Isotopic labelling of Quercetin 4'-O-β-D-glucoside

Article abstract of DOI:10.1016/S0040-4020(00)00325-2

[2-¹³C]-Quercetin-4'-O-β-glucoside was synthesised in four steps and 28% yield from barium [¹³C]-carbonate. This short route will be applicable to the synthesis of radiolabelled quercetin-4'-O-β-D-glucoside from barium [¹⁴

Full text of DOI:10.1016/S0040-4020(00)00325-2

TETRAHEDRON
Pergamon
Tetrahedron 56 (2000) 4101±4106
Isotopic Labelling of Quercetin 40-O-b-d-Glucoside
Stuart T. Caldwell,a Alan Crozierb and Richard C. Hartleya,
*
aDepartment of Chemistry, University of Glasgow, Glasgow G12 8QQ, UK
bDivision of Biochemistry and Molecular Biology, University of Glasgow, Glasgow G12 8QQ, UK
Received 1 March 2000; revised 23 March 2000; accepted 13 April 2000
AbstractÐ[2-13C]-Quercetin-40-O-b-glucoside was synthesised in four steps and 28% yield from barium [13C]-carbonate. This short route
will be applicable to the synthesis of radiolabelled quercetin-40-O-b-d-glucoside from barium [14C]-carbonate. The most important feature is
control of the regiochemistry and stereochemistry of glycosylation before introduction of the isotopic label. The synthesis also uses only
benzyl protecting groups allowing global deprotection in the last step. q 2000 Elsevier Science Ltd. All rights reserved.
Introduction
steps and 28% yield from barium [13C]-carbonate
(Scheme 1). Barium [14C]-carbonate is one of the cheapest
compounds containing this radioactive isotope of carbon
and our route is applicable to the synthesis of [2-14C]-
quercetin-40-O-b-glucoside. [4-14C]-Quercetin has been
made commercially,13 but no details of the method of
synthesis have been reported. There have been no other
syntheses of ¯avonols with a carbon isotope label in the
A, B or C rings, but such compounds have been extracted
from plants grown under [14C]-carbon dioxide.14 Progress
has been made in the deuterium-labelling of ¯avonols.15±18
Flavonols are polyphenolic secondary metabolites produced
by plants.1 They are found in high concentrations in foods
such as tomatoes and onions2 and in beverages such as tea
and red wine.3,4 They are normally present in fruits, vege-
tables and teas as sugar conjugates.5,6 Many possess anti-
oxidant and free radical scavenging activity7 and
epidemiological studies indicate that consumption of ¯avo-
nols is associated with a reduced risk of coronary heart
disease.8,9 The average intake of ¯avonols in the Nether-
lands diet is approximately 23 mg d21 of which 29%
comes from onions.8 Quercetin 1 is a strong dietary anti-
oxidant10 found in high concentrations in onions primarily
as its 40-O-b-d-glucoside and 3,40-di-O-b-d-glucoside (Fig.
1).11 The absorption and metabolism of individual ¯avonols
in humans is very poorly understood, but a recent study with
onions has shown that ¯avonols are absorbed into the blood-
stream as glucosides, and that minor structural differences
affect both the level of accumulation in plasma and the
extent to which conjugates are excreted in urine.12 Further
studies are required to determine the bioavailability of
¯avonol conjugates and their role as in vivo antioxidants.
Ultimately, understanding the pharmacology of various
¯avonols would allow us to assess their relative importance
to a healthy diet and to recommend appropriate foods and
beverages to the public. As a ®rst step, we wished to track
the biological fate of quercetin-40-O-b-d-glucoside in rats
and for this we needed to synthesise the ¯avonol radio-
labelled with a label that cannot be exchanged.
Results and Discussion
In order to minimise the number of steps after the intro-
duction of the isotopic label and to avoid the dif®culty of
selectively glucosylating quercetin, we decided to introduce
the glucoside early in our synthesis. We also decided to use
only one type of protecting group: the benzyl group was
selected as deprotection does not affect glycosidic links.19
Monobenzylated catechol 2 was selectively iodinated para
to the hydroxyl group using iodine monochloride20 to give
iodophenol 3. This reagent proved superior to chloramine
T,21 which gives side-products that are dif®cult to remove.
Glucosylation of iodophenol 3 using Schmidt's imidate22,23
4 gave a 4:1 mixture of b- and a-anomers, and a single
recrystallisation gave the b-glucoside 5. All regiochemical
and stereochemical issues had thus been dealt with prior to
Here, we demonstrate our synthetic strategy with the
synthesis of [2-13C]-quercetin-40-O-b-glucoside 10 in four
Keywords: labelling; ¯avonoids; ¯avonols; glycosides.
* Corresponding author. Tel.: 144-141-330-4398; fax: 144-141-330-
4888; e-mail: richh@chem.gla.ac.uk
Figure 1.
0040±4020/00/$ - see front matter q 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020(00)00325-2

4102
S. T. Caldwell et al. / Tetrahedron 56 (2000) 4101±4106
Scheme 1.
introduction of the isotopic label. NOE experiments
con®rmed that glucoside 5 was the correct regioisomer:
irradiation of the doublet at dH 6.90, corresponding to H-5
of the benzene ring, gave strong enhancement of the ano-
meric proton at dH 4.96, and irradiation of the anomeric
proton gave strong enhancement of the H-5 signal.
pentabenzylation are superior to those previously
reported.26 The best method for cyclising and dehydrating
ester 8 used potassium carbonate and a phase transfer cata-
lyst.27 However, these conditions led to selective depro-
tection of the 5-hydroxy group to give ¯avonol 11.
