Masike et al, (2017b), demonstrated that caffeic acid-containing molecules undergo geometrical isomerization under UV light. The trans-CA molecule was shown to undergo isomerization to form two additional peaks representing mono-cis and di-cis isomers (Masike et al, 2017b). However, in a separate study, isomerization of trans di-caffeoylquinic acid (diCQA) resulted in the formation of three additional peaks representing two mono-cis and one di-cis isomers (Makola et al, 2016b; Masike et al, 2018). Interestingly in this study, we found the two mono-cis isomers diCQA were resolvable with LC-MS, whilst those of CA co-eluted. This could be attributed to the structural differences between the two respective anchoring molecules, namely quinic acid and tartaric acid for diCQA and CA, respectively. The hydroxyl groups on the quinic acid have different stereochemistry (asymmetrical), whereas those of the tartaric acid have similar stereochemistry (symmetrical). Hence, the formation of cis isomer on either caffeoyl moiety of chicoric acid (Fig. 4.1A) could not be resolved or distinguished by LC-MS. However, looking at the structure of RS-CA (Fig. 4.1B), the attachment of caffeic acid happens on two OH groups, which are asymmetrical. Thus, the two mono-cis are expected to elute at different retention times.
Indeed, in the current study, extracts of B. pilosa and S. olareceus were subjected to UV light for 3 hrs Photoproducts were evaluated using reverse phase chromatography (RP-HPLC) to assess the formation of cis-geometrical isomers. As seen in Fig. 4.2, extracts from B. pilosa resulted in four geometrical isomers, namely the trans, mono-cis 1, mono-cis 2 and the di-cis isomers. On the other hand, UV-treated extracts of S. oleraceus resulted in only three
94 peaks representing the di-trans, mono-cis and the di-cis isomer (Fig. 4.2). The formation of two distinctive mono-cis isomers from B. pilosa extracts is an indication that the two hydroxyl groups on the tartaric acid are asymmetrical, thus being RS-CA stereoisomer. However, the formation of one broad mono-cis peak on the S. oleraceus leaf extracts indicates that the two hydroxyl groups can be symmetrical, thus being RR-CA stereoisomer.
Figure 4.2: Chromatograms of chicoric acid in A) authentic standard, B) S. oleraceus, C) lettuce, and D) B. pilosa. Co-elution of 3 peaks can be observed at similar retention times for A and B. The elution profile of C shares similar characteristics to A, B and D.
95 The use of UHPLC-MS/MS MRM in combination with UV-induced geometrical isomerization, successfully allowed for an efficient differentiation between the stereochemistry of the two CA molecules found in S. oleraceus and B. Pilosa. To substantiate our findings, MS fragmentation patterns of the resulting isomers were generated. Interestingly, they were found to produce similar fragmentation patterns (Fig. 4.3). Furthermore, RR-CA was also used as an authentic standard to confirm the findings reported and as expected. It also produced two additional isomers (mono-cis and di-cis) post UV-irradiation (Fig. 4.2). Unfortunately, the authentic standard of RS-CA is not commercially available. Methanolic extracts of iceberg lettuce (Lactuca sativa) were used as a surrogate standard (Clifford and Madala, 2017), as meso-CA has been reported to exist in the species (Baur et al, 2004; Luna et al, 2012). The results herein, show that indeed B. pilosa does contain RS-CA (Fig. 4.2).
96 Figure 4.3: Product ion scan (PIS) spectra with a similar fragmentation pattern in A) S. oleraceus and B) B. pilosa. The fragments in both spectra show very similar characteristics, where the major m/z (311, 179, 149 and 135) can be observed in both.
As stated in Chapter 3, both S. oleraceus and B. pilosa belong to the Astereceae family and as such, they are expected to have similar metabolite content. However as seen herein, the
97 stereochemistry of the CA molecules found in these two plants varies. Not much research has been done on the LC-MS based identification of RS-CA probably owing to its misidentification as RR-CA. In another study, the identification of RS-CA was deemed to be by virtue isomerization of RR-CA, which might have occurred during sample extraction and/or purification (Perry et al, 2001). However, we successfully demonstrated that RS-CA is a natural molecule and not a mere structural artifact as shown by Lee and Scagel (2013). From a biological view point, isomers of natural compounds are very important and could exhibit greater biological activities via synergism (Lusa et al, 2016; Makola et al, 2016a). However, there is need to investigate the biological activity of RS-CA as those compared to RR-CA.
