Elucidating the formation pathway of the off-flavor compound 6-propylbenzofuran-7-ol

Article abstract of DOI:10.1021/jf302762j

In a previous work, we identified 6-propylbenzofuran-7-ol as an off-flavor compound formed from ascorbic acid and (E)-hex-2-enal in a test apple beverage. In this study, we elucidate the pathway by which 6-propylbenzofuran-7-ol formed. Isotope labeling studies revealed that the propyl group of 6-propylbenzofuran- 7-ol derives from (E)-hex-2-enal and that 6-propylbenzofuran-7-ol contains carbons 2-6 of ascorbic acid. Two compounds, namely, 2,3-dihydro-6- propylbenzofuran-3,7-diol and 3-(2-furoyl)hexanal, were identified as byproducts of a model reaction of ascorbic acid and (E)-hex-2-enal. Each of these compounds was dissolved in an aqueous solution of citric acid and stored at 60 °C for 1 week. After storage, 6-propylbenzofuran-7-ol was detected from a solution of 2,3-dihydro-6-propylbenzofuran-3,7-diol, but not from 3-(2-furoyl)hexanal. 6-Propylbenzofuran-7-ol was formed by isolating tricyclic hemiacetal lactone derived from the Michael addition of ascorbic acid to (E)-hex-2-enal, mixing the tricyclic hemiacetal lactone with the aqueous solution of citric acid, and applying heat. This confirmed that 6-propylbenzofuran-7-ol was formed via the Michael adduct.

Full text of DOI:10.1021/jf302762j

Article
pubs.acs.org/JAFC
Elucidating the Formation Pathway of the Off-Flavor Compound
6‑Propylbenzofuran-7-ol
Kensuke Sakamaki,* Susumu Ishizaki, Yasutaka Ohkubo, Yoshiyuki Tateno, Akira Nakanishi,
and Akira Fujita
Technical Research Institute R&D Center, T. Hasegawa Co., Ltd., 29-7, Kariyado, Nakahara-ku, Kawasaki-shi, 211-0022, Japan
S
* Supporting Information
ABSTRACT: In a previous work, we identified 6-propylbenzofuran-7-ol as an off-flavor compound formed from ascorbic acid
and (E)-hex-2-enal in a test apple beverage. In this study, we elucidate the pathway by which 6-propylbenzofuran-7-ol formed.
Isotope labeling studies revealed that the propyl group of 6-propylbenzofuran-7-ol derives from (E)-hex-2-enal and that 6-
propylbenzofuran-7-ol contains carbons 2−6 of ascorbic acid. Two compounds, namely, 2,3-dihydro-6-propylbenzofuran-3,7-diol
and 3-(2-furoyl)hexanal, were identified as byproducts of a model reaction of ascorbic acid and (E)-hex-2-enal. Each of these
compounds was dissolved in an aqueous solution of citric acid and stored at 60 °C for 1 week. After storage, 6-propylbenzofuran-
7-ol was detected from a solution of 2,3-dihydro-6-propylbenzofuran-3,7-diol, but not from 3-(2-furoyl)hexanal. 6-
Propylbenzofuran-7-ol was formed by isolating tricyclic hemiacetal lactone derived from the Michael addition of ascorbic acid
to (E)-hex-2-enal, mixing the tricyclic hemiacetal lactone with the aqueous solution of citric acid, and applying heat. This
confirmed that 6-propylbenzofuran-7-ol was formed via the Michael adduct.
KEYWORDS: apple beverage, off-flavor, ascorbic acid, (E)-hex-2-enal, isotope labeling study, 6-propylbenzofuran-7-ol,
Michael addition
hex-2-enal were identified as important constituents of the off-
flavor compound formed. This off-flavor may develop in any of
the various foods found to contain the compounds ascorbic
acid and (E)-hex-2-enal. By clarifying the mechanisms by which
6-propylbenzofuran-7-ol forms, we may have better guidance
for preventing the occurrence of this off-flavor in foods with
these compounds.
This study seeks to clarify the pathway by which 6-
propylbenzofuran-7-ol forms. We describe our investigations
using isotope-labeled precursors and the isolation of reaction
intermediates for the formation of 6-propylbenzofuran-7-ol.
INTRODUCTION
■
Ascorbic acid is a water-soluble vitamin that plays many
biological roles and is contained in many foods. Ascorbic acid is
often added to processed foods as an antioxidant or nutritional
supplement. But on the other hand, it is known that
degradation of ascorbic acid causes problems such as
nonenzymatic browning and off-flavor .1−11
Kurata et al.1 reported that L-ascorbic acid was degraded to
furfural with the formation of 3-deoxy-L-pentose as an
intermediate in an acidic condition. This acid-catalyzed
degradation reaction took place without oxygen and under
the storage or cooking condition of foodstuffs. Tatum et al.2
identified fifteen compounds as degradation products from
ascorbic acid.
