Synthesis of Isotope-Labeled Deoxynivalenol-15-O-Glycosides

Article abstract of DOI:10.1002/ejoc.201700934

A versatile and efficient protocol for the Schmidt glycosylation of 3-acetyldeoxynivalenol (3-ADON) and subsequent deprotection has been developed to gain access to deoxynivalenol-15-O-glycosides in reasonable amounts for biological analysis and further investigations. By using this protocol with [¹³C₆]glycosyl donors, we were able to prepare isotope-labeled deoxynivalenol-15-O-glycosides, which are pivotal to enabling accurate quantification of masked mycotoxins by LC–MS.

Full text of DOI:10.1002/ejoc.201700934

A Journal of
Accepted Article
Title: Synthesis of Isotope Labeled Deoxynivalenol-15-O-Glycosides
Authors: Julia Weber, Philipp Fruhmann, Christian Hametner, Alois
Schiessl, Georg Häubl, Johannes Fröhlich, and Hannes
Mikula
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To be cited as: Eur. J. Org. Chem. 10.1002/ejoc.201700934
Link to VoR: http://dx.doi.org/10.1002/ejoc.201700934
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10.1002/ejoc.201700934
European Journal of Organic Chemistry
FULL PAPER
Synthesis of Isotope Labeled Deoxynivalenol-15-O-Glycosides
Julia Weber,[a] Philipp Fruhmann,[a,b] Christian Hametner,[a] Alois Schiessl,[c] Georg Häubl,[c] Johannes
Fröhlich[a] and Hannes Mikula*[a]
Abstract: A versatile and efficient protocol for Schmidt glycosylation
of 3-acetyldeoxynivalenol (3-ADON) and subsequent deprotection
has been developed to gain access to deoxynivalenol-15-O-
glycosides in reasonable amounts for bioanalysis and further
investigations. Applying this protocol in combination with
[13C6]glycosyl donors we were able to prepare isotope labeled
deoxynivalenol-15-O-glycosides, which are pivotal to enable accurate
quantification of masked mycotoxins by LC-MS.
have recently reported on the formation of DON-15-O-ß,D-
glucoside (2, Fig. 1b) in wheat.6 These altered forms, often called
masked mycotoxins, escape routine detection but can release the
parent toxin during food processing or digestion.7 Therefore, the
presence of such metabolites can cause an underestimation of
the potential toxicity of a particular sample and thus represent a
risk for food and feed safety.8
a
unreactive and least favorite
position for modification
O
plant metabolism:
HO
Introduction
O
phase I: hydrolysis, oxidation, etc.
phase II: glycosylation, sulfation, etc.
7
15
3
Deoxynivalenol (DON 1, Fig. 1a) is a trichothecene mycotoxin
produced by various Fusarium species, including Fusarium
graminearum and Fusarium culmorum. These species colonize
mainly wheat, barley, corn and oat in temperate regions of
Europe.1 Infection of the crop by Fusarium fungi is favored during
prolonged cool, moist growing and harvest season.2 DON is the
most commonly detected trichothecene with the highest
concentration in cereal-based food. It is a potent protein synthesis
inhibitor, and has an acute toxicity resulting in vomiting, nausea,
abdominal pain and diarrhea. Chronic exposure to low doses of
DON can cause anorexia, growth retardation, immune
dysregulation, impaired reproduction and development.3,4 To
decrease the exposure to DON for humans and animals, the
European Union set maximum levels, guidance values and
started monitoring programs. In 2002 the Scientific Committee for
Food (SCF) defined a provisional tolerable daily intake for DON
and its acetylated derivatives of 1 µg/kg body weight.2
As Fusarium mycotoxins are plant pathogens, they are prone to
metabolization by plants.5 Infected plants are capable of
biochemically modifying DON during the xenobiotic metabolism.
In phase I of this detoxification process, xenobiotics are oxidized
or hydrolyzed, while in phase II conjugates are formed by
glycosylation, sulfation, addition of glutathione, etc. The most
prominent conjugate of DON is DON-3-O-ß,D-glucoside (Fig. 1b).
Until recently, this metabolite was the only DON conjugate proven
to occur in naturally infected cereals (e.g. wheat).5 Schmeitzl et al.
