2242
LETTER
Synthesis of Isotopically Labeled Fusarium Mycotoxin 13C2-Moniliformin
[1-Hydroxycyclobut-1-ene-3,4-dione]
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S
ynthesis of Iso
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-Monilifo
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min hrey,a Takeshi Murata,b Daisuke Uemura,b,c Hans-Ulrich Humpf*a
2
a
Institut für Lebensmittelchemie, Westfälische Wilhelms-Universität Münster, Corrensstr. 45, 48149 Münster, Germany
Fax +49(251)8333396; E-mail: humpf@uni-muenster.de
b
c
Department of Chemistry, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8602, Japan
Department of Biosciences and Informatics, Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi,
Yokohama 223-8655, Japan
Received 13 June 2011
tion analysis by using isotopically labeled standards is
often applied.9 However, until now no isotopically labeled
MON is available and no strategy for the introduction of
stable isotopes has been proposed. Therefore our aim was
Abstract: The total synthesis of isotopically labeled [13C2]-1-hy-
droxycyclobut-1-ene-3,4-dione (moniliformin) a fungal toxic sec-
ondary metabolite to be used as internal standard for mycotoxin
analysis is described. The synthesis proceeds in four steps starting
from 1,4-dioxane, which was converted to 2,3-dihydro-1,4-dioxine to synthesize isotopically labeled MON to be used as in-
followed by a [2+2]-cycloaddition with trichloroacetyl chloride-
ternal standard in stable isotope dilution analysis. So far
1,2-13C2 as 13C source. The 13C2-labeled cyclobutanone precursor
several synthetic routes to MON have been reported and
was transformed to [13C2]-moniliformin by acid-catalyzed hydroly-
all rely on the generation of cyclobutane derivatives as
sis. The successful incorporation of two 13C atoms was proven by
precursors.10 The most recent synthesis was reported by
detailed NMR and mass spectrometric studies of labeled monili-
Fétizon et al. in 1990 based on a [2+2] cycloaddition of
2,3-dihydro-1,4-dioxine and dichloroketene via formation
of a four-membered ring precursor in 34% yield.11 By hy-
drolysis of the cyclobutanone precursor Fétizon and co-
workers were able to obtain MON in 82% yield. Based on
this straightforward approach we developed a modified
strategy for the first preparation of isotopically labeled
MON by using trichloroacetyl chloride-1,2-13C2 as 13C
source for isotope introduction.
formin and its precursor.
Key words: Fusarium, moniliformin, [2+2] cycloaddition, stable
isotope dilution analysis, HPLC–MS/MS
Moniliformin (MON) is a toxic secondary fungal metabo-
lite in cereals and maize worldwide,1 which is produced
by a number of Fusarium species, including the common
corn pathogen F. avenaceum, F. proliferatum, F. subglu-
tinans, F. tricinctum, and F. verticillioides.2 Naturally it
occurs as sodium or potassium salt of 1-hydroxycyclobut-
1-ene-3,4-dione and was first isolated and characterized
by Cole et al. in 1973.3,4 Toxicity studies with different
animal species indicate that MON is a potent cardiotox-
in.5,6 Thiel et al. suggested that the molecular mechanism
for the toxic effect of MON involves selective inhibition
of mitochondrial pyruvate and a-ketoglutarate oxida-
tions.7
SO2Cl2
Zn, NMP
O
O
45–70 °C, 16 h
O
O
Cl
Cl
O
O
95 °C, 4 h
71%
58%
1
2
3
Cl313C13COCl
))), Zn, Et2O
15–20 °C, 2 h
6 M HCl
O
O
H
O
O
O
13C
13C
45 °C, 2 d
13C
13C
3
Cl
15%
47%*
11%
47%*
OH
Cl
Due to the potential health risk for humans and animals
posed by MON, the establishment of fast and reliable
methods for the detection of this mycotoxin in different
food and feed as well as physiological samples is of great
importance. For this purpose the use of high-performance
liquid chromatography (HPLC) coupled to tandem mass
spectrometry (HPLC–MS/MS) is the method of choice.
Advantages of HPLC–MS/MS analysis are the high selec-
tivity and sensitivity. Thus this technique has a wide ap-
plication in food and feed analysis. However, the use of
HPLC–MS/MS is mostly limited by the influence of co-
eluting matrix compounds on the ionisation of the ana-
lyte.8 To overcome this limitation the stable isotope dilu-
4
5
Scheme 1 Synthetic route for the preparation of 13C2-moniliformin
(* yields for unlabeled compounds)
Since MON is known to be among the strongest naturally
occurring organic acid (pKa = 0.88), the two hydrogen
atoms in the molecule would undergo rapid hydrogen–
deuterium atom exchange in methanol or water, such that
deuterium-labeled MON would not be useful as internal
standard.12,13 To overcome this challenge we decided to
synthesize instead a 13C-labeled standard.
The synthesis started with the preparation of 2,3-dihydro-
1,4-dioxine (3) for which several procedures are pub-
lished.14,15 We synthesized 3 in two steps starting from
1,4-dioxane 1 (Scheme 1) following a modified procedure
by Shinohara et al.16 Firstly 2,3-dichloro-1,4-dioxane (2)
was generated by chlorination of 1 with sulfuryl chloride
SYNLETT 2011, No. 15, pp 2242–2244
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Advanced online publication: 12.08.2011
DOI: 10.1055/s-0030-1261189; Art ID: D18211ST
© Georg Thieme Verlag Stuttgart · New York