Termitomycesphins A-D, novel neuritogenic cerebrosides from the edible Chinese mushroom Termitomyces albuminosus

Article abstract of DOI:10.1016/S0040-4020(00)00548-2

Four novel cerebrosides, termitomycesphins A-D, were isolated from the edible Chinese mushroom Termitomyces albuminosus (Berk.) Heim. ('Jizong' in Chinese), shown to induce neuronal differentiation in rat PC12 cells. The absolute stereostructures were elucidated by spectroscopic methods and chemical derivatization. These new cerebrosides have a unique C19 hydroxylated sphingosine base with branching around the middle. Termitomycesphins A and C possessing a C16 α-hydroxy fatty acid showed a higher neuritogenic activity than did termitomycesphins B and D possessing a C18 α-hydroxy fatty acid. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0040-4020(00)00548-2

TETRAHEDRON
Pergamon
Tetrahedron 56 (2000) 5835±5841
Termitomycesphins A±D, Novel Neuritogenic Cerebrosides from
the Edible Chinese Mushroom Termitomyces albuminosus
*
Jianhua Qi, Makoto Ojika and Youji Sakagami
Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa-ku, Nagoya 464-8601, Japan
Received 24 April 2000; accepted 15 June 2000
AbstractÐFour novel cerebrosides, termitomycesphins A±D, were isolated from the edible Chinese mushroom Termitomyces albuminosus
(Berk.) Heim. (`Jizong' in Chinese), shown to induce neuronal differentiation in rat PC12 cells. The absolute stereostructures were elucidated
by spectroscopic methods and chemical derivatization. These new cerebrosides have a unique C19 hydroxylated sphingosine base with
branching around the middle. Termitomycesphins A and C possessing a C16 a-hydroxy fatty acid showed a higher neuritogenic activity than
did termitomycesphins B and D possessing a C18 a-hydroxy fatty acid. q 2000 Elsevier Science Ltd. All rights reserved.
Introduction
mycesphins A (1, 0.00026% of dry weight), B (2,
0.0003%), C (3, 0.00027%), and D (4, 0.00057%).
The nerve growth factor (NGF) is the ®rst and best
characterized neurotrophic factor that regulates the growth,
differentiation, and survival of neurons, and is thought to be
a candidate for treating Alzheimer's disease, one of neuro-
degenerative diseases of humans.1±3 Since the NGF
molecule is too large in size and too hydrophilic to pass
through the blood-brain barrier, there are no other safe
ways to be used as a drug except for its intraventricular
infusion.4 NGF-like low-molecular-weight compounds are
thus considered to be good candidates for the treatment of
Alzheimer's disease. A clonal rat pheochromocytoma cell
line, PC12, which expresses neuronal properties, has been
used as a model system for such studies.5
Structural analysis was started with termitomycesphin B (2).
The molecular formula of termitomycesphin B (2),
C43H81NO10, was determined by HRESIMS measurement
[m/z 772.5937 (M1H)1, D20.2 mmu]. The IR spectrum
of 2 suggests the presence of a secondary amide group
(1648 and 1535 cm21) and hydroxyl groups (3390 cm21).
1
The H and 13C NMR spectra of 2 showed the presence of
eight oxymethines and two oxymethylenes (dH 3.9±5.0, dC
62.5±78.5 and 105.5), a trans disubstituted ole®n (dH 6.01
and 6.05, Jˆ16.6 Hz), an exomethylene (dH 4.99 and 5.35,
dC 153.8/153.9 (1:1 ratio) and 108.6), and eight exchange-
able protons (broad singlets or doublets in the range of d
6.21±7.61), inculding an NH [dH 8.35 (d, Jˆ8.6 Hz)]. The
signals at d 0.84 (t), 0.85 (t) and many aliphatic methylenes
(dH 1.25, dC 22.8±36.0) suggest that there are two long fatty
chains in this molecule. The signal at d 175.6 supports the
presence of the amide group.
In the course of our search for such substances using the
PC12 cell line system, novel cerebrosides termed termi-
tomycesphins A±D (1±4) were isolated from the EtOH
extract of an edible Chinese mushroom, Termitomyces
albuminosus (Berk.) Heim. (`Jizong' in Chinese), as neurito-
genic substances. We report herein the isolation, structures,
and biological activity of these novel cerebrosides.
