Direct Synthesis of ESBO Derivatives-18O Labelled with Dioxirane

This work addresses a new approach developed in our laboratory, consisting in the application of isolated dimethyldioxirane (DDO, 1a) labelled with 18O for synthesis of epoxidized glyceryl linoleate (Gly-LLL, 2). We expect that this work could contribute in improving analytical methods for the determination of epoxidized soybean oil (ESBO) in complex food matrices by adopting an 18O-labelled-epoxidized triacylglycerol as an internal standard.


Introduction
The packaging has an essential rule in the food manufacture: it increases the life of food, while guarantees protection from physical and microbiological contaminations [1].
In order to improve the packaging materials performances, in recent decades the use of different types of additives has been significant development (plasticizers, stabilizers, etc.) [2][3][4], like vegetable oils, that represent an interesting renewable source for the production of useful chemicals and new materials [5][6][7][8]. In particular, the use of epoxidized soybean oil (ESBO; see Figure 1) has increased in recent years as a valid alternative to phthalates, since it shows relatively good compatibility with PVC and low toxicity to humans [9]. In this respect ESBO is listed as an authorized substance according to food packaging materials legislation (European Commission 2002).
However, a known potential is the migration of ESBO from the packaging into the food, especially oily foods and fats. This risk in food contamination was investigated by the European Food Safety Authority (EFSA), which set a specific migration limit for ESBO in baby foods [10]. Several analytical methods have been reported for the analysis of ESBO in PVC gaskets and food, although they are very timeconsuming and not so user-friendly, especially due to low concentrations of migrants and the complexity of the food matrix [10,11].
The aim of this work was to obtain a selective synthesis of exa-epoxy-three linoleate glyceryl-18 O labelled (2a) in one or multiple positions. This target compound could be used as internal standard in quantitative ESBO analysis, in order to simplify and increase the accuracy of our previous LC-MS/MS method [10].
Despite the fact that the synthesis of ESBO [12] of epoxy esters of unsaturated fatty acids [13] could be obtained by different methods yielding conversion and selectivity higher than 90%, the preparation of its 18 O labelled epoxide is quite difficult [14]. A possibility is to prepare the corresponding peroxide compound labelled R 18 O 18 OR , generated from hydrogen peroxide-18 O-18 O (Scheme 1) [15].
The standard procedure now mentioned is very arduous and delicate if one takes into account the fact that hydrogen peroxide- 18 Figure 1: Structure of (Gly-LLL-ox 2a): one of chemical species in ESBO (from linoleic in sn-1, sn-2, and sn-3). In this context, we have shown that the dioxiranes R 1 R 2 CO 2 (1) [18,19] three-member ring strained peroxides, generated in situ, were able to transfer electrophilic oxygen to a variety of donor substrates (S:), yielding the corresponding oxidation products (SO) and the parent ketone ( Figure 2).
Undoubtedly very attractive applications of these reagents are regio-, stereo-, and enantioselective epoxidations of alkenes. In fact simple [18] or supported ketones [20] and optical active ketones as precursors of chiral dioxiranes in situ [21,22] could be used in organic synthesis.
Further a possible alternative to obtain labelled epoxides consists of using DDO (1a) 18 O labelled, generated in situ, from acetone and potassium monoperoxysulfate ( 18 O-labelled-as reported in Scheme 2 [23].
This procedure requires a careful preparation, since the caroate is also able to give molecular oxygen, with loss of 18 O 2 , as background reaction [19].
In continuation of our work on selective polyepoxidations [28,29] we now report on the epoxidation of glyceryltrilinoleate (2) using the isolated 18 O DDO (1a). Our preliminary results are described herein.
Curox triple salt 2KHSO 5 ⋅KHSO 4 ⋅K 2 SO 4 (a gift by Peroxid-Chemie GmbH, Munich, Germany) was our source of potassium peroxymonosulfate (caroate); it was used as received for the synthesis of dioxiranes 1a. . Dioxiranes in solution of the parent ketones (i.e., acetone for 1a) could be obtained by following a procedure already reported in detail [24,30] starting with buffered (pH 7, NaHCO 3 ) aqueous potassium peroxomonosulfate KHSO 5 and the ketone. A 500 mL fournecked round-bottomed vessel is equipped with a mechanical stirrer, a splash-guard adapter connected to an air-cooled straight condenser, a solids addition funnel, a gas inlet tube extending into the reaction mixture, and a thermometer. The air condenser is connected laterally to the top entry of an efficient jacketed spiral condenser cooled at −70 ∘ C; the bottom exit of this condenser is fitted to a two-way fraction-collector adapter carrying two 50 mL receiving flasks and is also cooled at −70 ∘ C. In succession to the adapter, two cold traps are placed, the first is cooled at −70 ∘ C and the second at liquid nitrogen temperature. The main vessel is charged with 35 mL of bidistilled water, the mixture of 18 O acetone (30 mL) and H 18 2 O (6 mL) Na 2 EDTA 2 (0.1 g), and NaHCO 3 (22 g). Mechanical stirring is initiated,

Preparation of
and the solid potassium peroxomonosulfate (0.189 mol, 66 g of corresponding to 2.86 mmol/g of Caroat triple salt iodometry) is quickly added during 1-2 minutes to the reaction vessel, while passing a gentle stream of Ar (or N 2 ) gas through the mixture. Shortly after the addition is initiated, the end two-way adaptor is switched to insert the main collection flask and during 10 min ca. 25 mL of weak-yellow solution of 1a in acetone is collected. This solution had a 0.083 M concentration in dioxirane (iodometry).

