Structural characterization by Nuclear Magnetic Resonance of ozonized triolein

In the present study ozonized triolein with 739 mmolequiv/ kg peroxide index is characterized by NMR.The triolein and ozonized triolein show very similar 1H NMR spectra except for the resonances at δ 9.74 ppm, which correspond to aldehydic protons and δ 5.14 ppm (ozonides methylic protons). Other new signal assignments are based on the connectivities provided by the proton scalar coupling constants δ 2.41 ppm (methylenic group allylic to aldehydic protons and ozonides methynic protons) and δ 1.67 ppm (methylenic protons in position with respect to ozonides methylic protons). From the 13 C and 1 H- 13 C spectrum of the ozonized triolein, the presence of ozonides was confirmed by the signals δ 104.2 and 104.3 ppm, respectively. Other new signals in δ 43.9 ppm confirm the presence of methylenic carbon ozonides. From the structural elucidation of ozonated triglycerides, relevant chemical information about ozonated vegetable oil can be found .


INTRODUCTION
Most of the fatty acids in vegetable oil are present as triacylglycerols (triglycerides).The triolein is one of the components of vegetable oils and lipids (Vajda, 1976;Pryor, 1995).
The analytical tools which have been available in recent years include chromatographic techniques.The analysis of triacylglycerol by liquid chromatography is one of the most difficult applications because of the physicochemical properties of these oils such as their thermal instability due to the presence of unsaturated chains.Nowadays, the high-resolution nuclear magnetic resonance (NMR) spectroscopy is a technique used to provide valuable information about the acyl distribution of triacylglycerols of different vegetable oils (.Mannina, 1999;Vlahov, 1998).However, information on the characterization by NMR spectroscopic of the products generated by ozonolysis of triolein is limited.
The reaction of ozone with unsaturated compounds takes place through the known Criegee mechanism (Criegee, 1975;Pryor, 1991).This consists of: an electrophilic attack of ozone at the carbon-carbon double bond, resulting in the 1,2,3 trioxolane or primary ozonide, which rapidly decomposes to give a carbonyl oxide or zwitterion and a carbonylic compound (aldehyde or ketone).In nonparticipating organic solvents, these last two species recombine to give the Criegee ozonides.If water is present, a part of carbonyl oxide reacts with them giving hydroxyhydroperoxides, hydrogen peroxide, and aldehyde and the other carbonyl oxide part recombines with aldehydes to form the Criegee ozonides, but in a smaller amount compared with the reaction in organic solvents (Rietjens, 1987;Freeman, 1979).
Triacylglycerols represent difficult compounds to be studied, due to the presence of three different reaction sites of glycerol backbone which can be esterified with different fatty acids thus producing a complex mixture.
Due to triolein has the same esterified fatty acid in the third position, it was chosen as a model compound to be ozonized.In this study, the products of ozonized triolein were analyzed at a peroxide value of 739 mmol-equiv/kg applying 1 H, 13 C and 2D COSY NMR spectroscopy.

General ozonization procedure
Triolein (3 mL) were introduced into a reactor with bubbling ozone gas and placed in a water bath at room temperature.The reaction with ozone was continued for 8 minutes at applied ozone doses of 147.7 mg/g.Ozonized triolein was stored between 2-8 0 C before further analysis for NMR.

Ozone generation
Ozone was generated by passing oxygen through a 12-02 model ozone generator from the Trailigaz Company (France) at a fixed voltage (170 V) and a constant flow rate of 42 L/h.The resulting initial ozone concentration was 69 mg L -1 determined by an Ozomat model equipment from the Anseros Company (Germany).

Measurement of NMR spectra
1 H, 13 C, DEPT 135 and 2D COSY NMR spectra were obtained in a BRUKER 9.4 Tesla AVANCE Spectrometer with CDCl 3 as solvent and tetramethylsilane (TMS) as internal reference.The 1 H NMR spectra were obtained using a 5 KHz spectral width, 60 degree pulse width (5 ms), 8 scans, and 64 Kbytes of memory . 13C NMR spectra were recorded operating at 100 MHz and were obtained using the following acquisition parameter: 64 K acquisition point; spectral width 220 ppm; relaxation delay, 2 s; a total of 800 scans were collected for the sample with a 45 0 excitation pulse.The experiment (distortionless enhancement by polarization transfer, DEPT) was carried out using variable pulse θ ϭ 135 0 .The 2D 1 H-1 H correlation spectroscopy (COSY) and Heteronuclear Simple Quantum Correlation (HSQC) spectra were obtained with a digital resolution of 5.425 Hz after zero filling.Zero filling (one) was done in the F1 dimension of a 512 x 512 matrix, the data were 2D transformed, and the magnitude spectra multiplied by a sine window in each dimension and made symmetrical along the diagonal (Croasmun, 1987).

