Chemical interaction between polyphenols and a cysteinyl thiol under radical oxidation conditions

Article abstract of DOI:10.1021/jf3008822

Chemical interaction between polyphenols and thiols was investigated under radical oxidation conditions using a model cysteinyl thiol derivative, N-benzoylcysteine methyl ester. The radical oxidation was carried out with a stoichiometric amount of 2,2-diphenyl-1-picrylhydrazyl (DPPH), and the decreases in the amounts of polyphenols and the thiol were measured by HPLC analysis. Cross-coupling products between various polyphenols and the thiol were examined by LC-MS in reactions that showed decreases in both the polyphenols and the thiol. The LC-MS results indicated that three phenolic acid esters (methyl caffeate, methyl dihydrocaffeate, and methyl protocatechuate) and six flavonoids (kaempferol, myricetin, luteolin, morin, taxifolin, and catechin) gave corresponding thiol adducts, whereas three polyphenols (methyl ferulate, methyl sinapate, and quercetin) gave only dimers or simple oxidation products without thiol substituents. Thiol adducts of the structurally related compounds methyl caffeate and methyl dihydrocaffeate were isolated, and their chemical structures were determined by NMR analysis. The mechanism for the thiol addition was discussed on the basis of the structures of the products.

Full text of DOI:10.1021/jf3008822

Article
pubs.acs.org/JAFC
Chemical Interaction between Polyphenols and a Cysteinyl Thiol
under Radical Oxidation Conditions
Aya Fujimoto and Toshiya Masuda*
Graduate School of Integrated Arts and Science, University of Tokushima, Tokushima 770-8502, Japan
ABSTRACT: Chemical interaction between polyphenols and thiols was investigated under radical oxidation conditions using a
model cysteinyl thiol derivative, N-benzoylcysteine methyl ester. The radical oxidation was carried out with a stoichiometric
amount of 2,2-diphenyl-1-picrylhydrazyl (DPPH), and the decreases in the amounts of polyphenols and the thiol were measured
by HPLC analysis. Cross-coupling products between various polyphenols and the thiol were examined by LC-MS in reactions
that showed decreases in both the polyphenols and the thiol. The LC-MS results indicated that three phenolic acid esters
(methyl caffeate, methyl dihydrocaffeate, and methyl protocatechuate) and six flavonoids (kaempferol, myricetin, luteolin, morin,
taxifolin, and catechin) gave corresponding thiol adducts, whereas three polyphenols (methyl ferulate, methyl sinapate, and
quercetin) gave only dimers or simple oxidation products without thiol substituents. Thiol adducts of the structurally related
compounds methyl caffeate and methyl dihydrocaffeate were isolated, and their chemical structures were determined by NMR
analysis. The mechanism for the thiol addition was discussed on the basis of the structures of the products.
KEYWORDS: polyphenol, thiol adduct, radical oxidation, cross coupling, cysteine derivative
covalent bond formation between proteins and polyphenols via
thioether linkages has been found recently.12−15 This protein
modification is believed to be a result of a prooxidant effect of
polyphenols. The modification affected the functionality of the
proteins in food and living systems.16−19
INTRODUCTION
■
Polyphenols have been recognized as useful functional
constituents of plant-derived foods because of their potent
antioxidant activity. Recently, some polyphenols have been
found to possess various regulatory activities on signaling
pathways in living cells in addition to the antioxidant activity.1
Therefore, scientific interest in polyphenols continues to
increase, not only in food science but also in medical science.
It should be noted that antioxidant activity, a major function of
polyphenols, is based on sequential radical reactions including
radical trapping from radical species generated from bio-
molecules.2 More efficient antioxidative polyphenols trap
radicals more rapidly and then get converted to radicals
themselves. The polyphenol radicals are not very stable, so they
subsequently react with surrounding molecules. Foods and
living cells consume the polyphenols they contain via a similar
radical reaction process when they are subjected to oxidative
stress. Polyphenol radicals produced react with food or cell
constituents to give new stable products. These products
accumulate in foods or living cells and show both beneficial and
nonbeneficial functions.3,4 During lipid oxidation, some
polyphenol radicals have been reported to react with lipid
peroxyl radicals by a radical−radical coupling, and several
coupling products have been identified.5−8 Such radical-
coupling reactions are very fast and constitute an important
termination process for the oxidative deterioration of foods by
phenolic antioxidants. It is well-known that the thiol group
(sulfhydryl group) is easily converted to the thiyl radical and
that the thiyl radical has potent ability to react with other
radical species.9 Among various food constituents, a major
thiol-bearing compound is cysteine, an important constitutional
unit of peptides and proteins. Under radical oxidation
conditions, polyphenol radicals can react with the thiyl radical
to produce sulfur-bearing polyphenols,10,11 which are expected
to show various functions in foods or living cells. In addition,
In this investigation, we carried out 2,2-diphenyl-1-
picrylhydrazyl (DPPH)-induced radical reaction of polyphenols
in the presence of a cysteinyl thiol derivative (N-benzoylcys-
teine methyl ester). DPPH is a stable radical reagent and has
been frequently employed to evaluate the antioxidant capacity
of polyphenols.20 As a radical reagent, DPPH has the advantage
of allowing easy regulation of the amount of radicals in the
reaction. This is an advantage because an excess amount of
reactive radicals damages reaction products. The reaction
mixture produced by equimolecular DPPH radicals was then
analyzed by LC-TOFMS to determine the coupling products
between polyphenols and N-benzoylcysteine methyl ester.
Next, the chemical structures of the coupling products were
estimated from high-resolution MS results, and then the
structures were determined by NMR after the products had
been isolated. The results of this comprehensive study should
provide fundamental information about the chemical inter-
action between polyphenols and cysteine-bearing biomolecules
such as proteins and peptides in foods and living cells.
