Synthesis of deuterated isoflavone disulfates

Article abstract of DOI:10.1002/jlcr.1114

Efficient synthesis of polydeuterated di-O-sulfates of three isoflavones, daidzein (1), genistein (2), glycitein (3), is described. The isoflavones were first deuterated with CF₃COOD under microwave irradiation to yield daidzein-d₆ (

Full text of DOI:10.1002/jlcr.1114

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd. Radiopharm 2006; 49: 973–978.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.1114
Research Article
Synthesis of deuterated isoflavone disulfates
Otto Soidinsalo and Kristiina Wahala*
¨ ¨ ¨
Laboratory of Organic Chemistry, Department of Chemistry, University of Helsinki,
P.O. Box 55, 00014, University of Helsinki, Finland
Summary
Efficient synthesis of polydeuterated di-O-sulfates of three isoflavones, daidzein ð1Þ,
genistein ð2Þ, glycitein ð3Þ, is described. The isoflavones were first deuterated with
CF3COOD under microwave irradiation to yield daidzein-d6 ð4Þ, genistein-d4 ð5Þ and
glycitein-d6 ð6Þ in 90% yield and in a high isotopic purity. The deuterated isoflavones
were then sulfated with chlorosulfonic acid and pyridine to give the title compounds,
again in high yield and isotopic purity (>90%). The compounds are useful as internal
standards in LC-MS quantitation of isoflavones. Copyright # 2006 John Wiley &
Sons, Ltd.
Received 5 July 2006; Revised 11 July 2006; Accepted 12 July 2006
Key Words: isoflavones; sulfation; synthesis; deuteration; phytoestrogen; isoflavonoid
Introduction
Isoflavones are polyphenolic compounds possessing a wide variety of
biological activity.1 They exist in plants mainly as glycosides, and are
metabolized in man to a variety of compounds.2 However, neither the route
nor the mechanisms of the metabolic pathways are well known, leading to
uncertainty as to the exact structures of the metabolites. Members of the
known metabolites include isoflavonoid di-O-sulfates which have been
detected in human urine.3 Glucuronide, monosulfate and sulfoglucuronide
conjugates are other major metabolites.3 It has been previously shown that
daidzein sulfoconjugates are potent inhibitors of the sterol sulfatase enzyme
preventing the production of biologically active estrogenic steroids from their
inactive sulfoconjugates.4
*Correspondence to: K. Wahala, Laboratory of Organic Chemistry, Department of Chemistry, University
¨
of Finland, P.O. Box 55, 00014, University of Helsinki, Finland. Email: kristiina.wahala@helsinki.fi
¨
¨
Contract/grant sponsor: University of Helsinki
Contract/grant sponsor: Finnish Academy; contract/grant number: 210633
Copyright # 2006 John Wiley & Sons, Ltd.

