Regio- and stereoselective synthesis of the major metabolite of quercetin, quercetin-3-O-β-D-glucuronide

Article abstract of DOI:10.1016/S0040-4039(02)01264-9

Protected quercetin was first transformed selectively in its 3-O-β-D-glucoside. Further protection of the remaining phenolic hydroxyl group allows a clean oxidation of the glucoside by TEMPO/NaOCl/NaBr under phase transfer conditions into the corresponding glucuronide which was cleanly deprotected to quercetin-3-O-β-D-glucuronide.

Full text of DOI:10.1016/S0040-4039(02)01264-9

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 43 (2002) 6263–6266
Regio- and stereoselective synthesis of the major metabolite of
quercetin, quercetin-3-O-b- -glucuronide
D
Mohamed Bouktaib,a,b Aziz Atmanib,† and Christian Rolandoa,*
aUniversite´ des Sciences et Technologies de Lille, Equipe Polyphe´nols, UMR CNRS 8009, Baˆtiment C4,
59655 Villeneuve d’Ascq Cedex, France
bUniversite´ Sidi Mohamed Ben Abdellah, Faculte´ des Sciences D. Mehraz, De´partement de Chimie, PO Box 1796, Fe`s-Atlas,
Morocco
Received 29 May 2002; revised 26 June 2002; accepted 27 June 2002
Abstract—Protected quercetin was first transformed selectively in its 3-O-b-
D-glucoside. Further protection of the remaining
phenolic hydroxyl group allows a clean oxidation of the glucoside by TEMPO/NaOCl/NaBr under phase transfer conditions into
the corresponding glucuronide which was cleanly deprotected to quercetin-3-O-b-D-glucuronide. © 2002 Elsevier Science Ltd. All
rights reserved.
curonidated.11 The analysis of plasma of volunteers fed
with quercetin supplemented diet shows that quercetin is
mainly circulating as glucuronide, mainly quercetin-3-O-
Flavones and flavonols are plant secondary metabolites
found in many foods, especially in fruits and vegeta-
bles.1,2 The daily consumption of flavonoids ranges from
6 mg in Finland to 70 mg in Japan. Quercetin and its
glucosylated forms are the major individual molecules
among polyphenols which are not polymeric and repre-
sent 60–75% of the flavonoid intake.3
b-
D
-glucuronide and 3%-methylquercetin-3-O-b-D-glu-
curonide.12,13 The same metabolism is observed in rat and
pig with variation on the site of methylation.14–16
Quercetin is a very efficient antioxidant4 and appears to
be active in many diseases related to aging like cancer,5
cardiovascular6 and neurodegenerative7 diseases. How-
ever many questions are still open on the origin of this
activity.8,9 Both quercetin aglycone 1 and glucosylated
forms like quercitrin 2a (quercetin 3-O-a-rhamnopyran-
oside), rutin 2b (quercetin 3-O-b-D-rutinoside) or iso-
quercetin 3 (quercetin 3-O-b-D-glucoside), are absorbed
through the intestine.10 However their metabolism is
quite different as glucosylated forms are absorbed
directly whereas quercetin free aglycone is first glu-
In vitro studies showed that the activity of quercetin
derivatives on model enzymatic system is very depen-
dent upon the substitution pattern on quercetin.17,18
Keywords: quercetin; O-glucoside; O-glucuronide; TEMPO oxidation; chemical synthesis.
* Corresponding author. Tel.: +33-320434977 ; fax: +33 320336136; e-mail: christian.rolando@univ-lille1.fr
† E-mail: a.atmani@wanadoo.net.ma
0040-4039/02/$ - see front matter © 2002 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(02)01264-9

