New cerebrosides from a marine sponge Haliclona (Reniera) sp

Article abstract of DOI:10.1248/cpb.57.106

A chemical investigation of the MeOH extract of a marine sponge Haliclona (Reniera) sp., collected off the coast of Ulleung Island, Korea, led to the isolation of thirteen new cerebrosides (1-3, 5-14), along with a known analogue (4). Their structures wer

Full text of DOI:10.1248/cpb.57.106

1
06
Notes
Chem. Pharm. Bull. 57(1) 106—111 (2009)
Vol. 57, No. 1
New Cerebrosides from a Marine Sponge Haliclona (Reniera) sp.
a
b
a
a
Taeseong PARK, Tayyab Ahmad MANSOOR, Pramod Bapurao SHINDE, Baoquan BAO,
c
,a
Jongki HONG, and Jee Hyung JUNG*
a
b
College of Pharmacy, Pusan National University; Busan 609–735, Korea: Faculdade de Farmácia, Universidade de
c
Lisboa; Av. D. Forças Armadas, Lisboa 1600–083, Portugal: and College of Pharmacy, Kyung Hee University; Seoul
1
30–701, Korea. Received September 29, 2008; accepted October 27, 2008; published online October 28, 2008
A chemical investigation of the MeOH extract of a marine sponge Haliclona (Reniera) sp., collected off the
coast of Ulleung Island, Korea, led to the isolation of thirteen new cerebrosides (1—3, 5—14), along with a
known analogue (4). Their structures were elucidated on the basis of 1D and 2D NMR spectroscopy, MS spec-
trometry, and chemical method. The major new features of these glucocerebrosides are C15 and C19 acyl chains,
long (C –C ) acyl chains, or the S-configuration of the acyl chains. It is noteworthy that both R- and S-configu-
2
4
28
rations of the acyl chains were observed in the same specimen.
Key words Haliclona (Reniera) sp.; cerebroside; marine sponge
Sponges of the genus Haliclona have been extensively MeOH (LD50 27 mg/ml) and n-hexane (LD 45 mg/ml). Sep-
50
studied and afforded around 200 compounds belonging to aration of the n-hexane layer by medium pressure liquid
different chemical classes. These compounds include the cy- chromatography (MPLC) on a normal-phase flash column
1)
totoxic tertiary alkaloids haliclonacyclamines, antifungal yielded 20 fractions. Fraction 16 was found to contain sugar
2
)
1
pentacyclic alkaloid papuamine, antitumor alkaloid manza- moieties as observed in H-NMR spectra, and was subjected
3)
mine, )and anti-inflammatory cyclic depsipeptides hali- to repeated HPLC chromatographic separation using ODS
4
peptins, to name a few.
column to yield nine cerebrosides (1—9). And the mixture of
In our previous study on the marine sponge Haliclona (Re- fractions 10 to 15 afforded five analogues (10—14) through
niera) sp., we reported the isolation and structure elucidation HPLC separation using the same condition as for fraction 16.
5
)
6)
1
13
of eight new cerebrosides and a new sphingosine obtained Their structures were defined using NMR ( H, C, correla-
from the MeOH extract. Cerebrosides and sphingosine deriv- tion spectroscopy (COSY), heteronuclear single quantum
atives have been isolated from diverse natural sources, correlation (HSQC), heteronuclear multiple bond correlation
7)
8)
9)
including plants, marine sources, microorganisms, and (HMBC), and total correlation spectroscopy (TOCSY)) and
human. Phytosphingosine type cerebrosides are reported to MS analysis, optical rotation data, and chemical method.
exhibit bioactivities such as the antitumor, antiviral, inhi-
bition of histidine decarboxylase, cytotoxicity, and anti- solid. Its molecular formula was assigned as C H NO on
10)
11)
12)
Renieroside C (1) was obtained as a white, amorphous
1
13)
14)
3
9
78
10
14)
ꢀ
fungal activity.
the basis of HR-FAB-MS ([MꢀH] at m/z: 720.5600, D
In a continuing study on the same sponge, fourteen phy- ꢁ2.6 mmu) and 1D and 2D NMR spectroscopic analyses.
tosphingosine type cerebrosides (1—14) were isolated from The characteristic signals of a sugar (an anomeric proton at
the n-hexane layer. Nine (1—9) of them contained saturated d 4.30), an amide linkage (a nitrogenated methine proton at
H
acyl chains, while five (10—14) possessed unsaturated acyl d 4.27, and a carbonyl carbon at d 174.8), and a long acyl
H
C
chains. Their structures were established on the basis of
NMR and MS analyses, and chemical methods. Some of the
cerebrosides (1, 3, 5, 7, 11, 12) contained unusual acyl
chains with S-configuration. It is interesting to isolate
epimeric pairs (1 : 2, 3 : 4, 5 : 6, 7 : 8) from the same speci-
men. Herein, we report isolation and structural characteriza-
tion of cerebrosides from a marine sponge Haliclona (Re-
niera) sp.
The brine shrimp-active MeOH extract (LD50 126 mg/ml)
of the sponge was partitioned between CH Cl and H O. The
2
2
2
CH Cl layer was further partitioned between aqueous Fig. 1. Key COSY and HMBC Correlations of Compounds 1—14
2
2
∗
To whom correspondence should be addressed. e-mail: jhjung@pusan.ac.kr.
© 2009 Pharmaceutical Society of Japan

January 2009
107
chain (terminal methyl protons at dH 0.90 and methylene be the same as compound 1 on the basis of HR-FAB-MS
ꢀ
1
13
protons at d 1.55) were observed, indicating its nature as a ([MꢀH] at m/z: 720.5624, D ꢁ0.2 mmu), and H- and C-
H
1
3
glycosphingolipid. In the C-NMR data, the carbon reso- NMR data. The NMR data of 2 was found to be essentially
nances appeared at 103.5 (C-1ꢂ), 73.8 (C-2ꢂ), 77.0 (C-3ꢂ), identical to those of 1. Methanolysis of 2 gave rise to a satu-
7
0.5 (C-4ꢂ), 77.0 (C-5ꢂ), and 61.5 (C-6ꢂ), revealing the pres- rated FAME, which was analyzed to have the same molecular
15)
ꢀ
ence of a b-glucopyranoside. The coupling constant of the weight as that of 1 (C H O , [MꢀNa] at m/z: 295) but
1
6
32
3
1
3
27
anomeric proton at d 4.30 (d, J ꢃ8.0 Hz), and C chemical with opposite optical rotation ([a]D ꢁ3.7, MeOH). Hence, it
HH
shift value (d 103.5) further confirmed the b configuration was characterized as (2R)-2-hydroxypentadecanoic acid, and
C
of the glucose unit (in the case of a-glucopyranoside: the structure of 2 was defined as 1-O-b-D-glucopyranosyl-
16)
J
ꢃ3.7 Hz, d 98.5). Correlation spectroscopy (COSY) (2S,3S,4R)-2-[(2ꢄR)-2ꢄ-hydroxypentadecanoylamino]-16-
HH
C
showed correlations between the H-2 signal (d 4.27) and methyl-heptadeca-1,3,4-triol.
both the H-3 (d 3.63) and the H-1 (d 4.07, 3.82) signals.
