First synthesis of a stable isotope of Ochratoxin A metabolite for a reliable detoxification monitoring

Article abstract of DOI:10.1021/ol401630t

Due to its toxicity and presence in numerous food products, Ochratoxin A (OTA) has drawn attention for decades. This article summarizes the first synthesis of a labeled analogue of Ochratoxin α (OTα), one of the main products generated by the metabolization of OTA by microorganisms. This synthesis also led to a new labeled analogue of OTA with the deuteration located on the dihydroisocoumarin moiety allowing thus both the accurate quantification of OTA and OTα and the establishing of a reliable detoxification rate.

Full text of DOI:10.1021/ol401630t

ORGANIC
LETTERS
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Vol. XX, No. XX
000–000
First Synthesis of a Stable Isotope of
Ochratoxin A Metabolite for a Reliable
Detoxification Monitoring
‡
‡
Ana¨ıs Bouisseau,†,‡ Aurelie Roland, Florence Reillon, Remi Schneider,‡,§ and
ꢀ
ꢀ
Florine Cavelier*,†
ꢀ
IBMM, UMR-CNRS-5247, Universites Montpellier I and II, Place Eugene Bataillon,
34095 Montpellier, Cedex 5, France, Nyseos, Batiment 28, 2 place Pierre Viala,
ꢁ
^
34060 Montpellier Cedex 1, France, IFV UMT Qualinnov, 2 place Pierre Viala,
34060 Montpellier Cedex 1, France
florine@univ-montp2.fr
Received June 10, 2013
ABSTRACT
Due to its toxicity and presence in numerous food products, Ochratoxin A (OTA) has drawn attention for decades. This article summarizes the first
synthesis of a labeled analogue of Ochratoxin R (OTR), one of the main products generated by the metabolization of OTA by microorganisms. This
synthesis also led to a new labeled analogue of OTA with the deuteration located on the dihydroisocoumarin moiety allowing thus both the
accurate quantification of OTA and OTR and the establishing of a reliable detoxification rate.
The mycotoxin Ochratoxin A (OTA) (1, Figure 1) was
first isolated by van der Merwe et al. in 1965 from a South
African culture of Aspergillus ochraceus grown on a sterile
maize meal.1 In 1992, Marquardt and Frohlich assessed
the general toxicity of this toxin.2 Since, OTA has drawn
the attention of the International Agency for Research on
Cancer and belongs to the group 2B.3
toxic effects on animals such as carcinogenicity in mice
and rats kidneys and livers, hepatotoxicity, teratogenicity,
immunosuppressivity, and nephrotoxicity.4ꢀ7
This mycotoxin can contaminate different food pro-
ducts such as coffee, wheat, grapes, and wine.8,9 The
presence of OTA in wine is mainly due to the fungal
contamination of grapes by Aspergillus carbonarius, and
the occurrence depends on its geographic origin and on the
type of wine.10,11
OTA has been consequently classified as a possible
human carcinogenic substance and presents also many
(4) Krogh, P. Food Chem. Toxicol. 1992, 30, 213–224.
(5) Bendele, A. M.; Carlton, W. W.; Krogh, P.; Lillehoj, E. B. J. Natl.
Cancer Inst. 1985, 75, 733–742.
(6) Chopra, M.; Link, P.; Michels, C.; Schrenk, D. Cell. Biol. Toxicol.
2010, 26, 239–254.
(7) Haubeck, H. D.; Lorkowski, G.; Kolsch, E.; Roschenthaler, R.
Appl. Environ. Microbiol. 1981, 41, 1040–1042.
(8) Pittet, A. Rev. Med. Vet. 1998, 149, 479–492.
(9) Jørgensen, K. Food Addit. Contam. 2005, 22, 26–30.
(10) Fabiani, A.; Corzani, C.; Arfelli, G. Talanta 2010, 83, 281–285.
† IBMM.
‡ Nyseos.
§ IFV UMT Qualinnov.
(1) van der Merwe, K. J.; Steyn, P. S.; Fourie, L.; Scott, D. B.;
Theron, J. J. Nature 1965, 205, 1112–1113.
(2) Marquardt, R. R.; Frohlich, A. A. J. Anim. Sci. 1992, 70, 3968–
3988.
(3) IARC, IARC Monographs on the Evaluation of Carcinogenic
Risks to Humans 1993, 56, 489–521.
r
10.1021/ol401630t
XXXX American Chemical Society