Hydrogenolysis of ¯avonol 11 using the non-acidic palla-
dium hydroxide on carbon under 1 atm of hydrogen gave the
target compound 12 in four steps and 28% yield from
barium [13C]-carbonate. Compound 12 had a chemical
Carboxylation of the glucoside 5 using a variation of the
method described by Kratzel and Billek24 gave the benzoic
acid 6 in 83% yield based on glucoside 5 using 2 equiv. of
barium [13C]-carbonate, and in 76% yield based on carbon
dioxide using 1.5 equiv. of the glucoside 5 and 1 equiv. of
barium [13C]-carbonate. The glycosidic link was not
affected by either the reaction or the acidi®cation necessary
to isolate carboxylic acid 6. Esteri®cation using phenol 7
and a water-soluble coupling agent, 1-(3-dimethylamino-
propyl)-3-ethylcarbodiimide (EDCI), gave ester 8. Phenol
7 was prepared by degradation25 of pentabenzylated
quercetin 9 according to the literature procedure and was
fully characterised for the ®rst time (Scheme 2). The
carboxylic acid 10 was also isolated. Our conditions for
1
purity of $97% by HPLC, H and 13C NMR. Only one
step after the introduction of the isotopic label involves
chromatography and that is simply a ®ltration through
alumina. In summary, we have demonstrated a short high-
yielding route to [2-13C]-quercetin-40-O-b-glucoside 12 that
will also be useful for radiolabelling work.
Spectral assignments
1
1
All the signals that show H±13C coupling in the H NMR
spectra and those that show 13C±13C coupling in the 13C
NMR spectra of compounds 6, 8, 11 and 12 are presented
Scheme 2.

S. T. Caldwell et al. / Tetrahedron 56 (2000) 4101±4106
Table 1. 13C±X coupling in 13C NMR spectrum of carboxylic acid 6
4103
hexane and passed through a plug of silica eluting with
CH2Cl2±hexane (4:6). The resulting solid was recrystallised
from hexanes±ether several times to give phenol 3 as prisms
(18.81 g, 58%) mp 71±738C; RF [silica, CH2Cl2±hexane
(7:3)] 0.56; nmax(nujol)/cm21 3462 (OH) and 1495 (Ar);
dH (400 MHz, CDCl3) 5.03 (2H, s, ±OCH2±), 5.65 (1H, s,
OH), 6.69 (1H, d, Jˆ8.2 Hz, H-6) 7.17±7.19 (2H, m, H-3,
H-5) and 7.36±7.45 (5H, m, Ph-CH2); dC (100 MHz,
CDCl3) 71.3 (CH2), 80.8 (C±I), 116.6 (CH), 120.9 (CH),
128.0 (CH), 128.6 (CH), 128.7 (CH), 130.7 (CH), 135.5 (C),
145.8 (C) and 146.6 (C); m/z (EI): 326.0 (M1, 10%), and
91.1 (100); (Found: C, 47.96; H, 3.36; I, 38.77%; M1,
325.9803. C13H11O2I requires C, 47.87; H 3.40; I 38.91%,
M, 325.9804).
X
C-1
C-2
C-3
C-5
J (Hz)
C (ppm)
73.7
123.4
2.5
114.6
5.7
148.3
5.1
115.2
d
Table 2. 13C±X coupling in 13C NMR spectrum of ester 8
X
C-2
C-10
C-20
C-30
C-50
C-60
J (Hz)
d
3.0
150.6
78.6
123.1
2.8
114.8
6.1
148.5
5.4
115.3
1.8
124.7
C (ppm)
in Tables 1±4. The numbering of carbon and hydrogen
atoms of the different compounds is shown in Scheme 1.
Signals were assigned by comparison with the spectra of the
corresponding non-labelled compounds (synthesised by the
same route or reported in the literature28), by using HMQC
and HMBC spectroscopy and by inferring the relative
chemical shifts of the atoms in question from those in the
other labelled compounds.
1-Iodo-3-benzyloxy-4-(20,30,40,60-tetra-O-benzyl-b-d-glucos-
pyranosyloxy)benzene 5. A solution of boron tri¯uoride
etherate (0.46 ml, 3.70 mmol) in dry CH2Cl2 (8 ml) was
added over 1 h to a stirred solution of phenol 3 (3.99 g,
12.2 mmol), O-(2,3,4,6-tetra-O-benzyl-b-d-glucopyrano-
Ê
syl)trichloroacetimidate 4 (12.57 g, 18.4 mmol) and 4 A
molecular sieves (1.00 g) in dry CH2Cl2 (20 ml) under
nitrogen at 2788C. After stirring at 2788C for 3.5 h, the
solution was quenched with saturated aqueous Na2CO3 and
the molecular sieves were ®ltered off and washed with
CH2Cl2 (20 ml). The ®ltrate and washings were combined,
washed with brine (2£250 ml), dried (MgSO4) and concen-
trated under vacuum to give a solid, which was recrystal-
lised from EtOAc±pet. ether (1:4) to give the b anomer 5 as
needles. (5.22 g, 50%); mp 122±1238C; nmax(nujol)/cm21
1491 (Ar); dH (400 MHz, CDCl3) 3.57±3.78 (6H, m, H-20,
30, 40, 50, 60), 4.50 (1H, d, Jˆ12.0 Hz, ArOCHAHB), 4.54
(1H, d, Jˆ10.8 Hz, ArOCHCHD), 4.56 (1H, d, Jˆ12.0 Hz,
ArOCHAHB), 4.70 (1H, d, Jˆ11.2 Hz, ArOCHEHF), 4.77
(1H, d, Jˆ10.8 Hz, ArOCHGHH), 4.82 (1H, d, Jˆ10.8 Hz,
ArOCHCHD), 4.92 (1H, d, Jˆ10.8 Hz, ArOCHGHH), 4.96
(1H, d, Jˆ7.2 Hz, H-10), 4.99 (1H, d, Jˆ11.6 Hz,
Experimental
1H and 13C NMR spectra were obtained on a Bruker DPX/
400 spectrometer operating at 400 and 100 MHz, respec-
tively. All coupling constants are measured in Hz. DEPT
was used to assign the signals in the 13C NMR spectra as C,
CH, CH2 or CH3. Mass spectra (MS) were recorded on a Jeol
JMS700 (MStation) spectrometer. Infrared (IR) spectra
were obtained on a Perkin±Elmer 983 spectrophotometer.