The six of the oxygenated carbons at d 62.5 (t), 71.4 (d),
75.0 (d), 78.3 (d), 78.5 (d), and 105.5 (d) suggest the
presence of a b-glucoside moiety in 2 by comparison of
the observed and reported chemical shifts.6 The b con-
®guration of the glucoside bond was supported by the
coupling constant of 7.7 Hz for the anomeric proton at d
4.90. The 1H and 13C NMR data are summarized in Table 1,
in which the H±C direct connectivities were determined by
an HMQC experiment.
Results and Discussion
The EtOH extract of T. albuminosus was washed with
hexane, then partitioned between BuOH and H2O. The
active BuOH fraction was chromatographed on ODS, then
on silica gel to give a mixture of cerebrosides, which was
puri®ed by reversed-phase HPLC to yield termito-
The following partial structures were determined by a DQF-
COSY experiment: b-glucoside moiety, C1±C8, C10±C11,
C20 ±C40, and two terminal methyls connected to a methyl-
ene. An HMBC spectrum showed the correlations from
H7, H10, and H19 to the quaternary ole®nic carbon (C9),
Keywords: fungi; glycolipids; biologically active compounds.
* Corresponding author. Tel.: 181-52-789-4117; fax: 181-52-789-4118;
e-mail: ojika@agr.nagoya-u.ac.jp
0040±4020/00/$ - see front matter q 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020(00)00548-2

5836
J. Qi et al. / Tetrahedron 56 (2000) 5835±5841
Table 1. 1H and 13C NMR data for termitomycesphins A±D (1±4) in pyridine-d5
Carbon no.
1 and 2
3 and 4
1Ha
13Cb
1Ha
13Cb
Long-chain base
1a
1b
2
3
3 -OH
4
5
6
7
8
8-OH
9
9-OH
10
11
12±17
18
19
4.22 dd (10.7, 3.8)
4.69 dd (10.7, 4.3)
4.80 m
70.7 t
4.22 dd (10.6, 3.8)
4.68 dd (10.6, 5.7)
4.79 m
69.9 t
54.5 d
72.3 d
54.5 d
72.2 d
4.78 m
4.77 m
6.85 d (4.4)
6.05 dd (16.6, 3.5)
6.01 dd (16.6, 5.1)
2.38, 2.48 m
1.91 m
4.42 m
6.21/6.20 d (4.0)c
±
6.89 d (4.7)
6.05 dd(15.4, 5.1)
5.97 dt (15.4, 6.3)
2.92 t (6.3)
5.98 dt (15.5, 6.3)
5.88 d (15.5)
±
±
5.68 s
1.73 m
1.61 m
131.7 d
132.7 d
29.5 t
36.0 t
74.2/74.3 dc
132.4 d
131.0 d
35.5 t
124.9 d
140.2 d
153.8/153.9 sc
71.8 s
43.7 t
2.16, 2.32 m
1.57 m
31.7 t
28.4 t
24.5 t
d
f
1.25 m
1.24 m
0.85 t (7.5)e
4.99, 5.35 s
8.35 d (8.6)
14.2 q
108.6 t
0.85 t (7.5)e
1.48 s
8.35 d (8.4)
14.2 q
28.5 q
2-NH
Acyl
10
175.6 s
72.4 d
175.6 s
72.4 d
20
4.56 m
4.57 m
20-OH
30
40
50-150 (or 50-170)
160 (or 180)
7.61 d (4.3)
2.00, 2.21 m
1.69, 1.79 m
1.25 m
7.60 d (5.1)
2.00, 2.24 m
1.69, 1.78 m
1.24 m
35.5 t
25.9 t
35.5 t
25.8 t
d
f
0.84 t (7.7)e
14.2 q
0.84 t (7.7)e
14.2 q
Sugar
100
4.90 d (7.7)
4.02 dd (7.7, 7.1)
7.20 br s
4.20 m
7.17 br s
4.21 m
7.13 br s
3.89 m
4.34 dd (11.4, 4.5)
4.50 br d (11.4)
6.37 br s
105.5 d
75.0 d
4.90 d (7.7)
4.02 dd (7.7, 6.3)
7.19 br s
4.19 m
7.17 br s
4.21 m
7.13 br s
3.89 m
4.34 dd (11.6, 4.5)
4.50 br d (11.6)
6.35 br s
105.5 d
75.0 d
200
200-OH
300
78.3 d
71.4 d
78.4 d
71.5 d
300-OH
400
400-OH
500
78.5 d
62.5 t
78.5 d
62.6 t
600a
600b
600-OH
a 600 MHz, coupling constants (J, Hz) are in parentheses.
