Synthesis of zearalenone-16-β,D-glucoside and zearalenone-16-sulfate: A tale of protecting resorcylic acid lactones for regiocontrolled conjugation

Article abstract of DOI:10.3762/bjoc.10.112

The development of a reliable procedure for the synthesis of the 16-glucoside and 16-sulfate of the resorcylic acid lactone (RAL) type compound zearalenone is presented. Different protective group strategies were considered and applied to enable the preparation of glucosides and sulfates that are difficult to access up to now. Acetyl and p-methoxybenzyl protection led to undesired results and were shown to be inappropriate. Finally, triisopropylsilyl-protected zearalenone was successfully used as intermediate for the first synthesis of the corresponding mycotoxin glucoside and sulfate that are highly valuable as reference materials for further studies in the emerging field of masked mycotoxins. Furthermore, high stability was observed for aryl sulfates prepared as tetrabutylammonium salts. Overall, these findings should be applicable for the synthesis of similar RAL type and natural product conjugates.

Full text of DOI:10.3762/bjoc.10.112

Synthesis of zearalenone-16-β,D-glucoside and
zearalenone-16-sulfate: A tale of protecting
resorcylic acid lactones for
regiocontrolled conjugation
Hannes Mikula*1, Julia Weber1, Dennis Svatunek1, Philipp Skrinjar1,
Gerhard Adam2, Rudolf Krska3, Christian Hametner1
and Johannes Fröhlich1
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2014, 10, 1129–1134.
1Institute of Applied Synthetic Chemistry, Vienna University of
Technology (VUT), Getreidemarkt 9/163, A-1060 Vienna, Austria,
2Department of Applied Genetics and Cell Biology, University of
Natural Resources and Life Sciences, Vienna (BOKU), Konrad Lorenz
Str. 24, 3430 Tulln, Austria and 3Center for Analytical Chemistry,
Department for Agrobiotechnology (IFA-Tulln), University of Natural
Resources and Life Sciences, Vienna (BOKU), Konrad Lorenz Str. 20,
3430 Tulln, Austria
doi:10.3762/bjoc.10.112
Received: 03 January 2014
Accepted: 18 April 2014
Published: 15 May 2014
Associate Editor: S. Bräse
© 2014 Mikula et al; licensee Beilstein-Institut.
License and terms: see end of document.
Email:
Hannes Mikula* - hannes.mikula@tuwien.ac.at
* Corresponding author
Keywords:
glycosylation; masked mycotoxins; resorcylic acid esters; sulfation;
zearalenone
Abstract
The development of a reliable procedure for the synthesis of the 16-glucoside and 16-sulfate of the resorcylic acid lactone (RAL)
type compound zearalenone is presented. Different protective group strategies were considered and applied to enable the prepar-
ation of glucosides and sulfates that are difficult to access up to now. Acetyl and p-methoxybenzyl protection led to undesired
results and were shown to be inappropriate. Finally, triisopropylsilyl-protected zearalenone was successfully used as intermediate
for the first synthesis of the corresponding mycotoxin glucoside and sulfate that are highly valuable as reference materials for
further studies in the emerging field of masked mycotoxins. Furthermore, high stability was observed for aryl sulfates prepared as
tetrabutylammonium salts. Overall, these findings should be applicable for the synthesis of similar RAL type and natural product
conjugates.
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Beilstein J. Org. Chem. 2014, 10, 1129–1134.
