Chemoenzymatic synthesis and radical scavenging of sulfated hydroxytyrosol, tyrosol, and acetylated derivatives

Article abstract of DOI:10.1021/acs.jafc.9b01065

Potential metabolites of bioactive compounds are important for their biological activities and as authentic standards for metabolic studies. The phenolic compounds contained in olive oil are an important part of the human diet, and therefore their potenti

Full text of DOI:10.1021/acs.jafc.9b01065

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Bioactive Constituents, Metabolites, and Functions
Chemoenzymatic Synthesis and Radical Scavenging of
Sulfated Hydroxytyrosol, Tyrosol and Acetylated Derivatives
Paloma Begines, David Biedermann, Kate#ina Valentová, Lucie Petraskova,
Helena Pelantova, Inés Maya, Jose G. Fernandez-Bolaños, and Vladimir Kren
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b01065 • Publication Date (Web): 14 Jun 2019
Downloaded from http://pubs.acs.org on June 14, 2019
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Journal of Agricultural and Food Chemistry
Chemoenzymatic Synthesis and Radical Scavenging of Sulfated Hydroxytyrosol,
Tyrosol and Acetylated Derivatives
Paloma Begines†, David Biedermann‡, Kateřina Valentová‡, Lucie Petrásková‡, Helena
Pelantová‡, Inés Maya†, José G. Fernández-Bolaños†*, Vladimír Křen‡*
†Department of Organic Chemistry, Faculty of Chemistry, University of Seville, E-
41012, Spain
‡Institute of Microbiology, Czech Academy of Sciences, Vídeňská 1083, CZ 142 20
Prague, Czech Republic
* Corresponding author. (Tel.: +34-954550996. E-mail: bolanos@us.es)
* Corresponding author. (Tel.: +420-296442510. E-mail: kren@biomed.cas.cz)
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Abstract
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Potential metabolites of bioactive compounds are important for their biological
activities and as authentic standards for metabolic studies. The phenolic compounds
contained in olive oil are an important part of the human diet, and therefore their
potential metabolites are of utmost interest. We developed a convenient, scalable, one-
pot chemoenzymatic method using the arylsulfotransferase from Desulfitobacterium
hafniense for the sulfation of the natural olive oil phenols tyrosol, hydroxytyrosol and of
their monoacetylated derivatives. Respective monosulfated (tentative) metabolites were
fully structurally characterized using LC-MS, NMR and HRMS. In addition, Folin-
Ciocalteu reduction, 1,1-diphenyl-2-picrylhydrazyl radical scavenging and anti-
lipoperoxidant activity in rat liver microsomes damaged by tert-butylhydroperoxide
were measured and compared with the parent compounds. As expected, the sulfation
diminished the radical scavenging properties of the prepared compounds. These
compounds will serve as authentic standards of phase II metabolites.
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Keywords:
olive
phenols;
arylsulfotransferase;
hydroxytyrosol,
tyrosol;
chemoenzymatic; metabolites.
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Introduction
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Sulfation is one of the major pathways of the phase II of biotransformation and
detoxification of xenobiotics as well as eubiotics (e.g. steroid hormones). Sulfation
converts the compounds into more hydrophilic metabolites, facilitating their excretion.
In humans, sulfate conjugation is catalyzed by a superfamily (at least ten functional
genes)1 of membrane-associated and cytosolic sulfotransferases, which transfer a sulfate
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moiety (SO3 ) from the donor 3′-phosphoadenosine-5′-phosphosulfate (PAPS) onto a
wide variety of substrates. Sulfotransferases are present in tissues such as the brain,
kidney, liver, adrenal glands, gastrointestinal tissue and gut. Sulfotransferases are also
able to reverse the process of sulfation in cells (desulfation).2
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Recently, much attention has been focused on the phenolic components of olive fruit
and especially of olive oil for their beneficial effects on health. Extra virgin olive oil
(EVOO) is considered to be one of the main components of the Mediterranean diet; its
consumers have a reduced incidence of neurodegenerative diseases, coronary heart
disease, atherosclerosis and certain cancers.3 The phenolic compounds found in olive oil
with important bioactive properties include: simple phenols (phenolic acids and
phenolic alcohols such as tyrosol (1) and hydroxytyrosol (2), and their esters:
hydroxytyrosol-elenolic acid ester Hy-EA, tyrosol-elenolic acid ester Tyr-EA, and the
dialdehyde derivatives, oleocanthal and oleacein (Figure 1). Oleacein (2-(3,4-
dihydroxyphenyl)ethyl (3S,4E)-4-formyl-3-(2-oxoethyl)hex-4-enoate) and oleocanthal
(2-(4-hydroxyphenyl)ethyl (3S,4E)-4-formyl-3-(2-oxoethyl)hex-4-enoate) are the most
abundant dialdehydes in olive oil and have important biological properties.4-7
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Hydroxytyrosol has been reported to promote apoptosis in several tumor cell lines,8 it
inhibits their proliferation and, in addition, contributes to protecting humans in terms of
bone health, platelet function, oxidative damage, cellular aging and plasma lipoproteins
due to its anti-inflammatory, antimicrobial, anticancer, neuroprotective and antioxidant
activities.9 Hydroxytyrosol acetate was shown to be useful for cancer,10 systemic lupus
erythematous,11 and arthritis12 prevention. Tyrosol proved to be less active; however, it
was able to decrease LPS-stimulated cytokine production and increase mouse survival
in endotoxemia induced by LPS;13 tyrosol and its acetylated derivative inhibited the
synthesis of PAF (platelet-activating factor), a potent mediator of platelet aggregation
and inflammation.14
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The bioactivity of these phenolic compounds in vivo depends on their absorption and
metabolism. The study of the metabolic fate of olive oil polyphenols is an area of active
research.15-19 Due to low aqueous solubility of most polyphenols20, these compounds are
biotransformed in humans into more polar derivatives, typically sulfates and
glucuronides.21,22 Moreover, the process of conjugation reduces the amount of
polyphenols in the blood, increasing metabolite excretion, and also producing some
active metabolites;23 in this sense, sulfated polyphenols have been proven to be
biologically active.24 The sulfation process is considered to be reversible, involving
sulfotransferases, which catalyze the sulfation reaction and sulfatases, which catalyze
the hydrolysis of sulfate esters.25 Therefore, such conjugated metabolites are required as
reference compounds and standards for investigating their bioavailability in humans.
