Reaction products of γ-tocopherol with (E)-4-oxo-2-nonenal in acidic acetonitrile

Article abstract of DOI:10.1271/bbb.90681

γ-Tocopherol was reacted with (E)-4-oxo-2-nonenal (ONE) at 37 °C in an acidic acetonitrile solution. The reaction products were isolated by reversed-phase high-performance liquid chromatography and their structures were characterized to be 5-substituted γ

Full text of DOI:10.1271/bbb.90681

Biosci. Biotechnol. Biochem., 74 (1), 168–174, 2010
Reaction Products of ꢀ-Tocopherol with (E)-4-Oxo-2-nonenal
in Acidic Acetonitrile
1
1;2
1;2;y
Fumie SAITO, Satoshi IWAMOTO, and Ryo YAMAUCHI
1
The United Graduate School of Agricultural Science, Gifu University, Gifu 501-1193, Japan
Department of Applied Life Science, Faculty of Applied Biological Sciences, Gifu University,
Gifu 501-1193, Japan
2
Received September 17, 2009; Accepted October 27, 2009; Online Publication, January 7, 2010
[doi:10.1271/bbb.90681]
ꢀ
-Tocopherol was reacted with (E)-4-oxo-2-nonenal
ꢀ
inhibits the peroxidation of unsaturated lipids by trap-
11)
(
ONE) at 37 C in an acidic acetonitrile solution.
ping peroxyl radicals. On the contrary, ꢀ-tocopherol is
the most predominant homolog of vitamin E in dietary
The reaction products were isolated by reversed-phase
high-performance liquid chromatography and their
structures were characterized to be 5-substituted ꢀ-
tocopherols: 5-(1-(furan-2-yl)pentyl)-ꢀ-tocopherol (1),
-(1-butyl-4,5,7-trimethyl-7-(4,8,12-trimethyltridecyl)-8,9-
dihydro-7H-furo[3,2-f]chromen-2yl)propanal (2), and 1-
1-butyl-4,5,7-trimethyl-7-(4,8,12-trimethyltridecyl)-8,9-
dihydro-7H-furo[3,2-f]chromen-2-yl)-4-(furan-2-yl)octan-
-one (3). Compound 1 was the predominant product
1
2,13)
sources and acts as an antioxidant.
ꢀ-Tocopherol is
structurally different only by a single methyl group in
the 5-position of the chroman ring. The unsubstituted
5-position in ꢀ-tocopherol makes this vitamin E isomer
a better trap for such lipophilic electrophiles as reactive
nitrogen oxide species and thus counters the oxidative
3
(
1
4)
stress.
It has been demonstrated that ꢀ-tocopherol
3
was a target for nitration in vivo since the concentration
of 5-nitro-ꢀ-tocopherol was elevated in the plasma of
under the mild acidic conditions, whereas compounds 2
and 3 were relatively stable and accumulated during the
reaction. Compound 1 was detected as the product when
1
5)
subjects with coronary heart disease and in the plasma
1
6)
of smokers. Supplementation with ꢀ-tocopherol re-
sulted in a significant reduction of urinary nitrotyrosine,
a biomarker of nitrosative stress, in subjects with
ꢀ
-tocopherol and ONE were incubated in methyl
linoleate in the presence of dicetyl phosphate. The
results indicate that ꢀ-tocopherol may trap ONE under
acidic conditions of lipid peroxidation.
1
7)
metabolic syndrome. ꢀ-Tocopherol supplementation
also significantly reduced the biomarkers of oxidative
stress, plasma malondialdehyde and HNE, and lipid
peroxides. Therefore, the nucleophilic property of ꢀ-
tocopherol may make it act as a detoxifying agent
against lipophilic ꢁ,ꢂ-unsaturated aldehydes such as
HNE and ONE. However, the reaction of these alde-
hydes with ꢀ-tocopherol is not known.
Key words: ꢀ-tocopherol; (E)-4-oxo-2-nonenal; lipid
peroxidation; antioxidant; vitamin E
Lipid peroxidation in a biological system produces
lipid hydroperoxides as the primary products, which in
turn decompose to form ꢁ,ꢂ-unsaturated aldehydes and
other products.1 The ꢁ,ꢂ-unsaturated aldehydes are
bi-functional electrophiles which exhibit toxicity by
covalently modifying the nucleophilic moieties of
proteins and DNA. The most intensively studied lipid
hydroperoxide-derived aldehyde is (E)-4-hydroxy-2-
nonenal (HNE).1 HNE causes cytotoxicity in a number
of cell types. (E)-4-Oxo-2-nonenal (ONE, Fig. 1) has
recently been identified as one of the major hemolytic
This study describes the reaction of ꢀ-tocopherol with
ONE under acidic conditions. We chose ONE as the
ꢁ,ꢂ-unsaturated aldehyde for this study, because this
aldehyde is the most reactive in modifying and cross-
linking proteins and DNA among the electrophilic lipid
–3)
4
,5)
1
8–21)
peroxidation products.