Presence of both epimeric forms (1, 2) in the same speci-
Similarly, COSY correlations were observed between H-3 men is noteworthy. R-Configuration of a-hydroxyl group of
signal (d 3.63), H-4 (d 3.53), and H-5 (d 1.40) signals of the the acyl chain is common in nature. However, the S-configu-
sphingosine moiety. The iso-form terminal of the sphingo- ration is rare. Therefore, it was tried to confirm the configu-
18)
sine moiety was determined from the six-proton doublet at d ration by Mosher’s method using compounds 10 and 11.
1
0
.87 (Jꢃ6.5 Hz) in the H-NMR spectrum of the sphingosine However, the adjacent multiplet methylene protons were
base obtained by methanolysis of the mixture of 1—9 (vide crowded and not well resolved preventing accurate assign-
infra). COSY spectrum also showed correlations from H-2ꢄ ment.
(
d 4.04) to H-3ꢄ (d 1.58). HMBC spectrum showed correla-
The gross structures of compounds 3—9 were defined to
tions from H-2ꢄ and H-3ꢄ to carbonyl carbon C-1ꢄ at d 174.8. be analogous to compound 1, except differences in the length
Furthermore, the HMBC spectrum revealed the correlation of acyl chains and the configuration of the hydroxyl group.
from H-1 (d 4.07, 3.82) to anomeric carbon at d 103.5 (C- The acyl moieties of compounds 3 and 4 were characterized
H
1
ꢂ) (Fig. 1). Methanolysis of 1 yielded fatty acid methyl ester as (2S)-2-hydroxyhexadecanoic acid and (2R)-2-hydroxy-
(
FAME), and its molecular formula was established as hexadecanoic acid, respectively, on the basis of analysis of
ꢀ
ꢀ
ꢀ
27
D
C H O ([MꢀNa] at m/z: 295, [MꢀH] at m/z: 273). The the FAMEs thereof (FAME of 3: [MꢀNa] at m/z 309, [a]
1
6
32
3
2
7
ꢀ
27
D
optical rotation of the ester ([a] ꢀ3.2, MeOH) suggested it ꢀ3.8; FAME of 4: [MꢀNa] at m/z 309, [a] ꢁ6.0). The
D
17)
to be an S isomer. Thus, the fatty acid moiety was deduced molecular structure of 4 (renieroside C ) has been previously
4
19)
to be (2S)-2-hydroxypentadecanoic acid. On the basis of the reported from the starfish Linckia laevigata. Similarly, the
above mentioned data the structure of 1 was defined as 1-O- structures of the acyl moieties of compounds 5—9 were de-
b-D-glucopyranosyl-(2S,3S,4R)-2-[(2ꢄS)-2ꢄ-hydroxypentade- fined as (2S)-2-hydroxyheptadecanoic acid, (2R)-2-hydroxy-
canoylamino]-16-methyl-heptadeca-1,3,4-triol.
heptadecanoic acid, (2S)-2-hydroxyoctadecanoic acid, (2R)-
The molecular formula of renieroside C (2) was found to 2-hydroxyoctadecanoic acid, (2R)-2-hydroxynonadecanoic
2
acid, respectively (see Experimental).
Due to the paucity of individual samples, stereochemistry
1
13
Table 1. H- and C-NMR Data of 1 (CD OD, 500 MHz)
3
of the sphingosine and glucose units was determined by
using a mixture of 1—9. Methanolysis of the mixture fol-
lowed by silica-gel column chromatography afforded sphin-
a)
b)
Position
dH
dC
1
13
Lipid base unit
1
gosine base and methyl glucoside. H- and C-NMR data
(Fig. 2) of the tetraacetyl derivative of the sphingosine base
together with its specific rotation (Table 3) allowed the as-
4.07 (dd, 11.0, 6.5)
68.5
3
.82 (dd, 10.5, 4.0)
2
3
4
5
4.27 (m)
3.63 (m)
3.53 (m)
1.40 (m)
1.31 (m)
1.55 (m)
0.88 (m)
50.5
74.4
71.9
34.5
32—29
25.4
20)
signment of (2S,3S,4R) stereochemistry. The specific rota-
2
7
tion of the methyl glucoside ([a] ꢀ47.6, MeOH) indicated
D
21)
a D-configuration.
6
6
—15
Renieroside C10 (10) was obtained as a white amorphous
1
1
solid. Its molecular formula was assigned as C H NO on
4
8
94
10
7, 18
21.5
N-Acyl unit
1
2
3
4
ꢄ
ꢄ
ꢄ
174.8
71.9
32.0
32—29
13.0
4.04 (dd, 7.5, 4.0)
1.58 (m)
1.31 (m)
ꢄ—14ꢄ
1
5ꢄ
Glucose unit
0.90 (m)
1ꢂ
2ꢂ
3ꢂ
4ꢂ
5ꢂ
6ꢂ
4.30 (d, 8.0)
3.19 (m)
103.5
73.8
77.0
70.5
77.0
61.5
3.37 (m)
3.26 (m)
3.27 (m)
c)
c)
3.87 (d, 12.0)
3
.69 (dd, 12.0, 5.5)
1
13
Fig. 2. Characteristic H- and C-NMR Data of the Sphingosine Base in
CDCl (d Value)
a) Multiplicities and coupling constants are in parentheses. b) Assignments were
based on HMBC and HSQC experiments. c) Overlapped with solvent peak (assign-
ments were secured by HSQC experiment).
3
a
b
25)
c
Renieroside C, natural product isolated from Asterina pectinifera, and synthetic
26)
product. (* Assignment may be reversed.)

1
08
Vol. 57, No. 1
1
13
Table 2. H- and C-NMR Data of 10 (C D N, 500 MHz)
5
5
a)
b)
Position
dH
dC
Lipid base unit
1
4.74 (dd, 11.0, 6.5)
.55 (dd, 11.0, 4.5)
71.0
4
2
3
4
5
6
6
5.29 (m)
4.30 (m)
4.22 (m)
1.93 (m)
1.29 (m)
1.47
52.1
76.0
71.9
34.6
32—29
28.5
c)
c)
—15
1
1
7, 18
0.88 (m)
23.0
N-Acyl unit
NH
8.60 (d, 9.5)
1
2
3
4
ꢄ
ꢄ
ꢄ
176.0
72.8
32.0
32.0—29.0
28.0
130.4
14.7
4.60 (dd, 7.0, 3.0)
2.01 (m)
1.29 (m)
2.12 (m)
5.51 (m)
ꢄ—13ꢄ, 18ꢄ—23ꢄ
1
1
2
4ꢄ, 17ꢄ
5ꢄ, 16ꢄ
4ꢄ
0.88 (m)
Glucose unit
ꢀ
Fig. 3. Key FAB-CID Tandem Mass Fragmentations of the [MꢀNa] Ions
of the FAMEs Derived from 10—14
1ꢂ
2ꢂ
3ꢂ
4ꢂ
5ꢂ
6ꢂ
4.97 (d, 8.0)
106.2
75.3
78.9
72.4
79.0
63.0
4.03 (t, Jꢃ8.5)
c)
4.22 (m)
4.22 (m)
c)
ꢀ
3.89 (m)
4.48 (d, 12.0, 2.5)
to be C H NO on the basis of HR-FAB-MS ([MꢀH] at
4
9
94
10
1
13
m/z: 858.7019, D ꢁ1.5 mmu), and H- and C-NMR spec-
troscopic results. Again, the H- and C-NMR spectroscopic
4
.35 (dd, 12.0, 5.5)
1 13
data were found to be nearly identical to those of 10. The
a) Multiplicities and coupling constants are in parentheses. b) Assignments were
based on HMBC and HSQC experiments. c) J values could not be observed because methanolysis of 11 gave rise to a different FAME. Its molec-
of overlapping with other signals.