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The health benefits attributed to extra virgin olive oil (EVOO) such as antioxidant,
antimicrobial, anti-inflammatory, neuroprotective, cardioprotective and anti-cancer
properties can be mostly associated with its phenolic content.26-29 The bioactivity of the
phenolic compounds from EVOO has been widely studied. It is known than olive
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phenols undergo an extensive conjugation during their metabolism forming sulfated,
methylated and glucuronidated derivatives, and often before reaching the target tissues;
these metabolites are finally excreted in the urine. In previous literature, hydroxytyrosol
sulfate and hydroxytyrosol acetate sulfate have been detected as the main metabolites in
human plasma when consuming VOO.21 Moreover, these phase II metabolites has been
identified as the most suitable biomarkers for monitoring compliance with olive oil
intake.22 Therefore, sulfated metabolites are of great interest as standards to study their
biological properties.30,31
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Although there are number of chemical methods for the sulfation of phenols, they
generally suffer from the lack of regioselectivity and the products are often hard to
purify. The use of enzymes offers the way to improve the regioselectivity under mild
reaction conditions. The chemical methods for sulfation of small molecules was
reviewed recently.32
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The arylsulfate sulfotransferase33 (AST) from Desulfitobacterium hafniense catalyzes
the transfer of the sulfate group from various sulfate donors (typically p-nitrophenyl
sulfate) onto various acceptors with free OH groups. This commercially unavailable
enzyme has virtually no hydrolytic activity, i.e. it does not transfer the sulfate group to
water. This enzyme also exhibits some regioselectivity, as demonstrated e.g. on the
sulfation of quercetin and its derivatives34,35 or the flavonolignans from silymarin.36
This sulfotransferase using cheap donor p-nitrophenyl sulfate has a great advantage over
“classical sulfotransferases”, which employ very expensive and unstable PAPS.
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Sulfated derivatives of tyrosol and hydroxytyrosol were prepared previously
using chemical procedures, as regioisomeric mixtures of monosulfates in the case of
hydroxytyrosol.37, 38 As enzymatic procedures showed to be good methods of choice for
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the preparation of standards of sulfated metabolites of various xenobiotics, the aim of
the present study was to prepare pure isomers of sulfated natural olive oil phenolics
tyrosol (1), hydroxytyrosol (2) and their monoacetylated derivatives: tyrosol-2′-acetate
(3) and hydroxytyrosol-2′-acetate (4)39,40 using AST from D. hafniense. Although these
compounds are not the main phenols in olive oil, they are the metabolic precursors and
also the degradation products19 of the main phenolics in that oil, i.e., Tyr-EA, Hy-EA,
oleocanthal, and oleacein. The reducing, radical-scavenging and anti-lipoperoxidant
properties of the sulfated derivatives was compared with that of parent compounds.
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Experimental Section
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Materials. Tyrosol (1), hydroxytyrosol (2) and p-nitrophenyl sulfate potassium salt
were purchased from Sigma-Aldrich. Hydroxytyrosol-2′-acetate (4) was prepared by
refluxing 2 in ethyl acetate (EtOAc) in the presence of the strong acidic resin Amberlite
IR-120 H+ (The Dow Chemical Company, USA), i.e. by a chemoselective acid-
catalyzed acetylation reaction.41 Tyrosol-2′-acetate42 (3) was synthesized using the
above procedure.
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Methods. NMR spectra were recorded on a Bruker Avance III 400 MHz spectrometer
(Bruker BioSpin, Rheinstetten, Germany) in DMSO-d6 at 30 ºC, using the residual
1
solvent signal (δH 2.499 ppm, δC 39.46 ppm) as a reference. NMR experiments H
NMR, 13C NMR, gCOSY, gHSQC, and gHMBC were performed using the
manufacturer’s software.