MATERIALS AND METHODS
■
Chemicals. The following chemicals were obtained
commercially: (E)-hex-2-enal (PFW Aroma Chemicals B.V.,
Barneveld, Netherlands), ascorbic acid (DSM Nutrition Japan
K.K., Tokyo, Japan), citric acid (IWATA CHEMICAL CO.,
Ltd., Shizuoka, Japan), [13C6]-ascorbic acid and [1-13C]-
ascorbic acid (Omicron Biochemicals, Inc., South Bend, IN),
and [D10]-n-butanol (C/D/N ISOTOPES, Inc., Quebec,
Canada). [D6−8]-(E)-hex-2-enal was synthesized in our
laboratory. All other reagents and solvents were of analytical
grade.
Ascorbic acid reacts with other constituents of foods or
beverages and causes off-flavors. In experiments by Konig et
̈
al.12 with citrus soft drinks, the reaction of ascorbic acid with
ethyl alcohol during beverage storage produced 3-hydroxy-4,5-
dimethyl-2(5H)-furanone (sotolon) and caused on off-flavor.
In follow-up studies using isotopically labeled ethyl alcohol and
ascorbic acid, Konig et al.12 hypothesized that soloton could be
̈
formed by either of two pathways, that is, formation from one
molecule of ascorbic acid with two molecules of ethyl alcohol
or with one molecule of ethyl alcohol.
Synthesis. [D8]-n-Butanal (2). Dess-Martin periodinane
(75.6 g, 178 mmol) was added to a solution of [D10]-n-butanol
(1, 10.0 g, 118 mmol) and pyridine (23.5 g, 297 mmol) in
(E)-Hex-2-enal has a fresh, green odor, and occurs naturally
in many foods. In processed foods, (E)-hex-2-enal is used as a
flavor ingredient to impart freshness. The compound is
regarded as an important flavor component of apple juice.13,14
In a previous work, we identified 6-propylbenzofuran-7-ol as
a medicinal off-flavor compound present after the storage of a
test apple beverage at 40 °C for 8 weeks.15 Among the
constituents of the test apple beverage, ascorbic acid and (E)-
Received: June 27, 2012
Revised: September 12, 2012
Accepted: September 13, 2012
Published: September 13, 2012
© 2012 American Chemical Society
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CH2Cl2 (100 mL) at 0 °C. After 30 min of stirring at 0 °C, the
reaction mixture was warmed to room temperature and stirred
for 30 min, cooled back down to 0 °C (the temperature of the
mixture rose spontaneously to over 50 °C during the stirring),
and stirred for another 30 min at 0 °C. Then, the reaction
mixture was warmed to room temperature and stirred for 30
min. Next, more Dess-Martin periodinane (5.0 g, 11.9 mmol)
was added, the mixture was stirred for another 2.5 h, and
saturated aqueous Na2S2O3 and saturated aqueous NaHCO3
were added gradually, and stirred for a while. The mixture was
then extracted with CH2Cl2, washed with saturated aqueous
NaHCO3, and dried over MgSO4. This CH2Cl2 solution was
directly used in the next step.
1 h at 0 °C, and then stirred for 1 h at 0 °C. After the addition
of water, HCl (2 mol/L) was added, the organic layer was
separated, and the aqueous layer was extracted with diethyl
ether. The combined organic layer was washed with saturated
aqueous NaHCO3, dried over MgSO4, and concentrated by
distillation under atmospheric pressure. CH2Cl2 (100 mL) was
added to the residue and then directly used in that form in the
next step.
MS-EI (m/z: intensity in %) 108 (0.7), 107 (0.5), 89 (9), 88
(8), 87 (4), 59 (28), 58 (100), 45 (25), 44 (28).
[D6−8]-(E)-Hex-2-enal (6). Under N2 atmosphere, MnO2
(21.6 g) was added to CH2Cl2 solution of [D6−8]-(E)-hex-2-
enol (5) from the previous step and stirred at room
temperature for 14.5 h. MnO2 was added to the mixture
twice in equal portions of 21.6 g, with stirring for 4.5 and 2.5 h
after the first and second additions, respectively. After filtration,
a grain of 2,6-di-tert-butyl-4-methylphenol (BHT) was added,
and concentrated under reduced pressure (30 °C, 70 kPa). The
residue was purified by silica gel column chromatography
eluted with n-pentane/diethyl ether (50:1, v/v) into a crude
product (6, 3.2 g). The crude product was distilled under
reduced pressure in a Kugelrohr apparatus (110 °C, 2.0 kPa) to
give a colorless oil of [D6−8]-(E)-hex-2-enal (6, 2.4 g; yield,
57% for 2 steps; purity, 93.3%). Arbitrary numbering of carbon
atoms in NMR data refers to Figure 1.
MS-EI (m/z: intensity in %) 78 (54), 60 (20), 48 (55), 47
(11), 46 (100), 45 (36), 44 (35), 42 (13).
