An efficient method for the glycosylation of isoflavones

Article abstract of DOI:10.1002/ejoc.200800803

The isoflavone phytoestrogens are still of current interest for their positive and negative health benefits. However, there are still many unanswered questions regarding their absorption, metabolism and bioavailability. Studies in this area require access to samples of both the isoflavone 7-O-glucosides, the form found in plants and the 7-O-glucuronides, which are important mammaliam metabolites. A new efficient, high-yielding glycosylation procedure is described for isoflavones, which employs 2,2,2-trifluoro-N-(p-methoxyphenyl) acetamidates as the glycosyl donors. This methodology was used to prepare the 7-O-glycosides of the three main isoflavones, daidzein, genistein and glycitein. The isoflavones were protected with hexanoyl groups which improved their solubility in organic solvents and improved the efficiency of the reaction. The same methodology was then adapted for the synthesis of the analogous 7-O-glucuronides. The new synthesis will provide access to large quantities of these compounds for further biological studies. Wiley-VCH Verlag GmbH & Co. KGaA, 2008.

Full text of DOI:10.1002/ejoc.200800803

FULL PAPER
DOI: 10.1002/ejoc.200800803
An Efficient Method for the Glycosylation of Isoflavones
Nawaf Al-Maharik[a] and Nigel P. Botting*[a]
Keywords: Isoflavones / Phytoestrogens / Glucuronides / Glycosylation / Genistein
The isoflavone phytoestrogens are still of current interest for
their positive and negative health benefits. However, there
are still many unanswered questions regarding their absorp-
tion, metabolism and bioavailability. Studies in this area re-
quire access to samples of both the isoflavone 7-O-gluco-
sides, the form found in plants and the 7-O-glucuronides,
which are important mammaliam metabolites. A new ef-
ficient, high-yielding glycosylation procedure is described
odology was used to prepare the 7-O-glycosides of the three
main isoflavones, daidzein, genistein and glycitein. The iso-
flavones were protected with hexanoyl groups which im-
proved their solubility in organic solvents and improved the
efficiency of the reaction. The same methodology was then
adapted for the synthesis of the analogous 7-O-glucuronides.
The new synthesis will provide access to large quantities of
these compounds for further biological studies.
for isoflavones, which employs 2,2,2-trifluoro-N-(p-meth- (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
oxyphenyl)acetamidates as the glycosyl donors. This meth-
Germany, 2008)
(ODMA),[20] whereas genistein gives 6-hydroxy-ODMA.[21]
When the aglycones and their metabolites are absorbed,
they are then transported to the liver, where they can un-
dergo conjugation catalysed by hepatic enzymes to give O-
glucuronides,[22] and to a lesser extent O-sulfates.[23]
Introduction
The isoflavone phytoestrogens are a group of polypheno-
lic compounds with weak estrogenic and antiestrogenic ac-
tivity, present in the human diet.[1,2] The main dietary
sources are soybeans and soy-derived products.[3,4] Epide-
miological studies have shown that the consumption of an
isoflavone-rich diet is associated with a decrease in the inci-
dence of hormone-related cancers, such as breast[5] and
prostate cancer.[6] It has also been suggested that isofla-
vones may possess other health-promoting activities, in-
cluding chemoprevention of osteoporosis[7] and cardiovas-
cular disease[8] and lessening of menopausal symptoms.[9,10]
However, the biological effects of isoflavones on human
health cannot be fully understood until their absorption,
metabolism and bioavailability have been fully estab-
lished.[11–13]
In plants and foodstuffs isoflavones occur predominantly
as glycosides (2a–c),[14] and so these water-soluble deriva-
tives are the forms ingested by humans. The glycosides are
then hydrolysed by intestinal glucosidases, both from the
host[15] and intestinal bacteria,[16] to give the aglycones,
daidzein (1a), genistein (1b) and glycitein (1c). Some studies
have reported that the isoflavone aglycones are absorbed
more readily than the parent glycosides,[17,18] whereas data
from other studies have implied that the bioavailability of
genistein and daidzein glycosides is actually better than that
of the aglycones.[19] The aglycones can then be metabolised
to other biologically active metabolites. For example, daidz-
ein is converted to equol and O-demethylangolensin
In order to better understand the absorption, metabolism
and bioavailability of isoflavones, there is an urgent need
for the development of efficient synthetic routes for the syn-
thesis of pure standards of isoflavone O-glucosides and O-
glucuronides to allow their accurate quantification in bio-
logical fluids and to study their possible biological roles in
vivo. Recently, in our laboratory, Fairley et al. developed an
efficient synthesis of isoflavone O-sulfates,[24] and now we
wish to report herein a new, sophisticated and high-yielding
procedure for the synthesis of isoflavone 7-O-glucosides
and 7-O-glucuronides.[25]
Results and Discussion
Previous methods for the glycosidation of polyhy-
droxyisoflavones are low-yielding (Ͻ30%).[26,27] Reaction of
daidzein (1a) or its protected derivatives 4Ј-O-acetyldaidz-
ein and 4Ј-O-TBDMS-daidzein with α-acetobromoglucose,
employing Koenig–Knorr techniques, in CH2Cl2 with
Ag2CO3 as catalyst and in the presence of either lutidine or
[a] School of Chemistry, University of St Andrews,
St. Andrews, Fife, KY 16 9ST, Scotland
Fax: +44-1334-463800
E-mail: npb@st-andrews.ac.uk
5622
© 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2008, 5622–5629

An Efficient Method for the Glycosylation of Isoflavones
collidine as a base, afforded the glycosidation product in
low yields (10–15%). Lewis et al.[26] reported the synthesis
of daidzin (2a) in 40% yield from α-acetobromoglucose and
daidzein (1a) by using phase-transfer catalysis. However, in
our hands this procedure afforded daidzin (2a) in less than
1
10% yield as indicated by H NMR spectroscopy. Thus, it
Scheme 1. Reagents and conditions: (a) K2CO3, wet acetone, room
temp., 4 h.
was necessary to search for sophisticated glycosyl donors
outside the conventional glycosidation reagents. Alternative
glycosyl donors were prepared, allowing glycosidation to be
carried out under non-basic conditions, avoiding the use of
To overcome the regioselectivity problem and to increase
silver salts. Among these, treatment of phenyl selenoglyco- the solubility of the isoflavone in CH2Cl2, we sought to
side with daidzein in CH2Cl2, employing TMSOTf, Br2 or acylate the phenolic 4Ј-hydroxy group. The 7-hydoxy group
I2 as activator, afforded an intractable mixture of per-acety- in isoflavones is more acidic than that at C-4Ј because of
lated daidzin and per-acetylated daidzein-4Ј-yl glucoside in the electron-withdrawing effect of the para-carbonyl group
low yields. Glycosyl phosphates have emerged as useful gly- in ring C.[30] Therefore, use of an excess of KOtBu in DMF
cosyl donors for stereoselective glycosidic bond formation allows the generation of the 4Ј,7-diphenolate of either
in the synthesis of oligosaccharides and phenolic glyco- daidzein (1a) or glycitein (1c), or the 4Ј,5,7-triphenolate of
sides.[28] Unfortunately, attempts to adapt this approach for genistein (1b). The electron-withdrawing carbonyl groups
the glucosidation of 4Ј-O-acetyldaidzein proved to be low- para to the 7-oxy group and in the ortho position to the 5-
yielding and gave irreproducible results.
oxy group significantly diminish their nucleophilicity, thus
Recently, Li et al. reported an efficient and facile synthe- acylation with 1.0 equiv. of acyl chlorides produces the 4Ј-
sis of flavonoid 7-O-glycosides by employing a newly devel- acetylated isoflavone selectively. Acylation of daidzein (1a),
oped benzoyl-protected glycosyl trifluoroacetimidate do- and glycitein (1c) with an equivalent amount of hexanoyl
nor.[29] The method is based on a Lewis acid promoted chloride in the presence of KOtBu (2.1 equiv.) in dry DMF
coupling of a glycosyl trifluoroacetimidate donor with the at room temp. gave the isoflavone 4Ј-hexanoates 6a,c in 65–
7-OH group of flavonoid esters. 2,3,4,6-Tetra-O-acetyl-- 78% yield (Scheme 2).
glycopyranosyl 2,2,2-trifluoro-N-(p-methoxyphenyl)aceta-
midate (5a) and methyl 2,3,4-tri-O-acetyl--glycopyranosi- ceeded to investigate their glycosidation with the very reac-
duronyl 2,2,2-trifluoro-N-(p-methoxyphenyl)acetamidate tive trifluoroacetamidate donor 5a. The glycosyl donor
With the isoflavone 4Ј-hexanoates 6a,b,c in hand, we pro-
(5b) were readily prepared in 78 and 75% yield, respectively, α/β-O-acetylglycopyranosyl 2,2,2-trifluoro-N-(p-methoxy-
by treatment of the corresponding 1-hydroxy sugar, 3a or phenyl)acetamidate (5a) (1.5 equiv.) was thus treated with
3b, with 2,2,2-trifluoro-N-(p-methoxyphenyl)acetimidoyl the acceptor 6a,b,c (1.0 equiv.) by employing BF3·Et2O as a
chloride (4) in the presence of K2CO3 in wet acetone at promoter in dry CH2Cl2 and in the presence of freshly dried
room temperature for 4 h (Scheme 1). The glucuronyl tri- 4 Å molecular sieves at room temperature. The desired per-
fluoroacetimidate 5b was not stable for long periods at acetylated isoflavonoid 7-O-glycosides 7a,c were obtained
room temperature and had to be stored at –78 °C.
in 81 and 76% yield, respectively (Scheme 2).
