Dorsamin-A's, glycerolipids carrying a dehydrophenylalanine ester moiety from the seed-eating larvae of the bruchid beetle Bruchidius dorsalis

Article abstract of DOI:10.1021/np300713c

Using a TLC autographic assay for radical-scavenging activity with the ABTS radical, the presence of lipophilic antioxidants in the larvae of the wild bruchid seed beetle Bruchidius dorsalis was detected. Assay-guided fractionation of the CHCl₃-soluble fraction of the larvae resulted in the isolation of new glycerolipids, designated dorsamin-A763, -A737, -A765, -A739, and -A767, comprising 1,2-diacyl-sn-glycero-3-dehydrophenylalanine ester structural units. The ABTS radical scavenging activity of the dorsamin-A's was comparable with or stronger than that of Trolox.

Full text of DOI:10.1021/np300713c

Article
pubs.acs.org/jnp
Dorsamin-A’s, Glycerolipids Carrying a Dehydrophenylalanine Ester
Moiety from the Seed-Eating Larvae of the Bruchid Beetle Bruchidius
dorsalis
Yayoi Hirose,† Emi Ohta,‡ Yasushi Kawai,† and Shinji Ohta*,‡
†Nagahama Institute of Bio-Science and Technology, 1266 Tamura-Cho, Nagahama, Shiga 526-0829, Japan
‡Graduate School of Biosphere Science, Hiroshima University, 1-7-1 Kagamiyama, Higashi-Hiroshima 739-8521, Japan
S
* Supporting Information
ABSTRACT: Using a TLC autographic assay for radical-
scavenging activity with the ABTS radical, the presence of
lipophilic antioxidants in the larvae of the wild bruchid seed
beetle Bruchidius dorsalis was detected. Assay-guided fractio-
nation of the CHCl3-soluble fraction of the larvae resulted in
the isolation of new glycerolipids, designated dorsamin-A763,
-A737, -A765, -A739, and -A767, comprising 1,2-diacyl-sn-
glycero-3-dehydrophenylalanine ester structural units. The
ABTS radical scavenging activity of the dorsamin-A’s was
comparable with or stronger than that of Trolox.
xcessive generation of reactive oxygen species (ROS)
causes damage to proteins, nucleic acids, and lipids. Living
E
organisms have some antioxidant defense systems for
protection against ROS-induced oxidative damage. Phytopha-
gous insect species are subject to oxidative stress caused by
dietary pro-oxidants, extreme temperatures, and microbial
infection as well as by endogenously generated ROS during
metabolism.1 For protection against oxidative stress, insect
larvae possess antioxidant enzymes, such as superoxide
dismutase and thioredoxin,2−5 and also low-molecular-weight
antioxidants such as ascorbic acid, tocopherols, and plasmal-
ogens.6,7
Figure 1. Analysis by silica gel TLC (EtOAc−n-hexane = 1:9 v/v) of
the CHCl3 extract of Bruchidius dorsalis after spraying with an ABTS
radical solution (A) and treatment with vanillin−H2SO4 (B). The
major spots were dorsamin-A’s (d) and triacylglycerols (tg).
Larvae of the wild bruchid seed beetle Bruchidius dorsalis
infest dry mature seeds of the Japanese honey locust, Gleditsia
japonica.8,9 During our ongoing search for biologically active
compounds from natural sources,10−12 we observed that the
CHCl3 extract of B. dorsalis larvae exhibited radical-scavenging
activity. Activity-directed fractionation of the CHCl3 extract
resulted in the isolation of new antioxidant lipids, designated
dorsamin-A763 (1), -A737 (2), -A765 (3), -A739 (4), and
-A767 (5). Here we describe the isolation and structure
elucidation of the dorsamin-A’s.
of 302 nm and a positive response to ninhydrin spray,
suggesting the presence of conjugated double bonds and an
amino group. The CHCl3 extract was subjected to silica gel
column chromatography, to afford ABTS radical scavenging
compounds, designated dorsamin-A’s. The 50% scavenging
concentration (SC50) value of the dorsamin-A’s was 9.6 μg/mL.
