3.2.2 Nuclear magnetic resonance
Figure 5 displays the 1H-NMR of the products along with their resonance assignments. Similar to the ATR-FT-IR results, some general observations were seen from this analysis. Specifically, L-arginine ethyl hydrochloride, the starting material has two characteristic peaks noted at about 4.2 and 4.4 ppm that arise from the amine (-CH -NH2, position e ) and the methylene of the ethyl ester (-CO2CH2 CH3, position g ), respectively (spectrum not shown ). Upon formation of the amide bond by addition of the fatty acid, these two peaks have reversed their positions to 4.4 (-CH NH-, positione ) and 4.2 ppm (-CO2CH2 CH3, position g ), due to the deshielding effect of the new bond to the carbonyl group (Fig. 5 ). The 1H spectrum of the ethyl n -lauroyl arginate (Figure 5A) is consistent with the desired product. However, an apparent doubling is noted of the1H peaks associated with positions b and c. Close examination suggests that these likely arise from unreacted starting material, L-arginine ethyl hydrochloride. Since the solvent used for most of these NMR spectra was CDCl3, they also reveal the presence of the NH at 6.8-8 ppm. The NH protons are not observed in the 1H spectrum of ethyl n -stearoyl arginate (Fig. 5E ) due to their exchange with the hydroxyl protons of the methanol solvent. This reduction in NH peak intensity is also seen in the 1H spectrum of L-arginine ethyl hydrochloride, which was measured in D2O.
The 1H NMR spectrum of ethyl iso -oleoyl arginate (Fig. 5B ) is similar to that of ethyl n -lauroyl arginate, however, its 1H spectrum in CDCl3 reveals the presence of the methyl group attached to the ethylene group (CH3 CH=CH, position z’) at 1.58 ppm and ethylene group (CH3CH =CH -, position m ) at 5.38 ppm. The hydrogenated version of the ethyliso -oleoyl arginate, which is ethyl iso -stearoyl arginate spectrum (Fig. 5C ), reveals the evidence of the methyl group (CH3 CHCH2-, position z) at 0.82 ppm. This is upfield since it is not at the olefin position. However, there is clear evidence of olefin at 5.38 ppm, presumably from residual ethyl iso -oleoyl arginate. Although the reaction time of the hydrogenation step was prolonged for 24 hours, the olefin is still difficult to remove. It is also important to point out that the signals in both spectra are fairly broad due to the different isomers of the ethyl iso -oleoyl starting materials, wherein the methyl group is located at different positions of the alkyl chain (Ngo, Hoh, Foglia, 2012). The carbon spectra of ethyl n -oleoyl arginate (Fig. 5D ) and of ethyl n -stearoyl arginate (Fig. 5E ) are fully consistent with the clean desired products and display similar characteristic signals.
Figure 6 shows the 13C-NMR of the products. The 13C spectrum of the ethyl n -lauroyl arginate is fully consistent with the desired product (Fig. 6A ). The 13C spectrum of ethyl iso -oleoyl arginate also indicates the presence of the desired product, but close inspection reveals the likely presence of structural variants (Fig. 6B ). In particular, two ester carbonyl peaks (-C O2CH2CH3,position f ) are observed at 172.64 and 172.16 ppm. Similarly, two peaks are seen at 52.59 and 51.93 ppm arising from position e(-C HNH-). The intensity ratios of these two peaks suggests that the dominant product represents about 75% of the composition. The fact that these two resonances are close to the amide attachment site indicates that this is the location of the variation. It is possible that the smaller component is merely unreacted L-arginine ethyl hydrochloride. The spectra in Figures 6C to 6E of the synthesized arginate are fully consistent with the clean desired products and display similar characteristic signals.