MATERIALS AND METHODS
■
Chemicals, Instruments, and Software. Methyl caffeate, methyl
ferulate, methyl sinapate, methyl dihydrocaffeate, methyl protocatech-
uate, methyl vanillate, and methyl syringate (each purity > 95%) were
synthesized from corresponding carboxylic acids. Briefly, to a methanol
solution of 100 mg of each carboxylc acid were added 10 drops of
Received: February 29, 2012
Revised: May 2, 2012
Accepted: May 3, 2012
Published: May 3, 2012
© 2012 American Chemical Society
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concentrated sulfuric acid. After 2 h at 40 °C, the solution was
concentrated to ca. 2 mL, ethyl acetate (50 mL) was added, and the
mixture was washed twice with saturated NaCl aqueous solution and
then saturated NaHCO3 aqueous solution, subsequently. The washed
ethyl acetate layer was dried over anhydrous Na2SO4 and evaporated
to dryness to give each methyl ester. When purification was required,
silica gel column chromatography was carried out to obtain pure
sample. Kaempferol (purity ≥ 98%), morin (purity ≥ 90% as hydrate),
myricetin (purity ≥ 97%), and propyl gallate (purity ≥ 98%) were
purchased from Tokyo Kasei (Tokyo, Japan). Luteolin (purity not
specified) was purchased from Kanto Chemicals (Tokyo, Japan).
Taxifolin (purity ≥ 90% as hydrate) was purchased from Sigma-
Aldrich (St. Louis, MO, USA). DPPH was obtained from Wako Pure
Chemicals (Osaka, Japan). Quercetin (purity not specified, but no
other peak was observed by our HPLC analysis), catechin [(+)-form,
purity = 98% as hydrate], and all solvents (extra pure grade or HPLC
grade) were obtained from Nacalai Tesque (Kyoto, Japan). NMR
spectra were measured with an ECX-400 spectrometer (JEOL, Tokyo,
Japan) using the manufacturer-supplied pulse sequences [1H, 13C,
proton−proton correlated spectroscopy (HH−COSY), nuclear Over-
hauser enhancement spectroscopy (NOESY), heteronuclear multiple
quantum coherence (HMQC), and heteronuclear multiple bond
correlation (HMBC)]. Mass spectra were obtained with a XEVO
QtofMS spectrometer (Waters Japan, Tokyo, Japan) in ESI mode. A
LC-20AD low-pressure gradient system (Shimadzu, Kyoto, Japan)
equipped with an SPD-M20A photodiode array detector and a DGU-
20A3 degasser was employed for the analytical HPLC. PDA data were
analyzed with LC solution (ver. 6.10, Shimadzu). An LC-6AD system
(Shimadzu) equipped with a UV-970 detector (JASCO, Tokyo, Japan)
was used for preparative HPLC. Molecular orbital calculation was per-
formed by MOPAC2009 software using PM3 parameter via Chem &
Bio 3D ver. 12.0 (PerkinElmer, Boston, MA, USA) as interface software.
Preparation of N,N′-Dibenzoylcystine Dimethyl Ester and
N-Benzoylcysteine Methyl Ester. To a solution of L-cystine (10 g)
in 4.2% (w/w) NaOH aqueous solution (50 mL) were dropwise added
benzoyl chloride (9.8 mL) and 4.2% (w/w) NaOH aqueous solution
(50 mL) with stirring in a water bath (25 °C). The reaction continued
for 3 h, and 6 N HCl (22 mL) was added to the reaction mixture. The
produced precipitate was filtered and dried over P2O5 to give N,N′-
dibenzoylcystine. The dibenzoylcystine (11 g) was dissolved in
methanol (300 mL), and concentrated sulfuric acid (0.3 mL) was
added to the solution. After the solution had been kept overnight at
23 °C, it was poured into chloroform (300 mL), washed with saturated
NaCl aqueous solution, then washed with saturated NaHCO3 aqueous
solution, and partitioned. The chloroform layer was dried over
anhydrous Na2SO4 and evaporated to give N,N′-dibenzoylcystine
dimethyl ester (19, 8.0 g): ESI-MS (m/z) [M + Na]+, calcd for
2 h after the addition, aliquots of 50 μL each were taken from the
solution and diluted twice with acetonitrile. Five microliters of the
diluted solution was analyzed by HPLC under the following
conditions: column, 250 × 4.6 mm i.d., 5 μm, Cosmosil 5C18-AR-II
(Nacalai Tesque); flow rate, 1.0 mL/min; solvent A, 1% acetic acid in
water; solvent B, acetonitrile; linear gradient from 5% solvent B (0 min)
to 100% solvent B (40 min) and then 100% solvent B (until 50 min);
detection, absorbance at 245 and 280 nm; column temperature, 23 °C.
Two control experiments, which were the DPPH reaction of poly-
phenol only and the reaction of N-benzoylcysteine methyl ester only at
the same concentration, were carried out, and the products were
analyzed under the same conditions. The concentrations of each
polyphenol, N-benzoylcysteine methyl ester, and produced dimer
(N,N′-dibenzoylcystine) were calculated by calibration curves obtained
by HPLC using pure compounds.
LC-MS Analysis of Reaction Products from Polyphenols and
N-Benzoylcysteine Methyl Ester. Ten microliters of the acetonitrile
solution (1.25 mmol/L as starting polyphenol) of the DPPH oxidation
product was injected into an LC-MS instrument, Acquity UPLC and
Xevo QTofMS (Waters Japan, Tokyo, Japan), through a sample
injector, Acquity sample manager (Waters). The LC-MS analysis was
carried out under the following conditions: (separation conditions)
column, 250 × 4.6 mm i.d., 5 μm, Cosmosil 5C18-AR-II (Nacalai
Tesque); flow rate, 0.5 mL/min; solvent A, ultrapure water; solvent B,
acetonitrile (LC-MS grade, Merck, Darmstadt, Germany); (gradient
conditions) linear gradient from 5% solvent B (0 min) to 100% solvent
B (80 min) and then 100% solvent B until 100 min; UV absorbance
detection, 280 nm; column temperature, 23 °C; (MS conditions)
mode, ESI negative; capillary voltage, 2.4 kV; cone voltage, 40 V;
source temperature, 150 °C; desolvation temperature., 500 °C; cone
gas flow rate, 50 L/h; desolvation gas flow rate, 1000 L/h; MSE low
collision energy, 6 V; MSE high collision energy, from 20 to 30 V. The
elemental composition of each peak compound was calculated from
the high-resolution MS data of the protonated or ion-adducted
molecular ion by MassLynx software (V. 4.1, Waters).
