Synthesis and bioactivity profile of 5-S-lipoylhydroxytyrosol-based multidefense antioxidants with a sizeable (poly)sulfide chain

Article abstract of DOI:10.1021/jf302690c

Novel polyfunctionalized antioxidants, 5-S-lipoylhydroxytyrosol (1) and its disulfide 2, trisulfide 3, and tetrasulfide 4, were prepared from tyrosol and dihydrolipoic acid in the presence, when appropriate, of sulfur. Compound 1 exhibited significant activity in the ferric reducing/antioxidant power (FRAP) assay (1.60 Trolox equiv), whereas polysulfides 2-4 were more efficient in the DPPH reduction assay (88-93% reduction vs 68% by Trolox). At 10 μM concentration, all compounds 1-4 proved to be efficient hydroxyl radical scavengers (56-69% inhibition) in a Fenton reaction assay. When administered to human HepG2 cells, 1-4 proved to be nontoxic and exhibited marked protective effects against reactive oxygen species (ROS) generation (60-84% inhibition at 1 μM concentration) and cell damage induced by 400 μM tert- butylhydroperoxide. All compounds 1-4 exhibited overall greater antioxidant activity than hydroxytyrosol.

Full text of DOI:10.1021/jf302690c

Article
pubs.acs.org/JAFC
Synthesis and Bioactivity Profile of 5‑S‑Lipoylhydroxytyrosol-Based
Multidefense Antioxidants with a Sizeable (Poly)sulfide Chain
Lucia Panzella,*,† Luisella Verotta,‡ Luis Goya,§ Sonia Ramos,§ María Angeles Martín,§ Laura Bravo,§
Alessandra Napolitano,† and Marco d’Ischia†
†Department of Chemical Sciences, University of Naples “Federico II”, Via Cintia 4, I-80126 Naples, Italy
‡Natural Products Branch, Department of Chemistry, University of Milan, Via C. Golgi 19, I-20133 Milan, Italy
§Department of Metabolism and Nutrition, ICTAN, CSIC, Jose
́
Antonio Novais 10, 28040 Madrid, Spain
S
* Supporting Information
ABSTRACT: Novel polyfunctionalized antioxidants, 5-S-lipoylhydroxytyrosol (1) and its disulfide 2, trisulfide 3, and tetrasulfide
4, were prepared from tyrosol and dihydrolipoic acid in the presence, when appropriate, of sulfur. Compound 1 exhibited
significant activity in the ferric reducing/antioxidant power (FRAP) assay (1.60 Trolox equiv), whereas polysulfides 2−4 were
more efficient in the DPPH reduction assay (88−93% reduction vs 68% by Trolox). At 10 μM concentration, all compounds 1−
4 proved to be efficient hydroxyl radical scavengers (56−69% inhibition) in a Fenton reaction assay. When administered to
human HepG2 cells, 1−4 proved to be nontoxic and exhibited marked protective effects against reactive oxygen species (ROS)
generation (60−84% inhibition at 1 μM concentration) and cell damage induced by 400 μM tert-butylhydroperoxide. All
compounds 1−4 exhibited overall greater antioxidant activity than hydroxytyrosol.
KEYWORDS: hydroxytyrosol, lipoic acid, antioxidant, HepG2 cells, bioactive compounds
O−H bond dissociation enthalpy induced by chalcogens.20
INTRODUCTION
■
Furthermore, S-glutathionyl conjugates of catechols were
identified as most potent antinitrosating agents21,22 exceeding
the parent catechols, a finding that supported the important
role of chalcogen atoms, especially sulfur and selenium, in
enhancing the H-atom donating, metal chelating, and reducing
power of polyphenolic systems.
Hydroxytyrosol (2-(3,4-dihydroxyphenyl)ethanol, HTy), the
most representative phenolic constituent of extra virgin olive
oil, has been the focus of increasing interest over the past
decades because of a range of biological properties that have
been implicated to account for the lower risk of cardiovascular
diseases and malignant neoplasms observed in the populations
of Mediterranean countries.1,2 The beneficial role of HTy has
been ascribed to its potent antioxidant and scavenging
properties against reactive oxygen (ROS) and nitrogen
(RNS) species3−7 generated in settings of oxidative stress,
and its reactivity with oxidizing systems of physiological
relevance has been elucidated.8−10 In vitro, HTy has been
shown to exert protective effect against free radical-induced
oxidative stress in different cell lines, such as Caco-2,
melanoma, and human hepatoma HepG2 cells.11−13
Several efforts have been directed toward the preparation of
HTy derivatives and analogues with improved solubility
properties, particularly enhanced lipophilicity,14−16 and anti-
oxidant and pharmacological activities. The latter include alkyl
HTy ethers, which showed protective effects against oxidative
stress in HepG2 cells,17 and a novel ester of HTy with α-lipoic
acid (LA) exhibiting a significant antiproliferative effect on
human colon cancer HT-29 cells.18
On the basis of the above rationale, the aim of the present
study was the synthesis and characterization of a novel class of
HTy derivatives comprising 5-S-lipoylhydroxytyrosol (1), an
unprecedented S-conjugate of HTy with dihydrolipoic acid
(DHLA), the reduced form of LA or vitamin N, and a set of
dimers (2−4) linked by di-, tri-, and tetrasulfide chains, in that
order (Figure 1). DHLA features two SH groups onto a short
carbon chain ending with a carboxyl group, thus providing not
only the necessary SH functionality for conjugation with HTy
but also a second SH group as reactive site for secondary
functionalization as well as a flexible chain expected to favor
interaction with membranes, lipid bilayers, and other hydro-
phobic environments. Polysulfides exhibit diverse beneficial
properties for human health, including inhibition of prolifer-
ation and induction of apoptosis in cancer cells, suggesting a
chemopreventive action of these compounds for cancer
therapy.23−25 Natural polysulfides, such as diallyl disulfide,
diallyl trisulfide, and diallyl tetrasulfide from garlic, are also
In pursuing a research program on the design and synthesis
of novel bioinspired antioxidant agents, we recently became
interested in exploring the potential of chalcogen-containing
conjugates of HTy as prototypes of novel multifunctional
polyphenolic derivatives combining diverse scavenging and
inhibitory activities against oxidative stress inducing species and
systems. Organochalcogen-substituted phenols are efficient
scavengers of hydroperoxides,19 which is attributed to low
Special Issue: IX Italian Congress of Food Chemistry
Received: June 21, 2012
Revised: December 5, 2012
Accepted: December 20, 2012
Published: December 20, 2012
© 2012 American Chemical Society
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Figure 1. Synthetic route to 5-S-lipoylhydroxytyrosol (1) and polysulfides 2−4.