¨ ¨ ¨
O. SOIDINSALO AND K. WAHALA
974
Isotopically labeled di-O-sulfated isoflavone derivates are useful for further
studies of the biological activity and elucidation of their metabolism. They
may also be used as internal standards in LC-MS quantitation of the disulfate
conjugates. The conventional methods for deuteration of isoflavonoids involve
reactions under acidic or basic conditions.5,6 Previously daidzein-d4 has been
prepared by refluxing in CF3COOD for 9 days,6 while daidzein-d6 ð4Þ was
obtained by a D3PO4ꢀBF3/D2O deuteration in an autoclave at 1008C for 7
days.7 These methods suffer from long reaction times. Herein we report an
expedient synthesis in high yields of stable polydeuterated isoflavones and
their sulfate conjugates using CF3COOD as a source of deuterium under
microwave irradiation. By this method, CF3COOD allows H-D exchanges
even at the less activated ring sites.
Results and discussion
Isoflavones were first predeuterated by evaporation of their acetone/D2O
solutions to remove the phenolic protons from the ensuing equilibria. The
reaction of isoflavones with CF3COOD was performed under microwave
irradiation in pressure vessels. Reaction was followed by LC-MS ðESIþÞ,
establishing that the reaction proceeded in the order that first protons ortho to
the phenolic OH groups were exchanged, followed more slowly by the less
activated 20 and 60 protons. Repeating the reaction twice, the exchange of
protons with deuteriums was achieved in high yield (90%) with all the
compounds: daidzein-d6 ð4Þ, genistein-d6 and glycitein-d6 ð6Þ (Scheme 1).
1
According to the ESI-MS and H NMR data, the isotopic purity of the
deuterated compounds was over 90%. Attempted deuteration with
CH3COOD or DCl/d6-acetone under microwave irradiation gave no
significant H/D exchanges.
In genistein-d6, the deuterium atoms at C-6 and C-8 are potentially labile,8
and hence a back exchange was done by refluxing genistein-d6 in a solution of
0.5% CH3COCl/MeOH for 10 min to give genistein-d4 ð5Þ (Scheme 1). The
deuterium atoms at other positions were stable under this treatment.
The sulfation of the deuterated isoflavones was performed by our previously
described procedure for isoflavonoid di-O-sulfates,9 (Scheme 1) giving
daidzein-d6-di-O-sulfate ð7Þ, glycitein-d6-di-O-sulfate ð8Þ and genistein-d4-
di-O-sulfate ð9Þ in high yields. The deuterium labels were stable under these
reaction conditions including the work-up and the purification process, giving
the products in high isotopic purity. The deuterated di-O-sulfates were
analyzed by 1H ,13C NMR and ESIðþÞ. In ESIðþÞ only traces of M-1 (510%)
were detected and the isotopic pattern was the same as for the deuterated
isoflavones. Importantly, no undeuterated or other deutero species were
observed. The NMR spectra of sulfated polydeuterated isoflavones were
compared with our previously published spectra of isoflavone di-O-sulfates.9
Copyright # 2006 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2006; 49: 973–978
DOI: 10.1002/jlcr

SYNTHESIS OF DEUTERATED ISOFLAVONE DISULFATES
975
8
7
HO
8a O
2
2´
5´
6
R1
4
1´
3´
4´
3
4a
5
R2
O
6´
OH
1 R1=R2=H
2 R1=H R2=OH
3 R1=OMe R2=H
1) CF3COOD
MW
2) CH3COCl
MeOH
CF3COOD
MW
D
HO
R1
O
HO
O
D
D
D
D
D
R2
O
D
OH
O
OH
D
OH
D
4 R1=D R2=H
5
6 R1=OMe R2=D
ClSO3H
Pyridine
ClSO3H
Pyridine
O
O
NaO
D
O
NaO
S
S
O
O
O
O
O
D
D
D
D
D
R1
R2
O
OH
O
O
O
D
O
D
O
O
S
S
ONa
D
ONa
O
7 R1=D R2=H
8
9 R1=OMe R2=D
Scheme 1. Synthesis of deuterated isoflavonoid di-O-sulfates
The presence of deuterium atoms is clearly seen in the 13C spectrum as the low
intensity triplets of the deuterium carrying carbon atoms. Incidentally, a
reverse order procedure, i.e. the deuteration of preformed isoflavone sulfates
was not applicable as the substrates suffered ready desulfation on treatment
with CF3COOD. Also, the aromatic ring sites of the phenolic sulfate esters are
unlikely to have sufficient reactivity for electrophilic H/D exchanges.
Experimental
General
NMR spectra were measured on a Varian Gemini 2000 or a Bruker Avance
500 spectrometer with TMS as an internal standard. Chemical shifts are in d
Copyright # 2006 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2006; 49: 973–978
DOI: 10.1002/jlcr