Isolation and Identification of Reaction Products from
Methyl Caffeate and N-Benzoylcysteine Methyl Ester. To an
acetonitrile solution (40 mL) of methyl caffeate (1, 21 mg, 1.25 mmol/
L) and N-benzoylcysteine methyl ester (18, 25 mg, 1.25 mmol/L) was
added DPPH (81 mg, 2.5 mmol/L) in acetonitrile (40 mL). After the
mixture had been kept for 2 h at 23 °C, it was evaporated to give a
brown residue (118 mg). Part of the residue (80 mg) was purified by
preparative silica gel TLC (Merck) developed with ethyl acetate/
hexane (1:1, v/v) to afford 7.3 mg of a thiol adduct (20): ESI-MS
(m/z) [M + H]+, calcd for C21H20NO7S, 430.0960; found,
430.0956;1H NMR (400 MHz, CDCl3) δ 8.24 (1H, d, J = 16.0 Hz,
H3), 7.77 [2H, m (AA′BB′C), H2 and H6 of the benzoyl group of N-
benzoylcysteine methyl ester moiety], 7.55 [1H, m (AA′BB′C), H4 of
the benzoyl group of N-benzoylcysteine methyl ester moiety], 7.44
[2H, m (AA′BB′C), H3 and H5 of the benzoyl group of
N-benzoylcysteine methyl ester moiety], 7.20 (1H, d, J = 8.2 Hz,
H6′), 7.04 (1H, d, J = 7.6 Hz, 2″-NH), 6.91 (1H, d, J = 8.2 Hz, H5′),
6.34 (1H, d, J = 16.0 Hz, H2), 4.96 (1H, ddd, J = 7.6, 7.6, and 3.6 Hz,
H2 of N-benzoylcysteine methyl ester moiety), 3.80 (3H, s, 1-OCH3
or 1-OCH3 of N-benzoylcysteine methyl ester moiety), 3.74 (3H, s,
1-OCH3 of N-benzoylcysteine methyl ester moiety or 1-OCH3), 3.23
(1H, dd, J = 14.2 and 3.6 Hz, H3a of N-benzoylcysteine methyl ester
moiety), 3.16 (1H, dd, J = 14.2 and 7.6 Hz, H3b of N-benzoylcysteine
methyl ester moiety). 13C NMR (100 MHz, CDCl3) δ 170.8 (C1″),
168.1 (C1 or carbonyl of the benzoyl group of N-benzoylcysteine
methyl ester moiety), 167.7 (carbonyl of the benzoyl group of
N-benzoylcysteine methyl ester moiety or C1), 146.6 (C4′), 145.9 (C3′),
142.6 (C3), 133.0 (C1 of the benzoyl group of N-benzoylcysteine methyl
ester moiety), 132.5 (C4 of the benzoyl group of N-benzoylcysteine
methyl ester), 130.1 (C1′), 128.8 (C3 and C5 of the benzoyl group of N-
benzoylcysteine methyl ester), 127.3 (C2 and C6 of the benzoyl group of
N-benzoylcysteine methyl ester moiety), 120.2 (C6′), 119.6 (C2′), 118.1
(C2), 116.5 (C5′), 53.7 (C2 of N-benzoylcysteine methyl ester), 53.3
(1-OCH3 or 1-OCH3 of N-benzoylcysteine methyl ester moiety), 51.9
1
C22H24N2O6S2Na, 499.0974; found, 499.0968; H NMR (400 MHz,
acetone-d6) δ 8.15 (2H, br d, J = 7.6 Hz, 2- and 2′-NH), 7.90 (4H,
AA′BB′C, two benzoyl-H2 and H6), 7.54 (2H, AA′BB′C, two benzoyl-H4),
7.45 (4H, AA′BB′C, two benzoyl-H3 and H5), 5.00 (m, H2 and H2′), 3.71
(6H, s, 1-OCH3 and 1′-OCH3), 3.40 (2H, dd, J = 14.0 and 4.8 Hz, H3a
and H3′a), 3.27 (2H, dd, J = 14.0 and 9.2 Hz, H3b and H3′b).
To a methanol solution (570 mL) of the dibenzoylcystine dimethyl
ester (19, 7.05 g) was added dithiothreitol (4.56 g). After the solution
had been kept overnight at 23 °C, it was evaporated to dryness, and
the residue was purified by silica gel column chromatography eluted
with ethyl acetate/hexane (2:5, v/v) to give 5.5 g of N-benzoylcysteine
methyl ester (18): ESI-MS (m/z) [M + H]+, calcd for C11H14NO3S,
240.0694; found, 240.0672; 1H NMR (400 MHz, CDCl3) δ 7.84 (2H,
AA′BB′C, benzoyl-H2 and H6), 7.54 (1H, AA′BB′C, benzoyl-H4), 7.47
(2H, AA′BB′C, benzyol-H3 and H5), 7.06 (1H, br d, J = 2.0 Hz,
2-NH), 5.10 (1H, m, H2), 3.84 (6H, s, 1-OCH3), 3.15 (2H, m, H3),
1.40 (1H, t, J = 4.5 Hz, 3-SH).
HPLC Analysis of Radical Reactions of Polyphenols in the
Presence of Thiol Derivative N-Benzoylcysteine Methyl Ester.