Introduction
Resorcylic acid lactones (RALs, Figure 1), a compound class of glucoside 5 and the sulfate 6, were identified as ZEN metabo-
benzannulated macrolides, are pharmacologically active sec- lites in Arabidopsis thaliana [16]. These conjugates are easily
ondary metabolites produced by a variety of different fungal hydrolyzed back to the parental mycotoxin during digestion of
species [1]. Zearalenone (ZEN, 1) is a well-known RAL type contaminated grain, and should therefore be considered as
mycotoxin for which maximum tolerated levels in food and masked mycotoxins [12]. Recently it has been shown that ZEN
feed were enacted and recommended, respectively, in Europe treated barley, wheat and Brachypodium distachyon cells
[2,3]. ZEN is produced by several plant pathogenic Fusarium produce both the ZEN-14 and the ZEN-16-glucoside, with up to
species, including Fusarium graminearum and Fusarium 18-fold higher levels of ZEN-16-glucoside than ZEN-14-gluco-
culmorum. These species, which are the most frequently occur- side in barley roots [17]. We therefore intended to develop a
ring toxin-producing fungi of the northern temperate zone, are synthetic method for regiocontrolled conjugation of ZEN. Basi-
commonly found in cereals and crops throughout the world cally, the RAL type moiety of ZEN contains two possible sites
[4,5]. Significant levels of ZEN are prevalently found in grains for glycosylation/sulfation, but due to the higher reactivity of
such as maize, wheat, and rice [6]. It is known that ZEN can the phenol group at position 14, reactions at this site are
cause problems of the reproductive tract (e.g., impaired fertility) strongly favored compared to conjugation at C16–OH [18-20].
in animals [7]. Physiological studies revealed binding of ZEN to Although natural products containing a RAL type moiety conju-
recombinant human estrogen receptors [8] and have further- gated at the phenol group in ortho position to the carboxyl
more shown a ZEN-induced stimulation of the growth of human group (2’-OH) were already detected and identified [21-25], to
breast cancer cells [9].
the best of our knowledge there are no reliable synthetic pro-
cedures and strategies towards this class of compounds
described in the literature so far. The synthesis of the natural
glucoside delphoside by Saeed was performed using methyl
ether protection at O-6 of the isocoumarin core structure during
glucosylation and rather harsh unfavorable demethylation with
boron tribromide in the last step [26]. Without structure verifi-
cation and characterization ZEN-16-β,D-glucoside (7,
Figure 2B) was tentatively identified as a byproduct of the
Königs–Knorr glucosylation of ZEN for preparation of ZEN-
14-glucoside [27]. In the course of ongoing research in the
emerging field of masked mycotoxins, we were able to prepare
Figure 1: Structure of selected RAL type fungal secondary metabo-
lites.
Additionally, masked mycotoxins, especially altered deriva-
tives formed through conjugation to sugar moieties or sulfate,
emerge after metabolization by living plants. Due to changed
chemical structures and properties compared to the parent
mycotoxins, these conjugates can usually not be detected
applying standard analytical techniques [10,11]. Responsible
biochemical transformations are catalyzed usually by enzymes
within detoxification processes [12]. Schneweis et al. reported
the occurrence of ZEN-14-β,D-glucoside (5, Figure 2A) in
wheat [13] and the first chemical synthesis of this compound
applying phase transfer glycosylation has been reported by
Grabley et al. [14]. ZEN-14-sulfate (6, Figure 2A) was first
isolated from F. graminearum-inoculated rice [15] and both, the
Figure 2: Structures of zearalenone conjugates: (A) ZEN-14-β,D-
glucoside (5) and ZEN-14-sulfate (6), (B) ZEN-16-β,D-glucoside (7)
and ZEN-16-sulfate (8). Sulfates shown as sodium salts.
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Beilstein J. Org. Chem. 2014, 10, 1129–1134.
ZEN-16-β,D-glucoside (7) and ZEN-16-sulfate (8, Figure 2B) 2-OH group of the acceptor 10. This assumption was supported
in reasonable amounts after the development of reliable pro- by detection of the glucosyl acetamide 12, which is known to be
cedures that should be generally applicable to resorcylic acid formed by rearrangement of 11 when activated in the presence
lactones. Additionally the first chemical synthesis of the ZEN- of a weak acceptor (Scheme 2B) [31-34]. Königs–Knorr gluco-
derivative 14-O-acetylzearalenone (14-AcZEN, produced by sylation, which in general is most frequently used for the gluco-
some Fusarium strains) [28] is reported.
sylation of phenols, using commercially available bromo sugar
13 activated by silver(I) salts or under phase transfer conditions
led to complex product mixtures (Scheme 2C).
