A Convenient Synthesis of Hydroxytyrosol Monosulfate Metabolites

Article abstract of DOI:10.1021/acs.jafc.5b04307

The growing interest in the bioactivity of natural polyphenols and of their metabolites requires metabolites to be used in bioassays and as standards in research protocols. We report here on the synthesis of several hydroxytyrosol metabolite monosulfates achieved using a simplified protocol with improved yields. A synthetic solution based on avoidance of high temperature conditions during the synthesis and of low pressure conditions during purification has been established. Monosulfates of several phenolic compounds, namely, hydroxytyrosol, hydroxytyrosol acetate, homovanillyl alcohol, homovanillyl alcohol acetate, homovanillic acid, ferulic acid, and 3,4-dihydroxyphenylethanoic acid, were efficiently synthesized in 1-2 steps in good yield and isolated using simple procedures. The proposed protocol was shown to be relatively rapid, efficient, cheap, and widely applicable to a number of catechol scaffolds.

Full text of DOI:10.1021/acs.jafc.5b04307

Article
pubs.acs.org/JAFC
A Convenient Synthesis of Hydroxytyrosol Monosulfate Metabolites
Vania Patrícia Martins Gomes, Carmen Torres, Jose Enrique Rodríguez-Borges,
̂
́
and Fatima Paiva-Martins*
́
REQUIMTE, Faculdade de Cien
̂
cias, Universidade do Porto, Rua do Campo Alegre, 687, 4169-007 Porto, Portugal
S
* Supporting Information
ABSTRACT: The growing interest in the bioactivity of natural polyphenols and of their metabolites requires metabolites to be
used in bioassays and as standards in research protocols. We report here on the synthesis of several hydroxytyrosol metabolite
monosulfates achieved using a simplified protocol with improved yields. A synthetic solution based on avoidance of high
temperature conditions during the synthesis and of low pressure conditions during purification has been established.
Monosulfates of several phenolic compounds, namely, hydroxytyrosol, hydroxytyrosol acetate, homovanillyl alcohol,
homovanillyl alcohol acetate, homovanillic acid, ferulic acid, and 3,4-dihydroxyphenylethanoic acid, were efficiently synthesized
in 1−2 steps in good yield and isolated using simple procedures. The proposed protocol was shown to be relatively rapid,
efficient, cheap, and widely applicable to a number of catechol scaffolds.
KEYWORDS: polyphenols, olive oil, sulfates, hydroxytyrosol, homovanillyl alcohol, hydroxytyrosol acetate, metabolites
alcohol and its glucuronide and sulfate conjugates, it can be
found in human urine and plasma after olive oil consumption
(Figure 1).9,10,12−15 Until recently, there were very few studies
on the bioavailability for secoiridoids, except for oleuropein.
However, there is now considerable evidence showing that OL
aglycons are bioavailable. After VOO consumption, Hy
concentration increases more than expected if only the Hy
present in the oil was absorbed, and it is proportional to the
total phenol content in the oil.11 An initial study of 3,4-
DHPEA-EDA and 3,4-DHPEA-EA bioavailability showed that
both secoiridoid compounds are efficiently absorbed by Caco-2
cells and by the rat intestine and that they are hydrolyzed with
the production of hydroxytyrosol and its metabolites (Figure
1).16 In a recent study with the aim of identifying biomarkers
for olive oil consumption after a 3-week dietary intervention
with phenol-enriched olive oils as part of a randomized, double-
blind, crossover, and controlled nutrition intervention trial, two
olive oils were assessed, one virgin olive oil (VOO) and a
second one phenol-enriched with its own phenolic compounds
(FVOO).10 The metabolites hydroxytyrosol sulfate (Figure 1,
compounds 3/4) and hydroxytyrosol acetate sulfate (Figure 1,
compounds 5/6) were shown to be the metabolites most
suitable for monitoring FVOO intake compliance, which in
addition to a significant post-treatment increase in both urine
and plasma demonstrated a significant change compared to
VOO in urine.10
INTRODUCTION
■
Virgin olive oil (VOO) is the major source of fat in the
Mediterranean diet, and a number of studies indicate that
phenolic compounds, a minor constituent of VOO, are a
contributory factor to the healthy Mediterranean diet.1−3 Over
the past decade, many human intervention studies have been
carried out in order to determine the exact bioefficacy of several
different subclasses of olive oil polyphenols as protection
against chronic degenerative diseases4 and long-term clinical
intervention trials have provided evidence that the phenolic
compounds of virgin olive oil contribute to protecting humans
against lipid oxidation in a dose-dependent way.5,6