b 100 MHz.
c 1:1 ratio due to C-8 epimers.
d d 22.8, 29.3, 29.8, 29.9 and 32.0.
e Interchangeable.
f d 22.8, 29.5, 29.9, 30.6 and 32.0.
revealing the gross structure of a long-chain base (LCB)
(Fig. 2). The HMBC correlations between position 1 and
the anomeric position (H1/C100and H100/C1) led to the
connectivity between glucose and LCB. The correlation
between the exchangeable proton at d 8.35 and the carbonyl
carbon (d 175.6) showed the location of the amide group.
Although no correlation between position 20 and the amide
group was observed, the connectivity of C10 ±C20 is self-
evident, providing the gross structure of a cerebroside as
shown in Fig. 2. The number of other methylene groups
Figure 1. Structures of termitomycesphins A±D (1±4).

J. Qi et al. / Tetrahedron 56 (2000) 5835±5841
5837
alcohol systems, 2 was ®rst hydrogenated to give a tetra-
hydro derivative 5. Methanolysis of 5 gave methyl gluco-
side (2:1 mixture of a and b anomers), fatty acid methyl
ester 6, and free LCB 7. The con®guration of the methyl
glucoside was determined as D by comparison of the optical
rotation with the reported one.7 The methyl ester 6
[[a]2D5ˆ24.4 (c 0.092, CHCl3)] was determined as methyl
(R)-2-hydroxyoctadecanoate based on the optical rotation8
and FABMS data. To determine the absolute con®gurations
of 7, we applied a recent CD exciton chirality method.9
Thus, the benzoylation of LCB 7 gave tetrabenzoyl deriva-
tive 8. The stereochemistry of 8 was determined as 2S,3R by
Figure 2. Gross structure of 2 with selected HMBC correlations (arrows are
from H to C).
Figure 3. Degradation scheme of 2.
1
was determined as 19 from the molecular weight. Since the
signals assigned to C8 (d 74.3, 74.2), C9 (153.9, 153.8), and
8-OH (6.21, 6.20) split into two similar chemical shifts in
the ratio of 1:1, we presume that 2 is an inseparable 1:1
mixture of C8 epimers.
comparison of the CD spectrum (Fig. 3, Table 2) and H
NMR data (Table 2) with those reported.9 Since the exciton
interaction between the extra C8 benzoate and others can be
neglected due to the long range relation, the CD data are in
good agreement with the reported data. The absolute stereo-
structure of 2, except for position 8, was ®nally determined
as shown in Fig. 1.
The degradation of 2 was carried out to determine the
stereochemistry and the two carbon chain lengths (Fig. 3).
The ®rst trial of acidic methanolysis unsuccessfully led to a
complex mixture. To avoid decomposition of the allyl
The molecular formula of termitomycesphin A (1),
C41H77NO10, was determined by HRESIMS measurement
[m/z 744.5621 (M1H)1, D20.5 mmu]. The H and 13C
1
Table 2. Selected 1H NMR data (CDCl3, 400 MHz) and CD data (MeOH)
for tetrabenzoate 8 and tribenzoate 10
NMR spectra of 1 are superimposable on those of 2, and
its molecular formula is less than that of 2 by C2H4, indicat-
ing that 1 is a homologue of 2 possessing a shorter carbon
chain. It was reported that cerebrosides gave characteristic
fragment ions of (LCB1H2H2O)1 and (LCB1H)1 in the
positive mode FABMS measurements.10 The FABMS spec-
tra of both 1 and 2 showed the same fragment ions at m/z
292 due to (LCB1H2H2O)1 and at m/z 274 due to
(LCB1H22H2O)1, though (LCB1H)1 ion peak (m/z
310) was absent due to the instability of an extra OH
group at C-8 (Fig. 4), revealing that 1 and 2 possess the
1H NMRa
CD
(De)
222
JNH±H2 JH2±H3 dNH dH3 dH2
dH1
l(nm)
8
8.5
3.7
4.0
7.07 5.36 4.85 4.58/4.63
7.08 5.38 4.88 4.61/4.65
238
(27.8)
239
(28.4)
(17.4)
222
(17.2)
10 7.8
a Coupling constants J are in Hz, and chemical shifts d are in ppm.

