First chemical synthesis and in vitro characterization of the potential human metabolites 5-O-feruloylquinic acid 4'-sulfate and 4'-O-glucuronide

Article abstract of DOI:10.1021/jf200272m

Feruloylquinic acids are a major class of biologically active phenolic antioxidants in coffee beans, but their metabolic fate is poorly understood. The present study investigated the phase II metabolism of feruloylquinic acids with selected human sulfotra

Full text of DOI:10.1021/jf200272m

ARTICLE
pubs.acs.org/JAFC
First Chemical Synthesis and in Vitro Characterization of the Potential
Human Metabolites 5-O-Feruloylquinic Acid 40-Sulfate and 40-O-
Glucuronide
Candice Menozzi-Smarrito,† Chi Chun Wong,‡ Walter Meinl,§ Hansruedi Glatt,§ Renꢀe Fumeaux,†
Caroline Munari,† Fabien Robert,† Gary Williamson,†,‡ and Denis Barron*,†
†Nestlꢀe Research Center, P.O. Box 44, CH-1000 Lausanne 26, Switzerland
‡School of Food Science and Nutrition, University of Leeds, Leeds LS2 9JT, United Kingdom
§German Institute of Human Nutrition (DIfE) Potsdam-Rehbruecke, Arthur-Scheunert-Allee 114-116, 14558 Nuthetal, Germany
S Supporting Information
b
ABSTRACT: Feruloylquinic acids are a major class of biologically active phenolic antioxidants in coffee beans, but their metabolic
fate is poorly understood. The present study investigated the phase II metabolism of feruloylquinic acids with selected human
sulfotransferases (SULT1A1 and SULT1E1) and uridine 50-diphosphoglucuronosyltransferases (UGT1A1 and UGT1A9). For
unequivocal metabolite identification, the chemical synthesis of two potential human metabolites of 5-O-feruloylquinic acid, the 40-
sulfated and 40-O-glucuronidated conjugates, has been performed for the first time. Following incubation with human SULT1A1 or
SULT1E1, formation of 5-O-feruloylquinic acid 40-O-sulfate was confirmed by matching its HPLC and MS data with those of the
authentic standard. On the other hand, no glucuronide conjugates were detected after incubation with human uridine 50-
diphosphoglucuronosyltransferases. These results suggest that sulfation can take place on the ferulic acid moiety of feruloylquinic
acids and may be a major metabolic pathway for feruloylquinic acids in humans.
KEYWORDS: feruloylquinic acid glucuronide, feruloylquinic acid sulfate, human metabolites, coffee, synthesis, sulfotransferase
’ INTRODUCTION
hydroxyls of the quinic acid part, or both. Recently, the presence of
feruloylquinic acid sulfates and glucuronides has been demonstrated
in the ileal fluid of human volunteers with an ileostomy.23 However,
the position of conjugation was not determined. On the other hand, a
previous study performed on rats 24 suggested that glucuronidation
of 1,5-dicaffeoylquinic acid was directed to the quinic acid skeleton,
although no clear scientific evidence was provided to support this
hypothesis. In the absence of suitable chromatographic standards, the
identification and quantification of these conjugates is difficult.
Moreover, the availability of appropriate standards of chlorogenic
acid conjugates is essential for studying their biological effects.
Herein, we describe the first synthesis of two potential human
metabolites of 5-O-feruloylquinic acid, i.e., its 40-sulfate and 40-O-
glucuronide metabolites. The synthesized authentic standards
were used for unequivocal identification of 5-feruloylqinic acid
metabolites from in vitro incubations using human recombinant
UGTs and SULTs.
Chlorogenic acids are naturally occurring polyphenolic com-
pounds possessing a quinic acid unit and a trans-cinnamic acid
unit. The most widespread subgroup is the 5-monoester of caffeic
acid, present in coffee beans, potatoes, and many fruits and
vegetables.1,2 Chlorogenic acids possess many biological proper-
ties, such as antioxidant3ꢀ6 and antibacterial.7 However, the
human metabolism of chlorogenic acids is far from fully under-
stood. Chlorogenic acids are believed to be metabolized, mainly
in the colon, into a range of hydroxycinnamic, hydroxyphenyl-
propionic, and hydroxyphenylacetic acids.8ꢀ13 It has also been
demonstrated that chlorogenic acids could be absorbed in their
intact form in rats,14 and a number of recent studies have
indicated the presence of intact chlorogenic acid forms in human
plasma15ꢀ17 and human urine.18,19 However, the exact nature of
the human metabolites of feruloylquinic acids remained unclear
from these studies since their potential sulfated or glucuroni-
dated conjugates either were not investigated or were enzyma-
tically hydrolyzed prior to analysis.