The most common phenols found in olives include phenolic
alcohols, such as hydroxytyrosol (compound 1, 3,4-dihydrox-
yphenylethanol, 3,4-DHPEA or Hy) and tyrosol (4-hydrox-
yphenylethanol, 4-HPEA or Ty), secoiridoids, flavonoids, and
lignans. Secoiridoids are the major class of phenols found in
olives, with glycoside oleuropein (OL), one of the most
important secoiridoids, making up to 14% of the dry weight of
olives. Secoiridoid glycosides such as oleuropein are hydrolyzed
during VOO extraction by β-glucosidases. Therefore, the major
phenolic compounds found in VOO are the dialdehydic form of
elenolic acid linked to Hy (3,4-DHPEA-EDA), the oleuropein
aglycon (3,4-DHPEA-EA), and the dialdehydic form of elenolic
acid linked to Ty (4-HPEA-EDA).7 These compounds
represent as much as 80% of the total phenolic fraction.8
Although there have been a large number of studies
investigating the in vitro antioxidant properties of VOO
phenolics, as well as their protective effects against cell injury,
the biological properties of these phenols in vivo depends on
the extent to which they are absorbed and metabolized. Olive
oil polyphenols undergo extensive metabolism in the intestines
and liver and are thus chiefly found in biological fluids as phase
II metabolites.9,10 Bioavailability studies have shown that Hy is
efficiently absorbed in the small intestine11 and, together with
its acetate, glucuronide and sulfate conjugates, homovanillyl
Metabolic sulfation of compounds, or O-sulfonation, takes
place in the cytosol when one of the sulfotransferases from the
3′-phosphoadenosine-5′-phosphosulfate (PAPS) donates the
activated sulfonate group (SO3) to an acceptor alcohol, phenol,
or amine group.17This gives the molecule an anionic character,
Received: September 3, 2015
Revised: October 12, 2015
Accepted: October 13, 2015
© XXXX American Chemical Society
A
DOI: 10.1021/acs.jafc.5b04307
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Journal of Agricultural and Food Chemistry
Article
Figure 1. Olive oil polyphenol metabolites already identified in bioavailability studies. Arrows may represent a direct or an indirect metabolic
pathway.
which improves its excretion properties to avoid potential
adverse effects. However, sulfation is also an important means
of regulating the bioactivity of certain molecules. For example,
there is growing evidence that intracellular sulfation and
desulfation play an important role in regulating the availability
of active steroid hormones near the target sites.18 In fact, the
role of phase II conjugates as temporary deposits, from which
hydroxytyrosol and its phase I metabolites can be regenerated
by sulfatases and glucuronidases, has been attracting much
attention recently.19,20 Therefore, the potential health benefits
of Hy and its derivatives may be attributable to both parent
compounds together with their phase I and phase II
metabolites, although it is still unknown which components
play a major role in this protection. Investigations into these
topics require pure metabolites to be used in bioactivity assays
and as standards, but these metabolites are not widely available
commercially and are expensive. The aim of this paper is
therefore to report on the synthesis of the most important olive
oil polyphenol metabolites in animals and humans, i.e.,
hydroxytyrosol and its phase II monosulfate metabolites
(Figure 1, Table 1).
MATERIALS AND METHODS
■
All chemicals were obtained from chemical suppliers and used without
further purification, unless otherwise noted. All reactions were
monitored by thin layer chromatography (TLC) on precoated silica
gel 60 plates F254, and detected by iodine. Products were purified by
flash chromatography with silica gel 60 (200−400 mesh). NMR
spectra were recorded on 400 MHz NMR equipment at room
temperature for solutions in CD3OD. Chemical shifts are referred to
the solvent signal and are expressed in ppm. 2D NMR experiments
(COSY, TOCSY, ROESY, and HMQC) were carried out when
necessary to assign the corresponding signals of the compounds.
High resolution mass spectra were recorded on Hewlett-Packard
HP5988A. ESI-MS analyses were performed on a Finnigan LCQ
DECA XP MAX detector, equipped with an API source, using an
electrospray ionization (ESI) probe. The capillary temperature and
voltage used were 180 °C and 3 V, respectively, and spectra were
obtained in negative ion mode. When the molecular ion was detected,
the MS2 spectrum was obtained using a relative energy collision of 27.
B
DOI: 10.1021/acs.jafc.5b04307
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Article
mixture of sodium salts of 5 and 6 as a white solid (268 mg, 58%).
