Chemical synthesis of citrus Flavanone glucuronides

Article abstract of DOI:10.1021/jf1010403

Flavanone glucuronides are the major phenolic metabolites detected in human plasma after consumption of citrus fruits. As such, they might display significant cardioprotective effects. In this work, glucuronides of naringenin (4′- and 7-O-β-d-glucuronides

Full text of DOI:10.1021/jf1010403

J. Agric. Food Chem. 2010, 58, 8437–8443 8437
DOI:10.1021/jf1010403
Chemical Synthesis of Citrus Flavanone Glucuronides
M
UHAMMAD
K
AMRAN
K
HAN, NJARA
R
AKOTOMANOMANA, MICHELE
ANGLES
L
OONIS AND
,
O
LIVIER
D
*
University of Avignon, INRA, UMR408, F-84000 Avignon, France
Flavanone glucuronides are the major phenolic metabolites detected in human plasma after consumption
of citrus fruits. As such, they might display significant cardioprotective effects. In this work, glucuronides
of naringenin (40- and 7-O-β-
D
-glucuronides) and hesperetin (30- and 7-O-β-
D-glucuronides), the major
flavanone aglycones in grapefruit and orange, respectively, have been chemically synthesized. On the
one hand, the most reactive hydroxyl group, C7-OH, was protected by selective benzoylation to allow
subsequent glucuronidation of C40-OH (naringenin) or C30-OH (hesperetin) (B-ring). On the other hand,
the selective debenzoylation at C7-OH of the perbenzoylated flavanone aglycones allowed glucuronida-
tion at the same position (A-ring). After careful deprotection, the target compounds were purified and
characterized by nuclear magnetic resonance and mass spectrometry.
KEYWORDS: Flavanone; glucuronide; bioavailability; conjugation; chemical synthesis
INTRODUCTION
effects requires investigations on appropriate cell models (e.g.,
endothelial or smooth muscle cells) with the authentic circulating
metabolites instead of the commercially available glycosides and
aglycones that are frequently used as a first approach despite the
limited biological significance. As an alternative to the expensive,
inconvenient, and low-yielding extraction of conjugates from bio-
logical fluids, chemical synthesis appears as the most direct strategy
to obtain substantial amounts of these metabolites for bioavail-
ability and in vitro cell studies. In this paper, we report the synthesis
of the four circulating glucuronides of hesperetin and naringenin.
Because of the large and increasing worldwide consumption of
citrus fruits and juices, potentially bioactive citrus polyphenols,
which mainly belong to the flavanone class, are receiving increas-
ing attention from nutrition biologists. Among flavanones, the
naringenin and hesperetin glycosides are of particular interest
because of their high prevalence in grapefruit and orange, res-
pectively (1,2). Citrus flavanones may play a beneficial role in the
prevention of cardiovascular diseases and cancers due to their
anti-inflammatory, antioxidant, antimutagenic, and antitumor
activities (2-4). After ingestion, the flavanone glycosides typi-
cally reach the colon, where they are hydrolyzed by microflora
glycosidases into aglycones (naringenin, hesperetin), which are
partially absorbed through the intestinal barrier (5, 6). In con-
MATERIALS AND METHODS
All starting materials were obtained from commercial suppliers, mainly
Sigma-Aldrich (Steinheim, Germany), and were used without purification.
Solvents were distilled over CaH2. Analytical thin layer chromatography
(TLC) was performed on silica gel 60 F254 obtained from Merck KGaA
(Darmstadt, Germany). Detection was achieved by UV light (254 nm) and
by charring after exposure to a 5% H2SO4 solution in EtOH. Purifications
were performed by column chromatography on silica gel 60 (40-63 μm)
(Merck KGaA). Dowex 50WX4-50 ion-exchange resin was used for acid-
ification. Melting points were measured on a Barnstead Electrothermal
9100 apparatus and are uncorrected. Glucuronyl donor (1) (methyl 2,3,4-
trast, the partial absorption of flavanone aglycones and O-β-
glucosides (e.g., naringenin 7-O-β- -glucoside) can occur earlier
in the small intestine through passive diffusion for the former and
with preliminary hydrolysis of the latter by endothelial β-
D-
D
D-
glucosidases (6-8). In the intestinal and hepatic cells, flavanone
aglycones are extensively converted into glucuronides and sul-
fates so that the entire fraction of dietary flavanone that crosses
the intestinal barrier finally enters the general blood circulation
as conjugates, mainly glucuronides (5, 9-11). In particular, nar-
tri-O-acetyl-1-O-(trichloroacetimidoyl)-R-D-glucuronate) was synthesized
from glucurono-3,6-lactone according to a three-step procedure already
described in the literature (12, 13).
ingenin 40- and 7-O-β- -glucuronides and hesperetin 30- and 7-O-β-
D
NMR. 1D 1H and 13C NMR spectra of synthetic intermediates were
recorded at 300 MHz on a Bruker Advance DPX-300 apparatus. 1D 1H
and 13C NMR spectra and 2D 1H-1H (COSY) and 1H-13C (HMBC,
HSQC) spectra of the final products were recorded at 500 MHz on a
Bruker Advance DRX-500 apparatus. NMR chemical shifts (δ) are in
parts per million (ppm) relative to tetramethylsilane using the deuterium
D-glucuronides were all detected in the plasma and urine of
human volunteers having consumed a single portion of orange
fruit or juice (11).
Despite the detailed information now available on the bio-
availability of flavanones, there is still a great need of purified
metabolites for accurate titration and identification in biological
fluids. In addition, a better knowledge of the biochemical mech-
anisms by which dietary flavanones exert their potential health
1
signal of the solvent (CDCl3, CD3OD) for calibration. H-1H coupling
constants (J) are in hertz (Hz).
HR-MS. High-resolution mass analyses were carried out on a Qstar
Elite instrument (Applied Biosystems SCIEX, Foster City, CA) equipped
with an atmospheric pressure ionization (API) interface. Mass detec-
tion was performed in the positive electrospray ionization mode in the
*Author to whom correspondence should be addressed [phone (33) 490
14 44 46; fax (33) 490 14 44 41; e-mail Olivier.Dangles@univ-avignon.fr].