Scheme 2. Reagents and conditions: (a) 2.1 equiv. of KOtBu, DMF, 1 equiv. of hexanoyl chloride, room temp.; (b) 5a, 0.3 equiv. of
BF3·Et2O, CH2Cl2, 4 Å MS, room temp., 6 h; (c) K2CO3, H2O/MeOH, THF, 40 °C, 5 h.
Eur. J. Org. Chem. 2008, 5622–5629
© 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
5623

N. Al-Maharik, N. P. Botting
FULL PAPER
Unfortunately, glycosidation of genistein 4Ј-hexanoate
(6b) with the glycosyl trifluoroacetimidate donor 5a under
the above conditions afforded an intractable mixture.
Therefore, it was concluded that the 5- and 4Ј-OH groups
on genistein (1b) both had to be protected. Treatment of a
solution of genistein (1b) in pyridine with acetic anhydride
or hexanoyl chloride (4 equiv.) afforded 4Ј,5,7-tri-O-acyl-
genistein (8a) and 8b in 93 and 91% yield, respectively
(Scheme 3). The 7-O-acyl group on the peracylated
genistein 8a,b, which is in para position to the electron-
withdrawing pyrone carbonyl group, is the most electro-
philic entity. Taking advantage of this fact, regioselective
release of 7-OH was accomplished in good yield by nucleo-
philic attack of PhSH (1.2 equiv.) on the 7-ester group in
the presence of imidazole in N-methyl-2-pyrrolidinone
(NMP) as solvent at 0 °C (Scheme 3). Glycosylation of both
the acceptors 9a,b with the glycosyl donor 5a by employing
the conditions described above resulted in 10a and 10b in
72 and 87% yield, respectively.
This attractive synthetic route also allowed us to synthe-
sise the major three soybean isoflavone 7-O-glucuronides in
Scheme 3. Reagents and conditions: (a) Ac2O, Py, room temp., 8 h,
or hexanoyl chloride, Py, DMAP, CHCl3, 0 °C; (b) PhsH, NMP,
Im; (c) 5a, BF3·Et2O, CH2Cl2, 4 Å MS, room temp., 6 h; (d)
K2CO3, MeOH, THF, H2O, 40 °C, 5 h.
good
yields.
Isoflavon-7-yl-β--glucopyranosiduronic
methyl esters 11a–d were synthesised in excellent yields by
coupling of the acceptors 6a,c, 9a,b with glucuronyl 2,2,2-
trifluoro-N-(p-methoxyphenyl)acetamidate donor 5b under
standard glycosylation conditions described for their corre-
sponding isoflavone 7-O-glucosides (Scheme 4).
Conclusions
A new glycosylation procedure has been developed for
Final removal of the acyl groups to provide the isofla- isoflavones, which employs 2,2,2-trifluoro-N-(p-meth-
vone 7-O-glycosides and 7-O-glucuronides was achieved by oxyphenyl)acetamidates as the glycosyl donors. This meth-
using K2CO3 in MeOH/THF/H2O (5:5:1). The glycosides odology was used to prepare the 7-O-glycosides of the three
were purified by dissolving the crude material in a few main isoflavones, daidzein, genistein and glycitein. The iso-
drops of water and precipitating with ethanol, whereas the flavones were protected with hexanoyl groups which had
glucuronides were purified by means of C18 reversed-phase the added bonus of improving their solubility in organic
chromatography using water/acetonitrile (9:1) as eluant. solvents and improved the efficiency of the reaction. The
The purity of both glycosides and glucuronides was con- same methodology was then adapted for the synthesis of
firmed by HPLC using water/acetonitrile (70:30 for the gly- the analogous 7-O-glucuronides, which are mammalian me-
coside; 90:20 for the glucuronides) as eluant.
tabolites of the isoflavones. The new synthesis will provide
Scheme 4. (a) 5a, BF3·Et2O, CH2Cl2, 4 Å MS, room temp., 6 h; (d) K2CO3 MeOH, THF, H2O, room temp., 5 h.
5624 Eur. J. Org. Chem. 2008, 5622–5629
www.eurjoc.org
© 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

An Efficient Method for the Glycosylation of Isoflavones
extracts were dried with MgSO4, concentrated at reduced pressure,
and the residue was purified by flash chromatography on silica gel
by using DCM/EtOAc (5Ǟ15%) as eluant.
access to large quantities of these compounds and enable
further study of their biological properties. This efficient,
high-yielding methodology is also suitable for the synthesis
of 13C-labelled derivatives, and studies on isotopic labelling
are currently underway.
4Ј-O-Hexanoyldaidzein (6a): Compound 6a (0.514 g, 73%) was ob-
tained as a white solid; m.p. 190 °C. 1H NMR (300 MHz, CDCl3):
δ = 0.92 (t, J = 6.9 Hz, 3 H, CH2CH2CH3), 1.33–1.47 (m, 4 H,
CH2CH2CH3), 1.75 (quint, J = 7.2 Hz, 2 H, O2CCH2CH2CH2),
2.56 (t, J = 7.2 Hz, 2 H, O2CCH2), 6.84 (d, J = 2.3 Hz, 1 H, 8-H),
6.93 (dd, J = 8.8, 2.3 Hz, 1 H, 6-H), 7.13 (d, J = 8.7 Hz, 2 H, 3Ј-,
5Ј-H), 7.54 (d, J = 8.7 Hz, 1 H, 2Ј-, 6Ј-H), 7.91 (s, 1 H, 2-H), 8.10
(d, J = 8.8 Hz, 1 H, 5-H) ppm. 13C NMR (75 MHz, CDCl3): δ =
13.9 (C-CH3), 22.3 (C-CH2CH3), 24.6 (C-CH2CH2CH3), 31.3 (C-
O2CCH2CH2), 34.4 (C-O2CCH2CH2), 102.7 (C-8), 115.7 (C-6),
117.3 (C-4a), 121.7 (5, C-3), 124.2 (C-3), 127.8 (C-1Ј), 129.6 (C-5),
130.2 (C-2Ј, -6Ј), 150.6 (C-2), 153.0 (C-8a), 158.2 (C-4Ј), 162.7 (C-
7), 172.5 (C-4), 176.2 (C-CO2) ppm. MS (EI): m/z (%) = 352 (5)
[M]+, 254 (100), 137 (11). HRMS: calcd. for C21H20O5 352.1311;
found 352.1317.
Experimental Section
General: NMR spectra were recorded with a Varian Gemini 2000
(1H 300 MHz, 13C 75.45 MHz) or a Bruker Avance 300 (1H
300 MHz, 13C 75.45 MHz) spectrometer. Chemical shifts (δ) in
ppm are given relative to Me4Si, coupling constants (J) in Hz. Ele-
mental analyses were carried out in the departmental microanalyti-
cal laboratory. IR spectra were recorded with a Perkin–Elmer series
1420 FT IR spectrophotometer. The samples were prepared as Nu-
jol mulls or thin films between sodium chloride discs and recorded
in cm–1. EI mass spectra were recorded with a Micromass GC-T.
Electrospray mass spectra were recorded with a Micromass LC-T.
UV spectra were recorded with a Kontron Uvikon 930 spectrome-
ter. Melting points were recorded with an electrothermal melting
point apparatus and are uncorrected. Analytical TLC was carried
out on Merck 5785 Kieselgel 60F254 fluorescent plates. Flash
chromatography was performed according to the procedure of
Still[31] by using silica gel of 35–70 µm particle size. Dimethylform-
amide was distilled from magnesium sulfate. Diethyl ether and
tetrahydrofuran were distilled from sodium metal and benzophe-
none.
4Ј-O-Hexanoylgenistein (6b): Compound 6b (0.478 g, 65%) was ob-
1
tained as a white foam. H NMR (300 MHz, CDCl3): δ = 0.99 (t,
J = 6.9 Hz, 3 H, CH2CH2CH3), 1.38–1.43 (m, 4 H, CH2CH2CH3),
1.76–1.83 (m, 2 H, O2CCH2CH2CH2), 2.60 (t, J = 7.5 Hz, 2 H,
O2CCH2), 6.01 (s, 1 H, 7-OH), 6.23 (d, J = 2.1 Hz, 1 H, 6-H), 6.29
(d, J = 2.1 Hz, 1 H, 8-H), 7.16 (d, J = 8.7 Hz, 2 H, 3Ј-, 5Ј-H), 7.54
(d, J = 8.7 Hz, 1 H, 6Ј- ,2Ј-H), 7.84 (s, 1 H, 2-H), 12.80 (s, 1 H, 5-
H) ppm. 13C NMR (75 MHz, CDCl3): δ = 13.9 (C-CH3), 22.3 (C-
CH2CH3), 24.6 (C-O2CCH2CH2), 31.3 (C-O2CCH2CH2), 34.4 (C-
CH2CH2CH3), 94.2 (C-8), 99.8 (C-6), 105.9 (C-4a), 121.8 (C-3Ј,
-5Ј), 123.0 (C-3), 128.4 (C-1Ј), 130.0 (C-2Ј, -6Ј), 150.8 (C-4Ј), 153.3
(C-2), 157.9 (C-8a), 162.7 (C-5), 167.8 (C-7), 173.0 (C-O2C), 180.4
(C-4) ppm. MS (EI): m/z (%) = 368 (87) [M]+, 270 (100). HRMS:
calcd for C21H20O6 368.1260; found 368.1256.
