Hybrid of Resveratrol and Glucosamine: An Approach to Enhance Antioxidant Effect against DNA Oxidation

Article abstract of DOI:10.1021/acs.chemrestox.8b00136

Resveratrol exhibits various pharmacological activities, which are dependent upon phenolic hydroxyl groups. In this work, glucosamine, lipoic acid, or adamantanamine moiety was applied for attaching to ortho-position of hydroxyl group in resorcinol moiety of resveratrol (known as position-2). Antioxidant effects of the obtained hybrids were characterized using DNA oxidative systems mediated by ^?OH, Cu²⁺/glutathione (GSH), and 2,2′-azobis(2-amidinopropanehydrochloride) (AAPH), respectively. The glucosyl-appended imine and amine at position-2 of resveratrol were found to show higher inhibitory effects than other resveratrol derivatives against AAPH-induced DNA oxidation. The antioxidative effect was quantitatively expressed by stoichiometric factor (n, the number of radical-propagation terminated by one molecule of antioxidant). The stoichiometric factors of glucosyl-appended imine and amine of resveratrol increased to 4.74 (for imine) and 4.97 (for amine), respectively, higher than that of resveratrol (3.70) and glucoside of resveratrol (3.49). It was thereby concluded that the combination of resveratrol with glucosamine at position-2 represented a novel pathway for modifying resveratrol structure in the protection of DNA against peroxyl radical-mediated oxidation.

Full text of DOI:10.1021/acs.chemrestox.8b00136

Article
pubs.acs.org/crt
Cite This: Chem. Res. Toxicol. XXXX, XXX, XXX−XXX
Hybrid of Resveratrol and Glucosamine: An Approach To Enhance
Antioxidant Effect against DNA Oxidation
Liang-Liang Bao and Zai-Qun Liu*
Department of Organic Chemistry, College of Chemistry, Jilin University, Changchun 130021, China
S
* Supporting Information
ABSTRACT: Resveratrol exhibits various pharmacological
activities, which are dependent upon phenolic hydroxyl groups.
In this work, glucosamine, lipoic acid, or adamantanamine
moiety was applied for attaching to ortho-position of hydroxyl
group in resorcinol moiety of resveratrol (known as position-2).
Antioxidant effects of the obtained hybrids were characterized
using DNA oxidative systems mediated by •OH, Cu2+/
glutathione (GSH), and 2,2′-azobis(2-amidinopropane-
hydrochloride) (AAPH), respectively. The glucosyl-appended
imine and amine at position-2 of resveratrol were found to show
higher inhibitory effects than other resveratrol derivatives
against AAPH-induced DNA oxidation. The antioxidative effect
was quantitatively expressed by stoichiometric factor (n, the
number of radical-propagation terminated by one molecule of
antioxidant). The stoichiometric factors of glucosyl-appended imine and amine of resveratrol increased to 4.74 (for imine) and
4.97 (for amine), respectively, higher than that of resveratrol (3.70) and glucoside of resveratrol (3.49). It was thereby
concluded that the combination of resveratrol with glucosamine at position-2 represented a novel pathway for modifying
resveratrol structure in the protection of DNA against peroxyl radical-mediated oxidation.
moieties via the esterification with 4′-hydroxyl group or
amidation with the 4′-amino group (replacing the 4′-hydroxyl
group by amino group in advance). For example, aspirin,18
caffeic acid,19 p-dimethylaminophenol,20 and tacrine21 attached
to the 4′-position in resveratrol, which formed hybrids with
multiple pharmacological activities. In addition, some efforts
contributed to attaching sugar moieties to hydroxyl groups in
resveratrol (see Figure 1),22 owing to resveratrol glycoside
possessing high bioavailability.23 However, there has still been
scanty reports to-date on revealing the bioactivity of glucosyl-
appended imine or amine of resveratrol.24
INTRODUCTION
■
Resveratrol (3,4′,5-trihydroxystilbene) was found to be active
in the chemoprevention of cancer,1 and growing evidence
indicated that resveratrol was also effective in numerous
disease models including cardiovascular disease,2 diabetes,3
neurodegenerative disease,4 cancer,5 etc. Resveratrol acted as a
structural moiety of polyphenolic oligomers in plants6 and as a
basic scaffold in the discovery of novel drugs.7 However,
resveratrol might be a prooxidant to induce oxidative breakage
of cellular DNA in the presence of metal ions.8 Therefore,
burgeoning numbers of resveratrol derivatives were currently
found in the literature with the intensive goal of medicinal
application.9
The biological activity of resveratrol was related to its
hydroxyl groups, which were able to trap free radicals in vivo.10
In the preparation of resveratrol derivatives, both Heck-
Hiyama cross-coupling11 and Wittig reaction12 provided with
powerful strategies for achieving resveratrol derivatives with
various substituents attaching to the stilbene scaffold. As
shown in Figure 1, a typical resveratrol derivative was
produced by the substitution of one benzene ring in resveratrol
by bicyclo[1.1.1]-pentane13 or deferiprone,14 or the CC
bond in resveratrol was replaced by multiple CC bonds,15
1,4,2-dioxazole,16 or selenophene.17 A combination of two
pharmacological molecules was a popularly used strategy to
harvest duplicate functions within a single drug, in which 4′-
position in resveratrol usually bridged with other medicinal
In light of the recognition on the correlation of DNA
damage with diseases,25 we attempted to test the influence of
appended moieties on inhibitory effects of resveratrol
derivatives against DNA oxidative damage, which was
•
mediated by OH, Cu2+/glutathione (GSH), and 2,2′-azobis-
(2-amidinopropane hydrochloride) (AAPH, R-NN-R, R =
−CMe2C(NH) NH2), respectively, because •OH was
known as an important member in the family of reactive
oxygen species (ROS), while the decomposition of AAPH
provided with peroxyl radical (ROO•) to mimic DNA
undergoing peroxidation in vivo. The combination of Cu(II)
with glutathione in vivo might be harmful to health because of
Received: May 26, 2018
© XXXX American Chemical Society
A
DOI: 10.1021/acs.chemrestox.8b00136
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Chemical Research in Toxicology
Article
Figure 1. Recently published resveratrol derivatives.