© 2010 American Chemical Society
Published on Web 06/30/2010
pubs.acs.org/JAFC

8438 J. Agric. Food Chem., Vol. 58, No. 14, 2010
Khan et al.
following conditions: ion spray voltage, 5.5 kV; orifice voltage, 40 V;
nebulizing gas (air) pressure, 20 psi. The mass spectra were obtained with a
time-of-flight (TOF) analyzer. The accurate mass measurement was
performed in triplicate along with internal calibration. The ions chosen
for internal references were the ammonium adducts [MþNH4]þ of two
oligomers of polypropylene glycol (PPG425) (m/z 442.3374 and 500.3792).
Analytical HPLC. HPLC analyses were performed on a Waters 600
Controller HPLC system equipped with a Waters 2996 photodiode array
detector and monitored by the Waters Empower 2 chromatography data
software. The wavelength used for the HPLC analyses was 280 nm. The
chromatographic separation was carried out on a LiChrospher 100 RP-18
end-capped column (250ꢀ4 mm; 5 μm particle size) held at 37 °C. The
flow rate was set at 1 mL min-1. A mixture of 0.05% aqueous HCO2H and
MeCN (7:3) was used for the elution.
Semipreparative HPLC. Purifications were carried out on a Waters
600 chromatograph coupled to a UV-vis Waters 486 detector and equip-
ped with a Waters Atlantis PrepT3OBD 5 μm (19 mm ꢀ 150 mm) column.
The solvent was 0.05% aqueous HCO2H/MeCN (7:3). The flow rate was
18 mL min-1. Detection was carried out at 280 nm. Fractions obtained were
concentrated under vacuum and freeze-dried. Their purity was checked by
UPLC-MS analysis.
UPLC-MS. UPLC-MS analyses were performed on the Acquity
Ultra Performance LC (UPLC) apparatus from Waters, equipped with
an UV-visible diode array detector (DAD) and coupled with a Bruker
Daltonics HCT ultra ion trap mass spectrometer with a negative electro-
spray ionization (ESI) mode. The separation was conducted on a 1.7 μm
(2.1-50 mm) Acquity UPLC BEH C18 column thermostated at 30 °C
with an isocratic elution (0.05% aqueous HCO2H/MeCN (7:3)) at a flow
rate of 0.2 mL min-1. The mass spectra were generated in the Ultrascan
mode in the m/z range of 100-900. The ion source parameters were as
follows: nebulizer pressure, 40 psi; drying gas flow, 9 L min-1; drying gas
temperature, 350 °C.
7-O-Benzoylhesperetin (3b). The same procedure as for 3a was used by
starting from 2b: yield, 90%; light brown powder; mp, 146-147 °C;
Rf (cHex/EtOAc, 6:4) 0.50; 1H NMR (CD3OD), δ 8.20 (2H, d, J = 7.2,
H2Bz, H6Bz), 7.70 (1H, t, J = 7.2, H4Bz), 7.55 (2H, t, J = 7.2, H3Bz,
H5Bz), 6.93-6.92 (3H, m, H20, H50, H60), 6.46 (1H, d, J = 2.1, H6), 6.44
(1H, d, J = 2.1, H8), 5.49 (1H, dd, J = 12.7, 3.0, H2), 3.87 (3H, s, OMe),
3.07 (1H, dd, J = 12.7, 17.0, H3a), 2.82 (1H, dd, J = 3.0, 17.0, H3b); 13C
NMR (CD3OD), δ 197.22 (C4), 164.05, 163.37, 162.42 (OdC;Ph, C5,
C9), 158.74 (C7), 147.08 (C30 or C40), 145.97 (C30 or C40), 133.98 (C10 or
C4Bz), 131.15 (C10 or C4Bz), 130.32, 128.68 (C1Bz, C2Bz, C3Bz, C5Bz,
C6Bz), 118.20 (C60), 112.64 (C20 or C50), 110.70 (C20 or C50), 106.32
(C10), 103.39 (C6 or C8), 101.91 (C6 or C8), 79.07 (C2), 56.06 (OCH3),
43.43 (C3).
40,5,7-Tri-O-benzoylnaringenin (4a). Compound 2a (10 g, 37 mmol)
and benzoyl chloride (12 mL, 3 equiv) were dissolved in 700 mL of THF,
and the mixture was cooled in ice-water. Then, 50 mL of triethylamine
was added dropwise. The mixture was allowed to warm to room temp-
erature and stirred overnight. Then it was diluted with EtOAc (500 mL),
washed with saturated aqueous NaHCO3 (100 mL) and NaCl (100 mL),
dried over Na2SO4, filtered, and evaporated. The crude solid was
triturated in n-hexane several times to give 4a: yield, 85%; light brown
powder; mp, 112-113 °C; Rf (cHex/EtOAc, 6:4) 0.76; 1H NMR (CDCl3),
δ 8.27 - 8.19 (6H, m, 3H2bz, 3H6bz), 7.71-7.66 (3H, m, 3H4Bz),
7.66-7.48 (8H, m, 3H3Bz, 3H5Bz, H20, H60), 7.31 (2H, d, J = 8.6, H30,
H50), 7.02 (1H, d, J = 2.2, H6), 6.86 (1H, d, J = 2.2, H8), 5.61 (1H, dd,
J = 13.5, 2.7, H2), 3.12 (1H, dd, J = 13.5, 16.7, H3a), 2.84 (1H, dd, J =
2.7, 16.7, H3b); 13C NMR (CDCl3), δ 188.85 (C4), 165.05, 164.99, 163.91,
163.29 (3OdC;Ph, C5, C9), 156.33 (C7), 151.30 (C40), 135.78, 134.14,
133.76, 133.59 (3C4Bz, C10), 130.36, 128.64 (3C1Bz, 3C2Bz, 3C3Bz,
3C5Bz, 3C6Bz), 127.44 (C20, C60), 122.26 (C30, C50), 112.10, 111.13,
109.45 (C6, C8, C10), 79.18 (C2), 45.24 (C3).
