
was separated by FCC (205 g of silica gel) eluted with a CHCl3-MeOH
gradient (1:0 to 0:1) to give 10 fractions. A part (209.3 mg) of fraction
8 (263.2 mg) was separated by HPLC using an ODS column [Capselpak
C18 type SG120 A 5 µm; 10 mm × 250 mm; flow rate 2.0 mL/min;
MeOH-H2O (25:75) containing 0.1% CH3COOH] into 16 fractions.
Fraction 8-7 (107.5 mg) was identified as aspinonene (7). Fractions
8-11 (12.5 mg) and 8-15 (11.1 mg) were new compounds, aspinotriols
A (1) and B (3), respectively. Fraction 7 (187.7 mg) of the FCC was
separated by recycle-HPLC using the above-mentioned ODS column
[Capselpak C18 type SG120 A 5 µm; 20 mm × 250 mm; flow rate 6.0
mL/min; MeOH-H2O (30:70) containing 0.1% CH3COOH] into 10
fractions. Fraction 7-7 (24.1 mg) was identified as dihydroaspyrone
(9). Aspinonediol (5) was obtained as fraction 7-8 (7.5 mg).
acid chlorides, and after 30 min, trimethylsilyldiazaomethane (100 µL
of 10% n-hexane solution) was added to methylate acids that might
have formed by hydrolysis of the chlorides from moisture in the solvent.
Pyridine was removed in Vacuo, and the product was subjected to
preparative TLC with n-hexane-EtOAc (9:1) as the developing solvent.
The band at Rf 0.23 was scraped off to give di-(R)-MTPA ester (2)
(1.0 mg; 25% yield). The location of the pivaloyl and MTPA groups
was confirmed by HMBC analysis of 2. Esterification of 1 with (R)-
MTPA chloride was performed in the same manner, giving di-(S)-
1
MTPA ester (2) (Rf 0.15) (1.3 mg: 33%). Di-(R)-MTPA ester (2): H
NMR (400 MHz, CDCl3) δ 7.53–7.31 (10H, m, ArH), 6.15 (1H, d, J
) 15.9 Hz, H-4), 6.01 (1H, dq, J ) 9.0, 6.4 Hz, H-7), 5.87 (1H, dd, J
) 15.9, 6.8 Hz, H-3), 5.62 (1H, dq, J ) 6.8, 6.4 Hz, H-2), 5.50 (1H,
d, J ) 9.0 Hz, H-6), 5.03 (1H, d, J ) 12.5 Hz, H-9a), 4.71 (1H, d, J
) 12.5 Hz, H-9b), 3.54 (3H, brs, -OMe), 3.51 (3H, brs, -OMe), 1.43
(3H, d, J ) 6.4 Hz, H-8), 1.37 (3H, d, J ) 6.4 Hz, H-1), 1.14 (9H, s,
-Piv); HRTOFMS m/z 711.2352 [M + Na]+ (calc for C34H38O8F6Na
711.2369). Di-(S)-MTPA ester (2): 1H NMR (400 MHz, CDCl3) δ
7.53–7.31 (10H, m, ArH), 6.04 (1H, d, J ) 15.5 Hz, H-4), 6.01 (1H,
dq, J ) 9.4, 6.2 Hz, H-7), 5.80 (1H, dd, J ) 15.5, 6.5 Hz, H-3), 5.64
(1H, dq, J ) 6.5, 6.4 Hz, H-2), 5.54 (1H, d, J ) 9.4 Hz, H-6), 4.97
(1H, d, J ) 12.4 Hz, H-9a), 4.69 (1H, d, J ) 12.4 Hz, H-9b), 3.55
(3H, brs, -OMe), 3.51 (3H, brs, -OMe), 1.43 (3H, d, J ) 6.4 Hz,
H-1), 1.35 (3H, d, J ) 6.2 Hz, H-8), 1.12 (9H, s, -Piv); HRTOFMS
m/z 711.2386 [M + Na]+ (calc for C34H38O8F6Na 711.2369).