Results and Discussion
The general strategy for regiocontrolled conjugation at position
2’ of resorcylic acid esters and lactones is shown in Scheme 1.
Regioselective protection of the more nucleophilic 4’-phenol
and subsequent glucosylation or sulfation should lead to the
desired products.
Scheme 2: (A) Regioselective acetylation of resorcylic acid ester 9.
(B) Lewis acid mediated glucosylation; no conversion of 10, partially
rearrangement of 11 to glucosyl acetamide 12. (C) Königs–Knorr
glucosylation; Ag(I): Ag2CO3 or Ag2O, CH2Cl2 or MeCN; PTG: phase
transfer glucosylation, NaOH, tetrabutylammonium bromide, pH
10–11, CHCl3/H2O.
Scheme 1: General strategy for the synthesis of RAL-2’-conjugation
(Pg: protective group, pGlc: protected glucose moiety, pS: protected
sulfate, Glc: β,D-glucoside).
Nevertheless, the procedure for selective acetylation of resor-
For the development of a reliable protective group strategy and cylic acid esters and lactones was applied for the first synthesis
subsequent reaction optimization, 2,4-dihydroxybenzoic acid of 14-O-acetylzearalenone (14) (Scheme 3).
isopropyl ester (9) [29] was used as a RAL mimic. For protec-
tion of the 4-OH group we first considered an acetyl group that To avoid undesired cleavage of the acetyl group during gluco-
could be regioselectively introduced by reaction of 9 with acetic sylation of 10, p-methoxybenzyl (PMB) protection was applied
anhydride and catalytic amounts of 4-(dimethylamino)pyridine instead, since the PMB group was considered to be inert to the
(DMAP) to obtain the acetylated RAL mimic 10 (Scheme 2A). reactions conditions of the Königs–Knorr procedure, thus
Different methods for glycosylation were investigated using forcing conjugation at position 2. Regioselective p-methoxy-
acetyl-protected glucosyl donors since diastereoselective benzylation of 9 was achieved by reaction with PMB-Cl and
β-conjugation, which is needed for the preparation of gluco- Cs2CO3 in dry DMF after optimization in terms of base and
sides formed during phase II metabolism, is commonly solvent type (Scheme 4A). Königs–Knorr glucosylation of the
achieved applying the participation of acyl groups at O-2 of the PMB-protected mimic 15 afforded 16, which was deprotected
glycosyl donor (anchimeric effect) [30]. Lewis acid-mediated using 2,3-dichloro-5,6-dicyano-p-benzoquinone (DDQ) for
glucosylation using the trichloroacetimidate donor 11 according oxidative PMB cleavage and subsequent ester saponification to
to the procedure of Saeed [26] did not lead to the desired prod- yield the desired glucoside 17 in an overall yield of 30%
uct, which can be explained by the weak nucleophilicity of the (Scheme 4B).
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Beilstein J. Org. Chem. 2014, 10, 1129–1134.
Scheme 3: Regioselective acetylation of ZEN (1) affording 14-O-acetylzearalenone (14).
Scheme 4: (A) Regioselective p-methoxybenzylation of 9. (B) Syn-
thesis of the ZEN-16-Glc mimic 17.
Scheme 5: Regioselective protection of ZEN (1) and failed PMB
cleavage of the glucosylated intermediate 19.