The IR spectrum showed the presence of NH2, carbonyl, and
1
phenyl groups. The H and 13C NMR spectra indicated that
dorsamin-A’s are glycerol derivatives with long-chain fatty acyl
residues and a conjugated system containing a phenyl group.
The COSY, HMQC, and HMBC spectra suggested that the
conjugated system of dorsamin-A’s constitutes a dehydrophe-
nylalanine ester unit (Figure 2). This finding was supported by
RESULTS AND DISCUSSION
■
The CHCl3 extract of fourth instar B. dorsalis larvae was
analyzed using silica gel TLC (Merck Kieselgel 60 F254). After
development and drying, the TLC plate was sprayed with an
ABTS radical solution. Free radical scavenging constituents
with an Rf value of 0.29 appeared as a pale yellow spot (spot
“d”) against a dark green background (Figure 1A). The
constituents were more polar than triacylglycerols (spot “tg”)
(Figure 1B). The constituents gave a violet spot under UV light
1
the fact that the H and 13C NMR chemical shifts of the
dehydrophenylalanine ester unit were similar to those of
synthetic dehydrophenylalanine methyl and ethyl esters.13,14
The EIMS and CIMS spectra indicated that dorsamin-A’s are
Received: October 12, 2012
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
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Journal of Natural Products
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determined to be 1,2-dioleoylglycero-3-dehydrophenylalanine
ester.
The UV and 1H NMR spectra of dorsamin-A763 (1), -A737
(2), -A739 (4), and -A767 (5) were similar to those of 3,
indicating that the structures of 1, 2, 4, and 5 were 1,2-
diacylglycero-3-dehydrophenylalanine esters. The fatty acid
composition was determined based on analysis of the fragment
ions observed in the EIMS. The retention of dorsamin-A’s on
the ODS-HPLC column depended on the acyl carbon number
and the number of double bonds, similar to that of
triacylglycerols.16,17 The fatty acid composition of 1−5 is
summarized in Table 1.
Figure 2. Structures of dorsamin-A’s (1−5) and key HMBC
correlations (arrows).
glycerol derivatives consisting of two long-chain fatty acyl
residues and a single dehydrophenylalanine ester unit and have
a range of molecular weights from 737 to 767. The dorsamin-
A’s were separated by ODS-HPLC to yield five compounds, 1−
5 (Figure 3). The molecular weights of 1−5 were determined
Table 1. Fatty Acid Composition of 1−5
compound
MW
EIMS fragment ions
fatty acid composition
1
2
3
4
5
763
737
765
739
767
m/z 263, 265
m/z 239, 263
m/z 265
C18:1−C18:2
C16:0−C18:2
C18:1−C18:1
C16:0−C18:1
C18:0−C18:1
m/z 239, 265
m/z 265, 267
The similarity of the electronic circular dichroism (ECD)
spectra of 1−5 indicated that they have the same absolute
configuration, and therefore we used a mixture of the
compounds for determination of the absolute configuration.
The mixture was gradually decomposed in a CHCl3 solution to
yield 1,2-diacylglycerols. Such a selective hydrolysis of the
dehydrophenylalanine ester bond in dorsamin-A’s might be
attributed to inductive effects of the α-amino substituent and
the unsaturated bond.18−21 The 1,2-diacylglycerols were
converted into 1,2-diacylglycerol-3-O-TBDMS derivatives.
The ECD spectrum of these derivatives (Δε220 −0.18) was in
agreement with that of the 3-O-TBDMS derivative (Δε220
−0.14) derived from authentic 1,2-dioleoyl-sn-glycerol (Cay-
man Chemical), confirming the absolute configuration of
dorsamin-A’s to be R. Consequently, the structures of
dorsamin-A’s indicated that they were new 1,2-diacyl-sn-
glycero-3-dehydrophenylalanine esters 1−5.
Figure 3. ODS-HPLC-PDA analysis (Inertsil PREP-ODS, CH3CN−
CHCl3, 7:3 v/v) of dorsamin-A’s.