Aspinotriol B (3): colorless oil; [R]25D +6.1 (c 0.21, MeOH); UV
(MeOH) λmax (log ε) 231 (4.21) nm; 1H and 13C NMR data, see Tables
2 and 3; HRTOFMS m/z 195.1007 [M + Na]+ (calc for C9H16O3Na
195.0997).
Aspinotriol A (1): colorless oil; [R]25 -10.1 (c 0.23, MeOH);
D
1
UV (MeOH) λmax (log ε) 231 (4.22) nm; H and 13C NMR data, see
Tables 1 and 3; HRTOFMS m/z 195.1012 [M + Na]+ (calc for
C9H16O3Na 195.0997).
Di-(R)- and (S)-MTPA Esters (2). Pivaloyl chloride (2.6 µL; 3.6
equiv) was added to a solution of 1 (1.0 mg) in dry pyridine (0.1 mL),
and the mixture was allowed to stand at room temperature for 3 h,
when the TLC spot for the starting material spot had disappeared. Then,
without isolation of the monopivaloyl ester, (S)-MTPA chloride (11
µL) was added to the solution and the mixture was kept at room
temperature for 1 day. MeOH (0.1 mL) was added to destroy the excess
Di-(R)- and (S)-MTPA Esters (4). These esters were prepared as
for 2 (see above). Pivaloyl chloride (1.3 µL; 3.5 equiv) was added to
a solution of 3 (0.5 mg) in pyridine (50 µL), and after 2 h, (S)-MTPA
chloride (4.3 µL; 3.5 equiv) was added. The mixture was allowed to
stand at room temperature for 1 day, and N,N-dimethyl-1,3-propanedi-
amine (3.5 µL) was added to convert the excess chloride to polar
amides. After 10 min, the solvent was removed in Vacuo, and the residue
was separated by preparative TLC [n-hexane-EtOAc (9:1)], giving
di-(R)-MTPA ester (4) (Rf 0.20) (0.8 mg; 40%). Esterification of 3 (0.5
mg) with (R)-MTPACl gave di-(S)-MTPA ester (4) (Rf 0.15) (0.5 mg;
25%). Acetone-d6 was used as an NMR solvent to obtain well-separated
1
signals. Di-(R)-MTPA ester (4): H NMR (400 MHz, acetone-d6) δ
Figure 2. ∆δ values obtained for the MTPA esters, 2, 4, 6, 8, 10,
1
and 11. The H NMR spectra were recorded for CDCl3 solutions
except for 4, for which acetone-d6 was used as a solvent.
Table 4. NMR Spectroscopic Data (100/400 MHz, CD3OD) of
Aspinonediol (5)
position δC, mult.
δH (J in Hz)
COSY HMBC NOESY
2, 3
1
2
3
4
5
6
7
8
9
27.3, CH3
201.5, C
128.4, CH
147.5, CH
137.1, C
148.2, CH
64.8, CH
23.6, CH3
57.0, CH2
2.34, s
6.45, d (16.2)
7.24, d (16.2)
4
3
2, 5
2, 6, 9
9
6
6.09, d (8.5)
4.81, dq (8.5, 6.4)
1.33, d (6.4)
4.38 (s)
7
4, 9
7
6, 8
7
6, 8, 9
7
Figure 3. Biosynthetic pathway (Zeeck et al.) leading to 12a-c,
which are the precursors of 7 and aspyrone as well as 1, 3, 5, and
dihydroaspyrone (9). As for I and II, see text.