Isomer 5: H NMR (400 MHz, CD3OD) δ 7.24 (d, 1H, J = 8.2 Hz,
Table 1. Initial Polyphenols and Synthesized Compounds
(in Bold Italics)
1
Harom), 6.83 (d, 1H, J = 2.1 Hz, Harom), 6.73 (dd, 1H, 4J = 2.1 Hz, 3J =
8.2, Harom), 4.28 (t, 2H, J = 6.8 Hz, CH2OAc), 2.89 (t, 2H, J = 6.8 Hz,
Ar−CH2), 2.06 (s, 3H, CH3); 13C NMR (100 MHz, CD3OD) δ 173.0,
150.4, 140.0, 137.5, 124.1, 121.3, 118.8, 66.3, 35.5, 20.8. Isomer 6: 1H
NMR (400 MHz, CD3OD) δ 7.23 (d, 1H, J = 2.9 Hz, Harom), 6.86 (d,
1H, J = 8.2 Hz, Harom), 6.95 (dd, 1H, 4J = 2.1 Hz, 3J = 8.2, Harom), 4.25
(t, 2H, J = 6.8 Hz, CH2OAc), 2.87 (t, 2H, J = 6.8 Hz, Ar−CH2), 2.05
(s, 3H, CH3); 13C NMR (100 MHz, CD3OD) δ 172.9, 149.0, 141.2,
130.9, 127.5, 124.6, 118.2, 66.2, 35.1, 20.8; HRMS (EI) calcd for [M −
H]− 275.0198, found 275.0201; MS fragmentation pattern [M − H]−
= 275, [M − SO3H − H]− = 195, [2M − H]− = 551.
compound
R1
R2
R3
R4
1
H
H
H
H
2
COCH3
H
H
H
H
3
SO3H
H
H
H
2-(3-Hydroxy-4-hydroxysulfonyloxyphenyl)ethanol (3) and 2-(4-
Hydroxy-3-hydroxysulfonyloxyphenyl)ethanol (4), Sodium Salts. A
mixture of compound 5 and 6 DEA salts (200 mg, 0.72 mmol) and
K2CO3 (190 mg, 1.4 mmol) was dissolved in methanol (5 mL). The
reaction mixture was stirred at room temperature for 24 h. The solvent
was evaporated, and 10 mL of water was added and then acidified with
Amberlit IR 120 H+ resin. After filtration, the water phase was washed
with diethyl ether and immediately neutralized with DEA. The water
phase was concentrated to 2 mL, applied to a column of cation-
exchange resin (Dowex 50WX8, Na+ form, 10 g), and eluted with
water. Fractions containing the desired product were lyophilized to
afford a mixture of sodium salts of 3 and 4 as a cream-colored solid
4
H
SO3H
H
H
5
COCH3
COCH3
H
SO3H
H
H
6
SO3H
CH3
CH3
CH3
CH3
H
H
7
H
H
8
H
SO3H
H
H
9
COCH3
COCH3
SO3H
COCH3
COCH3
H
10
11
12
13
SO3H
H
H
H
SO3H
H
SO3H
H
H
SO3H
1
compound
R5
R6
(142 mg, 78%). Isomer 3: H NMR (400 MHz, CD3OD) δ 7.22 (d,
1H, J = 8.4 Hz, Harom), 6.82 (d, 1H, J = 2.4 Hz, Harom), 6.71 (dd, 1H, 4J
= 2.4 Hz, 3J = 8.4, Harom), 3.76 (t, 2H, J = 7.2 Hz, CH2O), 2.78 (t, 2H,
J = 7.2 Hz, Ar−CH2); 13C NMR (100 MHz, CD3OD) δ 150.4, 139.8,
14
H
CH3
CH3
H
15
SO3H
H
16
17
1
138.6, 124.0, 121.3, 118.9, 64.2, 39.8. Isomer 4: H NMR (400 MHz,
SO3H
H
H
CD3OD) δ 7.21 (d, 1H, J = 2.0 Hz, Harom), 6.95 (dd, 1H, 4J = 2.0 Hz,
3J = 8.4, Harom), (d, 1H, J = 8.4 Hz, Harom), 3.75 (t, 2H, J = 7.2 Hz,
CH2OH), 2.77 (t, 2H, J = 7.2 Hz, Ar−CH2); 13C NMR (100 MHz,
CD3OD) δ 148.9, 141.1, 131.9, 127.7, 124.5, 118.2, 64.3, 39.4; HRMS
(EI) calcd for [M − H]− 233.0101, found 233.0098; MS
fragmentation pattern [M − H]− = 233, [M − SO3H − H]− = 153,
[M − SO3 − CH2 − OH − H]− = 123.
18
SO3H
R8
compound
R7
19
H
CH3
CH3
20
SO3H
Phenolic Compounds. Hydroxytyrosol (Hy, 1) and hydroxytyr-
osol acetate (HyAc, 2) were purchased from Seprox Biotech (Madrid,
Spain). Homovanillyl alcohol acetate (HVAAc, 9) was synthesized
from homovanillyl alcohol (HVA, 7) (Sigma-Aldrich) according to the
procedure of Bernini et al.21 Homovanillic acid (HVAc, 14), ferulic
acid (19), and 3,4-dihydroxyphenylacetic acid (DOPAC, 16) were
obtained from Sigma-Aldrich.
Synthesis of Monosulfates (Table 1). General Procedure.
