Sulfation of deoxynivalenol, its acetylated derivatives, and T2-toxin

Article abstract of DOI:10.1016/j.tet.2014.05.064

The synthesis of several sulfates of trichothecene mycotoxins is presented. Deoxynivalenol (DON) and its acetylated derivatives were synthesized from 3-acetyldeoxynivalenol (3ADON) and used as substrate for sulfation in order to reach a series of five different DON-based sulfates as well as T2-toxin-3-sulfate. These substances are suspected to be formed during phase-II metabolism in plants and humans. The sulfation was performed using a sulfuryl imidazolium salt, which was synthesized prior to use. All protected intermediates and final products were characterized via NMR and will serve as reference materials for further investigations in the fields of toxicology and bioanalytics of mycotoxins.

Full text of DOI:10.1016/j.tet.2014.05.064

Accepted Manuscript
Sulfation of deoxynivalenol, its acetylated derivatives and T2-toxin
Philipp Fruhmann , Philipp Skrinjar , Julia Weber , Hannes Mikula , Benedikt Warth ,
Michael Sulyok , Rudolf Krska , Gerhard Adam , Erwin Rosenberg , Christian
Hametner , Johannes Fröhlich
PII:
S0040-4020(14)00758-3
10.1016/j.tet.2014.05.064
TET 25613
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 16 January 2014
Revised Date: 8 May 2014
Accepted Date: 20 May 2014
Please cite this article as: Fruhmann P, Skrinjar P, Weber J, Mikula H, Warth B, Sulyok M, Krska R,
Adam G, Rosenberg E, Hametner C, Fröhlich J, Sulfation of deoxynivalenol, its acetylated derivatives
and T2-toxin, Tetrahedron (2014), doi: 10.1016/j.tet.2014.05.064.
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Sulfation of deoxynivalenol, its acetylated derivatives and T2-toxin
a, ∗
a
a
a
b
Philipp Fruhmann , Philipp Skrinjar , Julia Weber , Hannes Mikula , Benedikt Warth , Michael
b
b
c
d
a
Sulyok , Rudolf Krska , Gerhard Adam , Erwin Rosenberg , Christian Hametner and Johannes
a
Fröhlich
a
Institute of Applied Synthetic Chemistry, Vienna University of Technology, Getreidemarkt 9/163-OC, A-1060 Vienna, Austria
University of Natural Resources and Life Sciences, Vienna (BOKU), Dept. for Agrarbiotechnology (IFA-Tulln), Center for Analytical Chemistry,
b
Konrad Lorenz Str. 20, A-3430 Tulln, Austria
c
University of Natural Resources and Life Sciences, Vienna (BOKU), Dept. of Applied Genetics and Cell Biology, Konrad Lorenz Str. 20, A-3430 Tulln,
Austria
d
Institute of Chemical Technologies and Analytics, Vienna University of Technology, Getreidemarkt 9/164, A-1060 Vienna, Austria

1
ACCEPTED MANUSCRIPT
Tetrahedron
journal homepage: www.elsevier.com
Sulfation of deoxynivalenol, its acetylated derivatives and T2-toxin
a, ∗
a
a
a
b
Philipp Fruhmann , Philipp Skrinjar , Julia Weber , Hannes Mikula , Benedikt Warth , Michael
b
b
c
d
a
a
Sulyok , Rudolf Krska , Gerhard Adam , Erwin Rosenberg , Christian Hametner and Johannes Fröhlich
a
Institute of Applied Synthetic Chemistry, Vienna University of Technology, Getreidemarkt 9/163-OC, A-1060 Vienna, Austria
University of Natural Resources and Life Sciences, Vienna (BOKU), Dept. for Agrarbiotechnology (IFA-Tulln), Center for Analytical Chemistry,
b
Konrad Lorenz Str. 20, A-3430 Tulln, Austria
c
University of Natural Resources and Life Sciences, Vienna (BOKU), Dept. of Applied Genetics and Cell Biology, Konrad Lorenz Str. 20, A-3430 Tulln, Austria
Institute of Chemical Technologies and Analytics, Vienna University of Technology, Getreidemarkt 9/164, A-1060 Vienna, Austria
d
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
The synthesis of several sulfates of trichothecene mycotoxins is presented. Deoxynivalenol
(DON) and its acetylated derivatives were synthesized from 3-acetyldeoxynivalenol (3ADON)
and used as substrate for sulfation in order to reach a series of five different DON-based sulfates
as well as T2-toxin-3-sulfate. These substances are suspected to be formed during phase-II
metabolism in plants and humans. The sulfation was performed using a sulfuryl imidazolium
salt which was synthesized prior to use. All protected intermediates and final products were
characterized via NMR and will serve as reference materials for further investigations in the
fields of toxicology and bioanalytics of mycotoxins.
Available online
Keywords:
Deoxynivalenol
T2-toxin
Masked Mycotoxins
Sulfation
2
009 Elsevier Ltd. All rights reserved.
Trichothecenes
1
. Introduction
Although these limits cover the toxins themselves, there is still
a lack of information regarding the occurrence and toxicity of
their conjugated forms. DON, for example, can undergo
glycosylation during late phase-II metabolism in the plant to end
up as DON-3-β-D-O-glucoside, which is classified as masked
Deoxynivalenol (DON), its acetylated derivatives which occur
as 3-acetyldeoxynivalenol (3ADON), 15-acetyldeoxynivalenol
15ADON) and 3,15-diacetyldeoxynivalenol (3,15-diADON) as
(
well as T2-toxin (Fig. 1) are very common and widespread
trichothecene mycotoxins. They are predominantly produced by
different Fusarium species and can contaminate food and feed.
They are known to act as a protein biosynthesis inhibitor ,
neurotoxin, immunosuppressive or nephrotoxin and can cause
acute and chronic symptoms after uptake. Based on this
knowledge, regulatory limits regarding the toxin concentration
for food and feed were established to minimize the daily uptake.
5
mycotoxin . A similar conjugation leads to the corresponding T2-
6
toxin glucoside which was recently discovered . These masking
mechanisms can also lead to di- and triglycosides, which are
difficult to investigate due to a lack of authentic reference
standards. The main concern regarding these masked forms is the
fact that the occurring conjugates can be cleaved in the stomach
after uptake, whereby releasing the parent toxin. In addition to
the stomach, this cleavage could also occur within the process of
1,2
3
4
7
malting , leading to an increase of free DON. Besides the
formation of glycosides, other masking processes like sulfation
or thia-michael addition can take place. Zearalenone is another
prominent mycotoxin produced by Fusarium species and its
8
9
metabolite zearalenone-14-sulfate was previously synthesized
in order to serve as reference material for contamination and
toxicity studies. Additionally, the synthesis of different
10
Alternaria toxin sulfates was recently published showing the
emerging research interest within the field of mycotoxin sulfur
conjugates. Besides the occurrence of different masked
mycotoxins in plants, many parent toxins also undergo phase-II
Fig. 1. Structures of DON, its derivatives and T2-toxin.
—
——
∗
Corresponding author. e-mail: philipp.fruhmann@tuwien.ac.at
Tel.: +43-1-58801-163737; fax: +43-1-58801-15499

2
Tetrahedron
eir MANIn
metabolism in living organisms, leading
A
m
C
ai
C
nlyEPtoTEth
D
U
a
S
dd
C
iti
R
on
I
,
P
a very mild protocol using sulfuryl imidazolium
T
11
12,13
corresponding glucuronides but also to their sulfates
Besides the occurring glucuronides, which are accessible and
were already used for the successful development of a biomarker
.
salts was recently employed in the synthesis of trichloroethyl-
protected sulfates on carbohydrates and offered a good possibility
of the sulfation of the fragile trichothecene scaffold.
1
4
15-17
method
for human deoxynivalenol exposure estimation, little
2
. Results and discussion
is known about possible occurring trichothecene sulfates.
Considering that sulfate conjugates are described for different
substance classes like steroids , pollutants and drugs , we
assume that there might be even more trichothecene-derived
sulfates occurring during metabolism.
26
After synthesis of the proper sulfuryl imidazolium salt
18
19
20,21
(Scheme 2a, 26), the method evaluation was done by the use of a
27
simple mimic (Scheme 2b, 27), which was selected because of
an incorporated primary alcohol group, a labile acetic ester group
(like T2-toxin) and its UV activity.
The objective of this work was the development of a reliable
synthetic strategy for the sulfation of trichothecenes to access all
possible DON-sulfates incorporating their acetylated derivatives
and T2-toxin-3-sulfate. Since the reactivity of the three hydroxyl
groups in deoxynivalenol is somewhat different in the order 15 >
3
>> 7 (Fig. 2), we expected all kinds of 3- and 15-sulfates to
occur. However, we were interested in evaluating the possibility
to obtain also some C7-sulfates.
Fig. 2. Structural numbering of trichothecenes.
2
2
3
ADON was isolated and crystallized from an extract of F.
graminearum and was directly used after comparison with a
ADON standard. Deprotection and the following acetylation of
3
23
DON led us to the four desired structures which we wanted to
utilize for sulfation (Scheme 1) to access a series of different
sulfates.
Scheme 2. Synthesis of the protected sulfate donor (A, 26) and method
evaluation with a simple mimic (B, 27). Conditions: (a) Pyridine, SO Cl , -
2
2
24
80°C to RT, 24 h, 97%, (b) 2-methyl imidazole, 2 h, 99%, (c) Et
MeOTf, 6 h, 94%, (d), 1,2-dimethylimidazole, 0°C, DCM, 24 h, 82%, (e)
MeOH, HCOONH , Zn, 20 min, 81%, (f) MeOH, NaOMe, 1 h, 42%.
2
O, 0°C,
Regarding sulfation itself, numerous synthetic methods can
be found within the literature mainly based on the application of
commercially available sulfur trioxide complexes. Since these
methods are limited concerning possible chemical modifications
after installation of the sulfate group as well as yield,
reproducibility and regioselectivity, several newer protective
groups for the sulfation of organic molecules are described in
literature. Besides the fact that we already made good
experiences in our group regarding the separation of
intermediates and yields by the use of 2,2,2-trichloroethyl (TCE)
protective groups within chemical sulfation and glycosylation
reactions, the use of this protective group also offers the
possibility of mild deprotection conditions. The cleavage could
therefore be done via catalytic transfer hydrogenation (Pd/C) or
via different mild reductive methods including ammonium
formate or Zn/ammonium formate.
4
The reaction of 26 and 27 to 28 proceeds smoothly if freshly
sublimed dimethylimidazole is used. The deprotection toward 29
was done in an ultrasonic bath at RT, which shortened the
reaction time to 20 minutes. This is a crucial achievement since
trichothecenes often own labile ester groups which are rapidly
cleaved in different solvents. The deprotection toward 30 was
done via NaOMe and was carried out as a proof of concept
regarding the deprotection of acetylated sulfates.
25
The reactions toward the different acetylated DON derivates
23
(
Scheme 3) were carried out as described in literature and led to
all desired products.
Scheme 1. Synthetic strategies toward all possible sulfate conjugates.
S=sulfation, D=deprotection, deAc=deacetylation.