2,3,4,6-Tetra-O-acetyl-D-glycopyranosyl 2,2,2-Trifluoro-N-(p-meth-
oxyphenyl)acetamidate (5a): N-(p-Methoxyphenyl)acetimidoyl
chloride (0.327 g, 1 mmol) was slowly added to a suspension of
sugar hemiacetal (1 mmol) and K2CO3 (0.276 g, 2 mmol) in wet
acetone at room temp. After 4 h of stirring at room temp., the solid
was filtered, and the solvent was evaporated under reduced pres-
sure. The residue was subjected to flash chromatography by using
petroleum ether/EtOAc (4:1) to give the title compound as an α/β
mixture (0.428 g, 78%) as white foam. 1H NMR (300 MHz,
CDCl3): δ = 2.017, 2.027, 2.032, 2.043, 2.051, 2.58, 2.66, 2.083 (8
s, 24 H, 8 Ac), 3.78, 3.79 (2 s, 6 H, OCH3), 4.07–4.15 (m, 4 H),
4.24 (d, J = 4.8 Hz, 1 H), 4.28 (d, J = 4.8 Hz, 1 H), 5.08–5.25 (m,
6 H), 5.52 (t, J = 9.9 Hz, 1 H), 5.74 (br. s, 1 H, 1-Hβ), 5.74 (br. s,
1 H, 1-Hα), 6.76–6.89 (m, 4 H, Ph) ppm. MS (ES): m/z (%) = 572
(100) [M + Na+], 353 (28). HRMS: calcd. for C23H26NF3O11Na
572.1356; found 572.1348.
4Ј-Hexanoylglycitein (6c): Compound 6c (0.481 g, 63%) was ob-
1
tained as a white foam. H NMR (300 MHz, CDCl3): δ = 0.93 (t,
J = 7.0 Hz, 3 H, CH3), 1.37–1.44 (m, 4 H, CH2CH2CH3), 1.74–
1.80 (m, 2 H, O2CCH2CH2), 2.57 (t, J = 7.5 Hz, 2 H, O2CCH2),
4.00 (s, 3 H, OCH3), 6.46 (s, 1 H, 7-OH), 6.98 (s, 1 H, 8-H), 7.15
(d, J = 8.7 Hz, 2 H, 3Ј-, 5Ј-H), 7.58 (d, J = 8.7 Hz, 1 H, 2Ј-, 6Ј-H),
7.64 (s, 1 H, 5-H), 7.95 (s, 1 H, 2-H) ppm. 13C NMR (75 MHz,
CDCl3): δ = 13.9 (C-CH3), 22.3 (C-CH2CH2CH2CH3), 24.6 (C-
O2CCH2CH2), 31.3 (C-CH2CH2CH3), 34.4 (C-O2CCH2), 56.5 (C-
OCH3), 102.7 (C-8), 104.7 (C-5), 117.8 (C-4a), 121.6 (C-3Ј, -5Ј),
123.7 (C-3), 129.6 (C-3), 130.0 (C-2Ј, -6Ј), 145.5 (C-6), 150.6 (C-7),
151.4 (C-8a), 152.5 (C-4Ј), 152.6 (C-2), 172.8 (C-O2C), 175.8 (C-4)
ppm. MS (EI): m/z (%) = 382 (10) [M]+ 284 (100). C22H22O6
(382.41): calcd. C 69.10; H, 5.80; found C 69.58, H 5.36.
2,3,4-Tri-O-acetyl-D-glycopyranosiduronyl
2,2,2-Trifluoro-N-(p-
methoxyphenyl)acetamidate (5b): Compound 5b (0.40 g, 75%) was
obtained as a yellow viscous oil from hemiacetal 3b and 4 by em-
5,7,4Ј-Triacetylgenistein (8a): Acetic anhydride (1.4 mL,
14.8 mmol) was added to a solution of genistein (1b, 1 g, 3.7 mmol)
in pyridine (10 mL) and dry chloroform (5 mL) at room temp. un-
der nitrogen. After stirring at room temp. overnight, the mixture
was diluted with CH2Cl2 (100 mL) and washed with 1  HCl
(3ϫ100 mL), and brine subsequently. The organic layer was dried
with MgSO4, and concentrated to give yellow solid, which was
recrystallised from MeOH to give 8a (1.35 g, 93%) as a white solid;
m.p. 137 °C (ref.[32] 139–141 °C). 1H NMR (300 MHz, CDCl3): δ
= 2.31, 2.34, 2.42 (3 s, 9 H, O2CCH3), 6.86 (d, J = 2.4 Hz, 1 H, 6-
H), 7.15 (d, J = 8.7 Hz, 2 H, 3Ј-, 5Ј-H), 7.25 (d, J = 2.4 Hz, 1 H,
8-H), 7.49 (d, J = 8.7 Hz, 2 H, 2Ј-, 6Ј-H), 7.89 (s, 1 H, 2-H) ppm.
13C NMR (75 MHz, CDCl3): δ = 21.2 (3 C-O2CCH3), 109.0 (C-8),
114.0 (C-6), 115.2 (C-4a), 121.7 (C-3Ј, -5Ј), 125.7 (C-3), 128.8 (C-
1Ј), 130.2 (C-2Ј, -6Ј), 150.64, 150.75 (C-8a, -4Ј), 152.0 (C-2), 153.8
(C-8a), 157.7 (C-5), 167.9, 169.4 (3 C-O2C), 174.2 (C-4) ppm.
1
ploying the procedure described for the synthesis of 5a. H NMR
(300 MHz, CDCl3): δ = 2.016, 2.019, 2.027, 2.035, 2.053 (5 s, 18
H, 6 Ac), 3.738, 3.765, 3.773 (3 s, 12 H, 2 CO2CH3, 2 OCH3), 4.42
(d, J = 10.5 Hz, 1 H), 5.14 (dd, J = 10.2, 3.9 Hz, 1 H), 5.21–5.34
(m, 3 H), 5.58 (m, 1 H), 5.85 (br. s, 1 H, 1-Hβ), 6.46 (br. s, 1 H, 1-
Hα), 6.72–6.85 (m, 8 H, Ph) ppm. MS (ES): m/z (%) = 558 (100)
[M + Na]+. HRMS: calcd. for C22H24NF3O11Na 558.1199; found
558.1203.
General Procedure for Regioselective Acylation of Isoflavones: A
solution of isoflavone (2 mmol) and dry tBuOK (4.2 mmol) in dry
DMF was stirred at room temp. under nitrogen for 10 min. Hexa-
noyl chloride (0.29 mL, 2.1 mmol) was added dropwise and the
mixture stirred for 2 h until TLC showed almost complete disap-
pearance of the substrate. After addition of ice-cold water, the mix-
ture was extracted with EtOAc (3ϫ50 mL). The combined organic
Eur. J. Org. Chem. 2008, 5622–5629
© 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
5625

N. Al-Maharik, N. P. Botting
FULL PAPER
4Ј,5,7-Trihexanoylgenistein (8b): To a solution of genistein (1b, 1 g,
3.7 mmol) in pyridine (10 mL) and dry chloroform (5 mL) was
added DMAP (0.045 g, 0.37 mmol), followed by hexanoyl chloride
(1.82 mL, 18.57 mmol) at –20 °C under nitrogen. After 9 h of stir-
ring at room temp., the mixture was diluted with CH2Cl2 (100 mL)
and washed with 1  HCl (2ϫ100 mL), and brine subsequently.
The organic layer was dried with MgSO4 and concentrated to give
a yellow solid, which was recrystallised from MeOH to give 8b
(1.95 g, 91%) as a white solid; m.p. 86 °C. 1H NMR (300 MHz,
CDCl3): δ = 0.90–0.96 (m, 9 H, 3 CH3), 1.38–1.43 (m, 12 H,
CH2CH2CH3), 1.74–1.80 (m, 6 H, CO2CH2CH2), 2.56 (t, J =
7.5 Hz, 2 H, O2CCH2), 2.59 (t, J = 7.5 Hz, 2 H, O2CCH2), 2.72 (t,
J = 7.5 Hz, 2 H, O2CCH2), 6.84 (d, J = 2.4 Hz, 1 H, 6-H), 7.13 (d,
J = 8.4 Hz, 2 H, 3Ј-, 5Ј-H), 7.23 (d, J = 2.4 Hz, 1 H, 8-H), 7.49 (d,
J = 8.4 Hz, 1 H, 2Ј-, 6Ј-H), 7.88 (s, 1 H, 2-H) ppm. 13C NMR
(75 MHz, CDCl3): δ = 13.88, 13.90, 113.94 (3 C-CH3), 22.3, 22.4,
24.5, 24.1, 24.4, 24.6 (3 C-CH2CH2CH3) 31.2, 31.3 (3 C-
O2CH2CH2), 34.1, 34.4 (3 C-O2CCH2), 108.9 (C-8), 114.0 (C-6),
115.2 (C-4a), 121.8 (C-3Ј, -5Ј), 125.7 (C-3), 128.8 (C-1Ј), 130.2 (C-
2Ј, -6Ј), 150.8 (C-7, -4Ј), 152.0 (C-2), 154.0 (C-8a), 157.7 (C-5),
170.9, 172.2, 172.3 (3 C-O2C), 174.2 (C-4) ppm. MS (EI): m/z =
564 (100) [M]+. C33H40O8 (564.68): calcd. C 70.19, H 7.14; found
C 70.35, H 6.98.
isoflavonoid acceptor 3 or 9 (.20 mmol) and freshly dried molecular
sieves 4 Å (2 g) in dry dichloromethane (20 mL), BF3·Et2O
(0.073 mL, 0.6 mmol) was added under nitrogen. After stirring at
room temperature for 5 h, Et3N (0.5 mL) was added and stirring
was continued for 15 min. The solids were removed by filtration
through a pad of Celite®, and the filtrate was concentrated. The
residue was purified by flash chromatography with petroleum ether/
EtOAc (20Ǟ40%) to give the title compound.
