Efficient synthesis of flavanone glucuronides

Article abstract of DOI:10.1021/jf9011467

The first efficient synthesis of flavanone glucuronides as potential human metabolites is described. The synthetic strategy is based on acetyl protection, followed by a combination of chemical and enzymatic deprotection steps. As an example, the method is

Full text of DOI:10.1021/jf9011467

7264 J. Agric. Food Chem. 2009, 57, 7264–7267
DOI:10.1021/jf9011467
Efficient Synthesis of Flavanone Glucuronides
‡
A
HCENE
B
OUMENDJEL,† MADELEINE
B
LANC,† GARY
,‡
WILLIAMSON
,
AND
DENIS
B
ARRON
*
†De
´
partment de Pharmacochimie Mole
B.P. 53, 38041 Grenoble Cedex 9, France, and ‡Nestle
CH-1000 Lausanne 26, Switzerland
´
culaire, UMR 5063, ICMG-FR 2607, Grenoble University,
´
Research Center, P.O. Box 44,
The first efficient synthesis of flavanone glucuronides as potential human metabolites is described.
The synthetic strategy is based on acetyl protection, followed by a combination of chemical and
enzymatic deprotection steps. As an example, the method is applied to a synthesis of 7,40-di-O-
methyleriodictyol 30-O-β-
D-glucuronide. The aglycone is a flavanone naturally present in tarragon
spice (Artemisia dracunculus) as well as in various Chinese, Brazilian, and Malaysian medicinal
plants.
KEYWORDS: Tarragon; Artemisia dracunculus; flavanone glucuronides; human metabolites; synth-
esis; enzymatic deprotection
INTRODUCTION
conjugates. Furthermore, in the absence of suitable chromato-
graphic standards, the quantification of conjugates is difficult.
Thus, the design of efficient methods for the preparation of
analytical standards of polyphenol conjugates is of the most
interest. Since the beginning of the century, a number of reports
on the synthesis of flavonoid conjugates have been made (for
reviews, see refs 16-18). In the case of glucuronide conjugates,
various glycosylation agents have been used, including acetobro-
Flavanones and their glycosides are phenolic compounds that
are common constituents of the diet, especially of citrus fruits (1).
The most classically encountered forms are the rhamnoglucosides
of naringenin 1, eriodictyol 2, and hesperetin 3, namely, narirutin
6, eriocitrin 7, and hesperidin 8 (Figure 1). Other naringenin or
eriodictyol derivatives are less common in edible plants. How-
ever, glycosides of isosakuranetin 4 are present in honeybush
tea (2), and persicogenin 5 has been reported in tarragon (3). On
the other hand, a number of biological activities have been
described for hesperetin and naringenin (4), as well as citrus
flavonoids (5). Consequently, the animal and human metabolism
offlavanoneshas received a lot ofscientificinterest. Inrats (6) and
humans (7), flavanones do not circulate as their aglycone forms.
Instead, they have been shown to be present in both plasma and
urine mainly in the form of glucuronide and/or sulfate conjugates.
For example, among the biliary metabolites of orally adminis-
mo-R-D-glucuronic acid methyl ester (19), more efficient glucur-
onyl trichloroacetimidates (20), and even more efficient
glucuronyl trifluoroacetimidates (21). All of these methods share
in common the fact that, in the final step of the synthesis, strong
aqueous alkaline conditions are necessary to remove the protec-
tive acetate and methyl ester groups of the glucuronic acid
residue. Such an approach was not expected to be applicable to
the preparation of flavanone glucuronides, because upon alkaline
treatment, the flavanone ring has been shown to open to its
corresponding chalcone form (22). Surprisingly, strong alkaline
deprotection conditions (NaOMe/MeOH/H2O) have been used
in a previous attempt to chemically prepare hesperetin 7-glucur-
onide (9). However, the yield of hesperetin 7-glucuronide was
only ∼20%, and the nature of the other products of the reaction
(which could have included appreciable amounts of hesperetin
chalcone glucuronide) was not specified. Thus, a more appro-
priate approach for the synthesis of flavanone glucuronides was
urgently required. In the present paper, we report a general and
efficient method for the preparation of flavanone glucuronides.
