Aspergillusol A, an α-glucosidase inhibitor from the marine-derived fungus Aspergillus aculeatus

Article abstract of DOI:10.1021/np9003883

A new tyrosine-derived metabolite, aspergillusol A (4), was isolated on a gram scale, together with a methyl ester of 4-hydroxyphenylpyruvic acid oxime (5) and secalonic acid A, from the marine-derived fungus Aspergillus aculeatus CRI323-04. The tetraol in 4 was identified as erythritol by comparison of the ¹H NMR spectrum of its benzoylated derivative with those of benzoylated erythritol (7) and D-threitol (8), as well as by cellulose-based chiral HPLC analysis. Aspergillusol A (4) selectively inhibited α-glucosidase from the yeast Saccharomyces cerevisiae, but it was inactive toward the α-glucosidase from the bacterium Bacillus stearothermophilus.

Full text of DOI:10.1021/np9003883

J. Nat. Prod. 2009, 72, 2049–2052
2049
Aspergillusol A, an r-Glucosidase Inhibitor from the Marine-Derived Fungus Aspergillus
aculeatus
Nattha Ingavat,† Jeffrey Dobereiner,‡ Suthep Wiyakrutta,§ Chulabhorn Mahidol,†,⊥,| Somsak Ruchirawat,†,⊥,| and
Prasat Kittakoop*,†,⊥
Chulabhorn Graduate Institute and the Center for EnVironmental Health, Toxicology and Management of Chemicals (ETM), Chemical Biology
Program, VibhaVadi-Rangsit Road, Laksi, Bangkok 10210, Thailand, Brandeis UniVersity, 415 South Street, Waltham, Massachusetts 02454,
Department of Microbiology, Faculty of Science, Bangkok 10400, Thailand, Chulabhorn Research Institute, VibhaVadi-Rangsit Road,
Laksi, Bangkok 10210, Thailand, and Chulabhorn Research Centre, Institute of Science and Technology for Research and DeVelopment,
Mahidol UniVersity, Thailand
ReceiVed July 8, 2009
A new tyrosine-derived metabolite, aspergillusol A (4), was isolated on a gram scale, together with a methyl ester of
4-hydroxyphenylpyruvic acid oxime (5) and secalonic acid A, from the marine-derived fungus Aspergillus aculeatus
1
CRI323-04. The tetraol in 4 was identified as erythritol by comparison of the H NMR spectrum of its benzoylated
derivative with those of benzoylated erythritol (7) and D-threitol (8), as well as by cellulose-based chiral HPLC analysis.
Aspergillusol A (4) selectively inhibited R-glucosidase from the yeast Saccharomyces cereVisiae, but it was inactive
toward the R-glucosidase from the bacterium Bacillus stearothermophilus.
Brominated tyrosine-derived metabolites, psammaplin A (1),
aerothionin (2), and homoaerothionin (3), are isolated from marine
sponges.1-4 These and related tyrosine-derived metabolites often
contain hydroxyphenylpyruvic acid oxime in their molecules, and
they are found in various marine sponges including Aplysinella rhax,
Psammaplinaplysilla sponges, Poecillastra sp., Jaspis sp., Verongia
aerophoba, Hymeniacidon sanguinea, Ianthella basta, and Suberea
claVata.1-10 In 1975, Cimino and colleagues reported the occur-
rence of 4-hydroxyphenylpyruvic acid oxime in marine sponges
and proposed that it was the biogenetic precursor of aerothionin
(2), homoaerothionin (3), and aeroplysinin-1.4 We have recently
isolated the marine-derived fungus Aspergillus aculeatus CRI323-
04 from a marine sponge, Xestospongia testudinaria. Surprisingly,
chemical exploration of the fungus A. aculeatus CRI323-04 led to
the isolation of tyrosine-derived metabolites 4 and 5. Aspergillusol
A (4) has structural similarities to psammaplin A (1), a metabolite
of marine sponges.1 R-Glucosidase and aromatase inhibitory
activities as well as cytotoxicity of the isolated compounds 4 and
5 are also presented in this paper.