,6)
Materials and Methods
7
)
decomposition products of lipid hydroperoxides. Both
HNE and ONE could modify covalently amino acids and
DNA bases primarily via Michael addition or Schiff
base formation.6 Furthermore, it has been demon-
strated that ascorbic acid, a water-soluble antioxidant,
acted as a biological nucleophile and formed Michael-
type conjugates with electrophilic lipid peroxidation
Materials. RRR-ꢀ-Tocopherol was prepared from mixed isomers of
tocopherol (J-Oil Mills, Tokyo, Japan) and purified by Sephadex LH-
0 column chromatography.22) ONE was synthesized from 2-pentyl-
2
furan by the procedure of Zhang et al.
–8)
23) 1
3
H-NMR (CDCl ) ꢃ: 0.91 (t,
J ¼ 6:8 Hz, 3H, H-9), 1.33 (m, 4H, H-7, H-8), 1.67 (dt, J ¼ 7:3,
7
.3 Hz, 2H, H-6), 2.70 (t, J ¼ 7:3 Hz, 2H, H-5), 6.78 (dd, J ¼ 7:3,
6.1, 1H, H-2), 6.89 (d, J ¼ 16:1 Hz, 1H, H-3), and 9.79 (d,
1
1
3
J ¼ 7:3 Hz, 1H, H-1, CHO); C-NMR (CDCl3) ꢃ: 13.8 (C-9), 22.4
9
,10)
products such as HNE and ONE.
(
C-8), 23.3 (C-6), 31.2 (C-7), 41.1 (C-5), 137.3 (C-2), 144.9 (C-3),
93.4 (C-1), and 200.1 (C-4). Methyl linoleate was purchased from
Tocopherols are generally present in edible oils and
are important as natural antioxidants. ꢁ-Tocopherol, the
most active form of vitamin E in biological systems,
1
Tokyo Kasei Co. (Tokyo, Japan) and purified by silica-gel column
chromatography. Dicetyl phosphate (dihexadecyl phosphate) was
y
To whom correspondence should be addressed. Fax: +81-58-293-2930; E-mail: yamautir@gifu-u.ac.jp
Abbreviations: APCIMS, atmospheric pressure chemical ionization mass spectrometry; COSY, correlation spectroscopy; EIMS, electron impact
mass spectrometry; HMBC, heteronuclear multiple-bond coherence; HMQC, heteronuclear multiple-quantum coherence; HNE, (E)-4-hydroxy-2-
nonenal; HPLC, high-performance liquid chromatography; NMR, nuclear magnetic resonance; ONE, (E)-4-oxo-2-nonenal; TLC, thin-layer
chromatography; UV, ultraviolet

Reaction of ꢀ-Tocopherol with 4-Oxo-2-nonenal
169
increasing proportions of diethyl ether in hexane. The products were
further purified by HPLC. Preparative reversed-phase HPLC was
conducted in an Inertsil PREP-ODS column (10 ꢁ 250 mm) developed
with methanol/ethanol (2:3, v/v) at a flow rate of 4.0 ml/min. The
purity of each isolated compound was checked by thin-layer
chromatography (TLC) on a pre-coated silica-gel 60 plate (Merck,
Darmstadt, Germany), developed with hexane/diethyl ether/acetic
acid (90:10:1, v/v/v). Spots on the TLC plate were visualized by
charring after spraying with 20% H2SO4. The spots with reducing
power were detected by spraying with a mixture of 0.2% FeCl3 and
0
24)
0
.5% 2,2 -bipyridyl in ethanol.
A different concentration of the HCl solution was prepared for
another experiment by diluting 12 M HCl with acetonitrile. To this
(
(
1.0 ml) was added a mixture of ꢀ-tocopherol (10 mM) and ONE
ꢀ
20 mM), and the mixture incubated at 37 C for 1.5 h. To the reaction
mixture was added 1.0 ml of distilled water, and the solution extracted
twice with 1.0 ml of hexane. The hexane layer was evaporated
in vacuo, and the residue dissolved in ethanol was injected for
reversed-phase HPLC as already described. The amounts of reaction
products were calculated from the peak area by monitoring the eluate
at 220 nm. A time-course experiment was conducted with a mixture of
ꢀ
-tocopherol (10 mM) and ONE (20 mM) dissolved in a 10 mM HCl
ꢀ
acetonitrile solution (2.0 ml) and incubated at 37 C. A 200-ml aliquot
of the sample solution was periodically withdrawn, and the amounts of
the reaction products were determined by HPLC as already described.
Reaction products of ONE in acidic acetonitrile. ONE (227 mg,
.4 mmol) was dissolved in 50 ml of acetonitrile containing 50 mM
1
ꢀ
HCl, and the solution incubated at 37 C for 10 min. The reaction
products were extracted with hexane and analyzed by TLC on a silica-
gel plate developed with hexane/diethyl ether/acetic acid (90:10:1,
v/v/v). Spots corresponding to ONE (Rf 0.14) and reaction product 4
Fig. 1. Structures of the Compounds Involved in the Study.
(
Rf 0.10) were detected on the TLC plate. Compound 4 was isolated by
obtained from Sigma-Aldrich (Tokyo, Japan). All other reagents used
were of analytical grade.
silica-gel column chromatography with increasing proportions of
diethyl ether in hexane, and obtained as a yellow oil (26.4 mg yield).