30,5,7-Tri-O-benzoylhesperetin (4b). The same procedure as for 4a was
used by starting from 2b rather than from 2a: yield, 85%; light brown
powder; mp, 101-102 °C; Rf (cHex/EtOAc, 6:4) 0.68; 1H NMR (CDCl3),
δ 8.21-8.17 (6H, m, 3H2Bz, 3H6Bz), 7.80-7.75 (3H, m, 3H4Bz),
7.75-7.50 (6H, m, 3H3Bz, 3H5Bz), 7.43 (1H, dd, J = 8.5, 2.1, H60),
7.37 (1H, d, J = 2.1, H20), 7.19 (1H, d, J = 8.5, H50), 7.07 (1H, d, J =
2.2, H6), 6.90 (1H, d, J = 2.2, H8), 5.66 (1H, dd, J = 13.0, 2.6, H2), 3.87
(3H, s, OMe), 3.07 (1H, dd, J = 13.0, 17.1, H3a), 2.82 (1H, dd, J = 2.6,
17.1, H3b); 13C NMR (CDCl3), δ 188.98 (C4), 164.98, 164.58, 163.89,
163.32 (3OdC;Ph, C5, C7, C9), 151.57 (C40), 140.19 (C30), 134.11,
133.59 (3C4Bz, C10), 130.70, 128.56 (3C1Bz, 3C2Bz, 3C3Bz, 3C5Bz,
3C6Bz), 124.94 (C20 or C60), 121.21 (C20 or C60), 112.64, 112.07, 111.03,
109.42 (C50, C6, C8, C10), 78.97 (C2), 56.12 (OCH3), 44.98 (C3).
40,5-Di-O-benzoylnaringenin (5a). Compound 4a (5 g, 8.5 mmol) and
imidazole (0.465 g, 0.7 equiv) were dissolved in N-methyl-2-pyrrolidinone
(NMP, 5 mL). Then, thiophenol (0.87 mL, 1 equiv) was slowly added at
0 °C. The resulting mixture was stirred for 1 h at room temperature and
then diluted with EtOAc (50 mL) and washed with saturated aqueous
NaCl (3 ꢀ 100 mL) and with 1 M HCl (100 mL). The organic layer was
dried over Na2SO4 and concentrated under reduced pressure. The crude
solid was triturated with cyclohexane and precipitated from a n-hexane/
AcOEt mixture to give 5a (4 g): yield, 95%; white powder; mp, 218-
219 °C; Rf (cHex/EtOAc, 6:4) 0.50; 1H NMR (CD3OD), δ 8.30-8.16 (4H,
m, 2H2Bz, 2H6Bz), 7.70-7.66 (2H, m, 2H4Bz), 7.64-7.50 (6H, m,
2H3Bz, 2H5Bz, H20, H60), 7.32 (2H, d, J = 8.6, H30, H50), 6.42 (1H, d,
J = 2.3, H6), 6.32 (1H, d, J = 2.3, H8), 5.59 (1H, dd, J = 12.9, 2.9, H2),
3.06 (1H, dd, J = 12.9, 16.7, H3a), 2.70 (1H, dd, J = 2.9, 16.7, H3b); 13C
NMR (CD3OD), δ 192.63 (C4), 178.79, 174.58 (2OdC;Ph, C5 or C7,
C9), 155.13 (C5 or C7), 151.82 (C40), 133.28, 132.28 (2C4Bz, C10), 130.61,
129.22 (2C1Bz, 2C2Bz, 2C3Bz, 2C5Bz, 2C6Bz), 128.74 (C20, C60), 122.50
(C30, C50), 103.61, 100.13, 96.92 (C6, C8, C10), 84.34 (C2), 39.62 (C3).
30,5-Di-O-benzoylhesperetin (5b). The same procedure as for 5a was
used by starting from 4b: yield, 95%; white powder; mp, 197-198 °C;
Rf (cHex/EtOAc, 6:4) 0.35; 1H NMR (CD3OD), δ 8.21-8.17 (4H, m,
2H2Bz, 2H6Bz), 7.80-7.75 (2H, m, 2H4Bz), 7.75-7.50 (4H, m, 2H3Bz,
2H5Bz), 7.43 (1H, dd, J = 8.5, 2.1, H60), 7.37 (1H, d, J = 2.1, H20), 7.19
(1H, d, J = 8.5, H50), 6.41 (1H, d, J = 2.4, H6), 6.32 (1H, d, J = 2.4, H8),
5.51 (1H, dd, J = 13.0, 2.8, H2), 3.87 (3H, s, OMe), 3.07 (1H, dd, J = 13.0,
17.0, H3a), 2.82 (1H, dd, J = 2.8, 17.0, H3b); 13C NMR (CDCl3), δ 188.79
(C4), 165.56, 164.92, 164.12, 163.79 (2OdC;Ph, C5, C7, C9), 151.65
Molecular Modeling. Semiempirical quantum mechanics calcula-
tions were performed in vacuum with Hyperchem software (Autodesk,
Sausalito, CA) using the PM3 program.
Synthesis. Naringenin/Hesperetin (2a/2b). 2a/2b are commercially
available compounds. 2a: mol wt, 272.25 g mol-1; light brown powder;
mp, 252-253 °C; Rf (cyclohexane (cHex)/EtOAc, 6:4) 0.36; 1H NMR
(CD3OD), δ 7.33 (2H, d, J = 8.5, H20, H60), 6.82 (2H, d, J = 8.5, H30,
H50), 5.90 (1H, d, J = 2.2, H6), 5.89 (1H, d, J = 2.2, H8), 5.33 (1H, dd,
J = 13.0, 3.0, H2), 3.12 (1H, dd, J = 13.0, 17.0, H3a), 2.69 (1H, dd, J =
3.0, 17.0, H3b); 13C NMR (CD3OD), δ 198.19 (C4), 168.76 (C7), 165.88
(C5 or C9), 165.30 (C5 or C9), 159.43 (C40), 131.50 (C10), 129.45 (2C, C20,
C60), 116.74 (2C, C30, C50), 103.77 (C10), 97.47 (C6 or C8), 96.58 (C6 or
C8), 80.89 (C2), 44.44 (C3). 2b: mol wt, 302.28 g mol-1; light yellow
powder; mp, 235-236 °C; Rf (cHex/EtOAc, 6:4) 0.27; 1H NMR
(CD3OD), δ 6.93-6.92 (3H, m, H20, H50, H60), 5.92 (1H, d, J = 2.2,
H6), 5.89 (1H, d, J = 2.2, H8), 5.32(1H, dd, J = 12.6, 3.1, H2), 3.87 (3H, s,
OCH3), 3.07 (1H, dd, J = 12.6, 17.1, H3a), 2.72 (1H, dd, J = 3.1, 17.1,
H3b); 13C NMR (CD3OD), δ 198.01 (C4), 168.77 (C7), 165.87 (C5 or C9),
165.17 (C5 or C9), 149.76 (C30 or C40), 148.19 (C30 or C40), 133.55 (C10),
119.40 (C60), 114.94 (C50), 112.97 (C20), 103.78 (C10), 97.47 (C6 or C8),
96.59 (C6 or C8), 80.69 (C2), 56.83 (OCH3), 44.49 (C3).