Separation of a cell extract by Sephadex LH-20 column chro-
matography and C18 reversed-phase HPLC yielded aspergillusol A
(4), a methyl ester of 4-hydroxyphenylpyruvic acid oxime (5), and
secalonic acid A.11 Although 5 is synthetically known,12 its NMR
data have never been well documented; the assignments of 1H and
13C resonances in 5 are listed in the Experimental Section.
Compound 5 was possibly an artifact derived from the methanolysis
of aspergillusol A (4) during extraction and separation.
methylene protons (δH 4.29 and 4.10; and 3.70), and one oxygenated
sp3 methine (δH 3.64). DEPT and HMQC spectra revealed that a
half-molecule of 4 contained two methylenes, five methines, and
four nonprotoned carbons. The13C NMR spectrum implied that C-1/
C-4 and C-2/C-3 are an oxygenated sp3 methylene (δC 67.1) and
an oxygenated methine (δC 69.4), respectively. A downfield shift
(δH 4.29 and 4.10) of H2-1/H2-4 implied that this methylene was
part of either an ester or an ether linkage. The 1H-1H COSY
spectrum of 4 demonstrated the connectivity of H2-1/H-2/2-OH,
establishing the fragment [-O-CH2-CH(OH)-]. Because the
molecule of 4 had symmetry, it possessed the fragment of a tetraol
moiety, [-O-CH2-CH(OH)-CH(OH)-CH2-O-]. The HMBC
spectrum of aspergillusol A (4) provided evidence of a 4-hydrox-
yphenylpyruvic acid oxime unit as a partial structure, showing
correlations (of a half-molecule) from dN-OH to C-2′; H-3′ to C-1′,
C-2′, C-4′, and C-5′ (or C-9′); H-5′ (or H-9′) to C-3′ and C-7′;
H-6′ (or H-8′) to C-4′ and C-7′; and Ar-OH to C-7′ and C-6′ (or
C-8′). In the NOESY spectrum of aspergillusol A (4), there were
correlations between H-5′/H-9′ (or H-5′′/H-9′′) and H2-3′ (or H2-
3′′) and between an aromatic hydroxy group (Ar-OH) and H-6′/
H-8′ (or H-6′′/H-8′′), suggesting the proximity among these protons.
However, there was no NOESY correlation observed between dN-
OH and H2-3′ (or H2-3′′), implying the presence of a (Z)-oxime in
4. It should be noted that, as in the case of an oxime with an amide
A 10 L culture of A. aculeatus CRI323-04 provided greater than
one gram (1262 mg) of aspergillusol A (4), which is a relatively
high yield. Compound 4 had, as revealed by the ESITOF-MS, a
molecular formula of C22H24N2O10. However, the 13C NMR
spectrum of 4 showed only 11 carbon signals; therefore 4 had
symmetry based on its molecular formula. The 1H NMR of 4
showed signals of a half-molecule including those of three
exchangeable protons (δH 12.38, 9.22, and 5.16), two pairs of
protons on a 1,4-disubstituted benzene (δH 7.00 and 6.63), two
* To whom correspondence should be addressed. Tel: +66-86-9755777.
Fax: +662-5740622, ext. 1513. E-mail: prasatkittakoop@yahoo.com.
† Chulabhorn Graduate Institute.
‡ Brandeis University.
§ Department of Microbiology, Mahidol University.
⊥ Chulabhorn Research Institute.
| Chulabhorn Research Centre, Mahidol University.