Ultraviolet (UV) spectra were recorded on a Shimadzu UV-
1601 spectrophotometer. Column chromatography was
carried out on silica gel, 70±230 mesh, or neutral alumina
(Brockmann grade III). Tetrahydrofuran and diethyl ether
were dried over sodium and benzophenone, and dichloro-
methane was dried over calcium hydride.
ArOCHIHJ), 5.05 (2H, Broad d, ˆ12.4 Hz, ArOCHEHF,
J
ArOCHIHJ), 6.90 (1H, d, Jˆ8.5 Hz, H-5) and 7.12±7.36
(27H, m, Ar±H); dC (100 MHz, CDCl3) 68.84 (CH2),
70.96 (CH2), 73.44 (CH2), 74.59 (CH2), 74.97 (CH2),
75.50 (CH), 75.64 (CH2), 77.57 (CH), 81.56 (CH), 84.38
(CH), 84.86 (C), 101.99 (CH), 118.60 (CH), 122.75 (CH),
127.38 (CH), 127.51 (CH), 127.54 (CH), 127.57 (CH),
127.63 (CH), 127.75 (CH), 127.80 (CH), 127.89 (CH),
127.97 (CH), 128.01 (CH), 128.11 (CH), 128.29 (CH),
128.30 (CH), 128.35 (CH), 128.41 (CH), 130.27 (CH),
136.19 (C), 137.95 (C), 138.03 (C), 138.27 (C), 138.38
(C), 147.22 (C) and 149.65 (C); m/z (FAB): 871.3
[(M1Na)1, 2%], 522.3 (3), 415.2 (5), 329.1 (3); [Found:
2-Benzyloxy-4-iodophenol 3. A solution of iodine mono-
chloride (24.35 g, 150 mmol) in dry diethyl ether (200 ml)
was added dropwise to a solution of 2-benzyloxyphenol 2
(20.00 g, 100 mmol) in dry diethyl ether (80 ml), shielded
from the light and under nitrogen. The resulting solution
was stirred overnight. The deep red solution was then
diluted with diethyl ether (300 ml) and washed with
aqueous sodium thiosulfate (3£600 ml). The organic layer
was dried over magnesium sulfate and the solvent removed
in vacuo. The resulting solid was dissolved in CH2Cl2±
1
Table 3. 13C±X coupling in 13C and H NMR spectra of ¯avonol 11
X
C-3
C-4
C-8
C-9
C-10
C-20
C-30
C-50
H-20
H-60
J (Hz)
d (ppm)
86.1
137.6
6.8
178.8
3.5
93.0
2.5
156.7
67.7
124.8
3.0
114.2
5.7
148.1
4.8
115.6
3.6
7.73
3.6
7.54
1
Table 4. 13C±X coupling in 13C and H NMR spectra of ¯avonol 12
X
C-3
C-4
C-8
C-9
C-10
C-20
C-30
C-40
C-50
H-20
H-60
J (Hz)
d (ppm)
90.1
136.7
5.7
176.4
3.1
93.9
2.7
156.6
68.0
125.5
1.7
115.5
5.6
146.7
1.3
147.1
5.0
116.2
4.0
7.71
4.0
7.62

4104
S. T. Caldwell et al. / Tetrahedron 56 (2000) 4101±4106
(M1Na)1, 871.2106. C47H45IO7Na requires (M1Na)1,
871.2107; (Found: C, 66.50; H, 5.43%. C47H45IO7 requires
C, 66.51; H, 5.34%); [a]1D9ˆ230.5 (cˆ40: CHCl3).
171.53 (13C label); m/z (FAB) 790.7 [(M1Na)1, 24%],
171.2 (23) and 91.6 (100); [Found: (M1Na)1, 790.3074.
C4713CH46O9Na requires (MˆNa), 790.3073]; (Found: C,
75.07; H, 6.26%. C4713CH46O9 requires C, 75.21; H,
6.04%); [a]1D9ˆ247.7 (cˆ0.044 g ml21, CHCl3).