[D6−8]-(E)-Hex-2-enoic Acid (3). In a flask equipped with
distillation apparatus, the CH2Cl2 solution of [D8]-n-butanal
(2) obtained in the previous step was added to a solution of
malonic acid (18.6 g, 178.2 mmol) in pyridine (22.0 g) at 50
°C. The mixture was then warmed to 80 °C as removing
CH2Cl2 by distillation. After stirring for 1.5 h at 80 °C, the
mixture was cooled to room temperature, malonic acid (9.3 g,
89 mmol), pyridine (11.0 g), and the previously removed
CH2Cl2 solution (containing unreacted [D8]-n-butanal) were
added, and the mixture was warmed to 80 °C again as removing
CH2Cl2. After stirring for 4 h at 80 °C, the mixture was cooled
to room temperature, the removed CH2Cl2 solution (contain-
ing unreacted [D8]-n-butanal) was added, and the mixture was
stirred for 3 h at 80 °C. The mixture was poured into water,
extracted with diethyl ether, washed with HCl (2 mol/L), and
concentrated under reduced pressure. The residue was
dissolved in n-hexane and extracted with 10% aqueous NaOH
(47.5 g, 119 mmol), and the aqueous NaOH layer was washed
with n-hexane, acidified with 50% aqueous H2SO4 (12.8 g, 131
mmol), and extracted with diethyl ether. The diethyl ether layer
was washed with water and brine, dried over MgSO4, and
concentrated under reduced pressure into a crude oil of [D6−8]-
(E)-hex-2-enoic acid (3, 15.7 g). This crude product was
directly used in the next step.
1
[D6−8]-(E)-hex-2-enal 6; H NMR (400 MHz, CDCl3): δ
2.25 and 2.27 (s, s, total 1H, H−C4 of [D6]-6 and [D7]-6);
6.09 (d, J = 8.0, 1H, H−C2); 9.48 (d, 1H, J = 8.0, H−C1). 13
C
NMR (100 MHz, CDCl3): δ 12.4 (septet, C6), 20.0 (m, C5),
33.3 (m, C4 of [D8]-6), 33.9 (t, C4 of [D7]-6), 34.3 (C4 of
[D6]-6), 133.0 (C2), 158.3 (t, C3), 194.1 (C1). MS-EI (m/z:
intensity in %) 106 (18), 105 (19), 104 (17), 88 (54), 87 (40),
86 (34), 76 (20), 75 (21), 73 (29), 72 (35), 71 (24), 60 (100),
59 (59), 58 (40), 57 (74), 56 (45), 48 (50), 47 (43), 46 (92),
45 (95), 44 (72), 42 (52), 41 (55), 40 (37).
Labeling Experiments. Three labeling experiments were
performed. Solutions with the following composition were
prepared: ascorbic acid (20 mg), citric acid (2 mg), (E)-hex-2-
enal (2 mg) and deionized water (20 mL). (E)-Hex-2-enal was
substituted with [D6−8]-(E)-hex-2-enal in one solution, ascorbic
acid was substituted with [13C6]-ascorbic acid in another
solution, and ascorbic acid was substituted with [1-13C]-
ascorbic acid in a third solution. The three solutions were
poured into glass bottles, and stored at 60 °C in darkness for 1
week, and extracted with diethyl ether (3 × 10 mL). The
organic layer was washed with brine, dried over anhydrous
sodium sulfate, filtered, concentrated with a Vigreux column
under atmospheric pressure, and analyzed by GC-MS.
Model Reaction. Ascorbic acid (200 mg), citric acid (20
mg) and (E)-hex-2-enal (20 mg) were dissolved in deionized
water (200 mL) to give a model solution with a pH of about
3.2. The solution was poured into glass bottle and stored at 60
°C in darkness for 1 week. After storage, the solution was
extracted with diethyl ether (3 × 20 mL). Diethyl ether
solution of tridecane (0.2 g/L, 1.0 mL) was added to the
organic layer as an internal standard (IS), and the organic layer
was dried over anhydrous sodium sulfate, filtered, concentrated
with a Vigreux column under atmospheric pressure, and
analyzed by GC-MS.
MS-EI (m/z: intensity in %) 122 (19), 121 (9), 120 (7), 104
(27), 103 (21), 102 (17), 77 (33), 76 (100), 75 (53), 74 (17),
62 (18), 61 (21), 60 (17), 57 (33), 56 (19), 48 (48), 47 (35),
46 (41), 45 (58), 44 (29).
[D6−8]-Ethyl (E)-Hex-2-enoate (4). Concd H2SO4 (1.6 g)
was added to a solution of crude [D8]-(E)-hex-2-enoic acid (3,
15.7 g) in EtOH (157 g) at room temperature and refluxed for
3 h. Then, EtOH was removed by distillation under
atmospheric pressure. After cooling, the mixture was poured
into water, extracted with diethyl ether, washed with saturated
aqueous NaHCO3, water and brine, dried over MgSO4, and
concentrated in a high vacuum. The residue (15.5 g) was
distilled (58−61 °C/1.0 kPa) into a colorless oil of [D6−8]-ethyl
(E)-hex-2-enoate (4, 6.2 g; yield, 35% for 3 steps; purity,
90.5%).