To a mixture of 5 mmol/L polyphenol solution (acetonitrile, 1 mL)
and 5 mmol/L N-benzoylcysteine methyl ester (18) solution
(acetonitrile, 1 mL) was added 5 mmol/L DPPH solution
(acetonitrile, 2 mL), and the solution was kept at 23 °C. At 1 and
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(1-OCH3 of N-benzoylcysteine methyl ester moiety or 1-OCH3), 40.4
(C3 of N-benzoylcysteine methyl ester moiety).
N-benzoylcysteine methyl ester moieties (II)], 2.46−2.28 (2H, m,
H2ab).
Isolation and Identification of Reaction Products from
Methyl Dihydrocaffeate and N-Benzoylcysteine Methyl Ester.
To an acetonitrile solution (200 mL) of methyl dihydrocaffeate
(4, 106 mg, 1.25 mmol/L) and N-benzoylcysteine methyl ester
(18, 121 mg, 1.25 mmol/L) was added DPPH (404 mg, 2.5 mmol/L)
in acetonitrile (207 mL). Two hours after the addition, the mixture
was concentrated to afford a product mixture (622 mg). Part of the
product (216 mg) was subjected to silica gel column chromatography
[silica gel, high-purity silica gel, Cosmosil 75SL-II-Prep 15 g, high-
purity silica gel elution solvent, ethyl acetate/hexane (1:1, v/v,
260 mL), ethyl acetate/hexane (3:2, v/v, 80 mL), and then ethyl
acetate/hexane (2:1, v/v, 250 mL)] to give three cysteine adducts, 21
(8.5 mg), 22 (10 mg), and 23 (17 mg).
RESULTS AND DISCUSSION
■
Radical Reaction of Polyphenols in the Presence of
N-Benzoylcysteine Methyl Ester. Six phenolic acids (in ester
form), seven flavonoids, and two phenolic diterpenoids (Figure 1)
were subjected to radical oxidation reaction in the presence
of 1 equiv of a thiol molecule. DPPH was employed as a radical
inducer. DPPH is a stable radical species and can be handled
stoichiometrically. As the thiol compound, we used N-
benzoylcysteine methyl ester (18). It is well-known that the
thiol group is highly nucleophilic and can be converted easily to
a reactive thiyl radical, so thiol compounds are possible reagents
for both nucleophilic and radical reactions. It should be noted
that the radical reactivity of thiols is influenced by the
surrounding functional groups. An amino group at the β-
position enhances reactivity,9 whereas an amide group at the
same position reduces reactivity. In food or living systems,
thiol-bearing molecules are mainly proteins and peptides
containing cysteine. Therefore, an N-benzoylated cysteine
derivative (18) is a suitable model for cysteine-containing
peptides and proteins, and it has the advantage of being
detectable with the UV detector of an HPLC instrument.
The radical reaction of polyphenols with equimolecular
N-benzoylcysteine methyl ester in acetonitrile was started by
the addition of 2 equiv of DPPH at 23 °C. After 2 h, the
constituents of the reaction mixture were analyzed by HPLC.
Quantitative analysis results for each polyphenol, N-benzoyl-
cysteine methyl ester, and the disulfide dimer of N-
benzoylcysteine methyl ester are summarized in Figure 2.
DPPH produces polyphenol radicals as well as thiyl radicals.
These radicals may react with each other to afford a coupling
product. In addition, polyphenol radicals have the ability to
form oxidation products such as quinone derivatives. The
quinone derivatives may also react with thiols by a nucleophilic
reaction.21 Therefore, the chemical interaction of polyphenols
and thiols is complicated and requires intensive analysis of not
only polyphenols and their products but also thiols and their
products.
By HPLC analysis, we examined the decrease in the amount
of each polyphenol and N-benzoylcysteine methyl ester from
each concentration of 1.25 mmol/L and the increase in the
amount of the disulfide derivative of N-benzoylcysteine methyl
ester (N,N′-dibenzoylcystine dimethyl ester), which is an
expected oxidative product from N-benzoylcysteine methyl
ester, in DPPH reactions. The amounts of methyl vanillate (6)
and methyl syringate (7) did not decrease during their
reactions with N-benzoylcysteine methyl ester,22 but the
amount of N-benzoylcysteine methyl ester decreased during
those reactions. On the other hand, the amounts of propyl
gallate (8) and carnosic acid (16) decreased during their
reaction with N-benzoylcysteine methyl ester, whereas the
decrease in the amount of N-benzoylcysteine methyl ester was
negligible during those reactions. These results indicate that
methyl vanillate (6) and methyl syringate (7) are too stable to
react with DPPH and that propyl gallate (8) and carnosic acid
(17) show probably potent antioxidant activity against thiol
oxidation by DPPH. In the polyphenol chemistry, strong
electron-withdrawing substituents at the phenolic part weaken
the activity for radicals, whereas some electron-donating groups
enhance the radical trapping activity.22 Much reactivity
difference between these polyphenols and the thiol probably
21 (monothiol adduct): ESI-MS (m/z) [M − H]−, calcd for
C21H22NO7S, 432.1096; found, 432.1117; 1H NMR (400 MHz,
CDCl3) δ 7.70 (2H, m, H2 and H6 of the benzoyl group of
N-benzoylcysteine methyl ester moiety), 7.51 (1H, m, H4 of benzoyl
group of N-benzoylcysteine methyl ester moiety), 7.32 (1H, m, H3
and H5 of the benzoyl group of N-benzoylcysteine methyl ester
moiety), 6.97 (1H, d, J = 7.6 Hz, 2-NH of N-benzoylcysteine methyl
ester moiety), 6.85 (1H, d, J = 2.0 Hz, H6′), 6.70 (1H, d, J = 2.0 Hz,
H2′), 5.04 (1H, dt, J = 7.6 and 3.6 Hz, H2″), 3.73 (3H, s, 1-OCH3 of
N-benzoylcysteine methyl ester moiety), 3.65 (3H, s, 1-OCH3), 3.39
(1H, dd, 14.8 and 3.6 Hz, H3″a), 3.18 (1H, dd, 14.8, 7.2 Hz, H3″b),
2.77 (2H, t, J = 7.2 Hz, H3), 2.53 (2H, t, J = 7.2 Hz, H2).