Phenolic compounds (1.0 equiv) and sulfur trioxide−pyridine
complex, SO3·Py (2.0 equiv), were placed in a round flask and
dissolved in 10 mL of dry dioxane in an ice bath. The mixture was left
under magnetic stirring for 15 min at 0 °C under argon and then
closed and stored at −20 °C for 24 h. After this period, 5 mL of water
was added and the mixture immediately neutralized with diethylamine
(DEA). The mixture was washed with ethyl ether, and the aqueous
phase was evaporated. The crude extract was purified by flash
chromatography on silica gel to afford monohydrogenosulfates (η =
20−30%) and a mixture of monohydrogenosulfates and their DEA
salts (η = 50−60%).
4-(2-Ethanoyloxyethyl)-2-hydroxyphenyl Hydrogenosulfate (5)
and 5-(2-Ethanoyloxyethyl)-2-hydroxyphenyl Hydrogenosulfate
(6) and Their Sodium Salts. Hydroxytyrosol acetate, 2 (300 mg,
1.55 mmol), and SO3·Py (3.10 mmol) were dissolved in 6 mL of
dioxane and submitted to sulfation conditions. TLC (ethyl
acetate:methanol, 9:1) showed the formation of a major product
and complete consumption of the initial material. Solvents were
removed, and the crude extract was purified by flash chromatography.
Fractions containing the desired product were evaporated to afford a
mixture of two isomers (5 and 6, in the proportion of 6:4) (130 mg,
31%) as a pale cream-colored solid and a mixture of the two isomers
and their DEA salts (320 mg). The mixture of DEA salts was then
dissolved in 2 mL of water, applied to a column of cation-exchange
resin (Dowex 50WX8, Na+ form, 10 g), and eluted with water.
Fractions containing the desired product were lyophilized to afford a
4-(2-Ethanoyloxyethyl)-2-methoxyphenyl Hydrogenosulfate (10).
Homovanillyl alcohol acetate, 9 (300 mg, 1.4 mmol), and SO3·Py (2.8
mmol) were dissolved in 6 mL of dioxane and submitted to sulfation
conditions. TLC (ethyl acetate:methanol, 9:1) showed the formation
of a major product and complete consumption of the initial material.
Solvents were removed, and the crude extract was purified by flash
chromatography. Fractions containing the desired product were
evaporated to afford compound 10 (120 mg, 25%) as a white solid
and a mixture of compound 10 and its DEA salt (260 mg). The
mixture of DEA salts was then dissolved in 2 mL of water, applied to a
column of cation-exchange resin (Dowex 50WX8, Na+ form, 10 g),
and eluted with water. Fractions containing the desired product were
lyophilized to afford the sodium salt of 10 as a white solid (238 mg,
1
54%): H NMR (400 MHz, CD3OD) δ 7.42 (d, 1H, J = 8.2 Hz,
Harom), 6.96 (d, 1H, J = 2.0 Hz, Harom), 6.82 (dd, 1H, 4J = 2.0 Hz, 3J =
8.2 Harom), 4.30 (t, J = 6.8 Hz, 2H, CH2OAc), 2.94 (t, J = 6.8 Hz, 2H,
Ar−CH2), 3.89 (s, 3H, O−CH3); 13C NMR (100 MHz, CD3OD) δ
172.9, 153.2, 141.6, 137.1, 123.7, 121.9, 114.9, 66.2, 56.6, 35.8, 20.8;
HRMS (EI) calcd for [M − H]− 289.0399, found 275.0402; MS
fragmentation pattern [M − H]− = 289, [M − SO3H − H]− = 209, [M
− SO3 − CH3 − CO2 − H]− = 149.
2-(4-Hydroxysulfonyloxy-3-methoxyphenyl)ethanol (8), Sodium
Salt. Compound 10 sodium salt (200 mg, 0.69 mmol) and K2CO3
(190 mg, 1.4 mmol) was dissolved in MeOH (5 mL). The reaction
mixture was stirred at room temperature for 24 h. The solvent was
evaporated, and 10 mL of water was added and then acidified with
Amberlit IR 120 H+ resin. After filtration, the water phase was washed
with ethyl ether and immediately neutralized with DEA. The water
phase was concentrated to 2 mL, applied to a column of cation-
exchange resin (Dowex 50WX8, Na+ form, 10 g), and eluted with
water. Fractions containing the desired product were lyophilized to
1
afford the sodium salt of 8 as a white solid (142 mg, 78%); H NMR
(400 MHz, CD3OD) δ 7.43 (d, 1H, J = 8.2 Hz, Harom), 7.05 (d, 1H, J
C
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4
3
= 2.0 Hz, Harom), 6.82 (dd, 1H, J = 2.2 Hz, J = 8.2, Harom), 3.87 (s,
3H, OCH3), 3,75 (t, 2H, J = 7.0 Hz, CH2OH), 2.74 (t, 2H, J = 7.0 Hz,
Ar−CH2); 13C NMR (100 MHz, CD3OD) δ 172.8, 153.1, 141.6,
137.1, 123.6, 121.9, 114.9, 66.1, 57.1, 39.7; HRMS (EI) calcd for [M −
H]− 247.0299, found 275.0303; MS fragmentation pattern [M − H]−
= 247, [M − SO3H − H]− = 167.