ular formula was established as C H O on the basis of
2
6
50
3
ꢀ
FAB-MS data ([MꢀNa] at m/z: 433). The optical rotation
2
7
Table 3. Comparison of Specific Rotations for Tetraacetyl Derivatives of of the FAME ([a] ꢀ22.3, CDCl ) suggested it as the S iso-
D
3
17)
the (2S,3S,4R)-Sphingosine Base
mer. The acyl moiety was defined as (2S,16Z)-2-hydroxy-
pentacos-16-enoic acid. The location of the double bond was
determined by FAB-CID-MS/MS analysis of the [MꢀNa]
ion at m/z: 433. Allylic cleavages were observed as enhanced
peaks at m/z: 333 and 279, indicating the location of the
double bond at C-16 (Fig. 3). All these data were in agree-
ment with the proposed structure of 11 as 1-O-b-D-glucopy-
ranosyl-(2S,3S,4R)-2-[(2ꢄS,16ꢄZ)-2ꢄ-hydroxypentacos-16ꢄ-
enoylamino]-16-methyl-heptadeca-1,3,4-triol.
2
7
Conc. (g/100 ml)
[a] (CHCl3)
D
a)
Renieroside C
0.1
1.5
1.5
ꢀ25.0
ꢀ28.9
ꢀ27.9
b)
Natural product
c)
Synthetic product
25)
a) Renieroside C. b) Natural product isolated from Asterina pectinifera.
c)
26)
Synthetic product.
ꢀ
the basis of HR-FAB-MS ([MꢀH] at m/z: 844.6907, D
The gross structures of compounds 12—14 were defin-
ꢀ
2.9 mmu) and 1D and 2D NMR spectroscopic analyses. ed to be analogous to compound 11, with only differences
1
13
The H- and C-NMR spectroscopic data were found to be in the length of acyl chain and the location of the double
nearly identical to those of 1, except for the presence of a bond thereon. The acyl moieties of compounds 12—14 were
double bond (d 5.51, d 2.12). The methanolysis of 10 gave characterized as (2S,17Z)-2-hydroxyhexacos-17-enoic acid,
H
H
rise to a differed FAME. Its molecular formula was estab- (2R,20Z)-2-hydroxyheptacos-20-enoic acid, and (2R,21Z)-2-
ꢀ
lished as C H O on the basis of FAB-MS data ([MꢀNa]
hydroxyoctacos-21-enoic acid, respectively, on the basis of
2
5
48
3
ꢀ
at m/z: 419). The location of the double bond was determined the analysis of FAMEs thereof (FAME of 12: [MꢀNa] m/z
ꢀ
27
ꢀ
27
D
by FAB-CID-MS/MS analysis of the [MꢀNa] ion at m/z: 447, [a]D ꢀ10.0; FAME of 13: [MꢀNa] at m/z 461, [a]
ꢀ
27
D
4
19. Allylic cleavages were observed as enhanced peaks at ꢁ6.38; FAME of 14: [MꢀNa] at m/z 475, [a] ꢁ22.0).
m/z: 319 and 265, indicating the location of the double bond
To the best of our knowledge, this is the first report on the
1
5ꢄ
at C-15ꢄ (Fig. 3). The geometry of D was assigned as Z on isolation of glucocerebrosides containing saturated C15 and
the basis of chemical shifts of allylic carbons C-14ꢄ and C- C acyl chains. This is also the first report on the isolation of
1
9
22)
1
7ꢄ, which appeared at d 28.0. The optical rotation of the isomeric pairs of cerebrosides. a-Hydroxy acyl chains with
C
2
7
2
1
)
FAME ([a] ꢁ5.82, CDCl ) indicated it as the R isomer.
On the basis of these data, the fatty acid moiety was deduced ported from bacterial genera Salmonella sp., Klebsiella sp.,
S-configuration were rather uncommon, and previously re-
D
3
2
3,24)
to be (2R,15Z)-2-hydroxytetracos-15-enoic acid. Therefore, Serratia sp., Escherichia coli, and so on.
the structure of compound 10 was defined as 1-O-b-D-glu-
Experimental
copyranosyl-(2S,3S,4R)-2-[(2ꢄR,15ꢄZ)-2ꢄ-hydroxytetracos-
General Experimental Procedures Optical rotations were measured
using a JASCO P-1020 polarimeter. 1D and 2D NMR spectra were recorded
1
5ꢄ-enoylamino]-16-methyl-heptadeca-1,3,4-triol.
The molecular formula of renieroside C11 (11) was found on Varian UNITY 400 and Varian INOVA 500 spectrometers. Chemical

January 2009
109
shifts were reported with reference to the respective residual solvent or NMR (500 MHz, CD OD) d: 4.12 (1H, dd, Jꢃ8.0, 4.8 Hz, H-2), 3.71 (3H, s,
3
deuterated solvent peaks (dH 3.30 and dC 49.0 for CD OD). FAB-MS data OCH ), 1.61 (2H, m, H-3), 1.28 (24H, m, H-4—H-15), 0.89 (3H, t,
3
3
1
3
were obtained on a JEOL JMS SX-102A. HR-FAB-MS data were obtained Jꢃ7.0 Hz, H-16); C-NMR (assignments based on HMBC and HSQC ex-
on a JEOL JMS SX-101A. HPLC was performed on a Gilson 370 pump
with an YMC packed J’sphere ODS-H80 column (250ꢅ10 mm, 4 mm, 80 Å)
using a Shodex RI-71 detector.
Animal Material The sponge, Haliclona (Reniera) sp., was collected hydroxyhexadecanoylamino]-16-methyl-heptadeca-1,3,4-triol White
off the coast of Ulleung Island, Korea in October, 2001, using SCUBA (20 m amorphous solid; [a] ꢀ5.8 (cꢃ0.23, MeOH); FAB-MS (ꢀve mode) m/z:
depth). The collected sample was frozen immediately. The specimen (sam- 756 [MꢀNa] , (ꢁve mode) m/z: 732 [MꢁH] ; HR-FAB-MS m/z: 734.5803
periments, 500 MHz, CD OD) d: 175.0 (C-1), 71.0 (C-2), 51.0 (OCH ), 34.5
3 3
ꢀ
(C-3), 28.0—31.0 (C-4—C-15), 13.0 (C-16); FAB-MS m/z: 309 [MꢀNa] .