The synthesis of the four diastereoisomers of OTA has
been described in order to assess the cytotoxicity of each
chiral compound separately.12 Another publication de-
scribes the synthesis of natural and d5-OTA with the
deuteration on the phenylalanine moiety.13
As contamination of crops by Aspergillus sp. is difficult
to avoid, due to the lack of infestation predictive models,
the food industry is looking for curative tools.
Figure 1. Natural OTA (1), d4-OTA (2), and d4-OTR (3).
In wine, different ways of detoxification have been
described, either by physical, chemical, or microbiological
means.14 The problem remains to determine the structure
and the potential toxicity of the degradation products. For
thermal processes, such as in coffee, OTA was reported
to be transformed into 14-(R)-OTA by isomerization
and 14-decarboxy-OTA by decarboxylation, both less
toxic.15 For microbiological processes, not only some
industrial yeast strains but also lactic acid bacteria or
filamentus fungi are able to degrade OTA into ochratoxin
R (OTR),14,16,17 which is less toxic.18,19 But it cannot be
excluded that other potentially toxic degradation products
could be produced according to a microbiological process.
Abrunhosa et al. described the two main metabolization
pathways involved for OTA when using microorganisms.
The first is the transformation of OTA into OTR which
represents a detoxification process because OTR is much
less toxic.20 The second main product is the compound
obtained after hydrolysis of the OTA lactone ring, but this
resulting opened-lactone molecule showed a high in vivo
toxicity.18
Thus, labeled OTR together with OTA could be of
interest to establish a precise quantification of the hydro-
lysis rate of the amide bond between phenylalanine and
OTR. By extension, this quantification of both OTA and
OTR after a microbiological process could represent a
reliable method for the determination of a real detoxifica-
tion rate since it is not only the disappearance of OTA
which will be studied but also the formation of the non-
toxic metabolization product, OTR.
Our study presents the synthesis of natural OTA (1,
Figure 1) and of a new isotope d4-OTA (2, Figure 1),
with the deuteration located on the dihydroisocoumarin
moiety. We also describe the first synthesis of d4-OTR
(3, Figure1) which could be used for degradation studies as
mentioned above.
The synthesis of OTA was previously described using
ꢀ~
CovarrubiasꢀZuniga’s method,21 which involves a reaction
between the sodium salt of dimethyl 3-oxopentanedioate
and but-2-ynal. We chose to synthesize the diethyl ester 5
instead of the dimethyl ester via Antus et al.’s method.22
Thus, the potassium salt was prepared by alkali-catalyzed
cleavage of 4,4-dimethoxybutan-2-one 4 which was then
reacted with diethyl 3-oxopentanedioate to afford 5.
Then, following Cramer et al.’s synthesis and as shown in
Scheme 1, the condensation step with acetaldehyde led
to the intermediate 6 as an enantiomeric mixture in high
yields.12 Racemic OTR 7 was prepared by chlorination and
saponification of 6.
To achieve the synthesis of OTA 1 from OTR 7, the next
step was the introduction of a phenylalanine moiety
(Scheme 1). Among all the conditions tested, including
BOP and HATU as coupling agents, the use of oxalyl chloride
appeared to be the most efficient for coupling OTR 7 with
L-phenylalanine tert-butyl ester.23,24 The tert-butyl group was
then removed using TFA.25 This deprotection step led to a
mixture of natural OTA 1 and its diastereoisomer 10.
In order to perform these quantifications accurately,
Stable Isotope Dilution Assay appears as a good alter-
native, and thus, syntheses of deuterated OTA and OTR
are required for their use as internal standards.
Scheme 1. Synthesis of Natural OTA 1 and Its Diastereoisomer 10
(11) Otteneder, H.; Majerus, P. Food Addit. Contam. 2000, 17, 793–
798.
(12) Cramer, B.; Harrer, H.; Nakamura, K.; Uemura, D.; Humpf,
H.-U. Biorg. Med. Chem. 2010, 18, 343–347.
(13) Gabriele, B.; Attya, M.; Fazio, A.; Di Donna, L.; Plastina, P.;
Sindona, G. Synthesis 2009, 11, 1815–1820.
(14) Quintela, S.; Villaran, M. C.; de Armentia, I. L.; Elejalde, E.
Food Control 2013, 30, 439–445.
(15) Cramer, B.; Konigs, M.; Humpf, H. U. J. Agric. Food. Chem.
2008, 56, 5673–5681.
(16) Cecchini, F.; Morassut, M.; Moruno, E. G.; Di Stefano, R. Food
Microbiol. 2006, 23, 411–417.
(17) Peteri, Z.; Teren, J.; Vagvolgyi, C.; Varga, J. Food Microbiol.
2007, 24, 205–210.
(18) Xiao, H.; Madhyastha, S.; Marquardt, R. R.; Li, S. Z.; Vodela,
J. K.; Frohlich, A. A.; Kemppainen, B. W. Toxicol. Appl. Pharmacol.
1996, 137, 182–192.
(19) Stander, M. A.; Steyn, P. S.; van der Westhuizen, F. H.; Payne,
B. E. Chem. Res. Toxicol. 2001, 14, 302–304.
(20) Abrunhosa, L.; Paterson, R. R. M.; Venancio, A. Toxins 2010, 2,
1078–1099.
ꢀ~
(21) Covarrubias-Zuniga, A.; Rıos-Barrios, E. J. Org. Chem. 1997,
62, 5688–5689.
ꢀ
ꢀ
(22) Antus, S.; Boross, F.; Nogradi, M. Liebigs Ann. 1978, 1978, 107–
117.
(23) Adams, R.; Ulich, L. H. J. Am. Chem. Soc. 1920, 42, 599–611.
(24) Montalbetti, C. A. G. N.; Falque, V. Tetrahedron 2005, 61,
10827–10852.
(25) Bryan, D. B.; Hall, R. F.; Holden, K. G.; Huffman, W. F.;
Gleason, J. G. J. Am. Chem. Soc. 1977, 99, 2353–2355.
B
Org. Lett., Vol. XX, No. XX, XXXX