The synthetic route is based on (i) the selective deprotection of the
acetyl groups of the glucuronyl residue using zinc acetate and (ii)
the enzymatic hydrolysis of the methyl ester of the glucuronyl
residue under the catalysis of pig liver esterase (PLE). We describe
here the preparation of a potential human metabolite of 7,40-di-
O-methyleriodictyol (persicogenin), a flavanone naturally present
in tarragon spice (Artemisia dracunculus) (3), as well as in various
Chinese, Brazilian, and Malaysian medicinal plants (23-26).
tered hesperetinin rats, hesperetin7-O-β-
D
-glucuronide 9, 30-O-β-
D
-glucuronide 10, and 30-O-β-
D
-glucuronide-7-O-sulfate 11 have
been identified (8). Metabolites 9 and 10 have been detected in rat
plasma after oral administration of hesperidin (9). On the other
hand, only very recently have human circulating flavanone
conjugates been documented in more detail (10, 11). This must
be related to the fact that, both in rats (6,12) and in humans (13-
15), most of the studies have been carried out after treatment of
the plasma by a mixture of glucuronidase and sulfatase. The main
interest of this enzymatic treatment is that the extraction from
plasma and the quantification of the aglycone are straightfor-
ward. In contrast, due to their polar nature, difficulties may be
encountered in the extraction of the glucuronide and sulfate
*Author to whom correspondence should be addressed (telephone
þ 41 21 785 9497; fax þ 41 21 785 8554; e-mail Denis.Barron@rdls.
nestle.com).
pubs.acs.org/JAFC
Published on Web 08/04/2009
© 2009 American Chemical Society

Article
J. Agric. Food Chem., Vol. 57, No. 16, 2009 7265
Figure 1. Structures of flavanones present in edible plants and their
metabolites: 1, R1 = R2 = R3 = H, naringenin; 2, R1 = R2 = H, R3 = OH,
eriodictyol; 3, R1 = H, R2 = Me, R3 = OH, hesperetin; 4, R1 = R3 = H, R2 =
Me, isosakuranetin; 5, R1 = R2 = Me, R3 = OH, 7,4-di-O-methyleriodictyol
(persicogenin); 6, R1 = β-rutinosyl, R2 = R3 = H, narirutin; 7, R1 = β-
rutinosyl, R2 = H, R3 = OH, eriocitrin; 8, R1 = β-rutinosyl, R2 = Me, R3 = OH,
Figure 2. Synthesis of persicogenin 30-O-β-
D-glucuronide 12. a) MeI,
iPr2NEt, DMF, 24 h at rt, 55%; b) Compound 13, BF3 etherate, dry CH2Cl2;
c) Zn(OAc)2, MeOH, 75 °C, 41% on two steps b þ c; d) PLE, aq.
phosphate buffer pH 7.2, 37 °C, 73%.
hesperidin; 9, R1 = β-
7-O-β-
-glucuronide; 10, R1 = H, R2 = Me, R3 = O-β-
hesperetin 30-O-β- -glucuronide; 11, R1 = SO3-, R2 = Me, R3 = O-β-
glucuronopyranosyl, hesperetin 30-O-β- -glucuronide-7-O-sulfate; 12, R1 =
R2 =Me, R3 =O-β- -glucuronopyranosyl, persicogenin 30-O-β-
-glucuronide.
D
-glucuronopyranosyl, R2 = Me, R3 = OH, hesperetin
heated at 75 °C for 24 h, then MeOH was evaporated, and the crude was
purified by column chromatography on silica gel (60 g) eluted with
CH2Cl2/MeOH (9:1), to provide compound 15 (177 mg, 0.35 mmol).
