Characterization by NMR of Ozonized Methyl Linoleate

No presente estudo o linoleato de metila ozonizado com índice de peróxidos de 1.800 mmol-equiv. kg foi caracterizado quimicamente. Os efeitos do ozônio sobre o linoleato de metila produz hidroperóxidos, ozonideos e aldeídos, os quais foram identificados por técnicas de ressonância magnética nuclear de H e C, monoe bidimensional. O linoleato de metila padrão e o linoleato de metila ozonizado mostram espectros muito similares, excetuando os valores de ressonância (δ 9,7 e δ 9,6) que correspondem aos hidrogênios aldeídicos (δ 5,7 e δ 5,5) e olefínicos de hidroperóxidos (δ 5,2). Outras atribuições estão baseadas nas conectividades fornecidas pelas constantes de acoplamento. Estes resultados indicam que a espectrometria de RMN pode oferecer informação valiosa a respeito da quantidade de compostos oxigenados formados à partir do linoleato de metila ozonizado para o uso em ozonoterapia e na química de óleos vegetais ozonizados.


Introduction
Linoleic acid is one of the most important components of vegetable oils and lipids. 1 The determination of molecular structure of ozonized fatty acids is one of aim of scientists which work with ozonized vegetable oils and ozone therapy. 2 Many studies have been carried out to develop new analytical techniques that, with very little or any manipulation of the sample, can afford the identification of the vegetable oil and lipids. 3,4Specifically, one spectroscopic technique with a high potential in this field is High-Resolution Nuclear Magnetic Resonance Spectroscopy.Both 1 H and 13 C NMR have already been employed in the analysis of vegetable oil and biological samples such as lipids. 5,6However, data on the spectra of ozonized fatty acids is scarce in the literature.
The reaction of ozone with vegetable oil and lipids occurs almost exclusively with the carbon-carbon double bonds present in unsaturated fatty acids.8][9] The mechanism of this reaction is well known (Criegee mechanism, Figure 1), as well as the conditions necessary to enhance the preferential formation of any of these oxygenated compounds. 10,11f all the natural fatty acids, linoleic acid is one of the most widely distributed and is present in practically all lipids.For this reason, methyl linoleate was chosen as a model compound to be ozonized and chemically

Experimental
General ozonization procedure A mixture of 1.6 mL (0.0048 mol L -1 ) of methyl linoleate (99%) and 0.16 mL of water were introduced into a bubbling reactor where ozone reaction took place at room temperature. 7,8The reaction was continued during 7.25 minutes and one sample was taken at applied ozone doses 245 mg g -1 .The samples were stored at -80 °C until NMR analysis.Methyl linoleate were purchased from Sigma Chemical Co.(St.Louis, MO).

Ozone generation
Ozone was generated by passing oxygen through a 12-02 model ozone generator of Trailigaz Company (France) at a fixed voltage (170 V) and constant flow rate of 42 L h -1 .The initial ozone concentration (69 mg L -1 ) was determined by an Ozomat model equipment of 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 at 5 kHz spectral width, 60 degree pulse width (5 μs), 8 scans, and 64 kbytes of memory were used to obtain the spectra. 13C NMR spectra were recorded operating at 100 MHz and were obtained using the following acquisition parameter: 64 k of acquisition point; spectral width 220 ppm; relaxation delay, 2 s; a total of 800 scans was collected for sample with a 45° excitation pulse.The experiment (distortionless enhancement by polarization transfer, DEPT) were obtained using variable pulse θ = 135°.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) were done in the F1 dimension of a 512×512 matrix, the data were 2D transformed and the magnitude spectra multiplied by a sine window in each dimension and symmetrized along the diagonal. 12

Peroxide index determination
The peroxide index represents the number of mmolequivalents of active oxygen that expresses the amount of peroxide contained in 1.000 g of the methyl linoleate.Briefly, a sample of 5 g was mixed with 30 volumes of glacial acetic, 20 volumes of chloroform and 0.5 mL of saturated potassium iodide solution.The mixture was shacked for exactly 1 minute, mixed with 30 mL of water and slowly titrated; shaking continuously, with 0.01 mol L -1 sodium thiosulphate until the yellow color almost disappears.The peroxide index values were obtained from the expression 10 v/m where v is the volume of sodium thiosulphate in mL consumed in the titration, and m is the weight in g, of substance taken. 13The peroxide index (PI) was expressed in mmol-equiv.kg -1 .