’ MATERIALS AND METHODS
The phase II metabolism in humans is carried out by uridine 50-
diphosphoglucuronosyltransferases (UGTs) and sulfotransferases
(SULTs). SULTs and UGTs are abundant in the human liver and
intestine, where they catalyze conjugation of xenobiotics.20 The
UGT1A subfamily are in general active toward phenolic hydroxyl
groups, with UGT1A1 and UGT1A9 being the most active
isoforms.21 A recent study by our group suggested that SULT1A1
and SULT1E1 are highly effective in the sulfation of phenolic acids.22
Theoretically, conjugation of chlorogenic acids could affect the
phenolic hydroxyls of the hydroxycinnamic acid moiety, the alcoholic
Chemicals and Instruments. The authentic standards of the 3-O-,
4-O-, and 5-O-feruloylquinic acids have been chemically synthesized
according to published methods.25,26 Thin-layer chromatography (TLC)
was carried out on silica 60 F254 (Merck) and RP-18 F254s (Merck) plates.
The TLC plates were visualized by short-wave UV light or ceric
Received: January 20, 2011
Revised:
March 18, 2011
Accepted: March 21, 2011
Published: March 21, 2011
r
2011 American Chemical Society
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ammonium molybdate stain. Flash chromatography was performed using
a Biotage SP1 high-performance flash chromatography system and
FLASH cartridges (25þM, 40þM, and 65i, KP-SIL). 1H NMR (360.13
MHz) and 13C NMR (90.56 MHz) spectra were recorded on a Bruker
DPX-360 spectrometer equipped with a broad-band multinuclear z-
gradient probehead. The chemical shifts (ppm) are expressed with respect
to an internal reference (TMS or TSP). Multiplicities are reported as
follows: s = singlet, d = doublet, t = triplet, q = quatruplet, m = multiplet, br
s = broad singlet. Melting points were recorded on a Buchi melting point
B-545 apparatus and are uncorrected. Optical rotations were measured
with a JASCO P2000 polarimeter. Elementary analyses were performed at
the University of Geneva, Switzerland (Service de Microanalyse). HPLC/
DAD/ESI-TOF-MS analyses were performed on an Agilent-1200 series
rapid resolution LC system including a binary pump SL, a high-perfor-
mance autosampler, a diode-array detector SL, and a thermostated
column compartment SL. The HPLC system was coupled to an Agilent
6210time-of-flightmassspectrometer. HPLC/DADanalyseswere carried
out using an Agilent 1200 series liquid chromatography system. Chro-
matograhy was performed with a Zorbax XDB-C18 column (4.6 ꢁ
50 mm, 1.8 μm) with 1% formic acid (A) and methanol (B) as the
mobile phase. The gradient started at 17% (B), which was kept for 2 min,
followed by an increase to 30% B in 13 min and to 60% in 2 min, and then
was returned to 17% B for 5 min at 0.5 mL/min. A 25 μL volume of the
supernatant was injected into the column. Isoferulic acid was used as the
internal standard. UV detection was carried out at 310 nm using a
photodiode array detector. Recombinant human UGT1A1 and UGT1A9
expressed in Sf9 insect cells (Supersomes) were obtained from BD
Gentest (Woburn, MA). The total protein content (5 mg/mL) of Super-
somes was specified on data sheets provided by the manufacturer. SULTs
were expressed in Salmonella typhimurium and purified as previously
described.27 All activities were adjusted to the expression levels of SULTs
measured by immunoblot analysis.
Chemical Synthesis of 5-O-Feruloylquinic Acid 40-Sulfate (1).