7-O-Benzoylnaringenin (3a). Benzoyl chloride (2.15 mL, 18.5 mmol)
was added dropwise to an ice-cold solution of 2a (1 equiv) in pyridine
(15 mL), and the reaction mixture was stirred overnight at room temp-
erature. The mixture was extracted with EtOAc (300 mL) and then washed
with water (200 mL), 2 M HCl (3 ꢀ 100 mL), saturated aqueous NaHCO3
(100 mL), and saturated aqueous NaCl (100 mL). The combined organic
extract was dried over Na2SO4, filtered, and concentrated. The product
was purified by precipitation from EtOAc/n-hexane to give 3a with a yield
of about 90%: light brown powder; mp, 138-139 °C; Rf (cHex/EtOAc,
6:4) 0.58; 1H NMR (CD3OD), δ 8.18 (2H, d, J = 7.2, H2Bz, H6Bz), 7.71
(1H, t, J = 7.2, H4Bz), 7.56 (2H, t, J = 7.2, H3Bz, H5Bz), 7.36 (2H, d, J =
8.6, H20, H60), 6.85 (2H, d, J = 8.6, H30, H50), 6.47-6.45 (2H, br s, H6,
H8), 5.50 (1H, dd, J = 13.0, 2.8, H2), 3.2 (1H, dd, J = 13.0, 17.2, H3a),
2.90 (1H, dd, J = 2.8, 17.2, H3b); 13C NMR (CD3OD), δ 197.24 (C4),
164.10, 163.41, 162.46 (OdC;Ph, C5, C9), 158.75 (C40 or C7), 156.17
(C40 or C7), 134.01 (C10, C4Bz), 130.32, 128.69 (C1Bz, C2Bz, C3Bz, C5Bz,
C6Bz), 127.97 (C20, C60), 115.73 (C30, C50), 106.31 (C10), 103.41 (C6 or
C8), 101.91 (C6 or C8), 79.06 (C2), 43.45 (C3).

Article
J. Agric. Food Chem., Vol. 58, No. 14, 2010 8439
(C40), 140.04 (C30), 133.67, 133.44, 131.29 (2C4Bz, C10), 130.38, 128.53
(2C1Bz, 2C2Bz, 2C3Bz, 2C5Bz, 2C6Bz), 125.08 (C20 or C60), 121.18
(C20 or C60), 112.59 (C50), 107.48, 105.92, 101.96 (C6, C8, C10), 78.65
(C2), 56.09 (OCH3), 44.86 (C3).
7-O-Benzoylnaringenin 40-O-[Methyl (200,300,400-tri-O-acetyl)-β-
D-gluco-
pyranosyl uronate] (6a). Compound 3a (0.5 g, 1.3 mmol) was added to a
H500), 3.87 (3H, s, OMe hesperetin), 3.74 (3H, s, OMe GlcU), 3.07 (1H, dd,
J = 13.0, 17.0, H3a), 2.82 (1H, dd, J = 2.9, 17.0, H3b), 2.08, 2.08, 2.07 (3 ꢀ
3H, br s, 3Ac); 13C NMR (CDCl3), δ 188.61 (C4), 169.97, 169.34, 169.11
(3OCOCH3, C600), 166.62, 164.60, 163.85, 161.69 (2OdC;Ph, C5, C7,
C9), 152.07 (C40), 140.21 (C30), 133.63, 130.67 (2C4Bz, C10), 130.36,
128.58 (2C1Bz, 2C2Bz, 2C3Bz, 2C5Bz, 2C6Bz), 124.98 (C20 or C60),
121.21 (C20 or C60), 112.63 (C50), 109.98, 106.39, 102.57, 97.71 (C100, C6,
C8, C10), 79.02 (C2), 72.73, 71.48, 70.67, 68.82 (C200, C300, C400, C500),
56.12 (OCH3 hesperetin), 53.06 (OCH3 GlcU), 43.73 (C3), 20.59
(3OCOCH3).
solution of glucuronyl donor (1) (0.45 g, 1 equiv) in dry CH2Cl2 (15 mL)
˚
containing 4 A molecular sieves. The reaction mixture was cooled in
an ice-water bath for 15 min under N2 atmosphere. Then, BF3 Et2O
3
(170 μL, 1 equiv) was added, and the mixture was stirred at room tempera-
ture for about 30 min. Then, the mixture was diluted with EtOAc, filtered
on Celite, concentrated, and purified by column chromatography (cHex/
EtOAc; 6:4) to provide 6a: yield, 50%; white solid; mp, 149-150 °C;
Rf (cHex/EtOAc, 6:4) 0.31; 1H NMR (CDCl3), δ 11.89 (1H, s, C5-OH),
8.18 (2H, d, J = 7.3, H2Bz, H6Bz), 7.67 (1H, t, J = 7.3, H4Bz), 7.53 (2H, t,
J = 7.3, H3Bz, H5Bz), 7.43 (2H, d, J = 8.7, H20, H60), 7.08 (2H, d, J =
8.7, H30, H50), 6.47 (1H, br d J = 2.0, H6 or H8), 6.45 (1H, m, H60o0 r H8)
5.48 (1H, dd, J = 13.0, 2.8, H2), 5.39 - 5.30 (3H, m, H200, H300, H4 ), 5.20
(1H, d, J = 7.1, H100), 4.23 (d, J = 7.0) plus 4.22 (d, J = 7.0, H500) (1H,
H500), 3.75 (3H, s, OMe), 3.15 (1H, dd, J = 13.0, 17.0, H3a), 2.9 (1H, dd,
Naringenin 40-O-β-
D-Glucuronide (8a). To a solution of 6a (0.85 mmol)
in MeOH (30 mL) under N2 atmosphere at 0 °C was slowly added a 0.5 M
aqueous solution of Na2CO3 (13 mL). The mixture was stirred at room
temperature with HPLC monitoring at 280 nm every hour. After 5 h, 8a
was detectedas a single product (estimatedanalytical yield ca. 60%). Then,
Dowex 50WX4-50 ion-exchange resin (Hþ form) was added under stir-
ring to lower the pH to ca. 6. The resin was filtered off and the filtrate
concentrated at 50 °C under vacuum. Purification was carried out by
semipreparative HPLC: overall yield, 30%; white amorphous powder;
Rf (n-BuOH/CH3CO2H/H2O, 10:1:1) 0.18; HPLC (0.05% aq HCO2H/
MeCN (7:3)), tR = 2.41 min, λmax = 290, 330 nm; 1H NMR (D2O), δ 7.37
(2H, d, J = 7.9, H20, H60), 7.07 (2H, d, J = 7.9, H30, H50), 5.91 (2H, br s,
H6, H8), 5.53 (1H, br d, J = 11.2, H2), 5.05 (1H, br d, J = 6.9, H100), 3.90
(1H, d, J = 8.4, H500), 3.69-3.61 (3H, m, H200, H300, H400), 3.26 (dd, J =
17.0, 11.2) plus 3.23 (dd, J = 17.0, 11.2) (1H, H3a), 2.86 (1H, br d, J =
17.0, H3b); DEPTQ 13C NMR (D2O), δ 196.56 (C4), 174.03 (C600), 170.22,
167.13, 164.22 (C5, C7, C9), 134.05 (C10), 129.65 (C20, C60), 118.30 (C30,
C50), 103.74 (C10), 101.47 (C100), 97.76, 96.97 (C6, C8), 79.96 (C2), 76.45,
76.24, 73.93, 72.64 (C200, C300, C400, C500), 42.98 (C3); HRMS-ESI, m/z
[M þ H]þ calcd for C21 H21O11, 449.1078; found, 449.1082; molar ratio of
epimers ca. 1:1 (based on H3a integration) (see the Supporting Information).