3-Benzyloxy-4-(20,30,40,60-tetra-O-benzyl-b-d-glucospy-
ranosyloxy)benzoic acid 6, [13C] at carbonyl. Carboxyl-
ation of the aryl iodide 5 was carried out using the apparatus
described by Kratzel and Billek and a variation of their
method.24 Butyl lithium (3.20 ml, 4.71 mmol) was added
to a solution of aryl iodide 5 (4.00 g, 4.71 mmol) in dry
THF (20 ml) at 2788C under nitrogen. The mixture was
stirred for 15 min and was then cooled to 21968C and the
whole system was evacuated to 4 mm of Hg. The system
was then sealed and an excess of concentrated sulfuric acid
was added dropwise onto [13C]-barium carbonate (98 at%
13C, 1.87 g, 9.43 mmol) in a separate reaction vessel in the
same system with occasional sonication of the vessel. Once
the majority of the carbon dioxide had been evolved, the
THF solution was allowed to warm to 2788C, was stirred
for 30 min and then recooled to 21968C. The [13C]-barium
carbonate/sulfuric acid mixture was sonicated for a further
15 min and the THF solution was then allowed to warm to
2788C and was stirred for 15 min. Throughout the pro-
cedure the pressure did not exceed 210 mm of Hg. The
THF solution was poured into a (1:1) two-phase mixture
of ether and aqueous HCl (1 M)/ice. The layers were sepa-
rated and the aqueous layer washed with ether (50 ml). The
combined organics were washed with brine (4£150 ml) and
concentrated to approximately 10 ml. Aqueous NaOH (2 M,
20 ml) was added and pet. ether was added until a white
solid formed. The sodium salt was ®ltered off, dissolved in
EtOAc and acidi®ed with aqueous HCl (1 M). The organic
layer was dried (MgSO4) and concentrated under vacuum to
give benzoic acid 6 as a purple solid (3.00 g, 83% from aryl
iodide 5) suf®ciently pure for the next step. 518 mg were
then recrystallised from iPrOH to give the acid 6 as a white
amorphous solid (297 mg, 57%). In a similar way, carbon
dioxide generated from only 1 equiv. of [13C]-barium
carbonate (0.545 g, 2.75 mmol), reacted with the aryl-
lithium generated from 1.5 equiv. of aryl iodide 5 (3.50 g,
4.12 mmol) and 1.4 equiv. of butyl lithium (2.62 ml,
3.85 mmol) to give benzoic acid 6 (1.60 g scale, 76%
from barium carbonate). mp 124±1268C; nmax(nujol)/cm21
3178 (COOH), 1646 (CvO), 1587 (Ar) and 1509 (Ar); dH
(400 MHz; CDCl3) 3.65±3.81 (6H, m, 20, 30, 40, 50, 60), 4.52
(1H, d, Jˆ11.6 Hz, ArOCHAHB), 4.55 (1H, d, Jˆ10.4 Hz,
ArOCHCHD), 4.58 (1H, d, Jˆ12 Hz, ArOCHAHB), 4.74 (1H,
d, Jˆ11.2 Hz, ArOCHEHF), 4.79 (1H, d, Jˆ11.2 Hz,
ArOCHGHH), 4.84 (1H, d, Jˆ10.4 Hz, ArOCHCHD), 4.95
(1H, d, Jˆ11.2 Hz, ArOCHGHH), 5.09 (2H, broad d,
Jˆ12.8 Hz, ArOCHEHF1ArOCHIHJ), 5.10 (1H, d, Jˆ
6.8 Hz, H-100), 5.14 (1H, d, Jˆ11.6 Hz, ArOCHIHJ), 7.13±
7.47 (26H, m, Ar±H), 7.69±7.73 (2H, m, Ar±H) and 11.0
(1H, v broad s, COOH); dC (100 MHz: CDCl3): 68.78
(CH2), 70.78 (CH2), 73.48 (CH2), 74.61 (CH2), 75.03
(CH2), 75.22 (CH), 75.47 (CH2), 77.52 (CH), 81.46 (CH),
84.32 (CH), 101.47 (CH), 114.64 (CH, d, Jˆ2.5 Hz), 115.19
(CH, d, Jˆ5.1 Hz), 123.40 (C, d, Jˆ73.7 Hz), 124.52 (CH),
127.38 (CH), 127.59 (CH), 127.65 (CH), 127.68 (CH),
127.82 (CH), 127.93 (CH), 127.99 (CH), 128.06 (CH),
128.16 (CH), 128.32 (CH), 128.34 (CH), 128.38 (CH),
128.43 (CH), 136.29 (C), 137.92 (C), 138.93 (C), 138.17
(C), 138.42 (C), 148.33 (C, d, Jˆ5.7 Hz), 151.66 (C), and
2-Hydroxy-v,4,6-tribenzyloxyacetophenone 7 and 3,5-
dibenzyloxybenzoic acid 10. Following the method of
Hauteville,25 150 ml of diethylene glycol was slowly
added to a solution of pentabenzylated quercetin 9
(15.00 g, 20.00 mmol) in 150 ml of pyridine and 20 ml
of 18 M potassium hydroxide. The heterogeneous solution
was stirred at 1208C for 6 h. After cooling, the solution was
poured into 1 l of iced water and the resulting solid was
®ltered off and the aqueous ®ltrate put aside (see below).