Compound 4 was identified as 2-(1-hydroxypentyl)furan.23,25) UV
Apparatus. High-performance liquid chromatography (HPLC) was
performed with a PU-2089 plus pump (Jasco, Tokyo, Japan) connected
to a Jasco UV-2075 plus UV/VIS detector or a FP-2020 plus intelligent
fluorescence detector. 1H nuclear magnetic resonance (NMR) spectra
were recorded at 600.17 MHz with a JNM-ECA-600 FT NMR
spectrometer (Jeol, Tokyo, Japan), using CDCl3 as the solvent and
(
(
(
(
(
(
hexane) ꢄmax 216 nm (" 5790); EIMS m=z: 71 (84%), 101 (100), 129
23), 144 (65), 155 (½M þ Hꢂ , 40); 1H-NMR (CDCl3) ꢃ: 0.91
t, J ¼ 6:9 Hz, 3H, H-9), 1.33 (m, 2H, H-7), 1.35 (m, 2H, H-8), 1.85
m, 2H, H-6), 3.48 (s, 1H, 5-OH), 4.67 (t, J ¼ 6:8 Hz, 1H, H-5), 6.23
d, J ¼ 3:2 Hz, 1H, H-3), 6.33 (dd, J ¼ 1:8, 3.2 Hz, 1H, H-2), and 7.37
þ
1
3
d, J ¼ 1:8 Hz, 1H, H-1); C-NMR (CDCl3) ꢃ: 14.1 (C-9), 22.6 (C-8),
1
1
tetramethylsilane as the internal standard. H- H chemical shift-
1
2
7.8 (C-7), 35.4 (C-6), 67.9 (C-5), 105.8 (C-3), 110.2 (C-2), 141.9
C-1), and 157.0 (C-4). A mixture of compound 4 (20 mM) and
correlation spectroscopy (COSY) was employed to assign the H shifts
(
and coupling. 13C-NMR spectra were recorded at 150.91 MHz.
Heteronuclear multiple-bond coherence (HMBC) and heteronuclear
multiple-quantum coherence (HMQC) techniques were used to assign
the correlations between 1H and 13C signals. Atmospheric pressure
chemical ionization mass spectra (APCIMS) were obtained by an
LCMS-QP8000ꢁ instrument (Shimadzu Co., Kyoto, Japan). Samples
were delivered into the ion source by using methanol/ethanol (2:3, v/v)
at 0.2 ml/min. Electron impact mass spectra (EIMS) were obtained with
a JMS-AMSUN 200/GI instrument (Jeol, Tokyo, Japan) operated at
ꢀ
ꢀ
-tocopherol (10 mM) in acetonitrile (1.0 ml) was incubated at 37 C
for 3 h in the presence or absence of 50 mM HCl. The reaction products
were analyzed by HPLC as already described.
Reaction of ꢀ-tocopherol with ONE in methyl linoleate. Methyl
linoleate containing ꢀ-tocopherol (1.5 mg, 3.6 mmol), ONE (5.5 mg,
3
6 mmol), and dicetyl phosphate (3.8 mg, 7.2 mmol) was incubated at
0 C for 3 h. The reaction mixture was dissolved in ethanol and
ꢀ
6
analyzed by HPLC as already described, except that detection was
performed by fluorescence (ex 293 nm, em 336 nm).
70 eV. Ultraviolet (UV) spectra were measured with a U-2810
spectrophotometer (Hitachi Co., Tokyo, Japan), and fluorescence
spectra were recorded with an FP-6200ST spectrofluorometer (Jasco,
Tokyo, Japan).
Results
Reaction of ꢀ-tocopherol with ONE. ꢀ-Tocopherol (166 mg,
.40 mmol) and ONE (123 mg, 0.80 mmol) were dissolved in 40 ml
Reaction products of ꢀ-tocopherol with ONE
A mixture of ꢀ-tocopherol and ONE in an acidic
acetonitrile was incubated at 37 C for 3 h, and the
0
of a 50 mM HCl acetonitrile solution which had been prepared by
ꢀ
diluting a concentrated HCl reagent (12 M) with acetonitrile. The
ꢀ
reaction products were analyzed by reversed-phase
HPLC (Fig. 2). Three peaks corresponding to the
reaction products of ꢀ-tocopherol with ONE, 1–3,
appeared on the chromatogram. ꢁ-Tocopherol did not
react with ONE under the same conditions (data not
shown). Each reaction product was isolated by silica-gel
column chromatography followed by preparative re-
versed-phase HPLC. The structures of compounds 1–3
were characterized as described next (Fig. 1).
reaction mixture was incubated at 37 C for 3 h with mechanical
shaking. To the reaction mixture was added 20 ml of distilled water,
and the solution extracted three times with 60 ml of hexane. The
hexane layer was evaporated to dryness in vacuo, and the extract was
dissolved in ethanol and injected into the HPLC instrument. Reversed-
phase HPLC was conducted in an Inertsil ODS-80A column
(
4:6 ꢁ 150 mm; GL Sciences, Tokyo, Japan) developed with methanol
at a flow rate of 1.0 ml/min. The reaction products were detected by
the absorbance at 220 nm.
This reaction was conducted five times to isolate the reaction
products of ꢀ-tocopherol with ONE. The reaction products were
separated by silica-gel column chromatography developed with
Compound 1 was obtained as a brown oil (R 0.58,
f
16.0 mg yield). Compound 1 had positive reducing

170
F. SAITO et al.
0
0
ꢃ 2.04 and 2.19 (H-6 ) and a carbon signal at ꢃ 122.9
(C-5). Compound 1 had a chiral carbon atom at the
0
0
5
-position of the pentylfuran moiety, although the
stereochemistry could not be resolved. The multiplicity
of the NMR signals of 1 might have been due to a
mixture of optical isomers, although we did not separate
each isomer.