4Ј-O-Hexanoyldaidzein-7-yl 2ЈЈ,3ЈЈ,4ЈЈ,6ЈЈ-Tetra-O-acetyl-β-D-gluco-
pyranoside (7a): Compound 7a (1.10 g, 81%) was obtained as a
white solid. m.p. 182 °C. [α]D = –24.4 (c = 1, CHCl3). 1H NMR
(300 MHz, CDCl3): δ = 0.92 (t, J = 7.0 Hz, 3 H, CH3), 1.37–1.42
(m, 4 H, CH2CH2CH3), 1.74–1.80 (m, 2 H, O2CCH2CH2), 2.05,
2.07, 2.02, 2.08, 2.10 (4 s, 12 H, 4 O2CCH3), 2.57 (t, J = 7.5 Hz, 2
H, O2CCH2), 3.96 (ddd, J = 10.0, 5.6, 2.5 Hz, 1 H, 5-H), 4.21 (dd,
J = 12.3, 2.5 Hz, 1 H, 6ЈЈ-Ha), 4.29 (dd, J = 12.3, 5.6 Hz, 1 H, 6ЈЈ-
Hb), 5.15–5.35 (m, 4 H, 1ЈЈ-, 2ЈЈ-, 3ЈЈ-, 4ЈЈ-H), 7.04 (d, J = 2.4 Hz,
1 H, 8-H), 7.05 (dd, J = 8.4, 2.4 Hz, 1 H, 6-H), 7.16 (d, J = 8.7 Hz,
2 H, 3Ј-, 5Ј-H), 7.57 (d, J = 8.7 Hz, 1 H, 2Ј-, 6Ј-H), 7.97 (s, 1 H,
2-H), 8.26 (d, J = 8.4 Hz, 1 H, 5-H) ppm. 13C NMR (75 MHz,
CDCl3): δ = 13.9 (C-CH3), 20.69, 20.74 (4 Ac), 22.3 (C-
CH2CH2CH3), 24.6 (C-O2CCH2CH2), 31.2 (C-CH2CH2CH3), 34.4
(C-O2CCH2), 61.9 (C-6ЈЈ), 68.1 (C-4ЈЈ), 71.0 (C-2ЈЈ), 72.4, 72.5 (C-
3ЈЈ, -5ЈЈ), 98.3 (C-1ЈЈ), 104.3 (C-8Ј), 115.4 (C-6), 120.2 (C-4a), 121.8
(C-3Ј, -5Ј), 124.8 (C-3), 128.2 (C-5), 129.1 (C-1Ј), 130.0 (C-2Ј, -6Ј),
150.8 (C-8a), 152.8 (C-2), 157.4 (C-4Ј), 160.5 (C-7), 169.2, 169.4,
170.2, 170.5 (C-O2CCH3), 172.3 (C-O2CCH2), 175.4 (C-4) ppm.
MS (ES): m/z (%) = 705 (57) [M + Na]+, 703 (68), 661 (5), 413
(17), 371 (100). HRMS: calcd. for C35H38O14Na 705.2159; found
705.2164. C35H38O14 (682.68): calcd. C 61.58, H 5.61; found C
61.45, H 5.49.
General Procedure for the Regioselective Deacylation: Thiophenol
(0.309 mL, 3.03 mmol) was slowly added to a solution of peracyl-
ated isoflavone 7a,b (2.5 mmol) and imidazole (0.06 g, 0.88 mmol)
in NMP (3 mL) at 0 °C under nitrogen. The mixture was stirred at
room temp. until disappearance of the starting material as moni-
tored by TLC. The reaction mixture was diluted with CH2Cl2
(100 mL) and washed with 1  HCl and brine subsequently. The
organic layer was dried with MgSO4, filtered, and the solvent was
removed at reduced pressure. The yellow semisolid was subjected
to flash chromatography in DCM/EtOAc (5Ǟ15%) to give the title
compound.
4Ј-O-Hexanoylglycitein-7-yl 2ЈЈ,3ЈЈ,4ЈЈ,6ЈЈ-Tetra-O-acetyl-β-D-gluc-
opyranoside (7c): Compound 7c (1.08 g, 76%) was obtained as a
white solid, m.p. 86 °C. [α]D = –22.4 (c = 1, CHCl3). 1H NMR
(300 MHz, CDCl3): δ = 0.87 (t, J = 7.0 Hz, 3 H, CH3), 1.31–1.36
(m, 4 H, CH2CH2CH3), 1.71 (quint, J = 7.5 Hz, 2 H, CH2CH2),
1.99, 2.00, 2.03, 2.05 (4 s, 12 H, 4 O2CCH3), 2.50 (t, J = 7.5 Hz, 2
H, O2CCH2), 3.86 (s, 3 H, OCH3), 3.81–3.87 (m, 1 H, 5ЈЈ-H), 4.14–
4.25 (m, 2 H, 6ЈЈ-Ha, -Hb), 5.02–5.13 (m, 2 H, 2ЈЈ-, 3ЈЈ-H), 5.19–
5.20 (m, 2 H, 1ЈЈ-, 4ЈЈ-H), 7.16 (d, J = 8.7 Hz, 2 H, 3Ј-, 5Ј-H), 7.21
(s, 1 H, 8-H), 7.58 (d, J = 8.7 Hz, 1 H, 2Ј-, 6Ј-H), 7.66 (s, 1 H, 5-
H), 7.97 (s, 1 H, 2-H) ppm. 13C NMR (75 MHz, CDCl3): δ = 13.9
(C-CH3), 20.6, 20.7 (4 Ac), 22.3 (C-CH2CH2CH3), 24.6 (C-
O2CCH2CH2), 31.3 (C-CH2CH2CH3), 34.4 (C-O2CCH2), 56.4 (C-
OCH3), 61.9 (C-6ЈЈ), 68.3 (C-4ЈЈ), 70.9 (C-2ЈЈ), 72.3, 72.4 (C-3ЈЈ, -
5ЈЈ), 99.9 (C-1ЈЈ), 106.3 (C-8), 107.1 (C-5), 120.5 (C-4a), 121.7 (C-
3Ј, -5Ј), 124.1 (C-3), 129.4 (C-1Ј), 130.0 (C-2Ј, -6Ј), 148.8 (C-6),
150.7 (C-8a), 150.9 (C-7), 151.2 (C-4a), 152.8 (C-2), 169.3, 169.4,
170.2, 170.6 (C-O2CCH3), 172.3 (C-O2CCH2), 175.2 (C-4) ppm.
MS (ES): m/z (%) = 735 (100) [M + Na]+, 713 (17) [M + H]+, 661
(7), 405 (23), 383 (12), 371 (11). HRMS: calcd. for C36H40O15Na
735.2265; found 735.2247. C36H40O15 (712.70): calcd. C 60.67, H
5.66; found C 60.50, H 5.22.
5,4Ј-Diacetylgenistein (9a): Compound 9a (0.734 g, 83%) was ob-
tained as a white solid; m.p. 202 °C. 1H NMR (300 MHz, CDCl3):
δ = 2.33, 2.34 (2 s, 6 H, 2 O2CCH3), 6.60 (d, J = 2.1 Hz, 1 H, 6-
H), 6.77 (d, J = 2.1 Hz, 1 H, 8-H), 7.19 (d, J = 8.7 Hz, 2 H, 3Ј-,
5Ј-H), 7.55 (d, J = 8.7 Hz, 2 H, 2Ј-, 6Ј-H), 7.97 (s, 1 H, 2-H) ppm.
13C NMR (75 MHz, CDCl3): δ = 21.19 (2 C-O2CCH3), 101.0 (C-
8), 105.7 (C-6), 109.5 (C-4a), 122.0 (C-3Ј, -5Ј), 123.7 (C-3), 128.0
(C-1Ј), 130.0 (C-2Ј, -6Ј), 151.0 (C-4Ј), 153.9 (C-2), 156.1 (C-8a),
156.9 (C-7), 162.4 (C-5), 168.3, 169.4 (2 C-O2CCH3), 181.0 (C-4)
ppm. MS (EI): m/z (%) = 354 (4) [M]+, 312 (27), 270 (100). HRMS:
calcd. for C19H14O7 354.0740; found 354.0744. C19H14O7 (354.32):
calcd. C 64.41, H 3.98; found C 63.93, H 3.78.