Purified dorsamin-A763 (1), -A737 (2), -A765 (3), -A739
(4), and -A767 (5) exhibited ABTS radical scavenging activity,
with 50% scavenging concentration (SC50) values of 9.7−13.8
μM (Table 2). These values were comparable with or stronger
than that of a vitamin E analogue, Trolox (SC50 = 19 μM). The
SC50 values of dehydrophenylalanine methyl ester and methyl
phenylpyruvate were 9.6 and 8.8 μM, respectively. On the
contrary, methyl cinnamate showed no activity. Our findings
indicate that the α-amino or α-hydroxy functionality attached
to be 763, 737, 765, 739, and 767, respectively, on the basis of
EIMS and (+)-CIMS data, and the compounds were therefore
designated dorsamin-A763 (1), -A737 (2), -A765 (3), -A739
(4), and -A767 (5).
Using high-resolution APCITOFMS measurement of the [M
+ H]+ ion at m/z 766.5986 (Δ +0.6 mmu), the molecular
formula of dorsamin-A765 (3), [α]25D +61 (c 0.2, CHCl3), was
established as C48H79NO6. The prominent EIMS fragment ion
at m/z 265 indicated the presence of an oleoyl residue (Figure
4). The geometry of the olefinic bond in the oleoyl residue was
established to be cis on the basis of the 13C chemical shift of the
allylic carbons (δC 27.3).15 Consequently, the structure of 3 was
Table 2. ABTS Radical Scavenging Activity of 1−5 and
Related Compounds
a
compound
SC50 (μM)
1
2
3
4
5
12.1 0.9
13.8 0.4
11.0 0.4
9.7 0.6
10.5 0.8
9.6 0.3
8.8 0.4
>1000
dehydrophenylalanine methyl ester
methyl phenylpyruvate
methyl cinnamate
a
Figure 4. Structure of dorsamin-A765 (3) and EIMS fragmentations.
50% scavenging concentration.
B
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employing EtOAc in n-hexane gradient mixtures [0:1, 1:49, 1:19, 1:9,
1:4, 1:2, 1:1, and 1:0 (10 mL of each solvent mixture)] to give eight
fractions (A−H). Fraction D, which eluted with EtOAc−n-hexane
(1:9), was a mixture of dorsamin-A’s (9 mg).
to an α,β-unsaturated ester skeleton plays an important role in
ABTS radical scavenging activity.
A special class of ether lipids, plasmalogens (1-O-alk-1′-enyl
2-acyl glycerophospholipids), serves as endogenous antiox-
idants and have been found in several insect larvae, e.g., the
tobacco budworm, Heliothis virescens,22 mealworm beetle,
Tenebrio molitor,23 and silkworm, Bombyx mori.7 Another type
of glycerolipids, 1,2-diacyl-3-O-lysylglycerols, which contain an
amino acid ester unit, have been isolated from Mycobacterium
phlei, but their biological properties have not been described.24
Several glycerolipidic cationic surfactants such as 1,2-diacyl-3-
O-arginylglycerols have been widely used for DNA trans-
fections into cells;25,26 however, the antioxidant activity of these
surfactants has not been reported.
We have shown that dorsamin-A’s isolated from B. dorsalis
larvae constitute a new type of endogenous antioxidant lipids.
The structures were determined to be 1,2-diacylglycerol
derivatives, carrying a 3-dehydrophenylalanine ester function-
ality. Several naturally occurring compounds with a dehy-
droamino acid unit have been reported; however, most of these
compounds are dehydropeptides.27−30 Dehydroamino acid
residues of dehydropeptides have been shown to contribute
to conformational stability and antimicrobial activity.31 This is
the first report of the isolation of glycerol derivatives carrying a
dehydroamino acid ester unit. The ABTS radical scavenging
activity of the dorsamin-A’s was comparable with or stronger
than that of Trolox. Similar to tocopherols and carotenoids,
dorsamin-A’s are lipophilic antioxidants. In contrast to the
larvae of B. dorsalis, the adults contained minute quantities of
dorsamin-A’s (data not shown). Further experiments using
biological systems are required to demonstrate the physio-
logical role of dorsamin-A’s in B. dorsalis larvae. However, we
propose that they may play an important role in protecting cell
membranes against oxidative stress caused by toxic oxygen
radicals.