10.1021/np9003883 CCC: $40.75  2009 American Chemical Society and American Society of Pharmacognosy
Published on Web 10/13/2009

2050 Journal of Natural Products, 2009, Vol. 72, No. 11
Notes
linkage, e.g., psammaplin A (1), the benzylic C-3′ (or C-3′′) of a
(E)-oxime resonates upfield (δC 26-27), while that of a (Z)-oxime
exhibits downfield shifts (δC 35).1 Previous studies employed the
chemical shifts of the benzylic carbon to address the oxime
geometry.1,2,5,7-10 However, such carbon chemical shifts may not
be applicable for the oxime with an ester linkage as present in
aspergillusol A (4). The intermediate resonance (δC 29.6) observed
for the benzylic C-3′ (or C-3′′) in 4 is inconclusive with regard to
the oxime geometry. Further experiments were carried out in order
to secure the oxime geometry in 4. Aspergillusol A (4) was
subjected to methylation under basic conditions (MeI, K2CO3, and
DMF), yielding the tri-O-methyl product 6. The NOESY spectrum
of 6 showed a cross-peak between the dN-OMe and COOMe
protons, indicating the proximity of these O-methyl groups and,
thus, the presence of a (Z)-oxime in 4. The linkage between the
tetraol moiety and the 4-hydroxyphenylpyruvic acid oxime unit was
evident from the HMBC correlation from H-1/H-4 to C-1′/C-1′′.
On the basis of these spectroscopic data, a gross structure of
aspergillusol A (4) was successfully established as shown. However,
the two stereogenic centers of the tetraol unit in 4 have to be
clarified. There are two natural tetraols, known as sugar alcohols,
threitol ((2R,3R)-butane-1,2,3,4-tetrol) and erythritol ((2R,3S)-
butane-1,2,3,4-tetraol), and the latter is widely found in microorgan-
isms. While threitol is optically active, erythritol is optically inactive
because it has a plane of symmetry (or meso forms).13 The specific
rotation for aspergillusol A (4) measured in MeOH was +3.1 (c
1.06), and the tetraol product obtained by enzymatic hydrolysis with
(human lung cholangiocarcinoma), A549 (human lung carcinoma),
and MOLT-3 (acute lymphoblastic leukemia) cell lines with
respective the IC50 values of 50, 74, and 19 µM. Interestingly,
compound 8 selectively inhibited the growth of HepG2 and HL-
60 (human promyelocytic leukemia) cancer cells with respective
IC50 values of 29 and 44 µM, while it was inactive (at 240 µM)
against HuCCA-1 and A549 cancer cells.
The fungus A. aculeatus CRI323-04 produces large quantities
of aspergillusol A (4), whose structure is similar to that of a
brominated tyrosine-derived metabolite, psammaplin A (1), a
metabolite of marine sponges. Brominated tyrosine-derived me-
tabolites are found in various classes of marine sponges,1-10
indicating that there is no chemotaxonomic significance of these
compounds in marine sponges. As particular secondary metabolites
in marine sponges originate from symbiotic cyanobacteria, bacteria,
and dinoflagellates,16 it is possible that marine sponges could utilize
a microbial tyrosine-derived metabolite similar to 4 for the
production of toxic brominated tyrosine-derived metabolites that
may act as chemical defenses in the sponge hosts. It should be
noted that the oxime unit in sponge metabolites has an E-geometry,
while that in the fungal metabolite (4) possesses a Z-geometry.
Because marine sponges and the fungus produce different geom-
etries of the oxime unit, their tyrosine-derived metabolites may not
be biogenetically related. In addition, there have been no reports
on the presence of brominated tyrosine-derived metabolites in the
sponge host Xestospongia testudinaria, from which the marine-
derived funugus was isolated. However, despite the inconsistencies
in the oxime geometries and the isolation of the fungus from a
nonproducing host sponge, the isolation of 4 does demonstrate that
fungi have the ability to synthesize tyrosine-derived oximes and
should be considered as potential sources of precursors for the
brominated tyrosine-derived sponge metabolites. A few natural
tyrosine derivatives have previously been isolated from fungi
including tyrosol carbamate from an Arthrinium sp. isolated from
a deep-water sediment,17 compound OF4949 from Penicillium
rugulosum,18 and the gymnastatins from a sponge-derived fungus
Gymnasella dankaliensis.19 However, these metabolites do not
contain an oxime unit in their molecules, and the present work is
the first report on a fungal tyrosine derivative with an oxime moiety.
lipase also had a small positive specific rotation ([R]27 +3.7 (c
D
2.15, MeOH)), which possibly suggested threitol as the tetraol core
of 4. However, the specific rotations for D-threitol previously
reported were +4.5 (H2O)14 and -14.0 (EtOH),15 indicating that
the sign and magnitude of rotation can vary in different solvents.