O
HO
HO
Deoxynivalenol (DON, 1)
b
O
O
HO
HO
O
O
HO
15
HO
3
O
HO
HO
O
O
O
OH
HO
O
HO
HO
O
HO
HO
DON-3-O-ß,
D
-glucoside
DON-15-O-ß,D
-glucoside (2)
Figure 1. (a) Chemical structure of deoxynivalenol (DON, 1) and reaction sites
for metabolic modification; (b) naturally occurring DON-glucosides
Awareness of these DON-metabolites is increasing, but reference
compounds for structure elucidation, analytics and toxicity testing
are still scarce.8,9 For example, 2 could only be tentatively
identified by Schmeitzl et al. via mass spectrometry analysis and
thus, a method for accurate quantification could not be achieved.6
For the development of improved and accurate analytical
methods, and toxicological risk assessment sufficient quantities
of these compounds are required in high purity. Furthermore,
isotope labeled conjugates are crucial for accurate quantification
of mycotoxin metabolites by LC-MS.6,10
Previously described glycosylation reactions of DON were carried
out under Königs-Knorr conditions (CdCO3 in toluene under
reflux) with 38% yield.11 Recent advances have been made in the
preparation of DON-3-O-ß,D-glucoside via an enzymatic
approach. Michlmayr et al. reported on a DON-conjugating UDP-
glucosyltransferase from rice that produces DON-3-O-ß,D-
glucoside in 69% under optimized conditions.12 The synthesis of
13C-labeled DON-3-O-ß,D-[13C6]glucoside was accomplished via
Königs-Knorr glycosylation (Ag2CO3 in dichloromethane) by
Habler et al. very recently. However, the authors reported several
drawbacks of their method: (i) an excess of isotope labeled
glucosyl donor, (ii) a very long reaction time (5 days), and (iii)
failed upscaling of the reaction.10
[a]
J. Weber, P. Fruhmann, C. Hametner, J. Fröhlich and H. Mikula*
Institute of Applied Synthetic Chemistry
Vienna University of Technology (TU Wien)
Getreidemarkt 9, 1060 Vienna, Austria
E-mail: hannes.mikula@tuwien.ac.at
P. Fruhmann
Center for Electrochemical Surface Technology (CEST)
Viktor-Kaplan Straße 2, 2700 Wiener Neustadt, Austria
A. Schiessl, G. Häubl
[b]
[c]
Romer Labs
Technopark 1, 3430 Tulln/Donau, Austria
Herein we present the development of an efficient general
procedure for the preparation of DON-15-O-glycosides and the
Supporting information for this article is given via a link at the end of
the document.
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10.1002/ejoc.201700934
European Journal of Organic Chemistry
FULL PAPER
synthesis of isotope labeled analogs applying a 13C6-labeled N-
phenyltrifluoroacetimidoyl glucosyl donor.
leading to orthoester formation the steric bulk of the acyl residue
at O-2 needs to be increased.19 Thus, to improve the glycosylation
reaction, a more complex glycosyl donor would have to be
synthesized. As the aim of this study was to develop a simple
procedure (starting from commercially available peracetylated
glucose), we did not consider any further modification of the
glycosyl donor. In principal, orthoesters can often be converted
under acidic conditions to form the glucoside, but in that case acyl
transfer again emerges as undesired side reaction.20,21
Results and Discussion
For glycosylation at position 15, 3-acetyldeoxynivalenol (3-ADON,
3)13,14 was used as starting material as the second free OH group
at position 7 is known to be biochemically and chemically
inert.5,15,16 Considering previously described procedures for the
glycosylation of DON, we tested Königs-Knorr conditions
(acetobromo-α,D-glucose (4), Ag2O, ACN) to synthesize DON-15-
O-β,D-glucoside, but only observed exclusive formation of the
undesired orthoester 5 (Scheme 1).
Applying Schmidt glucosylation using N-phenyltrifluoro-
acetimidoyl donor 617 at 0 °C in a second approach, successfully
yielded the acetyl-protected 3-ADON-glucoside 7 in good yield
(67%) with only two minor side products 8 and 9 (Scheme 2).
O
HO
glycosylation
O
favored
O
HO
O
O
O
O
AcO
AcO
OAc
0°C
O
7 (65%)
HO
AcO
3
OAc
OAc
TMSOTf
MS 3Å, CH2Cl2
OAc
O
HO
O
O
AcO
AcO
O
AcO
-78°C
HO
O
O
NPh
O
O
HO
O
AcO
6
O
O
OAc
OAc
CF3
O
O
O
HO
Ag2O
O
favored formation
of orthoester
AcO
O
AcO
3
AcO
O
OAc
5 (78%)
OAc
OAc
MS 3Å, MeCN
65%
O
O
O
AcO
AcO
AcO
Scheme 3. Temperature-dependent formation of orthoester vs. glycosylation.
AcO
Br
4
orthoester 5
Removal of acyl groups is usually done by saponification (basic
hydrolysis) or base-catalyzed transesterification (Zemplén
conditions).22–25 Using sodium methoxide in methanol for
deacetylation of 7 led to the formation of a mixture of desired
DON-15-O-β,D-glucoside (2) and isoDON-15-O-β,D-glucoside
(10) in a ratio of 1:0.6, which was confirmed by NMR.26
Preparative separation of the two isomers by column
chromatography did not work in our hands, but the isomers can
be analytically distinguished based on different UV absorption
maxima (2: 228 nm, 10: 280 nm; Figure 2).
Scheme 1. Königs-Knorr glucosylation of 3-ADON leading to undesired
orthoester formation.
OAc
O
HO
O
O
AcO
AcO
TMSOTf, 0°C
AcO
NPh
CF3
O
O
MS 3Å, CH2Cl2
65%
HO
AcO
3
6
O
O
HO
HO
O
O
O
O
O
HO
O
HO
O
OH
O
O
O
O
O
O
O
AcO
AcO
AcO
OAc
OH
O
O
O
O
O
O
O
AcO
AcO
HO
HO
OH
OH
AcO
3,15-diADON (8)
9
OAc
OAc
OAc
OAc
HO
HO
2
10
OH
OH
OH
OH
3-ADON-15-O-(tetra-O-
acetyl-β,D-glucoside) (7)
minor side products
DON-15-O-β,
D
-glucoside
isoDON-15-O-β,D-glucoside
Scheme 2. Schmidt glucosylation of 3-ADON.
