Convenient syntheses of metabolically important quercetin glucuronides and sulfates

Article abstract of DOI:10.1016/j.tet.2006.04.102

Synthetic approaches to the major human plasma metabolites of quercetin, quercetin 3′-sulfate and the β-d-glucopyranosiduronic acid derivatives 3′-methylquercetin 3-glucuronide (isorhamnetin 3-glucuronide), quercetin 3-glucuronide and quercetin 3′-glucuronide are described. This is the first report of the chemical synthesis of quercetin 3′-glucuronide. All procedures start from the same precursor, 4′,7-di-O-benzylquercetin, and all are more convenient than existing methods.

Full text of DOI:10.1016/j.tet.2006.04.102

Tetrahedron 62 (2006) 6862–6868
Convenient syntheses of metabolically important quercetin
glucuronides and sulfates
*
Paul W. Needs and Paul A. Kroon
BBSRC Institute of Food Research, Norwich Research Park, Colney Lane, Norwich, Norfolk NR4 7UA, UK
Received 7 November 2005; revised 18 April 2006; accepted 28 April 2006
Available online 19 May 2006
Abstract—Synthetic approaches to the major human plasma metabolites of quercetin, quercetin 30-sulfate and the b-D-glucopyranosiduronic
acid derivatives 30-methylquercetin 3-glucuronide (isorhamnetin 3-glucuronide), quercetin 3-glucuronide and quercetin 30-glucuronide are
described. This is the first report of the chemical synthesis of quercetin 30-glucuronide. All procedures start from the same precursor,
40,7-di-O-benzylquercetin, and all are more convenient than existing methods.
Ó 2006 Elsevier Ltd. All rights reserved.
3'
OH
1. Introduction
4'
OH
5'
2'
1
The human diet includes several classes of plant flavonoids;
many appear to be protective against coronary heart disease
and/or a variety of carcinomas.1 Quercetin 1 (Scheme 1) is
the major flavonol found in plants (though usually in glyco-
sylated forms)2 and is thus a ubiquitous part of the human
diet. In the past, the majority of evidence for the health bene-
fits of quercetin came from in vitro experiments on free quer-
cetin. Recent work has studied the human absorption and
metabolism of quercetin derivatives and has shown that
quercetin itself is not present in human plasma, but is found
instead in various glucuronidated and sulfated forms.3 In
order to better determine the biological effects of quercetin,
we required 100 mg quantities of the most abundant circulat-
ing forms—the b-D-glucopyranosiduronic acid derivatives
quercetin 3-glucuronide 2, quercetin 30-glucuronide 3 and
30-methylquercetin 3-glucuronide (isorhamnetin 3-glucuro-
nide) 4, together with quercetin 30-sulfate 5. Although
quercetin glucuronides have been synthesised using liver
microsomal preparations,4,5 this is not convenient if larger
quantities of glucuronides are needed; and existing chemical
syntheses of both the sulfate and the glucuronides, where
available, are either involved and/or low yielding. We
describe convenient syntheses from a common easily pre-
pared precursor, 40,7-di-O-benzylquercetin 6 (Scheme 2).
8
HO
O
6'
7
6
OH
3
5
OH
O
1
Scheme 1. Structure of quercetin 1 (showing ring numbering).
2. Results and discussion
2.1. 40,7-Di-O-benzylquercetin, 6
40,7-Di-O-benzylquercetin, 6, was prepared as described by
Jurd.6 Although the yields were modest (17–29%), gram
quantities of pure 6 were easily obtained without the need
for chromatography.
2.2. Quercetin 3-glucuronide, 2
Wagner et al. first reported the synthesis of 2 in 1970.7 Glu-
curonidation of 6 with methyl 2,3,4-tri-O-acetyl-a-D-gluco-
pyranosyluronate bromide 7 in the presence of silver oxide
(Ag2O), gave 9 in 44% yield (Scheme 2); debenzylation
and deacetylation afforded 2 in 24% overall yield. When
we attempted this procedure, we failed to obtain any glu-
curonidated products, or to recover 6. To ensure anhydrous
conditions, we had stirred 6, Ag2O and a desiccant (either
Keywords: Quercetin glucuronide; Quercetin sulfate; Quercetin 3-glucuro-
nide; Quercetin 30-glucuronide; Isorhamnetin 3-glucuronide; Quercetin
30-sulfate; Glucuronidation; Sulfation; Synthesis.
* Corresponding author. Tel.: +44 1603 255066; e-mail: needsp@bbsrc.
ac.uk
˚
calcium sulfate, CaSO4, or 3 A molecular sieves) in pyridine
for 2 h before adding 7. Conversely, when we added 7 imme-
diately, the reaction gave two mono-glucuronidated prod-
ucts, 8a and 8b, in 18 and 19% yields, respectively, after
0040–4020/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2006.04.102

P. W. Needs, P. A. Kroon / Tetrahedron 62 (2006) 6862–6868
OR
6863
OR
OAc
OBn
MeOOC
AcO
OBn
+
pyridine
Ag2O,
OAc
OAc
BnO
O
O
O
O
BnO
O
Br
O
OH
O
COOMe
9
OAc
OH
OAc
OAc
OH
OAc
7
O
6; R = H
MeOOC
12; R = Me
OAc
8a; R = Ac
8b; R = H
13; R = Me
EtOH,
Pd(OH)2,
OR
OR
cyclohexene
OH
OH
Na2CO3
O
HO
O
HO
aq. MeOH
O
O
OH
OH
OH
O
OAc
OAc
OH
O
O
O
HOOC
MeOOC
OH
OAc
2; R = H
4; R = Me
Scheme 2. Synthesis of quercetin 3-glucuronide 2 and isorhamnetin 3-glucuronide 4.
