Acremomannolipin A, the potential calcium signal modulator with a characteristic glycolipid structure from the filamentous fungus Acremonium strictum

Article abstract of DOI:10.1016/j.bmcl.2012.08.085

By the newly developed assay method, the glycolipid Acremomannolipin A (1) was isolated from a filamentous fungus Acremonium strictum as a potential calcium signal modulator. The structure of 1 elucidated on the basis of intensive spectroscopic analyses as well as its degradation studies is quite unique: the d-mannopyranose is connected to d-mannitol through a β-glycoside linkage; all the hydroxyls in the mannose are highly masked as peresters with aliphatic acids, and this moiety is made hydrophobic, whereas the mannitol part exhibits a highly hydrophilic property. The compound (1) showed the characteristic bioactivity property, enabling calcineurin deletion cells to grow in the presence of Cl^-, which would be caused by calcium signal modulating. The activity was so potent as to exert the effect at a concentration of 200 nM.

Full text of DOI:10.1016/j.bmcl.2012.08.085

Bioorganic & Medicinal Chemistry Letters 22 (2012) 6735–6739
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Acremomannolipin A, the potential calcium signal modulator
with a characteristic glycolipid structure from the filamentous fungus
Acremonium strictum
Reiko Sugiura a, , Ayako Kita a, Nozomi Tsutsui b, Osamu Muraoka b, Kanako Hagihara a, Nanae Umeda a,
⇑
Tatsuki Kunoh a, Hirofumi Takada a, Dai Hirose c
a Laboratory of Molecular Pharmacogenomics, School of Pharmacy, Kinki University, 3-4-1 Kowakae, Higashi-Osaka, Osaka 577-8502, Japan
b Laboratory of Pharmaceutical Organic Chemistry, School of Pharmacy, Kinki University, 3-4-1 Kowakae, Higashi-Osaka, Osaka 577-8502, Japan
c Laboratory of Molecular Cell Biology, School of Pharmacy, Nihon University, 7-7-1 Narashinodai, Funabashi, Chiba 274-8555, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
By the newly developed assay method, the glycolipid Acremomannolipin A (1) was isolated from a fila-
mentous fungus Acremonium strictum as a potential calcium signal modulator. The structure of 1 eluci-
dated on the basis of intensive spectroscopic analyses as well as its degradation studies is quite
Received 14 July 2012
Revised 11 August 2012
Accepted 22 August 2012
Available online 31 August 2012
unique: the D-mannopyranose is connected to D-mannitol through a b-glycoside linkage; all the hydrox-
yls in the mannose are highly masked as peresters with aliphatic acids, and this moiety is made hydro-
phobic, whereas the mannitol part exhibits a highly hydrophilic property. The compound (1) showed the
characteristic bioactivity property, enabling calcineurin deletion cells to grow in the presence of Clꢀ,
which would be caused by calcium signal modulating. The activity was so potent as to exert the effect
at a concentration of 200 nM.
Keywords:
Glycolipid
Acremomannolipin A
Calcium signal modulator
Acremonium strictum
Phenotypic screen
Ó 2012 Elsevier Ltd. All rights reserved.