A separation of the nondeuterated diastereoisomers
on preparative HPLC allowed us to isolate (ꢀ)-OTA 1
(natural form).
Scheme 2. Saponification Step
Since (þ)-OTA does not occur in food products, the
mixture of deuterated diastereoisomers can be directly
used as an internal standard for OTA quantification in
wine samples upon the condition that the same diastereo-
meric mixture be used for calibration. The analysis study
of wine samples was based on LC/MS. Using a mixture of
deuterated diastereoisomers, we observed an overlapping
of (ꢀ)-OTA and one of the diastereoisomers of d4-OTA by
liquid chromatography. But since the quantification was
determined by mass spectrometry, the detection was based
on the 4 Da shift in the molecular mass corresponding to
the introduction of four deuteriums.
In conclusion, we designed a synthetic pathway that led to
both d4-OTR and d4-OTA. We achieved the synthesis of a
new labeled analogue of natural OTA bearing the deuteration
on the dihydroisocoumarin moiety of the molecule, opposite
to phenylalanine. In addition, our strategy allowed us to
produce for the first time a labeled analogue of OTR, which
can be used for establishing a reliable detoxification rate.
As shown in Scheme 2, it should be noted that HPLC
and LC/MS monitorings showed that the saponification
step was actually a disaponification, and the lactone 7
was reformed under acidic conditions. Indeed, while the
chlorinated intermediate 8 presents a retention time of
2.01 min, we obtained, after lithium hydroxide addition
and ammonium chloride neutralization, the opened lac-
tone form 9 of OTR with a retention time of 1.19 min
([MHþ] = 275.1), which led to compound 7 ([MHþ] =
257.2) with a retention time of 1.48 min under acidic
conditions. Compound 9 unfortunately could not be pur-
ified by silica column because of its high polarity or by
preparative HPLC because of its instability under acidic
conditions.
Acknowledgment. Raymond Baumes (INRA) is kindly
thanked for helpful discussions at the beginning of the
ꢀ
study. France AgriMer and Region Languedoc Roussillon
are thanked for their financial support.
Supporting Information Available. Experimental pro-
cedures for the preparation of natural and deutarated
OTA and characterizations. This material is available free
of charge via the Internet at http://pubs.acs.org.
Using the appropriate labeled reagent, the same pro-
cedure was used to prepare d4-OTR 3 and d4-OTA 2 as a
mixture of deuterated enantiomers and diastereoisomers
respectively. Those three compounds were fully character-
ized after separation of d4-OTA diastereoisomers.
The authors declare no competing financial interest.
Org. Lett., Vol. XX, No. XX, XXXX
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