Ar−CH2); 13C NMR (100 MHz, CD3OD) δ 176.5, 149.5, 141.3,
128.5, 128.2, 125.3, 118.4, 42.7; HRMS (EI) calcd for [M − H]−
246.9921, found 246.9926; MS fragmentation pattern [M − H]−
=
247, [M − CO2 − H]− = 203, [M − SO3 − H]− = 167, [M − CO2 −
SO3 − H]− = 123.
(E)-3-(4-Hydroxysulfonyloxy-3-methoxyphenyl)prop-2-enoic Acid
(20). Ferulic acid, 19 (300 mg, 1.54 mmol), and SO3·Py (3.1 mmol)
were dissolved in 6 mL of dioxane and submitted to sulfation
conditions. Solvents were removed, and the crude extract was purified
by flash chromatography (ethyl acetate:methanol, 9:2.5). Fractions
containing the desired product were evaporated to afford compound
2-(3,4-Dihydroxyphenyl)ethyl Hydrogenosulfate (11). Hydroxy-
tyrosol, 1 (300 mg, 1.95 mmol), and SO3·Py (3.1 mmol) were
dissolved in 6 mL of dioxane and submitted to sulfation conditions.
TLC (ethyl acetate:methanol, 9:2.5) showed the formation of a major
product and complete consumption of the initial material. Solvents
were removed, and the crude extract was purified by flash
chromatography. Fractions containing the desired product were
evaporated to afford compound 11 (115 mg, 25%) as a white solid
and a mixture of compound 11 and its DEA salt (285 mg). The
mixture of DEA salts was then dissolved in 2 mL of water, applied to a
column of cation-exchange resin (Dowex 50WX8, Na+ form, 10 g),
and eluted with water. Fractions containing the desired product were
lyophilized to afford the sodium salt of 11 as a pale cream-colored
1
20 (95 mg, 23%) as a pale cream-colored solid: H NMR (400 MHz,
CD3OD) δ 7.64 (d, 1H, J = 16.0 Hz, Ar−CH) 7.53 (d, 1H, J = 8.3 Hz,
Harom), 7.27 (d, 1H, J = 2.0 Hz, Harom), 7.17 (dd, 1H, 4J = 2.0 Hz, 3J =
8.3, Harom), 6.45 (d, 1H, J = 16.0 Hz, Ar−CH-CH), 3.90 (s, 3H,
OCH3); 13C NMR (100 MHz, CD3OD) δ 170.8, 153.3, 145.7, 144.9,
133.2, 123.6, 122.1, 119.2, 113.1, 56.7; HRMS (EI) calcd for [M −
H]− 273.0102, found 273.0106; MS fragmentation pattern [M − H]−
= 273, [M − SO3 − H]− = 193.
1
solid (248 mg, 52%): H NMR (400 MHz, CD3OD) δ 6.74 (d, 1H, J
= 2.1 Hz, Harom), 6.72 (d, 1H, J = 8.0 Hz, Harom), 6.61 (dd, 1H, 4J = 2.1
Hz, 3J = 8.0, Harom), 4.15 (t, 2H, J = 7.3 Hz, CH2OSO3H), 2.87 (t, 2H,
J = 7.3 Hz, Ar−CH2); 13C NMR (100 MHz, CD3OD) δ 146.1, 144.8,
130.8, 121.3, 117.1, 116.3, 70.1, 36.2; HRMS (EI) calcd for [M − H]−
RESULTS AND DISCUSSION
■
Although the sulfation reaction has a long history and seems to
be accomplished in one step, sulfated small molecules are
difficult to chemically synthesize as their physicochemical
properties are radically changed by the introduction of a sulfate
group, making them water-soluble and therefore difficult to
isolate in a pure form. Typical problems include the presence of
sulfating complexes, the required proportion of which is usually
high, the presence of inorganic salts, the lability of sulfate
groups to acidic conditions and high temperatures,22,23 and the
need for purification protocols with expensive reagents and
preparative RP-HPLC.24−26
233.0100, found 233.0098; MS fragmentation pattern [M − H]−
=
233, [M − SO3 − H]− = 153, [O−SO3−H]− = 97, [2M + Na − H]− =
489.