Mixture of Dorsamin-A’s: colorless oil; UV (n-hexane) λmax
(E1 cm1%) 309 (113) nm; IR (KBr) νmax 3456, 3373 (N−H), 1743,
1716 (CO), 1635 (CC), 1593, 1508, and 1494 cm−1 (aromatic
1
ring); H NMR (400 MHz, CDCl3) δH 0.86 (t, J = 6.4 Hz, CH3),
1.19−1.35 (m, CH2), 1.51−1.64 (m, H2-3′ and H2-3″, 1.95−2.12 (m,
C−CH2−), 2.26−2.35 (m, H2-2′ and H2-2″), 2.73−2.77 (m, C−
CH2−C), 4.20 (dd, J = 12.1, 5.9 Hz, H-1a), 4.34 (dd, J = 12.1, 6.2
Hz, H-3a), 4.35 (dd, J = 12.1, 4.4 Hz, H-1b), 4.43 (dd, J = 12.1, 4.0 Hz,
H-3b), 5.31 (m, olefinic H), 5.37 (m, H-2), 6.43 (s, H-3‴), 7.21 (t, J =
7.7 Hz, H-7‴), 7.36 (t, J = 7.7 Hz, H-6‴ and H-8‴), 7.42 (d, J = 7.7
Hz, H-5‴ and H-9‴); 13C NMR (100 MHz, CDCl3) δC 173.1 and
172.6 (C-1′ and C-1″), 165.1 (C-1‴), 135.8 (C-4‴), 131.5 (C-2‴),
129.9 and 129.6 (CHCH), 128.7 (C-6‴ and C-8‴), 128.2 (C-5‴
and C-9‴), 126.9 (C-7‴), 109.8 (C-3‴), 68.8 (C-2), 63.5 (C-3), 62.1
(C-1), 34.3−22.8 (CH2), 14.3 (CH3).
Preparative HPLC of Dorsamin-A’s. The dorsamin-A’s (7 mg)
were separated using ODS-HPLC (CH3CN−CHCl3, 7:3 v/v) to
afford dorsamin-A763 (1; 0.3 mg), -A737 (2; 0.2 mg), -A765 (3; 2
mg), -A739 (4; 3 mg), and -A767 (5; 0.3 mg).
Dorsamin-A763 (1): colorless oil; UV (n-hexane) λmax (log ε) 309
(4.24) nm; ECD (n-hexane) Δε260 −0.50, Δε310 +0.10; 1H NMR (400
MHz, CDCl3) δH 0.86 (t, J = 6.4 Hz, CH3), 1.20−1.35 (m, CH2),
1.51−1.63 (m, H2-3′ and H2-3″), 1.95−2.05 (m, C−CH2−), 2.29−
2.34 (m, H2-2′ and H2-2″), 2.72−2.76 (m, C−CH2−C), 4.20
(dd, J = 12.1, 5.9 Hz, H-1a), 4.34 (dd, J = 12.1, 6.2 Hz, H-3a), 4.35
(dd, J = 12.1, 4.4 Hz, H-1b), 4.42 (dd, J = 12.1, 4.0 Hz, H-3b), 5.29−
5.35 (m, CH), 5.37 (m, H-2), 6.43 (s, H-3‴), 7.21 (t, J = 7.7 Hz, H-
7‴), 7.36 (t, J = 7.7 Hz, H-6‴ and H-8‴), 7.42 (d, J = 7.7 Hz, H-5‴
and H-9‴); EIMS (70 eV) m/z (rel int) 763 (4, M+), 601 (24), 265
(17), 263 (20), 55 (100); CIMS (isobutane) m/z (rel int) 764 (39,
[M + H]+), 601 (100); HRAPCITOFMS m/z 764.5825 [M + H]+
(calcd for C48H78NO6, 764.5829).
