Sulfation of β-resorcylic acid esters - First synthesis of zearalenone-14-sulfate

Article abstract of DOI:10.1016/j.tetlet.2013.04.059

The chemical sulfation of β-resorcylic acid esters was investigated by applying state of the art procedures for the synthesis and deprotection of 2,2,2-trichloroethyl protected sulfates as appropriate intermediates. The selectivity of monosulfation was st

Full text of DOI:10.1016/j.tetlet.2013.04.059

Tetrahedron Letters 54 (2013) 3290–3293
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Tetrahedron Letters
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Sulfation of b-resorcylic acid esters—first synthesis of zearalenone-14-sulfate
Hannes Mikula a,c, , Barbara Sohr a, Philipp Skrinjar a, Julia Weber a, Christian Hametner a, Franz Berthiller b,
⇑
Rudolf Krska b, Gerhard Adam c, Johannes Fröhlich a
a Institute of Applied Synthetic Chemistry, Vienna University of Technology, Getreidemarkt 9/163-OC, 1060 Vienna, Austria
b Christian Doppler Laboratory for Mycotoxin Metabolism and Center for Analytical Chemistry, Department for Agrobiotechnology (IFA-Tulln), University of Natural Resources and
Life Sciences, Vienna (BOKU), Konrad Lorenz Str. 20, 3430 Tulln, Austria
c Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Vienna (BOKU), Konrad Lorenz Str. 24, 3430 Tulln, Austria
a r t i c l e i n f o
a b s t r a c t
Article history:
The chemical sulfation of b-resorcylic acid esters was investigated by applying state of the art procedures
for the synthesis and deprotection of 2,2,2-trichloroethyl protected sulfates as appropriate intermediates.
The selectivity of monosulfation was studied and reaction optimization was performed considering the
effect of the solvent, different bases as well as the sulfation reagent itself. Finally the obtained protocols
were applied for the first synthesis of zearalenone-14-sulfate (ammonium salt), an important conjugated
(masked) mycotoxin, as reference material for further investigations in the field of bioanalytics as well as
toxicology.
Received 9 March 2013
Revised 9 April 2013
Accepted 16 April 2013
Available online 22 April 2013
Keywords:
Zearalenone
Masked mycotoxin
Sulfate
Ó 2013 Elsevier Ltd. All rights reserved.
Phase II metabolite
Resorcylic acid lactone
Zearalenone (ZEN, 1, Fig. 1) is a mycotoxin produced by several
plant pathogenic Fusarium species, including Fusarium graminea-
rum, Fusarium culmorum, and Fusarium cerealis. This mycotoxin is
common in maize, but also barley, oats, wheat, and rice are suscep-
tible to contamination with ZEN.1 Fusarium species are probably
the most prevalent toxin-producing fungi of the northern temper-
ate regions and are commonly found in cereals grown in America,
Europe, and Asia, but also in Africa.2,3 ZEN and its phase I metabo-
lites possess estrogenic activity in mammals, including pigs, cattle,
and sheep.4 Problems of the reproductive tract as well as impaired
fertility and abnormal fetal development in farm animals can be
OH O
OH O
16
O
O
HO14
HO
R
R'
O
-Zearalenol (2a): R = H, R' = OH
α
β-Zearalenol (2b): R = OH, R' = H
Zearalenone (1)
Figure 1. Structures of zearalenone (ZEN) and its main phase I metabolites
a-
caused by ZEN.5 Furthermore ZEN and its metabolites
-ZEL, 2a) and b-ZEL (2b, Fig. 1) can interfere with various en-
a-zearalenol
zearalenol (
a-ZEL) and b-ZEL.
(a
zymes involved in steroid metabolism, which was recently investi-
gated.6–8 Therefore ZEN is of significant importance from an
agricultural, economic, and health perspective.9
HO
Additionally, conjugated mycotoxins, for example, glucosides
and sulfates can emerge after metabolization by living plants.10
O
O
HO
HO
Na+
O
OH
O
OH
S
HO
-O
The occurrence of ZEN-14-b,D-glucoside (3, Fig. 2) in wheat was
O
shown by Schneweis et al.11 and preparation of this conjugate to
obtain reasonable amounts of reference material for further inves-
tigations has been first reported by Grabley et al. by applying an
optimized Königs–Knorr procedure under phase transfer condi-
tions.12 Basically there are two sites for glycosylation present in
O
O
O
O
3
4
O
O
⇑
Corresponding author. Tel.: +43 1 58801 163721; fax: +43 1 58801 16399.
E-mail address: hannes.mikula@tuwien.ac.at (H. Mikula).
Figure 2. Structures of conjugated zearalenone (ZEN) metabolites: ZEN-14-b,
glucoside (3) and ZEN-14-sulfate, sodium salt (4).
D-
0040-4039/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetlet.2013.04.059