chromatography. Compound 8a was contaminated with a
trace of the glycal 9 (Scheme 2), a known by-product of glu-
curonidation of phenols.8 Both 8a and 8b gave only 2 after
debenzylation9 and ester hydrolysis10 in a combined purified
yield of 23% from 6. Compound 8b was the expected prod-
uct; but8awas, in addition, 30-O-acetylated. (In contrast, glu-
cosylation of 6 with the corresponding tetraacetylglucosyl
bromide gave only the expected 3-glucosylated product—
data not shown. We noted the tendency of 7 to transfer an
acetyl group to a less acidic, unprotected phenolic hydroxyl
during the development of our synthesis of the isoflavone
conjugate daizein 7-glucuronide.)11 When the reaction was
repeated at 0 ꢀC, 8a and 8b were obtained in an improved
combined yield of 52%. Debenzylation and hydrolysis
gave, after purification, 2 in 40% overall yield in three steps
from 6. This represents a considerable improvement over
both the original report, and a more recent procedure involv-
ing selective oxidation of a glucoside derivative.12
Debenzylation and de-esterification gave, after purification,
3 in 11% yield. Although this yield is low, this is the first
reported chemical synthesis of this compound. The different
regioselectivities of 7 and 10 are noteworthy. Characteristic
downfield shifts of the 20 and 60-protons were evident in the
1H NMR spectrum (see Table 1 for comparative data). We
confirmed the structure of 3 by HMBC, when the expected
long range 1H–13C connectivities were observed. Thus
H00-1 showed 3J correlation to C-30, C-30 showed 3J correla-
tion to H-50 and 2J correlation to H-20.
A synthesis of 3 using liver microsomes has been described.4
Table 2 shows comparative NMR data for this material (the
ionic form of the material was not specified) and chemically
synthesised 3 (as both free acid and sodium salt).
2.4. 30-Methylquercetin 3-glucuronide, 4 (isorhamnetin
3-glucuronide)
We briefly investigated why delayed addition of 7 was dele-
terious to product yield. A 1H NMR spectrum after workup
of a mixture of 6, Ag2O, pyridine and CaSO4 after 2 h
showed no signs of debenzylation. The flavonol signals
were greatly broadened, suggesting complexation to silver
had occurred, which might account for the failure to react
with 7. Thus the problem might be anticipated in other
Ag2O-catalysed glycosylations where the substrate has a
suitable site for complexation, unrelated to the intended
reactive site (such as vicinally positioned 5-hydroxy and
4-oxo groups here). No problem occurred in the synthesis
of quercetin 7-glucuronide,13 where all hydroxyls—except
the 7—were protected.
We reported the first synthesis of this compound, from 6,10
but the yield was very low (5%). The procedure was based
on the treatment of 30-O-methyl-40,7-O-dibenzylquercetin
12 (derived from 6) with 10. Our synthesis of 3 suggested
that reaction with 7 would be a better approach, and indeed
treatment of 12 with 7 and Ag2O in pyridine at 0 ꢀC gave
crude 13 in 51% yield (Scheme 2). Debenzylation, hydroly-
sis and purification of 13 gave 4 in 33% yield from 12 (see
Table 1). We confirmed the structure of 4 by HMBC, when
1
the expected long range H–13C connectivities were ob-
3
served. Thus H00-1 showed J correlation to C-3 and C-30
showed 3J correlation to the protons of the methyl group.
2.5. Quercetin 30-sulfate 5
2.3. Quercetin 30-glucuronide, 3
Although treatment of 1 with sulfamic acid gives 5,3 it is as
part of a complex mixture, which is difficult to purify. We
thus considered alternative approaches based on sulfation
of 3,40,7-tri-O-benzylquercetin 14. Compound 14 was pre-
pared in 66% yield by treatment of 6 with 1.6 equiv of
Treatment of 6 with methyl 2,3,4-tri-O-acetyl-a-D-gluco-
pyranosyluronate trichloroacetimidate 10 gave 11 and re-
covered 6 (Scheme 3), which was partially purified by
chromatography. No 3-O-glucuronidation was observed.

6864
P. W. Needs, P. A. Kroon / Tetrahedron 62 (2006) 6862–6868
OAc
O
Cl
MeOOC
OAc
OAc
OBn
MeOOC
AcO
O
O
OH
Cl
Cl
O
OBn
NH
OAc
BnO
O
O
OAc
10
CH Cl
BF3.Et2O,
2
2
OH
O
BnO
OH
OH 11
6
OH
O
OH
OAc
HOOC
OH
OH
OH
Pd(OH)2
cyclohexene
EtOH
MeOOC
OAc
O
O
OAc
OH
O
O
Na2CO3
O
O
HO
aq. MeOH
O
HO
OH
OH
OH
OH
O
3
Scheme 3. Synthesis of quercetin 30-glucuronide 3.
Table 1. 1H NMR shifts/d (CD3OD) for quercetin glucuronides (H+ form)
Proton Quercetin Quercetin Quercetin Isorhamnetin
Table 2. 1H NMR shifts/d (DMSO-d6) for quercetin 30-glucuronide 3
Proton
Quercetin (1)a
3
3a,b
(1)
3-glucuronide 30-glucuronide 3-glucuronide
H+ form
Na+ form
(2)
(3)
(4)
20
60
50
8
7.67
7.53
6.87
6.40
6.18
7.85
7.88
6.99
6.48
6.20
7.93
7.85
6.97
6.50
6.19
7.98
7.90
6.96
6.59
6.16
20
60
50
8
7.72
7.62
6.87
6.37
6.17
7.61
7.64
6.84
6.38
6.19
8.03
7.89
6.95
6.39
6.15
7.95
7.54
6.86
6.37
6.17
6
6
a
Ref. 4.