5,4Ј-Dihexanoylgenistein (9b): Compound 9b (0.909 g, 78%) was
obtained as a white solid; m.p. 145 °C. 1H NMR (300 MHz,
CDCl3): δ = 0.88–0.97 (m, 6 H, 2 CH3), 1.31–1.42 (m, 8 H,
CH2CH2CH3), 1.77 (quint, J = 7.4 Hz, 4 H, O2CCH2CH2), 2.56
(t, J = 7.5 Hz, 2 H, O2CCH2), 2.71 (t, J = 7.7 Hz, 2 H, O2CCH2),
6.40 (d, J = 2.4 Hz, 1 H, 6-H), 6.47 (d, J = 2.4 Hz, 1 H, 8-H), 7.08
(d, J = 8.7 Hz, 2 H, 3Ј-, 5Ј-H), 7.44 (d, J = 8.7 Hz, 2 H, 2Ј-, 6Ј-H),
7.75 (s, 1 H, 2-H) ppm. 13C NMR (75 MHz, CDCl3): δ = 13.95,
13.99 (2 C-CH3), 22.3, 22.4, 24.1, 24.6 (2 C-CH2CH2CH3) 31.2,
31.3 (2 C-O2CCH2CH2), 34.2, 34.4 (2 C-O2CCH2), 101.4 (C-8),
109.6 (C-6), 111.2 (C-4a), 121.7 (C-3Ј, -5Ј), 125.0 (C-3), 129.1 (C-
1), 130.3 (C-2Ј, -6Ј), 150.7, 150.8 (C-4Ј, 8a), 152.0 (C-2), 158.7 (C-
7), 161.2 (C-5), 172.9, 173.4 (2 C-O2C), 174.8 (C-4) ppm. MS (EI):
m/z = 466 (100) [M]+. C27H30O7 (466.53): calcd. C 69.51, H 6.48;
found C 69.22, H 6.59.
5,4Ј-O-Diacetylgenistein-7-yl 2ЈЈ,3ЈЈ,4ЈЈ,6ЈЈ-Tetra-O-acetyl-β-D-gluc-
opyranoside (10a): Compound 10a (0.985 g, 72%) was obtained as
1
a white solid, m.p. 169 °C. [α]D = –24.7 (c = 1, CHCl3). H NMR
(300 MHz, CDCl3): δ = 2.05, 2.07, 2.08, 2.11 (4 s, 12 H, 4
O2CCH3), 2.31 (s, 3 H, 4Ј-O2CCH3), 2.41 (s, 3 H, O2CCH3), 3.96
(ddd, J = 12.5, 5.7, 2.7 Hz, 1 H, 5ЈЈ-H), 4.20 (dd, J = 12.3, 2.6 Hz,
1 H, 6ЈЈ-Ha), 4.29 (dd, J = 12.3, 5.7 Hz, 1 H, 6ЈЈ-Hb), 5.13–5.23
(m, 2 H, 3ЈЈ-, 2ЈЈ-H), 5.31–5.34 (m, 2 H, 1ЈЈ-, 4ЈЈ-H), 6.69 (d, J =
2.4 Hz, 1 H, 8-H), 6.93 (d, J = 2.4 Hz, 1 H, 6-H), 7.15 (d, J =
General Procedure for the Glycosylation: To a mixture of the per-
acetylated glycosyl trichloroacetimidate donor 4 or 11 (3.0 mmol),
5626
www.eurjoc.org
© 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2008, 5622–5629

An Efficient Method for the Glycosylation of Isoflavones
8.7 Hz, 2 H, 3Ј-, 5Ј-H), 7.49 (d, J = 8.7 Hz, 1 H, 2Ј-, 6Ј-H), 7.85 (C-1Ј), 130.2 (C-2Ј, -6Ј), 150.9 (C-4Ј), 151.4 (C-8a), 151.8 (C-2),
(s, 1 H, 2-H) ppm. 13C NMR (75 MHz, CDCl3): δ = 20.6, 21.2 (6
158.4 (C-7), 159.6 (C-5), 166.7 (C-CO2Me), 169.2, 169.3, 170.0,
O2CCH3), 61.90 (C-6ЈЈ), 68.0 (C-4ЈЈ), 70.8 (C-2ЈЈ), 72.4 (C-3ЈЈ, - 172.3, 172.4 (3 O2CCH3, 3 O2CCH2), 174.1 (C-4) ppm. MS (ES):
5ЈЈ), 98.0 (C-8), 102.5 (C-6), 109.8 (C-1), 113.7 (C-4a), 121.7 (C- m/z (%) = 805 (100) [M + Na]+, 783 (12). HRMS: calcd. for
3Ј, -5Ј), 125.6 (C-3), 128.8 (C-1Ј), 130.2 (C-2Ј, -6Ј), 150.7 (C-8a),
C40H46O16Na 805.2684; found 805.2665. C40H46O16 (782.79):
151.7 (C-4Ј), 151.8 (C-2), 158.4 (C-7), 159.8 (C-5), 169.2, 169.4, calcd. C 61.37, H 5.92; found C 60.89, H 5.50.
170.1, 170.5, 172.4 (C-O2CCH3), 175.8 (C-4) ppm. MS (ES): m/z
Methyl (4Ј-O-Hexanoylglycitein-7-yl β-D-2ЈЈ,3ЈЈ,4ЈЈ-triacetylgluco-
(%) = 707 (100) [M + Na]+, 661 (48), 371 (38). HRMS: calcd. for
C33H32O16Na 707.1563; found 707.1588. C33H32O16 (684.61):
calcd. C 57.90, H 4.51; found C 57.92, H 4.41.
pyranosid)urinate (11c): Compound 11c (1.23 g, 88%) was obtained
as a white solid; m.p. 129 °C. [α]D = –39.1 (c = 1, CHCl3). 1H NMR
(300 MHz, CDCl3): δ = 0.92 (t, J = 7.0 Hz, 3 H, CH3), 1.37–1.51
(m, 4 H, CH2CH2CH3), 1.72–1.85 (quint, J = 7.5 Hz, 2 H,
CH2CH2), 2.06, 2.07, 2.1 (3 s, 9 H, 3 O2CCH3), 2.60 (t, J = 7.5 Hz,
2 H, O2CCH2), 3.93 (s, 3 H, OCH3), 4.16–4.24 (m, 1 H, 5ЈЈ-H),
5.28–5.43 (m, 4 H, 1ЈЈ-, 2ЈЈ-, 3ЈЈ-, 4ЈЈ-H), 7.16 (d, J = 8.7 Hz, 2 H,
3Ј-, 5Ј-H), 7.21 (s, 1 H, 8-H), 7.58 (d, J = 8.7 Hz, 2 H, 2Ј-, 6Ј-H),
7.66 (s, 1 H, 5-H), 7.97 (s, 1 H, 2-H) ppm. 13C NMR (75 MHz,
CDCl3): δ = 13.9 (C-CH3), 20.5, 20.6, 20.08 (3 O2CCH3), 22.3 (C-
CH2CH2CH3), 24.6 (C-O2CCH2CH2), 31.2 (C-CH2CH2CH3), 34.4
(C-O2CCH2), 53.0, (C-CO2CH3), 56.4 (C-OCH3), 68.9 (C-4ЈЈ), 70.3
(C-5ЈЈ), 71.7, 72.7 (C-2ЈЈ, -3ЈЈ), 99.6 (C-1ЈЈ), 106.3 (C-8), 107.7 (C-
5), 120.7 (C-4a), 121.7 (C-3Ј, -5Ј), 124.0 (C-3), 129.4 (C-1Ј), 130.0
(C-2Ј, -6Ј), 148.9 (C-5), 150.5 (C-8a), 150.7 (C-7), 151.1 (C-4a),
152.8 (C-2), 166.8, 169.2, 169.3, 170.0, 170.6 (C-O2CCH3), 172.3
(C-O2CCH2), 175.2 (C-4) ppm. MS (ES): m/z (%) = 735 (100) [M
+ Na]+, 713 (17) [M + H]+, 661 (7), 405 (23), 383 (12), 371 (11).
HRMS: calcd. for C36H40O15Na 735.2265; found 735.2247.
C35H38O15 (698.68): calcd. C 60.17, H 5.48; found C 60.50, H 5.05.
5,4Ј-O-Dihexanoylgenistein-7-yl 2ЈЈ,3ЈЈ,4ЈЈ,6ЈЈ-Tetra-O-acetyl-β-D-
glucopyranoside (10b): Compound 10b (1.38 g, 87%) was obtained
as a white solid, m.p. 174 °C. [α]D = –26.3 (c = 1, CHCl3). 1H NMR
(300 MHz, CDCl3): δ = 0.85–0.90 (m, 6 H, 2 CH3), 1.35–1.42 (m,
8 H, 2 CH2CH2CH3), 1.72–1.82 (m, 4 H, 2 O2CCH2CH2), 2.05,
2.07, 2.08, 2.12 (4 s, 12 H, 4 O2CCH3), 2.56 (t, J = 7.5 Hz, 2 H,
O2CCH2), 2.72 (t, J = 7.8 Hz, 2 H, O2CCH2), 3.96 (ddd, J = 12.3,
5.7, 2.6 Hz, 1 H, 5ЈЈ-H), 4.20 (dd, J = 12.3, 2.6 Hz, 1 H, 6ЈЈ-Ha),
4.29 (dd, J = 12.3, 5.6 Hz, 1 H, 6ЈЈ-Hb), 5.13–5.23 (m, 2 H, 4ЈЈ-,
2ЈЈ-H), 5.31–5.34 (m, 2 H, 1ЈЈ-, 3ЈЈ-H), 6.67 (d, J = 2.4 Hz, 1 H, 8-
H), 6.92 (d, J = 2.4 Hz, 1 H, 6-H), 7.13 (d, J = 8.7 Hz, 2 H, 3Ј-,
5Ј-H), 7.48 (d, J = 8.7 Hz, 2 H, 2Ј-, 6Ј-H), 7.84 (s, 1 H, 2-H) ppm.