MS-EI (m/z: intensity in %) 150 (2), 149 (2), 148 (2), 122
(8), 121 (7), 120 (6), 105 (78), 104 (74), 103 (57), 100 (100),
77 (18), 76 (40), 75 (26), 57 (100), 56 (54), 45 (39).
[D6−8]-(E)-Hex-2-enol (5). Under N2 atmosphere, a solution
of AlCl3 (8.0 g, 60 mmol) in diethyl ether (100 mL) was added
dropwise to a stirred suspension of LiAlH4 (1.5 g, 40 mmol) in
diethyl ether (100 mL) for 30 min at 0 °C. After stirring for 30
min at 0 °C, a solution of (E)-hex-2-enoate (4, 6.0 g, 40 mmol)
in diethyl ether (50 mL) was added dropwise to the mixture for
Isolation of Diol (8) and Ketoaldehyde (9). Ascorbic
acid (1.0 g, 5.7 mmol), citric acid (0.1 g, 0.52 mmol) and (E)-
hex-2-enal (1.0 g, 10.2 mmol) were dissolved in deionized
water (1000 mL). The solution was poured into a glass bottle,
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Figure 1. Synthesis of labeled (E)-hex-2-enal.
stored at 70 °C in darkness for 1 week, and extracted with
diethyl ether (3 × 200 mL). The organic layer was washed with
brine, dried over anhydrous sodium sulfate, filtered, and
concentrated under reduced pressure. The residue (0.52 g)
was purified by silica gel chromatography using n-hexane/ethyl
acetate (5:1, v/v) and n-hexane/ethyl acetate (2:1, v/v) in
sequence into a yellow oil of 3-(2-furoyl)hexanal (9, 152 mg,
yield: 13.8%, purity: 93.7%) and a colorless solid of 2,3-
dihydro-6-propylbenzofuran-3,7-diol (8). This solid was
recrystallized in CH2Cl2 to give colorless needles of 2,3-
dihydro-6-propylbenzofuran-3,7-diol (8, 38 mg, yield: 3.4%,
purity: 98.7%). Arbitrary numbering of carbon atoms in NMR
data refers to Figure 6.
in the electron impact (EI) mode were recorded at 70 eV
ionization energy.
Gas Chromatography (GC). The GC analyses were
performed with an Agilent 6890 GC equipped with a flame
ionization detector (FID; 250 °C). The column, sample
volume, split ratio, injection temperature, oven temperature
program, carrier gas, and flow rate were all the same as those set
for the GC-MS analysis described above. The purity of the
compounds was calculated by integration of the chromatogram
obtained by the FID.
1
Nuclear Magnetic Resonance (NMR) Spectra. H, 13C,
HMQC, and HMBC experiments were performed on a JEOL
JNM-ECX400 spectrometer, using CDCl3 or DMSO-d6 as
solvent. The chemical shifts were measured from the signal of
tetramethylsilane used as an internal standard. The chemical
shifts (δ) and coupling constants (J) are expressed in parts per
million (ppm) and hertz (Hz), respectively.
Fourier Transformation Infrared Spectroscopy (FTIR).
The IR spectrum was recorded on a JUSCO FT/IR-4100 FTIR
spectrometer from 4000 to 400 cm−1.
High Resolution fast atom bombardment Mass
Spectra (HR-FAB-MS). The HR-FAB-MS were recorded on
a JEOL JMS-700 with a matrix of glycerol and PEG200.
Reaction of Diol (8). Diol (8, 20 mg) and citric acid (1.0 g)
were dissolved in deionized water (100 mL) to give a solution
with a pH of about 3. The solution was poured into a glass
bottle, stored at 60 °C in darkness for 1 week and extracted
with diethyl ether (3 × 20 mL). The organic layer was washed
with brine, dried over anhydrous sodium sulfate, filtered,
concentrated with a Vigreux column under atmospheric
pressure, and analyzed by GC-MS.
Reaction of Ketoaldehyde (9). Ketoaldehyde (9, 20 mg)
and citric acid (1.0 g) were dissolved in deionized water (100
mL) to give a solution with a pH of about 3. The solution was
stored and the volatile fractions were isolated by the method
described above.