In order to obtain additional information regarding the nature of
the tetraol, 1H and 13C NMR spectra of the tetraol were compared
with those of authentic erythritol and D-threitol. Unfortunately, these
comparisons did not unambiguously assign the identity of the
tetraol. Subsequently, erythritol and D-threitol were benzoylated,
1
and the H NMR spectrum of benzoylated erythritol (7) could be
clearly distinguished from that of a benzoylated D-threitol (8)
(Supporting Information). Accordingly, benzoylation of the tetraol
in aspergillusol A (4) was carried out to yield the corresponding
Experimental Section
1
benzoylated product, whose H NMR spectrum was identical to
General Experimental Procedures. Optical rotations were measured
with the sodium D line (590 nm) on a JASCO DIP-370 digital
polarimeter. UV spectra were recorded on a Shimadzu UV-vis
spectrometer (UV-1700 Pharma Spec). FTIR were obtained using a
universal attenuated total reflectance (UATR) attached on a Perkin-
Elmer Spectrum One spectrometer. 1H and 13C NMR spectra were
recorded on a Bruker AM 400 instrument (operating at 400 MHz for
1H and 100 MHz for 13C). ESITOF-MS were determined using a Bruker
MicroTOFLC spectrometer.
Fungal Material and Identification. Aspergillus aculeatus CRI323-
04 was isolated from the marine sponge Xestospongia testudinaria
(specimen no. CRI323), which was collected in November 2006, by
scuba diving at 35-40 feet, from Ton Sai Bay, Phi Phi Islands, Krabi
Province. The marine-derived fungus isolate CRI323-04 was identified
on the basis of both morphology on Czapek solution agar (CzA) and
analysis of the DNA sequences of the ITS1-5.8S-ITS2 rRNA gene
region and the calmodulin gene (Supporting Information). The ITS1-
5.8S-ITS2 and partial cmdA gene DNA sequences of the CRI323-04
fungus have been submitted to GenBank with the accession numbers
FJ525443 and FJ525444, respectively. A culture of the CRI323-04 strain
has been deposited at Chulabhorn Research Institute (CRI) and MIM
Laboratory, Department of Microbiology, Mahidol University, Thailand.
Extraction and Isolation. The fungus CRI323-04 was cultured in
potato dextrose broth (PDB) using seawater (Andaman Sea, Krabi
Province) instead of distilled water. The fungus was inoculated into 1
L Erlenmeyer flasks, each containing 250 mL of PDB, and further
incubated under static condition for 30 days. Fungal cells were separated
from broth (10 L) by filtration. Fungal cells were macerated sequentially
in MeOH and CH2Cl2, each for two days. The CH2Cl2 extract contained,
that of 7, indicating the presence of erythritol in 4. Furthermore,
the benzoylated products 7 and 8 behaved differently on a cellulose-
based chiral HPLC column; compounds 7 and 8 had respective
retention times (tR) of 10.57 and 3.83 min. The benzoylated tetraol
showed a tR similar to that of 7 (10.60 min), thus confirming the
presence of erythritol in 4. On the basis of these data, the stereogenic
centers in aspergillusol A (4) were conclusively assigned. With the
tetraol linker firmly established as erythritol, it seems likely that
the small optical rotations measured for 4 and the tetraol were due
to minor optically active impurities.