4-(2-Ethanoyloxyethyl)phenyl 1,2-Dihydrogenosulfate (12). Hy-
droxytyrosol acetate, 2 (300 mg, 1.55 mmol), and SO3·Py (12 mmol)
were dissolved in 10 mL of dioxane and submitted to sulfation
conditions. Solvents were removed, and the crude extract was purified
by flash chromatography (ethyl acetate:methanol, 9:2.5). Fractions
containing the desired product were evaporated to afford compound
1
12 (184 mg, 33%) as a white-colored solid: H NMR (400 MHz,
There are a number of reported procedures for the sulfation
of catechols.27−31 The majority of these perform sulfation
according to the method described by Kawai et al.,31 using
sulfur trioxide pyridine complex as a sulfating agent in excess
pyridine at 80 °C. However, this procedure leads to a mixture
of unreacted catechol with sulfates and has low reaction yields,
and purification is relatively difficult and expensive. A number
of strategies have therefore been developed to convert hydroxyl
groups to sulfate diesters, creating a protected sulfate
monoester that can then be deprotected at a later point in
time. However, this approach always involves two steps, and the
formation of sulfate diesters and the deprotection steps yield
moderate yields. Furthermore, the deprotection conditions are
incompatible with the synthesis of most aryl sulfate
monoesters.22,32
Recently a microwave-based protocol has been developed to
increase the sulfation rate of phenolic structures.33 It was
thought that microwaves would likely induce significant rate
increases because the sulfated intermediates would couple to
microwaves through ionic conduction. Optimal results were
achieved by using SO3−amine complex (−NR3 or −Py, where
R = Me or Et) at approximately 6−10 times molar proportion
per phenolic group in the presence of microwaves at 100 °C for
10−20 min. The method is especially suitable for isolating small
amounts of the persulfated products, and it may be possible to
scale up. Since most of the natural metabolites are
monosulfates, however, it is important to avoid disulfated
products as they are difficult to separate from the monosulfated
compounds. As a result, protection of one of the catechol
groups needs to be carried out before the sulfation reaction.
This limitation led us to seek an alternative approach to
sulfation, which should be relatively rapid, efficient, cheap, and
widely applicable to a number of catechol scaffolds.
CD3OD) δ 7.54 (d, 1H, J = 8.4 Hz, Harom), 7.53 (d, 1H, J = 1.9 Hz,
Harom), 7.04 (dd, 1H, 4J = 1.9 Hz, 3J = 8.4, Harom), 4.27 (t, 2H, J = 6.8
Hz, CH2OAc), 2.93 (t, 2H, J = 6.8 Hz, Ar−CH2), 2.05 (s, 3H, CH3);
13C NMR (100 MHz, CD3OD) δ 172.9, 145.1, 143.8, 136.9, 126.7,
123.8, 123.3, 66.0, 33.4; HRMS (EI) calcd for [M − H]− 354.9801,
found 354.9806; MS fragmentation pattern [M − H]− = 355, [M −
SO3 − H]− = 275.
2-(3-Methoxy-4-hydroxysulfonyloxyphenyl)ethanoic Acid (15). 4-
Hydroxy-3-methoxyphenylethanoic acid, 14 (300 mg, 1.64 mmol), and
SO3·Py (3 mmol) were dissolved in 6 mL of dioxane and submitted to
sulfation conditions. Solvents were removed, and the crude extract was
purified by flash chromatography (ethyl acetate:methanol, 9:1).
Fractions containing the desired product were evaporated to afford
compound 15 (115 mg, 27%) as a white-colored solid: 1H NMR (400
MHz, CD3OD) δ 7.43 (d, 1H, J = 8.2 Hz, Harom), 7.02 (d, 1H, J = 2.0
4
3
Hz, Harom), 6.87 (dd, 1H, J = 2.0 Hz, J = 8.2, Harom), 3.87 (s, 3H,
CH3O), 3.59 (s, 2H, Ar−CH2); 13C NMR (100 MHz, CD3OD) δ
175.6, 153.1, 141.9, 133.8, 123.6, 122.4, 115.4, 56.6, 42.6; HRMS (EI)
calcd for [M − H]− 261,0101, found 261,0105; MS fragmentation
pattern [M − H]− = 261, [M − CO2 − H]− = 217, [M − SO3 − H]−
= 181.
2-(3-Hydroxy-4-hydroxysulfonyloxyphenyl)ethanoic Acid (17)
and 2-(4-Hydroxy-3-hydroxysulfonyloxyphenyl)ethanoic Acid (18).