To a solution of vanillin (3; 400 mg, 2.62 mmol) in anhydrous THF
(6 mL) were added triethylamine (0.546 mL, 3.93 mmol) and DMAP
(320 mg, 2.62 mmol). After the resulting solution was stirred for 10 min,
neopentyl chlorosulfate28 (678 mg, 3.63 mmol) was added, and the
mixture was stirred for 2 h at room temperature. The solution was
diluted with ethyl acetate and water. The organic layer was washed with
water and brine, dried over sodium sulfate (Na2SO4), filtered, concen-
trated, and purified by flash chromatography on silica gel using a gradient
of ethyl acetate in petroleum ether. 4-Formyl-2-methoxyphenyl neo-
pentyl sulfate (4) was isolated as a white solid (530 mg, 1.85 mmol,
71%). To a solution of 4 (214 mg, 1.41 mmol) and malonyl quinic
fragment 8 (353 mg, 1.26 mmol) in anhydrous DMF (4 mL) were added
DMAP (55 mg, 0.45 mmol) and piperidine (71 μL) at room tempera-
ture. The details of the synthesis of compound 8 are displayed in the
Supporting Information. The mixture was stirred for 7 days at room
temperature in a sealed tube. It was then concentrated and purified by
flash chromatography on RP-18 using a gradient of methanol in water.
After lyophilization, the white solid 9 (550 mg) was directly deprotected
in anhydrous DMF (5 mL) using sodium azide (96 mg, 1.45 mmol). The
solution was heated at 55 °C for 12 h, concentrated, purified by flash
chromatography on RP-18 using a gradient of methanol in water,
and lyophilized to give the sodium salt of 1 as a white solid (315 mg,
0.67 mmol, 53% from compounds 4 and 8 over the two steps).
(2 ꢁ 50 mL), dried over sodium sulfate (Na2SO4), filtered, concentrated,
and purified by flash chromatography on silica gel using a gradient of ethyl
acetate in petroleum ether to give a yellow foam. Recrystallization and
trituration in diethyl ether gave a white solid of compound 6 (480 mg, 1.02
mmol, 69%). Alkaline hydrolysis of 6 was carried out in MeOH (11 mL)
and aqueous NaOH (1 N, 10 mL). The mixture was stirred for 5 h at room
temperature and then acidified to pH 6 by addition of Amberlite IRC 50
ion-exchange resin. The resulting suspension was filtered, concentrated
under reduced pressure, and purified by flash chromatography on reversed-
phase RP-18 using a gradient of methanol in water. Compound 7 was
isolated as a white foam (183 mg, 0.56 mmol, 64%). To a solution of 7
(150 mg, 0.45 mmol) and malonyl quinic fragment 8 (115 mg, 0.41 mmol)
in anhydrous DMF (1.3 mL) were added DMAP (20 mg, 0.16 mmol) and
piperidine (22 μL). The mixture was stirred for 7 days at room temperature
in a sealed tube. The solution was then concentrated under reduced
pressure and purified by flash chromatography on RP-18 using a gradient
of methanol in water to give, after freeze-drying, 2 as a white solid (20 mg,
0.037 mmol, 9%).
Data for 1. 1H NMR (360 MHz, MeOD-d4) δ 7.72 (d, J = 15.9 Hz,
1H), 7.54 (d, J = 8.3 Hz, 1H), 7.31 (d, J = 2.0 Hz, 1H), 7.19 (dd, J = 8.5, 1.9
Hz, 1H), 6.55 (d, J = 15.9 Hz, 1H), 5.42 (ddd, J = 11.8, 10.3, 5.4 Hz, 1H),
4.17 (dt, J = 6.2, 3.0 Hz, 1H), 3.91 (s, 3H), 3.72 (dd, J = 9.9, 3.1 Hz, 1H),
2.22ꢀ1.96 (m, 4H); 13C NMR (90 MHz, MeOD-d4) δ 182.44, 170.08,
154.72, 147.31, 146.34, 134.54, 125.96, 123.69, 120.16, 114.48, 79.22, 76.34,
74.40, 74.22, 58.08, 42.13, 40.40; LC/ESI-TOF-MS obsd m/z ([M þ 1]þ)
471.06 (M þ Na), 391.11, 177.05; obsd m/z ([M ꢀ 1]ꢀ) 468.99 (M þ
Na ꢀ 2H), 447.10 (M ꢀ H), 367.16, 193.04. Anal. Calcd for C17H18Na2-
O12S: C, 37.99; H, 4.31. Found: C, 38.08; H, 4.20.