Determination of the peroxide index (IP)
The peroxide index represents the number of mmol-equivalents of active oxygen which expresses the amount of peroxide contained in 1 000 g of the substance.Briefly, 5 g of sample were mixed with 30 volumes of glacial acetic, 20 volumes of chloroform and 0.5 mL of saturated potassium iodine solution.The mixture was shaken for exactly 1 minute, mixed with 30 mL of water and slowly titrated; shaking continuously, with 0.01 M sodium thiosulphate until the yellow color almost disappeared.The peroxide index values were obtained from the expression 10v/w, where v is the volume of sodium thiosulphate in mL consumed in the titration, and w is the weight in g of substance taken (B.P., 2000).The peroxide index (PI) was expressed in mmol-equiv/kg.

RESULTS AND DISCUSSION
The aim of this study was to assess the products of ozonized triolein, specifically, oleic acids from glycerol backbone.Since vegetable oils consist almost entirely of triglyceride molecules, no appreciable difference was anticipated between the chemical shift values of these vegetable oils and their component fatty acids (Woodbury, 1998).In our experiment for preparation of ozonated triolein, about 147.7 mg/g of ozone was absorbed per 3 mL of triolein which seemed to be enough to obtain ozonized triolein with 739 mmol-equiv/kg of peroxide index.Scheme 1 shows that possible ozonides, and aldehydes can be obtained in ozonized triolein.For a better explanation, table I shows the chemical shift and probable structures of the functional group of these oxygenated compounds.
Figure 1a shows the 1 H NMR spectrum from triolein, presenting a single peak at δ 7.2 ppm which belongs to the chloroform-d, multiplet peaks at δ 5.3 ppm which belong to olefinic signals from the fatty acid and, δ 5.2 ppm (triplet from methynic group of glycerol in sn-2 position).Other signals present are δ 4.1 to δ 4.2 ppm (doublets belonging to glycerol protons in sn 1,3 position); δ 2.3 ppm (triplet from methylenic groups in α position with respect to carbonylic group); δ 1.9 ppm (multiplet from methylenic group in both sides of olefinic protons); δ 1.6 ppm (multiplet from methylenic group in β position with respect to carbonylic group); δ 1.2 ppm (signal from methylenic groups in fatty acid chain); and δ 0.87 ppm (triplet from terminal methyl group).These signals coincided with those reported in the NMR study of olive oil (Vlahov, 1999).
The 1 H NMR spectrum from ozonated triolein is displayed in figure 1b.This spectrum has the same observed signals in triolein (figure 1a) and additionally other eight signals at δ 9.7 ppm y ppm (triplet from aldehydic protons), δ 5.1 ppm (multiple from ozonides), δ 2.41 ppm, δ 1.67 ppm, and δ 1.39 ppm (multiple from formed ozonides protons), see table 1.These additionally formed signals are oxygen compounds responsible for the germicide effect of coconut oil and ozonized sunflower oils (Díaz, 2001;Díaz, 2005a).The proton signals of olefinic double bond, ozonides and glycerol in the sn-2 and sn-1,3 positions can be observed in figure 2 with the wide spectral range of 3.6-5.4ppm.The  (Díaz, 1997;Díaz, 2003), but they did not disappear completely.This result indicates that some double bonds did not react with ozono.For this reason, Scheme 1 shows how possible ozonides, and aldehydes can be obtained in ozonized triolein with 739 mmol-equiv/kg of peroxide index.Another aspect observed in the figure 2 is the assignation of doublets signals belonging to glycerol in sn-1,3 positions at δ 4.14 to δ 4.29 ppm.These signals presented the same value of 2 J coupling constant 11.89 Hz.This result demonstrates that both chemical shifts correspond to the same methylenic signals in position sn-1,3 of glycerol (Table 1).