¨ ¨ ¨
O. SOIDINSALO AND K. WAHALA
976
values (ppm) and coupling constants in Hz (s, singlet; d, doublet; dd, double
doublet; m, multiplet). LC-MS(ESI+) was performed on a HP 1100 equipped
with Mariner ESI-TOF. High-resolution mass spectra were measured on a
Bruker Mikrotof on the negative mode with Tunemix as the internal standard.
TLC was conducted on Merck RP-18 F254s plates. Microwave irradiation was
carried out with a CEM Discover1 instrument. The starting materials
daidzein ð1Þ and genistein ð3Þ were synthesized according to our published
procedures.10 Glycitein ð2Þ was prepared by Houben-Hoesch synthesis from
4-methoxyresorcinol and 4-hydroxyphenylacetonitrile to give 2,4,40-tri-
hydroxy-5-methoxydeoxybenzoin, followed by the ring closure as previously
described.10
General procedure for the deuteration of isoflavones
Predeuterated (D2O/acetone) isoflavone (50 mg) was placed in a 10 ml
pressure-proof reaction vial. CF3COOD (2 ml) was added and the vial was
sealed. The vial was irradiated for 5 h with 50 W microwave power. The
pressure in the vial during the reaction was 12 bar and temperature 1808C.
After 5 h, the solvent was removed in vacuo and fresh CF3COOD was added.
This was repeated twice. After completion of the reaction, the solvent was
removed in vacuo and the residue recrystallized.
[6,8,20,30,50,60-d6]-Daidzein ð4Þ [7-hydroxy-3-(4-hydroxyphenyl-2,3,5,6-d4)-4H-
1-benzopyran-4-one-6,8-d2]. Recrystallization from EtOH/H2O gave off white
crystals (46 mg, 90%), m.p. 335–3368C.
[20,30,50,60-d4]-Genistein ð5Þ [5,7-dihydroxy-3-(4-hydroxyphenyl-2,3,5,6-d4)-4H-
1-benzopyran-4-one]. The crude product (50 mg) was refluxed in 0.5%
CH3COCl/MeOH (10 ml) for 2 h and poured into ice water. The product
was extracted with EtOAc (4 ꢁ 20 ml), dried with MgSO4 and the solvent was
removed in vacuo. Recrystallization from EtOH/H2O gave off white crystals
(46 mg, 90%), m.p. 301–3028C.
[5,8,20,30,50,60-d6]-Glycitein ð6Þ [7-hydroxy-3-(4-hydroxyphenyl-2,3,5,6-d4)-
6-methoxy-4H-1-benzopyran-4-one-5,8-d2]. Recrystallization from EtOH/
H2O gave off white crystals (45 mg, 89%), m.p. 344–3458C. 1H NMR
(200 MHz, DMSO-d6): d ¼ 3:88 (s, 3H, OCH3), 8.28 (s, 1H, H-2); 13C NMR
(50 MHz, DMSO-d6) 55.8 (CH3), 102.6(C-8)D, 104.4(C-5)D, 114.8 (C-30,
C-50)D, 116.1 (C-4a), 122.6 (C-10), 122.9 (C-3), 129.6 (C-20, C-60)D, 146.8 (C-6),
151.6 (C-8a), 152.4 (C-2), 152.8 (C-7), 157.0 (C-40), 174.3 (C-4).
HRMS calculated for C16H6D6O5: 290.1061; found: 290.1069.
Copyright # 2006 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2006; 49: 973–978
DOI: 10.1002/jlcr