13
J = 2.8, 17.0, H3b), 2.09, 2.08, 2.07, 2.06 (9H, 4bs, 3Ac); C NMR
(CDCl3), δ 196.90 (C4), 170.10, 169.34, 169.22 (3OCOCH3, C600), 166.80,
164.04, 163.40, 162.25 (OdC;Ph, C5, C9), 158.79 (C40 or C7), 157.04
(C40 or C7), 134.02, 133.08 (C4Bz, C10), 130.31, 128.70 (C1Bz, C2Bz,
C3Bz, C5Bz, C6Bz), 127.75 (C20, C60), 117.38 (C30, C50), 106.29, 103.53,
101.91, 98.97 (C100, C6, C8, C10), 78.76 (C2), 72.71, 71.82, 71.05, 69.07
(C200, C300, C400, C500), 53.02 (OCH3 GlcU), 43.45 (C3), 20.62 (3OCOCH3).
7-O-Benzoylhesperetin 30-O-[Methyl (200,300,400-tri-O-acetyl)-β-
D-gluco-
pyranosyl uronate] (6b). The same procedure as for 6a was used by starting
from 3b: yield, 50%; light yellow powder; mp, 115-116 °C; Rf (cHex/
EtOAc, 6:4) 0.19; 1H NMR (CDCl3), δ 11.89 (s) plus 11.88 (s) (1H, C5-
OH), 8.18 (2H, d, J = 7.3, H2Bz, H6Bz), 7.67 (1H, t, J = 7.3, H4Bz), 7.53
(2H, t, J = 7.3, H3Bz, H5Bz), 7.28 (1H, br s, H20), 7.19 (dd, J = 8.5, 2.3)
plus 7.18 (dd, J = 8.5, 2.3) (1H, H60), 6.96 (d, J = 8.5) plus 6.95 (d, J =
8.5) (1H, H50), 6.44-643 (2H, m, H6, H8) 5.51-5.20 (4H, m, H400, H200,
H300, H2), 5.10 (d, J = 6.5) plus 5.08 (d, J = 6.5) (1H, H100), 4.15 (d, J =
9.2) plus 4.12 (d, J = 9.2) (1H, H500), 3.87 (3H, s, OMe hesperetin),
3.74 (3H, s, OMe GlcU), 3.07 (1H, dd, J = 12.6, 17.0, H3a), 2.82 (1H, dd,
J = 2.9, 17.0, H3b), 2.1 (3H, s, Ac), 2.06 (3H, s, Ac), 2.04 (s) plus 2.01 (s)
(3H, Ac); 13C NMR (CDCl3), δ 196.99 (C4), 170.16, 169.37, 169.29
(3OCOCH3, C600), 166.87, 163.40, 162.21 (OdC;Ph, C5, C9), 158.76
(C7), 151.24 (C40), 145.75 (C30), 134.02, 130.50, 130.37, 130.32, 128.70
(C10, C1Bz, C2Bz, C3Bz, C4Bz, C5Bz, C6Bz), 123.07 (C50 or C60), 118.81
(C50 or C60), 112.76 (C20), 106.32, 103.50, 101.93, 100.58 (C100, C6, C8,
C10), 78.62 (C2), 72.56, 71.87, 71.12, 69.27 (C200, C300, C400, C500), 56.14
(OCH3 hesperetin), 52.98 (OCH3 GlcU), 43.03 (C3), 20.66 (3OCOCH3).
Hesperetin 30-O-β-
D-Glucuronide (8b). The same procedure as for 8a
was used by starting from 6b: overall yield, 30%; white amorphous
powder; Rf (n-BuOH/CH3CO2H/H2O, 10:1:1) 0.21; HPLC (0.05% aq
HCO2H/MeCN (7:3)), tR = 4.67 min, λmax = 288, 333 nm; 1H NMR
(D2O), δ 7.30 (d, J = 8.4) plus 7.28 (d, J = 8.4) (1H, H60), 7.23 (1H, br s,
H20), 7.19 (d, J = 8.4) plus 7.16 (d, J = 8.4) (1H, H50), 6.02 (1H, br s, H6
or H8), 6.00 (1H, br s, H6 or H8), [5.55 (1H, dd, J = 3.1, 12.0) plus 5.47
(dd, J = 3.1, 12.0)] (1H, H2), 4.7 (br d, J = 5.5, partly masked by the water
peak), 3.78 (3H, s, OMe), 3.88 (d, J = 9.8) plus 3.83 (d, J = 9.8) (1H, H500),
3.48-3.23 (3H, m, H200, H300, H400), 3.27 (dd, J = 12.0, 17.0) plus 3.19 (dd,
J = 12.0, 17.0) (1H, H3a), 2.85 (dd, J = 3.1, 17.0) plus 2.84 (dd, J = 3.1,
17.0) (1H, H3b); DEPT 13C NMR (D2O), δ 200.48 (C4), 167.08, 164.16
(C5, C7, C9, C600), 146.59 (C30, C40), 131.93 (C10), 123.22 (C60), 117.08
(C20), 114.38 (C50), 103.63 (C10), 101.72 (C100), 97.92, 97.00 (C6, C8),
80.34 (C2), 76.67, 76.56, 73.78, 72.76 (C200, C300, C400, C500), 57.20 (OCH3),
42.86 (C3); molar ratio of epimers ca. 7:3 (based on H2 integration);
HRMS-ESI, m/z [M þ H]þ calcd for C22H23O12, 479.1184; found, 479.1185.