2
10
To avoid undesired formation of 3,15-diacetyl-DON (3,15-
diADON, 8), the reaction temperature was lowered to -78°C fully
preventing acyl transfer. However, no desired glucoside was
formed. Instead, orthoester 5 was isolated in excellent yield
(Scheme 3). As rarely described in the literature,18 temperature-
dependent orthoester- vs. glucoside-formation was observed,
also confirming the results of DFT calculations done by Berces et
al. showing that acyl transfer is a related but separate reaction
from orthoester formation. To prevent acyl transfer without
Time (min)
Figure 2. HPLC analysis of an inseparable mixture of DON-15-O-β,D-glucoside
(2) and isoDON-15-O-β,D-glucoside (10).
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10.1002/ejoc.201700934
European Journal of Organic Chemistry
FULL PAPER
We tested several base-catalyzed and saponification methods
such as K2CO3 in MeOH,27 KOH in THF/H2O,23 and Mg(OMe)2 in
MeOH28 all leading to formation of an inseparable mixture of 2 and
10. Finally, selective deprotection of 7 was successfully carried
out applying a very mild procedure using potassium cyanide in
MeOH29 affording DON-15-O-β,D-glucoside (2) in excellent yield
(Scheme 4).
Gentiobiosyl donor 1130 was reacted with
3 followed by
deprotection of the intermediate 12 to obtain DON-15-β-
gentiobioside (13) (Scheme 5). Even though a lower yield of 32%
was achieved in the glycosylation step (compared to 65% for the
synthesis of 7) due to a more complex product mixture, and thus
more difficult separation, the final product was obtained in an
overall yield of 30%.
As the availability of isotope labeled compounds as internal
standards is essential for the development of robust and accurate
analytical methods, we have prepared 13C-labeled glycosyl
donors to enable the preparation of isotope labeled DON-15-O-
glycosides. In comparison to using [13C15]DON31 the application of
[13C6]glycosyl donors makes the synthesis of isotope labeled
DON-glycosides significantly easier and cheaper. The resulting
partially isotope labeled DON-15-O-[13C6]glycoside can be used
as a standard for reliable quantification in LC-MS analysis.
The 13C6-labeled glucosyl donor [13C6]6 was synthesized starting
from [13C6]glucose (14) by acetylation with Ac2O and a catalytic
amount of FeCl3 under ultrasonic irradiation to afford 15.32
Anomeric deprotection with benzyl amine33 yielded the OH-sugar
16 that was subsequently reacted with N-phenyltrifluoro-
acetimidoyl chloride34 to obtain the [13C6]glucosyl donor [13C6]6 in
high yield (Scheme 6a). [13C6]Gentiobiosyl donor [13C6]11 was
synthesized by preparing [13C6]gentiobiose octaacetate (18) via
Schmidt glycosylation of 1,2,3,4-tetra-O-acetyl-β,D-glucose (17)
with [13C6]glucosyl donor [13C6]6. Anomeric deprotection of 17 with
ammonium acetate35 to obtain OH-sugar 19 followed by
O
O
HO
OH
HO
O
O
O
O
KCN
O
O
O
O
HO
AcO
OAc
MeOH
90%
HO
AcO
OH
OH
OAc
OAc
7
2
Scheme 4. Deprotection of 7 affording DON-15-O-β,D-glucoside (2).
AcO
O
O
O
AcO
AcO
TMSOTf, 0°C
MS 3Å, CH2Cl2
HO
O
AcO
AcO
AcO
O
+
32%
O
AcO
HO
O
NPh
AcO
3
11
CF3
HO
AcO
O
O
introduction of
a N-phenyltrifluoroacetimidoyl leaving group
HO
HO
O
O
O
O
KCN
MeOH
HO
HO
AcO
AcO
afforded the 13C6-labeled gentiobiosyl donor [13C6]11 in good
overall yield (Scheme 6b).
O
O
HO
HO
HO
AcO
AcO
AcO
AcO
O
O
94%
O
O
O
OH
O
Following our developed procedure, Schmidt glycosylation of 3
using the 13C6-labeled glycosyl donors [13C6]6 and [13C6]11, and
subsequent deprotection afforded DON-15-O-β,D-[13C6]glucoside
OAc
HO
12
13
([13C6]2)
and
DON-15-O-β-[13C6]gentiobioside
([13C6]13),
Scheme 5. Synthesis of DON-15-O-β-gentiobioside (13).
respectively. Furthermore, to avoid laborious purification of the
intermediate acetyl-protected 3-ADON-glycosides, a ‘one-pot’
procedure was developed. Applying this fast and efficient
Having developed an optimized procedure, we tested its
application for the synthesis of other DON-15-O-glycosides.