droperoxide (t-BOOH), reduced nicotine adenine dinucleotide
(NADH), 2′,7′-dichlorofluorescin diacetate, gentamycin, penicillin G,
and streptomycin were purchased from Sigma-Aldrich (Madrid,
Spain). Other chemicals used were of analytical grade. ( )-Dihy-
drolipoic acid (DHLA)28 and 2-iodoxybenzoic acid (IBX)29 were
prepared as described.
known for their potent antioxidant activities, which increase
with the increasing number of S atoms.26,27
Specific goals of the study were therefore (a) to develop an
expeditious and versatile entry to the new HTy derivatives
through one-pot oxygenation/oxidation of commercially
available tyrosol in the presence of DHLA followed by
controlled exposure to elemental sulfur to provide polysulfide
bridges when required; (b) to compare the effects of the
different sulfur functionalities (SH vs polysulfides) in a battery
of chemical tests for antioxidant properties as well as in in vitro
assays on cultured HepG2 cells; and (c) to draw simple and
preliminary structure−property relationships that may guide
the rational design of new members of this class of antioxidants
with improved activity for diverse applications.
General Experimental Methods. UV−vis spectra were recorded
1
on a Beckman (Milan, Italy) DU 640 spectrophotometer. H and 13C
NMR spectra were recorded in CD3OD at 400 and 100 MHz,
respectively, on a Bruker Avance NMR spectrometer. H−H COSY,
HSQC, and HMBC data were collected using standard pulse
programs. Chemical shifts are reported in δ values downfield from
TMS. HPLC analysis was carried out on an Agilent (Santa Clara, CA,
USA) instrument equipped with a UV detector set at 280 nm.
Chromatographic separation was achieved on a Phenomenex (Castel
Maggiore, Italy) Sphereclone ODS column (250 mm × 4.6 mm, 5
μm) using binary gradient elution conditions as follows: 0.1%
trifluoroacetic acid (solvent A), acetonitrile (solvent B), from 5 to
90% B, 0−45 min, flow rate 0.7 mL/min (eluent system I); 10 mM
phosphoric acid (pH 2.5)/acetonitrile 80:20 v/v, flow rate 0.7 mL/min
(eluent system II). For preparative purposes an Alltech (Passirana di
Rho, Milan, Italy) Econosil C18 column (250 mm × 10 mm, 10 μm)
was used, with 0.1% trifluoroacetic acid/acetonitrile 60:40 v/v as
eluent, at a flow rate of 3 mL/min. LC-MS analysis was carried out
with an LC-MSD 1100 VL system (Agilent, Santa Clara, CA, USA)
equipped with a UV−vis detector (G1314A) and a quadrupole mass
spectrometer with electrospray ionization source (G1956A) operating
in positive ionization mode (ESI+): nebulizer pressure, 50 psi; drying
gas (nitrogen), 10 L/min, 350 °C; capillary voltage, 4000 V;
fragmentor voltage, 50 V. Chromatographic separation was performed
MATERIALS AND METHODS
■
Caution. IBX is explosive under impact or heating >200 °C.30
Chemicals. 2-Iodobenzoic acid, oxone, ( )-lipoic acid (LA),
tyrosol, 2,4,6-tris(2-pyridyl)-s-triazine (TPTZ), neocuproine, ethyl-
enediaminetetraacetic disodium salt (Na2EDTA) dihydrate, hydrogen
peroxide (30% w/w solution in water), 2-hydroxybenzoic acid
(salicylic acid), ( )-6-hydroxy-2,5,7,8-tetramethylchromane-2-carbox-
ylic acid (Trolox), and catalase from bovine liver (EC 1.11.1.6) were
purchased from Sigma-Aldrich (Milan, Italy). 2,2-Diphenyl-1-picrylhy-
drazyl (DPPH) was purchased from CalbioChem (Darmstadt,
Germany). Ascorbic acid was purchased from AppliChem (Darmstadt,
Germany). DMEM F-12 medium and fetal bovine serum (FBS) were
purchased from Biowhitaker (Lonza, Madrid, Spain); tert-butylhy-
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on an Eclipse XDB-C18 (150 × 4.60 mm, 5 μm, Agilent) column, at a
flow rate of 0.4 mL/min, with eluent system I. HRMS analysis were
performed on an FT-ICR Bruker Daltonics mass spectrometer using
an ESI source.