5838
J. Qi et al. / Tetrahedron 56 (2000) 5835±5841
Figure 4. FABMS (1) Fragmentations of 1 and 2 giving the same LCB-derived fragment ions.
same LCB moiety. Thus, the structural difference between 1
and 2 is that 2 possesses two more methylene groups in the
fatty acyl chain than 1. The stereochemistry of 1 seems to be
the same as that of 2, because both compounds showed not
only the same NMR data but also quite similar optical
rotations.
(Fig. 6). LCB 9 was converted to the tribenzoate 10 for
puri®cation and stereochemical analysis, being found to
be converted into an 8,9-dehydro and/or 9,10-dehydro
derivative due to dehydration during methanolysis. The
1
CD spectrum (Fig. 6, Table 2) and H NMR data (Table
2) again agree with those reported for benzoates of (2S,
3R)-LCB,9 revealing the 2S,3R stereochemistry of 10. The
absolute stereostructure of 4, except for position 9, was thus
determined as shown in Fig. 1
The molecular formula of termitomycesphin D (4),
C43H81NO10, was determined by a HRFABMS measurement
[m/z 794.5753 (M1Na)1, D20.6 mmu]. The IR and NMR
spectra of 4 are similar to those of 1 and 2, indicating that 4
is also a cerebroside. The most notable difference in the
NMR data between 4 and 2 is that the signals due to the
exomethylene (C9±C19) and a secondary alcohol (C8) in 1
are replaced by a trans ole®n (C7±C8 at dH 5.98 and 5.88)
and methyl carbinol (C9±C19 at dH19 1.48, dC9 71.8 and
The molecular formula of termitomycesphin C (3),
C41H77NO10, was determined by HRFABMS measurement
1
[m/z 766.5421 (M1Na)1, D22.4 mmu]. The H and 13C
NMR of 3 are superimposable on those of 4, and the
molecular formula of 3 is less than that of 4 by C2H4,
indicating that 3 is a homologue of 4 possessing a shorter
carbon chain. The positive FABMS spectra of both 3 and 4
displayed the same fragment ions at m/z 292
(LCB1H2H2O)1 and at m/z 274 (LCB1H22H2O)1 as in
the case of 1 and 2, revealing that 3 and 4 possess the same
LCB moiety. Thus, the structural difference between 3 and 4
is only the presence of two more methylenes in the fatty acid
chain moiety of 4 than that of 3. The absolute con®guration
of 3 was assumed to be the same as that of 4 by comparison
of the optical rotation and NMR data between 3 and 4
(Fig. 1).
d
C19 28.5). The other NMR data are quite similar to those of
1 (Table 1). The DQF-COSY spectrum of 4 revealed the
following partial structures: the b-glucoside moiety, C1±
C8, C10±C11, C20 ±C40, and two terminal methyls
connected to methylenes. The HMBC correlations shown
in Fig. 5 allowed us to propose the gross structure of 4.
The absolute stereochemistry and the two carbon-chain
lengths of 4 were determined by degradation experiments
using the same conditions as those for 2. The hydrogenation
of 4 yielded a saturated cerebroside, acidic methanolysis of
which gave methyl d-glucopyranoside (2:1 mixture of a and
b anomers), fatty acid methyl ester 6 and free LCB 9
Fig. 7 shows the time course of the neuronal differentiation
induced in PC12 cells by termitomycesphins A±D (1±4).
Termitomycesphins A (1) and C (3) showed the maximum
activity of 30% at the concentration of 10 mg/ml, which
were similar to the activity of an active control, dibutyryl
cAMP, at the concentration of 60 mg/ml, while termi-
tomycesphins B (2) and D (4) showed the maximum activity
of 10% for the same concentration and time. The control
cells cultured without the compounds did not show any
neuronal differentiation. It is noteworthy that the termi-
tomycesphins possessing a C16-fatty acyl moiety (1 and
3) showed a much higher activity than those possessing a
longer C18-fatty acyl moiety (2 and 4).