3-(2-Furoyl)hexanal 9; 1H NMR (400 MHz, CDCl3): δ 0.87
(t, J = 7.6, 3H, H−C12); 1.30 (m, 2H, H−C11); 1.47 (m, 1H,
H−C10); 1.69 (m, 1H, H−C10); 2.61 (dd, J = 4.4, 18.8, 1H,
H−C8); 3.07 (dd, J = 8.8, 18.8, 1H, H−C8); 3.71 (dddd, J =
4.4, 6.8, 6.8, 8.8, 1H, H−C9); 6.53 (dd, J = 1.6, 3.4, 1H, H−
C5); 7.23 (d, J = 3.6, 1H, H−C4); 7.59 (d, J = 1.6, 1H, H−
C6); 9.73 (s, 1H, H−C7). 13C NMR (100 MHz, CDCl3): δ
14.0 (C12), 20.3 (C11), 34.5 (C10), 40.6 (C9), 45.2 (C8),
112.4 (C5), 117.8 (C4), 146.6 (C6), 152.4 (C3), 191.2 (C2),
200.5 (C7). MS-EI (m/z: intensity in %) 194 (M+, 0.2), 152
(44), 123 (76), 95 (100), 55 (10), 39(12).
1
2,3-Dihydro-6-propylbenzofuran-3,7-diol 8; H NMR (400
MHz, CDCl3): δ 0.94 (t, J = 7.3, 3H, H−C12); 1.60 (tq, J =
7.3, 7,3, 2H, H−C11); 2.56 (t, J = 7.3, 2H, H−C10); 3.8 (m,
2H, H−C6); 4.86 (dd, J = 5.0, 5.92, 1H, H−C5); 5.75 (br, 1H,
HO-C2); 6.43 (d, J = 7.8, 1H, H−C7); 6.60 (d, J = 7.8, 1H, H−
C8); 8.39 (br, 1H, HO-C5). 13C NMR (100 MHz, CDCl3): δ
14.1 (C12), 22.8 (C11), 31.7 (C10), 66.3 (C6), 75.8 (C5),
117.7 (C7), 120.7 (C4), 121.1 (C8), 128.8 (C9), 142.3 (C3),
143.3 (C2). MS-EI (m/z: intensity in %) 194 (M+, 36), 176
(3), 165 (100), 147 (13), 137 (13), 123(12), 91 (12), 77 (7).
Gas Chromatography−Mass Spectrometry (GC-MS).
The GC-MS analyses were performed with an Agilent 7890 gas
chromatograph (GC) combined with an Agilent MSD5975
quadrupole mass spectrometer equipped with a TC-1 capillary
column (0.25 mm i.d. × 60 m, 0.25 μm film thickness; GL
Sciences Co., Tokyo, Japan). Each sample was injected in 0.2
μL volumes in a split mode (30: 1) at a constant temperature of
250 °C. The oven temperature was held at 40 °C for the initial
3 min and then increased to 250 °C at a rate of 3 °C/min, with
a constant carrier helium gas flow of 1.8 mL/min. Mass spectra
Synthesis of Tricyclic Hemiacetal Lactone (11). Under
nitrogen atmosphere, ascorbic acid (17.6 g, 0.10 mol) was
dissolved in deionized water (100 mL). Hydrochloric acid (0.5
mL) and (E)-hex-2-enal (10.8 g, 0.11 mol) were added and
then stirred for 1 week at room temperature. The reaction
mixture was extracted by n-hexane (30 mL), and then extracted
by ethyl acetate (6 × 30 mL). The ethyl acetate layers were
combined, dried over MgSO4, and concentrated under high
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Figure 2. Mass spectra of (a) unlabeled 6-propylbenzofuran-7-ol, (b) [D7]-6-propylbenzofuran-7-ol, (c) [D6]-6-propylbenzofuran-7-ol, and (d)
[D5]-6-propylbenzofuran-7-ol formed from [D6−8]-(E)-hex-2-enal and unlabeled ascorbic acid.
vacuum to yield a yellow oil (16.8 g). The crude product was
recrystallized from methyl tert-butyl ether (MTBE)/n-hexane
to give colorless needles of 1,3,7-trioxa-8-oxo-5,9,12-trihydroxy-
10-propyltricyclo-[4.3.2.02,6.02,9]-dodecane (11, 10.1 g, yield:
36.9%). Arbitrary numbering of carbon atoms in NMR data
refers to Figure 6.
concentrated with a Vigreux column under atmospheric
pressure, and analyzed by GC-MS.
RESULTS AND DISCUSSION
■
Synthesis of Labeled (E)-Hex-2-enal. The synthesis of
labeled (E)-hex-2-enal is outlined in Figure 1. [D10]-n-Butanol
(1) was oxidized with Dess-Martin periodinane to give [D8]-n-
butanal (2), whereupon the Doebner modification of [D8]-n-
butanal (2) gave [D6−8]-(E)-hex-2-enoic acid (3). This step
was thought that to form not only [D8]-(E)-hex-2-enoic acid,
but also [D6−7]-(E)-hex-2-enoic acid, because the α-deuterium
of [D8]-n-butanal (2) was abstracted by pyridine and
protonated by malonic acid. Next, 3 was esterified with ethanol
to produce [D6−8]-ethyl (E)-hex-2-enoate (4), whereupon 4
was reduced with AlH3 prepared from LiAlH4 and AlCl3 to give
[D6−8]-(E)-hex-2-enol (5), then 5 was oxidized with MnO2 to
give [D6−8]-(E)-hex-2-enal (6). The carbon atom on which
deuterium was attached was confirmed by NMR. This [D6−8]-
(E)-hex-2-enal (6) was used for the labeling experiment.