6, 7
4, 5, 6
3, 7

Notes
Journal of Natural Products, 2007, Vol. 70, No. 12 2025
7.56–7.41 (10H, m, ArH), 6.16 (1H, d, J ) 16.0 Hz, H-4), 6.09 (1H,
dq, J ) 9.5, 6.6 Hz, H-7), 5.91 (1H, dd, J ) 16.0, 6.6 Hz, H-3), 5.69
(1H, dq, J ) 6.6, 6.5 Hz, H-2), 5.54 (1H, d, J ) 9.5 Hz, H-6), 5.04
(1H, d, J ) 12.2 Hz, H-9a), 4.74 (1H, d, J ) 12.2 Hz, H-9b), 3.59
(3H, brs, -OMe), 3.57 (3H, brs, -OMe), 1.47 (3H, d, J ) 6.6 Hz,
H-8), 1.47 (3H, d, J ) 6.5 Hz, H-1), 1.13 (9H, s, Piv); HRTOFMS m/z
711.2424 [M + Na]+ (calc for C34H38O8F6Na 711.2369). Di-(S)-MTPA
ester (4): 1H NMR (400 MHz, acetone-d6) δ 7.57–7.41 (10H, m, ArH),
6.42 (1H, d, J ) 16.1 Hz, H-4), 6.14 (1H, dq, J ) 9.8, 6.6 Hz, H-7),
6.07 (1H, dd, J ) 16.1, 7.1 Hz, H-3), 5.87 (1H, d, J ) 9.8 Hz, H-6),
5.70 (1H, dq, J ) 7.1, 6.6 Hz, H-2), 5.06 (1H, d, J ) 12.4 Hz, H-9a),
4.85 (1H, d, J ) 12.4 Hz, H-9b), 3.54 (3H, brs, -OMe), 3.52 (3H,
brs, -OMe), 1.39 (3H, d, J ) 6.6 Hz, H-8), 1.39 (3H, d, J ) 6.6 Hz,
H-1), 1.14 (9H, s, Piv); HRTOFMS m/z 711.2318 [M + Na]+ (calc
for C34H38O8F6Na 711.2369).
7.1 Hz, H-5), 4.01 (1H, m, H-9), 2.68 (1H, brd, J ) 7.1 Hz, 5-OH),
2.47 (1H, dd, J ) 14.3, 4.0 Hz, H-8a), 2.40 (1H, dd, J ) 14.3, 8.0 Hz,
H-8b), 2.28 (1H, brs, 9-OH), 1.45 (3H, d, J ) 6.4 Hz, H-7), 1.22 (3H,
d, J ) 6.2 Hz, H-10); 13C NMR (100 MHz, CDCl3) δ 165.2 (C, C-2),
144.3 (CH, C-4), 129.1 (C, C-3), 79.4 (CH, C-6), 67.6 (CH, C-5), 66.9
(CH, C-9), 39.7 (CH2, C-8), 23.4 (CH3, C-10), 18.2 (CH3, C-7). These
NMR data agree well with those reported previously.6
(R)- and (S)-MTPA Esters (10). A solution of 9 (2.4 mg) and
(S)-MTPA chloride (2.9 µL) in pyridine (0.1 mL) was kept at room
temperature for 1 day. The solvent was removed in Vacuo, and the
residue was directly applied to preparative TLC developed with
CHCl3-MeOH (1:50) to give (R)-MTPA ester (10; Rf 0.23) (1.1 mg:
1
21%): H NMR (400 MHz, CDCl3) δ 7.56–7.36 (5H, m, ArH), 6.25
(1H, brs, H-4), 5.33 (1H, m, H-9), 4.15 (1H, dq, J ) 8.8, 6.2 Hz, H-6),
3.86 (1H, brdd, J ) 8.8, 7.0 Hz, H-5), 3.55 (3H, brs, OMe), 2.59 (1H,
dd, J ) 14.8, 3.9 Hz, H-9a), 2.48 (1H, dd, J ) 14.8, 9.3 Hz, H-9b),
1.66 (1H, d, J ) 7.0 Hz, 5-OH), 1.40 (6H, d, J ) 6.2 Hz, H-7, H-10).
Esterification of 9 (1.8 mg) with (R)-MTPA chloride (2.2 µL) was
performed in the same manner, affording (S)-MTPA ester (10) (2.0
Aspinonediol (5): yellow oil; [R]24 +2.4 (c 0.63, MeOH); UV
D
(MeOH) λmax (log ε) 269 (4.43) nm; 1H and 13C NMR data, see Table
4; HRTOFMS m/z 193.0869 [M + Na]+ (calc for C9H14O3Na
193.0841).