40,5-Di-O-benzoylnaringenin 7-O-[Methyl (200,300,400-tri-O-acetyl)-β-
D-
glucopyranosyl uronate] (7a). The same procedure as for 6a was used by
starting from 5a: yield, 50%; white solid; mp, 165-166 °C; Rf (cHex/
Naringenin 7-O-β-D-Glucuronide (9a). The same procedure as for 8a
1
EtOAc, 6:4) 0.32; H NMR (CDCl3), δ 8.14 (2H, br d, J = 7.0, H2Bz,
was used by starting from 7a: overall yield, 30%; white amorphous
powder; Rf (n-BuOH/CH3CO2H/H2O, 10:1:1) 0.18; HPLC (0.05% aq
HCO2H/MeCN (7:3)), tR = 2.38 min, λmax = 283, 329 nm; 1H NMR
(D2O), δ 7.43 (2H, d, J = 8.0, H20, H60), 6.96 (2H, d, J = 8.0, H30, H50),
6.27 (1H, br s, H6 or H8), 6.24 (1H, br s, H6 or H8),5.49 (1H, br d, J =
14.1, H2), 5.12 (1H, m, H100), 3.77 (1H, br d, J = 9.3, H500), 3.67-3.54
(3H, m, H200, H300, H400), 3.27 (1H, br dd, J = 16.7, 14.1, H3a), 2.85 (1H,
H6Bz), 8.06 (2H, m, H2Bz, H6Bz), 7.60-7.50 (2H, m, H4Bz), 7.46-7.40
(4H, m, H3Bz, H5Bz, H20, H60), 7.22 (2H, d, J = 8.6, H30, H50), 6.10 (d,
J = 2.0, H6 or H8) plus 6.09 (d, J = 2.0) (1H, H6 or H8), 6.07 (d, J = 2.0,
H6 or H8) plus 6.07 (d, J = 2.0) (1H, H6 or H8), 5.40 (dd, J = 13.0, 2.9,
H2), 5.30-5.13 (4H, m, H100, H200, H300, H400), 4.14 (d, J = 8, H500) plus
4.13 (d, J = 8, H500), 3.75 (3H, s, OMe GlcU), 3.0 (1H, dd, J = 13.0, 18.0,
H3a), 2.8 (1H, dd, J = 2.9, 18.0, H3b), 1.18 (3 ꢀ 3H, bs, 3Ac); 13C NMR
(CDCl3), δ 196.07 (C4), 170.04, 169.35, 169.10 (3OCOCH3, C600), 166.61,
165.06, 163.96 (2OdC;Ph, C5 or C7, C9), 154.72 (C5 or C7), 151.33
(C40), 133.80, 133.70 (2C4Bz, C10), 130.24, 129.20 (2C1Bz, 2C2Bz, 2C3Bz,
2C5Bz, 2C6Bz), 127.43 (C20, C60), 122.30 (C30, C50), 110.55, 104.53, 97.58,
96.32 (C100, C6, C8, C10), 78.80 (C2), 72.74, 71.55, 70.70, 68.91 (C200, C300,
C400, C500), 53.09 (OCH3 GlcU), 43.40 (C3), 20.50 (3OCOCH3).
13
br d, J = 16.7, H3b); C NMR (D2O), δ 198.32 (C4), 172.15 (C600),
164.63, 163.27, 162.59, 162.59 (C5, C7, C9), 156.69 (C40), 134.13 (C10),
129.98 plus 129.06 (C20, C60, 2 epimers), 115.91 (C30, C50), 104.13 (C10),
99.02 (C100), 97.47, 96.27 (C6, C8), 79.20 (C2), 75.28, 74.87, 72.53, 71.37
(C200, C300, C400, C500), 42.63 plus 41.59 (C3, 2 epimers); HRMS-ESI, m/z
[M þ H]þ calcd for C21 H21O11, 449.1078; found, 449.1078.
Hesperetin 7-O-β-D-Glucuronide (9b). The same procedure as for 8a
30,5-Di-O-benzoylhesperetin 7-O-[Methyl (200,300,400-tri-O-acetyl)-β-
D-
was used by starting from 7b: overall yield, 30%; light brown amorphous
powder; Rf (n-BuOH/CH3CO2H/H2O, 10:1:1) 0.17; HPLC (0.05% aq
HCO2H/MeCN (7:3)), tR = 4.51 min, λmax = 284, 328 nm; 1H NMR
(D2O), δ 6.83-6.77 (3H, m, H50, H60, H20), 6.07 (1H, br s, H8), 6.03 (1H,
br s, H6), 5.04 (1H, br d, J = 14.8, H2), 4.80 (1H, d, J = 7.5, H100), 3.70
(3H, s, OMe), 3.59-3.34 (4H, m, H200,H300, H400, H500), 2.83 (1H, br t (dd),
J = 16.0, 14.8, H3a), 2.54 (1H, br d, J = 16.0, H3b); 13C NMR (D2O),
δ 199.31 (C4), 165.78, 165.62, 164.22, 163.62 (C5, C7, C9, C600), 146.35
glucopyranosyl uronate] (7b). The same procedure as for 6a was used by
starting from 5b: yield, 50%; white powder; mp, 119-120 °C; Rf (cHex/
EtOAc, 6:4) 0.25; 1H NMR (CDCl3), δ 8.21-8.17 (4H, m, H2Bz, H6Bz),
7.78 (2H, m, H4Bz), 7.75-7.50 (4H, m, H3Bz, H5Bz), 7.33 (1H, dd, J =
2.1, 9.0, H60), 7.37 (1H, d, J = 2.1, H20), 7.19 (1H, d, J = 9.0, H50), 6.59
(1H, d, J = 2.4, H8), 6.50 (1H, d, J = 2.4, H6), 5.51 (1H, dd, J = 13.0, 2.9,
H2), 5.40-5.20 (4H, m, H100, H200, H300, H400), 4.23 (1H, br d, J = 7.6,

8440 J. Agric. Food Chem., Vol. 58, No. 14, 2010
Khan et al.
