The synthesis of daidzein sulfates

Article abstract of DOI:10.1016/S0040-4020(03)00869-X

The first syntheses of the urinary isoflavone metabolites, daidzein 4′-sulfate, daidzein 7-sulfate and daidzein 4′,7-disulfate are described. These syntheses employ a key protecting group strategy, allowing regiospecific sulfation.

Full text of DOI:10.1016/S0040-4020(03)00869-X

Tetrahedron 59 (2003) 5407–5410
The synthesis of daidzein sulfates
b
Brian Fairley,a Nigel P. Bottinga and Aedin Cassidy
,
*
aSchool of Chemistry, University of St. Andrews, St. Andrews, Fife KY16 9ST, UK
bUnilever Research Colworth, Colworth House, Sharnbrook, Bedford MK44 1LQ, UK
Received 7 April 2003; revised 2 May 2003; accepted 29 May 2003
Abstract—The first syntheses of the urinary isoflavone metabolites, daidzein 40-sulfate, daidzein 7-sulfate and daidzein 40,7-disulfate are
described. These syntheses employ a key protecting group strategy, allowing regiospecific sulfation. q 2003 Elsevier Science Ltd. All rights
reserved.
1. Introduction
transported around the body as its sulfate conjugate and it
has been proposed that some of the biological effects
attributed to isoflavones may result from the effect of
isoflavone sulfates on the enzymes that control this
transport. Pure samples of the isoflavone sulfates would
allow this theory to be addressed.
The isoflavones are phytoestrogens with weak estrogenic
activity,1 which are present in the human diet in soybeans
and soy derived products. There are now many studies on
the health benefits of these compounds including relief of
menopausal symptoms,2 improvement in blood cholesterol
levels3 and reducing the risk of certain hormone related
cancers.4 However, although many studies have been
carried out5–9 there are still questions to be answered
concerning the absorption, metabolism and bioavailability
of dietary isoflavones. Also the mechanisms of transport
around the body are unclear as most work has been
conducted on the free isoflavone aglycons. In the soya
plant, and food products, the isoflavones exist as glucosides
but following ingestion these are hydrolysed and the
aglycons are then converted to glucuronide and sulfate
conjugates, mainly in the liver. In order for studies to
progress further there is a need for pure standards of the
isoflavone conjugates both to allow their accurate determi-
nation in biological samples (blood, urine etc.) and for
examination of their biological role in vivo. Estradiol is also
We wish to report herein the first chemical syntheses of
daidzein 40-sulfate 1, daidzein 7-sulfate 2 and daidzein 40,
7-disulfate 3 in a pure form for use in analytical and
biological studies (Scheme 1).
2. Results and discussion
Several procedures for the sulfation of flavonoids have been
reported including sulfation with sulfur trioxide,10 sulfuric
acid/DCC and tetrabutylammonium hydrogen sulfate/
DCC.11 These methods, however, afford complex mixtures
of flavonoid mono- and di-sulfates. We have utilised an
alternative sulfation procedure for our synthesis, coupled
Scheme 1.
Keywords: daidzein sulfates; isoflavone; sulfation.
*
Corresponding author. Tel.: þ44-1334-463856; fax: þ44-1334-433808; e-mail: npb@st-andrews.ac.uk
0040–4020/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0040-4020(03)00869-X