the generation of glutathione radical (GS•) via the oxidation of
GSH by Cu(II).
mmol 1 (256 mg) and 10 mmol TBDMSCl (1.51 g); then 10 mmol
imidazole (680 mg dissolved in 4 mL of THF) was added under ice
bath and stirred for 10 h. After the reaction mixture was concentrated
under reduced pressure, the residue was purified by silica gel column
chromatography with petroleum ether as eluent to give (E)-2,4-
di(tert-butyldimethylsilyl)oxy-6-(4′-(tert-butyldimethylsilyl)-
oxylstyryl) benzaldehyde (1a) (430 mg, yield 72%) as a green oil.
Then −CHO in the aforementioned compound (1a) was reduced by
NaBH4 to afford −CH2OH. To 5 mL of THF solution of 1a (120 mg,
0.2 mmol) was added NaBH4 (7.6 mg, 0.2 mmol) and stirred at 0 °C
for 20 min, followed by the addition of 0.1 mL of water and stirring
for 5 min. The aqueous solution of NH4Cl (54 mg dissolved in 0.3
mL of water) was added and stirred for 20 min and then diluted with
THF. After the solid in the suspension was removed, the liquid phase
was concentrated under reduced pressure, and crude product was
purified by silica gel column chromatography with petroleum ether/
ethyl acetate mixture (v/v = 20:1) as eluent to give (E)-2,4-di(tert-
butyldimethylsilyl)oxy-6-(4′-(tert-butyldimethylsilyl)oxylstyryl)benzyl
alcohol (2a) as a colorless oil in a yield of 69% (83 mg). Finally, the
protective groups (TBDMS) at hydroxyl groups were removed under
acidic condition. To 3 mL of THF solution of 2a (36 mg, 0.06 mmol)
was added 200 μL of trifluoroacetic acid and stirred at 0 °C for 5 h in
the atmosphere of N2. After the organic media were evaporated under
reduced pressure, the residue was purified by silica gel column
chromatography with ethyl acetate/methanol mixture (v/v = 10:1) as
MATERIALS AND METHODS
■
Materials and Instrumentation. 2,2′-Azobis(2-amidinopropane
hydrochloride) (AAPH) and naked DNA sodium salt were products
of ACROS Organics, Geel, Belgium, and used as received. Solvents
and reagents used in the synthesis were obtained commercially and
used as such unless noted otherwise. All of the products were
1
identified by H and 13C NMR spectroscopy (Bruker Avance III 400
MHz spectrometer) that were outlined in the Supporting Information.
The molecular weight was detected by high resolution mass spectra
(HRMS) equipped with ESI as the ionization mode (Agilent 1290-
micrOTOF Q II).
Synthesis of Resveratrol Derivatives. (E)-2,4-Dihydroxyl-6-(4′-
hydroxylstyryl)benzaldehyde (1). The formylation of resveratrol was
carried out in 60 mL of acetonitrile solution of resveratrol (456 mg, 2
mmol), to which 2 mmol N,N-dimethylformamide (DMF, 146 mg)
and 2.3 mmol POC13 (352 mg) were added at −5 °C and stirred for
3 h at the same temperature. After the produced solid was collected
and washed with cold acetonitrile, 30 mL of water was added and
heated at 55 °C under stirring for 2 h. Then the solid was collected
and washed with cold water. After dried over reduced pressure, 1 was
obtained as yellowish solid in a yield of 62% (320 mg). MP 209 °C.
1H NMR (400 MHz, DMSO-d ): δ 10.27 (s, 1H), 7.69 (d, J = 16.0
1
6
eluent to give 2 as a brown oil in a yield of 71% (11 mg). H NMR
Hz, 1H), 7.50 (d, J = 8.0 Hz, 2H), 7.02 (d, J = 16.0 Hz, 1H), 6.79 (d,
J = 7.9 Hz, 2H), 6.62 (s, 1H), 6.22 (s, 1H). 13C NMR (101 MHz,
CD3OD): δ 192.9, 165.5, 157.8, 145.6, 134.6, 128.2, 127.4, 119.6,
115.2, 111.6, 106.1, 104.4, 100.9. HRMS (ESI): calcd for [M + H]
of C15H13O4, 257.0814; found, 257.3239.
(E)-2,4-Dihydroxyl-6-(4′-hydroxylstyryl)benzyl alcohol (2). Before
−CHO in 1 was reduced to form −CH2OH, phenolic hydroxyl
groups in 1 were protected by tert-butyldimethylsilyl chloride
(TBDMSCl). To 5 mL of tetrahydrofuran (THF) was added 1
(400 MHz, CD3OD): δ 7.29 (d, J = 8.3 Hz, 2H), 7.22 (d, J = 16.1 Hz,
1H), 6.81 (d, J = 16.1 Hz, 1H), 6.67 (d, J = 7.9 Hz, 2H), 6.14 (s, 1H),
6.05 (s, 1H), 4.48 (s, 2H). 13C NMR (101 MHz, CDCl3): δ 155.9,
155.5, 154.9, 139.9, 131.2, 130.3, 129.7, 126.6, 122.7, 119.8, 113.9,
110.2, 58.0. HRMS (ESI): calcd for [M + H]+ of C15H15O4,
259.0970; found, 259.2835.
