Notes
Isola tion of Cer a m id e (1). Cells of C. monotis were
J ournal of Natural Products, 1998, Vol. 61, No. 5 687
P r ep a r a tion of Tr is(br om o)ben zoyl sp h in gosin e
6. Sphingosine 3 (200 µg) obtained by methanolysis was
hydrogenated with H2 on Rh-Al2O3 in MeOH for 12 h.
After removal of the catalyst and solvent, the product
was dissolved in pyridine (100 µL) containing p-bro-
mobenzoyl chloride (3 mg) and (dimethylamino)pyridine
(2 mg), and left to stand for 12 h at room temperature.
After the addition of H2O, the product was extracted
with EtOAc and subjected to HPLC on a Si gel column
(YMC-pack A-024 SIL; φ 10 × 250 mm; flow rate, 2.0
mL/min) and eluted with hexane-EtOAc (8:1). The UV-
positive fraction at 17 min was separated and used for
CD measurements. Authentic sphingosine (D-erythro)
was derivatized to the corresponding bromobenzoate
and purified by the same procedure.
harvested by filtering a culture medium through a glass
fiber filter (Advantech, GP-100) and stored at -30 °C
until extraction. The algal cells obtained from 180 L of
the culture were extracted with MeOH four times and
then with Me2CO three times. The combined extracts
were evaporated to dryness and suspended in MeOH-
H2O (1:1). The suspension was washed with n-hexane
and extracted with CH2Cl2. The hexane solution was
again extracted with MeOH-H2O (98:2), and the MeOH
phase was combined with the CH2Cl2 extracts. After
removal of the solvents, the extracts were dissolved in
MeOH and passed through a column of HP-20 to remove
nonpolar constituents. The eluate was then loaded onto
a DIOL column (Lobar Pre-packed, Merck, Size B) and
eluted with MeOH. The fraction of 70-110 mL was
subjected to further purification with HPLC. Reversed-
phase chromatography was carried out with an ODS
column (YMC, AM-323-7, φ 10 × 250 mm, flow rate,
2.0 mL/min) and linear gradient elutions from MeOH-
H2O (4:1) to 100% MeOH (0.8% per min). A series of
ceramides were eluted at 128 min-156 min. Final
purification was carried out on the same column with
the following elution program; CH3CN-H2O-AcOH (17:
3:0.04) for 30 min and then a linear gradient of the same
mobile phase to 100% CH3CN at 0.6%/min at a flow rate
of 2.0 mL/min. Several ceramides were obtained, among
which 1 (2.4 mg) was eluted at 120-128 min as a major
constituent.
Cer a m id e (1): IR bands (liquid film) νmax 3360, 2852,
1650, 1538, 1463, 1050 cm-1 1H NMR (CD3OD, 500
;
MHz) δ 7.48 (1H, d, J ) 8.5 Hz, N-H), 6.00 (2H, m,
H-9, H-10), 5.84, (1H, dtd, J ) 1.6, 6.8, 15.5 Hz, H-4′),
5.71 (1H, dt, J ) 5.9, 15.3 Hz, H-5), 5.55 (2H, m, H-8,
H-11), 5.52 (1H, m, H-3′), 5.49 (1H, m, H-4), 4.44 (1H,
d, J ) 7.1 Hz, H-2′), 4.08 (1H, t, J ) 4.4 Hz, H-3), 3.83
(1H, m, H-2), 3.80 (1H, dd, J ) 3.5, 11.1 Hz, H-1), 3.68
(1H, dd, J ) 3.1, 11.1, H-1), 2.12 (2H, m, H2-7), 2.08
(2H, m, H2-6), 2.05 (4H, m, H2-12, H2-5′), 1.38 (5H, m,
H2-13, H2-6′, H-15′), 1.25-1.35 (24H, m, H2-14, H2-15,
H2-16, H2-17, H2-7′, H2-8′, H2-9′, H2-10′, H2-11′, H2-12′,
H2-13′, H2-14′), 1.10 (2H, m, H2-16′), 0.88 (6H, t, J )
6.7 Hz, H3-18, H3-18′), 0.85 (3H, d, J ) 6.8 Hz, H3-
C15′); 13C NMR (CD3OD, 125 MHz) δ 174.7 (s, C1′),
134.3 (d, C4′), 133.3 (d, C5), 132.8 (d, C11a), 131.6 (d,
C9), 131.6 (d, C10), 131.1 (d, C8a), 130.8 (d, C4), 128.5
(d, C3′), 73.3 (d, C2′), 72.7 (d, C3), 61.1 (t, C1), 55.5 (d,
C2), 40.1 (t, C16′b), 37.5 (t, C14′b), 32.5-33.2, 29.2-30.5,
27.6 (t, C13′), 23.2 (t, C17), 20.8 (t, C17′), 19.6 (q, CH3-
C15′), 14.2 (q, C18′), 13.8 (q, C18). a,bAssignments with
the same letter may be interchanged.