Labeling Experiments. [D6−8]-(E)-Hex-2-enal (6) was
added to an aqueous solution of unlabeled ascorbic acid, and
the mixture was stored at 60 °C for 1 week. GC-MS analysis
detected [D5−7]-6-propylbenzofuran-7-ol (Figure 2). A frag-
ment ion of unlabeled 6-propylbenzofuran-7-ol at m/z 147 [M
− 29] indicated cleavage of an ethyl group. Meanwhile, [D7]-6-
propylbenzofuran-7-ol gave a corresponding fragment ion at m/
z 149 [M − 34], which indicated cleavage of an [D5]-ethyl
1,3,7-Trioxa-8-oxo-5,9,12-trihydroxy-10-propyltricyclo-
20
[4.3.2.02,6.02,9]-dodecane 11; mp, 77 °C. [α]D = +18.2° (c
1
0.01, MeOH). H NMR (400 MHz, DMSO-d6): δ 0.88 (t, J =
6.8, 3H, H−C12); 1.18−1.26 (m, 2H, H−C11); 1.30−1.42 (m,
2H, H−C10); 1.72 (ddd, J = 5.5, 12.4, 12.4, 1H, H−C8); 1.92
(dd, J = 7.3, 12.4, 1H, H−C8); 2.73−2.82 (m, 1H, H−C9);
3.81 (dd, J = 5.0, 9.6, 1H, H−C6); 4.15 (dd, J = 6.8, 9.6, 1H,
H−C6); 4.25 (ddd, J = 4.1, 5.0, 6.8, 1H, H−C5); 4.41 (s, 1H,
H−C4); 5.49 (dd, J = 5.5, 5.5, 1H, H−C7); 5.58 (d, J = 4.1,
1H, HO-C5); 6.44 (d, J = 5.5, 1H, HO-C7); 6.73 (s, 1H, HO-
C2). 13C NMR (100 MHz, DMSO-d6): δ 14.1 (C12), 21.4
(C11), 30.9 (C10), 38.5 (C9), 40.3 (C8), 74.1 (C6), 74.4
(C4), 88.4 (C5), 89.0 (C2), 98.1 (C7), 106.0 (C3), 174.3
(C1). IR (KBr): cm−1 3419, 2961, 2935, 2874, 1793, 1634,
1467, 1341, 1023. HRMS (FAB) found 275.1133, calculated for
C12H19O7 [M + H]+ 275.1131.
Reaction of Tricyclic Hemiacetal Lactone (11). Tricyclic
hemiacetal lactone (11, 200 mg) and citric acid (2.0 g) were
dissolved in deionized water (200 mL) to give a solution with a
pH of about 3. The solution was poured into a glass bottle,
stored at 60 °C in darkness for 1 week, and extracted with
diethyl ether (3 × 40 mL). The organic layer was washed with
brine, dried over anhydrous sodium sulfate, filtered, and
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Figure 3. Mass spectra of 6-propylbenzofuran-7-ol formed from: (a) [13C6]-ascorbic acid and unlabeled (E)-hex-2-enal; (b) [1-13C]-ascorbic acid
and unlabeled (E)-hex-2-enal.
group. This indicated deuterium atoms must be located at the
propyl group.
Table 1. Compounds Identified from a Model Solution of
Ascorbic Acid and (E)-Hex-2-enal after Storage at 60 °C for
1 Week
Next, when we carried out the same reaction using solution
of [13C6]-ascorbic acid with unlabeled (E)-hex-2-enal, GC-MS
analysis revealed the incorporation of five labeled carbons in 6-
propylbenzofuran-7-ol (Figure 3a). Thus, one carbon was lost
from the ascorbic acid. In earlier investigations on the
degradation pathway of ascorbic acid in an acidic condition,1,5
the pathway involved a decarboxylation step resulting in the
loss of carbon 1 of the ascorbic acid. Similarly, we postulated
that carbon 1 of the acid was lost as CO2 via decarboxylation in
our reaction. To confirm this hypothesis, we carried out the
reaction using a solution of [1-13C]-ascorbic acid with
unlabeled (E)-hex-2-enal. GC-MS analysis detected unlabeled
6-propylbenzofuran-7-ol from this reaction (Figure 3b), which
confirmed that 6-propylbenzofuran-7-ol contained carbons 2−6
of ascorbic acid. Compared with the spectra of unlabeled and
[13C5]-6-propylbenzofuran-7-ol, three labeled carbons were
incorporated in the benzene ring of [13C5]-6-propylbenzofuran-
7-ol (m/z 91 versus 94). Therefore, two remaining labeled
carbons were corresponded to the two tertiary carbons in the
furan ring.