Yield = 41% from 5 (two steps). 1H NMR (400 MHz, DMSO-d6) δ 2.78
(dd, 1, J = 17.1 and 2.9 Hz, H3), c.a. 3.29-3.52 (m, 4, H3 þ H2” þ H-3” þ
H4”), [3.64 (s), plus 3.65 (s)] (1, H7”), 3.78 (s, 3, 4’-OMe), 3.80 (s, 3, 7-
OMe), 4.05 (d, 1, J = 9.6 Hz, H500), [5.20 (d, J = 7.8 Hz) plus 5.22 (d, J =
8.2 Hz)] (1, H100), ca. 5.47-5.55 (m, 1, H2), 6.10 (d, 1, J = 2.3 Hz, H6 or
H8), 6.13 (d, 1, J = 2.3 Hz, H6 or H8), 7.03(d, 1, J = 8.5 Hz, H50), 7.11(m,
1, H60), [7.26 (d, J = 2.0 Hz) plus 7.29 (d, J = 2.0 Hz)] (1, H20), 12.11 (s, 1,
5-OH); 13C NMR (100 MHz, DMSO-d6) δ 42.35 (C3), 42.40 (C3), 52.37
(C700), 56.18 (40-OMe), 56.39 (7-OMe), 71.83 (C400), 73.28 (C200), 75.62
(C500), 76.36 (C300), 78.75 (C2), 78.84 (C2), 94.32 (C6 or C8), 95.15 (C6 or
C8), 99.80 (C100), 99.91 (C100), 103.06 (C10), 112.82 (C50), 114.35 (C20),
114.44 (C20), 121.30 (C60), 121.39 (C60), 131.10 (C10), 146.18 (C30), 146.26
(C30), 149.73 (C40), 149.81 (C40), 163.16 (C5 or C9), 163.66 (C5 or C9),
167.94 (C7), 169.70 (C600), 197.26 (C4); MS (ESIþ) 529 (M þ Na).
D
D-glucuronopyranosyl,
D
D-
D
D
D
MATERIALS AND METHODS
Chemicals and Instruments. Thin layer chromatography (TLC) was
carried out on RP-18 F254s (Merck). Analytical HPLC was performed on a
C18 reverse-phase 2 μm ꢀ 250 mm column. The mobile phase was a
mixture of (A) methanol and (B) 0.1% aqueous trifluoroacetic acid. The
gradient program was as follows: 35% A þ 65% B for 10 min, followed by
a linear gradient to 95% A þ 5% B in 60 min. Column chromatography
was carried out using Merck silica gel 60, 200-400 mesh. Vacuum liquid
chromatography (VLC) was performed on bonded phase octadecyl
(C18, Chromabond) from Macherey-Nagel. 1H NMR and 13C NMR
spectra were recorded on a Bruker Advance 400 operating at 400.13 and
100.03 MHz for 1H and 13C, respectively. The chemical shifts (in ppm)
were expressed with respect to tetramethylsilane (TMS) as an internal
reference. Mass spectra (ESIþ) were recorded on either a JEOL HX-110
spectrometer or a Varian MAT 311. Liquid chromatography-high
resolution mass spectra (ESI-) were recorded using an Agilent-1200 Series
Rapid Resolution LC System including a binary pump SL, a high-
performance autosampler, a diode array detector SL, and a thermostated
column compartment SL. The HPLC system was coupled to an Agilent
6210 time-of-flight mass spectrometer. Elemental analysis was performed
at the University of Geneva, Switzerland.