13C NMR (75 MHz, CDCl3): δ = 13.9, 14.0 (C-CH3), 20.59, 20.63
(4 O2CCH3), 22.3, 22.4 (C-CH2CH2CH3), 24.1, 24.6 (C-
O2CCH2CH2), 31.27, 31.31 (C-CH2CH2CH3), 34.1, 34.4 (C-
O2CCH2), 61.90 (C-6ЈЈ), 68.1 (C-4ЈЈ), 70.9 (C-2ЈЈ), 72.5 (C-3ЈЈ, -
5ЈЈ), 98.1 (C-1ЈЈ), 102.3 (C-8), 109.8 (C-6), 113.7 (C-4a), 121.7 (C-
3Ј, -5Ј), 125.6 (C-3), 128.8 (C-1Ј), 130.2 (C-2Ј,6Ј), 150.9 (C-8a),
151.4 (C-4Ј), 151.7 (C-2), 158.4 (C-7), 159.8 (C-5), 169.2, 169.4,
170.1, 170.5 (C-O2CCH3), 172.4 (C-O2CCH2), 175.8 (C-4) ppm.
MS (ES): m/z (%) = 819 (100) [M + Na]+, 803 (23). HRMS: calcd.
for C41H48O16Na 819.2840; found 819.2854. C41H48O16 (796.82):
calcd. C 61.60, H 6.07; found C 61.22, H 5.69.
Methyl (5,4Ј-O-Diacetylgenistein-7-yl β-D-2ЈЈ,3ЈЈ,4ЈЈ-triacetylgluco-
pyranosid)urinate (11d): Compound 11d (1.00 g, 75%) was obtained
as a white foam; m.p. 205 °C. [α]D = –19.4 (c = 1, CHCl3). 1H
NMR (500 MHz, CDCl3): δ = 2.07 (s, 6 H, 2 O2CCH3), 2.08, 2.31,
2.42 (3 s, 9 H, 3 O2CCH3), 3.74 (s, 3 H, CO2CH3), 4.28 (d, J =
9.2 Hz, 1 H, 5ЈЈ-H), 5.31–5.33 (m, 2 H, 2ЈЈ-, 3ЈЈ-H), 5.37–5.39 (m,
2 H, 1ЈЈ-, 4ЈЈ-H), 6.70 (d, J = 2.4 Hz, 1 H, 8-H), 6.94 (d, J = 2.4 Hz,
1 H, 6-H), 7.15 (d, J = 8.6 Hz, 2 H, 3Ј-, 5Ј-H), 7.49 (d, J = 8.6 Hz,
1 H, 2Ј-, 6Ј-H), 7.86 (s, 1 H, 2-H) ppm. 13C NMR (75 MHz,
CDCl3): δ = 20.55, 20.64, 20.65, 21.2 (5 O2CCH3), 53.2 (C-OCH3),
68.7 (C-5ЈЈ), 70.7 (C-2ЈЈ), 71.4 (C-3ЈЈ), 72.8 (C-4ЈЈ), 98.0 (C-1ЈЈ),
102.5 (C-6), 109.8 (C-8), 113.7 (C-4a), 121.8 (C-3Ј, -5Ј), 125.6 (C-
3), 128.9 (C-1Ј), 130.3 (C-2Ј, -6Ј), 150.8 (C-8a), 151.2 (C-7), 151.9
(C-2), 158.4 (C-4Ј), 159.7 (C-5), 166.3 (C-O2CCH3), 169.2, 169.3,
169.4, 169.6, 170.0 (5 O2CCH3), 174.2 (C-4) ppm. MS (ES): m/z
(%) = 693 (100) [M + Na]+, 671 (12). HRMS: calcd. for
C32H30O16Na 693.1432; found 693.1436. C32H30O16 (670.58):
calcd. C 57.32, H 4.51; found C 56.91, H 4.92.
Methyl (4Ј-O-Hexanoyldaidzein-7-yl β-D-2ЈЈ,3ЈЈ,4ЈЈ-triacetylgluco-
pyranosid)urinate (11a): Compound 11a (1.08 g, 81%) was obtained
as a white solid, m.p. 205 °C. [α]2D0 = –24.2 (0.001 gmL–1 in CHCl3).
1H NMR (300 MHz, CDCl3): δ = 0.94 (t, J = 6.9 Hz, 3 H,
CH2CH3), 1.37–1.42 (m, 4 H, CH2CH2CH3), 1.72–1.82 (m, 2 H,
CH2CH2), 2.04, 2.06, 2.08 (3s, 3ϫ3 H, 3 COCH3), 2.57 (t, J =
7.5 Hz, 2 H, O2CCH2CH2), 3.73 (s, 3 H, CO2CH3), 4.28 (d, J =
9 Hz, 1 H, 5ЈЈ-H), 5.31–5.40 (m, 4 H, 1ЈЈ,2ЈЈ3ЈЈ,4ЈЈ-H), 7.04 (d, J =
2.4 Hz, 1 H, 8-H), 7.06 (dd, J = 9.3, 2.4 Hz, 1 H, 6-H), 7.15 (d, J
= 9.0 Hz, 2 H, 3Ј,5Ј-H), 7.57 (d, J = 9.0 Hz, 2 H, 2Ј,6Ј-H), 7.97 (s,
1 H, 2-H), 7.25 (d, J = 9.3 Hz, 1 H, 5-H) ppm. MS (ES): m/z (%)
= 691 (87) [M + Na]+, 669 (14) [M + H]+, 357 (100). C34H36O14
(668.65): calcd. C 61.07; H, 5.43; found C 60.70; H, 5.58.
General Procedure for Deprotection of the Peracylated Isoflavone
Glycosides: A mixture of peracylated isoflavone glycoside and
K2CO3 (2 equiv.) was dissolved in a solution of MeOH/THF/H2O
at room temp. under nitrogen. After stirring at 40 °C for 5 h, the
mixture was cooled to room temp., neutralised with Dowex-50 H+,
and then filtered and concentrated. The pale yellow solid was puri-
fied by preparative HPLC using H2O/CH3CN (7:3) for the glyco-
sides and H2O/CH3CN (9:1) for the glucuronides.
Methyl (5,4Ј-O-Dihexanoylgenistein-7-yl β-D-2ЈЈ,3ЈЈ,4ЈЈ-triacetylglu-
copyranosid)urinate (11b): Compound 9b (1.38 g, 88 %) was ob-
tained as a white solid; m.p. 94 °C. [α]D = –25.6 (c = 1, CHCl3).