Compound 2 was obtained as a brown oil (Rf 0.33,
2.0 mg yield) and had negative reducing power. The
5
structure of 2 was determined to be 3-(1-butyl-4,5,7-
trimethyl-7-(4,8,12-trimethyltridecyl)-8,9-dihydro-7H-
furo[3,2-f]chromen-2-yl)propanal from its spectral data.
UV (hexane) ꢄmax: 260 (" 16300), 295 (4490), and
3
05 nm (4410); fluorescence (EtOH) ꢄex/em 265/328 nm;
þ
APCIMS m=z: 287 (12%) and 553 (½M þ Hꢂ , 100);
1
H-NMR (CDCl ) ꢃ: 0.84 and 0.85 (d, J ¼ 6:8 Hz, 6H,
3
Fig. 2. HPLC of the Reaction Products of ꢀ-Tocopherol with ONE
under Acidic Acetonitrile Conditions.
0
0
0
0
H-4 a, H-8 a), 0.86 (m, 6H, H-12 a, H-13 ), 0.94
0
0
0
A reaction mixture of ꢀ-tocopherol (10 mM) and ONE (20 mM) in
(t, J ¼ 6:8 Hz, 3H, H-9 ), 1.03–1.70 (m, 18H, H-2 to
ꢀ
0
0
0
0
0
acetonitrile was incubated at 37 C for 3 h in the presence of 50 mM
HCl. Reversed-phase HPLC was conducted in an Inertsil ODS-80A
H-12 , H-8 ), 1.27 (s, 3H, H-2a), 1.53 (m, 2H, H-7 ),
1.57 (m, 2H, H-1 ), 1.79 (m, 1H, H-3), 1.85 (m, 1H,
H-3), 2.16 (s, 3H, H-7a), 2.33 (s, 3H, H-8b), 2.65 (t,
0
column (4:6 ꢁ 150 mm) developed with methanol at a flow rate of
1.0 ml/min. The eluate was monitored at 220 nm.
0
0
00
J ¼ 7:6 Hz, 2H, H-6 ), 2.86 (t, J ¼ 7:6 Hz, 2H, H-2 ),
0
0
3
.01 (t, J ¼ 7:6 Hz, 2H, H-3 ), 3.02 (m, 2H, H-4), and
00 13
power, indicating the presence of a phenolic hydroxyl
2
9.88 (s, 1H, H-1 ); C-NMR (CDCl3) ꢃ: 11.8 (C-7a),
4)
00 00
11.9 (C-8b), 14.0 (C-9 ), 19.1 (C-3 ), 19.7 and 19.8
group in the molecule.
The structure of 1 was
0 0 0 0
(C-4 a, C-8 a), 21.0 (C-4), 22.6 (C2, C-12 a, C-13 ), 22.7
determined to be 5-(1-(furan-2-yl)pentyl)-2,7,8-trimeth-
yl-2-(4,8,12-trimethyltridecyl)chroman-6-ol(5-(1-(furan-
0
00
0
0
(2C, C-2 , C-8 ), 23.9 (C-2a), 24.5 (C2, C-6 , C-10 ),
24.8 (C-6 ), 28.0 (C-12 ), 31.2 (C-3), 32.7 and 32.8
0 0 00 0 0
(C-4 , C-8 ), 34.5 (C-7 ), 37.3 and 37.5 (C4, C-3 , C-5 ,
00 0
2
-yl)pentyl)-ꢀ-tocopherol) from its spectral data. UV
hexane) ꢄmax 295 nm (" 4040); fluorescence (EtOH)
(
ꢄex/em 293/336 nm; APCIMS m=z: 137 (18%), 287 (67),
0
0
0
0
00
C-7 , C-9 ), 39.4 (C-11 ), 39.9 (C-1 ), 42.3 (C-2 ), 74.9
00
þ
4
85 (56), 535 (90), 551 (100), and 553 (½M þ Hꢂ , 98);
(C-2), 109.4 (C-4a), 116.2 (C-5 ), 118.0 (C-7), 121.5
(C-8), 123.4 (C-5), 147.2 (C-6), 147.5 (C-8a), 150.7
1
0
0
0
H-NMR (CDCl3) ꢃ: 0.87 (m, 15H, H-4 a, H-8 a, H-12 a,
0
00
0
0
00
00 00
(C-4 ), and 201.4 (C-1 ). The structural assignment was
H-13 , H-9 ), 1.06–1.70 (m, 23H, H-1 to H-12 , H-8 ),
.21 (s, 3/2H, H-2a), 1.26 (s, 3/2H, H-2a), 1.78 (m, 1H,
1
1
1
H-3), 1.82 (m, 1H, H-3), 2.04 (m, 1H, H-6 ), 2.10 (s, 6H,
confirmed by H- H COSY, HMQC, and HMBC analy-
ses (data not shown). HMBC showed correlations
00
00
00