(E)-2,4-Dihydroxy-6-(4′-hydroxystyryl)benzyl 5-(1,2-dithiolan-3-
yl)pentanoate (3). The esterification of −CH2OH in 3 with lipoic
acid was carried out with 2a being the starting material. To 10 mL of
+
B
DOI: 10.1021/acs.chemrestox.8b00136
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CH2Cl2 were added 206 mg of lipoic acid (1 mmol), 237 mg of 1,3-
dicyclohexylcarbodiimide (DCC, 1.15 mmol), and 12.2 mg of 4-
dimethylaminopyridine (DMAP, 0.1 mmol) and stirred at 0 °C for 15
min, followed by the addition of 5 mL of CH2Cl2 solution of 2a (600
mg, 1 mmol) and stirring for 15 min at the same temperature. The
solution was allowed to increase to room temperature and stirred for
18 h. After the reaction mixture was washed with water (3 × 50 mL),
the organic layer was dried with MgSO4, filtered, and evaporated. The
residue was purified by flash chromatography with petroleum ether/
ethyl acetate mixture (v/v = 20:1) as eluent to afford 3a as yellowish
oil in a yield of 38% (300 mg). 1H NMR (400 MHz, CD3OD): δ 7.45
(d, J = 8.0 Hz, 2H), 7.24 (d, J = 15.6 Hz, 1H), 6.96 (d, J = 16.2 Hz,
1H), 6.87 (d, J = 8.0 Hz, 2H), 6.59 (s, 1H), 6.33 (s, 1H), 5.32 (s,
1H), 3.43 (m, 1H), 3.13 (m, 2H), 2.33 (m, 3H), 1.62 (m, 7H), 1.03
(s, 27H), 0.28 (s, 18H). Following the same procedure as removing
the protective group in 2a, 3a (197 mg, 0.25 mmol) and 500 μL of
trifluoroacetic acid in 3 mL of THF furnished 3 as a brown oil in a
1H), 5.31 (d, J = 5.0 Hz, 1H), 5.12 (d, J = 4.6 Hz, 1H), 5.05 (d, J =
5.2 Hz, 1H), 4.81 (d, J = 7.5 Hz, 1H), 4.66 (t, J = 5.6 Hz, 1H), 3.74
(dd, J = 10.7, 4.8 Hz, 1H), 3.50 (dt, J = 11.7, 5.9 Hz, 1H), 3.25 (m,
4H). 13C NMR (101 MHz, DMSO-d6): δ 159.3, 158.8, 157.8, 139.8,
128.9, 128.4, 125.7, 115.9, 107.6, 105.2, 103.2, 101.1, 77.6, 77.2, 73.7,
70.2, 61.2.
N-2,4-Dihydroxy-6-((E)-4-hydroxystyryl)benzylidene-2-deoxy-α-
D-glucosamine (6). The reaction of D-glucosamine with 1 was
capable of furnishing 6 directly. To 5 mL of stirred aqueous solution
of D-glucosamine (179 mg, 1 mmol) was added 256 mg of 1 (1 mmol,
dissolved in 6 mL of THF) and one drop of formic acid. The reaction
mixture was stirred at room temperature for 24 h. The solvent was
removed in vacuo, and the crude product was purified by thin layer
chromatography with ethyl acetate/methanol mixture (v/v = 25:1) as
1
eluent to afford 6 in a yield of 6% (25 mg). H NMR (400 MHz,
DMSO-d6): δ 8.54 (s, 1H), 7.42 (d, J = 8.6 Hz, 2H), 7.29 (d, J = 16.0
Hz, 1H), 6.86 (d, J = 16.0 Hz, 1H), 6.77 (d, J = 8.5 Hz, 2H), 6.57 (d,
J = 2.3 Hz, 1H), 6.24 (d, J = 2.3 Hz, 1H), 5.21 (d, J = 3.5 Hz, 1H),
3.98 (q, J = 7.1 Hz, 1H), 3.66−3.56 (m, 2H), 3.52 (dd, J = 11.7, 5.1
Hz, 1H), 3.19 (t, J = 9.4 Hz, 1H), 2.91 (dd, J = 10.5, 3.6 Hz, 1H). 13C
NMR (101 MHz, DMSO-d6): δ 171.8, 159.3, 159.1, 157.5, 148.7,
139.9, 132.4, 128.8, 122.4, 115.9, 107.3, 105.2, 102.1, 89.1, 72.4, 70.2,
69.9, 60.6, 54.5. HRMS (ESI): calcd for [M + H]+ of C21H24NO8,
418.1502; found, 418.4623.
1
yield of 23% (26 mg). H NMR (400 MHz, CDCl3): δ 7.39 (d, J =
8.5 Hz, 1H), 7.16 (d, J = 16.0 Hz, 1H), 6.90 (d, J = 16.1 Hz, 1H),
6.85 (d, J = 8.5 Hz, 1H), 6.76 (d, J = 2.1 Hz, 1H), 6.30 (d, J = 2.1 Hz,
1H), 5.28 (s, 1H), 3.51 (dd, J = 14.0, 6.9 Hz, 1H), 3.13 (ddd, J =
17.8, 11.6, 4.9 Hz, 2H), 2.48−2.28 (m, 3H), 1.94−1.80 (m, 1H),
1.73−1.52 (m, 6H). 13C NMR (101 MHz, CDCl3): δ 173.7, 156.6,
155.9, 155.7, 140.5, 131.1, 130.6, 127.9, 123.6, 120.4, 117.1, 110.5,
109.7, 56.2, 40.2, 38.5, 34.6, 34.2, 28.7, 24.8, 18.3. HRMS (ESI): calcd
for [M + H]+ of C23H27O5S2, 447.1300; found, 447.1755.