1
(R)- and (S)-MTPA Esters (6). These esters were prepared as for
2 (see above). Pivaloyl chloride (3.4 µL; 5.8 equiv) was added to a
solution of 5 (0.8 mg) in pyridine (80 µL), and after 16 h, (S)-MTPA
chloride (8.8 µL; 10 equiv) was added. The mixture was allowed to
stand at room temperature for 10 h, and N,N-dimethyl-1,3-propanedi-
amine (7.1 µL) was added to convert the excess chloride to polar
amides. After 10 min, the solvent was removed in Vacuo, and the residue
was separated by preparative TLC [n-hexane-EtOAc (85:15)], giving
(R)-MTPA ester (6) (Rf 0.17) (0.7 mg; 32%). Esterification of 5 (0.8
mg) with (R)-MTPACl gave (S)-MTPA ester (6) (Rf 0.17) (0.8 mg;
mg; 36%): H NMR (400 MHz, CDCl3) δ 7.51–7.37 (5H, m, ArH),
6.45 (1H, brs, H-4), 5.36 (1H, m, H-9), 4.23 (1H, dq, J ) 8.8, 6.5 Hz,
H-6), 4.01 (1H, brdd, J ) 8.8, 7.3 Hz, H-5), 3.47 (3H, brs, -OMe),
2.62 (1H, dd, J ) 14.0, 4.3 Hz, H-8a), 2.56 (1H, dd, J ) 14.0, 7.8 Hz,
H-8b), 1.82 (1H, d, J ) 7.3 Hz, 5-OH), 1.42 (3H, d, J ) 6.5 Hz, H-7),
1.34 (3H, d, J ) 6.3 Hz, H-10).
(R)- and (S)-MTPA Esters (11). A solution of 9 (5.8 mg), TBDMS
chloride (5.6 mg), and imidazole (6.4 mg) in DMF (0.5 mL) was stirred
at 0 °C for 15 min and then at room temperature for 4 h. The mixture
was diluted with diethyl ether and H2O. The organic layer was separated.
The aqueous layer was again extracted with diethyl ether three times,
and the diethyl ether extract was combined with the first extract.
Evaporation of the solvent gave the TBDMS ether (5.6 mg): 1H NMR
(400 MHz, CDCl3) δ 6.55 (1H, brs, H-4), 4.26 (1H, m, H-6), 4.21
(1H, m, H-5), 4.06 (1H, m, H-9), 2.39 (1H, dd, J ) 13.5, 4.4 Hz, H-8a),
2.31 (1H, dd, J ) 13.5, 7.0 Hz, H-8b), 1.47 (3H, d, J ) 6.4 Hz, H-7),
1.15 (3H, d, J ) 6.4 Hz, H-10), 0.86 (9H, s, t-Bu), 0.03 (3H, s, SiMe),
0.02 (3H, s, SiMe). Equal sized portions of the TBDMS ether were
esterified with (S)- and (R)-MTPA chlorides in pyridine to afford (R)-
(1.6 mg: 33% yield) and (S)-MTPA (0.7 mg: 15% yield) esters (11),
respectively. (R)-MTPA ester (11): 1H NMR (400 MHz, CDCl3) δ
7.54–7.37 (5H, m, ArH), 6.54 (1H, d, J ) 2.8 Hz, H-4), 5.45 (1H, dd,
J ) 8.1, 2.8 Hz, H-5), 4.49 (1H, dq, J ) 8.1, 6.6 Hz, H-6), 4.05 (1H,
m, H-9), 3.53 (3H, brs, OMe), 2.46 (1H, dd, J ) 13.3, 4.2 Hz, H-8a),
2.31 (1H, dd, J ) 13.3, 7.8 Hz, H-8b), 1.32 (3H, d, J ) 6.6 Hz, H-7),
1.14 (3H, d, J ) 6.1 Hz, H-10), 0.86 (9H, s, t-Bu), 0.03 (3H, s, SiMe),
0.00 (3H, s, SiMe). (S)-MTPA ester (11): 1H NMR (400 MHz, CDCl3)
δ 7.54–7.33 (5H, m, ArH), 6.43 (1H, d, J ) 2.9 Hz, H-4), 5.45 (1H,
dd, J ) 7.8, 2.9 Hz, H-5), 4.56 (1H, dq, J ) 7.8, 6.6 Hz, H-6), 4.02
(1H, m, H-9), 3.53 (3H, brs, OMe), 2.43 (1H, dd, J ) 13.4, 4.2 Hz,
H-8a), 2.27 (1H, dd, J ) 13.4, 8.0 Hz, H-8b), 1.42 (3H, d, J ) 6.6 Hz,
H-7), 1.11 (3H, d, J ) 6.0 Hz, H-10), 0.86 (9H, s, t-Bu), 0.02 (3H, s,
SiMe), 0.01 (3H, s, SiMe).