H-7a, H-8b), 2.19 (m, 1H, H-6 ), 2.70 (m, 1H, H-4),
between the proton signal at ꢃ 9.88 (H-1 ) and a carbon
00
00
2
1
7
1
.78 (m, 1H, H-4), 4.29 (br. m, 1H, H-5 ), 5.06 (br. s,
00
signal at ꢃ 42.3 (C-2 ); between the proton signal at ꢃ
3.02 (H-4) and carbon signals at ꢃ 31.2 (C-3), 74.9 (C-2),
00
H, 6-OH), 6.21 (m, 1H, H-3 ), 6.31 (m, 1H, H-2 ), and
13
00
.33 (d, J ¼ 1:4 Hz, 1H, H-1 ); C-NMR (CDCl ) ꢃ:
109.4 (C-4a), 123.4 (C-5), and 147.5 (C-8a); between
00
3
00
2.0 (2C, C-7a, C-8b), 14.0 (1/2C, C-9 ), 14.1 (1/2C,
0
the proton signal at ꢃ 3.01 (H-3 ) and carbon signals at ꢃ
00
0
0
0
00
00
00
C-9 ), 19.6 and 19.7 (2C, C-4 a, C-8 a), 21.0 (1/2C,
0
42.3 (C-2 ), 116.2 (C-5 ), 150.7 (C-4 ), 201.4 (C-1 );
00
C-4), 21.2 (1/2C, C-4), 22.6 and 22.7 (2C, C-12 a,
0
between the proton signal at ꢃ 2.86 (H-2 ) and carbon
00
0
00
00
00
C-13 ), 22.81 and 22.84 (2C, C-2 , C-8 ), 23.2 (1/2C,
0
signals at ꢃ 19.1 (C-3 ), 150.7 (C-4 ), and 201.4 (C-1 );
00
C-2a), 24.2 (1/2C, C-2a), 24.5 and 24.8 (2C, C-6 ,
0
between the proton signal at ꢃ 2.65 (H-6 ) and carbon
00
0
00
00
00
C-10 ), 28.0 (1C, C-12 ), 30.1 (1C, C-7 ), 30.9 (1C,
0
signals at ꢃ 22.7 (C-8 ), 34.5 (C-7 ), 116.2 (C-5 ), 123.4
00
0
C-6 ), 31.7 (1/2C, C-3), 31.8 (1/2C, C-3), 32.6, 32.7,
0
(C-5), and 150.7 (C-4 ); between the proton signal at ꢃ
0
and 32.8 (2C, C-4 , C-8 ), 37.3 (1C), 37.4 (1C), and
3
2.33 (H-8b) and carbon signals at ꢃ 118.0 (C-7), 121.5
(C-8), and 147.5 (C-8a); and between the proton signal
at ꢃ 2.16 (H-7a) and carbon signals at ꢃ 118.0 (C-7),
121.5 (C-8), and 147.2 (C-6). The results indicate that
the 4- and 5-positions of the ONE molecule were
respectively attached to the 6-hydroxy and 5-positions of
ꢀ-tocopherol.
0 0 0 0 00
7.5(2C) (C-3 , C-5 , C-7 , C-9 ), 37.4 (1C, C-5 ), 39.1
0 0 0
1/2C, C-1 ), 40.6 (1/2C, C-1 ), 39.4 (1C, C-11 ), 74.3
1C, C-2), 105.6 (1C, C-3 ), 110.1 (1C, C-2 ), 116.6
1C, C-4a), 122.8 (1C, C-7), 122.9 (1C, C-5), 124.7 (1C,
(
(
(
00
00
00
C-8), 142.0 (1C, C-1 ), 145.5 (1C, C-8a), 145.6 (1C,
C-6), and 157.2 (1C, C-4 ). The structural assignment
was confirmed by H- H COSY, HMQC, and HMBC
analyses (data not shown). HMBC showed correlations
between the proton signal at ꢃ 7.33 (H-1 ) and carbon
signals at ꢃ 105.6 (C-3 ), 110.1 (C-2 ), and 157.2
(C-4 ); between the proton signal at ꢃ 6.31 (H-2 ) and
00
carbon signals at ꢃ 142.0 (C-1 ) and 157.2 (C-4 );
between the proton signal at ꢃ 6.21 (H-3 ) and carbon
signals at 142.0 (C-1 ) and 157.2 (C-4 ); between the
proton signal at ꢃ 2.10 (H-7a and H-8b) and carbon
signals at 122.8 (C-7), 122.9 (C-5), 145.5 (C-8a),
and 145.6 (C-6); and between the proton signals at
00
1
1
Compound 3 was also obtained as a brown oil (Rf
0.67, 36.0 mg yield) and had negative reducing power.