(C-α), 52.9 (C-6′), 65.1 (C-β), 117.0 (C-2), 121.5 (C-5), 126.0 (C-6),
132.1 (C-1), 144.9 (C-4), 146.2 (C-3), 178.2 (C-1′).
Tetrasulfide 4. To a reaction flask charged with 1 (250 mg, 0.69
mmol) in methanol solution (270 mL) was added S (80 mg, 2.5
mmol), and the resulting reaction mixture was taken under vigorous
stirring at room temperature and periodically analyzed by HPLC and
LC-MS (eluent system I). After ca. 48 h, when consumption of the
starting material was complete, the mixture was washed with n-hexane
(3 × 100 mL) to remove excess sulfur and taken to dryness, and the
residue obtained was dissolved in methanol and fractionated by
semipreparative HPLC to give 4 as a light yellow oil in pure form (85
mg, 31% yield): HRESIMS (negative), m/z 781.1362 ([M − H]−),
calcd for C32H45O10S6, m/z 781.1342; UV, λmax (CH3OH) 256, 294
nm; 1H NMR (400 MHz, CD3OD), δ 1.38 (m, 1 × 2, H-4′), 1.52 (m,
1 × 2, H-5′), 1.60 (m, 4 × 2, H-3′, H-4′, H-5′), 1.87 (m, 1 × 2, H-7′),
1.93 (m, 1 × 2, H-7′), 2.28 (t, 2 × 2, J = 7.2 Hz, H-2′), 2.65 (t, 2 × 2, J
= 7.2 Hz, H-α), 2.94 (m, 1 × 2, H-8′), 3.03 (m, 1 × 2, H-6′), 3.13 (m,
1 × 2, H-8′), 3.69 (t, 2 × 2, J = 7.2 Hz, H-β), 6.62 (d, 1 × 2, J = 1.6
Hz, H-2), 6.72 (d, 1 × 2, J = 1.6 Hz, H-6); 13C NMR (100 MHz,
CD3OD), δ 25.8 (C-3′), 27.5 (C-4′), 32.3 (C-8′), 34.8 (C-2′), 35.1
(C-5′, C-7′), 39.6 (C-α), 52.9 (C-6′), 64.5 (C-β), 116.9 (C-2), 121.3
(C-5), 125.7 (C-6), 131.9 (C-1), 145.0 (C-4), 146.3 (C-3), 178.0 (C-
1′).
Preparation of 5-S-Lipoylhydroxytyrosol (1). To a reaction
flask charged with tyrosol (1.0 g, 7.2 mmol), methanol (100 mL), and
a stir bar at −25 °C was added IBX (2.9 g, 10.4 mmol, 1.4 equiv), and
the resulting reaction mixture was stirred for 1 h. Then, DHLA (6.0 g,
28.8 mmol, 4.0 equiv) in methanol solution (100 mL) was added.
Stirring was continued at room temperature for 15 min, and then the
mixture was diluted 1:5 with water, acidified to pH 3 with 6 M HCl,
and extracted with toluene (6 × 500 mL) to remove 2-iodobenzoic
acid, with chloroform (3 × 500 mL) to obtain 1 and finally with ethyl
acetate (3 × 500 mL) to recover HTy. Residual IBX in the water phase
was destroyed by adding sodium dithionite. The combined chloroform
extracts were dried over Na2SO4 and taken to dryness to give 1 as a
light yellow oil in pure form (752 mg, 29% yield): HRESIMS
(negative), m/z 359.0985 ([M − H]−), calcd for C16H23O5S2 m/z
1
359.0992; UV, λmax (CH3OH) 256, 291 nm; H NMR (400 MHz,
CD3OD), δ 1.44 (m, 3, H-4′, H-5′), 1.59 (m, 4, H-3′, H-5′, H-7′),
1.85 (m, 1, H-7′), 2.27 (t, 2, J = 7.2 Hz, H-2′), 2.65 (t, 2, J = 7.2 Hz,
H-α), 2.92 (m, 2, H-6′, H-8′), 3.02 (m, 1, H-8′), 3.68 (t, 2, J = 7.2 Hz,
H-β), 6.63 (d, 1, J = 2.0 Hz, H-2), 6.72 (d, 1, J = 2.0 Hz, H-6); 13C
NMR (100 MHz, CD3OD), δ 25.9 (C-3′), 27.8 (C-4′), 32.6 (C-8′),
35.1 (C-2′), 39.6 (C-α), 39.7 (C-5′), 39.8 (C-7′), 40.3 (C-6′), 64.5
(C-β), 116.8 (C-2), 121.7 (C-5), 125.5 (C-6), 132.0 (C-1), 144.9 (C-
4), 146.4 (C-3), 178.0 (C-1′).
DPPH Assay. The assay was performed as described.31 Briefly, to
1.98 mL of 200 μM DPPH in methanol was added 20 μL of a 5 mM
solution of compounds 1−4, HTy, or LA. The reaction was followed
by spectrophotometric analysis measuring the absorbance at 515 nm
every 30 s for 10 min. Trolox was used as standard.