3
ACCEPTED MANUSCRIPT
Scheme 3. Synthesis of different deoxynivalenol derivatives. Conditions:
a) NaOMe, MeOH, 2 h, 94%, (b) Ac O, DMAP, 18 h, 47% for 3, 23% for 4,
sum=70%.
(
2
The sulfation of 1 yielded (as expected) only one product,
which was identified as the protected 3ADON-15-sulfate, and
sulfation of 3 yielded only the protected 15ADON-3-sulfate.
Both substances were successfully deprotected to the free sulfates
as ammonium salt. In case of 4 no reaction was observed at all,
even with four equivalents of 26 and prolonged reaction time
(Scheme 4).
Scheme 5. Synthesis of DON-3-sulfate and DON-3,15-disulfate as their
ammonium salts. Conditions: (a) DCM, 4 eq. 1,2-dimethylimidazole, 2 eq.
2
6, 0°C, 18 h, 24% for 9, 30% for 8, (b) MeOH, 18 eq. HCOONH
h, 54%, (c) MeOH, 9 eq. HCOONH , 3 eq. Zn, 30 min, 98%.
4
, 6 eq. Zn,
2
4
Position 7 was not sulfated during any reaction. Of the
remaining five theoretically possible sulfates, only DON-15-
sulfate was not isolated after direct sulfation (Scheme 1). For this
reason we used deacetylation of 3ADON-15-sulfate (Scheme 6)
to complete the set of sulfates.
Scheme 6. Synthesis of DON-15-sulfate as ammonium salt. Conditions:
(a) MeOH, NaOMe, 2 h, 69%.
Having proven that the method is well working for
trichothecenes, we aimed also for the sulfation of T2-toxin,
which was carried out in a similar way, whereby leading to the
corresponding 3-sulfate as ammonium salt (Scheme 7).
Scheme 4. Synthesis of different acetyldeoxynivalenol-derived sulfates.
Conditions: (a) DCM, 2.5 eq. 1,2-dimethylimidazole, 1.25+0.75 eq. 26, 0°C,
1
2
8 h, 52%, (b) MeOH, 9 eq. HCOONH
.7 eq. 1,2-dimethylimidazole, 1.35 eq. 26, 0°C, 18 h, 34%, (d) MeOH, 9 eq.
, 3 eq. Zn, 20 min, 89%.
4
, 3 eq. Zn, 20 min, 72%, (c) DCM,
HCOONH
4
In case of 2 we isolated protected DON-3,15-disulfate
surprisingly together with DON-3-sulfate instead of the expected
1
5-sulfate (Scheme 5). Additionally, these two products were the
only ones which were isolated. In general, we were expecting
position 7 to be somewhat unreactive due to steric hindrance and
poor nucleophilicity. Nevertheless, since acetylation resulted in a
2
:1 mixture of the 15- to the 3-product, we also expected a
Scheme 7. Synthesis of T2-toxin-3-sulfate as ammonium salt. Conditions:
similar regioselectivity and ratio of the sulfation reaction. The
reaction itself has not undergone any decomposition (observed
via TLC) during workup, so we could also exclude the theory of
degradation from the disulfate species toward the monosulfate.
Deprotection of both substances yielded DON-3-sulfate as
ammonium salt and DON-3,15-disulfate as the corresponding
diammonium salt.
(
a) DCM, 4 eq. 1,2-dimethylimidazole, 2 eq. 26, 0°C, 18 h, 49%, (b) MeOH,
9
eq. HCOONH , 3 eq. Zn, 1 h, 29%.
4
Finally, all isolated protected intermediates as well as all
1
ammonium sulfates were characterized via NMR, and H shifts
28,29
were assigned using several NMR references
for DON and its

4
Tetrahedron
1
derivatives. For the assignment of the H shi
A
fts
C
o
C
f T
E
2
P
-to
T
xin-3- MApu
E
D
N
rit
U
y
S
ch
C
ec
R
k.
I
All other chemicals were purchased from ABCR
P
T
sulfate, COSY and HMBC spectra were recorded. We tried to
purify all intermediates very quickly over short columns (10–15
g silica), since we have encountered deacetylation of all types of
trichothecenes during column chromatography (silica as well as
RP). Therefore, we ended up with traces of 1,2-
dimethylimidazole within the protected intermediates. After the
deprotection step, column chromatography was done with very
polar solvents (DCM:MeOH:NH OH=10:4:1 or 10:2.5:0.5),
which led to HCOONH impurities in our products. Because of
this we usually made a second column chromatography, followed
by lyophilization, and continued drying for several days to
(Germany) and Sigma Aldrich (Austria/Germany).
4
.2. 2,2,2-Trichloroethyl 2-(4-acetoxyphenyl)ethylsulfate (28)
7 (121.8 mg, 0.68 mmol, 1.0 eq.) was dissolved in 3 mL
2
DCM and a solution of 1,2-dimethylimidazole (194.9 mg, 2.03
mmol, 3.0 eq.) in 1 mL DCM was added at 0°C. Then, 26 (464.0
mg, 1.01 mmol, 1.5 eq.) was added in one portion and the
reaction was allowed to warm to room temperature over night.
After TLC indicated full conversion the reaction was directly
purified via column chromatography (hexane:EtOAc = 1:1) to
4
4
1
yield 28 (215.8 mg, 82%) as white solid. H NMR (200 MHz,
CDCl ) δ 7.28 (s, 4H), 4.82 (s, 2H), 4.27 (t, J=6.8 Hz, 2H), 2.95
1
remove remaining HCOONH . All NMR spectra and
chemical shift assignments can be found in the supporting
information.
H
4
3
13
(t, J=6.8 Hz, 2H), 2.02 (s, 3H); C NMR (50 MHz, CDCl ) δ
3
1
71.0 (s, 1C), 148.9 (s, 1C), 138.1 (s, 1C), 130.6 (d, 2C), 121.2
(
d, 2C), 92.5 (s, 1C), 80.5 (t, 1C), 64.5 (t, 1C), 34.5 (t, 1C), 21.0
3
. Conclusion
+
+
(q, 1C). HRMS m/z calcd for C H O SCl [M+H] , 390.9571,
12 14 6 3
found 390.9578.
Considering the proven unreactivity of the C7 position to
chemical sulfation, we have synthesized, isolated and
characterized all possible DON-sulfates including its acetylated
derivatives. In case of T2-toxin we were also able to synthesize
the desired T2-toxin-3-sulfate. Therefore, we have proven that
the utilized method is well working for trichothecenes and
provides a good way to access the class of trichothecene sulfates
via sulfation of the parent toxins including their acetylated
derivatives. Separation of the protected intermediates was done
using column chromatography, followed by fast deprotection by
4
.3. 2-(4-Acetoxyphenyl)ethylsulfate, ammonium salt (29)
28 (190.0 mg, 0.49 mmol, 1.0 eq.) was dissolved in 5 mL
MeOH. Ammonium formate (276.5 mg, 4.39 mmol, 9.0 eq.) and
Zn dust (95.6 mg, 1.46 mmol, 3.0 eq.) were added and the
reaction was placed in an ultrasonic bath. After 20 minutes, TLC
revealed complete conversion of the starting material and the
reaction mixture was filtered through celite and concentrated to 1
mL. Direct purification of this solution using column
the use of Zn/HCOONH within an ultrasonic bath. All products
chromatography (DCM:MeOH:NH OH = 10:2.5:0.5) yielded 29
4
4
1
13
1
and intermediates were characterized by H and C NMR, and
(109.5 mg, 81%) as white solid. H NMR (200 MHz, methanol-
1
+
all H chemical shifts were assigned to the substances. The
d ) δ = 7.22 (b, 4H), 4.93 (b, NH , H O), 4.23 (t, J=6.9, 2H),
4
4
2
1
13
gathered H NMR information will serve as a valuable reference
2.91 (t, J=6.9 Hz, 2H), 2.00 (s, 3H); C NMR (50 MHz,
for naturally isolated material. Finally, all standards will be used
for identification and quantification of their occurrence and
formation within human and plant metabolism.
methanol-d ) δ 172.9 (s, 1C), 152.5 (s, 1C), 136.0 (s, 1C), 130.6
4
(d, 2C), 122.5 (d, 2C), 66.2 (t, 1C), 35.3 (t, 1C), 20.8 (q, 1C).
-
-
HRMS m/z calcd for C H O S [M-NH ] , 259.0282, found
10
11
6
4
2
59.0278.
.4. 2-(4-Hydroxyphenyl)ethylsulfate, ammonium salt (30)
9 (69.0 mg, 0.25 mmol, 1.00 eq.) was dissolved in 2 mL dry
4
. Experimental section
4
4
.1. General remarks
2
CAUTION: All used toxins are strong protein biosynthesis
MeOH and NaOMe (14.1 mg, 0.26 mmol, 1.05 eq.) was added.
Since no reaction occurred after 30 minutes we added the same
amount of NaOMe again. After stirring for 30 minutes, TLC
indicated full conversion and the reaction was directly purified
inhibitors and can cause a series of acute and chronic symptoms.
Therefore, we strongly recommend considering their toxicity
within all reactions!
via column chromatography (DCM:MeOH:NH OH = 10:2.5:0.5)
4
All reactions were carried out under an argon atmosphere and
the progress of all reactions was monitored using thin-layer
chromatography (TLC) over silica gel 60 F254 (Merck, Germany).
All chromatograms were visualized by heat staining using ceric
ammonium molybdate/Hanessian’s stain in ethanol/sulfuric
acid. Chromatographic separation was done on silica gel 60 (40–
1
to yield 9 (28.7 mg, 42%) as white solid. H NMR (200 MHz,
+
methanol-d ) δ = 7.21 (s, 4H), 4.90 (b, NH , H O), 3.73 (t,
4
4
2
13
J=6.9, 2H), 2.80 (t, J=6.9 Hz, 2H); C NMR (50 MHz,
30
methanol-d ) δ 152.3 (s, 1C), 137.0 (s, 1C), 130.6 (d, 2C), 122.5
4
-
(
[
d, 2C), 64.2 (t, 1C), 39.5 (t, 1C). HRMS m/z calcd for C H O S
M-NH ], 217.0176, found 217.0174.
8
9
5
+
4
6
3 µm) using a SepacoreTM Flash System (Büchi, Switzerland)
or glass columns. All samples were measured via LC-ESI-
MS/MS and LC-APCI-MS/MS and in a negative ionization
mode. These measurements were performed on an HCT ion trap
4
.5. 15-ADON (3) and 3,15-diADON (4)
3
-ADON (1) (95.6 mg, 0.28 mmol, 1.0 eq.) was dissolved in 5
mass spectrometer (Bruker, Germany). A TLC-MS interface
mL methanol, followed by the addition of NaOMe (13.7 mg, 0.25
mmol, 0.9 eq.). After 2 h, TLC showed full conversion of the 1.
The reaction was concentrated to 1 mL and directly purified by
1
(
Camag, Germany) was used for ESI-MS analysis after TLC. H
13
and C spectra were recorded upon using a Bruker DPX-200
spectrometer as well as an Avance DRX-400 MHz spectrometer
the use of column chromatography (CHCl :MeOH = 9:1), which
3
(
both Bruker, Germany). Data were recorded and evaluated using
yielded deoxynivalenol (2, 79.0 mg, 94%) as white solid. The
reaction product was proven to be identical to an authentic
sample by TLC and, thus, was directly used for acetylation. For
this purpose, 2 (79.0 mg, 0.27 mmol) was dissolved in 50 mL dry
dichloromethane. Pyridine (1 mL) and 4-DMAP (app. 10 mg)
were added, followed by the dropwise addition of acetic
anhydride (27.2 mg, 0.27 mmol). The reaction was stirred over
night, treated with 20 mL 2N HCl, and extracted with
dichloromethane. After drying with Na SO , filtration and
TOPSPIN 1.3 (Bruker Biospin). All chemical shifts are given in
ppm relative to tetramethylsilane. The calibration was done using
31
residual solvent signals . Multiplicities are abbreviated as s
singlet), d (doublet), t (triplet), q (quartet) and b (broad signal).
-ADON was obtained from BOKU, Dept. for
(
3
Agrarbiotechnology (IFA-Tulln) as crude fermentation extract,
purified via column chromatography, and used after the H NMR
1
2
4