7-O-Methylhesperetin-30-O-β-
D-glucuronide (12). A solution of 15
(116 mg, 0.23 mmol) was made in 1.5 mL of DMSO, and 35 mL of 20 mM
NaH2PO4/Na2HPO4 buffer (pH 7.2) was added. The mixture was warmed
in a water bath at 37 °C for 5 min, and then 80 mg of PLE (2149 units) was
added. The solution was stirred at 37 °C for 8 h. Purification of the medium
was carried out by VLC on RP-18 (30 g), using a gradient of MeOH in
H2O (from 10 to 100%), to give 83 mg (0.16 mmol) of previously
D
unreported 7-O-methylhesperetin-30-O-β- -glucuronide (persicogenin 30-
O-β- -glucuronide) 12, as a white powder (73%). Rf, 0.5 (RP-18, H2O/
D
MeOH 1:1); HPLC retention time, 26.16 min; 1H NMR (400 MHz,
DMSO-d6) δ 2.76 (m, 1, H3), ca. 3.22-3.48 (m, 4, H3 þ H200 þ H300
þ
7-O-Methylhesperetin 30-O-[Methyl-β-
D-glucopyranosyluronate]
H400), 3.51 (m, 1, H500), 3.78 (s, 6, 7-OMe þ 40-OMe), 5.07 (br s, 1, H100),
5.49 (br d, 1, J = 10.0 Hz, H2), 6.08 (br s, 1, H6 or H8), 6.14 (br s, 1, H6 or
H8), 7.02 (d, 1, J = 8.4 Hz, H50), 7.10 (d, 1, J = 8.1 Hz, H60), 7.29 (br s, 1,
H20); 13C NMR (100 MHz, DMSO-d6) δ 42.59 (C3), 56.19 (7-OMe þ 40-
OMe), 72.42 (C400), 73.50 (C200), 74.82 (C500), 77.12 (C300), 79.03 (C2),
94.30 (C6 or C8), 95.41 (C6 or C8), 100.03 (C100), 112.79 (C50), 114.62
(C20), 121.09 (C60), 131.31 (C10), 146.69 (C30), 149.79 (C40), 163.19 (C5),
167.83 (C600), 173.63 (C7); MS (ESIþ) 492 (M þ Na); HRMS (ESI-), calcd
for C23H23O12 491.1189 [M - H]-, found 491.1193.
(15). To a solution of 7,40-di-O-methyleriodictyol 5 (273 mg, 0.86 mmol)
in dry CH2Cl2 (25 mL in the presence of mol sieve) was added the
glucuronic acid donor 13 (27) (1.08 g, 2.15 mmol) followed by BF3 (OEt)2
3
(32 μL). The mixture was stirred overnight under nitrogen and at room
temperature. The solution was diluted with a mixture of CH2Cl2/H2O 1:1
(50 mL). The organic layer was separated, washed with H2O, dried over
MgSO4, and evaporated. The crude product 14 was directly submitted to
the subsequent deacetylation reaction. However, an aliquot of 14 was
isolatedand characterized. 1H NMR (400 MHz, CDCl3) δ 2.03 (s, 3, OAc),
2.05 (s, 3, OAc), 2.09 (s, 3, OAc), 2.17 (s, 3, OAc), [2.78 (dd, J = 17.2 and
3.1 Hz) plus 3.04 (dd, J = 17.2 and 12.8 Hz), plus 3.06 (dd, 1, J = 17.2 and
12.8 Hz) ] (2, H3), 3.73 (s, 1, H700), 3.81 (s, 3, 7-OMe), 3.84 (s, 3,40-OMe),
[4.10 (d, J = 9.4 Hz) plus 4.12 (d, J = 9.6 Hz)] (1, H500), [5.06 (d, J =
6.8 Hz) plus 5.08 (d, J = 6.8 Hz)] (1, H100), ca. 5.30-5.40 (m, 4, H2 þ H200
þ H300 þ H400), 6.03 (br d, 1, J = 2.3 Hz, H8), 6.06 (d, 1, J = 2.2 Hz, H6),
[6.94 (d, J = 8.4 Hz) plus 6.95 (d, J = 8.4 Hz)] (1, H50), ca. 7.15-7.19 (m,
1, H60), 7.28 (m, 1, H20), 12.00 (s, 1, 5-OH); 13C NMR (100 MHz, CDCl3)
δ 20.50 (OAc), 20.63 (OAc), 42.89 (C3), 43.04 (C3), 52.95 (C700), 55.70 (7-
OMe), 56.10 (40-OMe), 69.20 (C400), 71.08 (C200), 71.82 (C300), 72.53 (C500),
78.47 (C2), 94.29 (C8), 95.08 (C6), 100.56 (C100), 100.65 (C100), 103.09
(C10), 112.69 (C50), 118.92 (C20), 119.34 (C20), 122.86 (C60), 123.02 (C60),
130.88 (C10), 130.97 (C10), 145.58 (C30), 145.65 (C30), 151.11 (C40), 162.65
(C9), 164.10 (C5), 166.89 (C600), 167.98 (C7), 169.32 (OAc), 169.39 (OAc),
170.18 (OAc), 195.78 (C4). To the solution of crude 14 in dry methanol
(20 mL) was added zinc acetate (787 mg, 4.3 mmol). The solution was
RESULTS AND DISCUSSION
The objective of the work was the optimization of a glucur-
onidation method, compatible with the stability of the flavanone
ring. Thus, we chose 7,40-di-O-methyleriodyctiol (persicogenin) 5
as substrate for glucuronidation, because it did not involve a
complex protection/deprotection strategy of the aglycone moiety.