N-2,4-Dihydroxy-6-((E)-4-hydroxystyryl)benzyl-2-deoxy-D-glu-
cosamine (7). To 2 mL of stirred methanolic solution of acetyl-
appended glucosamine (173 mg, 0.5 mmol) was added 1.5 mL of
methanolic solution of 1a (299 mg, 0.5 mmol) and one drop of formic
acid, and the stirring was continued at room temperature for 2.5 h to
achieve imine (1,3,4,6-tetra-O-acetyl-2-N-2,4-di(tert-
butyldimethylsilyl)oxy-6-((E)-4′-(tert-butyldimethylsilyl)oxystyryl)-
benzylidene-2-deoxy-β-D-glucosamine, 6a) as green solid in a yield of
58% (270 mg). To 3 mL of stirred THF solution of 6a (232 mg, 0.25
mmol) was added 19 mg of NaBH4 (0.5 mmol). The mixture was
stirred at 0 °C for 20 min, and 0.1 mL of water was added and stirred
for 5 min. After an aqueous solution of NH4Cl (54 mg dissolved in
0.3 mL of water) was added and stirred for 20 min, the solution was
evaporated under reduced pressure. The ethyl acetate and water were
added to dissolve the residue, and the organic layer was collected and
dried with MgSO4. After the organic solvent was evaporated, 1,3,4,6-
tetra-O-acetyl-2-N-(2,4-di(tert-butyldimethylsilyl)oxy-6-((E)-4′-(tert-
butyldimethylsilyl)oxystyryl)benzyl)-2-deoxy-D-glucopyranose (7a)
was achieved as brown oil in a yield of 44% (102 mg). To 2 mL of
vigorously stirred methanolic solution of 7a (93 mg, 0.1 mmol) was
added 69 mg of K2CO3 (0.5 mmol), and the stirring was continued at
room temperature for 6 h, followed by the addition of 0.5 mL of
trifluoroacetic acid and stirring for 2 h. The solvent was removed in
vacuo, and the crude product was purified by thin layer
chromatography with ethyl acetate/methanol mixture (v/v = 3:1) as
2-((Adamantan-1-ylamino)methyl) resveratrol (4). The adaman-
tan-1-yl group was directly furnished by the reaction of 1 with 1-
adamantanamine, followed by the reduction with NaBH4. To 8 mL of
methanol was dissolved 151 mg of 1-adamantanamine (1 mmol) and
256 mg of 1 (1 mmol), as well as one drop of formic acid, and the
stirring was continued at room temperature overnight. The solvent
was removed to afford imine as brown oil, which was diluted with
methanol and cooled in ice bath. Then 38 mg of NaBH4 (1 mmol)
was added and stirred for 20 min, and three drops of water was added
and stirred for 5 min. After an aqueous solution of NH4Cl (162 mg
dissolved in 1.5 mL of water) was added and stirred for 20 min, the
solution was evaporated under reduced pressure. The ethyl acetate
and water were added to dissolve the residue, and the organic layer
was collected and dried with MgSO4. After the organic solvent was
evaporated, 4 was obtained as brown solid in a yield of 17% (69 mg).
1
MP 218 °C. H NMR (400 MHz, CD3OD): δ 7.41 (d, J = 8.6 Hz,
2H), 7.34 (d, J = 16.1 Hz, 1H), 6.93 (d, J = 16.1 Hz, 1H), 6.79 (d, J =
8.6 Hz, 2H), 6.62 (d, J = 2.3 Hz, 1H), 6.27 (d, J = 2.3 Hz, 1H), 4.76
(s, 2H), 2.18 (s, 3H), 1.89 (d, J = 2.4 Hz, 6H), 1.75 (dd, J = 33.3, 12.4
Hz, 6H). 13C NMR (101 MHz, CD3OD): δ 157.4, 157.1, 157.0,
139.7, 130.2, 129.3, 127.6, 123.0, 116.0, 115.1, 103.2, 101.2, 51.5,
+
40.1, 38.0, 35.0, 29.0. HRMS (ESI): calcd for [M + H]
C25H30NO3, 392.2226; found, 392.5237.
of
1
eluent to give 7 as yellowish oil in a yield of 36% (15 mg). H NMR
trans-Resveratrol-3-O-β-D-glucopyranoside (5, Piceid). 2,3,4,6-
Tetra-O-acetyl-β-D-glucopyranosyl bromide was selected to be the
glucosidation agent in the preparation of piceid. To a stirred 50 mL of
acetonitrile were dissolved 228 mg of resveratrol (1 mmol) and 411
mg of the aforementioned glucosidation agent (1 mmol), and then
276 mg of Ag2CO3 (1 mmol) was added and stirred at ambient
temperature for 8 h in dark. The reaction mixture was then filtered
through a short Celite pad, the solvent was removed in vacuo, and the
obtained residue was purified by silica gel column chromatography
with petroleum ether/ethyl acetate mixture (v/v = 3:1) as eluent to
afford the precursor of 5. The acetyl groups in glucose moiety were
removed by reacting with NaOCH3. To 20 mL of methanolic solution
of the precursor of 5 (140 mg, 0.25 mmol) was added 67.5 mg of
NaOCH3 and stirred at room temperature for 6 h in the atmosphere
of N2. The reaction mixture was acidified to pH = 2 and filtered. The
liquid phase was concentrated in vacuo, and the residue was purified
by thin layer chromatography with ethyl acetate/methanol mixture
(v/v = 3:1) as eluent to give 5 as a white solid in a yield of 6% (25
(400 MHz, DMSO-d6): δ 7.37 (d, J = 8.5 Hz, 2H), 7.08 (d, J = 16.3
Hz, 1H), 7.01 (d, J = 15.9 Hz, 1H), 6.84 (d, J = 8.5 Hz, 2H), 6.78 (d,
J = 15.9 Hz, 1H), 6.75 (d, J = 16.3 Hz, 1H), 6.54 (d, J = 2.2 Hz, 1H),
6.51 (d, J = 2.5 Hz, 1H), 6.31 (d, J = 2.2 Hz, 1H), 6.30 (d, J = 2.5 Hz,
1H), 5.38 (d, J = 3.3 Hz, 1H), 5.06 (t, J = 9.6 Hz, 1H), 4.38−2.87
(10H), 1.29−1.22 (4H).