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Mass spectra in negative-ion mode were measured in an Orbitrap Elite (Thermo Fisher)
equipped with an electrospray ion source (HESI), using a spray voltage of 3,500 V (+)
and a resolution of 60,000. The acquisition range was from 60 to 900. The samples were
dissolved in methanol/water 50% (v/v) with 0.1% formic acid.
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The enzymatic sulfation was monitored by thin-layer chromatography (TLC) [silica gel
60 F254 plates (Merck, DE); mobile phase EtOAc/MeOH/HCO2H, 4:1:0.2, v/v and
EtOAc/MeOH 9:2.5, v/v.
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All analytical HPLC analyses were performed in a Shimadzu Prominence LC analytical
system consisting of a Shimadzu LC-20AD binary HPLC pump, Shimadzu SIL-
20ACHT cooling auto sampler, Shimadzu CTO-10AS column oven, Shimadzu CBM-
20A system controller and Shimadzu SPD-20MA diode array detector (Shimadzu, JP);
there was no coupling to a MS detector. The sample (0.5 mg) was dissolved in the
mobile phase A (50 µL) and analyzed in a Kinetex PFP (150 × 4.6 mm, 5 µm) column
(Phenomenex, USA) coupled with a PFP security guard cartridge kit (4 × 3 mm).
Binary gradient elution was used: mobile phase A = 0.1% trifluoroacetic acid in water;
mobile phase B = 100% methanol; gradient: 0 min 10% B, 20 min 40% B, 21 min 10%
B. The flow rate was 0.6 mL/min at 45 °C and the injection volume was 1 µL; the peaks
were detected at 275 nm (compounds 7, 8, 9, 10) or at 254 nm (compounds 5, 6).
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Preparative HPLC separations were performed using an ASAHIPAK GS-310 20F
column (Shodex, Munich, Germany), with the mobile phase specified for each
experiment, flow rate 5 mL/min and detection at 254 and 369 nm. The
preparative HPLC (Shimadzu, Kyoto, Japan) system consisted of an LC-8A
high-pressure pump with an SPD-20A dual-wavelength detector (with semi-
preparative cell), and fraction collector FRC-10A. The system was connected to
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a PC using a CBM-20A command module and controlled via the LabSolution
1.24 SPI software suite supplied with the machine.
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Preparation of the Enzyme. Frozen cells transformed with the plasmid containing the
AST gene33,34 (100 µL, the plasmid was kindly provided by Dr. van der Horst,
University of Amsterdam, The Netherlands), were incubated in LB (Luria-Bertani)
medium (100 mL) with kanamycin (KNM, 25 µM, 100 µL) at 37 ºC and 200 rpm, to an
optical density (OD) of 600. Isopropyl β-D-1-thiogalactopyranoside (IPTG, 0.4 mM,
160 µL) was then added. The mixture was incubated at 25 ºC overnight, at 200 rpm, and
was centrifuged (5000 g, 20 min, 8 ºC). The cells were resuspended in Tris-Gly buffer
(100 mM, pH 8.9, 2 mL), then they were sonicated for 4 × 4 min in an ice bath. The cell
debris were then centrifuged (5000 g, 20 min, 8 ºC), thus obtaining the enzyme as a
crude cell lysate.34
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Preparation of 4-Hydroxyphenethyl Acetate (Tyrosol-2′-Acetate) (3). Tyrosol (1)
(1.0 g, 7.24 mmol) in EtOAc (25 mL) containing Amberlite IR-120 H+ (2 g) was
refluxed and stirred thoroughly for 11 h under argon atmosphere (Ar from cylinder),
followed by filtration with a Buchner funnel in vacuum, the solvent was eliminated in a
rotary evaporator under reduced pressure, and then the residue was purified by column
chromatography (cyclohexane/EtOAc 5:1 to cyclohexane/EtOAc 2:1) yielding 3 as a
1
white solid (1.14 g, 87 %). RF 0.65 (cyclohexane/EtOAc 1:1). H NMR (399.87 MHz,
DMSO-d6, 30 ºC ) δ: 7.02 (m, 2H, o-H), 6.68 (m, 2H, m-H), 4.12 (t, 2H, J2′,1′ = 7.06
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Hz, OCH2), 2.75 (t, 2H, J1 ′ ,2 ′ = 7.08 Hz, ArCH2), 1.97 (s, 3H, CH3); C NMR
(100.55 MHz, DMSO-d6, 30 ºC) δ: 170.28 (CO), 155.79 (p-C), 129.74 (o-C), 127.86 (i-
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C), 115.09 (m-C), 64.67 (C-2′), 33.54 (C-1′), 20.62 (CH3); HRESIMS m/z calcd for
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C10H12O3Na [M+H]+ 203.0679, found 203.0676.