NPh
a
HO
AcO
AcO
AcO
FeCl3, Ac2O
MeCN, 98%
BnNH2
F3C
Cl
O
OH
O
OAc
O
O
HO
HO
AcO
AcO
AcO
AcO
AcO
AcO
THF, 87%
K2CO3
OH
HO
AcO
AcO
AcO
O
NPh
CF3
CH2Cl2, 83%
14
15
16
[
13C6]6
=
13C
AcO
AcO
NPh
Cl
b
AcO
HO
O
O
AcO
AcO
AcO
AcO
TMSOTf
F3C
O
O
O
AcO
AcO
O
OAc
AcO
AcO
AcO
AcO
AcO
AcO
AcO
AcO
K2CO3
O
O
O
MS 3Å, CH2Cl2
64%
AcO
AcO
CH2Cl2, 83%
O
NPh
CF3
R
AcO
AcO
NPh
CF3
17
[
13C6]6
18 (R = β-OAc)
19 (R = OH)
[
13C6]11
CH3COONH4, 85%
Scheme 6. Synthesis of isotope labeled glycosyl donors [13C6]6 and [13C6]11 for Schmidt glycosylation starting from [13C6]glucose (14).
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10.1002/ejoc.201700934
European Journal of Organic Chemistry
FULL PAPER
(dichloromethane, tetrahydrofuran, methanol and diethyl ether) were dried
using
a PureSolv system (inert technology, Amesbury, MA, USA).
Molecular sieves (3 Å) were activated under vacuum at 200°C before use.
The progress of the reactions was monitored by thin layer chromatography
(TLC) over silica gel 60 F254 (visualization either by using UV light or by
heat staining using ceric ammonium molybdate in ethanol/sulfuric acid).
LC-ESI-MS/MS was performed on an HCT ion trap mass spectrometer
(Bruker, Germany) in full scan mode. Chromatographic separation was
done on a 1200 series HPLC system (Agilent Technologies, Germany)
using a Luna RP-C18 column (3.0 x 150 mm, 3 µm particle size,
Phenomenex, Germany). Preparative column chromatography was
performed on silica gel 60 (Merck, 40-63 µm) using a SepacoreTM Flash
O
1.TMSOTf, 0°C
MS 3Å, CH2Cl2
2.KCN, MeOH
HO
O
HO
O
O
O
HO
AcO
no purification
of intermediate
required
O
O
O
NPh
CF3
3
HO
G
G
DON-15-O-[13C6]glycosides
isotpe labeled glycosyl
donor [13C6]6 or [13C6]11
HO
System or
a
Grace Reveleris Prep Purification System (Büchi,
Grace
O
O
HO
HO
Switzerland). Preparative HPLC separation was done on
a
O
O
O
O
HO
HO
HO
Reveleris Prep system (Büchi, Switzerland) using a Luna Prep C18(2), 10
µm, 250x10 mm column (Phenomenex, Germany). NMR spectra were
recorded on an Avance IIIHD 600Mhz spectrometer equipped with a
Prodigy BBO cryo probe (Bruker, Germany) or on an Avance DRX-400
MHz spectrometer (Bruker, Germany) at 20°C. Data was recorded and
evaluated using TOPSPIN 3.5 (Bruker Biospin, Germany). All chemical
shifts are given in ppm relative to tetramethylsilane. The calibration was
done using residual solvent signals.
HO
HO
HO
O
HO
HO
O
O
O
OH
O
O
OH
HO
HO
DON-15-O-β,D-[13C6]glucoside ([13C6]2)
DON-15-O-β-[13C6]gentiobioside ([13C6]13)
30% (over 2 steps)
32% ('one-pot')
49% (over 2 steps)
47% ('one-pot')
Scheme 7. Synthesis of isotope labeled DON-15-O-[13C6]glycosides.
General procedure A: Preparation of O-glycosyl N-phenyl trifluoro-
procedure, we were able to obtain both products ([13C6]2 and
[13C6]13) in sufficient quantities (up to 40 mg per batch; only
limited by the amount of 3) in less than 8 h (including final
purification by preparative HPLC) (Scheme 7).
acetimidates
To a solution of 1-hydroxy sugar (1 equiv) in CH2Cl2 (9 mL/mmol) was
added K2CO3 (2 eq.) followed by N-phenyl-2,2,2-trifluoroacetimidoyl
chloride (2 eq.). The reaction mixture was stirred at room temperature for
24 h, filtered and concentrated to afford the crude product. Purification by
flash chromatography (gradient elution, hexanes-EtOAc) gave the
corresponding trifluoroacetimidate.
Conclusions
General procedure B: glycosylation of 3-ADON (3)
In summary, we have developed an efficient method for the
synthesis of DON-15-O-glycosides by Schmidt-glycosylation of 3-
ADON (3) followed by deprotection under mild reaction conditions
avoiding the formation of undesired byproducts. This procedure
was successfully applied for the preparation of isotope labeled
[13C6]glycosides. The method was further optimized and simplified
by the development of a ‘one-pot’ protocol avoiding troublesome
and time-consuming purification of the intermediate protected
glycosides. We are convinced that this method will find application
towards the synthesis of further (isotope labeled) DON-15-O-
glycosides in reasonable amounts for bioanalysis and ongoing
investigations in the field of masked and conjugated mycotoxins.