Data for 2. 1H NMR (400 MHz, D2O) δ 7.53 (d, J = 16.2 Hz, 1H),
7.08ꢀ7.02 (m, 3H), 6.35 (d, J = 16.1 Hz, 1H), 5.27 (br s, 1H, OH), 5.19
(td, J = 15.8, 4.7 Hz, 1H), 5.08 (d, J = 5.4 Hz, 1H), 4.19 (m, 1H, OH),
4.15 8 m, 1H), 3.87ꢀ3.70 (m, 5H), 3.61ꢀ3.55 (m, 3H), 3.28 (m, 1H,
OH), 3.06 (m, 1H, OH), 2.10ꢀ1.86 (m, 4H); 13C NMR (90 MHz,
D2O) δ 181.00, 170.87, 168.86, 148.54, 147.42, 145.61, 129.30, 122.88,
116.16, 115.49, 111.35, 99.78, 76.89, 76.43, 75.27, 72.78, 72.63, 71.72,
71.28, 70.78, 55.84, 38.52, 38.13; LC/ESI-TOF-MS obsd m/z ([M þ
1]þ) 471.06 (M þ Na), 567.16, 177.05; obsd m/z ([M ꢀ 1]ꢀ) 543.06
(M ꢀ H), 193.04.
Data for 4. Mp 49ꢀ51 °C; 1H NMR (360 MHz, CDCl3, TMS as
reference) δ 9.99 (s, 1H), 7.58ꢀ7.48 (m, 3H), 4.18 (s, 2H), 3.97 (s, 3H),
1.01 (s, 9H); 13C NMR (90 MHz, CDCl3, TMS as reference) δ 190.67,
152.03, 143.55, 135.87, 124.49, 123.24, 111.35, 83.93, 56.25, 31.88,
25.25; LC/ESI-TOF-MS obsd m/z 289.0000 (289.0000 calcd for [M ꢀ
H]þ), obsd m/z 311.0000 (311.0000 calcd for [M þ Na]þ).
Data for (3R,4S,5S,6S)-2-(4-Formyl-2-methoxyphenoxy)-6-
(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl Triacetate
1
(6). Mp 137ꢀ139 °C; [R]2D5 = ꢀ47.17° (c = 1.06 g/L, MeOH); H
NMR (360 MHz, CDCl3, TMS as reference) δ 9.89 (s, 1H), 7.47 (s, 1H),
7.43 (dd, J = 6.6, 1.9 Hz, 1H), 7.25 (d, J = 8.6 Hz, 1H), 5.38ꢀ5.29 (m, 3H),
5.18 (m, 1H), 4.17 (m, 1H), 3.89 (s, 3H), 3.73 (s, 3H), 2.06 (s, 3H), 2.06 (s,
3H), 2.04 (s, 3H); 13C NMR (90 MHz, CDCl3, TMS as reference) δ
190.97, 170.11, 169.20, 166.81, 151.07, 133.02, 125.57, 118.81, 110.65,
99.66, 72.73, 71.52, 70.89, 69.01, 56.10, 53.00, 20.64, 20.61, 20.53; LC/ESI-
TOF-MS obsd m/z ([M þ 1]þ) 491 (M þ Na). Anal. Calcd for
C21H24O12 0.4H2O: C, 53.03; H, 5.26. Found: C, 52.85; H, 5.19.
3
Data for (2S,3S,4S,5R)-Methyl 6-(4-Formyl-2-methoxy-
phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxy-
late (7). [R]2D5 = ꢀ93.27° (c = 1.04 g/L, MeOH); 1H NMR (360 MHz,
D2O, TSP as internal reference) δ 9.70 (s, 1H), 7.50 (d, J = 8.3 Hz, 1H),
7.42 (s, 1H), 7.23 (d, J = 8.6 Hz, 1H), 5.24 (d, J = 7.1 Hz, 1H), 3.93 (d, J =
8.6 Hz, 1H), 3.88 (s, 3H), 3.72ꢀ3.58 (m, 3H); 13C NMR (90 MHz,
D2O, TSP as internal reference) δ 194.29, 174.70, 150.65, 148.32,
130.46, 126.37, 114.35, 110.63, 98.91, 76.00, 74.72, 72.02, 71.15, 55.33;
LC/ESI-TOF-MS obsd m/z ([M ꢀ 1]ꢀ) 327.07 (M ꢀ H).