3,4-Dihydroxyphenylethanoic acid, 16 (300 mg, 1,78 mmol), and SO3·
Py (3,5 mmol) were dissolved in 6 mL of dioxane and submitted to
sulfation conditions. TLC (ethyl acetate:methanol, 9:2.5) showed the
formation of a major product. Solvents were removed, and the crude
extract was purified by flash chromatography. Fractions containing a
mixture of the desired products were evaporated to afford compounds
17 and 18 (6:4) (115 mg, 26%) as a white-colored solid. Isomer 17:
1H NMR (400 MHz, CD3OD) δ 7.22 (d, 1H, J = 8.2 Hz, Harom), 6.88
(d, 1H, J = 2.1 Hz, Harom), 6.76 (dd, 1H, 4J = 2.1 Hz, 3J = 8.2, Harom),
3.50 (s, 2H, Ar−CH2); 13C NMR (100 MHz, CD3OD) δ 176.5, 150.5,
1
140.5, 135.1, 124.2, 122.0, 119.5, 43.7. Isomer 18: H NMR (400
4
MHz, CD3OD) δ 7.25 (d, 1H, J = 2.1 Hz, Harom), 6.99 (dd, 1H, J =
3
2.1 Hz, J = 8.2, Harom), 6.85 (d, 1H, J = 8.2 Hz, Harom), 3.50 (s, 2H,
D
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As the sulfating agent in this work, we started by using
chlorosulfonic acid in ethyl ether, which allows for milder
reaction conditions and shorter reaction times with respect to
amine sulfate complexes. Chlorosulfonic acid has been
successfully used for the sulfation of daidzein and other
phenols and polyphenols such as quercetin, genistein, and
equol.34 However, in none of our attempts could we obtain the
sulfated compound. In fact, after 24 h at room temperature with
a ratio ClSO3H/phenol of 8, only the 2,3-dihydroxy-5-(2-
hydroxyethyl)benzenesulfonic acid was obtained (η = 32% after
purification, please see Supporting Information, compound 13,
Spectra A).
Scaffolds based on phenolic structures and containing more
acidic −OH groups seem to be more efficiently sulfated with
SO3 complexes with weaker bases such as pyridine and DMF,
which suggests that the partial positive charge on the sulfur
atom of the SO3 complex would be greater than that of the
SO3·DEA or TEA complexes.35 It is thus likely that the
nucleophilic attack on the SO3 complex would be more
favorable and result in higher yields. Therefore, we decided to
perform the direct sulfation of hydroxytyrosol acetate with SO3·
Py complex in dioxane using the conventional conditions
reported in the literature:36 temperature of 40 °C and a
proportion of phenol/SO3 of 8. TLC analysis showed the
formation of a major product after 15 min at room temperature.
However, when heated at 40 °C for 2 h according to the
method described in the literature,36 we observed that the
product disappeared. After 5 h of reaction (entry 1, Table 2)
systems of low water content. For example, the rate of the acid-
catalyzed solvolysis of methyl sulfate is increased by a factor of
107 in changing from pure water to 1.9% water/dioxane.39 This
unusual dioxane−water effect is in opposite direction to those
commonly observed for most hydrolytic reactions. The most
likely mechanism thus involves a rate determining unimolecular
sulfur−oxygen bond fission with the elimination of sulfur
trioxide (Figure 3).40 During solvent evaporation, SO3 is
eliminated from the mixture, shifting the reaction toward the
decomposition. We have found that this decomposition during
solvent evaporation can be avoided by previously using a
stronger base than pyridine to neutralize the acidic mixture.
Therefore, DEA was used, and this amine was chosen since its
excess could also be easily removed by evaporation.
After solvent evaporation the crude extract was purified by
flash chromatography on silica gel using polar organic solvents.
During this purification and probably because of the acidic
character of silica gel, TLC analysis of the collected fractions
showed the presence of two spots partially overlapped
corresponding to hydrogenosulfates and sulfates. Therefore,
during this purification, two sets of fractions could be obtained:
one with only hydrogenosulfates and another one correspond-
ing to a mixture of hydrogenosulfates and their salts. Fractions
containing only hydrogenosulfates were evaporated to dryness.
Apparently, hydrogenosulfates showed much more stability in
the organic solvents used (ethyl acetate/methanol) than in
dioxane/water.
In this second attempt (Table 2, entry 2), still with the
proportion between the phenol and the SO3·Py complex of 8,
the only product obtained was the dihydrogenosulfate 12 and
its salts. Therefore, this ratio was decreased to 2 (Table 2, entry
3), and finally only monohydrogenosulfates were obtained.
Under these conditions, there was a preference for the sulfation
at the 4-hydroxyl group (∼60%), whatever the initial catechol
being sulfated. Moreover, only monohydrogenosulfates were
obtained in a one-step reaction, without the need to protect
one of the hydroxyl groups at the aromatic ring. After solvent
evaporation the crude extract was purified by flash chromatog-
raphy on silica gel. Again, two sets of fractions could be
obtained: one with only hydrogenosulfates and another one
corresponding to a mixture of hydrogenosulfates and their salts.