The precipitate was dissolved in EtOAc (100 ml) and
washed with 1 M aqueous hydrochloric acid. The ethyl
acetate extract was dried and concentrated. The resulting
solid was recrystallised from methanol to yield the ortho-
hydroxy acetophenone 7 as a powder. (3.43 g, 38%). mp
104±1058C (lit.,25 102±1038C); RF [silica, EtOAc±hexane
(7:3)] 0.77; nmax(nujol)/cm21 1609 (CvO) and 1568 (Ar);
dH (400 MHz, CDCl3) 4.52 (2H, s, PhCH2), 4.56 (2H, s,
PhCH2), 4.98 (2H, s, PhCH2), 5.06 (2H, s, OvC±CH2),
6.06 (1H, d, Jˆ2.2 Hz, H-3), 6.18 (1H, d, Jˆ2.2 Hz, H-5),
7.20±7.41 (15H, m, Ar±H) and 13.76 (1H, s, ±OH); dC
(100 MHz, CDCl3) 70.2 (CH2), 71.1 (CH2), 72.9 (CH2),
75.4 (CH2), 92.1 (CH), 94.9 (CH), 104.4 (C), 127.5 (CH),
127.6 (CH), 127.9 (CH), 128.2 (CH), 128.3 (CH), 128.66
(CH), 128.68 (CH), 128.7 (CH), 135.0 (C), 135.6 (C), 137.5
(C), 161.6 (C), 165.2 (C), 167.4 (C) and 200.8 (C); m/z (CI):
455.2 [(M1H)1, 100%], 347.2 (12), 333 (9), 91.1 (11);
[Found: (M1H)1 455.1859. C29H27O5 requires (M1H),
455.1858] (Found: C, 76.5; H, 5.9%. C29H26O5 requires C
76.63, H 5.76%). The aqueous ®ltrate was acidi®ed to pH 1
and the resulting solid ®ltered off. The precipitate was
recrystallised several times from ethyl acetate to yield the
benzoic acid 10 as needles (2.88 g, 43%); mp 185±1868C
(Lit.,6 183±1848C; RF [Silica, EtOAc±hexane (7:3)] 0.34;
n
max(nujol)/cm21 1676 (CvO), 1600 (Ar) and 1520 (Ar);
dH (400 MHz; D-6 Acetone) 5.09 (2H, s, PhCH2), 5.13 (2H,
s, PhCH2), 6.89 (1H, d, Jˆ8.7 Hz, H-2) and 7.03±7.55
(12H, m, Ar±H); dC (100 MHz; D-6 DMSO) 70.1 (CH2),
70.3 (CH2), 113.4 (CH), 114.8 (CH), 123.6 (CH), 123.8 (C),
127.7(CH), 127.9 (CH), 128.1 (CH), 128.2 (CH), 128.7
(CH), 128.8 (CH), 137.0 (C), 137.3 (C), 147.9 (C), 152.4
(C) and 167.3 (C); m/z (CI): 335.2 [(M1H)1, 100%], 245.1
(8), 181.2 (9), 91.1 (13); [Found: (M1H)1 335.1286.
C21H19O4 requires (M1H), 335.1283] (Found: C, 75.55;
H, 5.56%. C21H18O4 requires C, 75.43; H, 5.42%).
2-[30-Benzyloxy-40-(200,300,400,600-tetrabenzyl-b-d-gluco-
pyranosyloxy)benzoyloxy]-v,4,6,-tribenzyloxy
aceto-
phenone 8, [13C] at ester carbonyl. DMAP (576 mg,
4.72 mmol), EDCI (1.18 g, 6.14 mmol) and the phenol 8
(2.14 g, 4.72 mmol, 1.0 equiv.) were added to benzoic
acid 7 (3.63 g, 4.72 mmol) in dry CH2Cl2 (30 ml) under
nitrogen. After stirring for 24 h, the solution was diluted
with CH2Cl2 (50 ml) and washed with brine (3£150 ml).
The organics were dried (MgSO4) and concentrated under
vacuum. The resulting brown oil was chromatographed on
alumina (6% water) eluting with diethyl ether to give the
ester 9 as an amorphous solid (3.88 g, 68%); mp 106±1098C

S. T. Caldwell et al. / Tetrahedron 56 (2000) 4101±4106
4105
(diethyl ether); RF(alumina, diethyl ether) 0.72; nmax(nujol)/
cm21 1733 (CO2) and 1608 (CvO); dH (400 MHz, CDCl3)
3.64±3.81 (6H, m, H-200, 300, 400, 500, 600), 4.42 (2H, s,
ArOCH2), 4.50 (2H, s, ArOCH2), 4.51 (1H, d, Jˆ12 Hz,
ArOCHAHB), 4.55 (1H, d, Jˆ11.2 Hz, ArOCHCHD), 4.58
(1H, d, Jˆ12.4 Hz, ArOCHAHB), 4.72 (1H, d, Jˆ11.2 Hz,
ArOCHEHF), 4.79 (1H, d, Jˆ10.8 Hz, ArOCHGHH), 4.83
(1H, d, Jˆ10.8 Hz, ArOCHCHD), 4.94 (1H, d, Jˆ10.8 Hz,
ArOCHGHH), 5.01 (2H, s, ArOCH2), 5.03 [2H, s, C(O)CH2],
5.07 (1H, d, Jˆ11.2 Hz, ArOCHIHJ), 5.08 (1H, d, Jˆ
11.2 Hz, ArOCHEHF), 5.09 (1H, d, Jˆ7.2 Hz, H-100), 5.11
(1H, d, Jˆ12 Hz, ArOCHIHJ), 6.51 (1H, d, Jˆ2.1 Hz, H-3),
6.53 (1H, d, Jˆ2.1 Hz, H-5), 7.13±7.39 (41H, m, Ar±H, H-
20) and 7.73±7.75 (2H, m, H-50, 60); dC (100 MHz, CDCl3)
68.71 (CH2), 70.48 (CH2), 70.85 (CH2), 71.07 (CH2), 73.00
(CH2), 73.51 (CH2), 74.62 (CH2), 75.03 (CH2), 75.25 (CH),
75.69 (CH2), 75.88 (CH2), 77.47 (CH), 81.46 (CH), 84.34
(CH), 98.38 (CH), 101.54 (CH), 101.67 (CH), 114.81 (CH,
d, Jˆ2.8 Hz), 114.95 (C), 115.35 (CH, d, Jˆ5.4 Hz), 123.08
(C, d, Jˆ78.6 Hz), 124.66 (CH, d, Jˆ1.8 Hz), 127.46 (CH),
127.60 (CH), 127.62 (CH), 127.71 (CH), 127.74 (CH),
127.77 (CH), 127.84 (CH), 127.93 (CH), 127.96 (CH),
128.11 (CH), 128.17 (CH), 128.25 (CH), 128.32 (CH),
128.35 (CH), 128.40 (CH), 128.70 (CH), 135.56 (C),
135.84 (C), 136.29 (C), 137.68 (C), 137.99 (2£C), 138.20
(C), 138.47 (C), 148.49 (C, d, Jˆ6.1 Hz), 150.55 (C, d, Jˆ
3.0 Hz), 151.71 (C), 158.47 (C), 161.67 (C), 164.12 (13C
label) and 198.62 (C); m/z (FAB) 1226.5 [(M1Na)1,
65%], 1136.4 (5), 1134.4 (5), 1044.4 (3), 650.2 (5), 560.2
(5), 439.2 (6), 318.1 (30); [Found: (M1Na)1, 1226.4744.