The preparation of persicogenin 30-O-β-
D-glucuronide 12 is
displayed in Figure 2. The structures of the compounds have
been established on the basis of their 1D NMR (1H, 13C), and 2D
NMR (direct and long-distance heteronuclear correlations) data.
7,40-Di-O-methyleriodyctiol 5 has been previously prepared
either from its corresponding protected chalcone (28) or by
methylation of hesperetin derivatives (29, 30). In the present
study, compound 5 was synthesized in 55% yield (Figure 2) by

7266 J. Agric. Food Chem., Vol. 57, No. 16, 2009
Boumendjel et al.
selective methylation of a commercial mixture of (S)- and (R)-
hesperetin 3. The 1H and 13C NMR data of our synthetic persi-
cogenin 5 (see the Supporting Information) were in accordance
with the published ones (31, 32). Because in 7,40-di-O-methyler-
iodictyol a strong hydrogen bond is established between the 5-
hydroxyl and the 4-carbonyl group, the glucuronidation of 5 was
expected to be directed to position 30. To achieve this, we chose
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the recently introduced 2,3,4-triacetyl-D-methyl glucurono-
pyranosyl-(N-phenyl)-2,2,2-trifluoroacetimidate 13 (Figure 2)
(27). The latter was treated with 5 in the presence of BF3 (OEt)2
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hesperetin in rats. Shoyakugaku Zasshi 1993, 47, 43–46.
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3
to yield the protected glucuronide 14. Because the resulting
glucuronide 14 was a mixture of two diastereoisomers (which
unfortunately did not separate on usual silica or RP-18 chroma-
1
tographic phases), some of the H and 13C NMR signals of 14
were split (see Materials and Methods), which rendered the
interpretation of the spectra more difficult. Nevertheless, the
regioselective glucuronidation at position 30 in 14 was unequi-
vocally confirmed by the presence, on its long-distance proton-
carbon correlation spectrum, of a 3J correlation between the
anomeric proton of the glucuronyl moiety (H100) and the aro-
matic C30 carbon of the flavanone residue. Compound 14 was
subsequently subjected to deprotection step c (Figure 2). The first
challenge was to achieve the deacetylation of 14 while preventing
the opening of the flavanone into its corresponding chalcone.
Nudelman et al. (33) have reported the deprotection of podo-
phylle glucuronides by zinc acetate in anhydrous conditions.
When using these conditions, we were pleased to see that
compound 14 yielded compound 15 with no opening to the
chalcone form (41% yield over two steps from 5). However, in
accordance with the previous study (33), the methyl ester of the
glucuronic acid moiety was not hydrolyzed under these condi-
tions. Furthermore, the hydrolysis of the methyl ester function
of 15 turned out to be difficult by chemical means. Fortunately, the
methyl ester hydrolysis proceeded smoothly under the catalysis of
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pig liver esterase (PLE) (34). Thus, persicogenin 30-O-β-
D-glucur-
onide 12 was successfully obtained in 73% yield from 15. In
conclusion, this is the first efficient chemical synthesis of a
flavanone glucuronide. The method can be applied to the
preparation of a wide range of flavanone glucuronides. The
resolution of the two enantiomers of the starting product hesper-
etin was out of the scope of this study. Nevertheless, none of the
steps of this newly reported synthesis are susceptible to inducing
flavanone-chalcone isomerization. Thus, the method can be
potentially applied to the preparation of pure (S)- and (R)-
flavanone glucuronides from the appropriate enantiomerically
pure aglycone. Its application to the synthesis of the glucuronides
of the dietarily relevant hesperetin is currently under investigation
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1
Supporting Information Available: Chemical synthesis, H
NMR, 13C NMR, and MS data, and elemental analysis of 7,40-di-
O-methyleriodictyol 5. This materialis available free ofchargevia
the Internet at http://pubs.acs.org.
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