4.4.2. Computation modeling for structural stability
The two lowest energy structures, one for RR- CA and the other for RS-CA were obtained from in silico simulations. As noted, the two structures do not only differ with respect to their absolute configurations, but they are also structurally different. It is worth mentioning that with a thorough analysis of the structural features of these two conformers, we were able to establish many facets (Fig. 4.4). One of the interesting features of both RR-CA and RS-CA is the availability of many intramolecular interaction sites that are prominent with very strong intramolecular hydrogen bonding (H-bonding) interactions (Mammino & Kabanda, 2011).
Such interactions seem to have a very strong influence on the differential structural features observed for both conformers.
98 Figure 4.4: Geometry optimized structures of intramolecular and intermolecular H-bonds in the RR-CA (A) and RS-RR-CA (B) conformers. The H-bonds and the corresponding lengths are represented by dotted lines and (Å) respectively.
From the analyses of the H-bonding patterns of the head or crown ends of both conformers, it can be seen that there is H-bonding interactions between the two –COOH units of the
99 tartaric acid and caffeic acid moieties of the RR-CA conformer, but such a situation is completely absent in the RS-CA conformer. These characteristic H-bonding features have a significant role on the stability of the overall molecule and structural symmetry, making RR-CA conformer more symmetrical compared to the RS-RR-CA conformer (Fig. 4.4). Noteworthy, the analysis of the H-bonding distances and the bonding angles of the crown regions of both conformers, indicates that the nature of H-bonding is extremely strong. Besides these strong primary H-bonding interactions, we also observed some secondary H-bonding interactions (also known as bifurcated H-bonding interactions) as earlier noted by Matta et al (2003). For the RS-CA conformer, there was only one bifurcated H-bonding interaction observed, whereas for the RR-CA conformer, two bifurcated H-bonding interactions were observed and can be established to be responsible for its stability (Fig. 4.4). From the analysis of the tail ends of the RR- and RS-CA, it can be seen that in both cases, there are prominent H-bonding interactions between the two vicinal –OH groups present in each arm or tail. Whereas for the RR-conformer, we observed an extra inter-tail H-bonding interaction arising, due to the close proximity of the two tail-end rendering a feasible path for such an interaction. This brings an extra stability not only because of an extra H-bonding site, but also because of the formation of chain like H-bonding interaction site, which can strongly contribute to the exchange energy of the stabilization. Such a chain-like arrangement of H-bonding interactions in the RR- conformer is also capable of making this molecule to attain a cage-like molecular structure.
From the analysis of the structure of the RS-CA conformer, it can be seen that it adopts a V-shaped conformation with extremely large separation between the tail-ends (with the two arms parallel to each other, but separated by a large distance), thus eliminating any possible interaction between the two tail-ends. However from the analysis of the RR-CA conformer, it can be seen that it adopts a more symmetrical orientation of the two arms, with both arms arranged in a pseudo-parallel type of orientation and in close proximity to each other.
Visualizing the positions of the tail-end benzene rings, it was observed they are not exactly on top of one another, rather their planes have been shifted by a distance of >2.8 Å, making it possible for other stabilizing forces such as C-H---p interactions (Nishio et al, 1998). As discussed above, the differential H-bonding patterns observed in the RS-CA and RR-CA conformers are reflected in orienting the two arms of the conformers. Thus, making RR-CA conformer more symmetrical than the RS-CA conformer. The symmetrical nature of the RR-
100 CA conformer can also be well explained by how it is capable of forming only three possible geometrical isomers upon UV-irradiation, whereas the RS-CA molecule is capable of forming four upon UV excitation (Fig. 4.2), owing to its asymmetrical orientation.