Applying this procedure to ZEN (1) afforded the glucosylated
intermediate 19 after Königs–Knorr glucosylation of 14-PMB- hindrance and methods for selective deprotection under rela-
ZEN (18), but subsequent deprotection using DDQ did not tively mild conditions led us to the use of triisopropylsilyl
afford the desired product. Also an alternative procedure for (TIPS) protection for regiocontrolled glucosylation of resor-
oxidative PMB cleavage using cerium ammonium nitrate cylic acid esters and lactones. Regioselective silylation of 9 and
(CAN) did not lead to the formation of the deprotected com- ZEN (1) was readily achieved affording compounds 21 and 22
pound 20 (Scheme 5). Beside unreacted 19, LC–MS/MS (14-TIPS-ZEN), respectively, in nearly quantitative yields by
analysis showed the formation of a product with an m/z value reaction with TIPS-Cl and imidazole in dry CH2Cl2. Applying
16 amu higher than calculated for 19 indicating oxidation of this this strategy we were finally able to accomplish the synthesis of
intermediate but no deprotection.
the ZEN mimic glucoside 17 (Scheme 6A) as well as of the
target compound ZEN-16-β,D-glucoside (7) as shown in
Since the DDQ-promoted cleavage of phenolic PMB ethers can Scheme 6B. Reasonable yields of 41% (17) and 34% (7), res-
be complicated by overoxidation, especially with electron-rich pectively, were obtained applying an optimized purification
phenolic compounds [35], we assume a significant effect of the protocol.
conjugated olefinic double bond at C-6’ of the resorcylic acid
moiety being responsible for the observed different behavior of Additionally, TIPS protection was applied for the synthesis of
ZEN (1) and the ZEN mimic 9.
ZEN-16-sulfate (8) using a procedure that was successfully
applied for the synthesis of ZEN-14-sulfate (6) as described
After this second setback the protective group strategy was recently [20]. Reaction of 22 with the 2,2,2-trichloroethyl
changed again within a third approach. Considering steric (TCE) protected sulfuryl imidazolium salt 23 [36,37] gave the
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Beilstein J. Org. Chem. 2014, 10, 1129–1134.
Scheme 6: Synthesis of (A) ZEN mimic glucoside 17 and (B) ZEN-16-β,D-glucoside (7); a: TIPS-Cl, imidazole, 16 h, rt, >99% for 21 and 22; b: 13,
Ag2O, MeCN, 96 h, rt; c: TBAF, AcOH, THF, 24 h; d: KOH, THF/H2O, 4 h, rt, 41% for 17, 34% for 7 (3 steps).
protected sulfate 24. TIPS cleavage and subsequent TCE depro- terial and long term stability of appropriate standard solutions
tection using Zn/ammonium formate (HCOONH4) yielded the for further investigations.
desired product. Interestingly, when using the crude intermedi-
ate after TIPS deprotection without purification directly in the Conclusion
second step, we obtained the tetrabutylammonium salt of ZEN- In summary, different methods for the regioselective protection
16-sulfate (NBu4-8) in good yield (65% over 2 steps) as shown of resorcylic acid esters and lactones were investigated for
in Scheme 7. The stability of this compound was significantly subsequent regiocontrolled glucosylation and sulfation.
increased compared to the corresponding sodium salt, which is Whereas acetyl and p-methoxybenzyl protection led to unde-
of great importance in terms of preparation of reference ma- sired products, TIPS-protected RALs were successfully used as
intermediates for the preparation of corresponding glucosides
and sulfates applying the Königs–Knorr glucosylation and
chemical sulfation using TCE-protected sulfuryl imidazolium
salt 23, respectively. These methods were used for the first
chemical synthesis of the ZEN-16-conjugates 7 and 8 in reason-
able amounts for ongoing research and further investigations in
the field of conjugated/masked mycotoxins.
Supporting Information
Supporting Information File 1
Experimental details (including remarks and general
procedures), characterization data, copies of NMR spectra
of new compounds, 2D NMR spectra of glucoside 7.