Preparation of Polysulfides 2−4. Disulfide 2. To a reaction flask
charged with 1 (250 mg, 0.69 mmol) in methanol solution (15 mL)
was added 0.1 M phosphate buffer (pH 7.4) (255 mL), and the
resulting reaction mixture was taken under vigorous stirring at room
temperature and periodically analyzed by HPLC and LC-MS (eluent
system I). After ca. 60 h, when consumption of 1 was complete, the
mixture was acidified to pH 3 and extracted with ethyl acetate (3 ×
150 mL). The combined organic layers were dried over Na2SO4 and
taken to dryness. The residue obtained was dissolved in methanol and
fractionated by semipreparative HPLC to give 2 as a light yellow oil in
pure form (25 mg, 10% yield): HRESIMS (positive), m/z 741.1892
([M + Na]+), calcd for C32H46O10S4Na, m/z 741.1871; UV, λmax
(CH3OH) 256, 291 nm; 1H NMR (400 MHz, CD3OD), δ 1.39 (m, 2
× 2, H-4′), 1.57 (m, 4 × 2, H-3′, H-5′), 1.80 (m, 2 × 2, H-7′), 2.27 (t,
2 × 2, J = 7.6 Hz, H-2′), 2.65 (t, 2 × 2, J = 7.2 Hz, H-α), 2.80 (m, 1 ×
2, H-6′), 2.89 (m, 1 × 2, H-8′), 2.96 (m, 1 × 2, H-8′), 3.68 (t, 2 × 2, J
= 7.2 Hz, H-β), 6.63 (d, 1 × 2, J = 1.6 Hz, H-2), 6.72 (d, 1 × 2, J = 1.6
Hz, H-6); 13C NMR (100 MHz, CD3OD), δ 26.0 (C-3′), 27.4 (C-4′),
32.1 (C-8′), 34.9 (C-2′), 35.0 (C-5′, C-7′), 39.6 (C-α), 51.9 (C-6′),
64.5 (C-β), 116.8 (C-2), 121.5 (C-5), 125.6 (C-6), 131.9 (C-1), 144.9
(C-4), 146.3 (C-3), 177.7 (C-1′).
Trisulfide 3. To a reaction flask charged with 1 (250 mg, 0.69
mmol) in methanol solution (15 mL) and 0.1 M phosphate buffer (pH
7.4) (180 mL) was added S (80 mg, 2.5 mmol) in methanol solution
(90 mL), and the resulting reaction mixture was taken under vigorous
stirring at room temperature and periodically analyzed by HPLC and
LC-MS (eluent system I). After ca. 6 h, when consumption of the
starting material was complete, the reaction mixture was diluted 1:4
with water, acidified to pH 3, washed with n-hexane (3 × 700 mL) to
remove excess sulfur, and extracted with ethyl acetate (3 × 700 mL).
The combined organic layers were dried over Na2SO4 and taken to
dryness. The residue obtained was dissolved in methanol and
fractionated by semipreparative HPLC to give 3 as a light yellow oil
in pure form (40 mg, 15% yield): HRESIMS (positive), m/z 773.1611
([M + Na]+), calcd for C32H46O10S5Na, m/z 773.1592; UV, λmax
(CH3OH) 260, 293 nm; 1H NMR (400 MHz, CD3OD), δ 1.43 (m, 2
× 2, H-4′), 1.59 (m, 4 × 2, H-3′, H-5′), 1.84 (m, 1 × 2, H-7′), 1.94
(m, 1 × 2, H-7′), 2.27 (t, 2 × 2, J = 7.2 Hz, H-2′), 2.66 (t, 2 × 2, J =
7.2 Hz, H-α), 2.93 (m, 1 × 2, H-8′), 3.01 (m, 2 × 2, H-6′, H-8′), 3.68
(t, 2 × 2, J = 7.2 Hz, H-β), 6.63 (d, 1 × 2, J = 1.6 Hz, H-2), 6.72 (d, 1
× 2, J = 1.6 Hz, H-6); 13C NMR (100 MHz, CD3OD), δ 25.7 (C-3′),
27.5 (C-4′), 32.6 (C-8′), 34.6 (C-5′), 34.7 (C-7′), 35.0 (C-2′), 39.4
Ferric Reducing/Antioxidant Power (FRAP) Assay. The assay
was performed as described.32 To 3.6 mL of a solution of FRAP
reagent was added 5−150 μL of 5 mM solutions of compounds 1−4,
HTy, or LA (7−200 μM final concentration). After 10 min, the
absorbance at 593 nm was measured. Trolox was used as standard.
The FRAP reagent was prepared freshly by mixing 0.3 M acetate buffer
(pH 3.6), 10 mM TPTZ in 40 mM HCl, and 20 mM ferric chloride in
water, in the ratio 10:1:1, in that order.
Copper Reducing Antioxidant Capacity (CUPRAC) Assay.
The assay was performed as described.33 To a test tube containing 1
mL of 10 mM CuCl2 × 2H2O in water were added 1 mL of 7.5 mM
neocuproine in 96% ethanol, 1 mL of 1 M ammonium acetate buffer
(pH 7.0), 1 mL of water, and 2−25 μL of 5 mM solutions of
compounds 1−4, HTy, or LA (2.5−30 μM final concentration). After
1 h, the absorbance at 450 nm was measured. Trolox was used as
standard.