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M. Bouktaib et al. / Tetrahedron Letters 43 (2002) 6263–6266
However most of the in vitro biological studies for
assessing the properties of quercetin are performed on
quercetin and not on their metabolites as they are not
readily available from commercial sources. For example
other hand, glucosylation of phenols can be performed
in mild conditions using phase transfer catalyst such as
tetrabutylammonium bromide25 or tris(2-(2-methoxy-
ethoxy)ethyl)amine (TDA-1) in good to fair yields.26
Glucoside can be selectively oxidized to the correspond-
ing glucuronide by TEMPO in catalytic amount in the
presence of sodium hypochlorite which regenerates the
free radical.27,28 So we choose to synthesize quercetin
glucuronide by oxidation of the corresponding glu-
coside. This provides an easy access to both of these
compounds, including labeled molecules, needed in
metabolic studies, starting from commercially available
labeled glucose. Using the protecting strategy we devel-
oped to access selectively to each hydroxyl function of
quercetin,29 we synthesized selectively quercetin-3-O-b-
quercetin-3-O-b-D-glucuronide must be isolated from
green beans19 which contains in the most suitable vari-
ety 15 mg per kg of fresh beans or prepared in low yield
by direct glucuronidation of quercetin and separated by
tedious preparative HPLC procedures.16 As pointed out
by many authors, glucuronidation is by far more
difficult than glucosylation.20 This trend is enforced in
the case of phenols which requires special care even for
glucosylation. For example alkylation of 3,3%,4%,5-tetra-
benzoylquercetin by glucuronic acid methyl ester bro-
mide triacetate gave only a 8% yield of quercetin-7-
glucuronide.21 Similar results were reported previously
D
-glucoside, which was subsequently transformed in the
for the synthesis of normorphine-3 and 6-O-b-
D
-glu-
corresponding glucuronide.
curonide.22 The recently described efficient acid conden-
sation of methyl 2,3,4-tri-O-isobutyryl-trichloroaceti-
Selective protection of the north-east catechol of
quercetin 1 by dichlorodiphenylmethane led to the ketal
5 (Scheme 1) which gave an entry in the series substi-
tuted on the 3 position.29,30 Indeed protected quercetin
ketal 5 still exhibits three free hydroxyl groups. How-
midoyl-a-D-glucopyranuronate catalyzed by BF3–Et2O
for synthesizing morphine-3,6-diglucuronide23 is not a
general approach for flavonoids, as many of them like
catechin are very sensitive in acidic medium.24 On the
Scheme 1. Synthesis of quercetin-3-O-b-
D
-glucoside and quercetin-3-O-b-D-glucuronide.