Results and Discussion
The aim of this study was to characterize oxygenated products in ozonized methyl linoleate.Since vegetable oils and lipid consist almost entirely of triglycerides molecules, no appreciable difference was anticipated between the chemical shift values of these vegetable oils and their component esterified fatty acids. 14In our experiment for preparation of ozonized methyl linoleate, about 245 mg g -1 of ozone was absorbed per 1.6 mL of methyl linoleate which seemed to be enough to obtain ozonized methyl linoleate with 1,800 mmol-equiv.kg -1 of peroxide index.Scheme 1 shows possible ozonides, hydroperoxides and aldehydes can be obtained in ozonized methyl linoleate.
Figure 2 shows 1 H NMR spectrum from methyl linoleate, presenting a single peak at δ 7.3 which belongs to the chloroform-d, multiplet peaks at δ 5.3 which belong to olefinic signals from the fatty acid, δ 3,6 (single peak from methylic hydrogen of ester), δ 2.7 (triplet from methylenic group between olefinic hydrogen); δ 2.3 (triplet from methylenic groups in α position with respect to carbonylic group); δ 2.0 (multiplet from methylenic group in both sides of olefinic hydrogen); δ 1.6 (multiplet from methylenic group in β position with respect to carbonylic Scheme 1. Oxygenated compounds obtained of the reaction of ozone with methyl linoleate.group); δ 1.3 (signal from methylenic groups in fatty acid chain); and δ 0.9 (triplet from terminal methyl group). 1 H NMR spectrum from ozonized methyl linoleate is displayed in Figure 3.This spectrum has the same observed signals in methyl linoleate (Figure 2) and additionally other eight signals at δ 9.7 and δ 9.6 from aldehydic hydrogen, δ 5.7 and δ 5.5 (olefinic hydrogen signal from hydroperoxides), δ 5.2 (multiplet from ozonides), δ 3.2 (doublets from methylenic hydrogen alilic of olefinic hydrogen), δ 2.4 and δ 1.62 (multiplet from formed ozonides hydrogen).[17] The 13 C NMR spectrum of methyl linoleate contains resonance similar of carbons from the triglyceride fraction of vegetable oil, i.e., the fatty acid signals. 18,19However a new group of signals were found in the spectrum from ozonized methyl linoleate (Figure 4).The 13 C NMR espectrum are grouped in four sets of signals, aldehyde carbons resonating from δ 199.0 to δ 203.0, carbonyl carbon resonance from δ 174.4,The terminal methyl carbon shift C-18 is found at δ 14 ppm.Four methylene groups are readily identified: C-17 at δ 22.5 methylenic acylic chains; C-3 at δ 24.9, δ 24.3 and δ 24.2 methylenic group in β position with respect to carbonylic group; C-11 at δ 25.6 methylenic group between olefinic hydrogen of linoleic chains; C-11, C-14, and C-8, at δ 27.1, δ 27.5 allylic carbons of linoleic chains; δ 29.0-29.5 methylenic groups in fatty acid central chain; C-16 at δ 31.5 methylenic acylic chains ω; C-2 at δ 34.0 linoleic as a single signal but well resolved from saturated chains.New signals were observed at δ 24.9, δ 24.3, and δ 24.2 belong techniques 1 H-1 H and HSQC with 1 H and 13 C NMR spectra.In this study all functional groups of the ozonation products were well characterized as ozonides, hydroperoxides and aldehydes present in ozonized methyl linoleate.These reaction products were identified according to Criegee mechanism. 10The ozonides of methyl oleate have been considered compounds with biological activity, which has been demonstrated by Díaz et al. 15 The elucidation and chemical characterization of reaction products from ozonized methyl linoleate are important for ozone therapy follow up and new ozonation strategies with vegetable oils.

Figure 1 .
Figure 1.Mechanism of reaction of ozone with unsaturated fatty acids.