Starting from 15-O-acetyl-DON instead of 3-ADON (3) this
method might furthermore be useful for the synthesis of DON-3-
O-glycosides. Even though the glycosylation step might require
further optimization (considering the lower reactivity of the
secondary 3-OH in contrast to the primary 15-OH) the optimized
reaction conditions for deprotection avoiding the formation of
isoDON derivatives can be used for the synthesis of any DON
conjugate.
To a solution of 3-ADON (3) (1 equiv) and glycosyl donor (1.5 equiv) in dry
CH2Cl2 (8 mL/mmol) was added molecular sieve (3 Å, 0.1 g/mL), and the
reaction mixture was stirred at rt for 30 min. After cooling the reaction
mixture to 0 °C, TMSOTf (0.1 equiv) was added. The reaction mixture was
stirred at 0 °C for 2 h, and quenched by the addition of Et3N (0.15 equiv).
The reaction mixture was filtered through Celite and concentrated. The
crude product was purified by flash chromatography (MeOH in DCM,
gradient elution) and preparative HPLC (RP-C18, MeCN in H2O, gradient
elution).
General procedure C: Deprotection of acetyl-protected 3-ADON-15-
O-glycosides
To
a solution of acetyl-protected glycoside (1 equiv.) in MeOH
(14 mL/µmol) was added KCN (0.5 equiv.) at 0 °C. The reaction mixture
was slowly warmed to room temperature and stirred for 2-4 h. Water
(1 mL) was added and MeOH was evaporated under reduced pressure.
Purification by preparative HPLC (RP-C18, MeCN in H2O, gradient elution)
afforded the desired glycoside.
General procedure D (‘One-pot’)
To a solution of 3-ADON (3) (1 equiv) and glycosyl donor (1.5 equiv) in dry
CH2Cl2 (8 mL/mmol) was added molecular sieve (3 Å, 0.1 g/ mL), and the
reaction mixture was stirred at rt for 30 min. After cooling the reaction
mixture to 0 °C, TMSOTf (0.1 equiv) was added. The reaction mixture was
stirred at 0 °C for 2 h, and quenched by the addition of Et3N (0.15 equiv).
The reaction mixture was filtered through Celite and concentrated. The
crude product mixture was dissolved in MeOH (14 mL/µmol) and KCN
(0.5 equiv.) was added at 0 °C. The reaction mixture was slowly warmed
Experimental Section
All reactions were performed under an argon atmosphere. 3-
Acetyldeoxynivalenol (3-ADON, 3) was obtained from Romer Labs (Tulln,
Austria) and all other chemicals were purchased from ABCR (Germany),
Sigma-Aldrich (Germany) or Carbosynth (UK). Anhydrous solvents
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10.1002/ejoc.201700934
European Journal of Organic Chemistry
FULL PAPER
to room temperature and stirred for 2-4 h. Water (1 mL) was added and
MeOH was evaporated. Preparative HPLC (RP-C18, MeCN in H2O,
gradient elution) afforded the desired glycoside.
DON-15-O-β-gentiobioside (13)
General procedure C; starting from 12 (37.7 mg, 39.4 µmol) 13 was
obtained as a white solid (23 mg, 94 %); 1H NMR (600 MHz, MeOD): δ
6.62 (dq, J = 6.0, 1.6 Hz, 1H), 4.98 (d, J = 4.98 Hz, 1H), 4.84 (s, 1H), 4.39-
4.34 (m, 2H), 4.15 (d, J = 10.6 Hz, 1H), 4.12 (dd, J = 11.6, 1.9 Hz, 1H),
4.03 (d, J = 7.9 Hz, 1H), 3.86 (dd, J = 11.9, 2.2 Hz, 1H), 3.74 (dd, J = 11.9,
6.3 Hz, 1H), 3.66 (dd, J = 11.8, 5.5 Hz, 1H), 3.57 (d, J = 10.6 Hz, 1H), 3.53
(d, J = 4.4 Hz, 1H), 3.42-3.38 (m, 1H), 3.37 (t, J = 8.9, 1.9 Hz, 1H), 3.32-
3.23 (m, 4H), 3.20 (dd, J = 9.0, 8.0 Hz, 1H), 3.10 (d, J = 4.4 Hz, 1H), 3.07-
3.03 (m, 2H), 2.45 (dd, J = 14.7, 4.4 Hz, 1H), 1.96 (dd, J = 14.6, 11.2 Hz,
1H), 1.83 (s, 3H), 1.12 (s, 3H); 13C NMR (150 MHz, MeOD): δ 202.4 (s,
1C), 140.2 (d, 1C), 136.8 (s, 1C), 104.9 (d, 1C), 104.5 (d, 1C), 82.2 (d, 1C),
78.0 (d, 1C), 77.9 (d, 1C), 77.7 (d, 1C), 77.2 (d, 1C), 75.7 (d, 1C), 75.2 (d,
1C), 75.0 (d, 1C), 71.6 (d, 1C), 71.5 (d, 1C), 71.4 (d, 1C), 69.8 (t, 1C), 69.7
(d, 1C), 69.7 (t, 1C), 66.8 (s, 1C), 62.7 (t, 1C), 53.3 (s, 1C), 48.1 (t, 1C),
47.3 (s, 1C), 44.5 (t, 1C), 15.5 (q, 1C), 14.9 (q, 1C); HRMS calcd for
C27H40O16+ [M+Na]+ 643.2209, found 643.2221.