H. Mikula et al. / Tetrahedron Letters 54 (2013) 3290–3293
3291
ZEN, but conjugation in position 14 (ring system numbering fol-
OH O
lowing the scheme proposed by Metzler13) is strongly favored
compared to position 16.14 ZEN-14-sulfate (formerly known as
ZEN-4-sulfate) was first isolated by Plasencia and Mirocha from
rice inoculated with F. graminearum.15 El-Sharkawy et al. reported
the conversion of ZEN into ZEN-14-sulfate (4, Fig. 2) by various
microorganisms16 and Berthiller et al. identified 4 as phase II
metabolite of ZEN in the model plant Arabidopsis thaliana.17 The
conjugate most likely retains biological properties of the myco-
toxin, since the sulfate moiety is easily cleaved under acidic condi-
tions and in rats.16 Therefore, ZEN-14-sulfate was included in
analytical methods for the determination of free and masked
mycotoxins during the last years.18–21 Vendl et al. identified 4 as
the most abundant analyte in 84 cereal based food products by
LC–MS/MS measurements.22
, NEt3, DMAP or
2
5
O
4
HO
6
, 1,2-dimethylimidazole
7
O
Cl3C
O
O
S
OH O
O
O
O
O
O
O
O
Cl3C
Cl3C
O
S
S
O
O
O
O
9
8
Scheme 2. Chemical sulfation of b-resorcylic acid methyl ester (7), by applying
sulfuryl chloride 5 or imidazolium triflate 6 as reagent.
Nevertheless, reference material of 4 is still produced by time-
consuming F. graminearum inoculation of rice. Due to its low stabil-
ity the obtained sulfate is furthermore not suitable for long term
storage.
sulfur trioxide complexes (SO3ÁNMe3, SO3ÁPyr, and SO3ÁDMF) all
leading to complex product mixtures or low conversion of 7 as
indicated by HPLC. Therefore, chlorosulfuric acid 2,2,2-trichloro-
ethyl ester (5)31 was used as the sulfation reagent for further inves-
tigations. In a first attempt reaction of 7 and 5 (1.2 equiv) in the
presence of 4-(dimethylamino)pyridine (DMAP, 1.0 equiv) and
NEt3 (1.2 equiv), according to the original procedure of Taylor
and co-workers31 led to a conversion of 68% yielding the desired
monosulfated product 8 (44%), but also the disulfate 9 (24%,
Scheme 2). In contrast to glycosylation,14,12 acetylation34, silyla-
tion35, and benzylation36 no significant selectivity was observed
for monosulfation in position 4 of compound 7 by applying this
procedure. Since we have reasoned this outcome with the higher
reactivity of sulfuryl chlorides compared to glycosyl donors as well
as common acylation, silylation, and benzylation reagents, an opti-
mization study was performed considering the effect of the sol-
vent, different bases as well as the sulfation reagent itself (Table 1).
Starting by changing the solvent from tetrahydrofuran (THF) to
dichloromethane (DCM) the selectivity of monosulfation by reac-
tion of 7 with sulfuryl chloride 5 was increased to a 8:9 ratio of
3.5:1 using 0.5 M equiv of DMAP (Table 1, entry 5), whereas the
application of pyridine instead of DMAP basically led to no
(DCM) or only poor (THF) conversion of compound 7. Interestingly
sulfation of the monosulfate 8 (to form the disulfate 9) was ob-
served to occur faster than the sulfation of the ZEN mimic 7 when
using an excess of DMAP instead of NEt3/DMAP (entry 6). Basically
higher selectivity was observed in DCM compared to reactions car-
ried out in THF. Enhanced selectivity was achieved by applying
imidazolium salt 6 for sulfation of 7. This reaction was studied in
terms of varying the amount of the sulfating reagent starting from
1.05 to 2.0 M equiv leading to 8:9 ratios between 8:1 and 1:3. In