Results and Discussion
Considering remarkable success of methods of epoxidation using dioxirane [19,24,25], we decide to modify opportunely the technique which allowed for the first time the isolation of 18 O-DDO (1a). In view of the fact that we need to start 4 The Scientific World Journal The exchange reaction was monitored by GC-MS, quantifying the two species of acetone, labelled or not, by the signal intensity of the corresponding molecular ion ( / = 58 acetone, / = 60 18 O acetone). Finally we had a labelling for acetone equal to 69%. Dioxirane (1a) could be obtained by the standard procedure (Scheme 3). The only change is the amount of water, because we used the exchange mixture, without further purification.
Also in this case, it is of considerable importance that the dioxirane synthesis reaction must be perfectly buffered The Scientific World Journal 5 with NaHCO 3 , in order to minimize the loss of 18 O-acetone labelling due to water exchange process (acid and base catalyzed). Anyway, the rate of formation of dioxirane is faster than the exchange reaction with water, thanks to the high nucleophilicity of peroxide ((2) alpha effect) [32]: Before proceeding with the polyepoxidation of the triglyceride, in order to assess the amount of labelled of 18 O-DDO (1a), it appears necessary to explore its efficacy in the labelled reaction, as both 16 O and 18 O oxygen atoms of the dioxirane can be transferred to substrate [23]. We chose as a target reaction the oxidation of methyl-ptolyl-sulfide (3), a transformation well known for its efficiency [24]. The level of labelling on the corresponding sulfoxide (3a) was determined by GC-MS analysis, considering the increase of the ratio of the signal intensity M + 2/M (where M is the molecular ion), compared to the same ratio obtained by the GC-MS spectrum of a solution of methyl-p-tolyl sulfoxide standard unlabelled. In this case we obtained a labelled sulfoxide (3a) equal to 8% (3) Using the data regarding the oxidation of 3 it is possible to carry out a preliminary theoretical assessment of isotopic enrichment on M + 2 signals relative to our target compound, the 18 O-LLL ox (2a), for which there are six successive processes of oxygen insertion. We used the Bayes formula for the determination of probability to give the percent of labelled compound: , = !/ !( − )! × × ( − ) [33] where = 8/100 is the probability of the elemental event of epoxidation with 18 O, = 92/100 is the probability of the opposite elemental event, = 6, and is the number of 18 O constituent the compound, product of the reaction, from 0 to 6. Based on these calculations we expected an increase of isotope peak M + 2 of our product equal to 32%, and so the theoretical possibility of obtaining a yield of 32% as product (2a) containing a single Atom 18 O. The preliminary oxidation of triglyceride 2 was carried out with unlabelled DDO (1a) in order to optimize the reaction conditions and in particular to determine the best DDO (1a)/substrate ratio required for a complete substrate oxidation; afterwards, the reaction was performed using the 1a labelled.
The easy oxidation procedure involved the stepwise addition (20 min) of standardized dioxirane 1a solution to the glyceryl-three linoleate (2) dissolved in CH 2 Cl 2 (20 mL) and kept at 20 ∘ C. A moderate excess of dioxirane 18 O-DDO (1a) was applied in order to achieve complete substrate conversion as monitored by TLC (Scheme 4).
The GC-MS analysis of the reaction mixture showed that a significant residual of labelled acetone is still present. The precious acetone 18 O-enriched was recovered by a careful distillation of the crude reaction.
The polyepoxide 2a (oil) was fully characterized by 1 Hand 13 C-NMR, yielding spectra in complete agreement with the given structure, and the 1 H NMR spectrum of 2a shows inter alias the multiple at 2.90-3.08 (m, 12H) due to the C-H resonances of the epoxy ring.
In addition, the MALDI-TOF spectrum confirm the structure of the compound (noted as adducts Na + signal at / = 997.65) and also determine the isotopic enrichment of the M + 2 signal ( / = 999.66). We found that it is equal to 42%, and this is in agreement with the previously calculated according to the theory of probability (Figure 3).
Data that are representative of the enrichment attainable in the direct dioxirane oxidation of different substrates are collected in Table 1. These show that, using the procedure reported herein, labelled polyepoxide 2a can be synthesized up to 42% labelled yield.
In summary, by the easily prepared 18 O dimethyldioxirane (1a), natural products as glyceryl-three linoleate can be cleanly converted to the corresponding labelled epoxide in mild conditions. The high efficiency and selectivity of these oxidations provide highly pure products, which can be used directly in subsequent reaction steps, avoiding costly and 6 The Scientific World Journal time-consuming product purification procedures. Besides the oxidant precursor, the expensive acetone 18 O labelled can be recovered and reused for dioxirane regeneration, thus increasing the atom economy of the process.
In conclusion, we believe that these results contribute to reinforce the notion that the application of dioxiranes efficiently provides access to useful new application not only in organic synthesis but also for the setup of new analytical methods.