3-ADON-15-O-(tetra-O-acetyl-β,D-glucoside) (7)
General procedure B; starting from 3-ADON (3) (40 mg, 0.12 mmol) and
donor 617 (92 mg, 0.18 mmol) 7 was obtained as a white solid (54 mg,
1
68%); H NMR (400 MHz, CDCl3): δ 6.54 (dq, J = 5.8, 1.5 Hz, 1H), 5.20
(dt, J = 11.3, 4.5 Hz, 1H), 5.12 (t, J = 9.4 Hz, 1H), 5.04 (t, J = 9.6 Hz, 1H),
4.84 (dd, J = 9.4, 7.8 Hz, 1H), 4.78 (d, J = 2.0 Hz, 1H), 4.72 (d, J = 5.4 Hz,
1H), 4.29 (d, J = 8.2 Hz, 1H), 4.27 (dd, J = 12.4, 5.0 Hz, 1H), 4.11 (dd, J =
12.3, 2.5 Hz, 1H), 4.03 (d, J = 10.2 Hz, 1H), 3.88 (d, J = 4.3 Hz, 1H), 3.67
(d, J = 1.9 Hz, 1H), 3.66 – 3.60 (m, 1H), 3.48 (d, J = 10.1 Hz, 1H), 3.15 (d,
J = 4.5 Hz, 1H), 3.10 (d, J = 4.3 Hz, 1H), 2.42 (d, J = 15.2, 4.3 Hz, 1H),
2.17 (s, 3H), 2.11 (dd, J = 15.0, 11.2 Hz, 1H), 2.09 (s, 3H), 2.08 (s, 3H),
2.01 (s, 3H), 1.98 (s, 3H), 1.85 (s, 3H), 1.09 (s, 3H); 13C NMR (100 MHz,
CDCl3): δ 199.5 (s, 1C), 170.7 (s, 1C), 170.6 (s, 1C), 170.3 (s, 1C), 169.5
(s, 1C), 169.4 (s, 1C), 138.7 (d, 1C), 135.8 (s, 1C), 100.6 (d, 1C), 79.2 (d,
1C), 73.9 (d, 1C), 72.9 (d, 1C), 72.1 (d, 1C), 71.2 (d, 1C), 71.1 (d, 1C),
69.7 (t, 1C), 68.5 (d, 1C), 68.1 (d, 1C), 65.1 (s, 1C), 62.0 (t, 1C), 51.5 (s,
1C), 47.6 (t, 1C), 45.9 (s, 1C), 40.6 (t, 1C), 21.1 (q, 1C), 20.9 (q, 1C), 20.8
[
13C6]Glucose pentaacetate (15)
To a suspension of [13C6]glucose (14) (1 g, 5.37 mmol, 1 eq.) in acetonitrile
(3 mL) cooled to 0 °C was added FeCl3 (87 mg, 0.54 mmol, 0.1 eq.)
followed by dropwise addition of acetic anhydride (2.8 mL, 29.6 mmol,
5.5 eq.). The reaction mixture was warmed to room temperature and
treated with ultrasonic irradiation for 45 min. The reaction mixture was
diluted with CH2Cl2 (10 mL) and washed with water. The organic layer was
dried over Na2SO4 and concentrated. The crude product was filtered
through a pad of silica gel eluting with hexanes/EtOAc (1:1) to afford 15
(2.1 g, 98%) as a white solid. Analytical data matched those reported in
the literature.36
+
(q, 1C), 20.7 (q, 2C), 15.3 (q, 1C), 13.8 (q, 1C); HRMS calcd for C31H40O16
[M+Na]+ 691.2209, found 691.2206.
DON-15-O-β,D-glucoside (2)
General procedure C; starting from 7 (27.8 mg, 42 µmol) 2 was obtained
as a white solid white solid (18.7 mg, 98%); 1H NMR (600 MHz, MeOD): δ
6.50 (dq, J = 5.9, 1.5 Hz, 1H), 4.98 (d, J = 6.2 Hz, 1H), 4.85 (s, 1H), 4.37
(dt, J = 11.2, 4.4 Hz, 1H), 4.18 (d, J = 10.5 Hz, 1H), 4.03 (d, J = 7.9 Hz,
1H), 3.84 (dd, J = 11.9, 1.9 Hz, 1H), 3.63 (dd, J = 11.7, 5.6 Hz, 1H), 3.57
(d, J = 10.6 Hz, 1H), 3.53 (d, J = 4.7 Hz, 1H), 3.30 – 3.27 (m, 1H), 3.24 –
3.18 (m, 2H), 3.10 (d, J = 4.4 Hz, 1H), 3.07 (d, J = 4.4 Hz, 1H), 3.04 (dd, J
= 9.2, 7.8 Hz, 1H), 2.44 (dd, J = 14.8, 4.3 Hz, 1H), 1.96 (dd, J = 14.7, 11.2
Hz, 1H), 1.83 (s, 3H), 1.11 (s, 3H); 13C NMR (150 MHz, MeOD): δ 202.5
(s, 1C), 140.3 (d, 1C), 136.9 (s, 1C), 104.8 (d, 1C), 82.2 (d, 1C), 78.0 (d,
1C), 77.8 (d, 1C), 75.7 (d, 1C), 75.2 (d, 1C), 71.5 (d, 2C), 69.8 (t, 1C), 69.7
(d, 1C), 66.7 (s, 1C), 62.8 (t, 1C), 53.3 (s, 1C), 48.1 (t, 1C), 47.3 (s, 1C),
44.5 (t, 1C), 15.3 (q, 1C), 14.8 (q, 1C); HRMS calcd for C21H30O11+ [M+Na]+
481.1681, found 481.1685.