C77H70O13Na requires (M1Na), 1226.4747]; (Found: C,
76.83; H, 5.83%. C7613CH70O13 requires C, 76.87; H,
5.86%); [a]1D9ˆ29.5 (cˆ0.021 g ml21, CHCl3).
3,5,7,30,40-Pentabenzyloxy¯avone 9. Potassium carbonate
(45.7 g, 331.0 mmol) and benzyl bromide (39.3 ml,
331.00 mmol) were added to a solution of quercetin 1
(10.00 g, 33.1 mmol) in DMF (100 ml) under nitrogen,
and the resulting mixture solution was stirred at 708C for
4 d. After the appearance of a single highly UV active spot
in the TLC, the solution was allowed to cool and was then
acidi®ed to pH 1 with aqueous HCl (1 M). The solution was
diluted with water (500 ml), EtOAc (200 ml) was added and
the solution was stirred for 20 min. The resulting precipitate
was ®ltered and washed with H2O (100 ml) and EtOAc
(100 ml) to give pentabenzylated quercetin 9 as an off-
white solid (22.16 g, 89%) suf®ciently pure for the next
step. A small amount was recrystallised from CH2Cl2 to
give a white powder; mp 156±1598C; RF [silica, EtOAc±
hexane (7:3)] 0.62; nmax(nujol)/cm21 1631 (CvO) and 1601
(Ar); dH (400 MHz: CDCl3): 4.88 (2H, s, OCH2), 5.00 (2H,
s, OCH2), 5.01 (2H, s, OCH2), 5.15 (2H, s, OCH2), 5.19 (2H,
s, OCH2), 6.37 (1H, d, Jˆ2.2 Hz, H-6), 6.45 (1H, d, Jˆ
2.2 Hz, H-8), 6.87 (1H, d, Jˆ8.6 Hz, H-50), 7.12±7.54
(25H, m, Ar±H), 7.45 (1H, dd, Jˆ2.0 Hz, 8.3 Hz, H-60)
and 7.64 (1H, d, Jˆ2.0 Hz, H-20); dC (100 MHz: CDCl3):
70.4 (CH2), 70.8 (CH2), 70.9 (CH2), 71.0 (CH2), 74.1 (CH2),
93.9 (CH), 98.1 (CH), 110.1 (C), 115.2 (CH), 122.1 (CH),
123.9 (C), 126.9 (CH), 127.2 (CH), 127.3 (CH), 127.6 (CH),
127.7 (CH), 127.8 (CH), 127.9 (CH), 128.0 (CH), 128.1
(CH), 128.4 (CH), 128.6 (CH), 128.6 (CH), 128.7 (CH),
128.8 (CH), 135.7 (C), 136.4 (C), 136.8 (C), 137.0 (C),
139.8 (C), 148.2 (C), 150.5 (C), 153.2 (C), 158.7 (C),
159.7 (C), 162.7 (C) and 173.9 (C); m/z (EI): 752 [M1,
0.25%], 661.0 (5), 570.9 (4.5), 91.0 (100); (Found: M1,
752.2772. C50H40O7 requires M, 752.2774).
[2-13C]-(5-Hydroxy-3,7,30-tribenzyloxy-40-(200,300,400,600-
tetrabenzyl-b-d-glucopyranosyloxy)¯avone 11. Potas-
sium carbonate (1.65 g, 12.0 mmol) and tetrabutylammo-
nium bromide (1.44 g, 4.48 mmol) were added to the ester
8 (3.60 g, 2.99 mmol) in dry toluene (30 ml) under nitrogen
and the reaction mixture was then heated at 908C for 3 h.