5
evaporation of the solvent, the remaining resid
A
ue
C
w
C
as
E
s
P
ub
T
je
E
ct
D
ed MA(d
N
, 1
U
C)
S
,
C
80
R
.0
I
(t, 1C), 78.9 (d, 1C), 73.5 (d, 1C), 70.1 (d, 1C),
P
T
to column chromatography (CHCl :MeOH = 95:5) to yield 3
64.5 (s, 1C), 62.0 (t, 1C), 51.2 (s, 1C), 47.4 (t, 1C), 46.1 (s, 1C),
3
(
=
42.0 mg, 47%) and 4 (23.5 mg, 23%) as white solid. Total yield
70%, 93% conversion. 15-ADON (3): H NMR (200 MHz,
40.2 (t, 1C), 20.8 (q, 1C), 15.4 (q, 1C), 13.7 (q, 1C); HRMS m/z
1
+
+
calcd for C H O SCl [M+H] , 549.0150, found 549.0146.
19 24 10 3
CDCl ) δ 6.61 (dq, J=5.7, 1.6 Hz, 1H), 4.89 (d, J=5.7 Hz, 1H),
3
4
.8. 2,2,2-Trichloroethyl DON-3-sulfate (8) and bis(2,2,2-
4
2
.83 (d, J=1.6 Hz, 1H), 4.52 (dt, J=10.2, 4.7 Hz, 1H), 4.24 (s,
H), 3.78 (d, J=1.8 Hz, 1H), 3.63 (d, J=4.5 Hz, 1H), 3.13 (d,
trichloroethyl) DON-3,15-disulfate (9)
J=4.3 Hz, 1H), 3.08 (d, J=4.3 Hz, 1H), 2.22 (dd, J=14.8, 4.7 Hz,
2
(40.6 mg, 137 µmol, 1.0 eq.) was dissolved in 5 mL of DCM
1
1
1
6
1
H), 2.08 (dd, J=14.7, 10.4 Hz, 1H), 1.88 (s, 3 H), 1.87 (s, 3H),
and cooled to 0°C and 1,2-dimethylimidazole (52.7 mg, 548
µmol, 4.0 eq.) in 1 mL DCM was added to the reaction. Then, 26
13
.07 (s, 3H); C NMR (50 MHz, CDCl ) δ = 199.6 (s), 170.3 (s),
3
38.8 (d), 135.6 (s), 80.7 (d), 73.5 (d), 70.1 (d), 68.9 (s), 65.5 (s),
(125.4 mg, 274 µmol, 2.0 eq.) was added and the reaction was
2.2 (t), 51.4 (s), 47.4 (t), 46.3 (s), 43.3 (t), 20.7 (q), 15.4 (q),
allowed to reach room temperature over night. TLC after 24 h
showed nearly full conversion of the starting material and the
reaction was directly used for column chromatography
1
3.8 (q). 3,15-diADON (4): H NMR (200 MHz, CDCl ) δ 6.56
3
(
dq, J=5.8, 1.4 Hz, 1H), 5.20 (dt, J=10.9, 4.6 Hz, 1H), 4.80 (d,
J=2.0 Hz, 1H), 4.69 (d, J=5.8 Hz, 1H), 4.27 (d, J=12.1 Hz, 1H),
.20 (d, J=12.1 Hz, 1H), 3.89 (d, J=4.3 Hz, 1H), 3.80 (d, J=2.0
Hz, 1H), 3.14 (d, J=4.3 Hz, 1H), 3.09 (d, J=4.3 Hz, 1H), 2.31
(DCM:MeOH gradient from 100:0→95:5), yielding 8 (22.4 mg,
1
4
3
0%) and 9 (23.9 mg, 24%) as white solid. H NMR of 8 (200
MHz, CDCl ) δ 6.62 (dq, J=5.9, 1.4 Hz, 1H), 5.31 (dt, J=11.2,
3
(
3
dd, J=15.2, 4.8 Hz, 1H), 2.15 (dd, J=15.2, 10.9 Hz, 1H), 2.12 (s,
H), 1.88 (s, 3 H), 1.87 (s, 3H), 1.08 (s, 3H); C NMR (50 MHz,
4
.4, 1H), 4.81 (s, 1H), 4.78 (s, 2H), 4.74 (d, J=5.9 Hz, 1H), 3.99
d, J=4.5 Hz, 1H), 3.86 (d, J=11.5 Hz, 1H), 3.77 (b, 1H), 3.76 (d,
J=11.5 Hz, 1H), 3.22 (d, J=4.1 Hz, 1H), 3.14 (d, J=4.1 Hz, 1H),
13
(
CDCl ) δ 199.3 (s), 170.3 (s), 170.2 (s), 138.4 (d), 135.6 (s), 78.9
3
(
(
d), 73.4 (d), 71.1 (d), 70.1 (d), 64.9 (s), 62.1 (t), 51.5 (s), 47.4
t), 45.8 (s), 40.3 (t), 21.0 (q), 20.6 (q), 15.3 (q), 13.6 (q).
2
1
.80 (dd, J=15.7, 4.3 Hz, 1H), 2.25 (dd, J=15.7, 11.2 Hz, 1H),
13
.91 (b, 3H), 1.71 (b, 1H), 1.17 (s, 3H); C NMR of 8 (50 MHz,
CDCl ) δ 199.6 (s, 1C), 138.0 (d, 1C), 136.3 (s, 1C), 92.7 (s, 1C,
3
4
.6. 2,2,2-Trichloroethyl 3-acetyl-DON-15-sulfate (6)
-
CCl tiny signal!), 80.8 (d, 1C), 79.9 (t, 1C), 79.1 (d, 1C), 74.5
3
1
(45.1 mg, 133 µmol, 1.0 eq.) was dissolved in 2.5 mL of
(d, 1C), 70.1 (d, 1C), 64.8 (s, 1C), 62.0 (t, 1C), 51.8 (s, 1C), 47.7
(t, 1C), 46.1 (s, 1C), 40.3 (t, 1C), 15.4 (q, 1C), 14.1 (q, 1C);
DCM and cooled to 0°C and 1,2-dimethylimidazole (32.0 mg,
33 µmol, 2.5 eq.) in 1 mL DCM was added to the reaction.
-
+ -
3
HRMS m/z calcd for C17
504.9878. H NMR of 9 (200 MHz, CDCl
H
20
O
9
SCl
3
[M-H ] , 504.9899, found
) δ 6.69 (dq, J=5.9, 1.5
1
Then, 26 (76.2 mg, 167 µmol, 1.25 eq.) was added and the
reaction was allowed to reach room temperature over night. Since
TLC showed remaining starting material after 18 h, the reaction
was cooled again to 0°C and another 0.75 eq. of 26 (45.7 mg, 100
µmol) were added. After another 48 hours, TLC showed still
starting material, but also the formation of substantial amounts of
product. The reaction was directly used for column
3
Hz, 1H), 5.32 (dt, J=11.1, 4.4, 1H), 4.88 (s, 1H), 4.81 (dt, J=5.7,
1.5 Hz, 1H), 4.79 (s, 2H), 4.66 (d, J=11.0 Hz, 1H), 4.60 (d,
J=11.0 Hz, 1H), 4.48 (s, 2H), 4.04 (d, J=4.4 Hz, 1H), 3.83 (b,
1H), 3.21 (d, J=4.1 Hz, 1H), 3.17 (d, J=4.1 Hz, 1H), 2.57 (dd,
J=15.7, 4.2 Hz, 1H), 2.33 (dd, J=15.7, 11.1 Hz, 1H), 1.93 (b,
13
3H), 1.15 (s, 3H); C NMR of 9 (50 MHz, CDCl ) δ 198.5 (s,
3
chromatography (CHCl :MeOH = 95:5), yielding 6 (37.8 mg,
1C), 138.2 (d, 1C), 136.6 (s, 1C), 2 x 80.0 (d, 1C/t, 1C), 79.9 (t,
1C), 78.9 (d, 1C), 73.3 (d, 1C), 71.5 (t, 1C), 69.2 (d, 1C), 64.3 (s,
1C), 51.3 (s, 1C), 47.7 (t, 1C), 46.2 (s, 1C), 40.3 (t, 1C), 15.3 (q,
3
1
5
2%) as white solid. H NMR (200 MHz, CDCl ) δ 6.66 (dq,
3
J=5.9, 1.4 Hz, 1H), 5.24 (ddd, J=9.5, 6.0, 4.5, 1H), 4.89 (d, J=1.4
Hz, 1H), 4.80 (d, J=5.9 Hz, 1H), 4.64 (d, J=10.8 Hz, 1H), 4.57
1C), 13.6 (q, 1C), 2 x CCl between 92 and 93 ppm are missing,
but the corresponding CH groups are located at 80.0 and 79.9;
2
3
(
1
d, J=10.8 Hz, 1H), 4.56 (d, J=10.6 Hz, 1H), 4.43 (d, J=10.6 Hz,
H), 3.95 (d, J=4.5 Hz, 1H), 3.84 (d, J=1.4 Hz, 1H), 3.16 (d,
J=4.1 Hz, 1H), 3.13 (d, J=4.1 Hz, 1H), 2.10–2.36 (m, 2H), 2.16
-
-
HRMS m/z calcd for C17H O S Cl [M-TCE] , 584.9467, found
20 12 2 3
584.9505.
13
(s, 3H), 1.91 (b, 3H), 1.11 (s, 3H); C NMR (50 MHz, CDCl ) δ
3
4
.9. General deprotection procedure
1
1
1
1
98.8 (s, 1C), 170.2 (s, 1C), 138.7 (d, 1C), 136.1 (s, 1C), 92.4 (s,
C), 79.7 (t, 1C), 78.8 (d, 1C), 73.0 (d, 1C), 71.6 (t, 1C), 70.7 (d,
C), 69.1 (d, 1C), 64.6 (s, 1C), 51.3 (s, 1C), 47.4 (t, 1C), 45.8 (s,
C), 40.4 (t, 1C), 20.9 (q, 1C), 15.2 (q, 1C), 13.5 (q, 1C); HRMS
The protected intermediate was dissolved in MeOH (1 mL/10
µmol starting material). HCOONH (3 eq.) as well as Zn dust (9
4
eq.) were added and the reaction was placed in an ultrasonic bath
at room temperature. The reaction was followed via TLC until
substantial amounts of products were formed (20 to 90 min).
After filtration through celite the remaining residue was
subjected to column chromatography to end up with the
corresponding sulfates as ammonium salts. For all acetylated
+
+
m/z calcd for C H O SCl
[M+H] , 549.0150, found
19
24 10
3
5
4
49.0160.
.7. 2,2,2-Trichloroethyl 15-acetyl-DON-3-sulfate (11)
(76.5 mg, 226 µmol, 1.0 eq.) was dissolved in 3 mL of DCM
3
and cooled to 0°C and 1,2-dimethylimidazole (58.7 mg, 610
µmol, 2.7 eq.) in 1 mL DCM was added to reaction. Then 26
DON derivatives and T2-toxin, DCM:MeOH:NH
was used for purification. In case of DON-3- and 15-sulfate and
3,15-disulfate, a mixture of DCM:MeOH:NH OH = 10:4:1 was
used. Since all products contained accompanying HCOOHNH ,
4
we tried to purify some products via a second and third column
chromatography as well as via lyophilization. Nevertheless, we
obtained all desired products as a white misty veil.
4
OH=10:2.5:0.5
(139.7 mg, 305 µmol, 1.35 eq.) was added and the reaction was
4
allowed to reach room temperature over night. TLC showed
substantial amounts of product and the reaction was directly used
for column chromatography (DCM:MeOH = 95:5) yielding 11
1
(
42.6 mg, 34%) as white solid. H NMR (200 MHz, CDCl ) δ
3
6
1
1
1
.60 (dq, J=5.8, 1.5 Hz, 1H), 5.31 (dt, J=11.1, 4.3, 1H), 4.81 (s,
H), 4.79 (s, 2H), 4.70 (d, J=5.8 Hz, 1H), 4.27 (d, J=12.1 Hz,
H), 4.18 (d, J=12.1 Hz, 1H), 4.00 (d, J=4.3 Hz, 1H), 3.71 (s,
H), 3.21 (d, J=4.1 Hz, 1H), 3.15 (d, J=4.1 Hz, 1H), 2.64 (dd,
4
.10. 3-Acetyl-DON-15-sulfate, ammonium salt (13)
Following the general deprotection procedure, 6 (34.0 mg, 62
1
µmol) was converted into 13 (19.3 mg, 72%). H NMR (200
MHz, methanol-d ) δ 6.63 (dq, J=6.1, 1.4 Hz, 1H), 5.11 (dt,
J=11.3, 4.4 Hz, 1H), 4.92 (d, J=6.1 Hz, 1H), 4.89 (s, 1H), 4,87
J=15.7, 4.3 Hz, 1H), 2.29 (dd, J=15.7, 11.1 Hz, 1H), 1.93 (s, 3H),
1
1
4
13
.91 (b, 3H), 1.12 (s, 3H); C NMR (50 MHz, CDCl ) δ 198.9 (s,
3
+
C), 170.2 (s, 1C), 137.9 (d, 1C), 136.1 (s, 1C), 92.7 (s, 1C), 80.4
(b, NH , H O), 4.27 (d, J=11.0 Hz, 1H), 3.94 (d, J=11.1 Hz,
4
2