particular using 1.05 equiv of 6 and 1.5 equiv of 1,2-dimethylimid-
azole (1,2-DMIm), sulfation at 20 °C afforded 80% of the monosulf-
ated product 8 (entry 7). Applying these conditions at À10 °C
(entry 11) or using exactly one equivalent of the reagent (entry
12) did not lead to a significant improvement in terms of selectiv-
ity and conversion. Therefore conditions according to entry 7 were
applied for the sulfation of 7 affording compounds 8 and 9 in rea-
sonable isolated yields (75% and 10%, respectively). These TCE pro-
tected sulfates were readily separated and purified by silica gel
chromatography revealing an important advantage of this strategy
compared to common methods directly leading to nonprotected
sulfate salts.
Although alkyl and aryl sulfates as well as resorcylic acid lactones
(cyclic 2,4-dihydroxybenzoates) such as ZEN are widespread in bio-
logical systems,23–28 to the best of our knowledge there is still no
procedure reported for the synthesis of sulfated resorcylic acid lac-
tones or esters, which may be formed during plant or human metab-
olism (phase II detoxification). In general several synthetic methods
were developed for the preparation of aryl sulfates, mainly by apply-
ing commercially available sulfur trioxide amine and amide com-
plexes.29 Since these methods are limited in terms of chemical
modifications following installation of the sulfate group as well as
regioselectivity, yield, and reproducibility, protecting groups for
chemical sulfation were investigated during the last decade. Simp-
son and Widlanski introduced neopentyl and isobutyl chlorosulfat-
es for chemical sulfation of phenols,30 whereas the 2,2,2-
trichloroethyl (TCE) group was used by Taylor and co-workers.31
Application of the TCE group allows for efficient preparation (apply-
ing sulfuryl chloride 5 as reagent) and good stability of protected
aryl sulfates. Catalytic transfer hydrogenation using Pd/C, ammo-
nium formate (HCOONH4) as well as cleavage by reaction with Zn/
HCOONH4 was reported for the deprotection of the TCE group under
mild reductive conditions yielding arylsulfate ammonium salts
(Scheme 1a). Additionally sulfuryl imidazolium salt 6 was intro-
duced as reagent for incorporating trichloroethyl protected sulfate
esters to carbohydrates (Scheme 1b).32
Considering different methods for the synthesis of aryl sulfates,
we have started carrying out the chemical sulfation of b-resorcylic
acid methyl ester (7) as a mimic for ZEN14,33 by applying several
a
NEt3
DMAP Cl
Cl
O
R
O
R
O
Cl
HO
S
Cl
Cl
S
O
O
O
O
Cl
Cl
5
+ O
R
Pd/C or Zn,
N+
HCOONH4
NH4
O
S
-O
O
b
O
Deprotection of TCE protected sulfates was carried out by
applying both strategies known from the literature. Catalytic trans-
fer hydrogenation (Pd/C, HCOONH4) as well as cleavage by reaction
with zinc/ammonium formate (HCOONH4) yielded the desired
monosulfate 10 as well as the disulfate 11 as ammonium salts in
excellent yields after purification by simple filtration over a pad
of silica gel eluting with DCM/MeOH/NH4OH (10:4:1) (Scheme 3).
Both compounds were observed to be stable in solid form as well
O
-OTf
O
1) 1,2-DMIm
NH4+
N
HO
R'
Cl
Cl
S
'
R
S
-O
O
O
2) Pd/C or Zn,
HCOONH4
O
Cl
6
Scheme 1. (a) Synthesis of aryl sulfates by applying chlorosulfuric acid 2,2,2-
trichloroethyl ester (5),31 (b) sulfuryl imidazolium salt 6 for the preparation of alkyl
sulfates (e.g. carbohydrates),32 DMAP = 4-(dimethylamino)pyridine, and 1,2-
DMIm = 1,2-dimethylimidazole.