SYNTHESIS OF DEUTERATED ISOFLAVONE DISULFATES
977
General procedure for the synthesis of deuterated isoflavone 40,7-di-O-sulfates
Chlorosulfonic acid ClSO3H (10 eq.) was added dropwise and slowly
(CAUTION) to a stirred solution of the deuterated isoflavonoid (1 eq.) in
freshly distilled pyridine at ꢂ168C under argon atmosphere. After 14 h at
room temperature, 5% aq. NaHCO3 was added until the pH was 8. The
solvent was removed in vacuo and the obtained crude solid product was
purified on Sephadex LH-20 using water as an eluent.
[6,8,20,30,50,60-d6]-Daidzein 40,7-di-O-sulfate, disodium salt ð7Þ [7-sulfooxy-3-
(4-sulfooxyphenyl-2,3,5,6-d4)-4H-1-benzopyran-4-one-6,8-d2, disodium salt].
1
White solid: 90%; H NMR (200 MHz, DMSO-d6): d ¼ 8:05 (s, 1H, H-5),
8.45 (s, 1H, H-2); 13C NMR (50 MHz, DMSO-d6) 106.9 (C-8)D, 117.8 (C-6)D,
118.9 (C-4a), 120.0 (C-30, C-50)D, 123.3 (C-10), 126.2 (C-5), 126.3 (C-3), 129.3
(C-20, C-60)D, 153.2 (C-40), 153.9 (C-2), 156.4 (C-8a), 158.0 (C-7), 174.6 (C-4).
m/z (ESI-) 209 [M-2Na]2ꢂ
HRMS calculated for C15H2D6O10S2: 208.9973; found: 208.9972.
[20,30,50,60-d4]-Genistein 40,7-di-O-sulfate, disodium salt ð8Þ [5-hydroxy-7-
sulfooxy-3-(4-sulfooxyphenyl-2,3,5,6-d4)-4H-1-benzopyran-4-one, disodium salt].
White solid: 85%; 1H NMR (200 MHz, DMSO-d6): d ¼ 6:64 (d, 1H,
J ¼ 2:2 Hz, H-6), 6.90 (d, 1H, J ¼ 2:2 Hz, H-8), 8.48 (s, 1H, H-2), 12.80
(s, 1H, H-5); 13C NMR (50 MHz, DMSO-d6) 97.4 (C-8), 102.1 (C-6), 106.3
(C-4a), 119.8 (C-30, C-50)D, 122.1 (C-1), 124.9 (C-3), 129.2 (C-20, C-6)D, 153.4
(C-40), 155.1 (C-2), 156.6 (C-8a), 159.7 (C-7), 161.0 (C-5), 180.4 (C-4).
m/z (ESI-) 216 [M-2Na]2ꢂ
HRMS calculated for C15H4D4O11S2: 215.9885; found: 215.9876.
[5,8,20,30,50,60-d6]-Glycitein 40,7-di-O-sulfate, disodium salt ð9Þ [7-sulfooxy-
3-(4-sulfooxyphenyl-2,3,5,6-d4)-6-methoxy-4H-1-benzopyran-4-one-5,8-d2, dis-
1
odium salt]. White solid: 80%; H NMR (200 MHz, DMSO-d6): d ¼ 3:87
(s, 3H, OCH3), 8.43 (s, 1H, H-2); 13C NMR (50 MHz, DMSO-d6) 55.7 (CH3),
104.6 (C-8)D, 107.3 (C-5)D, 118.2 (C-4a), 120.0 (C-30, C-50)D, 122.6 (C-10),
126.4 (C-3), 128.7 (C-20, C-60)D, 148.2 (C-7), 148.2 (C-6), 150.5 (C-8a), 153.1
(C-40), 153.6 (C-2), 174.2 (C-4).
m/z (ESI-) 224 [M-2Na]2ꢂ
HRMS calculated for C16H4D6O11S2: 224.0026; found: 224.0018.
Conclusion
A rapid and efficient method for the preparation of polydeuterated di-
O-sulfates of three dietary isoflavones has been developed. In addition to use
as internal standards, these compounds are useful as monitoring tools during
the pretreatment process of biological samples and hence give more under-
standing about their behavior and chemical stability in such systems.
Copyright # 2006 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2006; 49: 973–978
DOI: 10.1002/jlcr

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O. SOIDINSALO AND K. WAHALA
978
Acknowledgements
We thank Dr Petri Heinonen for running the HRMS. Research Funds of the
University of Helsinki and Finnish Academy (210633) are acknowledged for
financial support.
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Copyright # 2006 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2006; 49: 973–978
DOI: 10.1002/jlcr