1H NMR (300 MHz, CDCl3): δ = 0.89–0.95 (m, 6 H, 3 CH3), 1.37–
1.45 (m, 8 H, 2 CH2CH2CH3), 1.72–1.82 (m, 4 H, 2 O2CCH2CH2),
2.06, 2.07, 2.08 (3 s, 9 H, 3 O2CCH3), 2.56 (t, J = 7.5 Hz, 2 H,
O2CCH2), 2.72 (t, J = 7.8 Hz, 2 H, O2CCH2), 3.73 (s, 3 H, -
CO2CH3), 4.29 (m, 1 H, 5ЈЈ-H), 5.28–5.43 (m, 4 H, 1ЈЈ-, 2ЈЈ-, 3ЈЈ-,
Daidzein-7-yl β-D-Glucopyranoside (Daidzin) (2a): Compound 2a
4ЈЈ-H), 6.67 (d, J = 2.4 Hz, 1 H, 8-H), 6.92 (d, J = 2.4 Hz, 1 H, 6- (0.387 g, 93%) was obtained as a white solid; m.p. 236–237 °C
H), 7.13 (d, J = 8.7 Hz, 2 H, 3Ј-, 5Ј-H), 7.48 (d, J = 8.7 Hz, 1 H,
(ref.[33] 235–237 °C). [α]D = –24.6 (c = 1, DMSO) {ref.[20] [α]D
2Ј-, 6Ј-H), 7.84 (s, 1 H, 2-H) ppm. 13C NMR (75 MHz, CDCl3): –24.6 (c = 1, DMSO)}. 1H NMR (300 MHz, [D6]DMSO): δ =
=
δ = 13.95, 14.0 (C-CH3), 20.5, 20.6 (3 O2CCH3), 22.3, 22.4 (C-
CH2CH2CH3), 24.1, 24.6 (C-O2CCH2CH2), 31.28, 31.32 (C-
3.17–3.21 (m, 1 H, 4ЈЈ-H), 3.28–3.36 (m, 2 H, 2ЈЈ-, 3ЈЈ-H), 3.45–
3.50 (m, 2 H, 5ЈЈ-, 6bЈЈ-H), 3.71–3.74 (m, 1 H, 6aЈЈ-H), 4.65 (t, J
CH2CH2CH3), 34.1, 34.4 (C-O2CCH2), 53.2 (C-CO2CH3), 68.7 (C- = 5.5 Hz, 1 H, 6ЈЈ-OH), 5.115 (d, J = 7.0 Hz, 1 H, 1ЈЈ-H), 5.12 (d,
4ЈЈ), 70.8 (C-5ЈЈ), 71.4 (C-3ЈЈ), 72.8 (C-2ЈЈ), 98.0 (C-1ЈЈ), 102.3 (C- J = 5.2 Hz, 1 H, 4ЈЈ-OH), 5.19 (d, J = 4.6 Hz, 1 H, 3ЈЈ-OH), 5.48
6), 109.8 (C-8), 113.8 (C-4a), 121.7 (C-3Ј, -5Ј), 125.6 (C-3), 128.8
(d, J = 4.7 Hz, 1 H, 2ЈЈ-OH), 6.83 (d, J = 8.6 Hz, 2 H, 3Ј-, 5Ј-H),
Eur. J. Org. Chem. 2008, 5622–5629
© 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
5627

N. Al-Maharik, N. P. Botting
FULL PAPER
7.15 (dd, J = 8.9, 2.2 Hz, 1 H, 6-H), 7.24 (d, J = 2.2 Hz, 2 H, 8-
H), 7.42 (d, J = 8.6 Hz, 2 H, 2Ј-, 6Ј-H), 8.06 (d, J = 8.9 Hz, 1 H,
5-H), 8.58 (s, 1 H, 2-H), 9.58 (s, 1 H, 4Ј-OH) ppm. 13C NMR
(75 MHz, [D6]DMSO): δ = 60.6 (C-6ЈЈ), 69.6 (C-4ЈЈ), 73.1 (C-2ЈЈ),
H), 3.50 (d, J = 10.0 Hz, 1 H, 5ЈЈ-H), 5.03 (d, J = 7.5 Hz, 1 H, 1ЈЈ-
H), 5.06 (d, J = 4.5 Hz, 1 H, 4ЈЈ-OH), 5.35 (d, J = 4.5 Hz, 1 H,
3ЈЈ-OH), 6.47 (d, J = 2.2 Hz, 1 H, 8-H), 6.72 (d, J = 2.2 Hz, 1 H,
6-H), 6.83 (d, J = 8.4 Hz, 2 H, 3Ј-, 5Ј-H), 7.38 (br. s, 1 H, COOH),
76.5 (C-3ЈЈ), 77.2 (C-5ЈЈ), 99.9 (C-1ЈЈ), 103.3 (C-8), 115.0 (C-3Ј, 7.38 (d, J = 8.4 Hz, 2 H, 2Ј-, 6Ј-H), 8.42 (s, 1 H, 2-H), 9.77 (s, 1
-5Ј), 115.6 (C-6), 118.4 (C-4a), 122.3 (C-1Ј), 123.7 (C-3), 127.0 (C-
H, 4Ј-OH), 12.94 (s, 1 H, 5-OH) ppm. 13C NMR (125 MHz, [D6]-
5), 130.1 (C-2Ј, -6Ј), 153.4 (C-2), 157.0 (C-8a), 157.3 (C-4Ј), 161.4 DMSO): δ = 72.0 (C-4ЈЈ), 72.9 (C-2ЈЈ), 73.5 (C-3ЈЈ), 76.5 (C-5ЈЈ),
(C-7), 174.7 (C-4) ppm. MS (ES): m/z (%) = 439 (100) [M + Na]+.
HRMS: calcd. for C21H20O9Na 439.1005; found 439.1010.
94.5 (C-8), 99.51, 99.54 (C-1ЈЈ, -6), 105.9 (C-4a), 115.1 (C-3Ј, -5Ј),
120.7 (C-1Ј), 122.4 (C-3), 130.0 (C-2Ј, -6Ј), 154.5 (C-2), 157.1 (C-
8a), 157.7 (C-4Ј), 161.5 (C-5), 163.1 (C-7), 171.7 (C-CO2H), 180.5
(C-4). MS (ES): m/z (%) = 445 (100) [M – H]–. HRMS: calcd. for
C21H17O11 445.0771; found 445.0768.
Genistein-7-yl β-D-Glucopyranoside (Genistin) (2b): Compound 2b
(0.406 g, 94%) was obtained as a white solid; m.p. 256 °C (ref.[27]
254–255 °C). [α]D = –28.7 (c = 1, DMSO) {ref.[26] [α]D = –32.0 (c
= 1, DMSO)}. 1H NMR (500 MHz, [D6]DMSO): δ = 3.13–3.17
(m, 1 H, 4ЈЈ-H), 3.26–3.32 (m, 2 H, 2ЈЈ-, 3ЈЈ-H), 3.43–3.48 (m, 2 H,
5ЈЈ-, 6ЈЈ-Hb), 3.67–3.72 (m, 2 H, 6ЈЈ-Ha), 4.63 (t, J = 5.5 Hz, 1 H,
6ЈЈ-OH), 5.07 (d, J = 7.0 Hz, 1 H, 1ЈЈ-H), 5.10 (d, J = 5.0 Hz, 1 H,
4ЈЈ-OH), 5.17 (d, J = 5.0 Hz, 1 H, 3ЈЈ-OH), 5.44 (d, J = 5.0 Hz, 1
H, 2ЈЈ-OH), 6.47 (d, J = 2.2 Hz, 1 H, 6-H), 6.72 (d, J = 2.2 Hz, 1
H, 6-H), 6.83 (d, J = 8.5 Hz, 2 H, 3Ј-, 5Ј-H), 7.40 (d, J = 8.5 Hz,
2 H, 2Ј-, 6Ј-H), 8.43 (s, 1 H, 2-H), 9.62 (s, 1 H, 4Ј-OH), 12.97 (s, 1
H, 5-OH) ppm. 13C NMR (125 MHz, [D6]DMSO): δ = 60.6 (C-
6ЈЈ), 69.6 (C-4ЈЈ), 73.1 (C-2ЈЈ), 76.4 (C-3ЈЈ), 77.2 (C-5ЈЈ), 94.5 (C-
8), 99.6 (C-6), 99.8 (C-1ЈЈ), 106.1 (C-4a), 115.1 (C-3Ј, -5Ј), 121.0
(C-1Ј), 122.5 (C-3), 130.2 (C-2Ј, -6Ј), 154.6 (C-2), 157.2 (C-8a),
157.5 (C-4Ј), 161.6 (C-5), 163.0 (C-7), 180.5 (C-4) ppm. MS (ES):
m/z (%) = 455 (100) [M + Na]+. HRMS: calcd. for C21H20O10Na
455.0954; found 455.0949.
(Glycitein-7-yl β-D-glucopyranosid)uronic Acid (12c): Compound
12c (0.42 g, 92%) was obtained as a white solid; m.p. 204–206 °C.
[α]D = –47.6 (c = 1, H2O). 1H NMR (500 MHz, D2O): δ = 3.57
(quint, J = 9.1, 7.2 Hz, 2 H, 3ЈЈ-, 4ЈЈ-H), 3.65 (t, J = 8.1 Hz, 1 H,
2ЈЈ-H), 3.70 (s, 3 H, OCH3), 3.83 (d, J = 11.5 Hz, 1 H, 5ЈЈ-H), 4.87
(d, J = 8.1 Hz, 1 H, 1ЈЈ-H), 6.83 (d, J = 8.2 Hz, 2 H, 3Ј-, 5Ј-H),
6.94 (s, 1 H, 8-H), 7.02 (s, 1 H, 5-H), 7.20 (d, J = 8.2 Hz, 2 H, 2Ј-,
6Ј-H), 7.87 (s, 1 H, 2-H) ppm. 13C NMR (125 MHz, D2O): δ =
58.3 (C-OCH3), 74.1 (C-4ЈЈ), 74.8 (C-2ЈЈ), 77.6 (C-3ЈЈ), 78.9 (C-5ЈЈ),
102.1 (C-1ЈЈ), 106.0 (C-8), 107.0 (C-4a), 117.8 (C-3Ј, -5Ј), 120.3 (C-
1Ј), 125.2 (C-5), 125.6 (C-3), 132.7 (C-2Ј, -6Ј), 149.3 (C-6), 153.3
(C-8a), 154.0 (C-7), 156.8 (C-2), 158.1 (C-4), 177.6 (CO2H), 179.1
(C-4) ppm. MS (ES): m/z (%) = 459 (100) [M – H]–. HRMS: calcd.
for C22H19O11 459.0927; found 459.0933.
Glycitein-7-yl β-
(0.419 g, 94%) was obtained as a white solid; m.p. 203–204 °C
(ref.[34] = 192–195 °C). [α]D = –39.4 (c = 1, DMSO) {ref.[34] [α]D
D-Glucopyranoside (Glycitin) (2c): Compound 2c
Acknowledgments
=
This work was funded by the Food Standards Agency (FSA) under
contract T05028.