Fractions containing DEA salts were evaporated, dissolved in 2
mL of water, applied to a column of cation-exchange resin, and
eluted with water. This allowed the exchange between DEA and
the physiological cation Na+. The experimental conditions used
for the sulfation reaction as well as their isolated yields are
summarized in Table 2.
The same reaction conditions were then applied to
compounds 9 (homovanillyl alcohol acetate), 14 (homovanillic
acid), 16 (3,4-dihydroxyphenyl acetic acid, DOPAC), and 19
(ferulic acid) with similar results.
In the first attempt to sulfate the hydroxytyrosol molecule,
the same conditions used above were applied. As expected, the
aliphatic hydroxyl group was preferred and sulfated, and only
the 2-(3,4-dihydroxyphenyl)ethyl hydrogenosulfate was ob-
tained. Therefore, another strategy was needed in order to
obtain these monohydrogenosulfates.
Table 2. Reaction Conditions
ratio
initial
entry reagent
phenol:
a
SO3·Py
T (°C)
t (h)
η (%)
product
1
2
3
2
2
2
1:8
1:8
1:2
0/50
0.25/5
65
33
31
58
13
12
0/−20 0.25/20
0/−20 0.25/20
5 and 6
Na+ salts of
5 and 6
a
After purification.
and following the workup described in Materials and Methods,
only compound 13 was again obtained. These results are in
accordance with early works performed by Cerfontain et al. in
the 1980s.37,38 They observed that the initial reaction of 1-
naphthol, 2,6-dialkylphenols, 2,6-dichlorophenol, phenol, and
2,6-dimethylanisole with SO3 in an aprotic solvent was sulfation
with formation of hydrogen sulfates. However, by increasing
the temperature, the initial phenyl hydrogen sulfate was slowly
converted into the phenolsulfonic acids via O-desulfonation
and subsequent C-sulfonation if the phenol was in excess and
via C-sulfonation and subsequent O-desulfonation if the SO3
was in excess. This probably happened in this first attempt
(entry 1, Figure 2). Therefore, after addition of reactants
(Table 2, entry 2), the mixture was kept at −20 °C, and a
complete consumption of the initial material was observed after
20 h.
The resulting acidic reaction mixture was then neutralized
with DEA to avoid sulfate decomposition during the solvent
evaporation. In fact, the decomposition of aryl sulfates to
alcohol and sulfur trioxide appears to proceed, at pH < 4, via a
rapid acid-catalyzed reaction where the leaving group is
protonated. It is interesting to note that the rate of acid-
catalyzed solvolysis of arylsulfates is subject to acceleration in
Using the DEA salts of compounds 5 and 6, acetyl
deprotection was carried out using K2CO3 in methanol to
obtain the mixture of monosulfated hydroxytyrosol derivatives
3 and 4. Solvent was then removed and water added, followed
by neutralization with Amberlite IR 120 H+ resin. This
procedure permitted the K2CO3 removal from the mixture by
E
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Journal of Agricultural and Food Chemistry
Article
Figure 2. Phenol sulfonic acid formation by O-desulfonation. (b, g, h) O-protodesulfonations. (c) Slow reaction of the arene CC with SO3 to give
a resonance stabilized hydroxy-carbocation. (d) Fast loss of H+ (intramolecular elimination) from the carbocation to restore the aromatic system. (c
+ d, e, f) Ring-sulfonation on the C3 position ascribed to steric hindrance reasons. Hydroxytyrosol acetate used as an example.
Figure 3. Sulfur−oxygen bond fission with the elimination of sulfur trioxide in acidic conditions.
the formation of CO2. The aqueous phase was then washed
with ethyl ether (to remove the formed acetic acid) and quickly
neutralized again with DEA. After concentration, the mixture
was then applied to a column of cation-exchange resin and
eluted with water. Once more, this allowed the exchange
between DEA and the physiological cation Na+. The same
procedure was then performed for the synthesis of compound 8
with similar results.
In conclusion, an efficient chemical method suitable for the
synthesis of mono-O-sulfate metabolic conjugates of several
simple phenolic compounds has been developed. The strategy
adopted is based on mild conditions of reaction: avoidance of
high temperature conditions during the synthesis and of low
pressure conditions during purification. Ten monosulfates have
been synthesized in 1−2 steps in a good yield and using simple,
cheap, and fast procedures. These compounds can be used as
standards for the determination of the metabolic profile of
hydroxytyrosol in various species, including humans. It is
foreseen that the proposed protocol will be useful for the
synthesis of conjugates of other compounds based on the
catechol scaffold.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jafc.5b04307.
■
S
1H- and 13C NMR spectra (PDF)
AUTHOR INFORMATION
Corresponding Author
*Tel: +351220402556. Fax: +351220402659. E-mail:
mpmartin@fc.up.pt.
■
Notes
The authors declare no competing financial interest.
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