Calcium signalling is an important regulator in all eukaryotic
cells for a wide variety of physiological processes. Cells have
evolved mechanisms to regulate cytoplasmic Ca2+ homeostasis in
response to external or internal stress. Small changes in cytoplas-
mic Ca2+ levels can activate various Ca2+-sensing proteins, which
then lead to the induction of various downstream signal transac-
tion pathways. Among the Ca2+-sensing proteins, calcineurin (pro-
tein phosphatase 2B) is considered to be one of the most important
mediators in signal transmission, connecting Ca2+-dependent sig-
nalling to a wide variety of cellular responses.1 The importance
of calcineurin signalling has been investigated in many cell types
and in various organisms because its activity is critical for many
Ca2+-regulated processes, including T-cell activation,2 neutrophil
chemotaxis, apoptosis, membrane trafficking,1a cardiac hypertro-
phy3 and angiogenesis.4 In this respect, calcineurin-mediated sig-
nalling pathways are often described as ‘druggable’ targets for a
number of pathological conditions including cardiac hypertrophy
and immune diseases.5 Calcineurin is conserved from yeast to
mammals, and is specifically inhibited by the immunosuppressant
drugs cyclosporin A and tacrolimus (FK506) in these organisms.6
We previously reported that disruption of the calcineurin gene
(ppb1+) in fission yeast resulted in a Clꢀ-sensitive growth defect. In
addition, we showed that calcineurin and the Pmk1 MAPK signal-
ling pathway play antagonistic roles in the regulation of the Ca2+
signal transduction pathway in fission yeast through the isolation
of several genes encoding regulators of MAPK signaling.7 For the
previous screen, we utilized the distinct Clꢀ-sensitive growth de-
fect of calcineurin deletion cells. Therefore, we developed a novel
cell-based yeast phenotypic screen for identifying compounds that
modulate Ca2+ signalling and have potential for regulating MAPK-
related processes in higher organisms through monitoring of the
suppression of the Clꢀ-sensitive growth defect of calcineurin dele-
tion cells.8
In this work, we describe the identification and structure eluci-
dation of the novel glycolipid Acremomannolipin A (1), which was
isolated from the extract of the filamentous fungus Acremonium
strictum, as a potential Ca2+ signalling modulator.
The structure elucidated on the basis of various 2D NMR tech-
niques and degradation studies is quite unique; a D-mannopyranose
OCO(CH2)6CH3
CH3(CH2)4COO
OH
OH
O
CH3(CH2)4COO
CH3(CH2)4COO
O
R
R
R
R
OH
OH
OH
⇑
Corresponding author.
E-mail address: sugiurar@phar.kindai.ac.jp (R. Sugiura).
Figure 1. Acremomannolipin A (1).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.08.085

6736
R. Sugiura et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6735–6739
is connected to a
D
-mannitol through a b-glycoside linkage. All the
O
O
6""'
1"
hydroxyls in the mannose are highly masked as peresters with ali-
phatic acids, and thus this is hydrophobic, whereas the mannitol
part exhibits a highly hydrophilic property (Fig. 1).
4"
1
6"
8"
1""'
3"
5"
7"
O
O
4'
6'
O
2"
OH OH
5'
O
H
1""
O
3'
1'
3
5
O
6""
O
H 2'
2
4
6
OH
Acremomannolipin A (1) was isolated as a colorless, amorphous
1"'
solid with negative optical rotation (½a D25
ꢁ
ꢀ31.1 in MeOH) from an
H
H
OH OH
O
6"'
extract of A. strictum after solvent partitioning and repeated chro-
matographies.9 The IR spectrum showed an absorption band at
1748 cmꢀ1 attributable to the ester carbonyls and broad bands at
3368 and 1069 cmꢀ1 that are suggestive of a glycoside moiety.
Its positive-ion electrospray ionization-mass spectrometry (ESI-
MS) analysis showed a quasi-molecular ion peak at m/z 787
[M+Na]+, and the high resolution MS (HRESIMS) analysis (ob-
served: m/z 787.4446, calculated for [M+Na]+; 787.4456) revealed
a molecular formula of C38H68O15. Thus, the degree of unsaturation
was calculated to be five. The 13C NMR (nuclear magnetic reso-
nance) spectrum contained 38 peaks corresponding to the molecu-
lar formula obtained by the HRESIMS measurement, which
included four ester carbonyl carbons (dC 175.0, 174.8, 173.85, and
173.76) (Table 1). Because no signals due to other olefinic carbons
were detected in the 13C NMR spectrum, a monocyclic structure
was deduced from the degree of unsaturation (Supplementary data
2). Through intensive spectroscopic analysis utilizing a variety 2D
NMR techniques, in particular a total correlation spectroscopy
(TOCSY) sequence, a quite novel and characteristic feature of 1
was revealed: an octanoate moiety (C100 to C800) and signals corre-
sponding to three pairs of hexanoate groups (C1000 to C6000, C10000 to
C60000 and C100000 to C600000). Based on the 13C NMR signals indicative
of eight methine and three methylene carbons, each bearing one
oxygen atom, the presence of two sugar moieties was speculated,
and a characteristic acetal carbon signal at dC 100.6 and the
correlation spectroscopy (COSY) spectrum (Supplementary data
3) suggested that one sugar moiety was a pyranose containing
the C10–C20–C30–C40–C50–C60 sequence, as shown in Figure 2.