6
Tetrahedron
13
1
H), 3.85 (d, J=4.5 Hz, 1H), 3.16 (d, J=4.3
A
Hz
C
,
C
1H
E
),
P
3
3
T
.1
E
2
D
(d, MA
C
NU
N
M
S
R
C
(1
R
0
I
0
P
MHz, methanol-d ) δ 201.3 (s, 1C), 139.6 (d, 1C),
4
T
J=4.3 Hz, 1H), 2.79 (dd, J=15.3, 4.3 Hz, 1H), 2.08 (dd, J=15.3,
137.2 (s, 1C), 81.3 (d, 1C), 75.8 (d, 1C), 75.7 (d, 1C), 70.5 (d,
1C), 67.4 (s, 1C), 66.1 (t, 1C), 52.4 (s, 1C), 48.4 (t, 1C), 47.1 (s,
1C), 42.4 (t, 1C), 15.3 (q, 1C), 14.5 (q, 1C); HRMS m/z calcd for
1
1
1.3 Hz, 1H), 2.13 (s, 3H), 1.85 (b, 3H), 1.18 (s, 3H); C NMR
(50 MHz, methanol-d ) δ 201.0 (s, 1C), 172.5 (s, 1C), 139.4 (d,
4
-
+
+ -
1
C), 137.1 (s, 1C), 80.5 (d, 1C), 75.8 (d, 1C), 72.7 (d, 1C), 70.7
C H O S [M-2xNH +H ] , 455.0323, found 455.0300.
15 20 12 2 4
(
(
d, 1C), 67.1 (t, 1C), 66.3 (s, 1C), 52.3 (s, 1C), 48.4 (t, 1C), 46.8
s, 1C), 41.7 (t, 1C), 20.8 (q, 1C), 15.3 (q, 1C), 14.4 (q, 1C);
4
.15. 2,2,2-Trichloroethyl T2-toxin-3-sulfate (21)
-
+ -
HRMS m/z calcd for C H O S [M-NH ] , 417.0861, found
4
17
21 10
4
5 (37.4 mg, 80 µmol, 1.0 eq.) was dissolved in 3 mL of DCM,
17.0834.
cooled to 0°C, and 1,2-dimethylimidazole (30.8 mg, 321 mmol,
.0 eq.) in 1 mL DCM was added to the reaction. Then, 26 (73.4
4
4
.11. 15-Acetyl-DON-3-sulfate, ammonium salt (18)
mg, 160 mmol, 2.00 eq.) was added and the reaction was allowed
to reach room temperature over night. After 18 hours, TLC
showed substantial amounts of product and the reaction was
directly used for column chromatography (DCM:MeOH = 95:5),
Following the general deprotection procedure, 11 (13.1 mg, 24
1
µmol) was converted into 18 (9.2 mg, 89%). H NMR (400 MHz,
methanol-d ) δ 6.65 (dq, J=5.9, 1.5 Hz, 1H), 4.70–5.10 (m, NH ,
C3-H, C7-H, C11-H, H O), 4.30 (d, J=12.1 Hz, 1H), 4.23 (d,
+
4
4
1
yielding 21 (26.4 mg, 49%) as white solid. H NMR (200 MHz,
2
J=12.1 Hz, 1H), 3.82 (d, J=4.5 Hz, 1H), 3.12 (s, 2H), 2.63 (dd,
J=15.3, 4.3 Hz, 1H), 2.11 (dd, J=15.3, 11.2 Hz, 1H), 1.90 (s, 3H),
CDCl ) δ 6.16 (d, J=3.1 Hz, 1H), 5.77 (dt, J=5.7, 1.4 Hz, 1H),
3
5.28 (d, J=5.5 Hz, 1H), 5.10 (dd, J=4.9, 3.1 Hz, 1H), 4.80 (s,
2H), 4.31 (d, J=12.7 Hz, 1H), 4.25 (d, J=5.7 Hz, 1H), 4.10 (d,
J=12.7 Hz, 1H), 3.96 (d, J=4.9 Hz, 1H), 3.09 (d, J=3.9 Hz, 1H),
2.85 (d, J=3.9 Hz, 1H), 2.35 (dd, J=15.2, 6.0 Hz, 1H), 2.11 (s,
3H), 2.09 (s, 3H), 2.00–2.25 (m, 3H), 1.80 (d, J=16.6 Hz, 1H),
13
1
2
1
1
1
.85 (b, 3H), 1.11 (s, 3H); C NMR (100 MHz, methanol-d ) δ
4
01.1 (s, 1C), 171.9 (s, 1C), 139.7 (d, 1C), 137.0 (s, 1C), 81.2 (d,
C), 75.2 (d, 1C), 75.0 (d, 1C), 71.3 (d, 1C), 65.9 (s, 1C), 63.3 (t,
C), 52.6 (s, 1C), 48.2 (t, 1C), 47.0 (s, 1C), 42.5 (t, 1C), 20.6 (q,-
C), 15.4 (q, 1C), 14.4 (q, 1C); HRMS m/z calcd for C H O S
1.76 (s, 3H), 0.96 (d, J=6.3 Hz, 3H), 0.95 (d, J=6.3 Hz, 3H), 0.74
17
21 10
+
-
13
[
M-NH ] , 417.0861, found 417.0824.
(s, 3H); C NMR (50 MHz, CDCl ) δ 172.8 (s, 1C), 170.4 (s,
4
3
1
C), 170.2 (s, 1C), 137.1 (s, 1C), 123.0 (d, 1C), 92.6 (s, 1C), 87.1
4
.12. DON-3-sulfate, ammonium salt (15)
(
(
d, 1C), 80.1 (t, 1C), 78.4 (d, 1C), 77.4 (d, 1C), 67.7 (d, 1C), 67.5
d, 1C), 64.6 (t, 1C), 63.8 (s, 1C), 48.7 & 47.5 (1t, 1s, 2x1C),
Following the general deprotection procedure, 8 (18.8 mg, 37
1
4
2
3.7 (t, 1C), 43.1 (s, 1C), 28.4 (t, 1C), 25.9 (d, 1C), 22.6 (q, 1C),
2.5 (q, 1C), 21.2 (q, 1C), 20.8 (q, 1C), 20.4 (q, 1C), 6.5 (q, 1C);
µmol) was converted into 15 (14.3 mg, 98%). H NMR (400
MHz, methanol-d ) δ 6.61 (dq, J=6.1, 1.4 Hz, 1H), 4.75–4.95 (m,
NH , C3-H, C11-H, H O), 4.79 (s, 1H), 3.80 (d, J=4.4 Hz, 1H),
3
4
-
-
+
HRMS m/z calcd for C H O S [M-TCE-group] , 545.1698,
found 545.1679.
24 33 12
4
2
.78 (d, J=12.3 Hz, 1H), 3.68 (d, J=12.3 Hz, 1H), 3.12 (d, J=4.5
Hz, 1H), 3.09 (d, J=4.5 Hz, 1H), 2.75 (dd, J=15.2, 4.4 Hz, 1H),
4
.16. T2-toxin-3-sulfate, ammonium salt (22)
13
2
.06 (dd, J=15.2, 11.4 Hz, 1H), 1.83 (b, 3H), 1.12 (s, 3H);
C
NMR (100 MHz, methanol-d ) δ 201.7 (s, 1C), 139.4 (d, 1C),
Following the general deprotection procedure, 21 (20.8 mg, 31
µmol) was converted into 22 (5.0 mg, 29%). H NMR (400 MHz,
4
1
1
1
1
37.0 (s, 1C), 81.2 (d, 1C), 75.8 (d, 1C), 75.4 (d, 1C), 71.6 (d,
C), 66.3 (s, 1C), 61.8 (t, 1C), 53.6 (s, 1C), 48.2 (t, 1C), 46.7 (s,
C), 42.5 (t, 1C), 15.4 (q, 1C), 14.6 (q, 1C); HRMS m/z calcd for
methanol-d ) δ 6.03 (d, J=3.1 Hz, 1H), 5.78 (dt, J=5.5 1H), 5.33
4
+
(d, J=5.5 Hz, 1H), 4.80–4.94 (m, NH , C3-H, H O), 4.33 (d,
4
2
-
+ -
C H O S [M-NH ] , 375.0755, found 375.0741.
J=12.1, 1H), 4.32 (d, J=5.5 Hz, 1H), 4.16 (d, J=12.1 Hz, 1H),
.79 (d, J=5.1 Hz, 1H), 3.04 (d, J=3.9 Hz, 1H), 2.87 (d, J=3.9
Hz, 1H), 2.38 (dd, J=15.3, 5.5 Hz, 1H), 2.13–2.18 (m, 2H), 2.07
s, 3H), 2.06 (s, 3H), 2.00–2.12 (m, 1H), 1.92 (d, J=15.3 Hz, 1H),
.74 (s, 3H), 0.97 (d, J=6.7 Hz, 3H), 0.96 (d, J=6.7 Hz, 3H), 0.72
15
19
9
4
3
4
.13. DON-15-sulfate, ammonium salt (14)
(
1
(
(
1
3 (12.0 mg, 28 µmol, 1.0 eq.) was dissolved in 5 mL MeOH
and NaOMe (3.0 mg, 55 mmol, 2.0 eq.) was added. After stirring
for 2 hours, TLC indicated full conversion of the starting material
and the reaction was subjected to column chromatography
13
s, 3H); C NMR (100 MHz, methanol-d ) δ 174.0 (s, 1C), 172.3
4
s, 1C), 172.2 (s, 1C), 137.2 (s, 1C), 125.1 (d, 1C), 82.3 (d, 1C),
8
6
2
2
1.5 (d, 1C), 79.7 (d, 1C), 69.4 (d, 1C), 68.5 (d, 1C), 65.8 (t, 1C),
5.0 (s, 1C), 50.1 & 47.8 (1t, 1s, 2x1C), 44.5 (t, 1C), 44.3 (s, 1C),
8.8 (t, 1C), 26.9 (d, 1C), 22.8 (q, 1C), 22.7 (q, 1C), 21.3 (q, 1C),
0.7 (q, 1C), 20.4 (q, 1C), 6.9 (q, 1C); HRMS m/z calcd for
(
DCM:MeOH:NH OH = 10:4:1), yielding 14 (7.5 mg, 69%) as
4
1
white solid. H NMR (200 MHz, methanol-d ) δ 6.65 (dq, J=6.1,
1
H O), 4.76 (dt, J=11.1, 4.5, 1H), 4.25 (d, J=11.0 Hz, 1H), 3.96
4
+
.4 Hz, 1H), 5.04 (d, J=6.1 Hz, 1H), 4.85–4.95 (m, NH , C7-H,
4
2
-
+ -
C H O S [M-NH ] , 545.1698, found 545.1682.
(d, J=11.0 Hz, 1H), 3.55 (d, J=4.5 Hz, 1H), 3.12 (d, J=4.5 Hz,
24 33 12
4
1
H), 3.06 (d, J=4.5 Hz, 1H), 2.57 (dd, J=14.8, 4.4 Hz, 1H), 1.99
13
Acknowledgements
(dd, J=14.8, 11.1 Hz, 1H), 1.84 (b, 3H), 1.14 (s, 3H); C NMR
(
50 MHz, methanol-d ) δ 201.1 (s, 1C), 139.9 (d, 1C), 136.9 (s,
4
The graduate school program Applied Bioscience Technology
of the VUT (Vienna University of Technology) in cooperation
with the BOKU Vienna is gratefully acknowledged for financial
support. This work was also funded by the Vienna Science and
Technology Fund (WWTF LS12-021) and the Austrian Science
Fund (SFB Fusarium F3702 and F3706).
1
1
1
C), 82.3 (d, 1C), 75.9 (d, 1C), 70.7 (d, 1C), 69.6 (d, 1C), 67.2 (s,
C), 66.7 (t, 1C), 52.5 (s, 1C), 48.2 (t, 1C), 47.5 (s, 1C), 45.0 (t,-
C), 15.3 (q, 1C), 14.4 (q, 1C); HRMS m/z calcd for C H O S
15
19
9
+
-
[
M-NH ] , 375.0755, found 375.0746.
4
4
.14. DON-3,15-disulfate, diammonium salt (16)
Following the general deprotection procedure, twice the
Supplementary Material
amount of HCOONH (6 eq.) and Zn dust (18 eq.) were used to
convert 9 (20.0 mg, 28 µmol) into 16 (7.4 mg, 54%). H NMR
4
1
NMR spectra of all protected and isolated sulfates as well as
(
5
1
400 MHz, methanol-d ) δ 6.67 (dq, J=6.0, 1.5 Hz, 1H), 4.80–
4
+
1
tables for the H chemical shifts of all substances can be found in
.00 (m, NH , C3-H, C7-H, C11-H, H O), 4.24 (d, J=10.9 Hz,
4 2
H), 3.98 (d, J=10.9 Hz, 1H), 3.84 (d, J=4.5 Hz, 1H), 3.15 (d,
the supporting information.
J=4.3 Hz, 1H), 3.10 (d, J=4.3 Hz, 1H), 2.97 (dd, J=15.3, 4.5 Hz,
H), 2.10 (dd, J=15.3, 11.4 Hz, 1H), 1.85 (b, 3H), 1.16 (s, 3H);
1