Aspergillusol A (4) inhibited R-glucosidase with IC50 values of
465 ( 2 (n ) 3) and 1060 ( 20 (n ) 3) µM toward R-glucosidases
from Saccharomyces cereVisiae and Bacillus stearothermophilus,
respectively. The reference drug, 1-deoxynojirimycin, inhibited
R-glucosidases from S. cereVisiae and B. stearothermophilus with
respective IC50 values of 222 ( 8 and 0.45 ( 0.01 µM. Therefore,
aspergillusol A (4) was ca. 2 times less active than the standard
drug toward R-glucosidase from the yeast S. cereVisiae; however,
it was 2300 times less active for R-glucosidase from the bacterium
B. stearothermophilus. These results indicated that the yeast
R-glucosidase was more susceptible to aspergillusol A (4) than the
bacterial R-glucosidase. Compound 4 was inactive toward aromatase
enzyme (IC50 > 100 µM). Aspergillusol A (4) was inactive (at 105
µM) against the HepG2 (human hepatocellular liver carcinoma)
cell line, while it showed weak cytotoxic activity toward HuCCA-1

Notes
Journal of Natural Products, 2009, Vol. 72, No. 11 2051
as revealed by a 1H NMR spectrum, triglycerides and fatty acids, while
the MeOH extract was partitioned with H2O and hexane in order to
remove triglycerides. A H2O layer was then extracted with EtOAc (10
times) to yield a crude extract (3.1 g), which was chromatographed on
Sephadex LH-20, eluted with MeOH, and nine fractions (F1-F9) were
obtained. Fraction F-6 contained aspergillusol A (4; 523 mg). Fraction
5 (1.37 g) was washed with a mixture of MeOH/H2O (40:60); the
residue obtained was aspergillusol A (4; 739 mg). Therefore, the total
amount of aspergillusol A (4) was 1262 mg. Fraction 4 was separated
by preparative HPLC, eluting with a mixture of MeOH/H2O (30:70),
to yield 73.6 mg of a methyl ester of 4-hydroxyphenylpyruvic acid
oxime (5). The residue that did not dissolve in the HPLC solvent system
was recrystallized from CH2Cl2 to afford secalonic acid A (1.9 mg).11
Aspergillusol A (4): yellow, amorphous solid; UV (MeOH) λmax
(log ε) 221 (2.7) and 278 (1.9) nm; FTIR (UATR) νmax 3404, 3185,
8.41 and 1.31 Hz), 7.56 (2H, t, J ) 7.54 Hz), 7.54 (2H, t, J ) 7.54
Hz), 7.43 (4H, t, J ) 7.90 Hz), 7.40 (4H, t, J ) 7.45 Hz); 6.00 (2H,
m); 4.78 (2H, dd, J ) 12.02 and 3.98 Hz); 4.69 (2H, dd, J ) 12.00
and 5.89 Hz); ESITOF-MS m/z 561.1519 [M + Na]+ (calcd for
C32H26NaO8, 561.1525).
Analysis of Benzoylated Tetraols by Chiral HPLC. Benzoylated
tetraols were analyzed by HPLC (SpectraSYSTEM P4000) equipped
with a UV detector (SpectraSYSTEM UV1000). The chiral column
was Lux Cellulose-1, 5 µm, 250 × 4.60 mm (solvent system 2-propanol/
hexane (20:80); flow rate 1 mL/min). The benzoylated tetraol in 4
showed tR ) 10.60 min, which was similar to that of a benzoylated
erythritol (tR ) 10.57 min), but significantly different from that of a
benzoylated threitol (tR ) 3.83 min).
Inhibition of Aromatase (CYP19). Inhibition of aromatase was
assayed following the method described by Stresser et al.20 Ketocona-
zole was a positive control exhibiting an IC50 value of 2.4 µM.
Inhibition of r-Glucosidase. An assay for R-glucosidase (Sigma,
G5003 from Saccharomyces cereVisiae; Sigma, G3651 from Bacillus
stearothermophilus) was performed in triplicate (n ) 3) using a
colorimetric method described by Wu et al.21 The substrate was
p-nitrophenyl R-D-glucoside (1.0 mM final concentration). R-Glucosi-
dase from the yeast S. cereVisiae and the bacterial B. stearothermophilus
was prepared at 0.1 and 0.3 U/mL (final concentration), respectively.
In our assay system, a standard drug, 1-deoxynojirimycin, showed IC50
values of 222 ( 8 and 0.45 ( 0.01 µM toward R-glucosidases from S.
cereVisiae and B. stearothermophilus, respectively.