After removing the toluene under vacuum, the residue was
dissolved in CH2Cl2 and washed with water and brine. The
organic layer was dried (MgSO4) and concentrated under
vacuum to give a brown solid, which recrystallised from
EtOAc to give ¯avonol 11 as an off-white solid (1.90 g,
58%); mp 163±1648C; RF (silica, EtOAc) 0.75; dH
(400 MHz, CDCl3) 3.66±3.82 (6H, m, 200, 300, 400, 600), 4.53
(1H, d, Jˆ12.0 Hz, ArOCHAHB), 4.56 (1H, d, Jˆ10.0 Hz,
ArOCHCHD), 4.59 (1H, d, Jˆ12.0 Hz, ArOCHAHB), 4.75
(1H, d, Jˆ11.2 Hz, ArOCHEHF), 4.80 (1H, d, Jˆ11.2 Hz,
ArOCHGHH), 4.82 (1H, d, Jˆ12.0 Hz, ArOCHIHJ), 4.85
(1H, d, Jˆ12.0 Hz, ArOCHCHD), 4.88 (1H, d, Jˆ12 Hz,
ArOCHIHJ), 4.96 (1H, d, Jˆ10.8 Hz, ArOCHGHH), 5.03
(1H, d, Jˆ10.8 Hz, ArOCHKHL), 5.08 (1H, d, Jˆ10.8 Hz,
ArOCHKHL), 5.09 (1H, d, Jˆ7.6 Hz, H-100), 5.12 (1H, d,
Jˆ10.8 Hz, ArOCHEHF), 5.13 (2H, s, ArOCH2), 6.44 (1H,
d, Jˆ2.1 Hz, H-5), 6.48 (1H, d, Jˆ2.1 Hz, H-7), 7.17±7.44
(36H, m, Ar±H), 7.54 [1H, ddd, Jˆ8.8 Hz, 3.6 Hz (13C±1H)
and 2 Hz, H-60], 7.73 [1H, dd, Jˆ3.6 Hz (13C±1H) and
2.0 Hz, H-20], 12.69 (1H, s, OH). dC (100 MHz, CDCl3):
68.87 (CH2), 70.41 (CH2), 70.60 (CH2), 73.47 (CH2), 74.45
(CH2), 74.61 (CH2), 75.06 (CH2), 75.25 (CH), 75.71 (CH2),
77.56 (CH), 81.52 (CH), 84.40 (CH), 93.02 (CH, d,
Jˆ3.5 Hz), 98.58 (CH), 101.70 (CH), 106.18 (C), 114.23
(CH, d, Jˆ 3.0 Hz), 115.56 (CH, d, Jˆ4.8 Hz), 122.13
(CH), 124.75 (C, d, Jˆ67.7 Hz), 127.41 (CH), 127.48
(CH), 127.53 (CH), 127.58 (CH), 127.61 (CH), 127.65
(CH), 127.83 (CH), 127.91 (CH), 127.93 (CH), 128.04
(CH), 128.17 (CH), 128.28 (CH), 128.33 (CH), 128.38
(CH), 128.39 (CH), 128.72 (CH), 128.77 (CH), 135.75
(C), 136.40 (C), 136.46 (C), 137.62 (C, d, Jˆ86.1 Hz),
137.93 (C), 138.05 (C), 138.26 (C), 138.43 (C), 148.14
(C, d, Jˆ5.7 Hz), 149.26 (C), 153.01 (C), 156.15 (13C
label), 156.69 (C, d, Jˆ 2.5 Hz), 162.07 (C), 164.48 (C)
and 178.82 (C, d, Jˆ 6.8 Hz); m/z (FAB) 1118.3
[(M1Na)1, 55%], 1096.3 (15), 1028.2 (6), 664.2 (5), 574.1
(18); [Found: (M1Na)1, 1118.4188. C6913CH62O12Na
requires (M1Na), 1118.4172]; (Found: C, 76.74; H, 5.75%.
C6913CH62O12 requires C, 76.78; H, 5.70%); [a]1D9ˆ226.0
(cˆ0.043 g ml21, CHCl3).
[2-13C]-Quercetin-40-b-d-glucoside monohydrate 12.
20% Palladium hydroxide on carbon (150 mg) was added
to a stirring suspension of ¯avonol 11 (1.06 g, 0.88 mmol) in
EtOAc±MeOH (1:1, 20 ml) under an atmosphere of hydro-
gen. The suspension was stirred overnight and then the solu-
tion was ®ltered through a plug of celite eluting with MeOH
(15 ml). The ®ltrate was concentrated under vacuum and the
resulting solid recrystallised from MeOH±water (4:1) to
give the monohydrate of the ¯avonol 12 as a yellow solid.
(400 mg, 94%); mp 200±2058C; nmax(nujol)/cm21: 3368
(OH), 1654 (CvO), 1592 (Ar) and 1505 (Ar); dH
(400 MHz, D6-DMSO) 3.17±3.51 (6H, m, H-200, 300, 400,

4106
S. T. Caldwell et al. / Tetrahedron 56 (2000) 4101±4106
600), 4.62 (1H, broad s, OH), 4.85 (1H, d, Jˆ7.1 Hz, H-100),
5.07 (1H, broad s, OH), 5.12 (1H, broad s, OH), 5.43 (1H,
broad s, OH), 6.20 (1H, d, Jˆ1.9 Hz, H-6), 6.45 (1H, d,
Jˆ1.9 Hz, H-8), 7.25 (1H, d, Jˆ8.7 Hz, H-50), 7.62 [1H,
ddd, Jˆ8.8 Hz, 4.0 Hz (13C±1H) and 2.0 Hz, H-60], 7.71
[1H, dd, Jˆ4.0 Hz (13C±1H) and 2.0 Hz, H-20], 8.95 (1H,
broad s, OH), 9.50 (1H, broad s, OH), 10.80 (1H, broad s,
OH) and 12.40 (1H, s, OH); dC (100 MHz, D6-DMSO):
61.05 (CH2), 70.13 (CH), 73.61 (CH), 76.29 (CH), 77.62
(CH), 93.85 (CH, d, Jˆ3.1 Hz), 98.60 (CH), 101.73 (CH),
103.43 (C), 115.49 (CH, d, Jˆ1.7 Hz), 116.18 (CH, d,
Jˆ5.0 Hz), 119.86 (CH), 125.45 (C, d, Jˆ68.0 Hz),
136.70 (C, d, Jˆ 90.1 Hz), 146.24 (13C label), 146.70 (C,
d, Jˆ5.6 Hz), 147.11 (C, d, Jˆ1.3 Hz), 156.57 (C, d,
Jˆ2.7 Hz), 161.05 (C), 164.41 (C), 176.38 (C, d,
Jˆ5.7 Hz); m/z (FAB): 466.1 [(M1H)1, 86%], 303.0
(100); (Found: (M1H)1, 466.1064. C2013CH21O12 requires
(M1H), 466.1066); (Found: C, 52.36; H, 4.71%.