Renieroside C4 (4), 1-O-b-D-Glucopyranosyl-[(2S,3S,4R)-2-(2ꢀR)-2ꢀ-
2
7
D
ꢀ
ꢁ
ꢀ
ple No. J01U-6) was identified as Haliclona (Reniera) sp. by Prof. Sim, C. J. [MꢀH] (Calcd for C H NO : 734.5782).
4
0
80
10
5)
and has been described elsewhere. A voucher specimen of the sponge (reg-
istry No. Spo. 45) was deposited in the Natural History Museum, Hannam
University, Daejon, Korea.
Methanolysis of 4 Cerebroside (2.0 mg) was subjected to methanolysis
2
7
1
similarly to 1 to yield a FAME (0.3 mg). [a]D ꢁ6.0 (cꢃ0.03, MeOH); H-
NMR (500 MHz, CD OD) d: 4.12 (1H, dd, Jꢃ8.0, 4.8 Hz, H-2), 3.71 (3H, s,
3
Extraction and Isolation The frozen sponge (7 kg) was extracted with OCH ), 1.61 (2H, m, H-3), 1.28 (24H, m, H-4—H-15), 0.89 (3H, t,
3
1
3
MeOH at room temperature. The MeOH extract (29.2 g) showed toxicity Jꢃ7.0 Hz, H-16); C-NMR (assignments based on HMBC and HSQC ex-
against brine shrimp larvae (LD50 126 mg/ml). The MeOH extract was parti- periments, 500 MHz, CD OD) d: 175.0 (C-1), 71.0 (C-2), 51.0 (OCH ), 34.5
3
3
ꢀ
tioned between CH Cl and H O. The CH Cl layer (6.6 g, LD 203 mg/ml)
(C-3), 28.0—31.0 (C-4—C-15), 13.0 (C-16); FAB-MS m/z: 309 [MꢀNa] .
Renieroside C5 (5), 1-O-b-D-Glucopyranosyl-[(2S,3S,4R)-2-(2ꢀS)-2ꢀ-
2
2
2
2
2
50
was further partitioned between aqueous MeOH (1.9 g, LD50 27 mg/ml) and
n-hexane layer (3.9 g, LD50 45 mg/ml). A portion of the n-hexane layer hydroxyheptadecanoylamino]-16-methyl-heptadeca-1,3,4-triol White
2
7
1
(
3.0 g) was subjected to Silica gel 60 (15—40 mm) column chromatography amorphous solid; [a]D ꢀ1.6 (cꢃ0.08, MeOH); H-NMR (500 MHz,
eluting with a gradient solvent system of 100% CH Cl to 100% MeOH, to
CD OD) d: 4.28 (1H, d, Jꢃ7.0 Hz), 4.25 (1H, m), 4.05 (1H, dd, Jꢃ10.5,
2
2
3
afford 20 fractions. Fraction 16 (64 mg) was subjected to RP-HPLC (YMC 6.0 Hz), 4.02 (1H, dd, Jꢃ8.0, 4.5 Hz), 3.87 (1H, br d, Jꢃ12.0 Hz), 3.80 (1H,
packed J’sphere ODS-H80 column, 250ꢅ10 mm, 4 mm, 80 Å), eluting with
6% aqueous MeOH to yield nine cerebrosides 1 (1.7 mg), 2 (0.5 mg), 3
1.7 mg), 4 (2.3 mg), 5 (0.8 mg), 6 (0.5 mg), 7 (0.9 mg), 8 (1.0 mg), and 9
0.5 mg). Compounds 10 (1.6 mg), 11 (2.4 mg), 12 (2.3 mg), 13 (3.2 mg), 14 746 [MꢁH] ; HR-FAB-MS m/z: 748.5951 [MꢀH] (Calcd for C H NO :
2.5 mg) were obtained by purification of combined fraction (10—15) by 748.5939).
dd, Jꢃ10.5, 4.0 Hz), 3.66 (1H, dd, Jꢃ12.0, 6.0 Hz), 3.61 (1H, m), 3.52 (1H,
9
m), 3.34 (1H, m), 3.26 (2H, m) 3.17 (1H, m), 1.80—1.10 (51H, m), 0.92—
ꢀ
(
(
(
0.85 (9H, m); FAB-MS (ꢀve mode) m/z: 748 [MꢀH] , (ꢁve mode) m/z:
ꢁ
ꢀ
41
82
10
RP-HPLC (YMC packed J’sphere ODS-H80 column, 250ꢅ10 mm, 4 mm,
Methanolysis of 5 Cerebroside (0.8 mg) was subjected to methanolysis
2
7
1
80 Å), eluting with 100% MeOH.
similarly to 1 to yield a FAME (0.3 mg). [a]D ꢀ3.7 (cꢃ0.03, MeOH); H-
Renieroside C1 (1), 1-O-b-D-Glucopyranosyl-(2S,3S,4R)-2-[(2ꢀS)-2ꢀ-
NMR (500 MHz, CD OD) d: 4.12 (1H, dd, Jꢃ8.0, 4.8 Hz, H-2), 3.71 (3H, s,
3
hydroxypentadecanoylamino]-16-methyl-heptadeca-1,3,4-triol White OCH ), 1.61 (2H, m, H-3), 1.28 (26H, m, H-4—H-16), 0.89 (3H, t,
3
2
7
1
13
13
amorphous solid; [a] ꢀ11.9 (cꢃ0.17, MeOH); H- and C-NMR see
Jꢃ7.0 Hz, H-17); C-NMR (assignments based on HMBC and HSQC ex-
D
ꢀ
Table 1; FAB-MS (ꢀve mode) m/z: 742 [MꢀNa] , (ꢁve mode) m/z 718
MꢁH] ; HR-FAB-MS m/z: 720.5600 [MꢀH] (Calcd for C H NO : (C-3), 28.0—31.0 (C-4–C-16), 13.0 (C-17); FAB-MS m/z: 323 [MꢀNa] .
periments, 500 MHz, CD OD) d: 175.0 (C-1), 71.0 (C-2), 51.0 (OCH ), 34.5
3
3
ꢁ
ꢀ
ꢀ
[
39 78 10
7
20.5626).