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General Method for the Preparation of Sulfated Phenolic Derivatives and their
Purification. A solution of potassium p-nitrophenyl sulfate (p-NPS, 1.2 eq) in Tris-Gly
buffer (100 mM, pH 8.9, 15 mL) and the enzyme (AST, 2.5 mL) were added to a
solution of the phenolic compound (100 or 150 mg, as indicated in each case) in acetone
(2 mL). The reaction mixture was purged with argon, incubated in the dark under a
positive pressure of argon (balloon) at 30 ºC, with stirring on an orbital shaker (Labnet)
at 220 rpm for 5 h. The organic solvent (acetone) was evaporated on a rotary evaporator
at room temperature under reduced pressure, and pH was adjusted to 7.5  7.7 (formic
acid). p-Nitrophenol and residual starting materials were extracted with ethyl acetate (3
× 20 mL) and the aqueous phase (15 mL) with the sulfated compounds was evaporated.
The residue was dissolved in the mobile phase, filtered on a 0.45-mm PTFE, injected
into preparative HPLC, and eluted with an isocratic flow (MeOH/H2O 1:4; 3:2; 0:1;
1:19, for the different experiments). The combined fractions containing the products
were evaporated and lyophilized from water.
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Antioxidant Activity Evaluation
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Folin-Ciocalteu Reduction Assay. Folin-Ciocalteu reduction (FCR) capacity was
measured according to a previously reported protocol,43,44 where 5 µL of the native
compounds 14 and the sulfated samples 510 (1 mM) or standards (gallic acid, 04
mM) in phosphate-buffered saline (PBS, pH 7.4) were mixed with 100 µL of Folin-
Ciocalteu reagent diluted tenfold with distilled water. It was incubated for 5 min, then
100 µL Na2CO3 (75 g/L) was added and the mixture was further incubated for 90 min at
room temperature. The absorbance was measured at 700 nm using a Tecan Sunrise plate
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reader (Tecan Group Ltd., Männedorf, Switzerland) and the reducing capacity was
expressed as gallic acid equivalents (GAE).
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DPPH Assay. The antiradical activity of the compounds was tested as the capacity to
reduce 1,1-diphenyl-2-picrylhydrazyl (DPPH, Sigma-Aldrich), thus decreasing the
violet color of DPPH as previously described44,45 with minor modifications. A 15 µL
solution of the tested substance (final concentration 0  5 mM in MeOH) was mixed
with 285 µL of a freshly prepared methanolic DPPH solution (final concentration 20
µM) in a microtiter plate well (total volume 300 µL). After 30 min at 25 °C, the
absorbance at 517 nm was measured. The concentration of the antioxidant required for
reducing the DPPH concentration to 50% (IC50) of its initial value was calculated.
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Inhibition of Microsomal Lipid Peroxidation. This assay was performed according to
the reported method.44 Pooled microsomes from male rat liver (M9066, Sigma-Aldrich)
were washed 5× using centrifugation (13,500 rpm, 5 min, 4 ºC) and PBS to remove
sucrose, and diluted to 0.625 mg protein/mL with PBS before use. The protein
concentration was determined using the Bradford method.46 A 0.4 mL solution of the
diluted microsomal suspension was then mixed with the compounds 110 (final
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concentration 5 µM  2 mM in 50 µL PBS), tert-butyl hydroperoxide (t-BH, a pro-
oxidant, 50 µL in PBS; final concentration 1 mM) was then added and the mixture was
incubated at 37 ºC for 60 min. The products of lipid peroxidation were determined as
thiobarbituric acid reactive substances (TBARS): 0.7 mL of trichloroacetic acid (26
mM) with thiobarbituric acid (918 mM) were added, the mixture was heated (90 ºC, 15
min), cooled, centrifuged (13,500 rpm, 10 min, 4 ºC) and the absorbance of the
supernatant at 535 nm was measured. The activity was calculated as the concentration
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of the analyzed compounds that inhibited the color reaction with the thiobarbiturate
(without the analyzed compounds) by 50% (IC50).
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Determination of log P Values. The hydrophobicity of the compounds (miLogP) was
calculated
using
the
Molinspiration
property
engine
v2016.10
(http://www.molinspiration.com, Molinspiration Cheminformatics, Slovensky Grob,
Slovakia, accessed on 26th September 2018).47
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Statistical Analysis. Data were expressed as means ± standard deviation (SD). Assays
were done in triplicate. The differences in mean values were analyzed by Student’s t-
tests. A p value of less than 0.05 was considered to be statistically significant.
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Results and Discussion
Preparation and Purification of the Synthesized Compounds
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Various methods based on chemical procedures have been used to synthesize sulfate
phenols, most of them consist of using sulfur trioxide-pyridine complex as the sulfating
reagent.38,48 Sulfation of hydroquinone derivatives with SO3·Py at 60 ºC gave the
disulfated compounds, whereas at room temperature led to monosulfated compounds;
no regioselectivity was described.48 Paiva Martins et al. described the sulfation of
hydroxytyrosol acetate with SO3·Py (2 equiv., -20 ºC) to afford a mixture of
monosulfated regioisomers on the phenolic hydroxyls with preference for 4-hydroxyl
group; whereas with 8 equiv. of the sulfating agent the disulfated product was
obtained.38 Furthermore, sulfation of unprotected hydroxytyrosol led to occurred
preferentially in the aliphatic hydroxyl group. The microwave-assisted O-sulfation
reaction was performed using SO3·NMe3 to prepare sulfate derivatives of olive
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polyphenols,49 however this method involves various protection-deprotection steps.