Hydroxyl Radical Scavenging Assay. The assay was performed
as described.34 The following solutions were prepared: 20 mM ferrous
chloride in 40 mM HCl; 20 mM Na2EDTA in water; 50 mM H2O2 in
water; 10 mM salicylic acid in 0.2 M phosphate buffer (pH 7.4). To
1.5 mL of 0.2 M phosphate buffer (pH 7.4) was added 500 μL of
salicylic acid solution, followed by 250 μL of EDTA solution, 250 μL
of Fe2+ solution, 2 mL of water or 25 μM solutions of compounds 1−
4, HTy, or LA in water, and 500 μL of H2O2 solution. After 10 min,
500 μL of 268 U/mL catalase solution was added, and the mixtures
were analyzed by HPLC (eluent system II).
Cell Culture and Treatment. Human hepatic HepG2 cells were
maintained in a humidified incubator containing 5% CO2 and 95% air
at 37 °C. They were grown in DMEM F-12 supplemented with 2.5%
FBS and 50 mg/L each of gentamicin, penicillin, and streptomycin.
Compound 1−4, Hty, or LA was dissolved at different concentrations
(1, 5, 10, and 20 μM) in serum-free culture medium and added to the
cell plates for 24 h (crystal violet assay) and 2 h (dichlorofluorescin
assay). In the experiments to evaluate the protective role of the
compounds against an oxidative insult, cells were pretreated with the
same concentrations of the compounds for 20 h, then the medium was
discarded and fresh medium containing 400 μM t-BOOH was added
for 3 h (lactate dehydrogenase (LDH) assay) or 2 h (dichloro-
fluorescin assay).
Evaluation of Cell Viability, Cell Damage, and ROS
Generation. Cell viability was determined using the crystal violet
assay.35 HepG2 cells were seeded at low density (104 cells/well) in 96-
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well plates, grown for 24 h, treated with the different compounds for
24 h, and then incubated with crystal violet (0.2% in ethanol) for 20
min. Plates were rinsed with water and allowed to dry, and 1% sodium
dodecyl sulfate was added. The absorbance of each well was measured
using a microplate reader at 570 nm.
trisulfides, in particular, are the main products in the case of
secondary thiols such as 1.38 Sulfur dissolved in methanol was
added to compound 1 dissolved in phosphate buffer at pH 7.4,
with a final methanol/phosphate buffer ratio of 1:2 v/v. Under
these conditions a fast consumption of 1 was observed (90%
after 6 h), with concomitant formation of a main product eluted
at 26 min (HPLC analysis, eluent system I). LC-MS/ESI+
analysis indicated for this product pseudomolecular ion peaks
[M + Na]+ and [M + K]+ at m/z 773 and 789, respectively, as
expected for the trisulfide derivative of 1; moreover, significant
fragmentation peaks at m/z 391 and 359 most likely related to
S−S bond cleavage were observed. The reaction mixture was
diluted 1:4 with water, taken to pH 3, washed with n-hexane to
remove any excess sulfur, and extracted with ethyl acetate. The
residue obtained from the combined organic layers was
fractionated by semipreparative HPLC to give 3 in pure form
(15% yield).
Tetrasulfide 4. Tetrasulfide 4 was prepared by reacting 1
under the conditions adopted for the preparation of 3 but in
pure methanol, to optimize solubilization of sulfur. A rather
slow consumption of 1 (90% after 48 h) was obtained, with
formation of a main product eluted at 27 min (HPLC analysis,
eluent system I). LC-MS analysis in ESI+ mode indicated for
this product pseudomolecular ion peaks [M + H]+ and [M +
Na]+ at m/z 783 and 805, respectively, with significant
fragmentation peaks at m/z 391 and 423. The reaction mixture
was washed with n-hexane to remove excess sulfur and
fractionated by semipreparative HPLC as previously described
to give 4 in pure form (31% yield).
Chemical Properties of 1−4. Compounds 1−4 were fairly
stable when stored dry or in organic solvent in the cold and
were fairly soluble in methanol, acetonitrile, and water.
However, they were rapidly degraded in alkaline media in the
presence of air or oxygen, consistent with the notorious
instability of catechol moieties. Interestingly, the observed mild
conversion of 1 to 2 in aerated phosphate buffer at pH 7.4
suggests that the SH functional group is more susceptible to
aerial oxidation than the catechol moiety, making an
equilibrium of the two compounds likely to occur in a
biological environment.
Evaluation of the Antioxidant Properties of Com-
pounds 1−4 by Chemical Assays. DPPH Assay. The
hydrogen donor capacity of compounds 1−4 was estimated
through the DPPH assay,31 which determines the ability of
molecules to reduce the colored and stable DPPH radical
against a suitable reference donor (typically Trolox). Figure 2
Cellular damage was evaluated by LDH leakage.13 Cells were seeded
(2 × 105 cells/plate) in 60 mm plates and grown for 20 h with the
different treatments, and then the cell culture medium was collected,
and the cells were scraped off in phosphate buffer saline. LDH activity
was determined by the disappearance of NADH at 340 nm. LDH
leakage was estimated from the ratio between the LDH activity in the
culture medium and the total LDH activity in the culture plus
intracellular medium.