Renieroside C6 (6), 1-O-b-D-Glucopyranosyl-[(2S,3S,4R)-2-(2ꢀR)-2ꢀ-
Methanolysis of 1 Cerebroside (1.6 mg) was dissolved in methanolic hydroxyheptadecanoylamino]-16-methyl-heptadeca-1,3,4-triol White
2
7
1
HCl (1.5 ml, 5% 1 N HCl in MeOH), and the mixture was refluxed on a mag-
amorphous solid; [a]D ꢀ27.6 (cꢃ0.05, MeOH); H-NMR (500 MHz,
netic stirrer for 18 h at 80 °C. It was cooled and extracted with n-hexane CD OD) d: 4.28 (1H, d, Jꢃ7.5 Hz), 4.25 (1H, m), 4.05 (1H, dd, Jꢃ11.0,
3
(
3 mlꢅ3). The n-hexane layer was evaporated under N to yield the FAME 6.5 Hz), 4.02 (1H, dd, Jꢃ7.5, 4.0 Hz), 3.87 (1H, br d, Jꢃ12.0 Hz), 3.80 (1H,
2
27 1
(0.7 mg). [a] ꢀ3.2 (cꢃ0.07, MeOH); H-NMR (500 MHz, CD OD) d: dd, Jꢃ10.5, 4.5 Hz), 3.67 (1H, dd, Jꢃ11.5, 4.5 Hz), 3.61 (1H, m), 3.51 (1H,
D
3
4
.12 (1H, dd, Jꢃ8.0, 4.8 Hz, H-2), 3.71 (3H, s, OCH ), 1.61 (2H, m, H-3),
.28 (22H, m, H-4—H-14), 0.89 (3H, t, Jꢃ7.0 Hz, H-15); C-NMR (assign- 0.85 (9H, m); FAB-MS (ꢀve mode) m/z: 770 [MꢀNa] , (ꢁve mode) m/z:
m), 3.34 (1H, m), 3.26 (2H, m) 3.17 (1H, m), 1.80—1.10 (51H, m), 0.92—
ꢀ
3
1
3
1
ꢁ
ꢀ
ments based on HMBC and HSQC experiments, 500 MHz, CD OD) d: 746 [MꢁH] ; HR-FAB-MS m/z: 748.5944 [MꢀH] (Calcd for C H NO :
3
41 82
10
175.0 (C-1), 71.0 (C-2), 51.0 (OCH ), 34.5 (C-3), 28.0—31.0 (C-4—C-14),
748.5939).
Methanolysis of 6 Cerebroside (0.5 mg) was subjected to methanolysis
3
ꢀ
ꢀ
13.0 (C-15); FAB-MS m/z: 295 [MꢀNa] , 273 [MꢀH] .
2
7
1
Renieroside C2 (2), 1-O-b-D-Glucopyranosyl-[(2S,3S,4R)-2-(2ꢀR)-2ꢀ-
similarly to 1 to yield a FAME (0.1 mg). [a]D ꢁ3.6 (cꢃ0.01, MeOH); H-
hydroxypentadecanoylamino]-16-methyl-heptadeca-1,3,4-triol White NMR (500 MHz, CD OD) d: 4.12 (1H, dd, Jꢃ8.0, 4.8 Hz, H-2), 3.71 (3H,
3
2
7
1
amorphous solid; [a] ꢀ5.0 (cꢃ0.05, MeOH); H-NMR (500 MHz,
s, OCH ), 1.61 (2H, m, H-3), 1.28 (26H, m, H-4—H-16), 0.89 (3H, t,
D
3
1
3
CD OD) d: 4.28 (1H, d, Jꢃ7.5 Hz), 4.25 (1H, m), 4.05 (1H, dd, Jꢃ11.0, Jꢃ7.0 Hz, H-17); C-NMR (assignments based on HMBC and HSQC ex-
3
7
.0 Hz), 4.03 (1H, dd, Jꢃ7.5, 4.0 Hz), 3.87 (1H, br d, Jꢃ11.5 Hz), 3.80 (1H,
periments, 500 MHz, CD OD) d: 175.0 (C-1), 71.0 (C-2), 51.0 (OCH ), 34.5
3 3
ꢀ
dd, Jꢃ10.5, 4.0 Hz), 3.67 (1H, dd, Jꢃ12.0, 6.0 Hz), 3.61 (1H, m), 3.52 (1H,
m), 3.37 (1H, m), 3.26 (2H, m), 3.17 (1H, m), 1.80—1.10 (47H, m), 0.92—
(C-3), 28.0—31.0 (C-4—C-16), 13.0 (C-17); FAB-MS m/z: 323 [MꢀNa] .
Renieroside C7 (7), 1-O-b-D-Glucopyranosyl-[(2S,3S,4R)-2-(2ꢀS)-2ꢀ-
ꢀ
0
7
7
.84 (9H, m); FAB-MS (ꢀve mode) m/z: 742 [MꢀNa] , (ꢁve mode) m/z: hydroxyoctadecanoylamino]-16-methyl-heptadeca-1,3,4-triol White
18 [MꢁH] ; HR-FAB-MS m/z: 720.5624 [MꢀH] (Calcd for C H NO : amorphous solid; [a]D ꢀ9.0 (cꢃ0.09, MeOH); H-NMR (500 MHz,
ꢁ
ꢀ
27
1
3
9
78
10
20.5626).
Methanolysis of 2 Cerebroside (0.5 mg) was subjected to methanolysis 6.0 Hz), 4.01 (1H, dd, Jꢃ7.5, 3.5 Hz), 3.86 (1H, dd, Jꢃ12.0, 1.5 Hz), 3.80
CD OD) d: 4.28 (1H, d, Jꢃ7.5 Hz), 4.24 (1H, m), 4.05 (1H, dd, Jꢃ10.0,
3
2
7
1
similarly to 1 to yield a FAME (0.3 mg). [a]D ꢁ3.7 (cꢃ0.03, MeOH); H-
(1H, dd, Jꢃ10.5, 4.5 Hz), 3.67 (1H, dd, Jꢃ11.5, 4.5 Hz), 3.61 (1H, m), 3.51
NMR (500 MHz, CD OD) d: 4.12 (1H, dd, Jꢃ8.0, 4.8 Hz, H-2), 3.71 (3H, s,
(1H, m), 3.34 (1H, m), 3.26 (2H, m) 3.18 (1H, m), 1.80—1.10 (53H, m),
3
ꢀ
OCH ), 1.61 (2H, m, H-3), 1.28 (22H, m, H-4—H-14), 0.89 (3H, t,
0.90—0.84 (9H, m); FAB-MS (ꢀve mode) m/z: 784 [MꢀNa] , (ꢁve mode)
3
13
ꢁ
ꢀ
Jꢃ7.0 Hz, H-15); C-NMR (assignments based on HMBC and HSQC ex-
m/z: 760 [MꢁH] ; HR-FAB-MS m/z: 762.6071 [MꢀH] (Calcd for
periments, 500 MHz, CD OD) d: 175.0 (C-1), 71.0 (C-2), 51.0 (OCH ), 34.5 C H NO : 762.6095).
3
3
42 84
10
ꢀ
(
C-3), 28.0—31.0 (C-4—C-14), 13.0 (C-15); FAB-MS m/z: 295 [MꢀNa] .