7
References and notes
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Mikula, H.; Skrinjar, P.; Sohr, B.; Ellmer, D.; Hametner,
C.; Fröhlich, J. Tetrahedron 2013, 69, 10322-10330.
Uhlig, S.; Ivanova, L.; Fæste, C. K. J. Agric. Food Chem.
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013, 61, 2006-2012.
Prelusky, D. B.; Veira, D. M.; Trenholm, H. L.; Foster, B.
C. J. Environ. Sci. Health, B 1987, 22, 125-148.
Wan, D.; Huang, L.; Pan, Y.; Wu, Q.; Chen, D.; Tao, Y.;
Wang, X.; Liu, Z.; Li, J.; Wang, L.; Yuan, Z. J. Agric.
Food Chem. 2013, 62, 288-296.
1
4.
Fruhmann, P.; Warth, B.; Hametner, C.; Berthiller, F.;
Horkel, E.; Adam, G.; Sulyok, M.; Krska, R.; Fröhlich, J.
World Mycotoxin J. 2012, 5, 127-132.
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1
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Warth, B.; Sulyok, M.; Berthiller, F.; Schuhmacher, R.;
Krska, R. Toxicol. Lett. 2013, 220, 88-94.
Warth, B.; Sulyok, M.; Fruhmann, P.; Berthiller, F.;
Schuhmacher, R.; Hametner, C.; Adam, G.; Fröhlich, J.;
Krska, R. Toxicol. Lett. 2012, 211, 85-90.
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7.
Warth, B.; Sulyok, M.; Fruhmann, P.; Mikula, H.;
Berthiller, F.; Schuhmacher, R.; Hametner, C.; Abia, W.
A.; Adam, G.; Fröhlich, J.; Krska, R. Rapid Commun.
Mass Spectrom. 2012, 26, 1533-1540.
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Saudan, C. Steroids 2009, 74, 359-364.
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M. W.; Robertson, L. W. Chem. Res. Toxicol. 2012, 25,
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ACCEPTED MANUSCRIPT
Supplementary Information
Sulfation of deoxynivalenol, its acetylated derivatives and T2-toxin
a, ∗
a
a
a
b
Philipp Fruhmann , Philipp Skrinjar , Julia Weber , Hannes Mikula , Benedikt Warth ,
b
b
c
d
a
Michael Sulyok , Rudolf Krska , Gerhard Adam , Erwin Rosenberg , Christian Hametner
and Johannes Fröhlich
a
a
Institute of Applied Synthetic Chemistry, Vienna University of Technology, Getreidemarkt 9/163-OC, A-1060 Vienna, Austria
University of Natural Resources and Life Sciences, Vienna (BOKU), Dept. for Agrarbiotechnology (IFA-Tulln), Center for Analytical
b
Chemistry,
Konrad Lorenz Str. 20, A-3430 Tulln, Austria
c
University of Natural Resources and Life Sciences, Vienna (BOKU), Dept. of Applied Genetics and Cell Biology, Konrad Lorenz Str. 20, A-
3
430 Tulln, Austria
d
Institute of Chemical Technologies and Analytics, Vienna University of Technology, Getreidemarkt 9/164, A-1060 Vienna, Austria
Table of content
1
Overview: H chemical shifts
Page 2
NMR Spectra mimic
Page 3 – 5
1
13
Compound 28
Compound 29
Compound 30
( H, C)
Page 3
Page 4
Page 4
1
13
( H, C)
1
13
( H, C)
NMR Spectra TCE protected intermediates
Page 6 – 10
1
13
Compound 6
Compound 8
Compound 9
Compound 11
Compound 21
( H, C)
Page 6
Page 7
Page 8
Page 9
Page 10
1
13
( H, C)
1
13
( H, C)
1
13
( H, C)
1
13
( H, C)
NMR Spectra toxin sulfates as their ammonium salts
Page 11 – 17
1
13
Compound 13
Compound 14
Compound 15
Compound 16
Compound 18
Compound 22
Compound 30
( H, C)
Page 11
Page 12
Page 13
Page 14
Page 15
Page 16
Page 17
1
13
( H, C)
1
13
( H, C)
1
13
( H, C)
1
13
( H, C)
1
13
( H, C)
(COSY, HSQC)
∗
Corresponding author. e-mail: philipp.fruhmann@tuwien.ac.at
Tel.: +43-1-58801-163737; fax: +43-1-58801-15499
1