1
36%). (R)-MTPA ester (6): H NMR (400 MHz, CDCl3) δ 7.60–7.34
(5H, m, ArH), 6.99 (1H, d, J ) 16.0 Hz, H-4), 6.36 (1H, d, J ) 16.0
Hz, H-3), 6.04 (1H, dq, J ) 9.3, 6.4 Hz, H-7), 5.87 (1H, d, J )
9.3 Hz, H-6), 5.08 (1H, d, J ) 12.6 Hz, H-9a), 4.75 (1H, d, J ) 12.6
Hz, H-9b), 3.53 (3H, brs, -OMe), 2.28 (3H, s, H-1), 1.47 (3H, d, J )
6.4 Hz, H-8), 1.17 (9H, s, -Piv); HRTOFMS m/z 493.1801 [M + Na]+
(calc for C24H29O6F3Na 493.1814). (S)-MTPA ester (6): 1H NMR (400
MHz, CDCl3) δ 7.60–7.34 (5H, m, ArH), 7.03 (1H, d, J ) 16.2 Hz,
H-4), 6.37 (1H, d, J ) 16.2 Hz, H-3), 6.07 (1H, dq, J ) 9.5, 6.1 Hz,
H-7), 6.00 (1H, d, J ) 9.5 Hz, H-6), 5.07 (1H, d, J ) 12.5 Hz, H-9a),
4.76 (1H, d, J ) 12.5 Hz, H-9b), 3.50 (3H, brs, -OMe), 2.29 (3H, s,
H-1), 1.40 (3H, d, J ) 6.1 Hz, H-8), 1.16 (9H, s, -Piv); HRTOFMS
m/z 493.1835 [M + Na]+ (calc for C24H29O6F3Na 493.1814).