5408
B. Fairley et al. / Tetrahedron 59 (2003) 5407–5410
Scheme 2. Reagents and reaction conditions: (a) (i) ClSO3H, pyridine, rt 12 h, (ii) K2CO3/H2O, .pH 10, (40%); (b) TBDMSOTf, 2,6-lutidine, DMF, 4 h, rt
(74%); (c) (i) ClSO3H, pyridine rt 12 h, (ii) K2CO3/H2O, .pH 10, (iii) TBAF, THF, CH3CN/H2O, (45%); (d) (i) KOBut (2.1 equiv.), DMF, rt, 2 h,
(ii) TBDMSCl, DMF, rt, 12 h (70%); (e) (i) ClSO3H, pyridine rt 12 h, (ii) K2CO3/H2O, .pH 10, (iii) TBAF, THF, CH3CN/H2O, (43%).
with a protecting group strategy, to afford the sulfated
regioisomers in both good yield and high purity.
acidic 7-position. This, however, was not observed. We had
previously shown formononetin 5, to be readily sulfated
under our standard conditions to give formononetin
7-sulfate. It was thus concluded that regiospecific sulfation
could only be achieved by employing a protecting group
strategy, whereby the protecting group could be readily
removed after sulfation. This protecting group should be
stable to both the harsh sulfation conditions and be easily
removable under mild conditions afterwards, leaving the
sulfate moiety intact. Of the functionalities investigated, a
TBDMS group was found to be most amenable to both these
criteria. The more acidic 7-position of daidzein 4 was thus
selectively protected with a TBDMS group to afford 6.
Sulfation was then carried out as before, followed by
treatment with TBAF to remove the silyl group and to afford
daidzein 40-sulfate01 (Scheme 2). The signatory downfield
shift of the 30 and 5 protons in was observed in the 1H NMR
spectrum (Table 1).
An alternative procedure was inspired by other work in our
laboratory on the synthesis of glucosinolates, an important
group of natural products possessing a sulfated oxime
moiety.12 Synthesis of these compounds employs a sulfation
step using a chlorosulfonic acid/pyridine mixture.13 Whe0n
daidzein 4 was reacted under these conditions the 7,4 -
disulfate 3 (Scheme 2) was obtained in quantitative yield
1
according to the H NMR data (Table 1). The aromatic
protons adjacent to the sulfate groups at the 6, 8, 30 and 50
positions were all shifted downfield as expected, due to the
electron withdrawing effect of the sulfates (Table 1). Mass
spectral analysis of 3 was most effective in ES 2ve mode
giving the required mass of 413 [M2H], although no
molecular ions could be observed in EI mode for any
sulfates. Unfortunately, microanalysis of 3 indicated that
large quantities of inorganic material were present.
Removal of the inorganic impurities by simple washing
was unsuccessful due to the very high polarity of the
daidzein disulfate, which meant it was very water soluble.
However, purification was eventually achieved using solid
phase extraction techniques. Even then, of the Sep-Packs
investigated, only a phenyl substituted support14 was found
to give sufficient retention of the isoflavone sulfate to allow
removal of the majority of the inorganic salts, although
some of the sulfate was still lost on washing. Indeed, the
sulfation step appeared to be quantitative based on 1H NMR
analysis and the overall yield determined by the losses on
purification. Purified 3 was obtained in greater than 95%
purity based on microanalysis but in only 40% yield.
Daidzein 7-sulfate 2 was prepared in a similar manner. In
this case the 40-position was protected selectively by
exploiting the increased reactivity of the di-phenolate
anion at the 40-position. Thus treatment of daidzein 4 with
two equivalents of potassium tert-butoxide followed by the
addition of one equivalent of TBDMSCl gave 40-tert-
butyldimethylsilyldaidzein 7 in 60% yield. Interestingly, no
selectivity was observed with the more reactive reagent,
TBDMSOTf. Finally, sulfation of intermediate 7 and
subsequent removal of the protecting group, furnished
daidzein 7-sulfate 2 in 43% yield.
In summary we have developed the first efficient syntheses
of daidzein 40-sulfate 1, daidzein 7-sulfate 2 and daidzein 40,
7-disulfate 3. These syntheses employ a key protecting
group strategy, allowing regiospecific sulfation. These
compounds have recently been identified in human urine
using LC-MS-MS methods.15 A similar strategy is currently
being employed in the preparation of the biologically
important, equol and genistein sulfates.
It was hoped that sulfation of daidzein 4 with one equivalent
of chlorosulfonic acid would give selectivity for the more
Table 1. 1H NMR shifts for daidzein and the respective sulfates
Proton
Daidzein (4)
(ppm)
Daidzein
Daidzein
Daidzein
40-sulfate (1) 7-sulfate (2) 7,40-disulfate (3)
(ppm)
(ppm)
(ppm)
2
5
6
8
8.30
7.98
6.95
6.89
7.40
6.89
8.35
7.98
6.95
6.90
7.46
7.21
8.40
8.03
7.25
7.45
7.40
6.80
8.30
8.02
7.23
7.40
7.45
7.16
3. Experimental
3.1. General procedures
200 and 60
3 and 5
Melting points were determined in open capillary tubes with