On the basis of the large coupling constants (J = 10.0) of the
three methine signals at C30, C40 and C50, and also on the small cou-
pling constant of the signal due to C20-H, the pyranose was consid-
ered to be mannose (Table 1). Consequently, the other sugar was
deduced to be a hexitol. The hexitol linkage (C1 to C6) was con-
firmed by heteronuclear multiple-bond correlation spectroscopy
(HMBC) (Supplementary data 5) via correlations between C4-H
and C2 and C3-H and C5, although the C3–C4 linkage was termi-
nated on the COSY spectrum due to a lack of coupling between
C3-H and C4-H.
COSY or HSQC-TOCSY
HMBC
NOESY
Figure 2. Selected 2D NMR correlations.
The hexitol moiety was shown to form a glycoside linkage with
the mannose, as indicated by the cross peak between C10-H and C1
in the HMBC spectrum. Overhauser effects (NOEs) with respect to
C10-H (between both C30-H and C50-H) showed the proton at C10 to
be in an axial orientation, and those to C20-H (between both C10-H
and C30-H) showed the proton at C20 to be equatorially oriented.
Thus, the b-glycoside structure of the mannose was ascertained.
The structure of the octanoate moiety was unambiguously clar-
ified on the basis of intensive heteronuclear single quantum coher-
ence (HSQC)–TOCSY studies. In the HSQC–TOCSY spectrum
measured at 60 ms mixing time, correlations were observed
among the signals corresponding to C200, C300, C400, C500, C600, C700
and C800, representing an octanoate chain. The position of the octa-
noate moiety was clarified on the basis of HMBC experiments in
which a cross peak was detected between the C100 carbonyl carbon
and the C20-H as shown in Figure 3.
The presence of three pairs of hexanoate was also detected by
both the TOCSY (Supplementary data 8) and the HSQC–TOCSY
spectra (Supplementary data 7). Their binding positions were also
determined on the basis of HMBC experiments (Supplementary
data 5).
Structural confirmation of the sugar, the hexitol and the acid
moieties attached to the sugar, was established after degradation
of 1 to the corresponding components. Thus, alkaline solvolysis of
1 with sodium methoxide in methanol gave a mixture of deacylated
Acremomannolipin A and two aliphatic acids. The acids liberated by
the solvolysis were confirmed as octanoic acid and hexanoic acid by
the HPLC analysis after derivatization to the corresponding p-nito-
robenzyl carboxylates [tR: 11.5 min (p-nitrobenzyl octanoate), tR:
7.2 min (p-nitrobenzyl hexanoate)].11 The hydrolysis of the deacy-
lated Acremomannolipin A with hydrochloric acid gave a mixture
Table 1
13C NMR (200 MHz) and 1H NMR (800 MHz) data of Acremomannolipin A (1) in CD3OD
Position
-Mannitol
dC
dH (J Hz)
Position
dC
dH (J Hz)
Position
dC
dH (J Hz)
20-O-octanoyl
100
200
40-O-hexanoyl
10000
20000
D
1
73.7
4.13 dd (10.6, 2.8)
3.69 dd (10.6, 6.6)
3.78 ddd (8.6, 6.6, 2.8)
3.72 dd (8.6, 0.9)
174.8
35.2
173.76
34.8
2.47 dt (15.4, 7.4)
2.41 dt (15.4, 7.4)
1.66–1.70 m
1.37–1.43 m
1.37–1.43 m
1.30–1.38 m
1.28–1.38 m
0.92 t (7.2)
2.31 dt (15.8, 7.4)
2.27 dt (15.8, 7.4)
1.55–1.59 m
1.24–1.31 m
1.28–1.38 m
2
3
4
5
6
71.7
71.1
71.2
73.0
65.2
300
26.3
30.3⁄
30.2⁄