Cytotoxicity Assay. Cytotoxic activity for HepG2, HuCCA-1, and
A549 cancer cell lines was evaluated with the MTT assay,22 while that
for MOLT-3 and HL-60 cell lines was assessed using the XTT assay.23
Doxorubicin was used as the reference drug, and respective IC50 values
of 0.37, 0.64, 0.40, and 0.04 µM were observed for HepG2, HuCCA-
1, A549, and MOLT-3 cell lines.
1
1708, 1612, 1513, 1414, 1307, 1257, 1201, 1096, 995, 800 cm-1; H
NMR (DMSO-d6, 400 MHz) δ 12.38 (1H, s, N-OH), 9.22 (1H, s, Ar-
OH), 7.00 (2H, d, J ) 8.5 Hz, H-5′/H-9′ or H-5′′/H-9′′), 6.63 (2H, d,
J ) 8.5 Hz, H-6′/H-8′ or H-6′′/H-8′′), 5.16 (1H, br d, J ) 5.1 Hz,
2-OH/3-OH), 4.29 (1H, br d, J ) 10.7 Hz, H-1a/H-4a), 4.10 (1H, dd,
J ) 11.2, 5.3 Hz, H-1b/H-4b), 3.70 (2H, s, H2-3′/H2-3′′), 3.64 (1H, m,
H-2/H-3); 13C NMR (DMSO-d6,100 MHz) δ 164.2 (qC, C-1′/C-1′′),
156.1 (qC, C-7′/C-7′′), 150.5 (qC, C-2′/C-2′′), 130.1 (CH, C-5′/C-9′
or C-5′′/C-9′′), 126.8 (qC, C-4′/C-4′′), 115.5 (CH, C-6′/C-8′ or C-6′′/
C-8′′), 69.4 (CH, C-2/C-3), 67.1 (CH2, C-1/C-4), and 29.6 (CH2, C-3′/
C-3′′); ESITOF-MS m/z 477.1513 [M + H]+ (calcd for C22H25N2O10,
477.1509).
1
Methyl Ester of 4-Hydroxyphenylpyruvic Acid Oxime (5): H
NMR (acetone-d6, 400 MHz) δ 7.10 (2H, d, J ) 8.5 Hz, H-5/H-9),
6.73 (2H, d, J ) 8.5 Hz, H-6/H-8), 3.82 (2H, s, H2-3), 3.72 (3H, s,
OMe); 13C NMR (acetone-d6,100 MHz) δ 164.3 (qC, C-1), 155.8 (qC,
C-7), 150.8 (qC, C-2), 129.9 (CH, C-5/C-9), 127.0 (qC, C-4), 115.0
(CH, C-6/C-8), 51.4 (OCH3), and 29.1 (CH2, C-3); ESITOF-MS m/z
210.0753 [M + H]+ (calcd for C10H12NO4, 210.0766).
Acknowledgment. P.K. is grateful to The Thailand Research Fund
(grant no. DBG5180014) and the Center for Environmental Health,
Toxicology and Management of Chemicals (ETM) for financial support.
We thank S. Sitthimonchai for aromatase inhibitory activity; P.
Intachote, S. Sengsai, and B. Saimanee for the cytotoxicity test; and
C. Kasettrathat for benzoylation of tetraols and chiral HPLC experi-
ments. The NSF-REU (INT-0123857) supported J.D. for a three-month
research study at CRI, Thailand.
Hydrolysis of Aspergillusol A (4) by Lipase. A reaction mixture
containing compound 4 (102 mg), 2.15 mL of acetone, 2.15 mL of
73.5 mM KH2PO4/K2HPO4 buffer (pH 6.8), and 6.1 mg of lipase was
stirred at 36 °C for 21 h. The mixture was evaporated to dryness, and
the dried material was extracted several times with EtOAc in order to
remove nonpolar compounds. The resulting residue was further
extracted twice with MeOH/H2O (3:1) to yield 27.4 mg of erythritol.