Cationic form unspecified.
b
compounds, tentatively identified as quercetin 7-sulfate
and quercetin 40-sulfate (data not shown). Purification of 5
in this case required preparative HPLC (see below); com-
plete separation of the closely running monosulfates was
difficult, and the yield of 5 was lowered as a result.
potassium tert-butoxide and benzyl bromide in DMF
(Scheme 4; 3,30,40,7-tetra-O-benzylquercetin 15 was also
produced, but these conditions maximised the yield of 14).
Treatment of 14 with a chlorosulfonic acid/pyridine mixture
at room temperature—conditions used to sulfate partially
protected daidzein derivatives14—or overnight at 80 ꢀC
was unsuccessful. Treatment of 14 in DMF with sulfur tri-
oxide-N,N-dimethylformamidecomplex16,ahighlyreactive
sulfating reagent15 for 4 d, gave 17, which was debenzylated
to give a mixture of 1 and 5 (Scheme 4). The latter were
easily separated on a C-18 reverse phase solid phase extrac-
tion (SPE) cartridge; elution with water gave pure 5 in 68%
yield from 14. (Alternatively, treatment of 14 with a slight
excess of sodium hydride for 10 min, to give the phenoxide
anion 18, followed by addition 16, also gave, after debenzy-
lation of 18, a mixture of 1 and 5, from which 5 was isolated
in 45% yield. Though this approach had the advantage of
a short reaction time, it was difficult to stoichiometrically
control NaH addition on the small scale employed, which
lowered the yield). Thus 5 was obtained directly as its
sodium salt, avoiding the need for the elaborate purification
procedures typically used in sulfate synthesis (see below);
but only if the 14 used was pure. We found 14, prepared
by benzylation of 1 as described,16 contained small amounts
of two isomeric tribenzylquercetins, which we were unable
to remove; and that these led, after sulfation and depro-
tection to a mixture of 5 and 1, together with two other
2.6. Purification of quercetin glucuronides and sulfates
by preparative HPLC
C-18 reverse phase columns, and gradients of 0.1% aq tri-
fluoroacetic acid (TFA) and acetonitrile, were effective for
preparative scale purification of glucuronides. It was neces-
sary, however, to load the crude glucuronides in their acid
forms in 50% methanol. Solubility in this solvent was
limited; this restricted loading capacity, but this was the
best compromise between solubility and solvent polarity.
Though sodium salts could be loaded in concentrated aque-
ous solution, two peaks—one early peak corresponding to
the sodium form, and a later one to the acid form—were
obtained for each glucuronide, making the approach suited
to simple mixtures only.
We also report a substantial improvement in the HPLC
purification of sulfates. Although it was not ultimately re-
quired for 5 prepared as above, it was used to obtain pure
sulfated products from several procedures that were ulti-
mately less efficient and are therefore not reported here.

P. W. Needs, P. A. Kroon / Tetrahedron 62 (2006) 6862–6868
6865
OH
OBn
OBn
OBn
BnO
O
BnO
O
O
tBuOK
BnBr
OH
OBn
OH
O
DMF
OH
6
15 (minor product)
O
OH
+
OBn
OBn
NaH, DMF,
10 min
O
O
BnO
BnO
O
OBn
OBn
OH
OH
O
18
14
SO3.DMF
16
16, 5 min
O
O
O
O
O
O
S
4 d
Pd(OH)2
S
O
O
OBn
OH
O
BnO
cyclohexene
O
HO
EtOH
OBn
OH
17
OH
O
OH
O
5
Scheme 4. Synthesis of quercetin 30-sulfate 5.
Aq TFA–acetonitrile gradients were found to be unsuitable.
Retention times were highly variable (due presumably to the
low pKa of sulfates) and fractions containing sulfates were
prone to acid catalysed sulfate cleavage during subsequent
evaporation (data not shown). We found that substituting
50 mM aq ammonium acetate for aq TFA overcame this
problem, and products were easily desalted by lyophilisa-
tion. Previously reported flavonoid sulfate purifications are
generally involved, employ several steps, and use techniques
such as size exclusion chromatography, normal phase
HPLC, preparative TLC and separation on polyamide.17–20
As far as we are aware, NH4OAc–CH3CN gradients and
reverse phase HPLC have not been used before for the iso-
lation of pure flavonoid sulfates.
pH 6.0 with aq sodium hydroxide, followed by immediate
evaporation. The resultant solids were stored at ꢁ20 ꢀC,
and have again been stable. The salt forms of these materials
are freely soluble in water, which is highly convenient for
use in cell culture studies, etc. Frozen, millimolar solutions
have also been stable. We are unable yet to fully comment on
long-term stability; but we have found solutions to be stable
for 48 h at 37 ꢀC in culture media.5
3. Experimental
3.1. General methods
˚
Solvents were dried over freshly activated 3 A molecular
2.7. Storage and handling of glucuronides and sulfates
sieves. Evaporations were performed in vacuo at 50 ꢀC.
Solids were dried overnight in vacuo over P2O5 before use.
TLC was performed on Macherey-Nagel Silica Gel 60/
UV254 plates using UV light, or 50% sulfuric acid and
charring, for visualisation. MPLC used pre-packed silica
cartridges (Isolute Flash Si, Argonaut Technologies) and
UV detection. Analytical HPLC and HPLC–ESMS used
a 5 mm Lunar column (250ꢂ4.4 mm) eluted at 1 mL minꢁ1
at 30 ꢀC. Eluant A 80% for 5 min (isocratic); to 10% A at
35 min (gradient). Eluate was monitored by UV detection
at 205 and 280 nm. Preparative HPLC of glucuronides
used a 5 mm Prodigy ODS3 column (Phenomenex Inc.,
We initially stored glucuronides in their acid forms, as pro-
tection against possible autooxidative reactions (to which 1
is increasingly susceptible as temperature, pH and/or salt
concentrations are raised),21 in solution in 50% aq methanol
at ꢁ20 ꢀC; they were indefinitely stable. Solutions were
warmed to room temperature and briefly sonicated before
use. Although the latter apparently redissolved any material
that had precipitated during refrigeration, subsequent filtra-
tion through a 0.2 mm membrane led to losses. Methanolic
solutions (100%) were filtered without loss, but under these
conditions the glucuronic acid moieties were prone to self-
catalysed methyl esterification. The addition of water suc-
cessfully prevented esterification during storage at ꢁ20 ꢀC,
or, for example, rotary evaporation. Sulfates were stored as
solid Na salts, or in solution in aq methanol or methanol,
at ꢁ20 ꢀC. More recently we have converted glucuronides
to their sodium salts by titration, in 50% aq methanol, to
250ꢂ21.2 mm+60ꢂ21.2 mm guard) eluted at 5 mL minꢁ1
.