Figure 1. Glucuronidation of citrus flavanones on the B-ring: (i) BzCl (1.1-1.5 equiv), pyridine; (ii) methyl 2,3,4-tri-O-acetyl-1-O-(trichloroacetimidoyl)-R-
D-
glucuronate (1), BF3-OEt2 (2 equiv), CH2Cl2; (iii) Na2CO3 (1.2equivper ester group), H2O/MeOH(2:5), thenacidification topH 6 by Dowex resin (Hþ form).
(C30 or C40), 144.07 (C30 or C40), 132.37 (C10), 120.210(0 C60), 114.94 (C20 or
C50), 113.50 (C20 or C50), 105.17 (C10), 100.11 (C1 ), 98.46, 97.17 (C6,
C8), 80.24 (C2), 76.52, 76.36, 73.59, 72.51 (C200, C300, C400, C500), 57.18
(OCH3 hesperetin), 43.59 (C3); HRMS-ESI, m/z [M þ H]þ calcd for
C22H23O12, 479.1184; found, 479.1185.
glucuronide (15), and a series of hydroxycinnamic acid O-β-
glucuronides (16). Catechin O-β- -glucuronides were also pre-
pared with methyl-2,3,4-tri-O-acetyl-1-O-bromo-R- -glucuro-
nate as the glucuronyl donor (17). Recently, the synthesis of a
flavanone glucuronide (persicogenin 30-O-β-
-glucuronide) was
carried out with methyl-2,3,4-tri-O-acetyl-1-O-(trifluoroacetimidoyl)-
R- -glucuronate, followed by a final deprotection step involving
pig liver esterase (PLE) for the hydrolysis of the methyl ester of
the glucuronyl residue (18). A synthesis of quercetin 3-O-β-
glucuronide was also performed by regioselective oxidation of the
corresponding 3-O-β- -glucoside (phenolic OH groups protected
D-
D
D
D
RESULTS AND DISCUSSION
D
Investigations over the past three decades have shown that
dietary polyphenols are only moderately bioavailable and that the
fraction crossing the intestinal barrier is typically extensively
metabolized in the intestinal and hepatic cells. Thus, the potential
cell effects of dietary polyphenols must be mainly mediated by
their metabolites, of which glucuronides make the largest con-
tribution. Hence, there is a growing interest for polyphenol
glucuronides as standards for identification and titration of in
vivo metabolites and as biologically pertinent compounds for cell
studies aiming at elucidating the potential health effects of poly-
phenols. Several works have been published about the chemical
synthesis of polyphenol glucuronides. For instance, the popular
procedure, based on the Lewis acid-activated coupling of methyl-
D-
D
as benzyl ethers) using TEMPO/NaOCl/NaBr under phase
transfer conditions (19).
In this particular work, an efficient chemical synthesis of the
four major metabolites of citrus flavanones (hesperetin 7- and
30-O-β-
D
-glucuronides, naringenin 7- and 40-O-β-
D-glucuronides)
is reported. The glucuronides were completely characterized by
NMR, HRMS, and UPLC-MS.
In a preliminary study, partial protection of the flavanone
OH groups was achieved by acetylation (20). However, the low
stability of the arylacetate groups in the glucuronidation step
resulted in low yields and tedious purifications. By contrast,
partial protection by benzoylation was found to be satisfactory.
2,3,4-tri-O-acetyl-1-O-(trichloroacetimidoyl)-R-
with partially protected polyphenols, was applied to the synthesis
of isoflavone 7-O-β-
-glucuronides (14), quercetin 30-O-β-
D-glucuronate (1)
D
D-

Article
J. Agric. Food Chem., Vol. 58, No. 14, 2010 8441
Figure 2. Glucuronidation of citrus flavanones on the A-ring: (i) BzCl (excess), NEt3, THF, 0 °C; (ii) PhSH (1 equiv), imidazole, NMP; (iii) methyl 2,3,4-tri-O-
acetyl-1-O-(trichloroacetimidoyl)-R- -glucuronate (1), BF3-OEt2 (2 equiv), CH2Cl2; (iv) Na2CO3 (1.2 equiv per ester group), H2O/MeOH (2:5), then
D
acidification to pH 6 by Dowex resin (Hþ form).
Among the three phenolic OH groups of hesperetin and nar-
ingenin, C7-OH is the most acidic because of the stabilization of the
corresponding phenolate with the keto group. Such an activation
is absent for the B-ring OH groups and is canceled out for C5-OH
by the strong hydrogen bond it forms with the keto group. Hence,
both hesperetin and naringenin can be selectively benzoylated at
C7-OH in the presence of one equivalent of benzoyl chloride at
0 °C (Figure 1). As expected, benzoylation at C7-OH induced a
deshielding of the C6, C8, and C10 NMR signals. A strong
shielding of the C7 signal was also noted. This first step allowed us
to carry out the regioselective glucuronidation of the B-ring phenolic
OH by compound 1 in the presence of the boron trifluoride-
diethyl ether complex as the Lewis acid. In the 1H NMR spectra
of the protected glucuronides 6a and 6b, most B-ring protons
(H30 and H50 in 6a, H20 and H50 in 6b) appeared to be strongly
deshielded with respect to the corresponding protons in 3a and 3b
as a consequence of the weakening of the electron-donating effect
of the O atom by the attached glucuronyl group. In the glucuro-
nidation step, the unprotected strongly H-bonded C5-OH group
remained inactive.
For A-ring conjugation, hesperetin and naringenin were first
converted into the corresponding tri-O-benzoates (Figure 2).
Then, the selective thiolysis of the most reactive 7-O-benzoate
group was successfully achieved using thiophenol as the nucleo-
phile. Displacements of NMR signals opposite those recorded
upon selective benzoylation at C7-OH were observed. The flava-
none dibenzoates thus formed are convenient substrates for
glucuronidation at C7-OH, which was performed under the same
conditions as for B-ring conjugation.
degradation of the flavanone nuclei (probably starting by a retro-
Michael reaction leading to opening of the pyrane ring and
concomitant formation of chalcones). In particular, strongly
alkaline conditions (NaOH/EtOH or KOH/EtOH) and even
the milder methanolysis of the acetate and benzoate groups
catalyzed by MeONa led to extensive degradation. Among the
deprotection reagents tested, the most satisfactory one was a
solution of sodium carbonate (1.2 equiv per ester group) in water/
methanol (2:5). Such conditions were successfully used for the
synthesis of 7-hydroxycoumarin O-β-D
-glucuronide (22). The 1H
and 13C NMR signals of the deprotected glucuronides were
assigned from 2D COSY, HMBC, and HSQC experiments.