Cellular ROS generation was quantified by the dichlorofluorescin
assay using a microplate reader.13 Cells were seeded in 24-well plates
(2 × 105 cells/well), treated with the noted conditions, and measured
in a fluorescent microplate reader at an excitation wavelength of 485
nm and an emission wavelength of 530 nm.
Statistical Analysis. One-way analysis of variance was used for
cellular assays followed by a Bonferroni test when variances were
homogeneous or by the Tamhane test when variances were not
homogeneous. Differences were considered to be statistically
significant if P < 0.05 (statistical package SPSS v. 19.0). Excel
Student’s t test was used for chemical antioxidant assays.
RESULTS AND DISCUSSION
■
Preparation of 5-S-Lipoylhydroxytyrosol (1). The
reaction of DHLA with quinones is known to lead to the S-
conjugation products.36,37 Access to the desired compound 1
was thus obtained by an expedient protocol involving
regioselective oxidation of tyrosol with IBX to HTy o-
quinone,10,22 followed by addition of DHLA (Figure 1).
Tyrosol (75 mM in methanol) was treated with 1.5 mol equiv
of IBX, at −25 °C, giving an instable o-quinone. After 1 h,
DHLA (300 mM) was added, and the mixture was taken at
room temperature for 15 min, diluted 1:5 with water, and then
acidified to pH 3 with 6 M HCl. HPLC analysis (eluent system
I) of the reaction mixture showed the presence of 1, 2-
iodobenzoic acid, and HTy, most likely deriving from a redox
exchange o-quinone/DHLA competing with the nucleophilic
addition. To isolate 1 in pure form, a sequential extraction with
solvents of increasing polarity was performed: removal of 2-
iodobenzoic acid, DHLA, and LA was achieved by extraction of
the mixture with toluene; subsequent extraction with chloro-
form allowed isolation of 1 in pure form in the organic phase,
whereas the more polar HTy was recovered by extraction with
ethyl acetate. This procedure led to isolation of 1 in 29% yield.
Preparation of Polysulfides 2−4. Disulfide 2. It is well-
known that thiols give corresponding disulfides by oxidation.
On this basis, 2 was prepared by aerial oxidation of 1 (2.5 mM)
under vigorous stirring in 0.1 M phosphate buffer (pH 7.4).
HPLC analysis (eluent system I) showed a complete
consumption of 1 after about 60 h with concomitant formation
of a main product eluted at 25 min. LC-MS/ESI+ analysis
showed for this product pseudomolecular ion peaks [M + Na]+
and [M + K]+ at m/z 741 and 757, respectively, suggestive of a
disulfide derivative. Consistently, an intense M/2 fragmentation
peak at m/z 359 was also observed. The reaction mixture was
taken to pH 3 and extracted with ethyl acetate, and the residue
obtained from the combined organic layers was fractionated by
semipreparative HPLC to give 2 in pure form (10% yield).
Trisulfide 3. For the preparation of the trisulfide derivative, 1
was reacted at pH 7.4 in the presence of elemental sulfur. In
fact, it is known that polysulfides can be obtained in good yields
from the reaction of thiols with sulfur at room temperature, and
Figure 2. Decrease in the absorbance of 200 μM DPPH (515 nm) in
the presence of 50 μM compounds 1−4, HTy, LA, or Trolox.
Reported are the mean SD values for two separate experiments.
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Table 1. H-Atom Transfer Reaction from Compounds 1−4 (50 μM Each) to DPPH (200 μM) in Comparison to Trolox, HTy,
a
and LA
b
c
d
e
antioxidant
DPPH reduced (%)
k1 (M−1 s−1
)
n
ntotal
1
82 4a
90 1ab
93 1b
88 1a
52 1c
515 17a
2.04 0.05a
2.81 0.07b
2.73 0.10b
2.65 0.02b
1.11 0.16cd
0.12 0.01d
2.81 0.14ab
3.10 0.03a
3.20 0.03a
3.01 0.02a
1.79 0.04c
0.12 0.02d
2.31 0.01b
2
1412 81bc
1067 35b
1531 22c
175 39de
3
4
HTy
LA
4
1d
4
1d
Trolox
68 2e
312 12e
1.67 0.07c
a
b
c
Values are the mean SD (n = 2). Values in a column without a common letter differ, P < 0.05. Calculated after 10 min of reaction. Rate
d
e
constant for the fast step. Number of H atoms transferred in the fast step. Number of H atoms transferred after 10 min.
shows the decrease in absorbance of DPPH at 515 nm in the
presence of compounds 1−4. HTy and LA were also tested for
comparison. DHLA was not tested because of its well-known
instability under oxidative conditions. Data obtained by kinetic
analysis are reported in Table 1. Polysulfides 2−4 were found
to have the strongest hydrogen donor ability. No significant
differences were observed related to the number of sulfur
atoms. Compound 1 displayed high DPPH reducing activity,
whereas HTy was less active than Trolox. Almost no reduction
was observed with LA. Higher rates for the fast hydrogen atom
transfer step were exhibited by the polysulfides 2−4 with
respect to 1.
FRAP and CUPRAC Assays. Whereas the reducing capacity
of thiol-containing compounds cannot be determined accu-
rately by the FRAP assay,32,39,40 the CUPRAC assay33 has been
proven to be suitable. Here, the reducing capacity of
compounds 1−4 was measured by the above two assays for
comparison. Table 2 reports the results for the two assays,
dihydroxybenzoic acids was calculated with respect to a control
reaction mixture run in the absence of antioxidant. As reported
in Table 3, all compounds 1−4 acted as efficient hydroxyl
radical scavengers at concentration as low as 10 μM.