22 (dithiol adduct): ESI-MS (m/z) [M − H]−, calcd for
C32H33N2O10S2, 669.1577; found, 669.1597; 1H NMR (400 MHz,
CDCl3) δ 7.75 (2H, m, H2 and H6 of the benzoyl group of one of
N-benzoylcysteine methyl ester moieties), 7.70 (2H, m, H2 and H6 of
the benzoyl group of one of N-benzoylcysteine methyl ester moieties),
7.53 (1H, m, H4 of the benzoyl group of one of N-benzoylcysteine
methyl ester moieties), 7.51 (1H, m, H4 of the benzoyl group of one
of N-benzoylcysteine methyl ester moieties), 7.44 (2H, m, H3 and H5
of the benzoyl group of one of N-benzoylcysteine methyl ester
moieties), 7.13 (1H, d, J = 7.2 Hz, 2-NH of one of N-benzoylcysteine
methyl ester moieties), 7.04 (1H, d, J = 8.0 Hz, 2-NH of one of
N-benzoylcysteine methyl ester moieties), 6.87 (1H, s, H6′), 4.99 (2H,
complex, H2 of the two N-benzoylcysteine methyl ester moieties),
3.70 (3H, s, 1-OCH3 of one of N-benzoylcysteine methyl ester
moieties), 3.69 (3H, s, 1-OCH3 of one of N-benzoylcysteine methyl
ester moieties), 3.65 (3H, s, 1-OCH3), 3.44 (1H, dd, J = 14.4 and 4.4
Hz, H3b of 5′-thiol), 3.33 (1H, dd, J = 14.0 and 6.0 Hz, H3a of
5′- thiol), 3.27 (2H, d, J = 5.2 Hz, H3 of 2′-thiol), 3.00 (2H, m, H3),
2.51 (2H, m, H2).
23 (trithiol adduct): ESI-MS (m/z) [M − H]−, calcd for
C43H44N3O13S3, 906.2036; found, 906.2058; 1H NMR (400 MHz,
CDCl3) δ 7.93 (1H, br, OH), 7.78 (2H, m, H2 and H6 of the benzoyl
group of one of N-benzoylcysteine methyl ester moieties), 7.77 (2H, m,
H2 and H6 of the benzoyl group of one of N-benzoylcysteine methyl
ester moieties), 7.72 (2H, m, H2 and H6 of the benzoyl group of one
of N-benzoylcysteine methyl ester moieties), 7.58−7.38 (9H, complex,
H3, H4, and H5 of the benzoyl groups of three N-benzoylcysteine
methyl ester moieties), 7.32 [1H, d, J = 7.2 Hz, 2-NH of one of
N-benzoylcysteine methyl ester moieties (I)], 7.25 [1H, d,
J = 7.2 Hz, 2-NH of one of N-benzoylcysteine methyl ester moieties
(II)], 7.14 [1H, d, J = 8.0 Hz, 2-NH of one of N-benzoylcysteine
methyl ester moieties (III)], 4.96 [1H, m, H2 of one of
N-benzoylcysteine methyl ester moieties (I)], 4.92 [1H, m, H2 of
one of N-benzoylcysteine methyl ester moieties (II)], 4.85 [1H, ddd,
J = 8.0, 6.4, and 4.4 Hz, H2 of one of N-benzoylcysteine methyl ester
moieties (III)], 3.71 (3H, s, 1-OCH3 of one of N-benzoylcysteine
methyl ester moieties), 3.65 (3H, s, 1-OCH3), 3.60 (6H, s, 1-OCH3 of
two N-benzoylcysteine methyl ester moieties), 3.53 (1H, m, H3a),
3.47 [1H, dd, J = 13.2 and 6.4 Hz, H3a of one of N-benzoylcysteine
methyl ester moieties (III)], 3.44 [2H, m, H3 of one of
N-benzoylcysteine methyl ester moieties (I)], 3.36 (1H, m, H3b),
3.27 [1H, dd, J = 13.2 and 4.4 Hz, H3b of one of N-benzoylcysteine
methyl ester moieties (III)], 3.23 [2H, m, H3 of one of
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Figure 1. Chemical structures of the investigated polyphenols and cysteinyl thiol derivatives.
made the cross-reaction impossible. Therefore, any chemical
interaction between these compounds and thiol molecules
including peptides and proteins is not expected. Very
interestingly, carnosol (16), a diterpenoid antioxidant, showed
a slight decrease of its concentration but very high disulfide
production during the reaction, when compared with the
control experiment (DPPH plus N-benzoylcysteine methyl
ester only). These results indicated that carnosol (16) has the
ability to catalyze thiol dimerization to form a disulfide.
Disulfide formation is important not only for the tertiary
structure formation of proteins but also for protein−protein
interaction in foods, for example, in wheat dough formation. It
is well-known that dehydroascorbic acid affects disulfide
formation and interchange of protein−glutathione disulfide in
wheat constituents to result in good-quality wheat flour
dough.23,24 The radical oxidation in the presence of carnosol
(16) may have similar possibilities for dough formation. The
other polyphenols showed decreases of both polyphenol and
N-benzoylcysteine methyl ester, which prompted us to seek
cross-coupling products of polyphenols and N-benzoylcysteine
methyl ester by using the LC-MS technique.