Eluant A—0.1% CF3COOH (TFA); Eluant B—CH3CN.
80% A for 15 min (isocratic); then to 10% A at 75 min (gra-
dient). Preparative HPLC of sulfates used the same column
and flow rate, but different elution conditions: Eluant A—
50 mM aq NH4OAc, pH 6.5; Eluant B—CH3CN. 80% A
for 15 min (isocratic); then to 40% A at 75 min (gradient).

6866
P. W. Needs, P. A. Kroon / Tetrahedron 62 (2006) 6862–6868
Solidphaseextraction(SPE)was performedonTechElut SPE
C-18 2000/12 mL cartridges, preconditioned withMeOH and
water. Melting points were determined with a Reichert 7905
hot stage microscope, and are uncorrected. 1H and 13C NMR
spectra were run on a JEOL EX-270 spectrometer at 21 ꢀC.
HMBC NMR spectra were run on a Bruker Avance 600 spec-
trometer at 27 ꢀC. NMR chemical shifts were referenced to
residual solvent absorption. ESMS, APCIMS and HPLC–
ESMS analyses were performed on a Micromass Quattro II
mass spectrometer. HRMS data were obtained by syringe
pump injection of solutions in aq CH3CN, using either
a Micromass LCT MS or a Bruker microTOF, equipped
with electrospray sources. IR spectra were measured by
Attenuated Total Reflectance (ATR), using a BioRad
FTS175C Fourier transform infrared spectrometer with
HgCdTe detector and Specac GoldenGate single reflection
diamond Horizontal ATR system; 128 scans at 2 cmꢁ1 reso-
lution, background spectrum empty crystal. Optical rotation
measurements were made on a Perkin Elmer 341 Polarimeter.
6.49 (d, 1H, J 2.0 Hz, H-8), 6.42 (d, 1H, J 2.0 Hz, H-6),
5.83 (s, 1H, 30-OH), 5.73 (d, 1H, J 7.9 Hz, H-100), 5.1–5.4
(m, 3H, H-200, H-300, H-400), 5.21, 5.11 (2ꢂs, 2ꢂ2H,
2ꢂCH2Ph), 4.00 (d, 1H, J 10.2 Hz, H-500), 3.60 (s, 3H,
OCH3), 2.10, 2.02, 2.00 (3ꢂs, 3ꢂ3H, 3ꢂCOCH3); m/z
(APCI, +ve mode) 821 [(M+Na)+].
Compounds 8a and 8b were not further purified or character-
ised but were debenzylated, de-esterified and purified by
HPLC as follows. The products were recombined and sus-
pended in a mixture of EtOH (80 mL) and cyclohexene
(20 mL). Twenty percent Pd(OH)2 on charcoal (200 mg)
was added. The mixture was refluxed under Ar for 30 min,
cooled and filtered through a 0.5 mm filter. The filtrate was
evaporated and the residue was suspended in 50% aq
MeOH (200 mL). Aq Na2CO3 (0.5 M, 6 mL) was added
and the mixture was stirred under Ar at room temperature
for 2.5 h. After cooling Dowex 50W resin (H+ form) was
added with stirring until the pH fell to below 3.0. The mix-
ture was filtered (0.5 mm), the resin was washed (50% aq
MeOH, 1ꢂ50 mL; MeOH, 1ꢂ50 mL) and filtrate and resin
washes were combined and evaporated. The residue was dis-
solved in 50% aq MeOH (8 mL). Compound 2 was isolated
by preparative HPLC (2ꢂ4 mL injections) as an orange
glass and was >95% pure by analytical HPLC. It was iden-
tical to authentic material.22 Yield 89 mg (41% from 6).
3.2. Synthesis of quercetin conjugates
3.2.1. 40,7-Di-O-benzylquercetin, 6. 40,7-Di-O-benzylquer-
cetin, 6, was prepared from quercetin pentaacetate (25 g) as
described by Jurd,6 except that the product was initially
purified by extraction with toluene at 80 ꢀC, rather than
with refluxing benzene. Yield of recrystallised 6 (as yellow
needles) 4.0–6.8 g, 17–29% (lit.6 13%), mp 181–182 ꢀC
(lit.6 181 ꢀC); dH (CDCl3) 11.70 (s, 1H, 5-OH), 7.77 (dd,
1H, J 9.6, 2.3 Hz, H-60), 7.78 (d, 1H, J 2.3 Hz, H-20),
7.29–7.46 (m, 10H, 2ꢂPh), 7.02 (d, 1H, J 9.6 Hz, H-50),
6.61 (s, 1H, 3-OH), 6.55 (d, 1H, J 2.3 Hz, H-8), 6.44 (d,
1H, J 2.3 Hz, H-6), 5.77 (s, 1H, 3-OH), 5.18, 5.13 (2ꢂs,
2ꢂ2H, 2ꢂCH2Ph).
Compound 2 (H+ form): dH (CD3OD) 7.64 (dd, 1H, J 8.9,
2.3 Hz, H-60), 7.61 (d, 1H, J 2.3 Hz, H-20), 6.84 (d, 1H,
J 8.9 Hz, H-50), 6.38 (d, 1H, J 2.0 Hz, H-8), 6.19 (d, 1H,
J 2.0 Hz, H-6), 5.32 (d, 1H, J 7.6 Hz, H-100), 3.74 (d,
1H, J 9.6 Hz, H-500), 3.42–3.62 (m, 3H, H-200, H-300, H-400).