33.0
23.8
14.5
30000
40000
50000
60000
25.63
3.75 dd (8.2, 0.9)
400
32.4⁄⁄
3.66 ddd (8.2, 6.0, 3.6)
3.61 dd (11.2, 6.0)
3.79 dd (11.2, 3.6)
500
23.38⁄⁄⁄
14.2⁄⁄⁄⁄
⁄⁄⁄⁄⁄
600
0.90 t (7.2)
700
800
30-O-hexanoyl
60-O-hexanoyl
100000
200000
D-Mannose
10
20
30
40
50
60
100.6
70.5
72.7
66.8
73.5
63.1
4.92 d (0.8)
1000
2000
173.85
35.0
175.0
34.9
5.51 dd (3.2, 0.8)
5.16 dd (10.0, 3.2)
5.30 dd (10.0, 10.0)
3.83 ddd (10.0, 4.2, 2.2)
4.28 dd (12.2, 4.2)
4.14 dd (12.2, 2.2)
2.19 dt (15.6, 7.4)
2.21 dt (15.6, 7.4)
1.51–1.57 m
2.37 dt (15.6, 7.4)
2.34 dt (15.6, 7.4)
1.62–1.66 m
3000
4000
5000
6000
25.5
300000
400000
500000
600000
25.59
32.3⁄⁄
23.36⁄⁄⁄
14.2⁄⁄⁄⁄
1.24–1.31 m
32.5⁄⁄
1.30–1.38 m
1.28–1.38 m
23.41⁄⁄⁄
14.3⁄⁄⁄⁄
1.28–1.38 m
0.90 t (7.2)⁄⁄⁄⁄⁄
0.91 t (7.2)⁄⁄⁄⁄⁄
⁄ ⁄⁄ ⁄⁄⁄ ⁄⁄⁄⁄ ⁄⁄⁄⁄⁄
are exchangeable with each other.
Values with
,
,
,
,

R. Sugiura et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6735–6739
6737
Figure 3. HSQC–TOCSY spectrum of 1 (aliphatic chain part). The abscissa axis in the spectrum represents 13C chemical shifts and the longitudinal axis represents 1H chemical
shifts, both in d. To clarify, assignment of signals and the 1H–13C bond correlation peaks were done only on the octanoate moiety at C20and was numbered from 200 to 800 in the
spectrum. Six black bold lines indicate correlations corresponding to this moiety, and the observed cross peak marked by a red arrow in the top line indicates that C800-H is in
an uninterrupted chain with C300. The cross peaks on the second and third lines from the top reflect the sequential mode of connectivity of all seven carbons from C200 to C800 in
the octanoate moiety.
Figure 4. Suppression of the Clꢀ-sensitive growth defect of calcineurin deletion cells by Acremomannolipin A. Wild-type (wt) cells (right panel) and calcineurin deletion
(D
ppb1) cells (left panel) were spread onto the YPD medium (upper) and YPD medium containing 0.1 M MgCl2 (lower). 5
ll of DMSO or 100 lM Acremomannolipin A in
DMSO was added to each filter paper disc placed at each section, and cells were grown at 27 °C for 3 days.
of the sugar and the hexitol, the NMR spectral properties of which
were in accord with those of a 1:1 mixture of -mannose and D-
mannitol. Structural confirmation of the hexitol was established
after derivatization of the degradation product to the correspond-
ing hexitol peracetate. Thus, reduction of the hydrolytic products
with NaBH4 and acetylation of the crude products with acetic anhy-
dride in pyridine produced peracetylated hexitols. Only one hexitol
derivative was detected on the GC analysis, which was identified as
peracetylated mannitol by comparison of its retention time with
that of an authentic specimen alternatively prepared (Figs. 5 and
D
Figure 5. Gas chromatogram of a mixture of peracetates of four hexitols (glucitol,
Figure 6. Gas chromatogram of the products obtained by the degradation of 1,
followed by reduction and acetylation under the same analytical conditions as
above.
galactitol, mannitol, and inositol). Column: SP-2380 (30 m ꢂ 0.25 mm, 0.25
lm film
thickness), Oven temp.: 80 °C (1 min hold)— >275 °C (25 °C/mim).