Moreover, these chemical syntheses are not chemoselective, generating a mixture of
monosulfates and disulfates, which is hard to separate.
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We employed a single-step, efficient and inexpensive chemoenzymatic methodology to
the transfer of a sulfate group from p-nitrophenyl sulfate (p-NPS) to the natural
unprotected and monoacetylated phenolic compounds.
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First, tyrosol-2′-acetate (3) and hydroxytyrosol-2′-acetate (4) were synthesized by
refluxing tyrosol 1 and hydroxytyrosol 2, respectively, in EtOAc, in strong acid resin.41
The resin can be easily removed from the reaction medium by filtration and reused, thus
constituting a green, economical, and effective method for the chemoselective
monoacetylation of phenolic alcohols.
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In the sulfation of tyrosol (1), a preference for the aromatic hydroxyl was observed, only
tyrosol-4-O-sulfate (5) was obtained as a product in a good yield (81%) and a
chemoselective fashion (Figure 2); in the same way, tyrosol-2′-acetate-4-O-sulfate (6)
was obtained in a 59 % yield. Both products were purified by preparative HPLC and
characterized by spectral techniques (see analytical HPLC chromatograms of 5
(supporting S7), and of 6 (supporting S12), after purification).
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With hydroxytyrosol (2), the formation of two monosulfated compounds (3-O-sulfate/4-
O-sulfate ca 2:1, calculated from 1H NMR integration) was observed in a
chemoselective manner, as no sulfation was detected in the side chain (Figure 3). The
enzymatic sulfation of hydroxytyrosol-2′-acetate (4) also yielded both regioisomers (3-
O-sulfate/4-O-sulfate ca 1:6), although the preferred reaction site changed from C3-OH
to C4-OH, with acetylation in the side chain. Again, the selectivity of the enzyme (AST)
for the aromatic hydroxyls was confirmed. Therefore, the regioselective outcome of the
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enzymatic sulfation of the catechol fragment of 2 and 4 depends on the features of the
side chain. No disulfated derivatives were formed, presumably because the charge and
the size of the sulfate group preclude subsequent sulfation.
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From the 1H NMR spectra of the reaction mixture we could deduce that hydroxytyrosol-
3-O-sulfate (7) and hydroxytyrosol-4-O-sulfate (8) were formed in a 1.3:1 ratio;
whereas, hydroxytyrosol-2′-acetate-3-O-sulfate (9) and hydroxytyrosol-2′-acetate-4-O-
sulfate (10) were in a 1:1.4 ratio. Due to their physicochemical properties, the
regioisomers 7 and 8 derived from hydroxytyrosol, and 9 and 10, derived from
hydroxytyrosol acetate were difficult to separate by preparative HPLC (ASAHIPAK
GS-310 20F column). After chromatography, partial separation was achived (7/8 in a
2:1 ratio, and 9/10 in a 1:6 ratio). At analytical scale (Kinetex PFP column), the best
conditions to separate both pairs of regioisomers involved the use of a gradient of 0.1%
trifluoroacetic acid in water (phase A) and methanol (phase B) using an analytical
Kinetex PFP HPLC column (Figure 4).
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Only monosulfate and glucuronide conjugates of olive phenols were found to be the
main metabolites in human plasma and excreted in urine after olive oil intake.50,51 In
accordance with this, no disulfated or trisulfated compounds were detected in any case,
even using up to four equivalents of p-NPS as the sulfate donor using the
chemoenzymatic method in this work. This is in contrast to the situation with flavonols
and flavonolignans.35,36 This might be due to the high polarity (low log P) of the
monosulfates of these small phenolics (see Table 1), which are therefore probably not
accepted as substrates for AST.
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4-(2-Hydroxyethyl)phenyl sulfate (tyrosol-4-O-sulfate; 5): Tyrosol (1, 150 mg, 1.08
mmol) was sulfated according to the general procedure and purified by preparative
HPLC in MeOH/H2O 1:4 to obtain 5 as a white solid (192 mg, (69%). RF 0.45
1
(EtOAc/MeOH/HCO2H 4:1:0.2). H NMR (399.87 MHz, DMSO-d6, 30 ºC) δ: 7.090
(2H, m, o-H), 7.050 (2H, m, m-H), 4.579 (1H, br t, J2 ′,2 ′-OH = 4.9 Hz, 2′-OH), 3.562
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(2H, dt, J2′,2′-OH = 4.9 Hz, J2′,1′ = 7.2 Hz, H-2′), 2.660 (2H, t, J1′,2′ = 7.2 Hz, H-1′);
13C NMR (100.55 MHz, DMSO-d6, 30 ºC) δ: 151.64 (p-C), 133.90 (i-C), 128.91 (o-C),
120.29 (m-C), 62.28 (C-2′), 38.30 (C-1′); HRESIMS m/z calcd for C8H9O5S [M-H]
217.0165, found 217.0168.