a
ratio to IS
RI on
TC-1
after
storage
compound
initial
furfural
804
834
nd
0.61
20.50
9.46
(E)-hex-2-enal
142.88
nd
3-hydroxypyran-2-one
(E)-hex-2-enoic acid
2-furoic acid
965
1032
1074
1440
1463
1627
3.80
nd
12.51
2.57
b
3-(2-furoyl)hexanal (9)
nd
1.02
b
6-propylbenzofuran-7-ol (10)
nd
0.26
2,3-dihydro-6-propylbenzofuran-3,7-diol
nd
0.08
b
(8)
a
nd, not detected. The IS was added to the extract at a 1.0 ppm
b
concentration. The compound numbering corresponds to that in
Figure 6.
not from the 3-(2-furoyl)hexanal (9). Our experiment thus
demonstrated that 2,3-dihydro-6-propylbenzofuran-3,7-diol (8)
was a precursor of 6-propylbenzofuran-7-ol (Figure 4).
Model Reaction. To gain insight on the formation of 6-
propylbenzofuran-7-ol, we identified other reaction products of
ascorbic acid with (E)-hex-2-enal. After storage of the model
solution at 60 °C for 1 week, the solution was extracted with
diethyl ether and analyzed. As shown in Table 1, eight
compounds were identified as major components. After storage,
(E)-hex-2-enal decreased to about one-seventh of its initial
amount. (E)-2-Hex-2-enoic acid was increased by oxidation of
(E)-hex-2-enal. Furfural, 3-hydroxy-2-pyrone, and 2-furoic acid
had been previously identified as degradation products of
ascorbic acid in aqueous solution.5 2,3-Dihydro-6-propylbenzo-
furan-3,7-diol (8) and 3-(2-furoyl)hexanal (9) had never been
reported and we identified as reaction products of ascorbic acid
with (E)-hex-2-enal. From the structure of these compounds, it
seems possible to form 6-propylbenzofuran-7-ol (10) by
dehydration of 2,3-dihydro-6-propylbenzofuran-3,7-diol (8) or
the intramolecular cyclization of 3-(2-furoyl)hexanal (9).
Hence, we dissolved each of these compounds in an aqueous
solution of citric acid and stored the solutions at 60 °C for 1
week. After storage, 6-propylbenzofuran-7-ol was detected from
the 2,3-dihydro-6-propylbenzofuran-3,7-diol (8) solution, but
Possible Pathways for the Formation of 6-Propylben-
zofuran-7-ol. It is known that ascorbic acid undergoes
Michael-type addition to α,β-unsaturated aldehydes such as
acrolein and gives the tricyclic hemiacetal lactone (Figure 5).16
This addition takes place below pH 4 of ascorbic acid, and no
basic catalyst is necessary. For this study, we assumed that the
Michael addition of ascorbic acid to (E)-hex-2-enal took place
in the same way and led, in turn, to the formation of 6-
propylbenzofuran-7-ol via tricyclic hemiacetal lactone. We thus
attempted to isolate the tricyclic hemiacetal lactone. (E)-Hex-2-
enal was added to an aqueous solution of ascorbic acid
containing a small amount of hydrochloric acid as catalyst, and
stirred at room temperature under nitrogen atmosphere for 1
week. According to an earlier report on the similar reaction
with acrolein, the tricyclic hemiacetal lactone formed via the
Michael addition was precipitated as a crystalline product.16 In
our experiment, no crystalline product was precipitated by the
reaction of ascorbic acid and (E)-hex-2-enal. Next, the reaction
mixture was extracted with n-hexane to remove unreacted (E)-
hex-2-enal, and then extracted with ethyl acetate. After the ethyl
acetate was removed, the crude product was recrystallized from
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Figure 4. Formation pathway of 6-propylbenzofuran-7-ol from ascorbic acid and (E)-hex-2-enal.
MTBE/n-hexane and the crystalline product could be isolated.
NMR analyses identified the crystalline product as tricyclic
hemiacetal lactone 11.
Next, 11 was dissolved in an aqueous solution of citric acid
and stored at 60 °C for 1 week. A GC-MS study after the 1
week of storage detected 6-propylbenzofuran-7-ol (10), 2,3-
dihydro-6-propylbenzofuran-3,7-diol (8), and 3-(2-furoyl)-
hexanal (9). All of these compounds were confirmed to have
been formed via tricyclic hemiacetal lactone 11.
On the basis of the foregoing, we proposed the following as a
possible reaction pathway (Figure 6). The first step is an acid-
catalyzed Michael addition of the ascorbic acid carbanion to
(E)-hex-2-enal to form lactone 11. After rearrangement into 12,
decarboxylation of 12 leads to diketone 13. The reaction
pathway is divided into two routes from this intermediate 13.
Figure 5. Michael addition of ascorbic acid to acrolein.16
Figure 6. Possible reaction pathway of the formation of 6-propylbenzofuran-7-ol from ascorbic acid and (E)-hex-2-enal.