6738
R. Sugiura et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6735–6739
OH
HO
HO
HO
O
OH
HO
HO
HO
OAc OAc
OH OH
O
1) NaBH4, H2O, rt
2) Ac2O, Py, 100 °C
2.5% HCl aq.
80 °C
OH
AcO
+
O
OAc
OH
OH OH
OAc OAc
OH OH
deacylated Acremomannolipin A
HO
NaOMe
OH
1
OH OH
CH3OH, rt
p-nitrobenzylation
+
CH3(CH2)6CO2CH2C6H4(p-NO2)
+
CH3(CH2)4CO2CH2C6H4(p-NO2)
CH3(CH2)4CO2H
CH3(CH2)6CO2H
Scheme 1. Degradation of 1 to the corresponding hexitol peracetate.
6).10 Absolute stereostructure of the mannose and the mannitol
were carried out by comparison of the retention times and optical
rotations of the hydrolytic products with those of authentic speci-
ties from Ministry of Education, Culture, Sports, Science and
Technology.
mens [tR: D-mannose 17.5 min (positive optical rotation) and D-
Supplementary data
mannitol 19.8 min (negative optical rotation)] by HPLC analysis
using an optical rotation detector.12 Based on the above-mentioned
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.
08.085.
evidence, the structure of 1 was determined as
D-mannitol-1-yl
3,4,6-tri-O-hexanoyl-2-O-octanoyl-b- -mannopyranoside (Scheme 1).
D
By the method independently developed by us, 1 has been iden-
tified as a potential calcium signal modulator. As described above,
disruption of the calcineurin gene (ppb1+) in fission yeast resulted
in a Clꢀ-sensitive growth defect, thus calcineurin gene deletion
References and notes
1. (a) Aramburu, J.; Rao, A.; Klee, C. B. Curr. Top. Cell. Regul. 2000, 36, 237; (b) Li, H.;
Rao, A.; Hogan, P. G. Trends Cell Biol. 2011, 21, 91.
cells (Dppb1) grew in YPD medium (Fig. 4Aa), but failed to grow
in YPD medium containing 0.1 M MgCl2 (Fig. 4Ba). As a control
vehicle, DMSO was added onto a filter paper disc at the centre of
the media (Fig. 4A–Da). When a solution of 1 in DMSO was added
2. (a) Clipstone, N. A.; Crabtree, G. R. Nature 1992, 357, 695; (b) O’Keefe, S. J.;
Tamura, J.; Kincaid, R. L.; Tocci, M. J.; O’Neill, E. A. Nature 1992, 357, 692.
3. (a) Wilkins, B. J.; Molkentin, J. D. Biochem. Biophys. Res. Commun. 2004, 322,
1178; (b) Wilkins, B. J.; Molkentin, J. D. J. Physiol. 2002, 541, 1.
4. Zachary, I. Biochem. Soc. Trans. 2003, 31, 1171.
5. Bastidas, R. J.; Reedy, J. L.; Morales-Johansson, H.; Heitman, J.; Cardenas, M. E.
Curr. Opin. Investig. Drugs 2008, 9, 856.
onto the filter paper disc,
Dppb1 cells grew well around the disc
(Fig. 4Bb). Wild type (wt) cells grew in YPD media containing
0.1 M MgCl2 (Fig. 4Da), and 1 did not affect the growth of wt cells
on the media, both with MgCl2 (Fig. 4Db) and without MgCl2
(Fig. 4Cb). Thus, 1 was found to enable calcineurin deletion cells
to grow in the presence of Clꢀ without noticeable cytotoxicity.
The activity was so potent as to exert the effect even at a concen-
tration of 200 nM.
Thus, our newly developed chemical-genetic method identified
the glycolipid which has potential to modulate Ca2+ signalling. The
structure of Acremomannolipin A (1) is quite novel and unique,
and exhibits the properties of both lipids and sugars due to the
hybridization of these two types of biomolecules. Once the func-
tion as the lipid is completed, the compound might be hydrolyzed
and exhibit the other function as a sugar. To the best of our knowl-
edge, this study provides the first evidence of a glycolipid influenc-
ing calcium signalling.