Sp h in gosin e 3: 1H NMR (CD3OD, 500 MHz,
CD2HOD taken as δ 3.35) 6.00 (2H, m, H-9, H-10), 5.77
(1H, dt, J ) 5.9, 15.3 Hz, H-5), 5.55 (2H, m, H-8, H-11),
5.53 (1H, m, H-4), 4.03 (1H, t, J ) 6.4 Hz, H-3), 3.72
(1H, dd, J ) 3.5, 11.1 Hz, H-1), 3.54 (1H, dd, J ) 3.5,
11.1 Hz, H-1), 2.82 (1H, m, H-2), 2.12 (2H, m, H2-7),
2.08 (2H, m, H2-6), 2.05 (2H, m, H2-12), 1.38 (2H, m,
H2-13), 1.25-1.35 (8H, m, H2-14, H2-15, H2-16, H2-17),
0.88 (3H, t, J ) 6.7 Hz, H3-18).
Meth a n olysis of Cer a m id e (1). Ceramide (1) (0.5
mg) was subjected to methanolysis in 0.8 mL of 0.75 N
HCl in MeOH at 80 °C for 18 h. Methyl ester 4 was
extracted from the MeOH solution with hexane. After
removal of MeOH, the residue was suspended in H2O,
and sphingosine 3 was extracted with EtOAc.
P r ep a r a t ion of Bis-b en zoylm et h ylglycer a t e 5.
Methyl ester 4 (200 µg) was dissolved in MeOH (800
µL) and oxidized with O3 at -78 °C followed by the
addition of NaBH4 in MeOH until the ozone color
disappeared. After removal of the solvent, the product
was treated with benzoyl chloride in pyridine for 48 h.
The products were subjected to HPLC using a Si gel
column (YMC-pack A-024 SIL; φ 10 × 250 mm; flow
rate, 2.0 mL/min) and eluted with hexane-EtOAc (3:
1). The UV-positive fraction at 15 min was separated
and used for chiral HPLC. Authentic R-glyceric acid
(2 mg) was dissolved in H2O and added along with
trimethylsilyldiazomethane (10% hexane solution). Af-
ter being allowed to stand for 1 h, the excess reagent
was quenched with AcOH, and the solvents were
removed to afford methylglycerate. The ester was
derivatized and purified by the same procedure as
described above. Authentic S-ester was prepared from
S-glyceric acid in the same manner.
F a tty a cid m eth yl ester 4: 1H NMR (CDCl3, 500
MHz, CHCl3 taken as δ 7.24) 5.86 (1H, dtd, J ) 1.6,
6.5, 15.5 Hz, H-4′), 5.48 (1H, m, H-3′), 4.58 (1H, dd, J )
6.5, 1.6 Hz, H-2′), 2.04 (2H, m, H2-5′), 1.25-1.38 (19H,
m, H2-6′, H2-7′, H2-8′, H2-9′, H2-10′, H2-11′, H2-12′, H2-
13′, H2-14′, H-15′), 1.05 (2H, m, H2-16′), 0.86 (3H, t, J
) 6.7 Hz, H3-18′), 0.81 (3H, d, J ) 6.8 Hz, H3-C15′).
Ch ir a l HP LC An a lysis. Comparison of the benzoyl
derivative 5 obtained from the natrual ceramide 1 with
authentic bis-benzoylmethylglycerate with R- and S-
configurations was carried out by chiral HPLC analysis;
column, Chiral Pak AD (L 4.6 × 250 mm, Daicel
Chemical Industory); mobile phase, hexane-iPrOH,
500:4; flow rate, 0.5 mL/min; monitor, UV at 254 nm.
Bis-benzoylmethylglycerate derived from 1 was eluted
at 12′53′′ and authentic R-ester at 12′56′′, while S-
derivative was detected at 20′34′′.
Ack n ow led gm en t. We are grateful to Prof. Y.
Fukuyo, the University of Tokyo, for identifying the
dinoflagellate species; to Dr. K. Yamada and Prof.
Higuchi, Kyushu University, for providing valuable
information; and to H. Kobayashi, Y. Kitamura, and H.
Nakagawa for their assistance in culturing the di-
noflagellates. This work was supported by Special
Coordination Funds from the Science and Technology
Agency J apan (Asian Network on Microbial Research),
and by the “Research for the Future” program of the