284
285
286
287
288
4-(2-Acetoxyethyl)phenyl sulfate (tyrosol-2′-acetate-4-O-sulfate; 6): Tyrosol-2′-
acetate (3, 100 mg, 0.55 mmol) was sulfated according to the general procedure and
purified by preparative HPLC in MeOH/H2O 3:2 to obtain 6 as a white solid (85 mg,
1
46%). RF 0.65 (EtOAc/MeOH 9:2.5). H NMR (399.87 MHz, DMSO-d6, 30 ºC) δ:
7.131 (2H, m, o-H), 7.082 (2H, m, m-H), 4.174 (2H, t, J1 ′,2 ′ = 6.9 Hz, H-2′), 2.822
289
(2H, t, J1
=
6.9 Hz, H-1′), 1.973 (3H, s, CH3);
′
,2
′
290
291
292
293
294
tris(hydroxymethyl)methylammonium: 7.25 (br s, NH3), 5.05 (s, OH), 3.46 (s, CH2);13C
NMR (100.55 MHz, DMSO-d6, 30 ºC) δ: 170.19 (CO), 152.03 (p-C), 132.28 (i-C),
128.92 (o-C), 120.36 (m-C), 64.40 (C-2′), 33.55 (C-1′), 20.62 (CH3);
tris(hydroxymethyl)methylammonium: 60.4 (C-N), 59.6 (C-O); HRESIMS m/z calcd
for C10H11O6S [M-H] 259.0271, found 259.0270.
295
296
297
298
2-Hydroxy-5-(2-hydroxyethyl)phenyl sulfate (hydroxytyrosol-3-O-sulfate; 7) and 2-
hydroxy-4-(2-hydroxyethyl)phenyl
sulfate (hydroxytyrosol-4-O-sulfate;
8):
Hydroxytyrosol (2, 100 mg, 0.64 mmol) was sulfated according to the general
procedure and purified by preparative HPLC in H2O to obtain a mixture of 7 and 8 as a
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300
301
302
303
304
305
colorless viscous substance (ratio 3-O-sulfate/4-O-sulfate 2:1, total yield: 86 mg, 49%).
RF 0.48 (EtOAc/MeOH/HCO2H 4:1:0.2). The position of sulfates was unambiguously
assigned in NMR using typical changes in carbon chemical shifts compared to the
parent compound.34 1H NMR (399.87 MHz, DMSO-d6, 30 ºC) δ: 3-O-sulfate (7) 8.669
(1H, br s, 4-OH), 6.933 (1H, d, J2,6 = 2.1 Hz, H-2), 6.793 (1H, dd, J6,2 = 2.1 Hz, J6,5
=
8.0 Hz, H-6), 6.710 (1H, d, J5,6 = 8.0 Hz, H-5), 4.553 (1H, br s, 2′-OH), 3.530 (2H, t,
J2′,1′ = 7.2 Hz, H-2′), 2.592 (2H, t, J1′,2′ = 7.2 Hz, H-1′); 4-O-sulfate (8) 8.669 (1H,
br s, 3-OH), 6.961 (1H, d, J5,6 = 8.0 Hz, H-5), 6.670 (1H, d, J2,6 = 2.1 Hz, H-2), 6.577
306
307
(1H, J6,2 = 2.1 Hz, J6,5 = 8.0 Hz, H-6), 4.553 (1H, br s, 2′-OH), 3.549 (2H, t, J2 ′,1 ′
=
308
7.2 Hz, H-2′), 2.601 (2H, t, J1′,2′ = 7.2 Hz, H-1′); 13C NMR (100.55 MHz, DMSO-d6,
309
310
311
312
30 ºC) δ: 3-O-sulfate (7) 147.25 (C-4), 140.41 (C-3), 130.40 (C-1), 125.17 (C-6),
123.45 (C-2), 116.83 (C-5), 62.26 (C-2′), 38.07 (C-1′); 4-O-sulfate (8) 148.78 (C-3),
138.95 (C-4), 136.34 (C-1), 122.84 (C-5), 119.74 (C-6), 117.65 (C-2), 62.14 (C-2′),
38.46 (C-1′); HRESIMS m/z calcd for C8H9O6S [M-H] 233.0114, found 233.0114.