3292
H. Mikula et al. / Tetrahedron Letters 54 (2013) 3290–3293
Table 1
Optimization study on the sulfation of b-resorcylic acid methyl ester (7) via Scheme 1
Entry
Reagent (equiv)
Conditionsa
8 (%)b
9 (%)b
Unreacted 7 (%)b
1
2
3
4
5
6
7
8
5 (1.20)
5 (1.20)
5 (1.20)
5 (1.20)
5 (1.20)
5 (1.20)
6 (1.05)
6 (1.25)
6 (1.50)
6 (2.00)
6 (1.05)
6 (1.00)
NEt3 (1.2 equiv), DMAP (1.0 equiv), THF, 20 °C
NEt3 (1.2 equiv), DMAP (1.0 equiv), DCM, 20 °C
NEt3 (1.2 equiv), Pyridine (1.0 equiv), THF, 20 °C
NEt3 (1.2 equiv), Pyridine (1.0 equiv), DCM, 20 °C
NEt3 (1.2 equiv), DMAP (0.5 equiv), DCM, 20 °C
DMAP (2.0 equiv), DCM, 20 °C
1,2-DMIm (1.5 equiv), DCM, 20 °C
1,2-DMIm (1.5 equiv), DCM, 20 °C
1,2-DMIm (1.5 equiv), DCM, 20 °C
1,2-DMIm (1.5 equiv), DCM, 20 °C
44
67
4
24
20
0
32
13
96
100
6
35
9
5
1
0
18
12
No conversion
73
17
80
71
52
23
64
80
21
48
11
24
47
77
18
8
9
10
11
12
1,2-DMIm (1.5 equiv), DCM, -10 °C
1,2-DMIm (1.5 equiv), DCM, 20 °C
a
Addition of reagent 5 or 6 at À10 °C to the reaction mixture and stirring over-night at the indicated temperature (DMAP = 4-(dimethylamino)pyridine, 1,2-DMIm = 1,2-
dimethylimidazole).
b
Determined by 1H NMR after aqueous work-up (integration of H-6 signals; see Supplementary data for detailed information).
ration of disulfated ZEN (14) which is why reaction conditions
according to entry 9 (see Table 1) were applied yielding both pro-
tected sulfates, 12 and 13 in an approximate ratio of 1:1. To avoid
undesired hydrogenation of the conjugated double bond present in
ZEN, Zn/HCOONH4 was applied for reductive cleavage of TCE
groups (Scheme 4).
The protected ZEN sulfates 12 and 13 were separated and puri-
fied by silica gel chromatography. Similar to compounds 10 and 11,
both ZEN sulfates, 4 and 14 were obtained as ammonium salts in
pure form after short filtration over a pad of silica gel eluting with
DCM/MeOH/NH4OH (10:4:1). The obtained ZEN-14-sulfate was
identical (NMR, ESI-MS) to natural occurring material that was pre-
viously isolated and characterized.15,37
In conclusion, the chemical sulfation of b-resorcylic acid esters
by applying TCE protection was investigated leading to improved
procedures for selective monosulfation as well as for simultaneous
preparation of mono- and disulfates. Sulfuryl imidazolium salt 6,
which can be easily prepared in large scale starting from 2,2,2-tri-
chloroethanol,32 was shown to be an appropriate reagent for con-
trolled synthesis of aryl sulfates and reductive conditions were
applied for efficient deprotection of the TCE group after simple
purification of the intermediates by silica gel chromatography.
These protocols were used for the first chemical synthesis of zea-
ralenone-14-sulfate (4). This fast and efficient procedure can easily
be reproduced in other labs to obtain the masked/conjugated
mycotoxin 4 in reasonable amounts and in short time for further
investigations, for example, as reference material for bioanalytical
studies or for toxicological testing. Additionally, the stability of
ammonium salts of these sulfates was reported to be adequate
for long term storage in solid or dissolved form.
OH O
OH O
O
i or ii
O
O
O
Cl3C
O
-O
O
+ O
S
S
NH4
O
O
8
10
i: 87 %
ii: 90 %
O
O
Cl3C
O
O
-O
S
S
NH4
O
O
O
O
O
+ O
O
O
O
O
i or ii
Cl3C
+-O
S
S
NH4
O
O
O
O
9
11
i: 93 %
ii: 91 %
Scheme 3. Deprotection of TCE sulfates 8 and 9 ((i) Pd/C, HCOONH4; (ii) Zn,
HCOONH4).
as dissolved in methanol at À20 °C and even room temperature
(RT) for several weeks and days, respectively. This outcome was
quite surprising, since these compounds were originally suspected
to be unstable in protic solvents. An NMR sample of the disulfate
11 (in MeOH) showed slight degradation to the monosulfate 10
after several days at RT indicating lower stability of the sulfate
moiety in position 2 compared to the 4-sulfate.
Based on these results and by applying the obtained optimized
procedures, ZEN-14-sulfate (4) as well as ZEN-14,16-disulfate (14)
were prepared in high overall yields. Due to the unexpected notice-
able stability of compound 11 we also got interested in the prepa-
OH O
OH O
OH O
O
Zn,HCOONH4
95 %
O
O
O
-O
Cl3C
O
S
S
O
+
NH4
O
O
O
O
HO
12
43 %
4
O
O
1,2-dimethylimidazole
20 °C, 15h
1
O
O
O
-O
Cl3C
O
S
S
NH4
O
O
O
O
N+
O
+ O
O
-OTf
N
O
O
Zn,HCOONH4
98 %
Cl
Cl
S
O
O
Cl3C
O
+-O
O
O
O
S
S
NH4
O
O
Cl
O
13
39 %
14
6 (1.5 equ.)
O
O
Scheme 4. Preparation of ZEN-14-sulfate (4) and ZEN-14,16-disulfate (14) using sulfuryl imidazolium salt 6 and an optimized procedure for the sulfation of ZEN. Reductive
conditions (Zn, HCOONH4) were applied for the cleavage of the 2,2,2-trichloroethyl group.

H. Mikula et al. / Tetrahedron Letters 54 (2013) 3290–3293
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