Dorsamin-A737 (2): colorless oil; UV (n-hexane) λmax (log ε) 308
(3.98) nm; ECD (n-hexane) Δε260 −0.35, Δε310 +0.15; 1H NMR (400
MHz, CDCl3) δH 0.86 (t, J = 6.4 Hz, CH3), 1.19−1.35 (m, CH2),
1.51−1.64 (m, H2-3′ and H2-3″), 1.96−2.04 (m, C−CH2−), 2.27−
2.34 (m, H2-2′ and H2-2″), 2.72−2.77 (m, C−CH2−C), 4.20
(dd, J = 12.1, 5.9 Hz, H-1a), 4.33 (dd, J = 12.1, 6.2 Hz, H-3a), 4.35
(dd, J = 12.1, 4.4 Hz, H-1b), 4.42 (dd, J = 12.1, 4.0 Hz, H-3b), 5.29−
5.34 (m, CH), 5.37 (m, H-2), 6.43 (s, H-3‴), 7.20 (t, J = 7.7 Hz, H-
7‴), 7.36 (t, J = 7.7 Hz, H-6‴ and H-8‴), 7.42 (d, J = 7.7 Hz, H-5‴
and H-9‴); EIMS (70 eV) m/z (rel int) 737 (4, M+), 575 (32), 263
(22), 239 (13), 55 (100); CIMS (isobutane) m/z (rel int) 738 (34,
[M + H]+), 575 (100); HRAPCITOFMS m/z 738.5670 [M + H]+
(calcd for C46H76NO6, 738.5673).
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotations were
measured on a Horiba SEPA-300 polarimeter. UV and IR spectra
were recorded on JASCO V-630 and Horiba FT720 spectrometers,
respectively. 1H and 13C NMR spectra were recorded on JEOL
1
ECX500 (500 MHz for H, 125 MHz for 13C) and JEOL AL400
spectrometers (400 MHz for 1H, 100 MHz for 13C) at 25 °C.
APCITOFMS and ESITOFMS were measured on a Shimadzu LCMS-
IT-TOF mass spectrometer. EIMS and CIMS were measured on a
Shimadzu GCMS-QP2010 Plus mass spectrometer. Electronic circular
dichroism spectra were recorded on a JASCO J-820 spectropolarim-
eter. Column chromatography was carried out using silica gel (Merck,
Darmstadt, Germany). Thin layer chromatography (TLC) was carried
out on precoated Kieselgel 60 F254 (Merck) and RP18 F254 plates
(Merck). High-performance liquid chromatography (HPLC)-photo-
diode array (PDA) analyses were performed with an Inertsil PREP-
ODS column (CH3CN−CHCl3, 7:3 v/v) on a JASCO LC-2000
instrument equipped with a JASCO MD-2015 multiwavelength
detector. Phenylpyruvic acid, methyl cinnamate, and 6-hydroxy-
2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) were purchased
from Tokyo Kasei Industry Co. (Tokyo, Japan).
Dorsamin-A765 (3): colorless oil; [α]25D +61 (c 0.2, CHCl3); UV
(n-hexane) λm1ax (log ε) 310 (4.02) nm; ECD (n-hexane) Δε260 −0.30,
Δε310 +0.25; H NMR (400 MHz, CDCl3) δH 0.85 (t, J = 6.4 Hz,
CH3), 1.20−1.32 (m, CH2), 1.53−1.64 (m, H2-3′ and H2-3″), 1.94−
2.01 (m, C−CH2−), 2.24−2.34 (m, H2-2′ and H2-2″), 4.19 (dd, J =
12.1, 5.9 Hz, H-1a), 4.33 (dd, J = 12.1, 6.2 Hz, H-3a), 4.35 (dd, J =
12.1, 4.4 Hz, H-1b), 4.42 (dd, J = 12.1, 4.0 Hz, H-3b), 5.29−5.34 (m,
CH), 5.36 (m, H-2), 6.43 (s, H-3‴), 7.21 (t, J = 7.7 Hz, H-7‴), 7.36
(t, J = 7.7 Hz, H-6‴ and H-8‴), 7.42 (d, J = 7.7 Hz, H-5‴ and H-9‴);
13C NMR (100 MHz, CDCl3) δC 173.2 and 173.1 (C-1′ and C-1″),
165.1 (C-1‴), 135.8 (C-4‴), 131.8 (C-2‴), 129.9 and 129.6 (CH
CH), 128.7 (C-6‴ and C-8‴), 128.2 (C-5‴ and C-9‴), 126.9 (C-7‴),
109.8 (C-3‴), 68.8 (C-2), 63.5 (C-3), 62.1 (C-1), 34.3 and 34.2 (C-2′
and C-2″), 32.0 (C-16′ and C-16″), 29.9−29.2 (CH2), 27.3 (allylic
CH2), 25.1 (C-3′ and C-3″),, 22.8 (C-17′ and C-17″), 14.3 (C-18′ and
C-18″); EIMS (70 eV) m/z (rel int) 765 (5, M+), 603 (100), 265
(20), 55 (80); CIMS (isobutane) m/z (rel int) 766 (57, [M + H]+),
603 (100); HRAPCITOFMS m/z 766.5986 [M + H]+ (calcd for
C48H80NO6, 766.5980).