Compound 2 (lit.22 H+ form): dH (CD3OD) 7.63 (H-60), 7.63
(H-20), 6.84 (H-50), 6.37 (H-8), 6.18 (H-6), 5.31 (H-100), 3.75
(H-500), 3.47–3.59 (H-200, H-300, H-400).
3.2.2. Quercetin 3-glucuronide, 2. Ag2O (0.27 g,
2.5 equiv), CaSO4 (dried at 120 ꢀC, 0.5 g) and dry pyridine
(4 mL) were stirred, in the dark, under Ar at 0 ꢀC for 5 min.
Methyl 2,3,4-tri-O-acetyl-a-D-glucopyranosyluronate bro-
mide 7 (0.23 g, 580 mmol, 1.25 equiv) was added; and, after
a further 5 min, 40,7-di-O-benzylquercetin 6 (0.22 g,
460 mmol). Stirring was continued at 0 ꢀC. After 16 h, aq
KCl (10%, 20 mL) and aq AcOH (10%, 100 mL) were added
and the mixture was filtered through Celite. The latter was
washed with water (2ꢂ100 mL), and the crude product
eluted with acetone (2ꢂ50 mL). The latter was evaporated
to give a light brown solid (0.35 g), which was purified
by MPLC (20 g silica, 4% acetone/96% toluene isocratic
elution) to give two main products 8a and 8b.
3.2.3. Quercetin 30-glucuronide, 3. Compound 6 (0.36 g,
˚
0.75 mmol), powdered 3 A molecular sieves (0.5 g) and dry
CH2Cl2 (5 mL) were stirred at room temperature under Ar
for 2 h. The trichloroacetimidate 10 (0.39 g, 1.1 equiv) was
added and the mixture was cooled to ꢁ15 ꢀC. BF3$Et2O
(freshly distilled, 120 mL, 1.25 equiv) was added drop wise
over 10 min. The mixture was allowed to warm to room tem-
perature and stirred overnight. The mixture was filtered
through Celite and the latter was washed with CH2Cl2,
acetone and methanol until the washings were colourless.
Filtrate and washings were combined and evaporated to
give a bright yellow glass. The crude product was purified
by MPLC (20 g silica, 5% acetone/95% toluene isocratic elu-
tion) to give recovered 6 (0.21 g, 58%) and crude 11 (0.10 g).
Compound 8a: dH (CDCl3) 12.42 (s, 1H, 5-OH), 8.01 (dd,
1H, J 8.9, 2.3 Hz, H-60), 7.85 (d, 1H, J 2.3 Hz, H-20),
7.31–7.42 (m, 10H, 2ꢂPh), 7.10 (d, 1H, J 8.9 Hz, H-50),
6.49 (d, 1H, J 2.0 Hz, H-8), 6.42 (d, 1H, J 2.0 Hz, H-6),
5.75 (d, 1H, J 7.9 Hz, H-100), 5.1–5.4 (m, 3H, H-200, H-300,
H-400), 5.26, 5.11 (2ꢂs, 2ꢂ2H, 2ꢂCH2Ph), 3.94 (d, 1H,
J 9.9 Hz, H-500), 3.58 (s, 3H, OCH3), 2.33 (s, 3H, 30-
COCH3), 2.10, 2.03, 2.01 (3ꢂs, 3ꢂ3H, 3ꢂsugar COCH3);
m/z (APCI, +ve mode) 863 [(M+Na)+].
Compound 11: dH (CDCl3) 11.65 (s, 1H, 5-OH), 8.05 (d, 1H,
J 2.3 Hz, H-20), 7.94 (dd, 1H, J 8.6, 2.3 Hz, H-60), 7.28–7.46
(m, 10H, 2ꢂPh), 7.02 (d, 1H, J 8.6 Hz, H-50), 6.69 (s, 1H,
3-OH), 6.58 (d, 1H, J 2.0 Hz, H-8), 6.41 (d, 1H, J 2.0 Hz,
H-6), 5.76 (d, 1H, J 7.9 Hz, H-100), 5.1–5.4 (m, 3H, H-200,
H-300, H-400), 5.36, 5.14 (2ꢂs, 2ꢂ2H, 2ꢂCH2Ph), 4.16 (d,
1H, J 10.2 Hz, H-500), 3.70 (s, 3H, OCH3), 2.05 (s, 3ꢂ3H,
3ꢂCOCH3).
Compound 8b: dH (CDCl3) 12.43 (s, 1H, 5-OH), 7.68 (dd,
1H, J 8.6, 2.3 Hz, H-60), 7.63 (d, 1H, J 2.3 Hz, H-20),
7.27–7.46 (m, 10H, 2ꢂPh), 7.02 (d, 1H, J 8.6 Hz, H-50),
Compound 11 was not further characterised or purified but
was debenzylated, de-esterified and purified in the same

P. W. Needs, P. A. Kroon / Tetrahedron 62 (2006) 6862–6868
6867
way as 8a and 8b (proportionately scaled down) to give 3 as
an orange glass. Yield 39 mg (11% from 6). The substitution
position was confirmed by HMBC (see text); 3 nmax (ATR of
solid) 3208br d, 1675, 1652, 1616, 1595, 1570, 1502, 1412,
1357, 1268, 1189, 1161, 1136, 1003; [a]2D5 ꢁ44 (c 1.0, water).
