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NMR assignments and the acid–base characterization of the pomegranate ellagitannin punicalagin in the acidic pH-range

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Abstract

In exploring the capability of nuclear magnetic resonance (NMR) spectroscopy for pomegranate juice analysis, the eight aromatic singlet resonances of α- and β-punicalagin were clearly identified in the 1H NMR spectra of juice samples. The four downfield resonances were found to be sensitive to small pH changes around pH 3.50 where the NMR spectra of the juice samples were recorded. To understand this unusual behavior, the 1H and 13C resonance assignments of the punicalagin anomers were determined in aqueous solution and pH titrations with UV and 1H NMR detection carried out to characterize the acid–base properties of punicalagin over the pH range 2–8. Simultaneous fitting of all of the pH-sensitive 1H NMR signals produced similar but significantly different pK a values for the first two deprotonation equilibria of the gallagic acid moiety of the punicalagin α- (pK a1 = 4.57 ± 0.02, pK a2 = 5.63 ± 0.03) and β- (pK a1 = 4.36 ± 0.01, pK a2 = 5.47 ± 0.02) anomers. Equivalent pK a values, (α : 6.64 ± 0.01, β : 6.63± 0.01) were measured for the third deprotonation step involving the ellagic acid group, in good agreement with a prior literature report. The punicalagin anomer equilibrium readjusts in parallel with the proton dissociation steps as the pH is raised such that β-punicalagin becomes the most abundant anomer at neutral pH. The unusual upfield shifts observed for the glucose H3 and H5 resonances with increasing pH along with the shift in the α/β anomer equilibrium are likely the consequence of a conformational rearrangement.

Titration of the punicalagin phenolate protons over the pH range 2–8 results in changes in the aromatic proton chemical shifts and a readjustment of the anomer equilibrium.

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Acknowledgments

Cynthia K. Larive acknowledges support of this work by PomWonderful, LLC. The authors gratefully acknowledge the assistance of Dr. Dan Borchardt with the constant time HMBC experiment.

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Authors and Affiliations

  1. Department of Chemistry, University of California, Riverside, Riverside, CA, 92521, USA

    Márta Kraszni, Attila Marosi & Cynthia K. Larive

  2. Department of Pharmaceutical Chemistry, Semmelweis University, Hőgyes E. u. 9, 1092, Budapest, Hungary

    Márta Kraszni & Attila Marosi

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  1. Márta Kraszni
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  2. Attila Marosi
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Correspondence to Cynthia K. Larive.

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Kraszni, M., Marosi, A. & Larive, C.K. NMR assignments and the acid–base characterization of the pomegranate ellagitannin punicalagin in the acidic pH-range. Anal Bioanal Chem 405, 5807–5816 (2013). https://doi.org/10.1007/s00216-013-6987-x

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  • DOI: https://doi.org/10.1007/s00216-013-6987-x

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