313
314
315
316
317
318
319
320
321
5-(2-Acetoxyethyl)-2-hydroxyphenyl
sulfate
(hydroxytyrosol-2′-acetate-3-O-
sulfate; 9) and 4-(2-acetoxyethyl)-2-hydroxyphenyl sulfate (hydroxytyrosol-2′-
acetate-4-O-sulfate; 10): Hydroxytyrosol-2’-acetate (4, 150 mg, 0.76 mmol) was
sulfated according to the general procedure and purified by preparative HPLC in
MeOH/H2O 1:19 to obtain a mixture of 9 and 10 as a colorless viscous substance (ratio
3-O-sulfate/4-O-sulfate 1:6; 96 mg, total yield 34%) RF 0.63 (EtOAc/MeOH/HCO2H
1
4:1:0.2). H NMR (399.87 MHz, DMSO-d6, 30 ºC) δ: 4-O-sulfate (10) 7.450 (1H, br s,
3-OH), 7.009 (1H, d, J5,6 = 8.0 Hz, H-5), 6.708 (1H, d, J2,6 = 2.1 Hz, H-2), 6.614 (1H,
dd, J6,2 = 2.1 Hz, J6,5 = 8.0 Hz, H-6), 4.161 (2H, t, J2 ′,1 ′ = 6.9 Hz, H-2′), 2.763 (2H,
322
J1 ′,2 ′ = 6.9 Hz, H-1′), 1.986 (3H, s, CH3); 3-O-sulfate (9): 8.630 (1H, br s, 4-OH),
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324
6.989 (1H, d, J2,6 = 2.1 Hz, H-2), 6.824 (1H, dd, J6,2 = 2.1 Hz, J6,5 = 8.0 Hz, H-6), 6.739
(1H, d, J5,6 = 8.0 Hz, H-5), 4.126 (2H, t, J2′,1′ = 6.9 Hz, H-2′), 2.751 (2H, J1′,2′ = 6.9
Hz, H-1′), 1.982 (3H, s, CH3); tris(hydroxymethyl)methylammonium: 7.45 (br s, NH3),
325
326
327
328
329
330
331
332
13
5.08 (s, OH), 3.46 (s, CH2); C NMR (100.55 MHz, DMSO-d6, 30 ºC) δ: 4-O-sulfate
(10) 170.23 (CO), 148.95 (C-3), 139.39 (C-4), 134.73 (C-1), 122.98 (C-5), 119.67 (C-
6), 117.65 (C-2), 64.32 (C-2′), 33.72 (C-1′), 20.67 (CH3); 3-O-sulfate (9) 170.24 (CO),
147.64 (C-4), 140.60 (C-3), 128.81 (C-1), 125.09 (C-6), 123.47 (C-2), 117.05 (C-5),
64.43 (C-2′), 33.37 (C-1′), 20.63 (CH3); tris(hydroxymethyl)methylammonium: 60.4
(C-N), 59.6 (C-O); signals for HRESIMS m/z calcd for C10H11O7S [M-H] 275.0220,
found 275.0218.
333
Antioxidant activity.
334
335
336
In order to better characterize the obtained compounds, their ability to reduce Folin-
Ciocalteu reagent, to scavenge DPPH radicals and to inhibit lipid peroxidation (ILP)
were determined and evaluated with respect of the calculated miLogP values (Table 1).
337
338
339
340
FCR assay is known as a total phenol determination, however it is based on reducing
capacity measurement, and thus it indicates the overall antioxidant status of the tested
compound.52 As expected, replacing hydroxyl groups with sulfate groups in the
aromatic ring decreased the reducing capacity (Table 1).
341
342
343
344
345
346
1,1-Diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging assay is one of the most
widely used methods to compare antioxidant activity of natural and (semi)synthetic
biologically active compounds. Although this assay has no direct physiological
relevance, it allows quick comparison of free radical scavenging potential as this
activity has been described for many compounds in the literature.53 In our experimental
setup, only hydroxytyrosol (2) and hydroxytyrosol-2′-acetate (4) displayed measurable
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348
349
350
activity with IC50 values of 11 and 9 µM, respectively. This is in contrast with
previously published activity of these two compounds,54 probably due to slightly
different experimental conditions. Tyrosol (1), tyrosol-2′-acetate (3) and all sulfate
conjugates 510 exhibited no detectable activity in this assay (IC50 ˃ 225 µM, Table 1).
351
352
353
354
355
356
357
358
359
360
361
362
363
364
365
366
367
368
369
370
Using a more biologically relevant system, we determined the ability of all compounds
to inhibit the lipid peroxidation of rat liver microsomes induced by the pro-oxidant tert-
butyl hydroperoxide (t-BH) in the ILP assay. The results are expressed as the
concentration of the tested compound needed to inhibit lipid peroxidation by 50 %
(IC50). The results for hydroxytyrosol (2) and hydroxytyrosol-2′-acetate (4) correlated
with their DPPH scavenging activity with IC50 values of 42 and 7 µM, respectively.
This is in good agreement with previously reported effect of both compounds on
microsomes from vitamin E deficient rats.55 Among the sulfated compounds, the best
inhibitors of the lipid peroxidation were the acetylated derivatives with more aromatic
hydroxyls; in this way, hydroxytyrosol-2′-acetate monosulfates (9, 10) were the most
active compounds (0.43 ± 0.04 mM). In addition, the hydroxytyrosol monosulfates (7,
8) (1.3 ± 0.3 mM) exhibited better activity than tyrosol (1) (2 ± 1 mM). Lipid
peroxidation can be considered as a process under which free radicals transform lipids
containing C=C bonds, especially polyunsaturated fatty acids.56 It has been shown that
the radical scavenging activity of lipophilic phenols is not linearly correlated with
hydrophobicity, and efforts to correlate lipophilicity (calculated log P) with antioxidant
capacity failed due to the influence of the antioxidant location in biphasic environments
on their properties.57 In agreement with this, no good correlation between ILP and
lipophilicity/hydrophilicity (miLogP) of the tested compounds was found in the present
work. However for hydroxytyrosol (2) and its derivatives (4, 9/10, 7/8), the more
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372
positive the values of miLogP (1.22, 0.52, -2.27, and -2.98), the stronger the lipid
peroxidation inhibition (IC50 7.0, 42.1, 433, and 1300 µM, respectively).