Compound 3 (H+ form): dH (CD3OD) 8.03 (d, 1H, J 2.0 Hz,
H-20), 7.89 (dd, 1H, J 8.6, 2.0 Hz, H-60), 6.95 (d, 1H,
J 8.6 Hz, H-50), 6.39 (d, 1H, J 2.0 Hz, H-8), 6.15 (d, 1H,
J 2.0 Hz, H-6), 4.92 (d, 1H, J 6.9 Hz, H-100), 4.04 (d,
1H, J 9.6 Hz, H-500), 3.52–3.70 (m, 3H, H-200, H-300, H-400).
H-6), 5.80 (d, 1H, J 7.9 Hz, H-100), 5.12–5.41 (m, 3H,
H-200, H-300, H-400), 5.21, 5.01 (2ꢂs, 2ꢂ2H, 2ꢂCH2Ph),
4.05 (d, 1H, J 9.9 Hz, H-500), 4.00 (s, 3H, OCH3), 3.55 (s,
3H, OCH3), 2.10, 2.02, 1.99 (3ꢂs, 3ꢂ3H, 3ꢂCOCH3).
Compound 13 was not further characterised or purified but
was debenzylated, de-esterified and purified in the same
way as 8a and 8b (proportionately scaled up) to give 4 as
an orange glass, identical in all respects to authentic mate-
rial,10 yield 198 mg (33% from 6). The substitution position
was confirmed by HMBC (see text); nmax (ATR of solid)
3223br d (OH), 1680, 1652, 1599, 1563, 1463, 1427, 1353,
1286, 1242, 1198, 1181, 1167, 1124; [a]2D5 ꢁ13 (c 1.0, water).
Compound 4 (H+ form): dH (CD3OD) 7.95 (d, 1H, J 2.4 Hz,
H-20), 7.54 (dd, 1H, J 8.2, 2.0 Hz, H-60), 6.86 (d, 1H,
J 8.2 Hz, H-50), 6.37 (d, 1H, J 2.0 Hz, H-8), 6.17 (d, 1H,
J 2.0 Hz, H-6), 5.48 (d, 1H, J 7.2 Hz, H-100), 3.93 (s,
3H, OCH3), 3.78 (d, 1H, J 9.2 Hz, H-500), 3.46–3.62 (m,
3H, H-200, H-300, H-400); dC (CD3OD) 179.1 (C4), 171.6
(C600), 166.0 (C7), 163.0 (C5), 158.7 (C2), 158.4 (C9),
150.9 (C30), 148.4 (C40), 135.1 (C3), 123.5 (C60), 122.8
(C10), 116.0 (C50), 114.4 (C20), 105.7 (C10), 104.0 (C100),
99.9 (C6), 94.8 (C8), 77.5 (C300), 77.2 (C500), 75.6 (C200),
73.0 (C400), 55.7 (OCH3).
Compound 3 (H+ form): dH (DMSO-d6) 12.43 (s, 1H, 5-OH),
9.45 (br d s, 1H, COOH), 7.88 (dd, 1H, J 8.2, 2.0 Hz, H-60),
7.85 (d, 1H, J 2.0 Hz, H-20), 6.99 (d, 1H, J 8.2 Hz, H-50),
6.48 (d, 1H, J 2.0 Hz, H-8), 6.20 (d, 1H, J 2.0 Hz, H-6),
5.2 (br d s, 1H, 3-OH), 4.92 (d, 1H, J 6.9 Hz, H-100), 3.79
(d, 1H, J 8.9 Hz, H-500), 3.3–3.5 (m, 3H, H-200, H-300, H-400).
Compound 3 (Na+ form, obtained by titration in aq MeOH to
pH 6.0 with aq NaOH, and evaporation): dH (DMSO-d6)
12.46 (s, 1H, 5-OH), 7.93 (d, 1H, J 2.3 Hz, H-20), 7.85
(dd, 1H, J 8.6, 2.3 Hz, H-60), 6.97 (d, 1H, J 8.6 Hz, H-50),
6.50 (d, 1H, J 1.6 Hz, H-8), 6.19 (d, 1H, J 1.6 Hz, H-6),
5.04 (br d s, 1H, 3-OH), 4.73 (d, 1H, J 6.9 Hz, H-100), 3.44
(d, 1H, J 8.9 Hz, H-500), 3.2–3.4 (m, 3H, H-200, H-300, H-
400); dC (DMSO-d6) 175.9 (C-4), 171.5 (C-600), 164.1 (C-7),
160.5 (C-5), 156.1 (C-9), 149.6 (C-40), 146.3 (C-2), 145.3
(C-30), 135.9 (C-3), 124.2 (C-60), 121.6 (C-10), 117.9 (C-
20), 116.5 (C-50), 103.3 (C-100), 102.9 (C-10), 98.2 (C-6),
93.7 (C-8), 74.2 (C-500), 74.5, 76.0, 73.0 (C-200, C-300, C-400).
Compound 4 (Na+ form): dH (CD3OD) 7.99 (d, 1H, J 2.0 Hz,
H-20), 7.56 (dd, 1H, J 8.3, 2.0 Hz, H-60), 6.89 (d, 1H,
J 8.3 Hz, H-50), 6.38 (d, 1H, J 2.0 Hz, H-8), 6.17 (d, 1H,
J 2.0 Hz, H-6), 5.38 (m, 1H, H-100), 3.93 (s, 3H, OCH3),
3.44–3.57 (m, 4H, H-200, H-300, H-400, H-500).
HRMS (ESI, ꢁve mode): (MꢁH)ꢁ, found 477.0686.
HRMS (ESI, +ve mode): (M+H)+, found 537.0642.
C22H19Na2O13 requires 537.0616.
C21H17O13 requires 477.0669.