Figure 5. Gross structure of 4 with selected HMBC correlations (arrows are
from H to C).
An outstanding structural feature of the termitomycesphins

J. Qi et al. / Tetrahedron 56 (2000) 5835±5841
5839
Figure 6. Degradation scheme of 4.
Figure 7. Time course of neuronal differentiation induced in PC12 cells by 10 mg/ml of termitomycesphins A±D (1±4) in comparison with 60 mg/ml of
dibutyryl cAMP as a positive control. The percentages of the cells with longer processes than the cell diameter were plotted.
is the presence of a branch allylic alcohol system in the
middle of the LCB, though the chain length of the fatty
acyl moiety is rather important for the activity than this
characteristic structure of LCB. To the best of our knowl-
edge, termitomycesphins are the ®rst cerebrosides not only
possessing such an unusual LCB but also inducing neurite
outgrowth in a neuronal cell line.11
recorded on a JASCO J-720 spectropolarimeter. FABMS
(positive) and high-resolution FABMS (positive) were
recorded on a JEOL DX-705L and a JEOL Mstation JMS-
700 mass spectrometer, respectively, using m-nitrobenzyl
alcohol as the matrix and PEG 600 as the calibration stan-
dard in the positive mode. HRESIMS (positive) were
recorded on a PE SCIEX QSTAR mass spectrometer,
using a mixture of CsI and sex pheromone inhibitor iPD1
(MW 829) (Bachem) as the calibration standard. One- and
two-dimensional (HMBC, HMQC, DQFCOSY) NMR spec-
tra were obtained in a CDCl3 or C5D5N solution using a
Bruker AMX-600 or a Bruker ARX-400 spectrometer.
Chemical shifts are reported in d (ppm) relative to TMS.
Experimental
General procedures
HPLC was performed on a JASCO high-pressure gradient
system with PU-980 pumps and a JASCO UV-970 detector
at 205 nm. Optical rotations were measured with a JASCO
DIP-370 digital polarimeter. IR spectra were recorded on a
JASCO FT/IR-7000S. UV spectra were recorded on a
JASCO Ubest-50UV/VIS spectrometer. CD spectra were
Extraction and puri®cation procedures
The fresh fruiting bodies of T. albuminosus were collected
in Yanyuan county, southwest of China in July 1999, then
dried and sent to Japan.

5840
J. Qi et al. / Tetrahedron 56 (2000) 5835±5841
The dried fruiting bodies (4.30 kg) of the mushroom were
powdered and immersed in EtOH with occasional stirring
for 10 days. The mixture was ®ltered and the ®ltrate was
concentrated to produce an EtOH extract (593.3 g) as a
viscous brown oil, which was divided into three portions
and subjected to solvent partition. In a typical case, a portion
(209.7 g) of the extract was dissolved in 90% aq. MeOH
(1 liter) and washed twice with hexane (0.8 l). The aq.
MeOH layer was concentrated, dissolved in H2O (0.8 l),
and then extracted three times with BuOH (0.4 l). The
BuOH fractions from the three portions were combined
and concentrated to give a yellowish viscous oil (74.9 g),
which was chromatographed on ODS (Cosmosil 75 C18-
OPN, 350 g, Nacalai Tesque) eluted with MeOH/H2O
(90:10, 95:5) and then MeOH to give four fractions. The
active third fraction (9.9 g), which was eluted with MeOH/
H2O (95:5), was separated by a silica gel (Silica gel 60 230±
240 Mesh ASTM, 150 g, Merck) open column eluted with
CHCl3/MeOH (95:5, 90:10) and MeOH to give four frac-
tions. The active third fraction (249 mg), which was eluted
with CHCl3/MeOH (90:10), was suspended in 90% aqueous
MeOH (12 ml) and ®ltered through a ®lter (DISMIC-13HP,
0.45 mm, Toyo Roshi). The ®ltrate (220 mg) was subjected
to HPLC [Develosil ODS-10 (f 20/250 mm), ¯ow rate:
8 ml/min; 4 injections] eluted by MeOH/H2O (90:10) to
yield termitomycesphins A (1) (11.5 mg, tRˆ93 min),
crude B (2) (25.0 mg, tRˆ162 min), C (3) (11.9 mg,
tRˆ100 min), D (4) (24.6 mg, tRˆ173 min). The pure
sample of 2 (13.0 mg, tRˆ250 min) was obtained from the
crude sample (20 mg) by further HPLC [Develosil ODS-10
(f 20/250), ¯ow rate: 8 ml/min] eluted by CH3CN/H2O
(82:18).