Table 3. Inhibition of Salicylic Acid Hydroxylation by
Compounds 1−4, HTy, and LA (10 μM Each)
a
antioxidant
inhibition (%)
1
69
56
57
59
64
61
9
2
2
2
4
6
2
3
4
HTy
LA
a
Values are the mean SD (n = 2).
Evaluation of the Antioxidant Properties of Com-
pounds 1−4 in Cell Culture. Following demonstration of the
marked antioxidant properties of compounds 1−4 in chemical
assays, further experiments were aimed at a screening of their
activities in validated cellular systems.41 The concentrations of
compounds 1−4 used in this study (1−20 μM) were in the
range of plasma concentrations of HTy in individuals who
consume extra virgin olive oil in Mediterranean countries. Daily
consumption of 25−50 mL of virgin olive oil, which contains
between 180 and 300 mg/kg of olive oil phenols, results in an
estimated daily intake of about 9 mg of olive oil phenols, which
is equivalent to 58 μmol of HTy equiv per day, of which 30−
90% could be absorbed,42 leading to plasma concentrations of
5−10 μM.
Table 2. Fe3+ and Cu2+ Reducing Activity of Compounds 1−
4 in Comparison to HTy and LA
a
a
antioxidant
Trolox equiv (FRAP)
Trolox equiv (CUPRAC)
1
1.60 0.15a
1.00 0.05ab
0.77 0.08bc
0.71 0.04c
0.92 0.04ab
0.03 0.01d
3.32 0.21a
2.35 0.08b
1.85 0.07c
1.75 0.04c
2.42 0.11b
0.02 0.01d
2
3
4
HTy
LA
a
Values are the mean SD (n = 2). Results are expressed as Trolox
equivalents. Values in a column without a common letter differ, P <
0.05.
Preliminarily, the effects of 1−4 (concentration range of 1−
20 μM) on liver cell viability were evaluated, and the results
showed that none of the compounds tested exerted significant
toxicity (data not shown). Figure 3 reports the effects of 1−4
on ROS production in HepG2 cells. The data show that all
compounds tested can efficiently abate ROS levels, with 2−4
being more effective than the parent HTy. These results were
in agreement with previous studies showing a direct effect on
ROS quenching in the same cell line by other natural olive oil
phenolics13,43 and also synthetic derivatives of HTy.17
Figures 4 and 5 show the effects of pretreating HepG2 cells
with compounds 1−4 at 1−20 μM concentrations before
exposure to 400 μM t-BOOH.41,44 Again, marked inhibitory
properties could be demonstrated against cell damage and ROS
generation under the stressing conditions of the assay. These
results demonstrate that the new compounds are efficient
protecting agents in in vitro models of oxidative stress,
preventing cell damage caused by potent oxidative insults.
expressed as Trolox equivalents. It is shown that 1 is endowed
with the best reducing capacity in both tests. The activity of
polysulfides 2−4 decreases apparently with increasing number
of sulfur atoms, whereas LA is, as expected, a very poor
reducing agent.
•
•OH Scavenging Assay. The OH scavenging activity of
compounds 1−4 was evaluated by a Fenton inhibition assay
based on HPLC determination of salicylic acid hydroxylation
products, which are catechol, 2,3-dihydroxybenzoic acid, and
2,5-dihydroxybenzoic acid.34 Briefly, salicylic acid at 1 mM
concentration was reacted with Fe2+ (1 mM), EDTA (1 mM),
and H2O2 (5 mM) in 0.2 M phosphate buffer (pH 7.4), in the
presence of compounds 1−4, HTy, or LA. After 10 min,
catalase (25 U/mL) was added to stop the reaction, and the
reaction mixture was analyzed by HPLC (eluent system II);
inhibition (%) of the formation of catechol and 2,3- and 2,5-
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Figure 5. Protective effect of HTy, LA, and compounds 1−4 on ROS
generation. HepG2 cells were treated with the noted concentrations of
mentioned compounds during 20 h, and then the cultures were
washed and 400 μM t-BOOH was added for 2 h to all cultures except
controls. Data are expressed as fluorescence units, and values are the
Figure 3. Direct effect of HTy, LA, and compounds 1−4 on ROS
generation. HepG2 cells were treated with the noted concentrations of
the mentioned compounds during 2 h. Results are expressed as
fluorescence units, and values are the mean
SD of four to six
different samples per condition. Means without a common letter differ,
P < 0.05.
mean
SD of four to six different samples per condition. Means
without a common letter differ, P < 0.05.