–13.4 (c = 3.65, DMF)}. 1H NMR (500 MHz, [D6]DMSO): δ =
3.17–3.20 (m, 1 H, 4ЈЈ-H), 3.26–3.33 (m, 2 H, 2ЈЈ-, 3ЈЈ-H), 3.44–
3.49 (m, 2 H, 5ЈЈ-H, 6ЈЈ-Hb), 3.68–3.72 (m, 2 H, 6ЈЈ-Ha), 3.88 (s, 3
H, OCH3), 4.56 (br. s, 1 H, 6ЈЈ-OH), 5.05 (br. s, 2 H, 3ЈЈ-, 4ЈЈ-OH),
5.17 (d, J = 7.5 Hz, 1 H, 1ЈЈ-H), 5.36 (br. s, 1 H, 4ЈЈ-OH), 6.82 (d,
J = 8.5 Hz, 2 H, 3Ј-, 5Ј-H), 7.32 (s, 1 H, 8-H), 7.41 (d, J = 8.5 Hz,
2 H, 2Ј-, 6Ј-H), 7.49 (s, 1 H, 5-H), 8.37 (s, 1 H, 2-H), 9.62 (s, 1 H,
4Ј-OH) ppm. 13C NMR (125 MHz, [D6]DMSO): δ = 55.8 (C-
OCH3), 60.6 (C-6ЈЈ), 69.6 (C-4ЈЈ), 73.0 (C-2ЈЈ), 76.7 (C-3ЈЈ), 77.2
(C-5ЈЈ), 99.6 (C-1ЈЈ), 103.4 (C-8), 104.7 (C-5), 114.9 (C-3Ј, -5Ј),
117.8 (C-4a), 122.5 (C-1Ј), 123.1 (C-3), 130.0 (C-2Ј, -6Ј), 147.4 (C-
6), 151.2 (C-8a), 151.5 (C-7), 153.0 (C-2), 157.2 (C-4), 174.3 (C-4)
ppm. MS (ES): m/z (%) = 469 (100) [M + Na]+. HRMS: calcd. for
C42H22O10Na 469.1111; found 469.1116.
[1] J. T. Dwyer, B. R. Goldin, N. Saul, L. Gualtieri, S. Barakat, H.
Adlercreutz, J. Am. Diet Assoc. 1994, 94, 739–743.
[2] S. Hendrich, K.-W. Lee, X. Xu, H.-J. Wang, P. A. Murphy, J.
Nutr. 1994, 124, 1789S.
[3] P. A. Murphy, T. Song, G. Buseman, K. Barua, J. Agric. Food
Chem. 1997, 45, 4635–4638.
[4] K. D. R. Setchell, L. Zimmer-Nechemias, J. N. Cai, J. E.
Heubi, Lancet 1997, 350, 23–27.
[5] M. Verheus, C. H. van Gils, L. Keinan-Boker, P. B. Grace, S. A.
Bingham, P. H. M. Peters, J. Clin. Oncol. 2007, 25, 648–655.
[6] J. M. Pendleton, W. W. Tan, S. Anai, M. Chang, W. Hou, K. T.
Shiverick, C. J. Rosser, BMC Cancer 2008, 8, 132.
[7] Y. Ikeda, M. Iki, A. Morita, E. Kajita, S. Kagamimori, Y. Ka-
gawa, H. Yoneshima, J. Nutr. 2006, 136, 1323–1328.
[8] F. M. Sacks, A. Lichtenstein, L. Van Horn, W. Harris, P. Kris-
Etherton, M. Winston, Circulation 2006, 113, 1034–1044.
[9] G. Cheng, B. Wilczek, M. Warner, J.-A. Gustafsson, B.-M.
Landgren, Menopause 2007, 14, 468–473.
(Daidzein-7-yl β-D-glucopyranosid)uronic Acid (12a): Compound
12a (0.39 g, 91%) was obtained as a white solid. m.p. 186–187 °C.
1
[α]D = –70.7 (c = 1, H2O). H NMR (500 MHz, D2O): δ = 3.48–
3.55 (m, 3 H, 2ЈЈ-, 3ЈЈ-, 4ЈЈ-H), 3.76 (d, J = 9.0 Hz, 1 H, 5ЈЈ-H),
4.86 (d, J = 7.5 Hz, 1 H, 1ЈЈ-H), 6.75 (d, J = 8.5, Hz, 2 H, 3Ј-, 5Ј-
H), 6.90 (d, J = 2.5 Hz, 1 H, 8-H), 6.94 (dd, J = 9.0, 2.5 Hz, 1 H,
6-H), 7.12 (d, J = 8.5 Hz, 2 H, 2Ј-, 6Ј-H), 7.72 (d, J = 9.0 Hz, 1 H,
5-H), 7.86 (s, 1 H, 2-H) ppm. 13C NMR (125 MHz, D2O): δ = 71.5
(C-4ЈЈ), 72.4 (C-2ЈЈ), 75.0 (C-3ЈЈ), 76.1 (C-4ЈЈ), 99.4 (C-1ЈЈ), 103.5
(C-8), 115.3 (C-3Ј, -5Ј), 115.8 (C-6), 118.1 (C-4a), 122.8 (C-1Ј),
123.4 (C-3), 126.8 (C-5), 130.3 (C-2Ј, -6Ј), 154.7 (C-2), 155.7 (C-
4Ј), 157.1 (C-8a), 161.0 (C-7), 175.1 (C-6ЈЈ), 177.8 (C-4) ppm. MS
(ES): m/z (%) = 429 (100) [M – H]–. HRMS: calcd. for C21H17O10
429.0822; found 429.0829.
[10] L. G. Howes, J. B. Howes, D. C. Knight, Maturitas 2007, 55,
203–211.
[11] S. de Pascual-Teresa, J. Hallund, D. Talbot, J. Schroot, C. M.
Williams, S. Bugel, A. Cassidy, J. Nutr. Biochem. 2006, 17, 257–
264.
[12] I. L. F. Nielsen, G. Williamson, Nutr. Cancer 2007, 57, 1–10.
[13] J. C. Espin, M. T. Garcia-Conesa, F. A. Tomás-Barberán, Phy-
tochemistry 2007, 68, 2986–3008.
[14] S. Barnes, M. Kirk, L. Coward, J. Agric. Food Chem. 1994, 42,
2466–2474.
[15] A. J. Day, F. J. Canada, J. C. Diaz, P. A. Kroon, R. Mclauch-
lan, C. B. Faulds, G. W. Plumb, M. R. Morgan, G. Williamson,
FEBS Lett. 2000, 468, 166–170.
[16] H. G. Hur Jr, R. D. Berger, J. P. Freeman, F. Rafii, Arch. Micro-
biol. 2000, 174, 422–428.
(Genistein-7-yl β-D-glucopyranosid)uronic Acid (12b): Compound
12b (0.414 g, 93%) was obtained as a white solid; m.p. 212–213 °C.
1
[α]D = –27.6 (c = 1, H2O). H NMR (500 MHz, [D6]DMSO): δ =
3.12 (dd, J = 10.0, 9.0 Hz, 1 H, 4ЈЈ-H), 3.23–3.30 (m, 2 H, 2ЈЈ-, 3ЈЈ-
5628
www.eurjoc.org
© 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2008, 5622–5629

An Efficient Method for the Glycosylation of Isoflavones
[17] M. Kano, T. Takayanagi, S. Sawada, F. Ishikawa, J. Nutr. 2006,
136, 2291–2296.
[18] A. Cassidy, J. E. Brown, A. Hawdon, M. S. Faughanan, L. J.
King, J. Milward, L. Zimmer-Nechamias, B. Wolfe, K. D. R.
Setchell, J. Nutr. 2006, 136, 45–51.
[19] K. D. R. Stechell, N. N. Brown, P. Desai, L. Zimmer-Necham-
ias, B. E. Wolfe, W. T. Brashear, A. S. Kirschner, A. Cassidy,
J. E. Heubi, J. Nutr. 2001, 131, 1362S–1375S.
[20] C. Atkinson, C. L. Frankenfeld, J. W. Lampe, Exp. Biol. Med.
2005, 230, 155–170.
[21] S. M. Heinonen, K. Wähälä, H. Adlercreutz, Anal. Biochem.
1999, 274, 211–219.
[22] J. Chen, S. Wang, X. Jia, S. Bajimaya, H. Lin, V. H. Tam, M.
Hu, Drug Metab. Dispos. 2005, 33, 1777–1784.
[23] H. Nakano, K. Ogura, E. Takahashi, T. Harada, T. Nishiyama,
K. Muro, A. Hiratsuka, S. Kadota, T. Watabe, Drug Metab.
Pharmacokinet. 2004, 19, 216–226.
[24] B. Fairley, N. P. Botting, A. Cassidy, Tetrahedron 2003, 59,
5407.
[25]
[26]
N. Al-Maharik, N. P. Botting, Tetrahedron Lett. 2006, 47, 8703.
P. Lewis, S. Kaltia, K. Wähälä, J. Chem. Soc. Perkin Trans. 1
1998, 2481–2484.
G. Zemplén, L. Farkas, Ber. Dtsch. Chem. Ges. 1943, 76, 1110–
1112.
G. Singh, H. Vankayalapati, Tetrahedron: Asymmetry 2000, 11,
125–138.
M. Li, X. Han, B. Yu, J. Org. Chem. 2003, 68, 6842–6845.
[27]
[28]
[29]
[30] N. I. Al-Maharik, S. A. Kaltia, K. Wähälä, Molecules 1999, 3,
20–24.
[31] W. C. Still, M. Kahn, A. Mitra, J. Org. Chem. 1978, 43, 2923–
2925.
[32] H.-Q. Li, H.-M. Ge, Y.-X. Chen, C. Xu, L. Shi, H. Ding, H.-
L. Zhu, R.-X. Tan, Chem. Biodivers. 2006, 3, 463–472.
[33] L. Farkas, J. Varady, Chem. Ber. 1959, 92, 819–821.
[34] M. Nógrádi, A. Szöllösy, Liebigs Ann. 1996, 1651–1652.
Received: August 18, 2008
Published Online: October 13, 2008
Eur. J. Org. Chem. 2008, 5622–5629
© 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
5629