Aspinonene (7): colorless oil; [R]24D -7.3 (c 0.38, MeOH). All of
the spectroscopic data except for optical rotation value were identical
with those reported in the literature.4
(R)- and (S)-MTPA Esters (8). A solution of 7 (15 mg) and
p-toluenesulfonic acid (1 mg) in 2,2-dimethoxypropane (2 mL) was
stirred for 30 min at room temperature, and the solvent was removed
by evaporation. The oily residue was separated by preparative TLC
developing with CHCl3-MeOH (14:1). The band at Rf 0.49 gave an
1
acetonide (7.2 mg; 83%) that showed a H NMR spectrum identical
with that reported.4 A solution of the acetonide (3.7 mg), (R)-MTPA
acid (7.6 mg), 2-methyl-6-nitrobenzoic anhydride (13.4 mg), and
4-(dimethylamino)pyridine (8.7 mg) in CH2Cl2 (1 mL) was stirred at
room temperature for 1 day.9 The mixture was shaken with 10%
aqueous citiric acid solution three times, washed with an aqueous
NaHCO3 solution and brine, and dried over Na2SO4. The crude reaction
product (10.0 mg) was separated by preparative TLC developed with
benzene-EtOAc (20:1). The band at Rf 0.38 gave (R)-MTPA ester (8;
4.1 mg) (57%): 1H NMR (400 MHz, CDCl3) δ 7.54–7.35 (5H, m, ArH),
5.83 (1H, dd, J ) 15.6, 6.1 Hz, H-3), 5.75 (1H, d, J ) 15.6 Hz, H-4),
5.63 (1H, dq, J ) 6.6, 6.1 Hz, H-2), 3.92 (1H, d, J ) 8.8 Hz, H-9a),
3.66 (1H, d, J ) 8.8 Hz, H-9b), 3.00 (1H, dq, J ) 5.1, 2.1 Hz, H-7),
2.62 (1H, d, J ) 2.1 Hz, H-6), 1.43 (3H, d, J ) 6.6 Hz, H-1), 1.38
(3H, s, J ) 6.6 Hz, -OC(CH3)2O-), 1.30 (3H, s, -OC(CH3)2O-),
1.30 (3H, d, J ) 5.1 Hz, H-8). (S)-MTPA ester (8) was obtained in the
same manner in 57% yield. (S)-MTPA ester (8): 1H NMR (400 MHz,
CDCl3) δ 7.54–7.35 (5H, m, ArH), 5.93 (1H, dd, J ) 15.8, 6.5 Hz,
H-3), 5.86 (1H, d, J ) 15.8 Hz, H-4), 5.63 (1H, dq, J ) 6.6, 6.5 Hz,
H-2), 3.95 (1H, d, J ) 8.9 Hz, H-9a), 3.70 (1H, d, J ) 8.9 Hz, H-9b),
3.03 (1H, dq, J ) 5.2, 2.1 Hz, H-7), 2.66 (1H, d, J ) 2.1 Hz, H-6),
1.40 (3H, s, -OC(CH3)2O-), 1.37 (3H, d, J ) 6.5 Hz, H-1), 1.36 (3H,
s, -OC(CH3)2O-), 1.30 (3H, d, J ) 5.2 Hz, H-8).
Supporting Information Available: Copies of 1H and 13C 1D and
2D NMR spectra of compounds 1, 3, and 5. This material is available
free of charge via the Internet at http://pubs.acs.org.
References and Notes
(1) Bhadury, P.; Mohammad, B. T.; Phillip, C. J. Ind. Microbiol. Bioth-
echnol. 2006, 33, 325–337.
(2) Jensen, P. R.; Fenical, W. In Drugs From the Sea; Fusetani, N., Ed.;
Karger: Basel, 2000; pp 6–29.
(3) Namikoshi, M.; Negishi, R.; Nagai, H.; Dmitrenok, A.; Kobayashi, H.
J. Antibiot. 2003, 56, 755–761.
(4) Fuchser, J.; Grabley, S.; Noltemeyer, M.; Phillips, R.; Tiericke, R.;
Zeeck, A. Liebigs Ann. Chem. 1994, 831–835.
(5) Fuchser, J.; Tiericke, R.; Zeeck, A. J. Chem. Soc., Perkin Trans. 1 1995,
1663–1666.
(6) Fuchser, J.; Zeeck, A. Liebigs Ann./Recl. 1997, 87–95.
(7) Kusumi, T.; Ooi, T.; Ohkubo, Y.; Yabuuchi, T. Bull. Chem. Soc. Jpn.
2006, 79, 1–16.
(8) Ohtani, I.; Kusumi, T.; Kashman, Y.; Kakisawa, H. J. Am. Chem. Soc.
1991, 113, 4092–4096.
(9) Shiina, I.; Kubota, M.; Ibuka, R. Tetrahedron Lett. 2002, 43, 7535–7539.
Dihydroaspyrone (9): 1H NMR (400 MHz, CDCl3) δ 6.62 (1H,
brs, H-4), 4.38 (1H, dq, J ) 7.9, 6.4 Hz, H-6), 4.20 (1H, dd, J ) 7.9,
NP070301N