Renieroside C3 (3), 1-O-b-D-Glucopyranosyl-[(2S,3S,4R)-2-(2ꢀS)-2ꢀ-
Methanolysis of 7 Cerebroside (0.8 mg) was subjected to methanolysis
similarly to 1 to yield a FAME (0.1 mg). [a]D ꢀ3.8 (cꢃ0.01, MeOH); H-
2
7
1
hydroxyhexadecanoylamino]-16-methyl-heptadeca-1,3,4-triol White NMR (500 MHz, CD OD) d: 4.12 (1H, dd, Jꢃ8.0, 4.8 Hz, H-2), 3.71 (3H,
3
2
D
7
1
amorphous solid; [a] ꢀ4.2 (cꢃ0.17, MeOH); H-NMR (500 MHz,
s, OCH ), 1.61 (2H, m, H-3), 1.28 (28H, m, H-4—H-17), 0.89 (3H, t,
3
1
3
CD OD) d: 4.28 (1H, d, Jꢃ8.0 Hz), 4.25 (1H, m), 4.05 (1H, dd, Jꢃ10.0, Jꢃ7.0 Hz, H-18); C-NMR (assignments based on HMBC and HSQC ex-
3
6
.0 Hz), 4.01 (1H, dd, Jꢃ8.0, 4.0 Hz), 3.86 (1H, br d, Jꢃ11.5 Hz), 3.80 (1H,
dd, Jꢃ10.5, 4.0 Hz), 3.66 (1H, dd, Jꢃ11.5, 5.0 Hz), 3.61 (1H, m), 3.52 (1H,
periments, 500 MHz, CD OD) d: 175.0 (C-1), 71.0 (C-2), 51.0 (OCH ), 34.5
3 3
ꢀ
(C-3), 28.0—31.0 (C-4—C-17), 13.0 (C-18); FAB-MS m/z: 337 [MꢀNa] .
Renieroside C8 (8), 1-O-b-D-Glucopyranosyl-[(2S,3S,4R)-2-(2ꢀR)-2ꢀ-
m), 3.37 (1H, m), 3.26 (2H, m), 3.17 (1H, m), 1.80—1.10 (49H, m), 0.92—
.84 (9H, m); FAB-MS (ꢀve mode) m/z: 756 [MꢀNa] , (ꢁve mode) m/z: hydroxyoctadecanoylamino]-16-methyl-heptadeca-1,3,4-triol White
32 [MꢁH] ; HR-FAB-MS m/z: 734.5771 [MꢀH] (Calcd for C H NO : amorphous solid; [a]D ꢀ14.4 (cꢃ0.1, MeOH); H-NMR (500 MHz,
34.5782).
Methanolysis of 3 Cerebroside (1.5 mg) was subjected to methanolysis 6.0 Hz), 4.01 (1H, dd, Jꢃ7.0, 3.5 Hz), 3.86 (1H, dd, Jꢃ11.5, 2.0 Hz), 3.79
ꢀ
0
7
7
ꢁ
ꢀ
27
1
4
0
80
10
CD OD) d: 4.27 (1H, d, Jꢃ7.5 Hz), 4.24 (1H, m), 4.05 (1H, dd, Jꢃ10.5,
3
2
7
1
similarly to 1 to yield the FAME (0.4 mg). [a]D ꢀ3.8 (cꢃ0.04, MeOH); H-
(1H, dd, Jꢃ10.5, 3.5 Hz), 3.66 (1H, dd, Jꢃ11.5, 5.0 Hz), 3.61 (1H, m), 3.51

1
10
Vol. 57, No. 1
2
7
1
(
0
1H, m), 3.34 (1H, m), 3.26 (2H, m) 3.17 (1H, m), 1.80—1.10 (53H, m),
1,3,4-triol White amorphous solid; [a]D ꢀ6.6 (cꢃ0.28, MeOH); H-
ꢀ
.91—0.84 (9H, m); FAB-MS (ꢀve mode) m/z: 784 [MꢀNa] , (ꢁve mode) NMR (500 MHz, C D N) d: 8.60 (1H, d, Jꢃ9.5 Hz), 5.50 (2H, m), 5.30 (1H,
5 5
ꢁ ꢀ
m/z: 760 [MꢁH] ; HR-FAB-MS m/z: 762.6110 [MꢀH] (Calcd for m), 4.97 (1H, d, Jꢃ8.0 Hz), 4.75 (1H, dd, Jꢃ10.5, 7.0 Hz), 4.61 (1H, dd,
C H NO : 762.6095).
Jꢃ7.5, 4.0 Hz), 4.55 (1H, Jꢃ11.0, 4.5 Hz), 4.51 (1H, d, Jꢃ11.5 Hz), 4.35
Methanolysis of 8 Cerebroside (0.8 mg) was subjected to methanolysis (1H, dd, Jꢃ11.5, 5.5 Hz), 4.30 (1H, m), 4.22 (3H, m), 4.04 (1H, t,
41
82
10
2
7
1
similarly to 1 to yield a FAME (0.3 mg). [a]D ꢁ3.6 (cꢃ0.04, MeOH); H-
Jꢃ8.5 Hz), 3.89 (1H, m), 2.12 (4H, m), 2.00—1.10 (63H, m), 0.91—0.84
ꢀ
ꢀ
NMR (500 MHz, CD OD) d: 4.12 (1H, dd, Jꢃ8.0, 4.8 Hz, H-2), 3.71 (3H, s,
(9H, m); FAB-MS m/z: 908 [MꢀNa] , 724 [(MꢀH)ꢁ162] ; HR-FAB-MS
3
ꢀ
OCH ), 1.61 (2H, m, H-3), 1.28 (28H, m, H-4—H-17), 0.89 (3H, t,
m/z: 886.7357 [MꢀH] (Calcd for C H NO : 886.7347).
Methanolysis of 13 Cerebroside (2.5 mg) was subjected to methanoly-
3
51 100
10
13
Jꢃ7.0 Hz, H-18); C-NMR (assignments based on HMBC and HSQC ex-
2
7
periments, 500 MHz, CD OD) d: 175.0 (C-1), 71.0 (C-2), 51.0 (OCH ), 34.5 sis similarly to 1 to yield a FAME (0.8 mg). [a] ꢁ6.4 (cꢃ0.08, CHCl3);
3
3
D
ꢀ
1
(C-3), 28.0—31.0 (C-4—C-17), 13.0 (C-18); FAB-MS m/z: 351 [MꢀNa] .
H-NMR (500 MHz, CD OD) d: 5.35 (2H, m, H-20, H-21), 4.24 (1H, dd,
Jꢃ8.5, 5.5 Hz, H-2), 3.79 (3H, s, OCH ), 2.01 (4H, m , H-19, H-22), 1.62
3
Renieroside C9 (9), 1-O-b-D-Glucopyranosyl-[(2S,3S,4R)-2-(2ꢀR)-2ꢀ-
3
hydroxynonadecanoylamino]-16-methyl-heptadeca-1,3,4-triol White (2H, m, H-3), 1.26 (34H, m, H-4—H-18, H-23—H-26), 0.88 (3H, t,
2
7
1
ꢀ
amorphous solid; [a] ꢀ10.2 (cꢃ0.05, MeOH); H-NMR (500 MHz,
Jꢃ7.0 Hz, H-27); FAB-MS m/z: 461 [MꢀNa] ; FAB-CID-MS/MS (ꢀve
ꢀ ꢁ
D
CD OD) d: 4.28 (1H, d, Jꢃ8.0 Hz), 4.24 (1H, m), 4.05 (1H, dd, Jꢃ10.5, mode) m/z: 461 [MꢀNa] , 307, 361, (ꢁve mode) m/z: 437 [MꢁH] , 311,
3
6
.0 Hz), 4.01 (1H, dd, Jꢃ7.5, 4.0 Hz), 3.86 (1H, dd, Jꢃ11.0, 1.5 Hz), 3.79
365.