•OH- and Cu2+/GSH-Induced Oxidations of DNA. DNA was
dissolved in phosphate buffered solution (PBS1: 6.1 mM Na2HPO4,
3.9 mM NaH2PO4) to reach 2.24 mg/mL as the stock solution,
tetrachlorohydroquinone (TCHQ) was dissolved in dimethyl
sulfoxide (DMSO) to reach 120 mM as the stock solution, and
H2O2 was diluted in PBS1 to reach 30.0 mM as the stock solution. To
13.4 mL of DNA solution was added 1.0 mL of H2O2, 0.5 mL of
TCHQ solution, and 0.1 mL resveratrol derivatives (dissolved in
DMSO as the stock solution). The aforementioned mixture was
aliquoted into test tubes, with each containing 2.0 mL (the final
concentrations of DNA, H2O2, TCHQ, and resveratrol derivatives
were 2.00 mg/mL, 1.5, 3.0 mM, and 200 μM, respectively. The same
volume of DMSO was contained in the control experiment to
eliminate the influence from DMSO). The test tubes were incubated
1
mg). H NMR (400 MHz, DMSO-d6): δ 9.60 (s, 1H), 9.46 (s, 1H),
7.41 (d, J = 8.4 Hz, 2H), 7.04 (d, J = 16.3 Hz, 1H), 6.88 (d, J = 16.3
Hz, 1H), 6.77 (d, J = 8.4 Hz, 2H), 6.74 (s, 1H), 6.57 (s, 1H), 6.34 (s,
C
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Figure 2. Modification of resveratrol by various structural features.
at 37 °C for initiating the oxidation of DNA, and three of them were
taken out at 30 min and cooled in ice water. Then 1.0 mL of PBS1
solution of 2-thiobarbituric acid (TBA) (1.00 g of TBA and 0.40 g of
NaOH were dissolved in 100 mL of PBS1) and 1.0 mL of 3.0%
aqueous solution of trichloroacetic acid were added and heated in
boiling water for 30 min. After the test tubes were cooled in ice water,
1.5 mL of n-butanol was added and shaken vigorously to extract
thiobarbituric acid reactive species (TBARS). The absorbance of
organic layer was measured at 535 nm (Adetect) and compared with
that in the control experiment (Aref).
CuSO4 and glutathione (GSH) were dissolved in PBS1 to reach
75.0 and 90.0 mM as stock solutions, respectively. To 13.4 mL of
PBS1 solution of DNA was added 1.0 mL of CuSO4 and 0.5 mL of
GSH solution, and 0.1 mL of DMSO solution of resveratrol
derivatives. The aforementioned mixture was aliquoted into test
tubes, with each containing 2.0 mL (the final concentrations of DNA,
Cu2+, GSH, and resveratrol were 2.00 mg/mL, 5.0, 3.0 mM, and 200
μM, respectively; the same volume of DMSO was contained in the
control experiment to eliminate the influence from DMSO). The test
tubes were incubated at 37 °C for initiating the oxidation of DNA,
and three of them were taken out at 90 min and cooled in ice water.
PBS1 solution of EDTA (1.0 mL, 30.0 mM) was added to chelate
Cu2+, and then 1.0 mL of PBS1 solution of TBA and 1.0 mL of 3.0%
aqueous solution of trichloroacetic acid were added and heated in
boiling water for 30 min. After the test tubes were cooled in ice water,
1.5 mL of n-butanol was added and shaken vigorously to extract
TBARS. The absorbance of organic layer was measured at 535 nm
(Adetect) and compared with that in the control experiment (Aref).
AAPH-Induced Oxidation of DNA. DNA was dissolved in
phosphate buffered solution (PBS2: 8.1 mM Na2HPO4, 1.9 mM
NaH2PO4, 10.0 μM EDTA, pH = 7.4) to reach 2.24 mg/mL as the
stock solution, and AAPH was also dissolved in PBS2 to reach 400
mM as the stock solution. To 13.4 mL of PBS2 solution of DNA was
added 1.5 mL of PBS2 solution of AAPH and a certain volume of
DMSO solution of resveratrol derivatives. The aforementioned
mixture was aliquoted into test tubes, with each containing 2.0 mL
(the final concentrations of DNA and AAPH were 2.00 mg/mL and
40.0 mM, respectively, and the final concentrations of resveratrol
derivatives were shown in the corresponding chart in the Supporting
Information; the same volume of DMSO was contained in the control
experiment to eliminate the influence from DMSO). The test tubes
were incubated at 37 °C for initiating the oxidation of DNA, and three
of them were taken out every 2 h and cooled immediately in ice water.
Then 1.0 mL of PBS2 solution of TBA (1.00 g of TBA and 0.40 g of
NaOH dissolved in 100 mL of PBS2) and 1.0 mL of 3.0% aqueous
solution of trichloroacetic acid were added, and the mixture was
heated in boiling water for 15 min. After the test tubes were cooled in
ice water, 1.5 mL of n-butanol was added and shaken vigorously to
extract TBARS, whose absorbance was measured at 535 nm and
plotted versus the reaction period.