The structure of 3 was determined to be 1-(1-butyl-4,5,7-
trimethyl-7-(4,8,12-trimethyltridecyl)-8,9-dihydro-7H-
furo[3,2-f]chromen-2-yl)-4-(furan-2-yl)octan-3-one from
its spectral data. UV (hexane) ꢄmax: 260 (" 13200), 295
(4240), and 305 nm (3910); fluorescence (EtOH) ꢄex/em
268/332 nm; APCIMS m=z: 423 (12%), 509 (19),
00
00
00
0
0
0
0
00
00
00
00
þ
1
and 689 (½M þ Hꢂ , 100); H-NMR (CDCl ) ꢃ: 0.85
3
0
0
0
0
000
(m, 15H, H-4 a, H-8 a, H-12 a, H-13 , H-9 ), 0.92
0
0
0
(t, J ¼ 7:5 Hz, 3H, H-9 ), 1.03–1.62 (m, 23H, H-2 to

Reaction of ꢀ-Tocopherol with 4-Oxo-2-nonenal
171
0
00
000
000
H-12 , H-8 , H-8 ), 1.19 (m, 2H, H-7 ), 1.26 (s, 3H,
0
00
H-2a), 1.49 (m, 2H, H-7 ), 1.59 (m, 2H, H-1 ), 1.78 (m,
000
1
H, H-3), 1.79 (m, 1H, H-6 ), 1.83 (m, 1H, H-3), 1.95
000
m, 1H, H-6 ), 2.16 (s, 3H, H-8b), 2.32 (s, 3H, H-7a),
(
2
(
00
00
.61 (t, J ¼ 7:5 Hz, 2H, H-6 ), 2.78 (m, 2H, H-2 ), 2.89
00
m, 2H, H-3 ), 3.00 (t, J ¼ 6:6 Hz, 2H, H-4), 3.70 (t,
000 000
J ¼ 7:5 Hz, 1H, H-5 ), 6.14 (d, J ¼ 3:4 Hz, 1H, H-3 ),
000
6
.30 (dd, J ¼ 2:1, 3.4 Hz, 1H, H-2 ), and 7.32 (d,
000 13
J ¼ 2:0 Hz, 1H, H-1 ); C-NMR (CDCl3) ꢃ: 11.7
000 00
C-8b), 11.9 (C-7a), 13.8 (C-9 ), 14.0 (C-9 ), 19.7 (2C,
(
C-4 a, C-8 a), 19.8 (C-4), 20.5 (C-3 ), 22.5 (3C, C-12 a,
0
0
00
0
0
00
000
0
C-13 , C-8 ), 22.6 (C-8 ), 22.7 (C-2 ), 23.9 (C-2a), 24.5
00 0 0 0
(
(
C-6 ), 24.4 and 24.8 (C-6 , C-10 ), 28.0 (C-12 ), 29.3
0
0
00
000
C-6 ), 29.5 (C-7 ), 31.3 (C-3), 32.7 and 32.8 (C-4 ,
0
00
0
0
0
C-8 ), 34.5 (C-7 ), 37.3 and 37.5 (3C, C-3 , C-5 , C-7 ,
0
0
00
0
C-9 ), 39.3 (C-11 ), 39.4 (C-2 ), 39.9 (C-1 ), 52.6
000 000
(
(
(
(
C-5 ), 74.9 (C-2), 107.2 (C-3 ), 109.4 (C-4a), 110.5
C-2 ), 115.9 (C-5 ), 117.8 (C-7), 121.2 (C-8), 123.4
0
00
00
000
Fig. 3. Effect of HCl Concentration on the Formation of the Reaction
Products from ꢀ-Tocopherol and ONE.
C-5), 142.0 (C-1 ), 147.1 (C-8a), 147.4 (C-6), 151.5
00
0
0
000
C-4 ), 152.4 (C-4 ), and 207.5 (C-1 ). The structural
A reaction mixture of ꢀ-tocopherol (10 mM) and ONE (20 mM) in
1
1
assignment was confirmed by H- H COSY, HMQC,
and HMBC analyses (data not shown). HMBC correla-
tions were observed between the proton signal at ꢃ 7.32
ꢀ
acetonitrile was incubated at 37 C in the presence of HCl for 1.5 h.
The reaction mixture was extracted with hexane and analyzed
by reversed-phase HPLC. The amounts of reaction products 1 ( ),
2 ( ) and 3 ( ) were calculated from the peak areas by monitoring
at 220 nm. Each value is expressed as the mean ꢃ S.D. of three
separate experiments.
000 000
H-1 ) and carbon signals at ꢃ 107.2 (C-3 ), 110.5
(
(
000 000
C-2 ), and 152.4 (C-4 ); between the proton signal at ꢃ
000 000
.30 (H-2 ) and carbon signals at ꢃ 107.2 (C-3 ), 142.0
6
(
0
00
000
C-1 ), and 152.4 (C-4 ); between the proton signal at ꢃ
.14 (H-3 ) and carbon signals at ꢃ 110.5 (C-2 ), 142.0
000 000
6
(
0
00
000
C-1 ), and 152.4 (C-4 ); between the proton signal at ꢃ
.70 (H-5 ) and carbon signals at ꢃ 29.3 (C-6 ), 107.2
000 000
3
0
00
000
00
(
proton signal at ꢃ 3.00 (H-4) and carbon signals at ꢃ 31.3
C-3 ), 152.4 (C-4 ), and 207.5 (C-1 ); between the
(
(
C-3), 74.9 (C-2), 109.4 (C-4a), 123.4 (C-5), and 147.1
0
0
C-8a); between the proton signal at ꢃ 2.89 (H-3 ) and
00
00
carbon signals at ꢃ 39.4 (C-2 ), 115.9 (C-5 ), 151.5
00 00
(
2
C-4 ), and 207.5 (C-1 ); between the proton signal at ꢃ
00 00
.78 (H-2 ) and carbon signals at ꢃ 20.5 (C-3 ), 151.5
0
0
00
(
2
C-4 ), and 207.5 (C-1 ); between the proton signal at ꢃ
.61 (H-6 ) and carbon signals at ꢃ 22.5 (C-8 ), 34.5
0
0
0
0
0
0
00
00
(
C-7 ), 115.9 (C-5 ), 123.4 (C-5), and 151.5 (C-4 );
between the proton signal at ꢃ 2.32 (H-7a) and carbon
signals at ꢃ 117.8 (C-7), 121.2 (C-8), and 147.4 (C-6);
between the proton signal at ꢃ 2.16 (H-8b) and carbon
signals at ꢃ 117.8 (C-7), 121.2 (C-8), and 147.1 (C-8a);
0
00
and between the proton signals at ꢃ 1.79 and 1.95 (H-6 )
000
000
and carbon signals at ꢃ 22.6 (C-8 ), 29.5 (C-7 ), 52.6
0
Fig. 4. Time-Course Characteristics for the Formation of the Reac-
tion Products of ꢀ-Tocopherol with ONE under Acidic Conditions.