3.2.4. 30-O-Methyl-40,7-di-O-benzylquercetin, 12. 30-O-
Methyl-40,7-di-O-benzylquercetin, 12, was prepared from
3,30,5,40,7-di-O-benzylquercetin triacetate (1.66 g) as de-
scribed by Jurd,6 except that the crude product after filtration
and acidification was purified by MPLC (silica, 2% acetone/
toluene). Yield of 12 (as a yellow powder) 0.18 g, 15% (lit.6
12%); mp 172–174 ꢀC (lit.6 175 ꢀC); dH (CDCl3) 11.71 (s,
1H, 5-OH), 7.79 (d, 1H, J 2.3 Hz, H-20), 7.74 (dd, 1H, J
8.9, 2.3 Hz, H-60), 7.33–7.46 (m, 10H, 2ꢂPh), 7.00 (d, 1H,
J 8.9 Hz, H-50), 6.60 (s, 1H, 3-OH), 6.56 (d, 1H, J 2.0 Hz,
H-8), 6.52 (d, 1H, J 2.0 Hz, H-6), 5.24, 5.14 (2ꢂs, 2ꢂ2H,
2ꢂCH2Ph), 3.98 (s, 3H, OCH3).
3.2.6. 3,40,7-Tri-O-benzylquercetin, 14. Compound 6 (2 g,
4.15 mmol) was dissolved with stirring in dry DMF (35 mL)
t
under Ar at 21 ꢀC. BuOK (0.745 g, 1.6 equiv) was added,
followed by BnBr (790 mL, 1.6 equiv). Stirring was contin-
ued overnight at 21 ꢀC for 17 h. The mixture was poured
into stirred 10% aq HCl (375 mL) and cooled to 5 ꢀC for
2 h. The resultant solid was recovered by filtration, washed
with water, dissolved in EtOAc (300 mL), washed twice
with water (2ꢂ100 mL) and dried (MgSO4). Evaporation
gave a solid (2.38 g) that was purified by MPLC (50 g silica,
100% toluene for 10 min, then to 5% acetone/95% toluene
after another 5 min) to give 14 (1.57 g, 66%) as yellow
needles, mp 155 ꢀC (lit.16 mp not given).
3.2.5. 30-Methylquercetin 3-glucuronide (isorhamnetin
3-glucuronide), 4. Ag2O (0.71 g, 2.5 equiv), CaSO4 (dried
at 120 ꢀC, 0.6 g) and dry pyridine (10 mL) were stirred, in
the dark, under Ar at 0 ꢀC. After 5 min, methyl 2,3,4-
tri-O-acetyl-a-D-glucopyranosyluronate bromide 7 (0.61 g,
580 mmol, 1.25 equiv) was added; and, after a further
5 min, 30-O-methyl-40,7-di-O-benzylquercetin 12 (0.61 g,
1.23 mmol). Stirring was continued at 0 ꢀC. After 16 h, the
reaction was worked up as described for 8a and 8b (on
2.5ꢂthe scale) to give 0.82 g of a light brown solid. MPLC
(50 g silica, 5% acetone/95% toluene, isocratic elution)
gave 0.42 g (51%) of 13; dH (CDCl3) 12.40 (s, 1H, 5-OH),
7.77 (d, 1H, J 2.0 Hz, H-20), 7.53 (dd, 1H, J 8.6, 2.0 Hz,
H-60), 7.29–7.46 (m, 10H, 2ꢂPh), 6.92 (d, 1H, J 8.6 Hz,
H-50), 6.41 (d, 1H, J 2.0 Hz, H-8), 6.35 (d, 1H, J 2.0 Hz,
Compound 14: dH (DMSO-d6) 12.67 (s, 1H, 5-OH), 9.44 (s,
1H, 30-OH), 7.57 (d, 1H, J 2.3 Hz, H-20), 7.53–7.29 (m, 16H,
H-60, 3ꢂPh), 7.13 (d, 1H, J 8.8 Hz, H-50), 6.80 (d, 1H,
J 2.0 Hz, H-8), 6.48 (d, 1H, J 2.0 Hz, H-6), 5.23–5.32
(br s, 4H, 2ꢂCH2Ph), 5.03 (s, 2H, CH2Ph).
Compound 14 (lit.16): dH (DMSO-d6) 12.67 (s, 1H, 5-OH),
9.47 (s, 1H, 30-OH), 7.56 (d, 1H, J 1.5 Hz, H-20), 7.52–
7.29 (m, 16H, H-60, 3ꢂPh), 7.13 (d, 1H, J 8.8 Hz, H-50),
6.80 (d, 1H, J 1.5 Hz, H-8), 6.47 (d, 1H, J 1.5 Hz), 5.23–
5.32 (br s, 4H, 2ꢂCH2Ph), 5.02 (s, 2H, CH2Ph).
3.2.7. Quercetin 30-sulfate, 5. Compound 14 (0.252 g,
440 mmol) was dissolved in dry DMF (5 mL) with stirring

6868
P. W. Needs, P. A. Kroon / Tetrahedron 62 (2006) 6862–6868
2. Williams, C. A.; Harborne, J. B. Flavone and Flavonol
Glycosides. In The Flavonoids: Advances in Research Since
1986; Harborne, J. B., Ed.; Chapman and Hall: London, 1994.
3. Day, A. J.; Mellon, F.; Barron, D.; Sarrazin, G.; Morgan,
M. R. A.; Williamson, G. Free Radical Res. 2001, 35,
941–952.
4. Boersma, M. G.; van der Woude, H.; Bogaards, J.; Boeren, S.;
Vervoort, J.; Cnubben, N. H. P.; van Iersel, M. L. P. S.; van
Bladeren, P. J.; Rietjens, I. M. C. M. Chem. Res. Toxicol.
2002, 15, 662–670.
5. O’Leary, K. A.; Day, A. J.; Needs, P. W.; Mellon, F. A.;
O’Brien, N. M.; Williamson, G. Biochem. Pharmacol. 2003,
65, 479–491.
6. Jurd, L. J. Org. Chem. 1962, 27, 1294–1297.
7. Wagner, H.; Danninger, H.; Seligmann, O.; Farkas, L. Chem.
Ber. 1970, 103, 3674–3677.