2,3,4,6-Tetra-O-acetyl-D-[13C6]glucopyranose (16)
To a solution of glucose pentaacetate (15) (2.1 g, 5.3 mmol, 1 eq.) in dry
THF (40 mL) was added benzylamine (625 mg, 5.8 mmol, 1.1 eq.). The
reaction mixture was stirred at room temperature for 24 h, concentrated
and dissolved in CH2Cl2 (40 mL). The solution was washed with 1 N HCl
(25 mL) and water (25 mL). The organic layer was dried over Na2SO4 and
concentrated. Flash chromatography (hexanes/EtOAc 1:1, 90 g silica gel)
afforded the title compound 16 (1.64 g, 87 %) as a yellowish oil, which
slowly crystallized during storage. Analytical data matched those reported
in the literature.36
3-ADON-15-(hepta-O-acetyl-β-gentiobioside) (12)
General procedure B; starting from 3-ADON (3) (50 mg, 148 µmol) and
donor 1130 (180 mg, 223 µmol) 12 was obtained as a white solid (46 mg,
32 %); 1H NMR (400 MHz, CDCl3): δ 6.55 (dq, J = 6.0, 1.4 Hz, 1H), 5.25-
5.19 (m, 1H), 5.16 (t, J = 9.4 Hz, 1H), 5.12 (t, J = 9.4 Hz, 1H), 5.06 (t, J =
9.6 Hz, 1H), 4.96 (dd, J = 9.3; 8.2 Hz, 1H), 4.90-4.82 (m, 3H), 4.76 (d, J =
5.9 Hz, 1H), 4.63 (d, J = 8.2 Hz, 1H), 4.28-4.22 (m, 2H), 4.19 (d, J = 10.1
Hz, 1H), 4.11 (dd, J = 12.5, 2.4 Hz, 1H), 3.94 (d, J = 1.96 Hz, 1H), 3.89 (d,
J = 4.2 Hz, 1H), 3.79 (dd, J = 11.7, 1.9 Hz, 1H), 3.68 (dd, J = 11.9, 7.2 Hz,
1H), 3.64-3.57 (m, 2H), 3.34 (d, J = 9.8 Hz, 1H), 3.20 (d, J = 4.3 Hz, 1H),
3.11 (d, J = 4.0 Hz, 1H), 2.60 (dd, J = 15.3, 4.3 Hz, 1H), 2.17 (s, 3H), 2.09
(s, 3H), 2.08 (s, 3H), 2.02 (s, 3H), 2.01 (s, 6H), 2.00 (s, 3H), 1.98 (s, 3H),
1.85 (s, 3H), 1.61 (s, 1H), 1.09 (s, 3H); 13C NMR (100 MHz, CDCl3): δ
199.6 (s, 1C), 170.7 (s, 1C), 170.5 (s, 1C), 170.4 (s, 1C), 170.3 (s, 1C),
169.6 (s, 1C), 169.5 (s, 1C), 169.4 (s, 1C), 169.2 (s, 1C), 138.3 (d, 1C),
136.0 (s, 1C), 100.8 (d, 1C), 100.6 (d, 1C), 79.4 (d, 1C), 74.4 (d, 1C), 74.3
(d, 1C), 73.1 (d, 1C), 72.7 (d, 1C), 72.1 (d, 1C), 71.5 (d, 1C), 71.3 (d, 1C),
71.2 (d, 1C), 69.4 (d, 1C), 68.9 (d, 1C), 68.4 (d, 1C), 68.3 (t, 1C), 67.5 (t,
1C), 65.3 (s, 1C), 61.9 (t, 1C), 51.5 (s, 1C), 47.7 (t, 1C), 45.9 (s, 1C), 40.7
(t, 1C), 21.1 (q, 1C), 20.9 (q, 2C), 20.8 (q, 1C), 20.7 (q, 4C), 15.3 (q, 1C),
2,3,4,6-Tetra-O-acetyl-α,D-[13C6]glucopyranosyl-1-(N-phenyl)-2,2,2-
trifluoroacetimidate ([13C6]6)
General procedure A; starting from 16 (1.54 g, 4.4 mmol) [13C6]6 was
obtained as a colorless oil (1.9 g, 83 %) that slowly crystallized during
storage in the freezer; 13C-decoupled 1H-NMR matched those reported in
the literature17; HRMS calcd for C16
found 548.1462.