M. Bouktaib et al. / Tetrahedron Letters 43 (2002) 6263–6266
6265
ever the higher reactivity of position 3 allows the selective
alkylation by methyl iodide or benzyl bromide in DMF
using 1 equiv. of K2CO3 as base along with the formation
of dialkylated products in 3, 7 positions (in 50 and 25%
ratio for methyl and benzyl, respectively).29 Under the
same conditions glucosylation take place very cleanly at
the 3 position leading to 6 in 54% yield. Only traces of
di-glucosylated quercetin are formed. On the other hand
we observed that glucosylation takes place in the 7
position as reported by Dangles and co-workers under
more basic conditions involving phase transfer by tris[2-
(2-methoxyethoxy)ethyl]amine, saturated hydrogenocar-
bonate and dichloromethane.30 Moreover, the cross peak
from H-1%% (l 5.77) to C-3 (l 136.29) in the HMBC
spectrum confirmed that the glucusodation takes place in
the 3 position. The large proton coupling (J=7.5 Hz)
observed for the anomeric proton demonstrates that the
glucoside has the expected b-configuration. Cleavage of
the ketal by hydrogenolysis catalyzed by palladium
protons H-6 and H-8 on the resorcinol A-ring, whereas
the catechol B-ring protons are characterized by the ABX
system (l 6.75, J=8.6 Hz; 7.57, J=8.6 and 2.0 Hz; 7.70,
1
J=2.0 Hz). The H NMR spectrum of compound 4 is
similar to that of compound 3. The complete attribution
of the glucuronic acid moiety as well as the structure of
the aglycone moiety was established by 1H and 13C NMR
and HMBC analyses. Besides the signal attributed to the
quercetin structure, the 13C NMR spectrum of 4 showed
six carbon signals assigned to the glucuronic acid,
including the anomeric carbon (l 104.3) and one car-
boxyl carbon (l 176.2) (Table 1). The obtained 13C NMR
spectrum for the synthesized quercetin-3-O-b-D-glu-
curonide 4 is very similar to the spectra previously
published from natural sources.16,19 This hemisynthesis
of quercetin-3-O-b- -glucoside 3 and quercetin-3-O-b-
D
D
-glucuronide 4 is very complementary of the total
synthesis of labeled quercetin-4%-O-b-D-glucoside
recently described.34
hydroxide on charcoal led to quercetin-3-O-b-D-glu-
coside (isoquercetin) 3 in 86% isolated yield. Before
oxidation by TEMPO of the glucoside to the glu-
curonide, the free hydroxyl functions in position 5 and
7 must be protected, as phenols are reactive under such
conditions. Treatment of the partially protected glu-
coside with benzyl bromide in excess (K2CO3, DMF,
room temperature) led to the fully protected quercetin-3-
glucoside 6. Classical deprotection of the acetoxy pro-
tecting group on the sugar residue (sodium methylate
then neutralization by anionic resin in protonated form)
led to the unprotected glucoside 7.
We are now studying the synthesis of other quercetin
glucuronides and labeled quercetin glucuronides and
glucosides, where the labels are on the sugar moiety
and/or in the polyphenol portion, as well as the extension
of the present methodology starting from selectively
protected catechin.
Acknowledgements
This work was supported by the European Community
(European Contract QLK1-CT-199-00505 POLYBIND)
and the Conseil Re´gional Nord, Pas-de-Calais. M.B.
thanks the Universite´ Sidi Mohamed Ben Abdel-
Oxidation of the protected quercetin-3-O-b-D-glucoside
to the corresponding glucuronic acid was performed by
NaOCl catalyzed by TEMPO in presence of NaBr. The
low solubility of protected quercetin glucoside 7 was
circumvented using phase transfer catalysis by tetra-
butylammonium between CH2Cl2 and saturated sodium
hydrogenocarbonate.27 After 2 hours, TLC showed a
complete disappearance of the starting glucoside. Then
the glucuronide was precipitated by the addition of ethyl
acetate. This phase transfer protocol avoids the use of an
oxidant soluble in organic medium as t-butyl hypochlo-
Table 1. 13C NMR spectra of isoquercetin 3 and 3-glucur-
onide quercetin 4 (75 MHz, CD3OD, l ppm) and com-
parison with spectra from natural products
Carbon
This work
Ref. 19
Ref. 16
3
4
4
4
2
3
4
5
6
159.00
135.65
179.41
162.93
99.98
159.22
135.76
179.46
163.02
99.90
159.01
134.84
179.19
163.06
99.87
158.5
135.4
166.0
159.1
99.9
rite required for the oxidation of phenyl-O-b-D-glu-
curonide.31 The crude product was purified by
chromatography on silica gel column. We plan to remove
both diphenylmethylene and benzyl protecting groups in
one step by catalytic hydrogenation. However, the depro-
tection of the ketal was more difficult than expected and
the diphenylmethylene group was not cleaved by the
usual catalyst (10% Pd–C). The cleavage required a more
reactive catalyst such as 30% palladium on charcoal.
Final purification on LC-18 SPE cartridge using a
methanol, water gradient as eluent, afforded analytically
7
8
165.91
94.75
166.06
94.70
165.71
94.83
163.0
94.8
9
158.37
105.67
123.04
117.62
145.76
149.81
116.00
123.23
104.32
75.73
158.52
105.73
122.72
118.12
145.89
149.85
116.17
122.81
104.33
75.62
158.38
105.61
122.83
117.21
145.90
149.86
116.00
123.52
104.32
75.37
158.5
105.7
122.9
117.3
145.9
149.9
116.1
123.5
104.3
75.4
10
1%
2%
3%
4%
5%
6%
1%%
2%%
3%%
4%%
5%%
6%%
pure quercetin-3-O-b- -glucuronide 4 according to
D
NMR and LC-MS/MS analysis in millimolar amount
(27% isolated yield from 5).
The quercetin-3-O-b- -glucoside 3 and quercetin-3-O-b-
D
78.34
71.19
78.10
62.55
78.13
73.41
77.68
176.46
77.58
72.82
77.02
172.14
77.1
72.9
77.6
179.3
D
-glucuronide 4 were fully characterized by their ESI
mass spectra and their 1H and 13C NMR spectra.32,33 The
1H NMR spectrum of compound 3 showed two doublets
(l 6.15 and 6.33, J=2.0 Hz) consistent with the meta

6266
M. Bouktaib et al. / Tetrahedron Letters 43 (2002) 6263–6266
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