Statistical Analysis. All of the data were the average values from
at least three independent measurements with the experimental error
within 10%. The equations were analyzed by one-way ANOVA in
Origin 7.0 professional Software, and p < 0.001 indicated a
significance difference.
RESULTS AND DISCUSSION
■
Attaching Substituents to Position-2 of Resveratrol.
The synthesis of resveratrol derivatives was mainly composed
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of constructing CC by the Heck11 or Wittig reaction12 and
of linking substituents with hydroxyl groups of resveratrol. We
initially envisioned that introducing substituents into resvera-
trol directly was a convenient protocol for the preparation of
resveratrol derivatives. Especially, attaching substituents to
hydroxyl group-free positions in resveratrol might preserve
biologically functional groups for resveratrol.26 A successful
protocol as reported in literature27 was the formylation at
position-2 via the Vilsmeier−Haack reaction; then −CHO
acted as the linkage with other moieties. In contrast to the
method reported in literature,27 as depicted in Figure 2, we
herein performed the Vilsmeier−Haack reaction to afford 1
with phenolic hydroxyl groups in resveratrol not being
protected by any groups (phenolic hydroxyl groups in
resveratrol were methylated before the Vilsmeier−Haack
reaction in the ref 27).
We have found that introducing glucosyl or adamantyl group
into an antioxidative molecule was able to enhance inhibitory
effect against DNA oxidation.28 Thus, we attempted to bridge
glucosamine or 1-adamantanamine with −CHO in 1 directly
to simplify the synthetic operation, during which one drop of
formic acid (as catalyst) and stirring at room temperature were
applied to furnish imine between −CHO in 1 and amino group
in glucosamine or 1-adamantanamine with mixed solution
(THF/H2O) applied. As claimed in a previous report,29 “a
synthetic nightmare” might unfortunately be met when
resveratrol performed a reaction in the absence of the
protection of hydroxyl groups, owing to the difficulty in the
isolation of the target product. Thus, yields of 4 and 6 were
just 17% and 6%, respectively, when thin-layer chromatography
was used to isolate the products. Interestingly, the config-
uration of glucosyl group in 6 was transformed to be α-type
although the configuration of D-glucosamine was β-type. A
hydrolysis might perform on the glucosamine under acidic
condition (one drop of formic acid as catalyst), while the
configuration of hydroxyl group at position-1 in glucose moiety
was changed via the recyclization. To avoid lowering yield in
the case of 1 being the reagent, we had to use 1a as the starting
material for preparing 6a, in which hydroxyl groups in
glucosamine were protected by acetylation as well. Another
benefit from the protection of hydroxyl groups in resveratrol by
tert-butyldimethylsilyl chloride (TBDMSCl) and by acetylation
in glucosamine was to dissolve all of the reagents in a
homogeneous solution (methanol used herein). Then we
attempted to reduce the CN in 6a to form amine
(−CH2NH−) by NaBH4, and the acetyl groups at glucose
moiety was removed under K2CO3, followed by the removal of
TBDMS group under acidic condition. Figure 3 outlined that
1H NMR peak of −CHN- in 6 (8.71 ppm) moved to 4.08
ppm, indicating that −CHN− in 6 was transformed to
−CH2NH− in 7.
1
Figure 3. Comparison on H NMR peak of imine in 6 and that of
amine in 7.
2 on the antioxidative effect of resveratrol. The lipoic acid was
a widely used antioxidant, and whether it could enhance
antioxidative effect after forming a hybrid with resveratrol was
concerned in this work. Thus, −CH2OH in 2a took place the
esterification with lipoic acid, and compound 3 was given after
the deprotection of TBDMS groups.
Inhibitory Effects against •OH- and Cu2+/GSH-
Induced Oxidations of DNA. The ribose moieties in DNA
were readily oxidized by hydroxyl radical (•OH), leading to the
degradation of DNA.31 Chemically, a mixture of tetrachlor-
ohydroquinone (TCHQ, 3.0 mM) and H2O2 (1.5 mM) was
able to generate •OH, which was applied to mimic DNA (2.00
•
mg/mL) undergoing OH-mediated oxidation. The products
resulting from DNA oxidation could be colorized by
thiobarbituric acid (TBA), and the absorbance of thiobarbi-
turic acid reactive species (TBARS, λmax = 535 nm) resulting
from the control experiment (performing 30 min) was assigned
as 100%. The absorbances of TBARS in the presence of
resveratrol derivatives (200 μM) were compared with that in
the control experiment and expressed as TBARS percentages.
As depicted in Figure 4, a low TBARS% meant that the
corresponding resveratrol derivative possessed high inhibitory
•
effect against OH-induced oxidation of DNA. However, data
in the upper panel of Figure 4 did not provide positive results
because TBARS percentages limited from 83.9% to 97.2%, not
different significantly. A recently published report suggested
•
that resveratrol functioned as OH-scavenger by all possible
mechanisms, but mainly followed sequential electron proton
transfer (SEPT) and radical adduct formation (RAF).26 This
might be helpful for understanding the results obtained in the
present study. The −CHO as an electron-withdrawing group
in 1 was not beneficial for electrons being transferring from
resveratrol skeleton to •OH (following SEPT mechanism),
while position-2 was an active site for taking place radical-
addition by •OH (following RAF mechanism). In the
presented work, position-2 was applied to link with other
structural moieties and thus could not play antioxidative role
For better comparison of the antioxidative effects of glucosyl
imine or amine of resveratrol (6 or 7) with that of resveratrol
glucoside (a glucosyl moiety attaching to one of hydroxyl
group in resveratrol), we synthesized piceid (5, with 2,3,4,6-
tetra-O-acetyl-β-D-glucopyranosyl bromide being the glycosyl
donor) as a reference compound. In contrast to the method
reported in a literature,30 we employed resveratrol to react with
2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl bromide directly.