[http://www.beilstein-journals.org/bjoc/content/
supplementary/1860-5397-10-112-S1.pdf]
Acknowledgements
The Theodor Körner fund (Vienna, Austria), the Austrian
Federal Ministry of Economy, Family and Youth as well as the
Scheme 7: Chemical sulfation using the 2,2,2-trichloroethyl (TCE)-
protected sulfuryl imidazolium salt 23 yielding ZEN-16-sulfate (8) as
tetrabutylammonium salt; a: 23, 1,2-dimethylimidazole, CH2Cl2, 12 h,
National Foundation for Research, Technology and Develop-
ment are gratefully acknowledged for financial support. GA and
rt, 91%, b: TBAF, AcOH, THF, 3 h, −10 °C, c: Zn, HCOONH4, MeOH,
16 h, rt, 65% (2 steps).
RK were supported by the Austrian Science Fund (FWF)
special research project SFB Fusarium (F3706 and F3708).
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Beilstein J. Org. Chem. 2014, 10, 1129–1134.
23.Zidorn, C.; Lohwasser, U.; Pschorr, S.; Salvenmoser, D.;
References
Ongania, K.-H.; Ellmerer, E. P.; Börner, A.; Stuppner, H.
Phytochemistry 2005, 66, 1691–1697.
1. Winssinger, N.; Barluenga, S. Chem. Commun. 2007, 22–36.
doi:10.1039/b610344h
doi:10.1016/j.phytochem.2005.05.004
2. European legislation (EC 1881/2006). Commission Regulation (EC) No
1881/2006 setting maximum levels for certain contaminants in foodstuff
– consolidated version 03.12.2012. European Community (EC):
Brussels, Belgium.
24.Xu, M.; Zhang, M.; Wang, D.; Yang, C.-R.; Zhang, Y.-J.
Chem. Biodiversity 2011, 8, 1891–1900. doi:10.1002/cbdv.201000220
25.Morikawa, T.; Wang, L.-B.; Ninomiya, K.; Nakamura, S.; Matsuda, H.;
Muraoka, O.; Wu, L.-J.; Yoshikawa, M. Chem. Pharm. Bull. 2009, 57,
853–859. doi:10.1248/cpb.57.853
http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=CONSLEG:200
6R1881:20121203:EN:PDF (accessed April 2, 2014).
3. European commission recommendation (2006/576/EC) on certain
mycotoxins in animal feed – 17.08.2006.. European Community (EC):
Brussels (Belgium).
26.Aamer, S. Chin. J. Chem. 2005, 23, 762–766.
doi:10.1002/cjoc.200590762
27.Versilovskis, A.; Huybrecht, B.; Tangni, E. K.; Pussemier, L.;
De Saeger, S.; Callebaut, A. Food Addit. Contam. 2011, 28,
1687–1693.
http://eur-lex.europa.eu/legal-content/EN/ALL/?uri=CELEX:32006H057
6 (accessed April 2, 2014).
28.Muñoz, L.; Castro, J. L.; Cardelle, M.; Castedo, L.; Riguera, R.
Phytochemistry 1989, 28, 83–85. doi:10.1016/0031-9422(89)85014-9
29.Mikula, H.; Hametner, C.; Fröhlich, J. Synth. Commun. 2013, 43,
1939–1946. doi:10.1080/00397911.2012.681827
30.Mydock, L. K.; Demchenko, A. V. Org. Biomol. Chem. 2010, 8,
497–510. doi:10.1039/b916088d
4. Creppy, E. E. Toxicol. Lett. 2002, 127, 19–28.
doi:10.1016/S0378-4274(01)00479-9
5. Sudakin, D. L. Toxicol. Lett. 2003, 143, 97–107.
doi:10.1016/S0378-4274(03)00116-4
6. Krska, R.; Josephs, R. Fresenius' J. Anal. Chem. 2001, 369, 469–476.
doi:10.1007/s002160100715
31.Wegmann, B.; Schmidt, R. R. J. Carbohydr. Chem. 1987, 6, 357–375.
doi:10.1080/07328308708057926
7. Tiemann, U.; Tomek, W.; Schneider, F.; Vanselow, J. Reprod. Toxicol.
2003, 17, 673–681. doi:10.1016/j.reprotox.2003.07.001
8. Miksicek, R. J. J. Steroid Biochem. Mol. Biol. 1994, 49, 153–160.
doi:10.1016/0960-0760(94)90005-1
32.Damager, I.; Olsen, C. E.; Møller, B. L.; Motawia, M. S.