A reaction mixture containing ꢀ-tocopherol (10 mM) and ONE
00
000
00
(C-5 ), 152.4 (C-4 ), and 207.5 (C-1 ). The results
indicate that the 5-position of ONE-derived pentylfuran
00
(20 mM) in acetonitrile containing 10 mM HCl was incubated at
ꢀ
molecule is attached to the C-1 position of compound 2
to form compound 3.
3
7 C. The reaction mixture was extracted with hexane and analyzed
by reversed-phase HPLC. Reaction products 1 ( ), 2 ( ) and 3 ( )
were calculated from the peak areas by monitoring at 220 nm. Each
value is expressed as the mean ꢃ S.D. of three separate experiments.
Reaction of ꢀ-tocopherol with ONE under acidic
conditions
A mixture of ꢀ-tocopherol and ONE was incubated at
different concentrations of HCl (0.05–500 mM) in
acetonitrile. After 1.5 h of incubation, the amounts of
compounds 1–3 produced were determined (Fig. 3).
ꢀ-Tocopherol was reacted with ONE in the presence
of 10 mM HCl in acetonitrile. Figure 4 shows the
formation of compounds 1–3 during the reaction
(Fig. 4). Compound 1 was produced first, with the
maximum amount after 1.5 h of incubation. After that,
compounds 2 and 3 accumulated with a decreasing
amount of compound 1. The amounts of compounds 1–3
in the 6-h reaction mixture were 0.32, 0.18, and
0.56 mM, respectively.
ꢀ
-Tocopherol could not react with ONE in the absence
of or at a low concentration (0.05 mM) of HCl.
Increasing the concentration of HCl in the reaction
mixture resulted in compound 1 appearing first, the
maximum amount of 1 being observed at 10 mM HCl.
Higher concentrations of HCl produced more of com-
pounds 2 and 3 in the reaction mixture.

172
F. SAITO et al.
Fig. 5. HPLC of the Reaction Products of ꢀ-Tocopherol with 2-(1-
Hydroxypentyl)furan (4).
Fig. 6. HPLC of the Reaction Products of ꢀ-Tocopherol with ONE in
Methyl Linoleate.
A reaction mixture of ꢀ-tocopherol (10 mM) and 2-(1-hydroxy-
pentyl)furan (20 mM) in acetonitrile was incubated at 37 C for 3 h in
the absence (A) or presence (B) of 50 mM HCl. The reaction mixture
was extracted with hexane and analyzed by reversed-phase HPLC as
described in Fig. 2.
Methyl linoleate containing ꢀ-tocopherol (1 mol%, based on
methyl linoleate), ONE (10 mol%, based on methyl linoleate), and
dicetyl phosphate (2 mol%, based on methyl linoleate) was
incubated at 60 C for 3 h. The reaction mixture in an ethanol
solution was analyzed by reversed-phase HPLC as described in
Fig. 2, except that detection was performed by fluorescence (ex
ꢀ
ꢀ
293 nm; em 336 nm). Arrows indicate the elution positions of
compounds 1–3.
Reaction of ꢀ-tocopherol with 2-(1-hydroxypentyl)-
furan (4)
Incubation of ONE in an acidic acetonitrile solution at
5-Nitro-ꢀ-tocopherol has been detected in human plasma
and in plant tissues, and is considered to be a biomarker
ꢀ
37 C for 10 min resulted in a cyclized reaction to give
2-(1-hydroxypentyl)furan (4). Resulting compound 4
1
5,16,26)
for oxidative stress. ꢀ-Tocopherol has also reacted
with hypochlorous acid, a potential biological oxidant
and electrophile, to form 5-chloro-ꢀ-tocopherol and
was thought to be an intermediate of the reaction
between ꢀ-tocopherol and ONE under acidic conditions.
We thus analyzed the reaction products of ꢀ-tocopherol
with compound 4 by reversed-phase HPLC (Fig. 5).