373
374
375
376
In general, the sulfated derivatives have a lower antioxidant activity than their
respective parent phenols as expected, as sulfation involves reduction in the number of
phenolic hydroxyl groups. The results of ILP and DPPH highlight the structural
importance of the catechol moiety in phenols for potent antioxidant activity.
377
378
379
380
381
382
383
384
385
386
In conclusion, chemoenzymatic sulfation using AST from D. hafniense is a relatively
rapid one-step method that is efficient and widely applicable to a number of catechol
scaffolds. We demonstrate that AST is chemoselective towards aromatic hydroxyls, so
it is not necessary to protect aliphatic hydroxyls allowing thus preparation of respective
metabolites in a single step. When comparing aromatic hydroxyls, the enzyme slightly
prefers the 3-position in hydroxytyrosol, and the 4-position in hydroxytyrosol-2′-
acetate. The sulfation decreased the anti-lipoperoxidant, radical scavenging and
reducing properties of the tested phenolics and increased their hydrophilicity. The
monosulfate metabolites synthesized here will be used as reference compounds and
standards to determine their bioavailability in humans and thus clarify their metabolism.
387
Acknowledgments
388
389
390
391
392
M.A. van der Horst, University of Amsterdam, The Netherlands, is gratefully
acknowledged for the plasmid containing the AST gene from D. hafniense. We wish to
thank to Prof. J. Cvačka from the Institute of Organic Chemistry and Biochemistry,
CAS, Prague, CZ for recording of the HRMS spectra and to Ben Watson-Jones, MEng
for providing linguistic assistance.
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394
Supporting Information
1
395
396
Supporting information contains: H NMR, 13C NMR, HMBC, MS-ESI and HPLC
chromatograms of the compounds.
397
398
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Funding
Financial support from Czech Science Foundation projects 18-00121S and 19-00043S,
MINECO (Spain)/FEDER project CTQ2016-75960-P and FQM134 (Junta Andalucía)
are gratefully acknowledged. P. Begines also wishes to thank the University of Seville
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Page 28 of 34
for the award of a predoctoral grant, and for financing a short stay at Czech Academy of
Sciences.
Figure captions
Figure 1. Structures of phenolic compounds in olive oil
Figure 2. Sulfation of tyrosol and tyrosol-2′-acetate.
Figure 3. Sulfation of hydroxytyrosol and hydroxytyrosol-2′-acetate.
Figure 4. HPLC chromatograms of (A) a mixture of hydroxytyrosol sulfate
regioisomers 7 and 8, and (B) a mixture of hydroxytyrosol-2′-acetate sulfate
regioisomers 9 and 10. Small signals to the right are not identified.
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Journal of Agricultural and Food Chemistry
Tables
Table 1: Lipoperoxidation Inhibition, Reducing Capacity and Lipophilicity of Olive Oil
Phenolic Compounds and their Sulfated Derivativesa
Compound
FCR (GAE)b
DPPH
ILP
miLogPe
(IC50 µM)bc (IC50 µM)b
Tyrosol (1)
0.82 ± 0.02
0.87 ± 0.01
0.38 ± 0.02
0.86 ± 0.03
0.04 ± 0.01**
0.04 ± 0.02**
0.42 ± 0.03**
˃ 225
11 ± 1
˃ 225
9 ± 1
˃ 2000
42 ± 26
˃ 3000
1.00
0.52
Hydroxytyrosol (2)
Tyrosol-2′-acetate (3)
1.71
Hydroxytyrosol-2′-acetate (4)
Tyrosol-4-O-sulfate (5)
Tyrosol-2′-acetate-4-O-sulfate (6)
Hydroxytyrosol-sulfate (7, 8)
7 ± 5
1.22
˃ 225
˃ 225
˃ 225
˃ 225
˃ 3000
-2.46
˃ 3000
-1.75
1300 ± 300*
433 ± 41**
-2.98, -2.98
-2.27, -2.27
Hydroxytyrosol-2′-acetate-sulfate (9, 10) 0.35 ± 0.03**
a
Values are given as means ± standard deviation calculated from three independent experiments. b Folin-
Ciocalteu reagent reduction (gallic acid equivalents), c 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical
d
scavenging,
Inhibition of lipoperoxidation of rat liver microsomal membranes induced by tert-
e
butylhydroperoxide, hydrophobicity of compounds. **p ˂0.001 statistically significant difference from
value obtained with corresponding non-sulfated phenol. *p ˂0.01 statistically significant difference from
value obtained with corresponding non-sulfated phenol.
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