LC-MS Analysis of Reaction Products of DPPH Radical
Reaction of Polyphenols and N-Benzoylcysteine Methyl
Ester. The LC-MS analytical results of the DPPH radical
reactions of polyphenols and N-benzoylcysteine methyl ester
(18) are summarized in Table 1. The high-resolution ESI-MS
(negative ion mode) data of the peaks observed in HPLC of
these reaction mixtures clarified that methyl caffeate (1),
methyl dihydrocaffeate (4), methyl protocatechuate (5),
kaempferol (9), myricetin (12), luteolin (13), morin (10),
taxifolin (14), and catechin (15) underwent coupling reactions
with N-benzoylcysteine methyl ester, whereas methyl ferulate
(2), methyl sinapate (3), and quercetin (11) did not afford any
coupling products under the conditions employed. LC-MS
analysis of the reactions of methyl ferulate (2) and methyl
sinapate (3) revealed that corresponding polyphenol dimers
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Figure 2. Concentrations of polyphenols, cysteinyl thiol (N-benzoylcysteine methyl ester) and its disulfide dimer in DPPH radical oxidation
reaction: (a) initial concentration of each polyphenol; (b) final concentration of each polyphenol in the presence of
N-benzoylcysteine methyl ester; (c) final concentration of each polyphenol after DPPH radical oxidation without N-benzoylcysteine methyl
ester; (d) initial concentration of N-benzoylcysteine methyl ester; (e) final concentration of N-benzoylcysteine methyl ester after DPPH radical
oxidation with each polyphenol; (f) concentration of disulfide dimer of N-benzoylcysteine methyl ester (N,N′-dibenzoylcystine dimethyl ester)
produced after DPPH radical oxidation reaction with each polyphenol. The bar graph set in the left side of each line, which is indicated as data for
the N-benzoylcysteine methyl ester, shows the results of DPPH oxidation of N-benzoylcysteine methyl ester without any polyphenol. All data are
presented as the mean SD (n = 3).
were produced along with a disulfide from N-benzoylcysteine
methyl ester. These data indicated that oxidation of the
phenolic acids was independent of thiol oxidation. In the case
of quercetin (11), Awad et al.25 have reported several cysteine
adducts of enzymatically oxidized quercetin; however, our MS
data showed the formation of three oxidation products and no
thiol adducts. The instability of cysteine adducts of quercetin
(11) was also mentioned by Awad et al.26 Hence, quercetin
(11) adducts might be too unstable to be detected by HPLC or
did not form under our conditions. Other flavonoids
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Table 1. MS Analytical Results of Major Product Peaks from the Reaction of Polyphenols and N-Benzoylcysteine Methyl Ester
observed typical fragment ions (m/z) in MS
retention time
(min) on HPLC
observed deprotonated expected molecular
estimated
product
polyphenol
molecular ion (m/z)
formula for the ion
and MSE and their molecular formulas
methyl caffeate (1)
23.2
430.0956
C21H20NO7S
225.0219 (C7H9O4S)
165.0005 (C9H5O2S)
133.0282 (C8H5O2)
monothiol
adduct
methyl ferulate (2)
methyl sinapate (3)
27.1
413.1201
C22H21O8
dimer
22.6
22.9
23.5
473.1470
473.1446
473.1447
C24H25O10
C24H25O10
C24H25O10
dimer
dimer
dimer
methyl dihydrocaffeate (4)
23.6
26.6
432.1139
669.1597
C21H22NO7S
153.0002 (C7H5O2S)
monothiol
adduct
C32H33N2O10S2
464.0833 (C21H22NO7S2)
259.0099 (C10H11O4S2)
225.0228 (C10H9O4S)
dithiol adduct
28.0
906.2058
C43H44N3O13S3
701.1314 (C32H33N2O10S3)
496.0565 (C21H22NO7S3)
trithiol adduct
methyl protocatechuate (5)
kaempferol (9)
21.8
20.3
404.0811
540.0923
C19H18NO7S
C26H22NO10S
199.0053 (C8H7O4S)
monothiol
adduct
522.0828 (C26H20NO9S)
283.0222 (C15H7O6)
255.0287 (C14H7O5)
monothiol and
water adduct
morin (10)
23.6
538.0776
C26H20NO10S
299.0167 (C15H7O7)
271.0220 (C14H7O6)
151.0022 (C7H3O4)
monothiol
adduct
quercetin (11)
20.2
20.3
317.0286
317.0283
C15H9O8
C15H9O8
299.0183 (C15H7O7)
271.0226 (C14H7O6)
151.0025 (C7H3O4)
299.0177 (C15H7O7)
271.0247 (C14H7O6)
151.0025 (C7H3O4)
271.0223 (C14H7O6)
oxidative water
adduct
oxidative water
adduct
24.1
19.1
299.0189
554.0761
C15H7O7
oxidation
product
myricetin (12)
C26H20NO11S
349.0006 (C15H9O8S)
315.0148 (C15H7O8)
287.0192 (C14H7O7)
151.0025 (C7H3O4)
monothiol
adduct
22.1
791.1218
C37H31N2O14S2
586.0458 (C26H20NO11S2)
380.9734 (C15H9O8S2)
346.9846 (C15H7O8S)
328.9749 (C15H5O7S)
dithiol adduct
luteolin (13)
taxifolin (14)
21.7
25.3
20.1
522.0859
759.1382
540.0942
C26H20NO9S
C37H31N2O12S2
C26H22NO10S
317.0103 (C15H9O6S)
165.0004 (C8H5O2S)
151.0022 (C7H3O4)
monothiol
adduct
554.0580 (C26H20NO9S2)
348.9864 (C15H9O6S2)
dithiol adduct
522.0846 (C26H20NO9S)
335.0225 (C15H11O7S)
317.0139 (C15H10O6S)
208.9904 (C9H5O4S)
522.0843 (C26H20NO9S)
335.0287 (C15H11O7S)
317.0147 (C15H9O6S)
208.9931 (C9H5O4S)
monothiol
adduct
20.6
540.0964
C26H22NO10S
monothiol
adduct
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Table 1. continued
observed typical fragment ions (m/z) in MS
retention time
(min) on HPLC
observed deprotonated expected molecular
estimated
product
polyphenol
molecular ion (m/z)
formula for the ion
and MSE and their molecular formulas
24.2
778.1449
C37H33N2O13S2
759.1334 (C37H31N2O12S2)
572.0678 (C26H22NO10S2)
446.0390 (C20H16NO7S2)
240.9625 (C9H5O4S2)
dithiol adduct
25.8
1014.1944
C48H44N3O16S3
809.1183 (C37H33N2O13S3)
775.1334 (C37H31N2O13S2)
683.0851 (C31H27N2O10S3)
570.0552 (C26H20NO11S2)
