Absolute stereochemistry of pentacecilides
H Yamazaki et al
318
the hydroxy group at C-4¢, synthesis of acyl derivatives at this position Preparation of 3S*-Hydroxy-pentacecilide A
To a solution of pentacecilide A (2.9mg, 0.0070 mmol) in CH3OH (500ml),
is now in progress.
NaBH4 (3.3mg, 0.087mmol) was added, and the resulting solution was stirred
at room temperature. After 10 h, the reaction mixture was diluted with H2O
and the aqueous phase was extracted with EtOAc. The organic layer was
recovered, dried over anhydrous Na2SO4, filtered and concentrated in vacuo to
dryness. The product was purified by preparative HPLC (column; PEGASIL
ODS, 20ꢀ250 mm; Senshu Scientific; solvent, 80% CH3CN; detection, UV at
210 nm; flow rate, 8.0 mlminꢁ1) to give 3 (2.5mg, 0.0060mmol, 85.7%) as a
white powder.
3S*-Hydroxy-pentacecilide A (3): 1H NMR (400MHz, CDCl3) d 0.86
(s, 3H), 1.02 (s, 3H), 1.09 (s, 3H), 1.26 (m, 1H), 1.29 (s, 3H), 1.46, 1.56
(m, 2H), 1.55 (d, 3H, J¼6.0 Hz), 1.66, 1.80 (m, 2H), 1.70 (m, 2H), 1.76
(m, 1H), 2.00, 2.08 (m, 2H), 2.40 (m, 2H), 2.68, 2.85 (dd, 2H, J¼17.0, 11.0Hz,
J¼17.0, 3.5 Hz), 3.26 (dd, J¼10.0, 4.0 Hz, 1H), 4.63 (m, 1H), 6.26 (s, 1H), 11.0
(s, 1H); HRESI-TOF-MS (m/z) found: 437.2290, calcd: 437.2304 [M+Na]+ for
C25H34NaO5.
METHODS
Materials
(S)-(+)- and (R)-(ꢁ)-MTPA chlorides, and sodium borohydride (NaBH4)
were purchased from Wako Pure Chemical Industries (Osaka, Japan). Acetic
anhydride (Ac2O) and 4-(dimethylamino) pyridine were purchased from Kanto
Chemical (Tokyo, Japan). Triethylamine was purchased from Nacalai Tesque
(Kyoto, Japan).
General experimental procedures
Fungal strain P. cecidicola FKI-3765-1 was used for the production of penta-
cecilides.1,2 Pentacecilide D was from 13-day-old culture broth fermented as
reported previously.1 For determination of the amounts of pentacecilide D in
culture broths, samples (ethyl acetate extracts) dissolved in methanol were
analyzed by the HP1100 system (Hewlett-Packard, Palo Alto, CA, USA) under
the following conditions: column, Symmetry (2.1ꢀ150 mm; Waters, Milford,
MA, USA); flow rate, 0.2 mlminꢁ1; mobile phase, a 20-min linear gradient
from 60% CH3CN to 100% CH3CN containing 0.050% H3PO4; detection, UV
at 210 nm. Under these conditions, pentacecilide D was eluted with a retention
time of 3.59min, respectively.
Biological assays
An assay for the synthesis of CE, triacylglycerol and phospholipids by mouse
macrophages was carried out according to the method described previously.11
An assay for ACAT1 and ACAT2 activities in ACAT1- and ACAT2-CHO cells
was carried out by our established method.12,13
SSC-ODS-7515-12 (Senshu Scientific) was used for ODS column chromato-
graphy. HPLC was carried out using the L-6200 system (Hitachi, Tokyo, Japan).
UV spectra were recorded on a spectrophotometer (8453 UV-Visible spectro-
photometer; Agilent Technologies, Santa Clara, CA, USA). IR spectra were
recorded on a Fourier transform infrared spectrometer (FT-710; Horiba, Kyoto,
Japan). Optical rotations were measured with a digital polarimeter (DIP-1000;
JASCO, Tokyo, Japan). ESI-TOF-MS and HRESI-TOF-MS spectra were recorded
on a mass spectrometer (JMS-T100LP; JEOL, Tokyo, Japan). Various NMR
spectra were measured with a spectrometer (XL-400; Varian, Palo Alto, CA, USA).
ACKNOWLEDGEMENTS
This study was supported in part by a Sasakawa Scientific Research Grant
(to HY) from The Japan Science Society. We express our thanks to Ms N Sato
for NMR experiments, and Dr K Nagai and Ms A Nakagawa for measuring
mass spectra.