6. (a) Rusnak, F.; Mertz, P. Physiol. Rev. 2000, 80, 1483; (b) Sugiura, R.; Sio, S. O.;
Shuntoh, H.; Kuno, T. Genes Cells 2002, 7, 619.
7. (a) Sugiura, R.; Toda, T.; Shuntoh, H.; Yanagida, M.; Kuno, T. EMBO J. 1998, 17,
140; (b) Takada, H.; Nishimura, M.; Asayama, Y.; Mannse, Y.; Ishiwata, S.; Kita,
A.; Doi, A.; Nishida, A.; Kai, N.; Moriuchi, S.; Tohda, H.; Giga-Hama, Y.; Kuno, T.;
Sugiura, R. Mol. Biol. Cell 2007, 18, 4794; (c) Sugiura, R.; Kita, A.; Shimizu, Y.;
Shuntoh, H.; Sio, S. O.; Kuno, T. Nature 2003, 424, 961; (d) Ma, Y.; Kuno, T.; Kita,
A.; Asayama, Y.; Sugiura, R. Mol. Biol. Cell 2006, 17, 5028; (e) Ma, Y.; Sugiura, R.;
Koike, A.; Ebina, H.; Sio, S. O.; Kuno, T. PLoS ONE 2011, 6, e22421.
8. Ishiwata, S.; Kuno, T.; Takada, H.; Koike, A. Sugiura, R. In Source-book of Models
for Biomedical Research; Conn, P. M., Ed.; Humana Press: New York, 2008; pp
439–443.
9. Acremomannolipin A (1) was isolated from an Acremonium strictum strain
collected at Tomakomai, Hokkaido, Japan by Dr. Seiji Tokumasu. Fermentation:
A slant culture of the fungus strain, Acremonium strictum, grown on the potato
dextrose agar medium was inoculated into a K-1 flask containing 100 mL of the
seed medium [Glucose 3.5%, Soy bean meal 2.0%, Soluble starch 1.0%,
Polypeptone 0.5%, Meat extract 0.5%, Yeast extract 0.3%, NaCl 0.2%, MgSO4/
7H2O 0.05%, KH2PO4 0.05%]. The flask was shaken on a rotary shaker at 25 °C for
3 days. The seed culture was transferred into a 30 L jar fermenter containing
20 L production medium [Glucose 0.5%, Glycerin 2.0%, Tomato paste 2.0%, Meat
extract 0.5%, Rice bran 2.0%, Tangle powder 0.2%, Anti-foam agent 0.05%]. The
fermentation was carried out at 25 °C for 5 days with an aeration of 10 L/min
and agitation of 250 rpm.
A plausible mechanism of action of 1 to modulate Ca2+ signal
transduction could be derived from our previous findings that
the Pmk1 MAPK and calcineurin pathways antagonistically regu-
late cytosolic Ca2+ concentration.7e,13 Alternatively, it would be
intriguing to speculate that this compound might inhibit the Clꢀ
channel thereby affecting the phenotypes of calcineurin deletion.
Intensive studies are in progress to identify the molecular target(s)
as well as to clarify the mechanism of action of this unique
molecule.
Isolation of 1: The culture broth of Acremonium strictum obtained above was
extracted with 18 L of n-butanol, and then the mixture was centrifuged to
separate the butanol layer. The butanol layer was concentrated to
approximately 1 L under reduced pressure, and the resulting concentrate was
extracted with equal volume of CHCl3. The CHCl3 phase was subjected to a
silica gel column (Wakogel C-200, 200 g). The column was washed with CHCl3/
MeOH (98:2, v/v), then eluted with CHCl3/MeOH (98:10, v/v). The elution was
concentrated under reduced pressure to produce dark green materials (6.54 g).
