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  • Chicoric acid CA is a di acylated hydroxycinnamoyl tartaric


    Chicoric Penicillin G Sodium (CA) is a di-acylated hydroxycinnamoyl tartaric acid ester [12] found in a variety of plant species, especially within the Astereceae family [13]. Of interest for this study, Sonchus oleraceus and Bidens pilosa plants, from Astereceae family, were used. Both S. oleraceus and B. pilosa plants have been reported to possess pharmaceutical properties aiding against ailments such as inflammation, bacterial infection, diabetes and anti-HIV activities [2,8,11]. The latter could be attributed to CA content. The ability of CA to inhibit activity of the HIV-1 integrase enzyme was a groundbreaking discovery [12,14]. The two fundamental characteristics of CA responsible for HIV-1 integrase inhibition include the two caffeoyl moieties and two free carboxyl groups [15,16]. Furthermore, in the study by Healy et al. [15], the biological activity of CA was noted to be isomer dependent, a phenomenon noted elsewhere with closely related molecules such as dicaffeoylquinic acid [17]. In nature, HCA derivatives are produced with trans configuration. However, exposure to sunlight results in the formation of cis geometrical isomers [7,18,19]. Similarly, CA is synthesized in plants with the trans configuration, and upon UV exposure, geometrical isomerization occurs in the olefin group of the caffeic acid moiety [16]. The stereochemistry of bioactive compounds is important since different isomers are expected to have different pharmacodynamics in humans [20]. Chemically, CA is formed by esterification between one molecule of tartaric acid and two molecules of trans-caffeic acid [16,21,22]. Stereochemically, though CA molecule occurs naturally as 2R, 3R O-dicaffeoyltartaric acid, this molecule also been reported to occur as meso-chicoric acid (2R, 3S O-dicaffeoyltartaric acid) [12]. To date, the identification and differentiation between the commonly occurring isomers (RR and RS) of CA has proven difficult. However, advanced analytical approaches such as liquid chromatography-mass spectrometry (LC-MS) have been applied to distinguish between geometrical isomers of closely related molecules. For instance, closely related geometrical isomers of diCQA can be positively distinguished through preferential metal ion binding [17]. We proposed a simple method based on controlled collision induced fragmentation as a feasible way to distinguish between geometrical isomers of CA [16]. Other advanced analytical methods, such as ion mobility mass spectrometry in combination with in silico modeling, have been used to distinguish between isomers of other plant metabolites [23]. In the current study, a simple LC-MS method based on resolution of UV-induced geometrical isomers was applied to distinguish between stereoisomers of CA. Additionally, DFT models were used to highlight subtle structural differences between the two stereoisomers and generate optimized structures for molecular docking studies with the HIV-1 integrase enzyme.
    Materials and Methods
    Results and Discussion LC–MS has become one of the most common techniques for annotation of plant metabolites, as well as discriminating between different isomers [7,16,18]. Despite analytical breakthroughs, differentiation of CA's stereoisomers by LC-MS still poses an undisputed analytical challenge. As previously stated (Introduction), there are two stereoisomers of CA, with the RR-CA being the more abundant in nature than RS-CA [12]. Structurally, the two stereoisomers of CA differ in the chemistry of the ester bond between the tartaric acid and caffeic acid, with the two caffeoyl moieties on the RR-CA being symmetrical and those of the RS-CA being asymmetrical (Fig. 1). This study found use of UHPLC-MS/MS MRM, in combination with UV-induced geometrical isomerization, allowed for an efficient differentiation between the stereochemistry of the two CA molecules found in S. oleraceus and B. pilosa.
    Conclusion This study successfully demonstrated that chicoric acid found in S. oleraceus can be putatively identified as a naturally occurring RR form, while the one found in B.pilosa is identified as the RS (meso) form. Although these plants are from the same family, both producing L-CA with the same mass and fragmentation pattern, UHPLC-QqQ-MRM in combination with UV-induced geometrical isomerization effectively distinguished between the different forms. Computational modeling results revealed that the RR-CA is more symmetrical and stable when compared to the RS-CA form, owing to the stronger hydrogen bonding interaction sites. In addition, the reported docking studies confirmed that RR-CA not only produces two distinct sets of binding positions, but also binds better than RS-CA. These results can help pave the way for further research into RR-CA's ability to block the HIV-1 integrase enzyme and, ultimately, help fight the spread of the Human Immunodeficiency Virus - a pressing medical, social, and economic problem facing communities around the world.