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

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.

S2 General procedure C: regioselective TIPS-protection of

General remarks
All reactions were performed under an argon atmosphere. The progress of reactions was monitored by thin-layer chromatography (TLC) over silica gel 60 F254 (Merck). The chromatograms were visualized by irradiation with ultraviolet light or by heat staining with 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 × 150 mm, 3 µm particle size, Phenomenex, Germany) and application of pure substances was achieved using a TLC-MS interface (Camag, Germany). Preparative column chromatography was performed on silica gel 60 (Merck, 40-63 µm) or RP-C18 silica gel (Merck, 40-63 µm) using a Büchi Sepacore TM Flash System. NMR spectra were recorded on a Bruker DPX-200 MHz or Avance DRX-400 MHz spectrometer. Data were recorded and evaluated using TOPSPIN 1.3 (Bruker Biospin). All chemical shifts are given in ppm relative to tetramethylsilane. The calibration was done using residual solvent signals. Multiplicities are abbreviated as s (singlet), d (doublet), t (triplet), q (quartet), b (broad signal). Zearalenone was obtained from Fermentek (Israel) and all other chemicals were purchased from ABCR (Germany) or Sigma-Aldrich (Austria/Germany).

General procedure A: regioselective acetylation of RAL type compounds
To a solution of the RAL type compound (1.0 mmol) and DMAP (1.2 mg, 10 µmol) in dry toluene (5 mL) at 0 °C was slowly added a solution of Ac 2 O (110 mg, 1.1 mmol) in dry toluene (1 mL). The reaction mixture was gradually warmed to room temperature and stirred for further 16 h. The solvent was removed under reduced pressure and the residue was purified by column chromatography (hexanes/EtOAc gradient elution) to yield the desired 4'-O-acetylated product. S4 4-Acetoxy-2-hydroxybenzoic acid isopropyl ester (10) Following general procedure A, 10 was obtained as a white solid (203 mg, 85%); R f 0.4 (hexanes/EtOAc,9/

General procedure C: regioselective TIPS-protection of RAL type compounds
To a solution of the RAL type compound (1 mmol) and imidazole (156 mg, 2.3 mmol) in dry CH 2 Cl 2 (10 mL) was slowly added TIPS-Cl (216 mg, 1.1 mmol). The reaction mixture was stirred at room temperature for 16 h, diluted with CH 2 Cl 2 (20 mL), washed with brine, dried over Na 2 SO 4 and concentrated. The residue was purified by column chromatography (hexanes/EtOAc gradient elution).

General procedure D: Königs-Knorr glucosylation
To a solution of the glucosyl acceptor (0.5 mmol) and bromoacetoglucose 13 (617 mg, 1.5 mmol) in dry MeCN (10 mL) was added molecular sieve 3 Å (1 g) and the resulting suspension was stirred at room temperature for 1 h. After addition of Ag 2 O (174 mg, 0.75 mmol), the reaction mixture was stirred in the dark for 48 h. Since TLC analysis indicated remaining glucosyl acceptor, 13 (411 mg, 1 mmol) and Ag 2 O (116 mg, 0.5 mmol) were added and stirring was continued in the dark for 24 h.
The reaction mixture was diluted with CH 2 Cl 2 , filtered through Celite and concentrated under reduced pressure. Silica gel filtration (hexane/EtOAc gradient elution) was performed to remove most the carbohydrate byproducts. Since we were not able to fully purify the crude glucosylated products, these compounds were directly subjected to deprotection within subsequent steps.

S9
to room temperature and stirred overnight. After dilution with CH 2 Cl 2 (10 mL), the solution was washed with water, dried over Na 2 SO 4 and concentrated. Column chromatography over silica gel