Renieroside C14 (14), 1-O-b-D-Glucopyranosyl-[(2S,3S,4R)-2-
(2ꢀR,21ꢀZ)-2ꢀ-hydroxyoctacos-21ꢀ-enoylamino]-16-methyl-heptadeca-
(1H, dd, Jꢃ10.5, 3.5 Hz), 3.66 (1H, dd, Jꢃ12.0, 5.0 Hz), 3.61 (1H, m), 3.52
(1H, m), 3.34 (1H, m), 3.26 (2H, m) 3.17 (1H, m), 1.80—1.10 (55H, m),
ꢀ
27
1
0
.91—0.84 (9H, m); FAB-MS (ꢀve mode) m/z: 798 [MꢀNa] , (ꢁve mode) 1,3,4-triol White amorphous solid; [a]D ꢀ11.9 (cꢃ0.1, MeOH); H-
ꢁ
ꢀ
m/z: 774 [MꢁH] ; HR-FAB-MS m/z: 776.6243 [MꢀH] (Calcd for NMR (500 MHz, C D N) d: 8.60 (1H, d, Jꢃ9.5 Hz), 5.51 (2H, m), 5.31 (1H,
5
5
C H NO : 776.6252).
m), 4.97 (1H, d, Jꢃ8.0 Hz), 4.75 (1H, m), 4.60 (1H, m), 4.56 (1H, Jꢃ11.5,
Methanolysis of 9 Cerebroside (0.5 mg) was subjected to methanolysis 3.0 Hz), 4.51 (1H, d, Jꢃ12.0 Hz), 4.36 (1H, m), 4.30 (1H, m), 4.22 (3H, m),
41
82
10
2
7
similarly to 1 to yield a FAME (0.13 mg). [a] ꢁ20.0 (cꢃ0.03, MeOH);
4.04 (1H, t, Jꢃ8.0 Hz), 3.89 (1H, m), 2.13 (4H, m), 2.00—1.10 (65H, m),
0.90—0.84 (9H, m); FAB-MS m/z: 922 [MꢀNa] , 738 [(MꢀH)ꢁ162] ;
HR-FAB-MS m/z: 900.7510 [MꢀH] (Calcd for C H NO : 900.7504).
Methanolysis of 14 Cerebroside (1.0 mg) was subjected to methanoly-
D
1
ꢀ
ꢀ
H-NMR (500 MHz, CD OD) d: 4.12 (1H, dd, Jꢃ8.0, 4.8 Hz, H-2), 3.71
3H, s, OCH ), 1.61 (2H, m, H-3), 1.28 (30H, m, H-4—H-18), 0.89 (3H, t,
3
ꢀ
(
3
52 102
10
13
Jꢃ7.0 Hz, H-19); C-NMR (assignments based on HMBC and HSQC ex-
2
7
periments, 500 MHz, CD OD) d: 175.0 (C-1), 71.0 (C-2), 51.0 (OCH ), 34.5 sis similarly to 1 to yield a FAME (0.5 mg). [a] ꢁ22.0 (cꢃ0.05, CHCl3);
3
3
D
ꢀ
1
(C-3), 28.0—31.0 (C-4—C-18), 13.0 (C-19); FAB-MS m/z: 337 [MꢀNa] .
H-NMR (500 MHz, CD OD) d: 5.34 (2H, M, H-21, H-22), 4.22 (1H, dd,
Jꢃ8.5, 6.0 Hz, H-2), 3.79 (3H, s, OCH ), 2.01 (4H, m, H-20, H-23), 1.62
3
Renieroside C10 (10), 1-O-b-D-Glucopyranosyl-[(2S,3S,4R)-2-
3
(
2ꢀR,15ꢀZ)-2ꢀ-hydroxytetracos-15ꢀ-enoylamino]-16-methyl-heptadeca- (2H, m, H-3), 1.25 (34H, m, H-4—H-19, H-24—H-27), 0.87 (3H, t,
2
7
1
ꢀ
1
,3,4-triol White amorphous solid; [a]D ꢀ6.6 (cꢃ0.16, MeOH); H- and
Jꢃ5.5 Hz, H-28); FAB-MS m/z: 475 [MꢀNa] ; FAB-CID-MS/MS m/z: 475
13
ꢀ
ꢀ
ꢀ
C-NMR see Table 2; FAB-MS m/z: 866 [MꢀNa] , 682 [(MꢀH)ꢁ162] ; [MꢀNa] , 321, 375.
ꢀ
HR-FAB-MS m/z: 844.6907 [MꢀH] (Calcd for C H NO : 844.6878).
Methanolysis of the Glycoside Mixture of 1—9 The mixture of 1—9
4
8
94
10
Methanolysis of 10 Cerebroside (1.5 mg) was subjected to methanoly-
was heated at 80 °C in 4.0 ml of 1 N HCl/MeOH (20 : 80) overnight. The re-
2
7
sis similarly to 1 to yield a FAME (1.1 mg). [a] ꢁ5.8 (cꢃ0.11, CHCl3); action mixture was concentrated by evaporation. The residue was subjected
D
1
H-NMR (500M Hz, CD OD) d: 5.35 (2H, m, H-15, H-16) 4.21 (1H, dd, to silica gel column chromatography with a MeOH/CHCl solvent system.
3
3
Jꢃ9.0, 6.0 Hz, H-2), 3.79 (3H, s, OCH ), 2.01 (4H, m, H-14, 17), 1.62 (2H, MeOH/CHCl (1 : 4) fraction afforded a mixture of sphingosine bases
3
3
1
m, H-3), 1.25 (32H, m, H-4—H-13, H-18—H-23), 0.87 (3H, t, Jꢃ5.5 Hz,
(1.5 mg). H-NMR (400 MHz, C D N) d: 6.37 (–NH, br s), 5.01, 4.83, 4.48,
5 5
13
ꢀ
ꢀ
H-24); FAB-MS m/z: 419 [MꢀNa] ; FAB-CID-MS/MS m/z: 419 [MꢀNa] , 2.18, 1.81, 1.26, 0.87 (6H, d, Jꢃ6.5 Hz); C-NMR (400 MHz, C D N) d:
5
5
2
65, 319.
73.7, 72.7, 59.3, 57.4, 39.2, 35.4, 34.6, 32.1, 30.2, 29.9, 28.1, 27.7, 26.0,
22.8. The MeOH elute yielded an anomeric mixture of the methyl glucoside
Renieroside C11 (11), 1-O-b-D-Glucopyranosyl-[(2S,3S,4R)-2-
2
7
1
(
1
2ꢀS,16ꢀZ)-2ꢀ-hydroxypentacos-16ꢀ-enoylamino]-16-methyl-heptadeca- (1.8 mg); [a]D ꢀ47.6 (cꢃ0.18, MeOH); H-NMR (MeOH) d 5.11 (d,
2
7
1
,3,4-triol White amorphous solid; [a]D ꢀ7.7 (cꢃ0.24, MeOH); H-
Jꢃ3.2 Hz, H-1, a-anomer), 4.47 (d, Jꢃ7.6 Hz, H-1, b-anomer).