ACCEPTED MANUSCRIPT
Name / Position
2
3
4 α
4 β
7 α
7 β
8 β
10
11
13 a
13 b
14
15 a
15 b
16
3 Ac/OH
7 Ac/OH
15 Ac/OH
3 TCE
15 TCE
DON (2)
in CDCl3
3.63
d [4.5]
4.53
bt
2.23
dd [14.8; 4.6]
2.08
dd [14.9; 10.6]
x
4.84
bs
x
6.62
dq [5.9; 1.4]
4.81
d [5.7]
3.08
d [4.3]
3.17
d [4.3]
1.1
s
3.74
d [11.5]
3.89
d [11.5]
1.9
bs
2.37
d [2.4]
3.86
bs, 1H
1.75
bs, 1H
x
x
DON (2)
in MeOD
3.53
d [4.5]
4.37
dt [11.0; 4.5]
2.39
dd [14.6; 4.5]
1.95
dd [14.6; 11.0]
x
x
x
x
4.72
s
x
x
x
x
6.50
dq [5.9; 1.5]
4.93
d [5.7]
3.05
d [4.3]
3.10
d [4.3]
1.1
s
3.73
3.73
2
bs
x
x
x
x
x
x
x
x
x
x
x
x
x
s, 2H
3
ADON (1)
in CDCl3
3.91
d [4.7]
5.23
dt [11.0; 4.5]
2.38
dd [15.1; 4.5]
2.17
dd [15.1; 11.2]
4.84
d [1.8]
6.61
dq [5.8; 1.5]
4.70
d [5.9]
3.12
d [4.3]
3.18
d [4.3]
1.2
S
3.72 - 3.88
m, 3H
1.90
bs
2.14
s, 3H
3.72 - 3.88
m, 3H
1.67
bt, 1H
1
5 ADON (3)
in CDCl3
3.60
d [4.5]
4.49
dt [9.8; 4.9]
2.20
dd [14.8; 4.8]
2.06
dd [14.8; 10.5]
4.81
bs
6.59
dq [5.9; 1.4]
4.88
d [5.7]
3.06
d [4.3]
3.11
d [4.3]
1.1
s
4.22
bs
4.22
bs
1.9
bs
2.92
bs, 1H
3.8
bs, 1H
1.86
bs
3
,15 diADON (4)
in CDCl3
3.89
d [4.5]
5.19
dt [10.9; 4.6]
2.30
dd [15.2; 4.8]
2.14
dd [15.2; 10.9]
4.79
d [2.0]
6.55
dq [5.7; 1.4]
4.68
d [5.7]
3.08
d [4.3]
3.13
d [4.3]
1.1
s
4.19
d [12.1]
4.27
d [12.1]
1.9
bs
2.11
s, 3H
3.79
d [2.0], 1H
1.86
bs
T2 toxin (5)
in CDCl3
3.64
d [4.9]
4.18
dd [4.9; 2.9]
5.34
d [2.9]
x
1.9-2.2
m
2.38
dd [15.1; 5.4]
5.3
d [5.5]
5.78
dt [5.9; 1.2]
4.35
d [6.3]
2.79
d [3.9]
3.05
d [3.9]
0.8
s
4.06
d [12.7]
4.30
d [12.7]
1.7
bs
2.00 - 2.20
2xs, 2x3H, 6H
x
2.00 - 2.20
2xs, 2x3H, 6H
3
ADON-15-sulfate [p] (6)
in CDCl3
3.95
d [4.5]
5.24
ddd [9.5; 6.0; 4.5]
2.10 - 2.36
m, 2 H
2.10 - 2.36
m, 2 H
x
x
x
x
4.89
d [1.4]
x
x
x
x
6.66
dq [5.9; 1.4]
4.8
d [5.9]
3.13
d [4.1]
3.16
d [4.1]
1.1
s
4.43
d [10.6]
4.56
d [10.6]
1.9
bs
2.16
s, 3H
3.84
d [1.4], 1H
x
x
4.64; 4.57
2 x d [10.8]
15 ADON-3-sulfate [p] (11)
in CDCl3
4.00
d [4.3]
5.31
dt [11.1; 4.3]
2.64
dd [15.7; 4.3]
2.29
dd [15.7; 11.1]
4.81
s
6.60
dq [5.8; 1.5]
4.70
d [5.8]
3.15
d [4.1]
3.21
d [4.1]
1.1
s
4.18
d [12.1]
4.27
d [12.1]
1.9
bs
x
x
x
3.71
s, 1H
1.93
s
4.79
s, 2H
x
DON-3,15-disulfate [pp] (9)
in CDCl3
4.04
d [4.4]
5.32
dt [11.1; 4.4]
2.57
dd [15.7; 4.2]
2.33
dd [15.7; 11.1]
4.88
bs
6.69
dq [5.9; 1.5]
4.81
dt [5.7; 1.5]
3.17
d [4.1]
3.21
d [4.1]
1.2
s
4.60
d [11.0]
4.66
d [11.0]
1.9
bs
3.83
bs, 1H
x
4.79
s, 2H
4.48
s, 2H
DON-3-sulfate [p] (15)
in CDCl3
3.99
[4.5]
5.31
dt [11.2; 4.4]
2.80
dd [15.7; 4.3]
2.25
dd [15.7; 11.2]
4.81
s
6.62
dq [5.9; 1.4]
4.74
d [5.9]
3.14
d [4.1]
3.22
d [4.1]
1.2
s
3.76
d [11.5]
3.86
d [11.5]
1.9
bs
3.77
bs, 1H
1.71
bs, 1H
x
4.78
s, 2H
T2 toxin-3-sulfate [p] (21)
in CDCl3
3.96
d [4.9]
5.10
dd [4.9; 3.1]
6.16
d [3.1]
x
1.80
d [16.6]
2.35
dd [15.2; 6.0]
5.3
d [5.5]
5.77
dt [5.7; 1.4]
4.25
d [5.7]
2.85
d [3.9]
3.09
d [3.9]
0.7
s
4.1
d [12.7]
4.31
d [12.7]
1.8
bs
2.00 - 2.20
2xs, 2x3H, 6H
x
2.00 - 2.20
2xs, 2x3H, 6H
4.80
s, 2H
x
3
1
ADON-15-sulfate (13)
in MeOD
3.85
d [4.5]
5.11
dt [11.3; 4.4]
2.79
dd [15.3; 4.3]
2.08
dd [15.3; 11.3]
x
x
x
x
x
4.89
s
x
x
x
x
x
6.63
dq [6.1; 1.4]
4.92
d [6.1]
3.12
d [4.3]
3.16
d [4.3]
1.2
s
3.94
d [11.0]
4.27
d [11.0]
1.9
bs
2.13
s, 3H
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
5 ADON-3-sulfate (18)
in MeOD
3.82
d [4.5]
4.70-5.10
m [+NH4, H2O]
2.63
dd [15.3; 4.3]
2.11
dd [15.3; 11.2]
4.70-5.10
m [+NH4, H2O]
6.65
dq [5.9; 1.5]
4.70-5.10
m [+NH4, H2O]
3.12
s
3.12
s
1.1
s
4.23
d [12.1]
4.30
d [12.1]
1.9
bs
x
x
x
x
1.90
s, 3H
DON-3-sulfate (15)
in MeOD
3.80
d [4.4]
4.90
dt [11.2; 4.5]
2.75
dd [15.2; 4.4]
2.06
dd [15.2; 11.4]
4.79
s
6.61
dq [6.1; 1.4]
4.86
d [5.8]
3.09
d [4.5]
3.12
d [4.5]
1.1
s
3.68
d [12.3]
3.78
d [12.3]
1.8
bs
x
x
x
DON-15-sulfate (14)
in MeOD
3.55
d [4.5]
4.76
dt [11.1; 4.45]
2.57
dd [14.8; 4.4]
1.99
dd [14.8; 11.1]
4.85-4.95
m [+NH4, H2O]
6.65
dq [6.1; 1.4]
5.04
d [6.1]
3.06
d [4.5]
3.12
d [4.5]
1.1
s
3.96
d [11.0]
4.25
d [11.0]
1.8
bs
DON-3,15-disulfate (16)
in MeOD
3.84
d [4.5]
4.80 - 5.00
m [+NH4, H2O]
2.97
dd [15.3; 4.5]
2.10
dd [15.3; 11.4]
4.80-5.00
m [+NH4, H2O]
6.67
dq [6.0; 1.5]
4.80-5.00
m [+NH4, H2O]
3.10
d [4.3]
3.15
d [4.3]
1.2
s
4.24
d [10.9]
3.98
d [10.9]
1.9
bs
T2 toxin-3-sulfate (22)
in MeOD
3.79
d [5.1]
5.10
dd [4.9; 3.1]
6.03
d [3.1]
x
1.92
d [16.6]
2.38
dd [15.3; 5.9]
5.3
d [5.5]
5.78
d [5.5]
4.32
d [5.5]
2.87
d [3.9]
3.04
d [3.9]
0.7
s
4.16
d [12.1]
4.33
d [12.1]
1.7
bs
2.00 - 2.20
2xs, 2x3H, 6H
2.00 - 2.20
2xs, 2x3H, 6H
2