However, the low yield of piceid (5) was due to the sensitivity
toward ambient oxidation when the acetyl groups at glycose
moiety in 5 were removed by CH3ONa. Moreover, it was
necessary to test the influence of −CHO in 1 and −CH2OH in
•
by absorbing OH at this position. However, compound 6,
which consisted of a CN bond to enlarge the conjugation
system of resveratrol, exhibited similar inhibitory effect
(TBARS% = 84.8%) as resveratrol (TBARS% = 83.9%) in
this experimental system.
Subsequently, we tested the inhibitory effects of these
resveratrol derivatives against DNA oxidation mediated by
copper ion (Cu2+) and glutathione (GSH).32 A mixture of 5.0
mM Cu2+ and 3.0 mM GSH was used to produce glutathione
radical (GS•) for oxidizing 2.00 mg/mL DNA at 37 °C. After
DNA underwent Cu2+/GSH-mediated oxidation for 90 min,
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substrate for a period, the inhibition period (tinh) was proved
to correlate proportionally with the concentration of the
antioxidant (see eq 1). In eq 1, Ri stood for the initiation rate
of the radical-induced oxidation, and n referred to the
stoichiometric factor, which meant the number of the
radical-propagation terminated by one molecule of the
antioxidant:36
tinh = (n/Ri) [antioxidant]
(1)
We have measured the relationship between the concen-
tration of resveratrol and the inhibition period (tinh) in the
experimental system of AAPH-induced oxidation of DNA (see
eq 2):37
tinh = 1.10 ( 0.04) [resveratrol] + 38.62 ( 3.05)
(2)
It was safely assumed that the initiation rate (Ri) of DNA
oxidation was equal to the radical-generation rate (Rg = (1.4
0.2) × 10−6 [AAPH] s−1 at 37 °C), namely, Ri = Rg = 1.4 ×
10−6 × 40 mM s−1 = 3.36 μM min−1 because DNA sodium salt
and AAPH were dissolved in the same water phase.38 The
coefficient in eq 2, 1.10, was equivalent to n/Ri in eq 1, and the
stoichiometric factor (n) of resveratrol was the product of the
coefficient in eq 2 multiplying Ri. Thus, the n of resveratrol was
3.70, indicating that one molecule of resveratrol was able to
terminate 3.70 radical-propagations in the protection of DNA
against AAPH-induced oxidation. We next attempted to
measure inhibition periods (tinh) with various concentrations
of resveratrol derivatives added (shown in Figure S8) and
obtained correlations of tinh with concentrations of resveratrol
derivatives (shown in Figure S9). Subsequently, equations of
tinh ∼ [resveratrol derivative] were collected in Table S1, and
stoichiometric factors (n) of resveratrol derivatives were
calculated and outlined in Figure 5. It was found in Figure 5
that the n values of 4 and 5 were similar to that of resveratrol,
Figure 4. TBARS percentages in the DNA oxidation mediated by
•OH (upper) and Cu2+/GSH (below) as well as structures of
resveratrol derivatives.
TBARS percentages in the presence of resveratrol derivatives
(200 μM) were measured and outlined in the below panel of
Figure 4. A previous report revealed that the combination of
Cu2+ with resveratrol could destroy DNA strand.33 We herein
found that TBARS% of resveratrol was 94.1%, almost closing
to the control experiment (100%). In addition, TBARS% in the
case of additions of other resveratrol derivatives limited from
85.5% (7) to 94.3% (4), not different significantly with each
other. As pointed out in the literature,33 DNA strand might be
oxidized in the mixture of Cu2+ and resveratrol. In the
presented study, we just appended a series of structural
moieties to position-2 of resveratrol and did not vary the
resveratrol skeleton. Hence, the resveratrol derivatives used
herein might play the same role as resveratrol in the case of
interacting with Cu2+ and functioned as resveratrol itself.
Therefore, the inhibitory effects of the synthetic resveratrol
derivatives approached to that of resveratrol in this
experimental system. This encouraged us to find a quantitative
index for characterizing the antioxidant effects of these
compounds.
Stoichiometric Factor (n) in AAPH-Induced Oxidation
of DNA. Of interest to us was to quantitatively express
inhibitory effects of these resveratrol derivatives against peroxyl
radical-mediated oxidation of DNA. The guanine base in DNA
was sensitive toward the oxidation by 2,2′-azobis(2-amidino-
propane hydrochloride) (AAPH, R-NN-R, R = −CMe2C-
(NH)NH2, ROO• resource),34 and oxidative products could
also be detected via the reaction with TBA.35 Chemically, if an
antioxidant was able to inhibit AAPH-induced oxidation of a
Figure 5. Stoichiometric factors (n) of resveratrol derivatives with
antioxidative activities being cataloged as low (1, 2, and 3), middle (4
and 5), and high (6 and 7) groups.