(
13C)6H24NO10 [M+Na]+ 548.1446,
3-ADON-15-O-(tetra-O-acetyl-β,D-[13C6]glucoside) ([13C6]7)
General procedure B; starting from 3-ADON (3) (80 mg, 0.24 mmol) and
donor [13C6]6 (189 mg, 0.36 mmol) [13C6]7 was obtained as a white solid
(85 mg, 53 %); 13C-decoupled 1H NMR data matched those of compound
7; 13C NMR (150 MHz, CDCl3): δ 199.5 (s, 1C), 170.7 (s, 1C), 170.6 (s,
1C), 170.3 (s, 1C), 169.5 (s, 1C), 169.4 (s, 1C), 138.7 (d, 1C), 135.8 (s,
1C), 100.9-100.1 (m, 1C), 79.2 (d, 1C), 73.9 (d, 1C), 73.5-70.5 (m, 4C),
69.7 (t, 1C), 68.8-67.8 (m, 2C), 65.1 (s, 1C), 62.4-61.5 (m, 1C), 51.5 (q,
1C), 47.6 (t, 1C), 45.9 (s, 1C), 40.6 (t, 1C), 21.1 (q, 1C), 20.9 (q, 1C), 20.8
(q, 1C), 20.7 (q, 2C), 15.3 (q, 1C), 13.8 (q, 1C); HRMS calcd for
+
13.8 (q, 1C); HRMS calcd for C43H56O24 [M+Na]+ 979.3054, found
C25(
13C)6H40O16 [M+Na]+ 697.2410, found 697.2430.
979.3060.
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10.1002/ejoc.201700934
European Journal of Organic Chemistry
FULL PAPER
DON-15-O-β,D-[13C6]glucoside ([13C6]2)
Keywords: glycosylation • orthoester • trichothecenes • masked
General procedure C; starting from [13C6]7 (62 mg, 92 µmol) [13C6]2 was
obtained as a white solid (40 mg, 93 %); 13C-decoupled 1H NMR data
matched those of compound 2; 13C NMR (150 MHz, MeOD): δ 202.5 (s,
1C), 140.3 (d, 1C), 136.9 (s, 1C), 104.0-102.9 (m, 1C), 82.2 (d, 1C), 77.3-
75.7 (m, 2C), 75.7 (d, 1C), 75.6-74.7 (m, 1C), 71.9-71.1 (m, 2C), 69.8 (t,
1C), 69.7 (d, 1C), 66.7 (s, 1C), 63.1-62.4 (m, 1C), 53.3 (s, 1C), 48.1 (t, 1C),
47.3 (s, 1C), 44.5 (t, 1C), 15.3 (q, 1C), 14.8 (q, 1C); HRMS calcd for
mycotoxin • isotope labeling
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[
dry CH2Cl2 (3 mL). MS 3Å (300 mg) was added and the mixture was stirred
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0.03 mmol, 0.15 eq) was added, the reaction mixture was slowly warmed
to rt and stirred for 1 h. The reaction was quenched by addition of Et3N,
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707.2101, found 707.2117.
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13C6]Gentiobiose heptaacetate (19)
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1 eq.) in dry DMF (1 mL) was added ammonium acetate (31 mg,
0,40 mmol, 2 eq.). The reaction mixture was stirred for 16 h at rt,
concentrated and purified by column chromatography to afford 19 (109 mg,
85 %) as a white solid. 13C-decoupled 1H-NMR matched those reported in
the literature38; HRMS calcd for C20
665.2007.
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in the freezer; 13C-decoupled 1H-NMR matched those of non-labeled 11 as
+
reported in the literature30; HRMS calcd for C28
836.2291, found 836.2283.
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13C)6H40F3NO18 [M+Na]+
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DON-15-O-β-[13C6]gentiobioside ([13C6]13)
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113 µmol) and donor [13C6]11 (138 mg, 170 µmol) [13C6]13 was obtained
as a white solid (23 mg, 32 %); 13C-decoupled 1H NMR data matched
those of non-labeled 13; 13C NMR (150 MHz, MeOD): δ 202.4 (s, 1C),
140.2 (d, 1C), 136.8 (s, 1C), 105.3-104.5 (m, 1C), 104.5 (d, 1C), 82.2 (d,
1C), 78.5-77.5 (m, 3C), 77.2 (d, 1C), 75.7 (d, 1C), 75.5-74.7 (m, 2C), 72.1-
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+
1C), 14.9 (q, 1C); HRMS calcd for C21
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We thank the Austrian Research Promotion Agency (FFG,
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10.1002/ejoc.201700934
European Journal of Organic Chemistry
FULL PAPER
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10.1002/ejoc.201700934
European Journal of Organic Chemistry
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Entry for the Table of Contents
FULL PAPER
Glycosylation
Julia Weber, Philipp Fruhmann,
Christian Hametner, Alois Schiessl,
Georg Häubl, Johannes Fröhlich, and
Hannes Mikula*
Page No. – Page No.
An efficient and fast procedure for the synthesis of deoxynivalenol-15-O-glycosides
was developed enabling access to isotope labeled compounds. Applying this
method DON-15-O-b,D-[13C6]glucoside and DON-15-O-b-[13C6]gentiobioside were
prepared in reasonable amounts for characterization and as reference materials for
further investigations.
Synthesis of isotope labeled
deoxynivalenol-15-O-glycosides
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