The 13 C NMR spectrum of triolein contains the resonance of carbons from the triglyceride fraction of vegetable oil, i.e. the fatty acid resonance (Vlahov, 1998), (Wollenberg, 1990).The different signals are resolved on the basis of chain double bond number: oleoyl chains (C18:1 9c).The chemical shift can be predicted by the additive relationship for the normal alkanes based upon the number of α, β and γ carbon atoms in the molecule (Giovanna, 1998).Figure 3 shows the 13 C and DEPT NMR spectra from ozonized triolein.The terminal methyl carbon shift of three chains C-18 is found at δ 14.1 ppm.Three methylene groups are readily identified: C-17 at δ 22.6 and δ 22.1 ppm methylenic acylic chains; C-3 at δ 24.7 ppm methylenic group in β position with respect to the carbonylic group; C-11(O) and C-8(O) at δ 27.1 ppm allylic carbons of oleoyl chains; δ 28.9-29.7 ppm methylenic groups in fatty acid central chain; C-16 at δ 31.8 methylenic acylic chains ω.The composition of mono-, di-and triglycerides was determined by means of the glycerol carbon resonance (Vlahov, 1996).The 1(3)-and 2-glycerol carbons of triglycerides resonate at δ ϭ 62.1 ppm and δ ϭ 68.9 ppm, respectively.The resonances C-9 and C-10 at δ 129.7 ppm and δ 130 ppm corresponding to oleoyl unsaturated carbons between 2-and 1(3) positions of glycerol and carbonyl carbons at δ 172.8 ppm belonging to 2glycerol chain positions and δ 173.2 ppm belonging to 1(3) glycerol chain positions were also identified.1).
The DEPT experiment was applied to obtain 13 C NMR spectra over the whole carbon-13 frequency range with the purpose of producing 13 C NMR resonance for a more precise interpretation.The only drawback was the loss of carbonyl carbon resonance, which is not detected by the DEPT sequence (Freeman, 1988).
The assignments of the various signals were accomplished by using a combination of 2D COSY techniques 1 H-1 H and HSQC (Figure 4 to 7).To explain the detailed interpretation of the 1 H-1 H NMR COSY spectra, the 1 H NMR spectrum showed four new signals: δ 5.1 ppm, δ 2.41 ppm, δ 1.67ppm and δ 1.39 ppm.From the 1 H-1 H NMR COSY correlation spectrum (Figure 4), it was clear that the multiplet a δ 2.41 ppm (from methylenic group) was correlated to the δ 9.7 ppm (aldehydic protons) and δ 5.1 ppm from ozonides was correlated to the δ 2.41 ppm, δ 1.67 and δ 1.39 ppm (multiplet from formed ozonides protons) (Figure 5).Also in figure 6 the 1 H-1 H NMR COSY spectra in the range the 4-6 ppm can be observed, where the triplet from methynic group of glycerol in sn-2 position at δ 5.2 ppm is correlated with the doublets belonging to glycerol protons in sn 1,3 position at δ 4.1 to δ 4.2 ppm.This result achieved one unequivocal assignment of glycerol signals.
In the structural elucidation of the ozonized triolein (Table 1), the assignments of various signals (ozonation products) were accomplished by using a combination of 2D COSY techniques ( 1 H-1 H and HSQC) with 1 H and 13 C NMR spectra.In this study the ozonation products were well characterized as ozonides and aldehydes present in ozonized triolein.The ozonides are oxygenated compound with biological activity which has been demonstrated by (Diaz, 2001).The elucidation and chemical characterization of this ozonized model compound is important for new ozonation strategies with vegetable oil.

ACKNOWLEDGEMENT
We wish to thank CYTED and CITMA-CNPq for providing financial support.Appreciation is expressed Scheme 1. Oxygenated compounds obtained of the reaction of ozone with triolein.
Figure 3. 13 C and DEPT 135 NMR spectra of ozonized triolein in CDCl 3 in a 9.4 Tesla equipment.

Table 1 Chemical shift of group functional of ozonized triolein. No. Señal Functional Groups Structures Chemical Shift Chemical Shift δ 1 H (ppm) δ 13 C (ppm)
1 and R 2 can be the represented structures in scheme 1 for ozonides and aldehydes.
R STRUCTURAL CHARACTERIZATION BY NUCLEAR MAGNETIC RESONANCE OF OZONIZED TRIOLEIN