trithiol adduct
catechin (15)
17.3
18.1
21.7
23.8
526.1191
526.1173
763.1630
1000.2159
C26H24NO9S
C26H24NO9S
C37H35N2O12S2
C48H46N3O15S3
321.0418 (C15H13O6S)
180.9972 (C8H5O3S)
165.0029 (C8H5O2S)
321.0433 (C15H13O6S)
183.0127 (C8H7O3S)
141.0004 (C6H5O2S)
558.0881 (C26H24NO9S2)
353.0147 (C15H13O6S2)
214.9837 (C8H7O3S2)
795.1360 (C37H35N2O12S3)
590.0613 (C26H24NO9S3)
monothiol
adduct
monothiol
adduct
dithiol adduct
trithiol adduct
[(myricetin (12), luteolin (13), morin (10), taxifolin (14),
kaempferol (9), and catechin (15)] gave corresponding thiol
adducts, consisting of mono-, di-, and/or a small amount of
trithiol adducts. Mono- and diglutathione adducts of taxifolin
(14) and luteolin (13) have been reported.25 They determined
from NMR analysis that substitutions occurred at the 2- and/or
5-positions of the B ring. Similar to their results, in our
monothiol adducts of myricetin (12), luteolin (13), and morin
(10), the substitution of N-benzoylcysteine methyl ester might
have occurred on the B ring, because MSE fragment analysis of
each monothiol adduct showed a typical fragment at m/z
151(C7H4O4) due to the unreacted A ring.27 Sang and co-
workers identified cysteine adducts of enzymatically oxidized
epigallocatechin, both at the 2-position of the B ring and at the
2-position of the 3-galloyl moiety by NMR analysis.28 Similar
adducts of epigallocatechin with a thiol-bearing protein were
also observed by Mori et al.; however, they observed a protein−
thiol adduct in only the galloyl part of gallocatechins and did
not observe any in catechin (15).29 A cysteine adduct of
catechin (15) at the 4-position has been found as a thiolysis
product from catechin polymer.30 Our MS results concerning
observation of MSE fragments from the thiol-substituted B ring
(C8H7O3S from a monothiol adduct and C8H7O3S2 from a
dithiol adduct) strongly indicated that N-benzoylcysteine
methyl ester reacted at the B ring (2′-, 5′-, and/or 6′-position)
under our radical conditions, although catechin (15) has no
galloyl moiety. The reaction of kaempferol (9) showed a
predominant peak in the HPLC data, which had a molecular-
related ion at m/z 540.0923 (M − H)− (estimated molecular
formula, C26H23NO10S). This MS unit indicated that the peak
compound was an adduct of both N-benzoylcysteine methyl
ester and a water molecule with kaempferol (9). Two major
fragment ions (C26H20NO9S and C15H7O6) in the MSE of the
peak compound, which correspond to a dehydrated ion and an
oxidized kaempferol ion, respectively, also suggested that the
compound was possibly formed by oxidative thiol addition and
subsequent water addition.
LC-MS analysis of their reaction products revealed several
dimers and their oxidation products. On the other hand, methyl
caffeate (1), methyl dihydrocaffeate (4), and methyl proto-
catechuate (5) yielded the corresponding thiol adducts, which
were also detected by LC-MS. Interestingly, methyl caffeate (1)
and methyl protocatechuate (5) gave only monothiol adducts,
whereas methyl dihydrocaffeate (4) afforded mono-, di-, and
trithiol adducts. Bassil et al. have tentatively identified a
2-cysteine adduct of caffeic acid in the reaction of cysteine and
caffeic acid by periodate oxidation.31 Saito and Kawabata have
also reported that several thiols reacted first at the 2-position of
a quinone from protocatechuic acid.32 Our thiol adduct might
be similar to the product reported by Saito and Kawabata in the
reaction position of N-benzoylcysteine methyl ester. Although
methyl dihydrocaffeate (4) is structurally related to methyl
caffeate (1), without a double bond at the 2-position, three
kinds of dihydrocaffeate thiol adducts were observed in its
reaction, in contrast with the reaction of methyl caffeate (1).
LC-MS analytical results of the reaction mixture revealed that
these adducts were mono-, di-, and trithiol adducts. Caffeates
and dihydrocaffeates have structures similar to the B−C ring
systems of flavones and flavanones, respectively. Detailed
analysis of the structures of the thiol derivatives of both
caffeates and dihydrocaffeates would afford valuable informa-
tion for the chemical interaction of thiols with not only
phenolic acids but also flavonoids.
Structure Identification of the Products of the
Reaction of Methyl Caffeate and Methyl Dihydrocaf-
feate with N-Benzoylcysteine Methyl Ester. As shown in
the panel A of Figure 3, methyl caffeate afforded one product,
whereas methyl dihydrocaffeate gave three products during
DPPH oxidation in the presence of N-benzoylcysteine methyl
ester (panel B of Figure 3). The DPPH reaction of methyl
caffeate (1) was carried out again, and the reaction mixture was
then purified by silica gel TLC to give a monothiol adduct (20)
[m/z 430.0956 (M − H)−]. The structure of the adduct,
especially the substituted position of the thiol, was determined
1
For phenolic acid esters, methyl ferulate (2), methyl sinapate
(3), and propyl gallate (8) did not yield any thiol adducts.
by NMR analysis of isolated 20. The H NMR data of 20
showed signals due to a benzoylcysteine moiety without a thiol
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N-benzoylcysteine methyl ester attaches at the 2′-position of
methyl caffeate (1). The structure was also confirmed by a C−H
long-range correlation between H3 of the thiol and C2′, which was
observed in the HMBC spectrum of 20.