The materials were dissolved in 65 mL of DMSO and applied on a preparative
HPLC column (Capcell-Pak C18 UG120, (Shiseido Co.) 20 ꢂ 250 mm; mobile
phase, 80% CH3CN containing 0.1%-acetic acid; flow rate 10 mL/min;
temperature 40 °C, injection volume 1 mL). Under these conditions,
Acknowledgments
We thank Drs. T. Toda, and M. Yanagida and the Yeast Resource
Centre (YGRC/NBRP; http://yeast.lab.nig.ac.jp/nig) for providing
strains and plasmids. Financial support for this study was provided
by Grant-in-Aid for Scientific Research on Innovative Areas, and re-
search Grants from the Ministry of Education, Culture, Sports, Sci-
ence and Technology of Japan (to R.S.). This work was also
financially supported by ‘Antiaging Center Project’ (to R.S.) and
‘High-Tech Research Center Project’ (to O.M.) for Private Universi-
Acremomannolipin
23.0 min, indicating biological activity resulting in suppression of Clꢀ-sensitive
growth of calcineurin deletion cells. The Acremomannolipin containing
A was eluted as a peak with a retention time 21.1–
A
fractions (1.2 L) were diluted to 40% CH3CN with water, and then applied to a
solid extraction system Sep-Pak C18 (Waters Inc.). The columns were washed
with 20 mL of 40% CH3CN and eluted with 20 mL of CH3CN. The elution was
concentrated under reduced pressure to produce yellow-green oily materials
(133 mg). Finally, the materials were purified by an HPLC column (Capcell-Pak
C8 UG120 (Shiseido Co.) 10 ꢂ 250 mm; mobile phase, 70% CH3CN; flow rate

R. Sugiura et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6735–6739
L). Under these
conditions, Acremomannolipin A was eluted as a peak with a retention time
of 14.3–16.0 min. The fraction was concentrated under reduced pressure to
produce white solid materials (47.3 mg).
6739
5 mL/min; temperature 40 °C, injection volume 100
l
residue, pyridine (0.5 mL) and acetic anhydride (0.5 mL) were added and the
mixture was heated at 100 °C for 3 h. After removing pyridine and acetic
anhydride by nitrogen blow, the residue was dissolved in chloroform (l mL).
The chloroform solution was washed with water (1 mL) three times, dried with
anhydrous sodium sulfate, and filtrated. The filtrate was evaporated to produce
pale brown oil, which was subjected to GC analysis.
10. A mixture of 1 (10.0 mg) and sodium methoxide (NaOMe, 3.4 mg) in MeOH
(0.8 mL) was kept at room temperature for 1 h. The reaction mixture was
diluted with MeOH (4 mL), and to the solution was added Amberlite IRA400 J
CL (Clꢀ form, 500 mg). After 5 h, the resin was removed by filtration, and
evaporation of the solvent under reduced pressure yielded the corresponding
deacylated product. The crude product was dissolved in 2.5% hydrochloric acid
(0.8 mL), and the resulting mixture was heated at 80 °C for 2 h. The reaction
mixture was neutralized with Amberlite IRA400J CL (OHꢀ form). After
filtration, the filtrate was concentrated in vacuo. The NMR spctroscopic
11. Analytical conditions [column: COSMOSIL 5C18-MS-II, 4.6 mm i.d. ꢂ 250 mm
(Nacalai Tesque Inc., Tokyo, Japan); detection: UV (254 nm, Shimadzu SPD-
10A, Shimadzu Corporation, Kyoto, Japan); mobile phase, MeOH-H2O (85:15, v/
v); flow rate 1.0 mL/min].
12. Analytical conditions [column: Kaseisorb LC NH2-60-5, 4.6 mm i.d. ꢂ 250 mm
(Tokyo Kasei Co., Ltd, Tokyo, Japan); detection: optical rotation (JASCO OR-
2090 Plus, JASCO Co., Tokyo Japan); mobile phase, CH3CN-H2O (82:18, v/v);
flow rate 1.0 mL/min].
properties of the product were in accord with those of a 1:1 mixture of
D-
mannose and -mannitol. To a solution of the residue in H2O (0.4 mL) was
added sodium borohydride (7 mg) at room temperature for 2 h, the reaction
mixture was quenched with acetone and concentrated in vacuo. To the dry
D
13. Deng, L.; Sugiura, R.; Takeuchi, M.; Suzuki, M.; Ebina, H.; Takami, T.; Koike, A.;
Iba, S.; Kuno, T. Mol. Biol. Cell 2006, 17, 4790.