ACCEPTED MANUSCRIPT
2
8.)
2,2,2-Trichloroethyl 2-(4-acetoxyphenyl)ethylsulfate
1
H NMR (200 MHz, CDCl ) δ 7.28 (s, 4H), 4.82 (s, 2H), 4.27 (t, J=6.8 Hz, 2H), 2.95 (t, J=6.8 Hz, 2H), 2.02 (s, 3H)
3
13
C NMR (50 MHz, CDCl ) δ 171.0 (s, 1C), 148.9 (s, 1C), 138.1 (s, 1C), 130.6 (d, 2C), 121.2 (d, 2C), 92.5 (s, 1C),
3
8
0.5 (t, 1C), 64.5 (t, 1C), 34.5 (t, 1C), 21.0 (q, 1C)
3

ACCEPTED MANUSCRIPT
2
9.)
2-(4-Acetoxyphenyl)ethylsulfate, ammonium salt
1
+
H NMR (200 MHz, methanol-d ) δ = 7.22 (b, 4H), 4.93 (b, NH , H O), 4.23 (t, J=6.9, 2H), 2.91 (t, J=6.9 Hz, 2H),
4
4
2
2
.00 (s, 3H);
13
C NMR (50 MHz, methanol-d ) δ 172.9 (s, 1C), 152.5 (s, 1C), 136.0 (s, 1C), 130.6 (d, 2C), 122.5 (d, 2C), 66.2 (t,
4
1
C), 35.3 (t, 1C), 20.8 (q, 1C)
4

ACCEPTED MANUSCRIPT
3
0.)
2-(4-Hydroxyphenyl)ethylsulfate, ammonium salt
1
+
H NMR (200 MHz, methanol-d ) δ = 7.21 (s, 4H), 4.90 (b, NH , H O), 3.73 (t, J=6.9, 2H), 2.80 (t, J=6.9 Hz, 2H)
4
4
2
13
C NMR (50 MHz, methanol-d ) δ 152.3 (s, 1C), 137.0 (s, 1C), 130.6 (d, 2C), 122.5 (d, 2C), 64.2 (t, 1C), 39.5 (t, 1C)
4
5

ACCEPTED MANUSCRIPT
6
.)
2,2,2-Trichloroethyl 3-acetyl-DON-15-sulfate
1
H NMR (200 MHz, CDCl ) δ 6.66 (dq, J=5.9, 1.4 Hz, 1H), 5.24 (ddd, J=9.5, 6.0, 4.5 Hz, 1H), 4.89 (d, J=1.4 Hz, 1H),
3
4
1
2
.80 (d, J=5.9 Hz, 1H), 4.64 (d, J=10.8 Hz, 1H), 4.57 (d, J=10.8 Hz, 1H), 4.56 (d, J=10.6 Hz, 1H), 4.43 (d, J=10.6 Hz,
H), 3.95 (d, J=4.5 Hz, 1H), 3.84 (d, J=1.4 Hz, 1H), 3.16 (d, J=4.1 Hz, 1H), 3.13 (d, J=4.1 Hz, 1H), 2.10-2.36 (m,
H), 2.16 (s, 3H), 1.91 (b, 3H), 1.11 (s, 3H)
13
C-NMR (50 MHz, CDCl ) δ 198.8 (s, 1C), 170.2 (s, 1C), 138.7 (d, 1C), 136.1 (s, 1C), 92.4 (s, 1C, -CCl tiny
3
3
signal!), 79.7 (t, 1C), 78.8 (d, 1C), 73.0 (d, 1C), 71.6 (t, 1C), 70.7 (d, 1C), 69.1 (d, 1C), 64.6 (s, 1C), 51.3 (s, 1C), 47.4
(t, 1C), 45.8 (s, 1C), 40.4 (t, 1C), 20.9 (q, 1C), 15.2 (q, 1C), 13.5 (q, 1C)
6

ACCEPTED MANUSCRIPT
8
.)
2,2,2-Trichloroethyl DON-3-sulfate
1
H NMR (200 MHz, CDCl ) δ 6.62 (dq, J=5.9, 1.4 Hz, 1H), 5.31 (dt, J=11.2, 4.4, 1H), 4.81 (s, 1H), 4.78 (s, 2H), 4.74
3
(d, J=5.9 Hz, 1H), 3.99 (d, J=4.5 Hz, 1H), 3.86 (d, J=11.5 Hz, 1H), 3.77 (b, 1H), 3.76 (d, J=11.5 Hz, 1H), 3.22 (d,
J=4.1 Hz, 1H), 3.14 (d, J=4.1 Hz, 1H), 2.80 (dd, J=15.7, 4.3 Hz, 1H), 2.25 (dd, J=15.7, 11.2 Hz, 1H), 1.91 (b, 3H),
1
.71 (b, 1H), 1.17 (s, 3H)
13
C-NMR (50 MHz, CDCl ) δ 199.6 (s, 1C), 138.0 (d, 1C), 136.3 (s, 1C), 92.7 (s, 1C, -CCl tiny signal!), 80.8 (d, 1C),
3
3
7
4
9.9 (t, 1C), 79.1 (d, 1C), 74.5 (d, 1C), 70.1 (d, 1C), 64.8 (s, 1C), 62.0 (t, 1C), 51.8 (s, 1C), 47.7 (t, 1C), 46.1 (s, 1C),
0.3 (t, 1C), 15.4 (q, 1C), 14.1 (q, 1C)
7

ACCEPTED MANUSCRIPT
9
.)
bis(2,2,2-Trichloroethyl) DON-3,15-disulfate
1
H NMR (200 MHz, CDCl ) δ 6.69 (dq, J=5.9, 1.5 Hz, 1H), 5.32 (dt, J=11.1, 4.4, 1H), 4.88 (s, 1H), 4.81 (dt, J=5.7,
3
1
.5 Hz, 1H), 4.79 (s, 2H), 4.66 (d, J=11.0 Hz, 1H), 4.60 (d, J=11.0 Hz, 1H), 4.48 (s, 2H), 4.04 (d, J=4.4 Hz, 1H), 3.83
(b, 1H), 3.21 (d, J=4.1 Hz, 1H), 3.17 (d, J=4.1 Hz, 1H), 2.57 (dd, J=15.7, 4.2 Hz, 1H), 2.33 (dd, J=15.7, 11.1 Hz, 1H),
1
.93 (b, 3H), 1.15 (s, 3H)
13
C-NMR (50 MHz, CDCl ) δ 198.5 (s, 1C), 138.2 (d, 1C), 136.6 (s, 1C), 2 x 80.0 (d, 1C/t, 1C), 79.9 (t, 1C), 78.9 (d,
3
1
1
C), 73.3 (d, 1C), 71.5 (t, 1C), 69.2 (d, 1C), 64.3 (s, 1C), 51.3 (s, 1C), 47.7 (t, 1C), 46.2 (s, 1C), 40.3 (t, 1C), 15.3 (q,
C), 13.6 (q, 1C), 2 x CCl between 92 and 93 ppm are missing, but the corresponding CH groups are located at 80.0
3
2
and 79.9
8

ACCEPTED MANUSCRIPT
1
1.)
2,2,2-Trichloroethyl 15-acetyl-DON-3-sulfate
1
H NMR (200 MHz, CDCl ) δ 6.60 (dq, J=5.8, 1.5 Hz, 1H), 5.31 (dt, J=11.1, 4.3, 1H), 4.81 (s, 1H), 4.79 (s, 2H), 4.70
3
(d, J=5.8 Hz, 1H), 4.27 (d, J=12.1 Hz, 1H), 4.18 (d, J=12.1 Hz, 1H), 4.00 (d, J=4.3 Hz, 1H), 3.71 (s, 1H), 3.21 (d,
J=4.1 Hz, 1H), 3.15 (d, J=4.1 Hz, 1H), 2.64 (dd, J=15.7, 4.3 Hz, 1H), 2.29 (dd, J=15.7, 11.1 Hz, 1H), 1.93 (s, 3H),
.91 (b, 3H), 1.12 (s, 3H); 1,2-DI = traces of 1,2-dimethylimidazole
1
1,2-DI
1,2-DI
13
C-NMR (50 MHz, CDCl ) δ 198.9 (s, 1C), 170.2 (s, 1C), 137.9 (d, 1C), 136.1 (s, 1C), 92.7 (s, 1C, -CCl tiny
3
3
signal!), 80.4 (d, 1C), 80.0 (t, 1C), 78.9 (d, 1C), 73.5 (d, 1C), 70.1 (d, 1C), 64.5 (s, 1C), 62.0 (t, 1C), 51.2 (s, 1C), 47.4
(t, 1C), 46.1 (s, 1C), 40.2 (t, 1C), 20.8 (q, 1C), 15.4 (q, 1C), 13.7 (q, 1C)
9