Chemical Synthesis of 5-O-Feruloylquinic Acid 40-O-Glucur-
onide (2). The synthesis of the glucuronic acid donor (2,3,4-tri-O-acetyl-D-
methylglucuronopyranosyl)-N-phenyl-2,2,2-trifluoroacetimidate (5) is de-
tailed in the Supporting Information. To compounds 5 (1.47 g, 2.91 mmol)
and 3 (225 mg, 1.48 mmol) in 20 mL of anhydrous dichloromethane, BF3
etherate (56 μL, 1.48 mmol), was added dropwise, and the mixture was
stirred overnight at room temperature. The solution was then diluted with
dichloromethane (50 mL), and the organic layer was washed with water
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Figure 1. HPLC/DAD/ESI-MS/MS analysis of 5-O-feruloylquinic acid incubated with SULT1E1. The product ion spectra of 5-O-feruloylquinic acid
(m/z 367.1) and 5-O-feruloylquinic acid 40-sulfate (m/z 447.1) were obtained in product ion mode.
Glucuronidation of Feruloylquinic Acids by Recombinant
Human UGTs. The contribution of individual isozymes to the glucur-
onidation of the feruloylquinic acids was assessed using human recombi-
nant UGT1A1 and UGT1A9. The reaction was performed in 100 mM
potassium phosphate buffer (pH 7.4) containing 100 μM vitamin C,
1 mM UDPGA, 5 mM saccharolactone, and 0.025 mg/mL alamethicin
(final volume 50 μL). Control Supersomes and UGTs were added at a
final concentration of 2 mg/mL. The reaction was started by the addition
of 100 μM 5-O-feruloylquinic acid, allowed to proceed at 37 °C for 1 h,
and finally stopped by the addition of 10 μL of ice-cold acetonitrile
containing 500 mM HCl. After centrifugation at 13000g for 5 min, the
supernatant was dried in vacuum and the residue redissolved in the initial
mobile phase and analyzed by HPLC and HPLC/DAD/ESI-MS.
Sulfation of Feruloylquinic Acids by Recombinant Human
SULTs. Sulfation of the feruloylquinic acids by SULT1A1 and
SULT1E1 was performed in 100 mM potassium phosphate buffer
(pH 7.4) containing 100 μM vitamin C, 100 μM 30-phosphoadenosine
50-phosphosulfate, and 1 mM DL-dithiothreitol (final volume 50 μL).
The control cytosolic fraction and cytosolic preparations from SULT-
expressing bacteria were added to a final concentration of 0.25 mg/mL
total protein. The reaction was initiated by the addition of 100 μM
feruloylquinic acid. The reaction was allowed to proceed at 37 °C for 1 h
and stopped by adding 10 μL of ice-cold acetonitrile with 500 mM HCl.
After centrifugation at 13000g for 5 min, the supernatant was dried in
vacuum and the residue redissolved in the initial mobile phase and
analyzed by HPLC/DAD/ESI-TOF-MS.
presence of intact 5-feruloylquinic acid, significant formation of
5-feruloylquinic acid sulfate was observed with SULT1A1 and
SULT1E1 (Figure 1). In the spectrum of 5-feruloylquinic acid,
two ions were present at m/z 367.1 ([M ꢀ H]ꢀ) and 191.1
(corresponding to the loss of the feruloyl part; Figure 1). In the
spectrum of the corresponding sulfated conjugate, three ions
were detected at m/z 447.1 ([M]ꢀ), 367.1 ([feruloyl quinic acid
ꢀ H]ꢀ), and 191.1 ([quinic acid ꢀ H]ꢀ). The two latter ions
originated after the loss of the sulfate group. Thus, the MS data
did not allow determination of which of the two ferulic or quinic
acid moieties of feruloylquinic acid was bearing the sulfate group.
Similar incubation of authentic standards of 3-O- and 4-O-
feruloyquinic acids with SULTs was carried out (Figure 2).