Preparation of the (S)-(+)- and (R)-(ꢁ)-MTPA ester derivatives
of pentacecilide C
1
2
3
4
Yamazaki, H. et al. Pentacecilides, new inhibitors for lipid droplet formation in mouse
macrophages produced by Penicillium cecidicola FKI-3765-1:I. Taxonomy, fermenta-
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To a solution of pentacecilide C (5.0mg, 0.010 mmol) in CH2Cl2 (500ml),
(R)-(ꢁ)-MTPA chloride (16.5 mg, 0.065 mmol), 4-(dimethylamino) pyridine
(5.3mg, 0.043 mmol) and triethylamine (10ml, 0.073mmol) were added. The
reaction mixture was stirred at room temperature. After 10h, the reaction
mixture was diluted with 1.0 N HCl and the aqueous phase was extracted with
EtOAc. The organic layer was recovered, dried over anhydrous Na2SO4, filtered
and concentrated in vacuo to dryness. The product was purified by preparative
HPLC (column; PEGASIL ODS, 20ꢀ250 mm; Senshu Scientific; solvent, 80%
CH3CN; detection, UV at 210nm; flow rate, 8.0 mlminꢁ1) to give 1 (7.40 mg,
0.0081 mmol, 80.6%) as a white powder. Similarly, 2 (4.63 mg, 0.0050 mmol,
61.5%) was obtained using (S)-(+)-MTPA chloride.
¯
Yamazaki, H., Omura, S.
& Tomoda, H. Pentacecilides, new inhibitors for lipid
droplet formation in mouse macrophages produced by Penicillium cecidicola
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functional expression of human acyl-coenzyme A:cholesterol acyltransferase cDNA in
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specific to liver and intestine in nonhuman primates. J. Biol. Chem. 273, 26747–
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5
6
7, 4¢-(S)-(+)-a-methoxy-a-(trifluoromethyl)phenylacetoxy pentacecilide C
(1): 1H NMR (400 MHz, CDCl3) d 1.05 (s, 3H), 1.06 (s, 3H), 1.10 (s, 3H), 1.36
(s, 3H), 1.52 (d, 3H, J¼7.0 Hz), 1.68, 2.28 (m, 2H), 1.85, 2.14 (m, 2H), 2.17
(s, 3H), 2.57 (m, 2H), 2.74, 2.82 (dd, 2H, J¼17.0, 11.0Hz, J¼17.0, 3.5Hz), 3.52
(s, 3H), 3.73 (s, 3H), 4.58 (m, 1H), 5.54 (m, 1H), 5.56 (m, 1H), 6.35 (s, 1H),
7.26 (m, 3H), 7.44 (m, 3H), 7.52 (m, 2H), 7.75 (m, 2H); HRESI-TOF-MS
(m/z) found: 941.2935, calcd: 941.2948 [M+Na]+ for C47H48 F8NaO12.
7, 4¢-(R)-(ꢁ)-a-methoxy-a-(trifluoromethyl)phenylacetoxy pentacecilide C
(2): 1H NMR (400 MHz, CDCl3) d 1.14 (s, 3H), 1.17 (s, 3H), 1.30 (s, 3H), 1.33
(s, 3H), 1.53 (d, 3H, J¼7.0Hz), 1.87, 2.16 (m, 2H), 2.31 (m, 2H), 2.17 (s, 3H),
2.50 (m, 2H), 2.74, 2.82 (dd, 2H, J¼17.0, 11.0 Hz, J¼17.0, 3.5 Hz), 3.53 (s, 3H),
3.76 (s, 3H), 4.57 (m, 1H), 5.60 (m, 1H), 5.61 (m, 1H), 6.31 (s, 1H), 7.32 (m,
3H), 7.44 (m, 3H), 7.52 (m, 2H), 7.74 (m, 2H); HRESI-TOF-MS (m/z) found:
941.2980, calcd: 941.2948 [M+Na]+ for C47H48 F8NaO12.
7
Parini, P. et al. ACAT2 is localized to hepatocytes and is the major cholesterol-
esterifying enzyme in human liver. Circulation 110, 2017–2023 (2004).
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¯
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12 Lada, A. T. et al. Identification of ACAT1- and ACAT2-specific inhibitors using a
novel, cell-based fluorescence assay: individual ACAT uniqueness. J. Lipid. Res. 45,
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¯
13 Ohshiro, T., Rudel, L. L., Omura, S. & Tomoda, H. Selectivity of microbial acyl-CoA:
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The Journal of Antibiotics