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Figure 6. Influence of structural factors on stabilities of radicals deriving from resveratrol derivatives.
while the n values of 1, 2, and 3 were lower than that of
resveratrol. Exceptionally, the n values of 6 and 7 were higher
than that of resveratrol, revealing that only 6 and 7 exhibited
higher antioxidative effects than resveratrol in this case. It was
reported that installation of substituent to hydroxyl group was
not beneficial for enhancing the antioxidative effect of
resveratrol.26 The stoichiometric factor (n) of 5 was only
3.49, lower than that of 6 and 7 (4.74 and 4.97, respectively),
and even lower than that of resveratrol, 3.70. Thus, the
glucoside of resveratrol did not show higher antioxidative effect
than resveratrol. This fact reminded us to equip substituents
with hydroxyl group-free positions in resveratrol. However, as
depicted in Figure 6, if a phenoxyl radical was produced at
position-5, the single electron could be transferred via a series
of resonance structures (from 8 to 10) to position-2,
encountering the electron-withdrawing group, −CHO, which
was not beneficial for stabilizing radical. Meanwhile, the
intramolecular hydrogen bond produced between −CHO and
adjacent −OH increased the bond dissociation enthalpy
(BDE) of O−H to 412.0 kJ mol−1, 59.0 kJ mol−1 (ΔBDE)
higher than that in resveratrol. The −CHO even increased
ΔBDE of O−H of 23.1 and 8.9 kJ mol−1 when −OH located at
5- and 4′-positions, respectively.39 Apparently, introducing
−CHO into position-2 made H atoms in all of the −OH not to
be abstracted readily. After −CHO in 1 was reduced to form
−CH2OH, stoichiometric factors (n) of 2 decreased to 1.99,
the lowest value among these resveratrol derivatives. The
intramolecular hydrogen bond between the oxygen atom in
−CH2OH and the hydrogen atom in the adjacent phenolic
−OH still hindered the hydrogen atom in phenolic −OH to be
transferred to radicals.
adamantanyl group with resveratrol just recovered stoichio-
metric factor (n) of 4 to 3.55, still lower than that of resveratrol
(3.70). The aforementioned results implied that both lipoic
and adamantanyl groups could not bring resveratrol with an
ability to affiliate DNA strand. On the other hand, compound
6, which was composed of a glucose moiety as an imine,
increased the n value to 4.74, higher than that of resveratrol
(3.70). The n value of 7 was further increased to 4.97;
therefore, attaching glucose moiety to hydroxyl group-free
position was able to enhance the inhibitory effect of resveratrol
against DNA oxidation.
A detail in the structures of 6 and 7 aroused our interests,
and we tried to use conformation analysis to give an
explanation. In compound 6, −OH at position-1 of glucosyl
moiety located at axial bond, while −OH at position-1 of
glucosyl moiety in compound 7 might locate at axial or
equatorial bond. As shown in Figure 6, the axial −OH in 6
might form a hydrogen bond with N atom in imine. The same
type of hydrogen bond could also be produced in compound 7.
Moreover, the amine group in 7 might form a hydrogen bond
with the −OH in resveratrol moiety. Thus, acidic and basic
conditions applied in the deprotection of hydroxyl groups (see
Figure 2) might change the configuration of −OH at position-
1 in glucosyl group.
CONCLUSIONS
■
Resveratrol derivatives endowed with unique antioxidative
properties. We herein provided with a protocol for the
synthesis of resveratrol derivatives via the formylation at
position-2 of resveratrol. Although both −CHO and −CH2OH
at position-2 of resveratrol did not exert positive effects against
DNA oxidation, and attaching lipoic acid and adamantanamine
to position-2 even showed negative effects, bridging glucose
moiety with −CHO at position-2 as an imine or amine
ameliorated the antioxidative property, and the inhibitory
effect against DNA oxidation was much higher than that of
resveratrol glucoside. Therefore, preserving hydroxyl groups
and attaching other modified moieties at position-2 might
As depicted in Figure S10, lipoic acid just decreased the
oxidative rate of AAPH-induced oxidation of DNA and cannot
result in tinh. The stoichiometric factor (n) of 3 was 2.04, lower
than that of resveratrol. In addition, we have found that
introducing adamantanyl group into antioxidative molecule
was able to ameliorate inhibitory effect against AAPH-induced
oxidation of DNA,28 but herein, the combination of
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(12) Nakao, S., Mabuchi, M., Wang, S., Kogure, Y., Shimizu, T.,
Noguchi, K., Tanaka, A., and Dai, Y. (2017) Synthesis of resveratrol
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become a promising protocol for ameliorating antioxidative
effect of resveratrol.
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.chemres-
tox.8b00136.
NMR and HRMS spectra of resveratrol derivatives;
inhibitory effects of resveratrol derivatives against
AAPH-induced oxidation of DNA (PDF)
(15) Tang, J.-J., Fan, G.-J., Dai, F., Ding, D.-J., Wang, Q., Lu, D.-L.,
Li, R.-R., Li, X.-Z., Hu, L.-M., Jin, X.-L., and Zhou, B. (2011) Finding
more active antioxidants and cancer chemoprevention agents by
elongating the conjugated links of resveratrol. Free Radical Biol. Med.
50, 1447−1457.
AUTHOR INFORMATION
Corresponding Author
*E-mail: zaiqun-liu@jlu.edu.cn.
■
(16) Poutiainen, P. K., Palvimo, J. J., Hinkkanen, A. E., Valkonen, A.,
̈
̈
Vaisanen, T. K., Laatikainen, R., and Pulkkinen, J. T. (2013)
Discovery of 5-benzyl-3-phenyl-4,5-dihydroisoxazoles and 5-benzyl-3-
phenyl-1,4,2-dioxazoles as potent firefly luciferase inhibitors. J. Med.
Chem. 56, 1064−1073.
ORCID
Zai-Qun Liu: 0000-0001-9573-0724
Notes
The authors declare no competing financial interest.
(17) Domazetovic, V., Fontani, F., Tanini, D., D’Esopo, V.,
Viglianisi, C., Marcucci, G., Panzella, L., Napolitano, A., Brandi, M.
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T. (2017) Protective role of benzoselenophene derivatives of
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(18) Zhu, Y., Fu, J., Shurlknight, K. L., Soroka, D. N., Hu, Y., Chen,
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ACKNOWLEDGMENTS
■
Financial support from the Jilin Provincial Science and
Technology Department, China, is acknowledged gratefully
(20160101317JC).
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