Among the feruloylquinic acids, 4-O-feruloylquinic acid was
the most highly sulfated isomer by both SULTs tested, followed
by 5-O-feruloylquinic and 3-O-feruloylquinic acids. In addition,
SULT1E1 was more active than SULT1A1 in the sulfation of all
feruloylquinic acids tested. This is in agreement with our
previous study, in which it was shown that SULT1E1 possessed
a unique regioselectivity toward the 4-hydroxyl group of ferulic
acid and catalyzed sulfation with high efficiency.22 In humans,
SULT1A1 and SULT1E1 are highly expressed in the intestine
and liver;29 hence, sulfation may be a major phase II metabolic
pathway for feruloylquinic acids in vivo.
To unequivocally determine the structure of the product of SULT
incubations, we embarked on the chemical synthesis of an authentic
standard of 5-feruloylquinic acid 40-sulfate (1), the preparation of
which has not been reported before. Classical approaches for the
synthesis of chlorogenic acids are based on the esterification of quinic
acid (A1) by a hydroxycinnamoyl fragment (A2; Figure 3, pathway
A).30ꢀ33 However, we followed the synthetic strategy we recently
designed for 5-O-feruloylquinic acid and its methyl ester derivative.34
In this case the elaboration of the (hydroxycinnamoyl)quinic acid
skeleton is based on a Knoevenagel condensation of a malonate ester
of quinic acid (B1) with a hydroxybenzaldehyde fragment
(B2; Figure 3, pathway B). This approach does not require any
’ RESULTS AND DISCUSSION
To gain insight into the potential feruloylquinic acid metabo-
lites that may be formed in vivo in man, we synthesized 40-O-
sulfated (1) and 40-O-glucuronidated (2) conjugates of 5-fer-
uloylquinic acid and studied the metabolism of feruloylquinic
acids using human recombinant UGT and SULT enzymes. The
feruloylquinic acid metabolites were analyzed by HPLC/DAD
and mass spectrometry following incubations with UGT1A1,
UGT1A9, SULT1A1, and SULT1E1, respectively. Besides the
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protective groups. Consequently, the deprotection step can be
carried out on the quinic and benzaldehyde fragments before the
formation of the hydroxycinnamoyl core. In this way, any hydro-
genating, basic or strongly acidic conditions, which could be detri-
mental to the sensitive feruloylquinic skeleton, were avoided.
The sulfation of vanillin (3) with neopentyl chlorosulfate28 afforded
the desired sulfate monoester 4 after stirring for 2 h at room
temperature (Figure 4). Vanillin derivative 4 and malonate ester 8
were treated with piperidine and 4-(dimethylamino)pyridine in
anhydrous DMF and stirred for 7 days at room temperature to give
the neopentyl sulfate conjugate9. Deprotection of the sulfate group
in 9 afforded 1 in fair 53% yield from the malonylquinic acid
derivative 8 (Figure 4).
The identity of the product of the incubation of 5-O-feruloyl-
quinic acid with SULT1E1 was definitively established as 5-O-
feruloylquinic acid 40-sulfate by comparison with the authentic
synthesized compound 1 using the HPLC retention time and the
electrospray ionization mass spectrum. This demonstrated that
sulfate conjugation was targeted to the ferulic acid moiety of
feruloylquinic acids by human SULTs. The synthetic 1 was
accompanied by minor amounts of isomers displaying the same
molecular ion in ESI-MS. We suspected that these could be the
3-O- and 4-O-feruloylquinic acid sulfate isomers, probably
formed during the last deprotection step of the sulfate group
by sodium azide (step e of Figure 4). This was confirmed by
matching their retention times with those of the corresponding
sulfates obtained by sulfation of authentic standards of 3-O- and
4-O-feruloyquinic acids with SULTs (Figure 2). Having identi-
fied the three components of the synthetic feruloyl quinic acid
sulfates, the 5-O-, 3-O-, and 4-O-feruloylquinic sulfate isomer
ratio of the synthetic standard was established as 116:3:2 on the
basis of HPLC/DAD/ESI-TOF-MS analysis.
