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.