lead structures for the development of innovative antioxidants
inspired by natural products. The compounds were synthesized
by a versatile and expedient synthetic protocol, which
capitalized on the IBX-mediated conjugation of tyrosol with
DHLA to afford the unprecedented adduct 1, from which the
di- to tetrasulfide derivatives 2−4 were easily obtained without
complex purification stages. Determination of the antioxidant
activities of 1−4 in the DPPH, FRAP, CUPRAC, and Fenton
reaction inhibition assays revealed significantly more potent
effects compared to the parent HTy, LA, or the reference
compound Trolox. Compounds 1−4 also exerted protective
effects against ROS generation and oxidative cell damage in
human liver HepG2 cells. On the basis of these results, it is
concluded that (1) insertion of the sulfur-containing chain
potentiates the overall antioxidant activity of HTy, conferring
to the core system a more pronounced lipophilic character and
a novel putative metal-chelating site;46 (2) compounds 1−4
behave as broad-spectrum, multidefense antioxidants acting
through different mechanisms, including H-atom release, free
radical quenching, ferric and cupric ion reduction, and OH
radical scavenging; (3) the antioxidant effects of 1−4 vary
within the product series with the nature of the assay, for
example, 1 being more efficient as a ferric reducing agent and
OH radical scavenger and polysulfides 2−4 being more active
as H-atom donors in DPPH reduction exhibiting a higher
protecting capacity in vitro against ROS generation; (4) the
overall properties of compounds 1−4 reflect both electronic
effects caused by the sulfur substituent on the catechol ring and
solubility effects induced both by the S-lipoyl side chain and the
bridging polysulfide chain, which may play a crucial role in cell
membrane permeation. On this basis, DHLA−HTy conjuga-
tion can be proposed as a convenient access route to versatile
multidefense antioxidant systems that can be finely tuned
through proper selection of molecular size (monomer 1 versus
dimers 2−4), functional groups (SH versus S−(S)n−S, with 0
≤ n ≤ 2 and possibly more) and lipophilicity. Pursuit of this
approach may benefit also from future work aimed at testing
new members of the series, for example, as antinitrosating and
hypochlorous acid scavengers as most natural polysulfides and
Figure 4. Protective effect of HTy, LA, and compounds 1−4 on cell
damage. HepG2 cells were treated with the noted concentrations of
mentioned compounds during 20 h, and then the cultures were
washed and 400 μM t-BOOH was added for 3 h to all cultures except
controls. Results of LDH leakage are expressed as the ratio of LDH
activity in the culture medium to that in culture plus intracellular
medium. A value of 100% was given to controls, and the other values
are referred to them. Values are the mean SD of four to six data.
Means without a common letter differ, P < 0.05.
Although antioxidant activity data in chemical assays and in
cell culture were similar for all compounds, it is worth noting
that in cells compounds 2, 3, and 4 were relatively more
efficient than compound 1, perhaps due to their higher
hydrophobicity, enabling them to penetrate the membranes and
reach the radicals formed within the cell. In line with this view,
Lima et al.44 evaluated the protective effects of phenolic acids
and their esters against oxidative damage in HepG2 cells and
emphasized the important role of hydrophobicity in the
hepatoprotective potential of the phenolic compounds. Like-
wise, Spencer et al.45 suggested that the antioxidant bioactivity
of polyphenols strongly depends on the extent to which they
interact with, and permeate, cell membranes.
In conclusion, we have reported herein the synthesis and
characterization of a new set of HTy derivatives as promising
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the rational formulation of suitable cocktail mixtures for a
broader activity spectrum.
ASSOCIATED CONTENT
* Supporting Information
NMR and MS spectra of compounds 1−4. This material is
available free of charge via the Internet at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
■
*Phone: +39(0)81 674131. Fax: +39(0)81 674393. E-mail:
panzella@unina.it.
Funding
This work was supported by the program “Finanziamento per
l’Avvio di Ricerche Originali” 2011 (“Coniugati lipidici di
molecole di interesse biologico: progettazione, sintesi e
caratterizzazione biofisica di nuovi agenti per la terapia e la
diagnostica”) and by Grants AGL2010-17579 and AGL2010-
18269 and Project CSD2007-00063 from Programa Consol-
ider-Ingenio from the Spanish Ministry of Science and
Innovation (CICYT).
Notes
The authors declare no competing financial interest.
(14) Bernini, R.; Mincione, E.; Barontini, M.; Crisante, F.
Convenient synthesis of hydroxytyrosol and its lipophilic derivatives
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ACKNOWLEDGMENTS
■
We thank Centro Interdipartimentale Grandi Apparecchiature
(CIGA), University of Milan, for ICR-FTMS experiments.
ABBREVIATIONS USED
■
HTy, hydroxytyrosol; ROS, reactive oxygen species; RNS,
reactive nitrogen species; LA, lipoic acid; DHLA, dihydrolipoic
acid; TPTZ, 2,4,6-tris(2-pyridyl)-s-triazine; Na2EDTA, ethyl-
enediaminetetraacetic disodium salt; DPPH, 2,2′-diphenyl-1-
picrylhydrazyl; FBS, fetal bovine serum; t-BOOH, tert-
butylhydroperoxide; NADH, reduced nicotine adenine dinu-
cleotide; IBX, 2-iodoxybenzoic acid; FRAP, ferric reducing/
antioxidant power; CUPRAC, copper reducing antioxidant
capacity; LDH, lactate dehydrogenase
(16) Bernini, R.; Crisante, F.; Barontini, M.; Tofani, D.; Balducci, V.
Synthesis and structure/antioxidant activity relationship of novel
catecholic antioxidants structurally analogues to hydroxytyrosol and its
lipophilic esters. J. Agric. Food Chem. 2012, 60, 7408−7416.
́
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M. C.; Velotti, F. Synthesis of a novel ester of hydroxytyrosol and α-
lipoic acid exhibiting an antiproliferative effect on human colon cancer
HT-29 cells. Eur. J. Med. Chem. 2011, 46, 439−446.
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Org. Lett. 2010, 12, 2326−2329.
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