Insects. Fourth instar larvae of the wild bruchid seed beetle
Bruchidius dorsalis were collected in Shiga Prefecture, Japan, in March
2010. Voucher specimens are kept at the Laboratory of Ecological
Biochemistry, Graduate School of Biosphere Science, Hiroshima
University.
Extraction, Isolation, and Characterization. The larvae (0.6 g)
of B. dorsalis were homogenized and extracted with MeOH. After
filtration, the residue was extracted with CHCl3. The CHCl3 extract
(123 mg) was separated on a silica gel column (1 cm i.d. × 25 cm)
Dorsamin-A739 (4): colorless oil; [α]25D +38 (c 0.3, CHCl3); UV
(n-hexane) λmax (log ε) 310 (4.24) nm; ECD (n-hexane) Δε260 −0.45,
C
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Journal of Natural Products
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1
Δε310 +0.30; H NMR (400 MHz, CDCl3) δH 0.86 (t, J = 6.4 Hz,
for 5 min, the mixture was concentrated under reduced pressure to
afford a crude product. The crude product was purified by silica gel
column chromatography (EtOAc−n-hexane, 1:9 v/v), to afford methyl
phenylpyruvate (3 mg).
CH3), 1.18−1.35 (m, CH2), 1.50−1.63 (m, H2-3′ and H2-3″), 1.94−
2.02 (m, C−CH2−), 2.26−2.35 (m, H2-2′ and H2-2″), 4.20 (dd, J =
12.1, 5.9 Hz, H-1a), 4.34 (dd, J = 12.1, 6.2 Hz, H-3a), 4.35 (dd, J =
12.1, 4.4 Hz, H-1b), 4.43 (dd, J = 12.1, 4.0 Hz, H-3b), 5.29−5.34 (m,
CH), 5.36 (m, H-2), 6.43 (s, H-3‴), 7.21 (t, J = 7.7 Hz, H-7‴), 7.36
(t, J = 7.7 Hz, H-6‴ and H-8‴), 7.42 (d, J = 7.7 Hz, H-5‴ and H-9‴);
EIMS (70 eV) m/z (rel int) 739 (6, M+), 577 (100), 265 (8), 239
(10), 55 (60); CIMS (isobutane) m/z (rel int) 740 (67, [M + H]+),
577 (100); HRAPCITOFMS m/z 740.5829 [M + H]+ (calcd for
C46H78NO6, 740.5829).
Dorsamin-A767 (5): colorless oil; UV (n-hexane) λmax (log ε) 309
(3.81) nm; ECD (n-hexane) Δε260 −0.10, Δε310 +0.15; 1H NMR (400
MHz, CDCl3) δH 0.86 (t, J = 6.4 Hz, CH3), 1.19−1.35 (m, CH2),
1.51−1.64 (m, H2-3′ and H2-3″), 1.95−2.02 (m, C−CH2−), 2.26−
2.35 (m, H2-2′ and H2-2″), 4.20 (dd, J = 12.1, 5.9 Hz, H-1a), 4.34 (dd,
J = 12.1, 6.2 Hz, H-3a), 4.35 (dd, J = 12.1, 4.4 Hz, H-1b), 4.43 (dd, J =
12.1, 4.0 Hz, H-3b), 5.29−5.34 (m, CH), 5.37 (m, H-2), 6.43 (s, H-
3‴), 7.21 (t, J = 7.7 Hz, H-7‴), 7.36 (t, J = 7.7 Hz, H-6‴ and H-8‴),
7.42 (d, J = 7.7 Hz, H-5‴ and H-9‴); EIMS (70 eV) m/z (rel int) 767
(4, M+), 605 (100), 267 (10), 265 (10), 55 (90); CIMS (isobutane)
m/z (rel int) 768 (45, [M + H]+), 605 (100); HRAPCITOFMS m/z
768.6142 [M + H]+ (calcd for C48H82NO6, 768.6142).