Similar synthesis of glucuronidated 5-feruloylquinic acid 40-O-
glucuronide (2) was carried out by reaction of malonyl ester 8
with glucuronidated vanillin 7, beforehand prepared by glucuroni-
dation of 3 followed by deprotection of the intermediate glucur-
onide 6 (Figure 4, steps b, c, and f). However, no product matching
the chromatographic behavior of 2 or corresponding to the mass of
a glucuronidated ferulic acid was detected following incubation of
5-O-feruloylquinic acid with UGT1A1 or UGT1A9 (data not
shown). Under the same conditions, isoferulic acid was in contrast
well conjugated.22 Since feruloylquinic acid glucuronides are present
in the ileostomy fluid after consumption of coffee by ileostomists,23
it is possible that isoforms other than UGT1A1 or UGT1A9 could
be responsible for the production of these conjugates.
In vivo sulfation of feruloylquinic acids has important impli-
cations for their bioactivity in humans. The phenolic group
of these compounds is critical for their biological properties,
such as antioxidant activity and photoprotective activities.35 In
some cases, conjugation of polyphenols may lead to an enhance-
ment of biological activities, such as the ability to interact with
transporter proteins.36 Thus, future research should emphasize the
potential bioactivity of the conjugates, especially sulfated
Figure 2. HPLC chromatograms of 3-O-feruloylquinic acid (top), 4-O-
feruloylquinic acid (middle), and 5-O-feruloylquinic acid (bottom)
incubated with SULT1A1: 3-FQA, 3-O-feruloylquinic acid; 4-FQA,
4-O-feruloylquinic acid; 5-FQA, 5-O-feruloylquinic acid; 3-FQA-S,
3-O-feruloylquinic acid 40-sulfate; 4-FQA-S, 4-O-feruloylquinic acid 40-
sulfate; 5-FQA-S, 5-O-feruloylquinic acid 40-sulfate; IS, internal stan-
dard. The two peaks around 2 min are the solvent front, and the peak at
about 3.5 min is 30-phosphoadenosine-50-phosphosulfate (PAPS), the
cofactor. Arrows indicate retention times of the standards.
Figure 3. Retrosynthetic scheme for the elaboration of the 5-feruloylquinic acid skeleton.
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Figure 4. Preparation of 5-O-feruloylquinic acid metabolites 1 and 2. Reagents and conditions: (a) DMAP, Et3N, THF, room temperature (rt), 10 min,
and then ClSO3C5H11, THF, rt, 2 h, 71%; (b) compound 5, BF3 etherate, CH2Cl2, rt, 12 h, 69%; (c) NaOH (1 N), MeOH, rt, 5 h, 64%; (d) compound 4,
DMAP, piperidine, DMF, rt, 7 days; (e) NaN3, DMF, 55 °C, overnight, (d) þ (e) 53%; (f) compound 7, DMAP, piperidine, DMF, rt, 7 days, 9%.
conjugates of feruloylquinic acids, which are the relevant metabo-
lites in vivo. The current work also shows that the sulfate conjugation
is on the ferulic acid moiety of 5-O-feruloylquinic acid. For 1,5-
dicaffeoylquinic acid, it was suggested that glucuronidation might
also occur on the quinic acid moiety.24 In contrast, the data presented
here support the ferulic acid moiety as a site for conjugation.
In conclusion, the first syntheses of sulfate and glucuronide
conjugates of 5-O-feruloylquinic acid have been achieved. In
vitro incubation with SULTs showed that sulfation at the ferulic
acid moiety may be a major metabolic pathway for feruloylqui-
nic acids in humans. These novel conjugates may prove valuable
for further investigating the bioavailability and bioactivity of
feruloylquinic acids.
diode-array detection/electrospray ionization time-of-flight
mass spectrometry; SULT, sulfotransferase; THF, tetrahy-
drofuran; TMS, tetramethylsilane; TSP, (trimethylsilyl)-
propionic acid; UDPGA, uridine 50-diphosphoglucuronic-
acid; UGT, uridine 50-diphosphoglucuronyltransferase.
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’ ASSOCIATED CONTENT
S
Supporting Information. Synthesis of 5 and 8 and the
b
NMR data for the synthetic intermediates. This material is
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’ AUTHOR INFORMATION
Corresponding Author
*Address: Nestlꢀe Research Center, Vers Chez Les Blanc, 1000
Lausanne 26, Switzerland. Fax: þ41 21 785 8554. Phone: þ41 21
785 9497. E-mail: denis.barron@rdls.nestle.com.
’ ABBREVIATIONS USED
BF3, boron trifluoride; calcd, calculated; DMF, dimethylfor-
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