A large-scale DPPH reaction in the presence of N-
benzoylcysteine methyl ester was also performed with
dihydrocaffeic acid methyl ester. The reaction mixture was
purified by silica gel column chromatography to give a
monothiol adduct (21), a dithiol adduct (22), and a trithiol
1
adduct (23) as major products. The H NMR analytical data
of the monoadduct 21 showed a signal set due to a benzoyl-
cysteine moiety and a signal set due to a methyl dihydrocaffeate
moiety. It was determined that the thiol is attached at the
5′-position of the dihydrocaffeate moiety on the basis of the
meta coupling constant (J = 2.0 Hz) between H2′ and H6′
(δ 6.85 and 6.70) on the benzene ring. The structure of the
1
dithiol adduct, 22, was also analyzed by H NMR. The NMR
data showed the presence of two N-benzoylcysteine methyl
ester moieties and the lack of two protons on the
dihydrocaffeate benzene moiety, indicating that the two thiols
were attached to the benzene moiety. The remaining benzene
ring proton was determined to be at the 6′-position, because
NOE correlations from the 6′-proton (δ 6.87) to the methylene
protons at the 3-position of the dihydrocaffeate (δ 3.00) and to
the 3-methylene protons of the 5′-thiol moiety (δ 3.44 and
3.33) were observed in the NOESY spectrum. Therefore, the
dithiol adduct 22 was determined to be the 2′,5′-dithiol adduct
of methyl dihydrocaffeate. The structure of the trithiol adduct,
Figure 3. HPLC analytical profile for DPPH reaction (2 h) products
of methyl caffeate (A) and methyl dihydrocaffeate (B) in the presence
of N-benzoylcysteine methyl ester.
hydrogen signal, and the data also showed signals due to methyl
caffeate (1) lacking one proton signal of the benzene moiety.
These results revealed that the thiol reacted at the benzene
moiety of methyl caffeate (1). The remaining two hydrogen
signals (δ 7.20 and 6.91) for the methyl caffeate (1) benzene
part showed an ortho relationship from their coupling con-
stant (J = 8.2 Hz). Therefore, it was concluded that the
1
23, was also determined by H NMR. The data showed only
two methylene signals [δ 3.53 (1H), 3.36 (1H), 2.37 (2H)] and
a methyl signal [δ 3.65 (3H, s)] as signals of the protons due to
methyl dihydrocaffeate. Therefore, three N-benzoylcysteine
Figure 4. Proposed reaction mechanism for the formation of the monothiol adduct of methyl caffeate and the mono-, di-, and trithiol adducts of
methyl dihydrocaffeate: route A, radical coupling mechanism for monothiol adduct 20; route B, nucleophilic addition mechanism for monothiol
adduct 20; route C, nucleophilic addition mechanism for thiol adducts 21, 22, and 23.
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methyl ester moieties should be attached to the dihydrocaffeate
benzene moiety. From these results, triadduct 23 was
determined to be the 2′,5′,6′-trithiol adduct of dihydrocaffeic
acid methyl ester.
AUTHOR INFORMATION
■
Corresponding Author
*Phone/fax: +81-88-656-7244. E-mail: masuda@ias.tokushima-
u.ac.jp.
From these NMR data, all structures of the thiol adducts of
methyl caffeate (1) and methyl dihydrocaffeate were success-
fully identified. Interestingly, thiol substitution positions are
different in monoadducts of caffeate and dihydrocaffeate.
Monoadduct 20 from methyl caffeate (1) has the thiol at the
2′-position, whereas monoadduct 21 from methyl dihydrocaf-
feate (4) has it at the 5′-position. In addition to the substituted
position, methyl caffeate (1) gave only the monoadduct under
the reaction conditions; however, methyl dihydrocaffeate gave
higher substituted thiol adducts under the same conditions.
The thiol adducts from methyl dihydrocaffeate (4) all have the
5′-position thiol substituent, which strongly suggested that the
first addition of the thiol to methyl dihydrocaffeate (4) occurs
at the 5′-position. A thiol can be easily converted to an active
thiyl radical in addition to it having potent nucleophilicity;
therefore, both types of reactions (radical and ionic) should be
considered in the production of these thiol adducts. We
previously reported that a lipid peroxyl radical reacted at the
3′-position of methyl caffeate (1).8 This position is a radical-
stabilizing position by the captodative effect. When the thiyl
radical is formed along with caffeate radicals, reaction should
occur at the 3′-position. After the radical coupling, the thiol can
migrate via a reaction such as a dienone−phenol rearrangement
to the 2′-position to form adduct 20 (route A in Figure 4). On
the other hand, when quinone formation (dehydration of the
caffeate) is faster than thiyl radical formation, the thiol can react
at any of the 2′-, 5′-, and 6′-positions by nucleophilic addition.
MO calculation using MOPAC2007 software showed that the
lowest unoccupied molecular orbital (LUMO) is distributed
mainly on the 2′-position of the quinone derivative of methyl
caffeate (1). Therefore, nucleophilic addition of the thiol can
occur at the 2′-position on the quinone derivative (route B in
Figure 4).
It should be noted that the quinone of methyl
dihydrocaffeate is more stable than that of methyl caffeate
(1) because the former has no electron-withdrawing group on
the benzene ring, in contrast to the latter. Hence, the
dihydrocaffeate quinone is probably produced much more
rapidly than the caffeate quinone and possibly than thiyl
radicals. The quinone produced reacts next with the thiol at the
5′-position by nucleophilic addition, because the 5′-position is
the least sterically hindered position, despite there being no
difference in the reactivity between the 2′-, 5′-, and 6′-positions
as estimated from the MO calculation results. The monothiol
adduct of the dihydrocaffeate probably has higher reactivity to
radicals than the original dihydrocaffeate to form another
quinone derivative. Therefore, the next thiol addition might
take place to yield the dithiol adduct 22 and the trithiol adduct
23 (route C in Figure 4).
Funding
Financial support from the Japan Society for the Promotion of
Science (Kakenhi No. 23800929) and the Iijima Memorial
Foundation for the Promotion of Food Science and
Technology is gratefully acknowledged.
Notes
The authors declare no competing financial interest.
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