Methyl phenylpyruvate: colorless oil; 1H NMR (400 MHz,
CDCl3) δH 3.91 (3H, s, OCH3), 6.40 (1H, d, J = 1.8 Hz, OH), 6.51
(1H, br d, J = 1.8 Hz, H-3), 7.26 (1H, t, J = 7.7 Hz, H-7), 7.36 (2H, t, J
= 7.7 Hz, H-6 and H-8), 7.74 (2H, d, J = 7.7 Hz, H-5 and H-9); 13C
NMR (100 MHz, CDCl3) δC 166.6 (C-1), 138.9 (C-4), 134.1 (C-2),
129.8 (C-6 and C-8), 128.4 (C-5 and C-9), 127.9 (C-7), 111.1 (C-3),
53.3 (OCH3); HRESITOFMS m/z 177.0551 [M − H]− (calcd for
C10H9O3, 177.0552).
ABTS Radical Scavenging Activity Assay. The assay was based
on the method of Re et al.,32 with slight modification. A stable stock
solution of 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) dia-
mmonium salt (ABTS) radical was prepared by mixing equal volumes
of aqueous solutions of 14 mM ABTS and 4.9 mM potassium
persulfate for 16 h in the dark at room temperature. The ABTS radical
solution was diluted with EtOH to an absorbance of 0.70 0.02 at
734 nm and equilibrated at room temperature. After addition of 1.0
mL of diluted ABTS radical solution to 10 μL of sample in EtOH, the
absorbance at 734 nm was recorded in the dark at room temperature
for 6 min. Trolox (SC50 = 19 2 μM) was used as a standard.
Conversion of Dorsamin-A’s into TBDMS Derivatives of 1,2-
Diacylglycerol. Treatment of the dorsamin-A’s (2 mg) with CHCl3
(0.6 mL) at room temperature for 1 month gave a degradation
mixture. The mixture was subjected to silica gel column chromatog-
raphy (0.6 cm i.d. × 10 cm) using EtOAc in n-hexane gradient
mixtures [0:1, 1:9, 1:4, 1:2, and 1:1 (3 mL of each solvent mixture)] to
give five fractions. The fraction that eluted with EtOAc−n-hexane
(1:2) was a mixture of 1,2-diacylglycerols. After drying, the fraction
was dissolved in Et3N (0.5 mL), and DMAP (10 mg) and TBDMS
chloride (10 mg) were added. The mixture was stirred at 25 °C for 1 h
to afford a crude product, which was purified by silica gel column
chromatography (0.6 cm i.d. × 10 cm) using EtOAc in n-hexane
gradient mixtures [0:1, 1:49, 1:19, 1:9, and 1:2 (3 mL of each solvent
mixture)] to give five fractions. The fraction that eluted with EtOAc−
n-hexane (1:19) was TBDMS derivatives of 1,2-diacylglycerol (1 mg).
The structure was confirmed by NMR and MS. The ECD spectra of
the derivative showed a negative Cotton effect (Δε220 −0.18). The
TBDMS derivative of the authentic 1,2-dioleoyl-sn-glycerol (Catalog
No. 62230, Cayman Chemical, Ann Arbor, MI, USA) was prepared in
a similar manner and also showed a negative Cotton effect (Δε220
−0.14).
ASSOCIATED CONTENT
* Supporting Information
This material is available free of charge via the Internet at
http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
*Tel: +81-82-424-6537. Fax: +81-82-424-0758. E-mail: ohta@
hiroshima-u.ac.jp.
■
Notes
The authors declare no competing financial interest.
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