derivative 5: colorless powder, FABMS m/z 798 (M1Na)1,
1H NMR (pyridine-d5, 400 MHz) d 8.42 (1H, d, Jˆ8.4 Hz,
NH), 5.46 to 7.66 (broad singlets or doublets, seven OH),
4.90 (1H, d, Jˆ7.7 Hz, H-100), 4.71 (1H, m, H-2), 4.69 (1H,
m, H-3), 4.67 (1H, m, H-1b), 4.59 (1H, m, H-20), 4.52 (1H,
d, Jˆ11.4 Hz, H-600b), 4.36 (1H, dd, Jˆ11.4, 5.5 Hz,
H-600a), 4.23 (1H, m, H-1a), 4.20 (1H, m, H-400), 4.18 (1H,
m, H-300), 4.02 (1H, dd, Jˆ7.7, 7.2 Hz, H-200), 3.91 (1H, m,
H-500), 3.77/3.67 (1H, m, H-8, 1:1 ratio), 2.21 (1H, m,
H-30b), 2.05 (1H, m, H-30a), 1.63 to 1.90 (m, methylenes),
1.25 (s, methylenes), 1.09/1.07 (3H, d, Jˆ7.4 Hz, H-19, 1:1
ratio), 0.86 (3H, t, Jˆ6.1 Hz, terminal methyl), 0.85 (3H, t,
Jˆ6.9 Hz, terminal methyl).
A solution of 5 (4.6 mg) in a mixture of MeOH (1 ml), water
(0.1 ml), and 12 N HCl (0.1 ml) was re¯uxed for 7 h. The
reaction mixture was cooled and dried by a N2 ¯ow. The
residue was dissolved in MeOH/H2O (90:10) (4 ml) and
extracted three times with hexane (4 ml). The hexane layers
were combined, concentrated, and puri®ed by preparative
TLC (silica gel, EtOAc/hexaneˆ1:3, Rfˆ0.44) to afford the
fatty acid methyl ester 6 (1.3 mg): colorless powder,
[a]D25ˆ24.4 (c 0.092, CHCl3) [lit.8 [a]D24ˆ23.6 (CHCl3)],
1
FABMS m/z 315 (M1H)1, H NMR (CDCl3, 400 MHz) d
4.19 (1H, m, H-2), 3.79 (3H, s, COOCH3), 2.66 (1H, d,
Jˆ5.8 Hz, OH), 1.78 (1H, m, H-3), 1.63 (1H, m, H-3),
1.26 (30H, br s, methylenes), d 0.88 (3H, t, Jˆ6.8 Hz,
terminal methyl).
The aqueous methanolic layer was concentrated, dissolved
in MeOH (0.2 ml), and neutralized by passing through an
Amberlite CG-4B (OH- form) resin column (3 ml), which
was then eluted with MeOH (20 ml). The eluate was
concentrated, dissolved in H2O (2 ml), and extracted twice
with EtOAc (2 ml). The aqueous phase was puri®ed by TLC
(silica gel, CHCl3/MeOHˆ4:1, developed twice, Rfˆ0.36)
to afford a mixture of methyl a- and b-d-glucopyranoside
(2:1): [a]2D4ˆ175 (c 0.035, MeOH) [lit.7 [a]2D5ˆ177.3 (c
0.1, MeOH)].
Termitomycesphin A (1). A colorless powder; [a]2D4ˆ16.0
(c 0.233, MeOH); IR (KBr) 3386, 2922, 2853, 1648, 1537,
1467, and 1078 cm21; HRESIMS m/z 744.5621, calcd for
1
C41H78NO10 (M1H) 744.5626, for H and 13C NMR see
Table 1.