ACCEPTED MANUSCRIPT
2
1.)
2,2,2-Trichloroethyl T2-toxin-3-sulfate
1
H NMR (200 MHz, CDCl ) δ 6.16 (d, J=3.1 Hz, 1H), 5.77 (dt, J=5.7, 1.4 Hz, 1H), 5.28 (d, J=5.5 Hz, 1H), 5.10 (dd,
3
J=4.9, 3.1 Hz, 1H), 4.80 (s, 2H), 4.31 (d, J=12.7 Hz, 1H), 4.25 (d, J=5.7 Hz, 1H), 4.10 (d, J=12.7 Hz, 1H), 3.96 (d,
J=4.9 Hz, 1H), 3.09 (d, J=3.9 Hz, 1H), 2.85 (d, J=3.9 Hz, 1H), 2.35 (dd, J=15.2, 6.0 Hz, 1H), 2.11 (s, 3H), 2.09 (s,
3
3
H), 2.00-2.25 (m, 3H), 1.80 (d, J=16.6 Hz, 1H), 1.76 (s, 3H), 0.96 (d, J=6.3 Hz, 3H), 0.95 (d, J=6.3 Hz, 3H), 0.74 (s,
H); 1,2-DI = traces of 1,2-dimethylimidazole
1,2-DI
1,2-DI
13
C NMR (50 MHz, CDCl ) δ 172.8 (s, 1C), 170.4 (s, 1C), 170.2 (s,1C), 137.1 (s, 1C), 123.0 (d, 1C), 92.6 (s, 1C), 87.1
3
(d, 1C), 80.1 (t, 1C), 78.4 (d, 1C), 77.4 (d, 1C), 67.7 (d, 1C), 67.5 (d, 1C), 64.6 (t, 1C), 63.8 (s, 1C), 48.7 & 47.5 (1t,
1
2
s, 2x1C), 43.7 (t, 1C), 43.1 (s, 1C), 28.4 (t, 1C), 25.9 (d, 1C), 22.6 (q, 1C), 22.5 (q, 1C), 21.2 (q, 1C), 20.8 (q, 1C),
0.4 (q, 1C), 6.5 (q, 1C)
1
0

ACCEPTED MANUSCRIPT
1
3.)
3-Acetyl-DON-15-sulfate, ammonium salt
1
H NMR (200 MHz, methanol-d ) δ 6.63 (dq, J=6.1, 1.4 Hz, 1H), 5.11 (dt, J=11.3, 4.4 Hz, 1H), 4.92 (d, J=6.1 Hz,
4
+
1
H), 4.89 (s, 1H), 4,87 (b, NH , H O) 4.27 (d, J=11.0 Hz, 1H), 3.94 (d, J=11.1 Hz, 1H), 3.85 (d, J=4.5 Hz, 1H), 3.16
4 2
(d, J=4.3 Hz, 1H), 3.12 (d, J=4.3 Hz, 1H), 2.79 (dd, J=15.3, 4.3 Hz, 1H), 2.08 (dd, J=15.3, 11.3 Hz, 1H), 2.13 (s, 3H),
1
.85 (b, 3H), 1.18 (s, 3H)
13
C NMR (50 MHz, methanol-d ) δ 201.0 (s, 1C), 172.5 (s, 1C), 139.4 (d, 1C), 137.1 (s, 1C), 80.5 (d, 1C), 75.8 (d,
4
1
1
C), 72.7 (d, 1C), 70.7 (d, 1C), 67.1 (t, 1C), 66.3 (s, 1C), 52.3 (s, 1C), 48.4 (t, 1C), 46.8 (s, 1C), 41.7 (t, 1C), 20.8 (q,
C), 15.3 (q, 1C), 14.4 (q, 1C)
1
1

ACCEPTED MANUSCRIPT
1
4.)
DON-15-sulfate, ammonium salt
1
+
H NMR (200 MHz, methanol-d ) δ 6.65 (dq, J=6.1, 1.4 Hz, 1H), 5.04 (d, J=6.1 Hz, 1H), 4.85-4.95 (m, NH , C7-H,
4
4
H O), 4.76 (dt, J=11.1, 4.5, 1H), 4.25 (d, J=11.0 Hz, 1H), 3.96 (d, J=11.0 Hz, 1H), 3.55 (d, J=4.5 Hz, 1H), 3.12 (d,
2
J=4.5 Hz, 1H), 3.06 (d, J=4.5 Hz, 1H), 2.57 (dd, J=14.8, 4.4 Hz, 1H), 1.99 (dd, J=14.8, 11.1 Hz, 1H), 1.84 (b, 3H),
1
.14 (s, 3H)
13
C NMR (50 MHz, methanol-d ) δ 201.1 (s, 1C), 139.9 (d, 1C), 136.9 (s, 1C), 82.3 (d, 1C), 75.9 (d, 1C), 70.7 (d, 1C),
4
6
9.6 (d, 1C), 67.2 (s, 1C), 66.7 (t, 1C), 52.5 (s, 1C), 48.2 (t, 1C), 47.5 (s, 1C), 45.0 (t, 1C), 15.3 (q, 1C), 14.4 (q, 1C)
Missing Signal (solvent
overlay) detected via DEPT
1
2

ACCEPTED MANUSCRIPT
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5.)
DON-3-sulfate, ammonium salt
1
+
H NMR (400 MHz, methanol-d ) δ 6.61 (dq, J=6.1, 1.4 Hz, 1H), 4.75-4.95 (m, NH , C3-H, C11-H, H O), 4.79 (s,
4
4
2
1
H), 3.80 (d, J=4.4 Hz, 1H), 3.78 (d, J=12.3 Hz, 1H), 3.68 (d, J=12.3 Hz, 1H), 3.12 (d, J=4.5 Hz, 1H), 3.09 (d, J=4.5
Hz, 1H), 2.75 (dd, J=15.2, 4.4 Hz, 1H), 2.06 (dd, J=15.2, 11.4 Hz, 1H), 1.83 (b, 3H), 1.12 (s, 3H)
13
C NMR (100 MHz, methanol-d ) δ 201.7 (s, 1C), 139.4 (d, 1C), 137.0 (s, 1C), 81.2 (d, 1C), 75.8 (d, 1C), 75.4 (d,
4
1
1
C), 71.6 (d, 1C), 66.3 (s, 1C), 61.8 (t, 1C), 53.6 (s, 1C), 48.2 (t, 1C), 46.7 (s, 1C), 42.5 (t, 1C), 15.4 (q, 1C), 14.6 (q,
C)
1
3

ACCEPTED MANUSCRIPT
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6.)
DON-3,15-disulfate, diammonium salt
1
+
H NMR (400 MHz, methanol-d ) δ 6.67 (dq, J=6.0, 1.5 Hz, 1H), 4.80-5.00 (m, NH , C3-H, C7-H, C11-H, H O),
4
4
2
4
1
.24 (d, J=10.9 Hz, 1H), 3.98 (d, J=10.9 Hz, 1H), 3.84 (d, J=4.5 Hz, 1H), 3.15 (d, J=4.3 Hz, 1H), 3.10 (d, J=4.3 Hz,
H), 2.97 (dd, J=15.3, 4.5 Hz, 1H), 2.10 (dd, J=15.3, 11.4 Hz, 1H), 1.85 (b, 3H), 1.16 (s, 3H)
13
C NMR (100 MHz, methanol-d ) δ 201.3 (s, 1C), 139.6 (d, 1C), 137.2 (s, 1C), 81.3 (d, 1C), 75.8 (d, 1C), 75.7 (d,
4
1
1
C), 70.5 (d, 1C), 67.4 (s, 1C), 66.1 (t, 1C), 52.4 (s, 1C), 48.4 (t, 1C), 47.1 (s, 1C), 42.4 (t, 1C), 15.3 (q, 1C), 14.5 (q,
C)
1
4

ACCEPTED MANUSCRIPT
1
8.)
15-Acetyl-DON-3-sulfate, ammonium salt
1
+
H NMR (400 MHz, methanol-d ) δ 6.65 (dq, J=5.9, 1.5 Hz, 1H), 4.70-5.10 (m, NH , C3-H, C7-H, C11-H, H O),
4
4
2
4
2
.30 (d, J=12.1 Hz, 1H), 4.23 (d, J=12.1 Hz, 1H), 3.82 (d, J=4.5 Hz, 1H), 3.12 (s, 2H), 2.63 (dd, J=15.3, 4.3 Hz, 1H),
.11 (dd, J=15.3, 11.2 Hz, 1H), 1.90 (s, 3H), 1.85 (b, 3H), 1.11 (s, 3H)
13
C-NMR (100 MHz, methanol-d ) δ 201.1 (s, 1C), 171.9 (s, 1C), 139.7 (d, 1C), 137.0 (s, 1C), 81.2 (d, 1C), 75.2 (d,
4
1
1
C), 75.0 (d, 1C), 71.3 (d, 1C), 65.9 (s, 1C), 63.3 (t, 1C), 52.6 (s, 1C), 48.2 (t, 1C), 47.0 (s, 1C), 42.5 (t, 1C), 20.6 (q,
C), 15.4 (q, 1C), 14.4 (q, 1C)
Missing Signal (solvent
overlay) detected via DEPT
1
5

ACCEPTED MANUSCRIPT
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2.)
T2-toxin-3-sulfate, ammonium salt
1
H NMR (400 MHz, methanol-d ) δ 6.03 (d, J=3.1 Hz, 1H), 5.78 (dt, J=5.5 1H), 5.33 (d, J=5.5 Hz, 1H), 4.80-4.94 (m,
4
+
NH , C3-H, H O), 4.33 (d, J=12.1, 1H), 4.32 (d, J=5.5 Hz, 1H), 4.16 (d, J=12.1 Hz, 1H), 3.79 (d, J=5.1 Hz, 1H), 3.04
4
2
(d, J=3.9 Hz, 1H), 2.87 (d, J=3.9 Hz, 1H), 2.38 (dd, J=15.3, 5.5 Hz, 1H), 2.13-2.18 (m, 2H), 2.07 (s, 3H), 2.06 (s, 3H),
2
.00-2.12 (m, 1H), 1.92 (d, J=15.3 Hz, 1H), 1.74 (s, 3H), 0.97 (d, J=6.7 Hz, 3H), 0.96 (d, J=6.7 Hz, 3H), 0.72 (s, 3H)
13
C NMR (100 MHz, methanol-d ) δ 174.0 (s, 1C), 172.3 (s, 1C), 172.2 (s, 1C), 137.2 (s, 1C), 125.1 (d, 1C), 82.3 (d,
4
1
1
1
C) 81.5 (d, 1C), 79.7 (d, 1C), 69.4 (d, 1C), 68.5 (d, 1C), 65.8 (t, 1C), 65.0 (s, 1C), 50.1 & 47.8 (1t, 1s, 2x1C), 44.5 (t,
C), 44.3 (s, 1C), 28.8 (t, 1C), 26.9 (d, 1C), 22.8 (q, 1C), 22.7 (q, 1C), 21.3 (q, 1C), 20.7 (q, 1C), 20.4 (q, 1C), 6.9 (q,
C)
1
6

ACCEPTED MANUSCRIPT
2
2.)
T2-toxin-3-sulfate, ammonium salt - COSY
2
2.)
T2-toxin-3-sulfate, ammonium salt – HMBC
1
7