B. Fairley et al. / Tetrahedron 59 (2003) 5407–5410
5409
electrothermal apparatus and are uncorrected. For 1H NMR
(300 MHz) spectra the residual peak of CHCl3 (7.26 ppm)
and CH3COCH3 (2.05 ppm) were used as the internal
reference, while for 13C NMR (75 MHz) spectra the central
peak of CDCl3 (77.0 ppm) and the central peak CD3COCD3
(29.95 ppm) were used as the reference. Chemical shifts are
given in d and J values in Hz.
water and the pH adjusted to 10 with solid potassium
carbonate. To the aqueous mixture was added MeOH
(5 mL) and TBAF (1 M soln in THF, 1.4 mL, 1.4 mmol) and
the solution stirred for a further 2 h. The solvent was
removed at reduced pressure, with the crude residue taken
up in a 1:1 mixture of acetonitrile/water and the solution
acidified to pH 5 with 1 M HCl. Evaporation gave the crude
sulfate which was subjected to flash column chromato-
graphy (reverse phase silica gel (C18), acetonitrile/water,
1:1) to remove organic impurities. Portions of the product
(50–100 mg) were then suspended in water and applied to
Sep-packs (Strata 50 m, Tri-Func., Phenyl) and the
inorganic salts washed off with water (3 volumes). The
sulfate was then removed by washing with acetonitrile (2
volumes) to give the product as a white solid (210 mg,
45%); mp 296–3008C (dec); nmax/cm21 (nujol) 3210br
(OH), 1630, 1590, 1170, 815; 1H NMR (300 MHz, d6-
DMSO) 6.90 (1H, d, J¼0.6 Hz, H-8), 6.95 (1H, dd, J¼0.6,
8 Hz, H-6), 7.21 (2H,0 d, J¼8 Hz, H-30 and 50), 7.46 (2H,
d,J¼8 Hz, H-20 and 6 ), 7.98 (1H, d, J¼8 Hz, H-5), 8.35
(1H, s, H-2); 13C NMR (75.4 MHz, d6-DMSO) 106.0 (CH),
119.0 (CH), 120.5 (C), 123.9 (CH), 127.2 (CH), 130.4 (C),
131.1 (CH), 133.1 (CH), 157.1 (CH), 161.3 (C), 166.2 (C),
166.4 (C), 177.2 (C); MS (ES, 2ve ion mode) 333 (M2H,
100%); HRMS (ES, 2ve mode) calcd for C15H9O15S
333.0069, found 333.0068; Anal. calcd for C15H9O7SK: C,
48.38; H, 2.44. Found: C, 46.45; H, 2.30 (data imply 96%
purity).
3.1.1. Daidzein 7,40-disulfate (3). Daidzein (250 mg,
0.98 mmol) was dissolved in dry pyridine (10 mL) and
cooled to 08C. To this solution was added chlorosulfonic
acid (0.80 g, 6.9 mmol) and the mixture allowed to warm to
room temperature overnight. Evaporation at reduced
pressure afforded a residue which was re-dissolved in
water and the pH adjusted to 10 with solid potassium
carbonate. The sulfate was then purified by chromatography
(C2-SiO2, MeCN/H2O 1:1) to remove organic impurities.
Portions of the product (50–100 mg) were then suspended
in water and applied to Sep-packs (Strata 50 m, Tri-Func.,
Phenyl) and the inorganic salts washed off with water (3
volumes). The sulfate was then removed by washing with
acetonitrile (2 volumes) to give the product as a white solid
(193 mg, 40%); mp .375 8C; nmax/cm21 (nujol) 1640,
1595, 1176, 814; 1H NMR (300 MHz, d6-DMSO) 7.16 (2H,
d, J¼8 Hz, H-30 and 50), 7.23 (1H, dd, J¼0.6, 8 Hz, H-6),
7.40 (1H, d, J¼0.6 Hz, H-8), 7.45 (2H, d, J¼8 Hz, H-20 and
60), 8.02 (1H, d, J¼8 Hz, H-5), 8.30 (1H, s, H-2); 13C NMR
(75.4 MHz, d6-DMSO) 116.6 (CH), 127.5 (CH), 128.6 (C),
129.6 (CH), 133.0 (C), 136.0 (CH), 136.1 (C), 139.0 (CH),
162.9 (C), 163.5 (CH), 166.0 (C), 167.7 (C), 184.3 (C); MS
(ES, 2ve ion mode) 413 (M2H, 46%), 333 (M2SO3,
100%), 206 ((M-2H)22); HRMS (ES) calcd for C15H8O10S2
((M-2H)22 ion) 205.9786, found 205.9785; Anal. calcd for
C15H8O10SK2: C, 36.73; H, 1.64. Found: C, 36.51; H, 1.50
(data imply 99% purity).
3.1.4. 40-tert-Butyldimethylsilyldaidzein (7). To a solution
of daidzein (500 mg, 2 mmol) in DMF (15 mL) was added
potassium t-butoxide (463 mg, 4 mmol) and the mixture left
to stir for 2 h. To this was then added tert-butyldimethylsilyl