Peaks corresponding to compounds 1–3 appeared on the
chromatogram when the reaction mixture was incubated
in the presence of 50 mM HCl. On the other hand,
2
7)
other chlorinated products. ꢀ-Tocopherol in the pres-
ent study reacted with ONE, the bi-functional electro-
phile produced from the hemolytic decomposition of
lipid hydroperoxides, in an acidic acetonitrile solution,
and three addition products, 1–3, were detected by
reversed-phase HPLC (Fig. 2). These products were
characterized as 5-substituted ꢀ-tocopherols: 5-(1-(furan-
2-yl)pentyl)-ꢀ-tocopherol (1), 3-(1-butyl-4,5,7-trimethyl-
7-(4,8,12-trimethyltridecyl)-8,9-dihydro-7H-furo[3,2-
f]chromen-2-yl)propanal (2), and 1-(1-butyl-4,5,7-
trimethyl-7-(4,8,12-trimethyltridecyl)-8,9-dihydro-7H-
furo[3,2-f]chromen-2-yl)-4-(furan-2-yl)octan-3-one (3)
(Fig. 1). Compound 1 formed initially, and was the most
predominant product under mild acidic conditions
(Figs. 3 and 4), while compounds 2 and 3 were relatively
stable and accumulated during the reaction.
ꢀ-tocopherol did not react with 4 without the acid. This
result indicates that the acid was essential for the
reaction of ꢀ-tocopherol with 2-(1-hydroxypentyl)furan
(4) and that the same products could be formed as those
for the reaction between ꢀ-tocopherol and ONE.
Formation of the addition products of ꢀ-tocopherol
with ONE in methyl linoleate
ꢀ
-Tocopherol and ONE in the bulk phase of methyl
linoleate were incubated for 3 h. The reaction was
performed in the presence of dicetyl phosphate, a model
compound for such acidic phospholipids as phosphati-
dylserine, phosphatidylinositol and phosphatidic acid. A
product peak corresponding to compound 1 appeared on
the HPLC chromatogram, together with some unknown
peaks and a minor peak corresponding to compound
It has been reported that the nucleophilic groups of
proteins can attack the C-1 aldehyde of ONE to form
Schiff base adducts, or attack the C-2 or C-3 position of
1
8,23,28–31)
the double bond to form Michael adducts.
The
addition by nucleobases also occurs at the C-1 position
of ONE with the subsequent reaction at the C-2 position
of the double bond to form 2-oxoheptyl-substituted
2
(Fig. 6). The identity of each peak was confirmed by
co-eluting against authentic compounds 1–3. This result
indicates that the reaction between ꢀ-tocopherol and
ONE may proceed in methyl linoleate under acidic
conditions.
1
9,32–36)
etheno adducts.
The acid-catalyzed reaction of
ꢀ-tocopherol with ONE in an acetonitrile solution
proceeded by a different mechanism (Fig. 7). The first
step would be isomerization of the double bond of ONE
from trans to cis, this being followed by the formation of
Discussion
2
3)
the enol tautomer, 4-hydroxy-2,4-nonadienal. Intra-
molecular nucleophilic addition between the 4-hydroxyl
and 1-carbonyl groups of the enol molecule would then
give a cyclic hemiacetal intermediate. If ꢀ-tocopherol is
present in the reaction mixture, the C-5 position of
ꢀ-tocopherol could attack the double bond at the C-5
ꢀ
-Tocopherol is a better nucleophile than ꢁ-tocopherol
and may scavenge electrophiles with high affinity.
Previous studies have revealed that ꢀ-tocopherol had
the capability to scavenge nitric oxide by nitration,
1
4)
resulting in the formation of 5-nitro-ꢀ-tocopherol.

Reaction of ꢀ-Tocopherol with 4-Oxo-2-nonenal
173
Fig. 7. Proposed Mechanism for the Reaction of ꢀ-Tocopherol (ꢀ-TH) with ONE under Acidic Acetonitrile Conditions.
position of the hemiacetal intermediate to give 5-(1-
furan-2-yl)pentyl)-ꢀ-tocopherol (1). In the absence of
-tocopherol, however, hydration at the C-5 position and
subsequent dehydration of the intermediate would yield
-(1-hydroxypentyl)furan (4). The reaction of ꢀ-toco-
(Fig. 6). However, the biological importance of this
(
ꢀ
reaction is still unknown. It has been reported that the
supplementation of ꢀ-tocopherol to humans with meta-
bolic syndrome reduced the plasma malondialdehyde
1
7)
2
and HNE levels, and that the nucleophilic property of
ꢀ-tocopherol might be important to trap such lipophilic
aldehydes. Further studies will be needed to elucidate
the scavenging ability of ꢀ-tocopherol against ꢁ,ꢂ-
unsaturated aldehydes in a biological system.
pherol with compound 4 gave the same addition
products, 1–3, as the reaction with ONE (Fig. 5). This
supports the notion that the cyclization of ONE might be
the first step of the addition reaction with ꢀ-tocopherol.
Compound 4 has already been reported to be the major
ONE-derived side product observed in reactions with
Acknowledgment
2
3)
some nucleophiles. When the reaction proceeds or the
reaction is performed under stronger acidic conditions,
cleavage of the furan ring of compound 1 occurs and the
attack by the 6-hydroxyl group of the ꢀ-tocopherol
moiety at the C-4 position of the opened furan ring
This work was supported in part by Grant-in-Aid for
Scientific Research (nos. 18580115 and 20580125) from
JSPS.
3
7)
results in the formation of compound 2. In addition,
incubation of compound 2 with ONE or compound 4 in
acidic acetonitrile resulted in the formation of compound
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