C2013CH22O13 requires C, 52.38; H, 4.59%); lmax (EtOH)
365.4 (eˆ18,116.4) and 253.2 (eˆ16,386.6); [a]1D9ˆ
265.6 (cˆ0.025 g ml21, MeOH).
8. Hertog, M. G. L.; Feskens, E. J. M.; Hollman, P. C. H.; Katan,
M. B.; Kromhout, D. Lancet 1993, 342, 1007±1011.
9. Hertog, M. G. L.; Kromhout, D.; Aravansis, C.; Blackburn, H.;
Buzina, R.; Fidanza, F.; Giampaoli, S.; Jansen, A.; Menotti, A.;
Nedeljkovic, S.; Pekkarinen, M.; Simic, B. S.; Toshima, H.;
Feskens, E. J. M.; Hollman, P. C. H.; Katan, M. Arch. Int. Med.
1995, 155, 381±386.
10. Rice-Evans, C. A.; Miller, N. J.; Paganga, G. Trends Plant Sci.
1997, 2, 152±159.
11. Tsushida, T.; Suzuki, M. Nippon Shokuhin Kagaku Kagaku
Kaishi 1995, 42, 100±108.
12. Aziz, A. A.; Edwards, C. A.; Lean, M. E. J.; Crozier, A. Free
Rad. Res. 1998, 29, 257±269.
13. Ueno, I.; Nakano, N.; Hirono, I. Jpn. J. Exp. Med. 1983, 53,
41±50.
14. Petrakis, P. L.; Kallianos, A. G.; Wender, S. H.; Shetlar, M. R.
Arch. Biochem. Biophys. 1959, 85, 264±271.
È È È
15. Rasku, S.; Wahala, K. Tetrahedron 2000, 56, 913±916.
16. Hiraoka, K.; Miyamato, T.; Baba, S.; Furuta, T. J. Labelled
Compd. Radiopharm. 1981, 18, 613±619.
17. Baba, S.; Furuta, T.; Masanobu, H.; Nakagawa, H. J. Pharm.
Sci. 1981, 70, 780±782.
18. Baba, S.; Furuta, T.; Fujioka, M.; Goromaru, T. J. Pharm. Sci.
1983, 72, 1155±1158.
Acknowledgements
19. Razanamahefa, B.; Demetzos, C.; Skaltsounis, A.-L.;
Andriantisiferana, M.; Tillequin, F. Heterocycles 1994, 38, 357±
373.
Thanks to the University of Glasgow for ®nancial support
from the Fleck Bequest. Thanks also to C. Andrew Wood-
land and Duncan Coutts for their preliminary work in this
area and to the Nuf®eld Foundation for the latter's summer
studentship.
20. Thomsen, I.; Torssell, K. B. G. Acta Chem. Scand. 1991, 45,
539±542.
21. Jung, M. E.; Starkey, L. S. Tetrahedron 1997, 53, 8815±8824.
22. El-Desoky, E.-S. I.; Abel-Rahman, H. A. R.; Schmidt, R. R.
Liebigs Ann. Chem. 1990, 877±881.
References
23. Schmidt, R. R.; Stump, M. Liebigs Ann. Chem. 1983, 1249±
1256.
1. The FlavonoidsÐAdvances in Research Since 1986, Harborne,
J. B., Ed.; Chapman & Hall: London, 1993.
24. Kratzel, K.; Billek, G. Holzforschung 1953, 7, 66±70.
25. Hauteville, M.; Chadenson, M.; Chopin, J. Bull. Soc. Chim. Fr.
1979, II, 125±131.
2. Hertog, M. G. L.; Hollman, P. C. H.; van de Pute, B. J. Agric.
Food Chem. 1993, 15, 1242±1246.
3. Crozier, A.; Lean, M. E. J.; McDonald, M. S.; Black, C. J. Agric.
Food Chem. 1997, 45, 590±595.
26. Picq, M.; Prigent, A. F.; Chabannes, B.; Pacheco, H.; Parent,
P.; Pichat, L. Tetrahedron Lett. 1984, 25, 2227±2230.
27. Deng, B. -L.; Lepoivre, J. A.; Lemiere, G.; Dommisse, R.;
Claeys, M.; Boers, F.; De Groot, A. Liebigs Ann./Recuiel 1997,
2169±2175.
4. McDonald, M. S.; Hughes, M.; Burns, J.; Lean, M. E. J.;
Matthews, D.; Crozier, A. J. Agric. Food Chem. 1998, 46, 368±
375.
5. Herrmann, K. J. Food Technol. 1976, 11, 433±448.
6. Herrmann, K. Z. Lebensm.-Unters. Forsch. 1988, 186, 1±5.
7. Vinson, J. A. Adv. Exp. Med. Biol. 1998, 439, 151±164.
28. Markham, K. R.; Ternai, B.; Stanley, R.; Geiger, H.; Mabry,
T. J. Tetrahedron 1978, 34, 1389±1397.