Acetylation of the Mixture of the Sphingosine Bases A mixture of the
sphingosine bases (1.5 mg) isolated from the methanolysis of a mixture of
NMR (500 MHz, C D N) d: 8.59 (1H, d, Jꢃ9.0 Hz), 5.51 (2H, m), 5.30 (1H,
5
5
m), 4.97 (1H, d, Jꢃ8.0 Hz), 4.74 (1H, dd, Jꢃ10.5, 6.5 Hz), 4.60 (1H, m),
.55 (1H, dd, Jꢃ11.0, 4.5 Hz), 4.50 (1H, d, Jꢃ12.0 Hz), 4.34 (1H, m), 4.30
1H, m), 4.22 (3H, m), 4.03 (1H, t, Jꢃ7.5 Hz), 3.89 (1H, m), 2.13 (4H, m),
4
(
2
6
glucosides of 1—9 was treated with Ac O/pyridine (1 : 1, 0.5 ml) overnight
2
at room temperature. The reaction mixture was diluted with 2 ml of H O and
2
ꢀ
.00—1.10 (59H, m), 0.90—0.84 (9H, m); FAB-MS: m/z: 880 [MꢀNa] , extracted with CHCl (2 mlꢅ3). The CHCl layer was evaporated under N
3
3
2
ꢀ
ꢀ
27
96 [(MꢀH)ꢁ162] ; HR-FAB-MS: m/z: 858.7019 [MꢀH] (Calcd for to yield the mixture of peracetylated sphingosine bases (4.0 mg). [a]
D
1
C H NO : 8587034).
ꢀ25.0 (cꢃ0.12, CHCl ); H-NMR (500 MHz, CDCl ) d: 5.99 (1H, d,
4
9
96
10
3
3
Methanolysis of 11 Cerebroside (2.2 mg) was subjected to methanoly- Jꢃ9.2 Hz, –NH), 5.08 (1H, dd, Jꢃ8.4, 2.8 Hz, H-3), 4.91 (1H, dt, Jꢃ9.6,
2
7
sis similarly to 1 to yield a FAME (0.15 mg). [a] ꢀ22.3 (cꢃ0.15, CHCl3);
3.6 Hz, H-4), 4.45 (1H, m, H-2), 4.27 (1H, dd, Jꢃ12.0, 5.2 Hz, H-1), 3.98
H-NMR (500 MHz, CD OD) d: 5.35 (2H, m, H-16, H-17), 4.22 (1H, dd, (1H, dd, Jꢃ11.6, 2.8 Hz, H-1), 2.06 (s), 2.03 (s), 2.01 (s), 1.63 (m), 1.49
D
1
3
1
3
Jꢃ8.5, 5.5 Hz, H-2), 3.79 (3H, s, OCH ), 2.02 (4H, m , H-15, H-18), 1.62 (m), 1.20—1.42 (m), 0.85 (6H, d, Jꢃ6.4 Hz); C-NMR (400 MHz, CDCl3)
3
(
2H, m, H-3), 1.26 (34H, m, H-4—H-14, H-19—H-24), 0.88 (3H, t,
d: 171.3, 171.1, 170.3, 169.9, 73.2 (C-3), 72.2 (C-4), 63.0 (C-1), 47.8 (C-2),
ꢀ
Jꢃ7.5 Hz, H-25); FAB-MS m/z: 433 [MꢀNa] ; FAB-CID-MS/MS m/z: 433 39.3, 32.1, 29.8, 29.7, 28.3, 25.7, 23.5, 22.9, 21.3, 21.0; FAB-MS m/z: 486
ꢀ
ꢀ
ꢀ
[MꢀNa] , 279, 333.
[MꢀH] ; HR-FAB-MS m/z: 486.3444 [MꢀH] (Calcd for C H NO :
26 48 7
Renieroside C12 (12), 1-O-b-D-Glucopyranosyl-[(2S,3S,4R)-2-
2ꢀS,17ꢀZ)-2ꢀ-hydroxyhexacos-17ꢀ-enoylamino]-16-methyl-heptadeca-
486.3431).
Preparation of S- and R-MTPA Derivatives of the Fatty Acid Methyl
(
2
7
1
1
,3,4-triol White amorphous solid; [a]D ꢀ8.5 (cꢃ0.23, MeOH); H- Esters (S)-a-Methoxy-a-(trifluoromethyl) phenylacetyl chloride (1 ml)
NMR (500 MHz, C D N) d: 8.60 (1H, d, Jꢃ9.5 Hz), 5.51 (2H, m), 5.31 (1H, was individually added to solution of FAME (0.5 mg/CDCl , 0.4 mg/CDCl )
5
5
3
3
m), 4.97 (1H, d, Jꢃ8.0 Hz), 4.75 (1H, m), 4.60 (1H, m), 4.56 (1H, Jꢃ11.5, of 10 in pyridine-d (10 ml). The mixture was then stirred at room tempera-
5
3
4
0
.0 Hz), 4.51 (1H, d, Jꢃ12.0 Hz), 4.36 (1H, m), 4.30 (1H, m), 4.22 (3H, m),
ture for 24 h, followed by evaporation under N to yield the R-MTPA deriva-
2
.04 (1H, t, Jꢃ8.0 Hz), 3.89 (1H, m), 2.13 (4H, m), 2.00—1.10 (61H, m),
tive. The progress of reaction was monitored by TLC. The same procedure
ꢀ
ꢀ
.90—0.84 (9H, m); FAB-MS m/z: 894 [MꢀNa] , 710 [(MꢀH)ꢁ162] ; was employed for S-MTPA derivative of FAME of 10. The FAME
ꢀ
HR-FAB-MS m/z: 872.7209 [MꢀH] (Calcd for C H NO : 872.7191).
(0.4 mg/CDCl ) of 11 also subjected to Mosher method similarly to FAME
5
0
98
10
3
Methanolysis of 12 Cerebroside (2.2 mg) was subjected to methanoly- of 10 to yield the S- and R-MTPA derivatives.
2
7
sis similarly to 1 to yield a FAME (1.3 mg). [a] ꢀ10.0 (cꢃ0.13, CHCl3);
D
1
H-NMR (500 MHz, CD OD) d: 5.35 (2H, m, H-17, H-18), 4.21 (1H, m, H-
Acknowledgement Thanks are due to Prof. C. J. Sim for the identifica-
3
2
), 3.79 (3H, s, OCH ), 2.01 (4H, m , H-16, H-19), 1.62 (2H, m, H-3), 1.26 tion of the sponge specimen.
3
(
34H, m, H-4—H-15, H-20—H-25), 0.89 (3H, t, Jꢃ7.0 Hz, H-26); FAB-MS
ꢀ
ꢀ
m/z: 447 [MꢀNa] ; FAB-CID-MS/MS m/z: 447 [MꢀNa] , 293, 347.
Renieroside C13 (13), 1-O-b-D-Glucopyranosyl-[(2S,3S,4R)-2-
(
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