Termitomycesphin B (2). A colorless powder; [a]2D4ˆ16.6
(c 0.119, MeOH); IR (KBr) 3390, 2923, 2853, 1648, 1535,
1467, and 1078 cm21; HRESIMS m/z 772.5937, calcd for
C43H82NO10 (M1H) 772.5939; 1H and 13C NMR were
superimposable on those of 1 (Table 1).
The EtOAc phase was concentrated and benzoylated with
benzoyl chloride (30 ml) and DMAP (1 mg) by stirring in
dry pyridine (1.5 ml) for 5 h at 508C. The reaction was
quenched by adding H2O (0.5 ml) and stirring for 5 min.
The reaction mixture was concentrated in vacuo, dissolved
in benzene (1 ml), and passed through an alumina (7 g)
column (Aluminiumoxid 90, II±III, Merck). The column
was eluted with hexane/EtOAc (9:1 and then 8:2), and the
combined eluates were concentrated and puri®ed by
preparative TLC (silica gel, EtOAc/hexaneˆ1:9, developed
two times, Rfˆ0.18) to afford (2S,3R)-2-benzoylamido-
1,3,8-tribenzoyloxy-9-methyloctadecane (8) (0.6 mg) as a
colorless powder: 1H NMR (400 MHz, CDCl3) d 8.02
(4H, m), 7.94 (2H, m), 7.77 (2H, m), 7.34±7.55 (12H, m),
7.08/7.06 (1H, d, Jˆ8.5 Hz, 1:1 ratio), 5.36 (1H, m), 5.07/
5.03 (1H, m, 1:1 ratio), 4.85 (1H, m), 4.63 (1H, m), 4.58
(1H, m), 1.22±1.94 (25H, m), and 0.84±0.96 (6H, m).
Termitomycesphin C (3). A colorless powder; [a]2D4ˆ17.6
(c 0.226, MeOH); IR (KBr) 3387, 2927, 2854, 1637, 1541,
1467, and 1079 cm21; HRFABMS m/z 766.5421, calcd for
C41H77NO10Na (M1Na) 766.5445; for 1H and 13C NMR see
Table 1.
Termitomycesphin D (4). A colorless powder; [a]2D4ˆ18.0
(c 0.400, MeOH); IR (KBr) 3375, 2924, 2854, 1646, 1540,
1467, and 1079 cm21; HRFABMS m/z 794.5753, calcd for
1
C43H81NO10Na (M1Na) 794.5759; H and 13C NMR were
superimposable on those of 3 (Table 1).
Degradation of 2
Degradation of 4
A solution of 2 (4.6 mg) in EtOH (1 ml) was stirred in the
presence of PtO2 (1 mg) under hydrogen atmosphere for 4 h
at room temperature. The reaction mixture was ®ltered, and
the ®ltrate was concentrated to afford 4.6 mg of tetrahydro
Termitomycesphin D (4) (6 mg) was subjected to
hydrogenation followed by acidic methanolysis in the
same conditions as those for 2, yielding methyl a- and

J. Qi et al. / Tetrahedron 56 (2000) 5835±5841
5841
b-d-glucopyranoside (0.7 mg) and 6 (2.0 mg): colorless
Acknowledgements
powder, [a]2D4ˆ25 (c 0.13, CHCl3), FABMS, m/z 315
(M1H)1. LCB from the EtOAc fraction was subjected to
benzoylation in the same conditions as those for 2. The
product was puri®ed by preparative TLC (silica gel,
EtOAc/hexaneˆ1:9, developed four times, Rfˆ0.25) to
afford (2S,3R)-2-benzoylamido-1,3-dibenzoyloxy-9-methyl-
8- or -9-octadecene (10) (0.3 mg) as a colorless powder:
1H NMR (400 MHz, CDCl3) d 8.03 (2H, m), 7.95 (2H,
m), 7.78 (2H, m), 7.35±7.58 (9H, m), 7.08 (1H, d,
Jˆ7.8 Hz), 5.38 (1H, m), 5.06 (1H, m), 4.88 (1H, m),
4.65 (1H, m), 4.61 (1H, m), 1.88±1.96 (6H, m), 1.25±1.62
(21H, m), and 0.86 (3H t, Jˆ5.9 Hz).
This work was supported by a grant, Research for the Future
Program, from JSPS.
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