Our results are consistent with the literature (9, 21).
The major citrus flavanones are naringin (7-O-β-
R-1,2- -glucosyl)-(2S)-naringenin) and hesperidin (7-O-β-
rhamnosyl-R-1,6- -glucosyl)-(2S)-hesperetin). In both compounds,
D-(
L-rhamnosyl-
D
D-(L-
D
the configuration of C2 is S (1) as a consequence of the enantio-
selectivity of the chalcone isomerase enzyme catalyzing the forma-
tion of the flavanone nucleus by intramolecular Michael addition
within the chalcone precursor (23). However, mixtures of (2R)-
and (2S)-hesperidin epimers in an approximate molar ratio of
1:6 can be detected in orange juice (25), possibly because of the
propensity of flavanones to undergo epimerization at C2 via the
chalcone form. Interestingly, the minor 2R epimer seems about
twice as bioavailable as the 2S epimer based on the excreted rates
in urine of 2R and 2S hesperetin conjugates (25). In this work, the
commercially available racemic naringenin and hesperetin were
used. After the glucuronidation step, the 1H NMR spectra of the
glucuronides (except 7b) are complicated by the splitting of several
signals in two signals of nearly equal intensity, thus confirming that
the compounds are equimolar mixtures of epimers. For instance,
the NMR spectrum of 6b in CDCl3 displays two strongly de-
shielded singlets at ca. 11.9 ppm for the C5-OH proton, which is
In this work, the final deprotection step consisted of the
alkaline hydrolysis of one or two benzoates (flavanone nucleus)
along with three acetates and one methyl ester (sugar moiety).
It was found to be particularly difficult to complete without

8442 J. Agric. Food Chem., Vol. 58, No. 14, 2010
Khan et al.
Figure 3. UPLC-MS analyses of flavanone glucuronides.
strongly hydrogen-bonded to the neighboring O4 atom. This
splitting is also frequently observed not only for other protons of
the flavanone nucleus but also for some benzoyl, acetyl, and GlcU
protons, in particular H500. After deprotection, only the B-ring
glucuronides displayed some split signals confirming the pre-
the A-ring and B-ring glucuronides can be distinguished from their
UV spectra. Indeed, C7-OH is conjugated with the keto group through
the A-ring and as such contributes to the main UV absorption
band. Hence, glucuronidation of the A-ring causes a hypsochromic
shift of 4-5 nm in comparison to the parent aglycones (λmax
≈
sence of the two epimers. The fraction of hesperetin 30-O-β-
D-
288 nm), whereas glucuronidation of the B-ring leaves the spectra
unchanged. Molecular modeling experiments with the deprotected
glucuronides yielded low-energy conformations showing a pseudo-
equatorial position for the B-ring (vs C-ring) and an anti arrange-
ment of H2 and one of the H3 protons, which is consistent with the
observed coupling constants in the NMR spectra.
glucuronide (8b) purified by semipreparative HPLC was enriched
in one of them (7:3 molar ratio) during this step. The UPLC-MS
analysis did not permit the two epimers to be distinguished. The
molecular ion of each pure glucuronide (MS1) was sequentially
fragmented into aglycone and
D
-glucuronic acid (MS2) and,
finally, into the aglycone fragments (MS3) (Figure 3). No signifi-
cant differences were observed between regioisomers, thus indicat-
ing that they possess the same fragmentation patterns. Further-
more, the aglycone fragmentation patterns were consistent with the
literature (24-28). Unfortunately, it is impossible to differentiate
between the A-ring and B-ring glucuronides on the basis of their
mass fragmentation patterns as both MS2 spectra give the agly-
In conclusion, simple and reasonably efficient procedures
(10-15% overall yield from the starting aglycones) have been
developed for the chemical synthesis of four major citrus flava-
none metabolites. After a one- or two-step partial protection of
the flavanone moiety by benzoyl groups, the glucuronidation step
was carried out either on the A- or B-ring and followed by the
removal of all protecting groups in optimized mild alkaline condi-
tions, thereby avoiding significant degradation of the glucuronides.
cone and D-glucuronic acid as the main fragments. By contrast,

Article
J. Agric. Food Chem., Vol. 58, No. 14, 2010 8443
The affinity of the flavanone glucuronides for serum albumin, their
likely carrier in the blood plasma, and their cell effects in relation
with the protection against cardiovascular diseases are currently
investigated.
(13) Pearson, A. G.; Kiefel, M. J.; Ferro, V.; Itzstein, M. V. Towards
the synthesis of aryl glucuronides as potential heparanase probes.
An interesting outcome in the glycosidation of glucuronic acid
with 4-hydroxycinnamic acid. Carbohydr. Res. 2005, 340, 2077–
2085.
(14) Al-Maharik, N.; Botting, N. P. A facile synthesis of isoflavone 7-O-
glucuronides. Tetrahedron Lett. 2006, 47, 8703–8706.
(15) Needs, P. W.; Kroon, P. A. Convenient syntheses of metabolically
important quercetin glucuronides and sulfates. Tetrahedron 2006, 62,
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(16) Galland, S.; Rakotomanomana, N.; Dufour, C.; Mora, N.; Dangles,
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ACKNOWLEDGMENT
Svitlana Poix-Shinkaruk (ENITA, Bordeaux) is gratefully
acknowledged for helpful discussions. The Higher Education
Commission of Pakistan is gratefullyacknowledged for providing
a grant to MKK.
Supporting Information Available: Optimized conforma-
tions of the naringenin glucuronides and high-resolution mass
spectra of the citrus flavanone glucuronides. This material is
available free of charge via the Internet at http://pubs.acs.org.
(17) Gonzalez-Manzano, S.; Gonzalez-Paramas, A.; Santos-Buelga, C.;
~
Duenas, M. Preparation and characterization of catechin sulfates,
glucuronides, and methylethers with metabolic interest. J. Agric.
Food Chem. 2009, 57, 1231–1238.
(18) Boumendjel, A.; Blanc, M.; Williamson, G.; Barron, D. Efficient
synthesis of flavanone glucuronides. J. Agric. Food Chem. 2009, 57,
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Received for review March 20, 2010. Revised manuscript received
May 20, 2010. Accepted June 9, 2010. This work was supported by
the National Agency of Research (ANR) through the AGRUVASC
program (2007-2009).