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(4) Robertson, G. L.; Samaniego, C. M. L. Effect of initial dissolved
oxygen levels on the degradation of ascorbic acid and the browning of
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Subsequent cyclization by an intramolecular aldol reaction
yields compound 14. Next, compound 15 is formed by the
further cyclization of 14 via the intramolecular addition of the
terminal hydroxyl group to the carbonyl group, and the
subsequent dehydration of 15 yields 6-propylbenzofuran-7-ol
via diol 8. If diketone 13 undergoes cyclization by an
intramolecular addition of the terminal hydroxyl group to the
carbonyl group, then compound 16 is formed. Subsequent
dehydration yields ketoaldehyde 9.
In conclusion, we elucidated the possible reaction pathway of
6-propylbenzofuran-7-ol which is an off-flavor compound
formed from (E)-hex-2-enal and ascorbic acid by the
investigations using isotope labeled precursors and by isolation
of the reaction intermediates. By learning details on the
generation of 6-propylbenzofuran-7-ol, its formation pathway,
and its reaction intermediates, we may be able to find ways to
control or prevent this off-flavor. (E)-Hex-2-enal and ascorbic
acid are widely found in natural foods, as well as in various
beverages. By confirming how reaction parameters influence the
formation of 6-propylbenzofuran-7-ol, we can better prevent
the characteristic off-flavor that appears in foods containing
(E)-hex-2-enal and ascorbic acid. Studies to clarify the
generation conditions of 6-propylbenzofuran-7-ol are in
progress.
(8) Shaw, P. E.; Tatum, J. H.; Kew, T. J.; Wagner, C. J.; Berry, R. E.
Taste evaluation of nonenzymic browning compounds from orange
powder and use of inhibitors. J. Agric. Food Chem. 1970, 18, 343−345.
(9) Nagy, S.; Randall, V. Use of furfural content as an index of
storage temperature abuse in commercially processed orange juice. J.
Agric. Food Chem. 1973, 21, 272−275.
(10) Nagy, S.; Dinsmore, H. L. Relationship of furfural to
temperature abuse and flavor change in commercially canned single-
strength orange juice. J. Food Sci. 1974, 39, 1116−1119.
(11) Handwerk, R. L.; Coleman, R. C. Approaches to the citrus
browning problem. A review. J. Agric. Food Chem. 1988, 21, 231−236.
(12) Konig, T.; Gutsche, B.; Hartl, M.; Hubscher, R.; Schreier, P.;
̈
̈
Schwab, W. 3-Hydroxy-4,5-dimethyl-2(5H)-furanone (sotolon) caus-
ing an off-flavor: elucidation of its formation pathways during storage
of citrus soft drinks. J. Agric. Food Chem. 1999, 47, 3288−3291.
(13) Su, S. K.; Wiley, R. C. Changes in apple juice flavor compounds
during processing. J. Food Sci. 1998, 63, 688−691.
ASSOCIATED CONTENT
* Supporting Information
■
(14) Steinhaus, M.; Bogen, J.; Schieberle, P. Key aroma compounds
in apple juicechanges during juice concentration. In Flavour Science:
Recent Advances and Trends; Bredie, W. L. P., Peterson, M. A., Eds.;
Elsevier: Amsterdam, The Netherlands, 2006; pp189−192.
(15) Sakamaki, K.; Ishizaki, S.; Ohkubo, Y.; Tateno, Y.; Fujita, A.
Identification of the medicinal off-flavor compound formed from
ascorbic acid and (E)-hex-2-enal. J. Agric. Food Chem. 2011, 59, 6667−
6671.
S
Total ion chromatogram and mass chromatogram of [D6−8]-
(E)-hex-2-enal synthesized according to the route outlined in
Figure 1. Total ion chromatogram and mass chromatogram of
[D5−7]-6-propylbenzofuran-7-ol formed from [D6−8]-(E)-hex-
2-enal and unlabeled ascorbic acid. This material is available
free of charge via the Internet at http://pubs.acs.org.
(16) Fodor, G.; Arnold, R.; Mohacsi, T. A new role for L-ascorbic
acid: Michael donor to carbonyl compounds. Tetrahedron 1983, 39,
2137−2145.
AUTHOR INFORMATION
Corresponding Author
■
*Tel: +81-44-411-0298. Fax: +81-44-434-5257. E-mail:
kensuke_sakamaki@t-hasegawa.co.jp.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Dr. Kikue Kubota and Dr. Yasujiro Morimitsu,
Ochanomizu University, for HRMS measurements.
ABBREVIATIONS USED
■
GC-MS, gas chromatography−mass spectrometry; NMR,
nuclear magnetic resonance; MTBE, methyl t-butyl ether; RI,
retention index; HMQC, heteronuclear multiple quantum
correlation; HMBC, heteronuclear multiple bond correlation;
FTIR, Fourier Transformation Infrared Spectroscopy; HRMS,
High Resolution Mass Spectra; FAB, fast atom bombardment.
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■
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