Preparation of Tri-O-methyl Derivative 6. A reaction mixture
containing aspergillusol A (1) (70 mg), K2CO3 (101 mg), DMF (1 mL),
and MeI (1.5 mL) was left stirring overnight at room temperature. After
removing DMF and MeI under vacuum, the reaction mixture was
washed several times with a mixture of MeOH/H2O (40:60). The
insoluble oily residue (8.1 mg) was the tri-O-methyl product 6: 1H NMR
(CDCl3, 600 MHz) δH 7.18 (2H, d, J ) 8.66 Hz, H-5/H-9), 6.81 (2H,
d, J ) 8.66 Hz, H-6/H-8), 4.10 (3H, s, N-OCH3), 3.87 (2H, s, H-3),
3.83 (3H, s, COOCH3), 3.78 (3H, s, 7-OCH3); 13C NMR (CDCl3, 150
MHz) δC 163.87 (-COO, C-1), 158.34 (qC, C-7), 150.83 (CdN, C-2),
130.07 (CH, C-5/C-9), 127.81 (qC, C-4), 113.93 (CH, C-6/C-8), 63.32
(CH3, N-OCH3), 55.22 (CH3, 7-OCH3), 52.73 (CH3, COOCH3), 30.33
(CH2, C-3); ESITOF-MS m/z 238.1082 [M + H]+ (calcd for C12H16NO4,
238.1079).
Supporting Information Available: 1H, 13C, HMQC, HMBC, and
NOESY spectra of 4; 1H and 13C NMR spectra of 6; 1H NMR spectra
of 7 and 8; and details of fungal identification. This material is available
free of charge via the Internet at http://pubs.acs.org.
References and Notes
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Benzoylation of meso-Erythritol and D-Threitol. A reaction
mixture consisting of the tetraol, pyridine (0.2 mL), and benzoyl
chloride (0.4 mL) was stirred overnight at room temperature. After
removing pyridine under vacuum, the reaction mixture was dissolved
in EtOAc and extracted sequentially with 0.5 M HCl (3 times) and
saturated Na2CO3 (3 times) and finally washed with H2O (3 times) to
give the benzoylated tetraol. With this procedure, meso-erythritol (52.1
mg) and D-threitol (50.6 mg) gave the benzoylated products 7 and 8
with respective yields of 129.9 and 132.0 mg. The tetraol (21.5 mg)
obtained from lipase hydrolysis of aspergillusol A (4) was benzoylated
in the same manner as that of authentic compounds to afford 6.9 mg
(7) Miao, S. C.; Andersen, R. J.; Allen, T. M. J. Nat. Prod. 1990, 53,
1441–1446.
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1
of the product, whose H NMR spectrum was identical to that of 7.
Benzoylated meso-erythritol (7): white powder; 1H NMR (CDCl3, 400
MHz) δH 8.04 (4H, dd, J ) 8.49 and 1.34 Hz), 8.01 (4H, dd, J ) 8.48
and 1.33 Hz), 7.57 (2H, t, J ) 7.44 Hz), 7.55 (2H, t, J ) 7.44 Hz),
7.44 (4H, t, J ) 8.27 Hz), 7.40 (4H, t, J ) 7.84 Hz); 5.97 (2H, m);
4.89 (2H, dd, J ) 12.23 and 2.74 Hz); 4.65 (2H, dd, J ) 12.19 and
5.42 Hz); ESITOF-MS m/z 561.1511 [M + Na]+ (calcd for C32H26NaO8,
(11) Hooper, J. W.; Marlow, W.; Whalley, W. B.; Borthwick, A. D.;
Bowden, R. J. Chem. Soc. 1971, C, 3580–3590.
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1
561.1525). Benzoylated D-threitol (8): white solid; H NMR (CDCl3,
400 MHz) δH 8.06 (4H, dd, J ) 8.46 and 1.33 Hz), 7.99 (4H, dd, J )

2052 Journal of Natural Products, 2009, Vol. 72, No. 11
Notes
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