8. Andersson, F. B.; Leaback, D. H. Chem. Ind. (London) 1960,
967–968.
9. Boers, F.; Lemiere, G.; Lepoivre, J. A.; Dommisse, R.;
De Bruyne, T.; Vlietinck, A. J.; Vanden Berghe, D. A.
Pharmazie 1998, 53, 512–517.
10. de Pascual-Teresa, S.; Johnston, K. L.; Dupont, M. S.; O’Leary,
K. A.; Needs, P. W.; Morgan, L. M.; Clifford, M. N.; Bao, Y. P.;
Williamson, G. J. Nutr. 2004, 134, 552–557.
11. Needs, P. W.; Williamson, G. Carbohydr. Res. 2001, 330,
511–515.
12. Bouktaib, M.; Atmani, A.; Rolando, C. Tetrahedron Lett. 2002,
43, 6263–6266.
13. O’Leary, K. A.; Day, A. J.; Needs, P. W.; Sly, W. S.; O’Brien,
N. M.; Williamson, G. FEBS Lett. 2001, 503, 103–106.
14. Fairley, B.; Botting, N. P.; Cassidy, A. Tetrahedron 2003, 59,
5407–5410.
15. Roy, A. B. The Chemistry of Sulfate Esters and Related
Compounds. In Sulfation of Drugs and Related Compounds;
Mulder, G. J., Ed.; CRC: Florida, FL, USA, 1981; pp 5–8.
16. Alluis, B.; Dangles, O. Helv. Chim. Acta 2001, 84, 1133–1156.
17. Ahmed, A. A.; Mabry, T. J. Phytochemistry 1986, 26, 1517–
1518.
under Ar. A solution of sulfur trioxide-N,N-dimethylforma-
mide complex 16 (0.340 g, 5 equiv, weighed and kept under
Ar) in DMF (5 mL) was added. After 4 d, the reaction mix-
ture was evaporated, and satd NaHCO3 (6 mL) was added
with stirring (final pH 7.6). The mixture was again evapo-
rated to dryness and the residue was dissolved in acetone
(150 mL) and filtered (sinter). The residual solid was washed
with acetone (50 mL). (Attempts to isolate more products
from the residual undissolved material were unsuccessful.)
The acetone washings and filtrate were combined and
evaporated to a yellow glass (0.27 g). This material was
not further purified but was dissolved in EtOH (50 mL);
cyclohexene (25 mL) and Pd(OH)2/C (250 mg) were added,
and the mixture was heated at reflux (30 min) and cooled.
The mixture was filtered (0.5 mm membrane) and evaporated
to give a yellow solid (0.34 g). The latter was redissolved in
water (10 mL), loaded onto an SPE cartridge and eluted with
water (100 mL) to give 5 (121 mg, 68% from 14) as a yellow
solid, identical to authentic material.23
Compound 5 (Na+ form): dH (DMSO-d6) 12.45 (br s, 1H, 5-
OH), 9.47 (br s, 2H, 2ꢂOH), 8.03 (d, 1H, J 2.0 Hz, H-20),
7.85 (dd, 1H, J 8.6, 2.0 Hz, H-60), 6.98 (d, 1H, J 8.6 Hz, H-
50), 6.44 (d, 1H, J 1.9 Hz, H-8), 6.19 (d, 1H, J 1.9 Hz, H-6).
Compound 5 (lit.23, Na+ form): dH (DMSO-d6) 7.99 (d, 1H,
J 2.4 Hz, H-20), 7.80 (dd, 1H, J 7.6, 2.4 Hz, H-60), 6.93 (d,
1H, J 7.6 Hz, H-50), 6.39 (d, 1H, J 1.5 Hz, H-8), 6.14 (d,
1H, J 1.5 Hz, H-6).
Compound 5 (Na+ form): dH (CD3OD) 8.20 (d, 1H, J 2.3 Hz,
H-20), 8.01 (dd, 1H, J 8.9, 2.3 Hz, H-60), 7.01 (d, 1H,
J 8.9 Hz, H-50), 6.42 (d, 1H, J 1.9 Hz, H-8), 6.16 (d, 1H,
J 1.9 Hz, H-6). m/z (ESI, ꢁve mode) 381 ((MꢁH)ꢁ).
Acknowledgements
This work was funded by the Biotechnology and Biological
Sciences Research Council (UK). We wish to thank Dr. Fred
Mellon and Mr. John Eagles for performing mass spectros-
copy, and Dr. Ian Colquhoun for obtaining the HMBC
NMR data. We wish to acknowledge the use of the
Engineering and Physical Sciences Research Council’s
Chemical Database Service at Daresbury.24
18. Mann, P.; Tofern, B.; Kaloga, M.; Eich, E. Phytochemistry
1999, 50, 267–271.
19. Seabra, R. M.; Alves, A. C. Phytochemistry 1991, 30, 1344–
1345.
20. Barron, D.; Ibrahim, R. K. Phytochemistry 1988, 27, 2362–
2363.
21. Dangles, O.; Fargeix, G.; Dufour, C. J. Chem. Soc., Perkin
Trans. 2 1999, 1387–1395.
22. Price, K. R.; Colquhoun, I. J.; Barnes, K. A.; Rhodes, M. J. C.
J. Agric. Food Chem. 1998, 46, 4898–4903.
References and notes
23. Jain, N.; Ahmad, M.; Kamil, M.; Ilyas, M. Phytochemistry
1991, 30, 1345–1347.
24. Fletcher, D. A.; Meeking, R. F.; Parkin, D. J. Chem. Inf.
Comput. Sci. 1996, 36, 746–749.
1. Kroon, P. A.; Clifford, M. N.; Crozier, A.; Day, A. J.; Donovan,
J. L.; Manach, C.; Williamson, G. Am. J. Clin. Nutr. 2004, 80,
15–21.