chloride (385 mg, 2.6 mmol) and the mixture stirred over-
night. Evaporation under reduced pressure afforded a
residue which was dissolved in water and acidified to pH
5 with 1 M HCl. The precipitate was then collected by
filtration and purified by chromatography (SiO2, hexane/
diethyl ether 1:1), furnishing 6 as a white crystalline solid
(438 mg, 60%); mp 242–2458C; nmax/cm21 (nujol) 3300br
3.1.2. 7-tert-Butyldimethylsilyldaidzein (6). To a solution
of daidzein (300 mg, 1.18 mmol) in DMF (10 mL) was
added 2,6-lutidine (137 mL, 1.18 mmol). After stirring for
15 min, tert-butyldimethylsilyl trifluoromethanesulfonate
(298 mL, 1.30 mmol) was added and the mixture stirred
for a further 8 h. Evaporation under reduced pressure
afforded a residue which was purified by chromatography
(SiO2, hexane/diethyl ether 1:1) furnishing 6 as a white
1
(OH), 1628, 1250, 835; H NMR (300 MHz, d6-acetone)
0.25 (6H, s, 2£CH3), 1.01 (9H, s, But), 6.92 (3H, m, H-8,
H-30and 50), 7.01 (1H, dd, J¼0.6, 8.0 Hz, H-6), 7.53 (2H, d,
J¼8 Hz, H-20and 60), 8.07 (1H, d, J¼8 Hz, H-5), 8.19 (1H, s,
H-2); 13C NMR (300 MHz, d6-acetone); 13C NMR
(75.4 MHz, d6-DMSO) 4.2 (CH3), 18.3 (C), 25.9 (CH3),
102.5 (CH), 115.5 (CH), 116.9 (C), 119.8 (CH), 123.4
(C),125.5 (C), 127.6 (CH), 130.5 (CH), 153.6 (CH), 155.2
(C), 157.8 (C), 162.9 (C), 174.9 (C); MS (EI) 368 (Mþ,
47%), 311 ((M2But)þ, 100%); HRMS (ES, 2ve mode)
calcd for C21H23O4Si 367.1366, found 367.1359.
crystalline solid (320 mg, 74%); mp 176–1788C; nmax
/
cm21 (nujol) 3300br (OH), 1624, 1250, 840; 1H NMR
(300 MHz, CDCl3) 0.28 (6H, s, 2£CH3), 1.01 (9H, s, But),
6.84 (2H, d, J¼8 Hz, H-30and 50), 6.87 (1H, d, J¼0.6 Hz,
H-8), 6.94 (1H, dd, J¼0.6, 8 Hz, H-5), 7.30 (2H, d, J¼8 Hz,
H-20and 60), 7.78 (1H, s, OH), 7.92 (1H, s, H-2), 8.20 (1H, d,
J¼8 Hz, H-5); 13C NMR (75.4 MHz, CDCl3) 24.0 (CH3),
18.7 (C), 26.0 (CH3), 107.9 (CH), 116.3 (CH), 119.1 (C),
119.8 (CH), 123.4 (C), 125.7 (C), 128.2 (CH), 130.6 (CH),
153.2 (CH), 157.1 (C), 158.3 (C), 161.3 (C), 171.4 (C); MS
(EI) 368 (Mþ, 47%), 311 ((M2But)þ, 100%); HRMS (EI)
calcd for C21H24O4Si 368.1444, found. 368.1450.
3.1.5. Daidzein 7-sulfate (2). This was prepared as for the
daidzein 40-sulfate, to give the product as a white solid in
43% yield; mp 242–2468C (dec); nmax/cm21 (nujol) 3200br
(OH), 1636, 1590, 1182, 820; 1H NMR (300 MHz, d6-
DMSO) 6.89 (2H, d, J¼8 Hz, H-30 and 50), 7.25 (1H, dd,
J¼0.6, 8 Hz, H-6), 7.40 (2H, d, J¼8 Hz, H-20 and 60), 7.43
(1H, d, J¼0.6 Hz, H-8), 8.03 (1H, d, J¼8 Hz, H-5), 8.4 (1H,
s, H-2); 13C NMR (75.4 MHz, d6-DMSO) 116.5 (CH), 124.5
(CH), 127.5 (CH), 128.5 (C), 131.9 (C), 133.2 (C), 135.9
(CH), 139.6 (CH), 162.9 (CH), 166.0 (C), 166.8 (C), 167.7
(C), 184.4 (C); MS (MALDI-TOF, 2ve ion mode) 333
3.1.3. Daidzein 40-sulfate (1). 7-tert-Butyldimethlsilyl-
daidzein (6) (516 mg, 1.4 mmol) was dissolved in dry
pyridine (8 mL) and cooled to 08C. To this solution was
added chlorosulfonic acid (1.63 g, 14 mmol) and the
mixture allowed to warm to room temperature overnight.
Evaporation afforded a residue which was re-dissolved in

5410
B. Fairley et al. / Tetrahedron 59 (2003) 5407–5410
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(M2H, 100%); HRMS (ES, 2ve mode) calcd for C15H9O7S
333.0069, found 333.0082; Anal. calcd for C15H9O7SK: C,
48.38; H, 2.44. Found: C, 46.20; H, 2.31 (data imply 95%
purity).
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Acknowledgements
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We acknowledge Unilever for funding for B. F. and G. J.
Langley (University of Southampton) for the accurate mass
measurement on daidzein 7,40-disulfate.
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