Carbohydr. Res. 1999, 320, 19–30.
doi:10.1016/S0008-6215(99)00131-7
9. Shier, W. T.; Shier, A. C.; Xie, W.; Mirocha, C. J. Toxicon 2001, 39,
1435–1438. doi:10.1016/S0041-0101(00)00259-2
33.Motawia, M. S.; Olsen, C. E.; Enevoldsen, K.; Marcussen, J.;
Møller, B. L. Carbohydr. Res. 1995, 277, 109–123.
doi:10.1016/0008-6215(95)00203-6
10.Berthiller, F.; Schuhmacher, R.; Adam, G.; Krska, R.
Anal. Bioanal. Chem. 2009, 395, 1243–1252.
34.Motawia, M. S.; Olsen, C. E.; Møller, B. L.; Marcussen, J.
Carbohydr. Res. 1994, 252, 69–84.
doi:10.1007/s00216-009-2874-x
11.Tran, S. T.; Smith, T. K. Anim. Feed Sci. Technol. 2011, 163, 84–92.
doi:10.1016/j.anifeedsci.2010.10.008
doi:10.1016/0008-6215(94)90006-X
35.Green, T. W.; Wuts, P. G. W. Protective Groups in Organic Synthesis;
John Wiley & Sons, Inc.: New York, 1999; pp 269–270.
doi:10.1002/0471220574
12.Berthiller, F.; Crews, C.; Dall'Asta, C.; De Saeger, S.; Haesaert, G.;
Karlovsky, P.; Oswald, I. P.; Seefelder, W.; Speijers, G.; Stroka, J.
Mol. Nutr. Food Res. 2013, 57, 165–186. doi:10.1002/mnfr.201100764
13.Schneweis, I.; Meyer, K.; Engelhardt, G.; Bauer, J.
J. Agric. Food Chem. 2002, 50, 1736–1738. doi:10.1021/jf010802t
14.Grabley, S.; Gareis, M.; Böckers, W.; Thiem, J. Synthesis 1992,
1078–1080. doi:10.1055/s-1992-26306
36.Ingram, L. J.; Desoky, A.; Ali, A. M.; Taylor, S. D. J. Org. Chem. 2009,
74, 6479–6485. doi:10.1021/jo9014112
37.Liu, Y.; Lien, I.-F. F.; Ruttgaizer, S.; Dove, P.; Taylor, S. D. Org. Lett.
2004, 6, 209–212. doi:10.1021/ol036157o
15.Plasencia, J.; Mirocha, C. J. Appl. Environ. Microbiol. 1991, 57,
146–150.
16.Berthiller, F.; Werner, U.; Sulyok, M.; Krska, R.; Hauser, M.-T.;
Schuhmacher, R. Food Addit. Contam. 2006, 23, 1194–1200.
doi:10.1080/02652030600778728
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J. Agric. Food Chem. 2014, 62, 1181–1189. doi:10.1021/jf405627d
18.Mikula, H.; Hametner, C.; Berthiller, E.; Warth, B.; Krska, R.; Adam, G.;
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21.Bunyapaiboonsri, T.; Yoiprommarat, S.; Khonsanit, A.; Komwijit, S.
J. Nat. Prod. 2008, 71, 891–894. doi:10.1021/np070689m
22.Paraschos, S.; Magiatis, P.; Kalpoutzakis, E.; Harvala, C.;
Skaltsounis, A.-L. J. Nat. Prod. 2001, 64, 1585–1587.
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