Toward the prediction of the activity of antioxidants: Experimental and theoretical study of the gas-phase acidities of flavonoids

  • Hugo F. P. Martins
  • J. Paulo Leal
  • M. Tereza Fernandez
  • Victor H. C. Lopes
  • M. Natália D. S. Cordeiro


The relative gas-phase acidities were determined for eight flavonoids, applying the kinetic method, by means of electrospray-ion trap mass spectrometry. The experimental acidity order, myricetin > luteolin > quercetin > (±)-taxifolin > kaempferol > apigenin > (+)-catechin > (±)-naringenin shows good agreement with the order obtained by theoretical calculations at the B3LYP/6-311 + G(2d,2p)//HF/6-31G(d) level. Moreover, these calculations provide the gas-phase acidities of the different OH groups for each flavonoid. The calculated acidity values (ΔacH), corresponding to the most favorable deprotonation, cover a narrow range, 314.82013;330.1 kcal/mol, but the experimental method is sensitive enough to differentiate the acidity of the various flavonoids. For all the flavones and the flavanol, catechin, the 4′-hydroxyl group is the most favored deprotonation site whereas for the flavanones studied, taxifolin and naringenin, the most acidic site is the 7-hydroxyl group. On the other hand, the 5-hydroxyl, in flavones and naringenin, and the 3-hydroxyl, in taxifolin and catechin, are always the less acidic positions. The acidity pattern observed for this family of compounds mainly depends on the following structural features: The ortho-catechol group, the 2,3 double bond and the 4-keto group.


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  1. 1.
    Jovanovic, S. V.; Steenken, S.; Simic, M. G.; Hara, Y. Antioxidant Properties of Flavonoids: Reduction Potentials and Electron Transfer Reactions of Flavonoid Radicals In Flavonoids in Health and Disease; Rice-Evans, C.; Packer, L., Eds.; Marcel Dekker: New York, 1998; p 137.Google Scholar
  2. 2.
    Brown, J. E.; Khodr, H.; Hider, R. C.; Rice-Evans, C. A. Structural Dependence of Flavonoid Interactions with Cu2+ Ions: Implications for Their Antioxidant Properties. Biochem. J. 1998, 330, 1173–1178.Google Scholar
  3. 3.
    van Acker, S. A. B. E.; van den; Berg, D. J.; Tromp, M. N. J. L.; Griffaen, D. H.; van Bennekom, W. P.; van Vijgh, W. J. F.; Bast, A. Structural Aspects of Antioxidant Activity of Flavonoids. Free Rad. Biol. Med. 1996, 20, 331–342.CrossRefGoogle Scholar
  4. 4.
    Mira, L.; Fernandez, M. T.; Santos, M.; Rocha, R.; Florêncio, M. H.; Jennings, K. R. Interactions of Flavonoids with Iron and Copper Ions: A Mechanism for Their Antioxidant Activity. Free Rad. Res. 2002, 36, 1199–1208.CrossRefGoogle Scholar
  5. 5.
    Fernandez, M. T.; Silva, M. M.; Mira, L.; Florêncio, M. H.; Gill, A.; Jennings, K. R. Iron and Copper Complexation by Angiotensin-Converting Enzyme Inhibitors. A Study by Ultraviolet Spectroscopy and Electrospray Mass Spectrometry. J. Inorg. Biochem. 1998, 71, 93–98.CrossRefGoogle Scholar
  6. 6.
    Fernandez, M. T.; Mira, L.; Florêncio, M. H.; Jennings, K. R. Iron and Copper Chelation by Flavonoids—An Electrospray Mass Spectrometry Study. J. Inorg. Biochem. 2002, 92, 105–111.CrossRefGoogle Scholar
  7. 7.
    Hotta, H.; Sakamoto, H.; Nagano, S.; Osakai, T.; Tsujino, Y. Unusually Large Numbers of Electrons for the Oxidation of Polyphenolic Antioxidants. Biochim. Biophys. Acta 2001, 1526(159), 167.Google Scholar
  8. 8.
    Nakanishi, I.; Miyazaki, K.; Shimada, T.; Ohkubo, K.; Urano, S.; Ikota, N.; Ozawa, T.; Fukuzumi, S.; Fukuhara, K. Effects of Metal Ions Distinguishing Between One-Step Hydrogen and Electron-Transfer Mechanisms for the Radical-Scavenging Reaction of (+)-Catechin. J. Phys. Chem. A 2002, 106, 11123–11126.CrossRefGoogle Scholar
  9. 9.
    Santos, R. M. B.; Simões, J. A. M. Energetics of the O-H Bond in Phenol and Substituted Phenols: A Critical Evaluation of Literature Data. J. Phys. Chem. Ref. Data 1998, 27, 707–739.CrossRefGoogle Scholar
  10. 10.
    Lias, S. G.; Bartmess, J. E.; Liebman, J. F.; Holmes, J. L.; Levin, R. D.; Mallard, G. W. Gas-Phase Ion and Neutral Thermochemistry. J. Phys. Chem. Ref. Data 1988, 17, Suppl. 1.Google Scholar
  11. 11.
    Silva, M. M.; Santos, M. R.; Caroço, G.; Rocha, R.; Justino, G.; Mira, L. Structure-Antioxidant Activity Relationships of Flavonoids: A Re-examination. Free Rad. Res. 2002, 36, 1219–1227.CrossRefGoogle Scholar
  12. 12.
    Cooks, R. G.; Patrick, J. S.; Kotiaho, T.; McLuckey, S. A. Thermochemical Determinations by the Kinetic Method. Mass Spectrom. Rev. 1994, 13, 287–339.CrossRefGoogle Scholar
  13. 13.
    Zhang, K.; Zimmerman, D. M.; Chung-Phillips, A.; Cassidy, J. C. Experimental and ab Initio Studies of the Gas-Phase Basicities of Polyglycines. J. Am. Chem. Soc. 1993, 115, 10812–10822.CrossRefGoogle Scholar
  14. 14.
    Gorman, G. S.; Speir, J. P.; Turner, C. A.; Amster, I. J. Proton Affinities of the 20 Common α-Amino-Acids. J. Am. Chem. Soc. 1992, 114, 3986–3988.CrossRefGoogle Scholar
  15. 15.(a)
    van Acker, S. A. B. E.; Bast, A.; van der Vijgh, W. J. F. The Structural Aspects in Relation to Antioxidant Activity of Flavonoids. Antioxidant in Health and Disease; Rice-Evans, C. A.; Packer, L., Eds.; Marcel Dekker: New York, 1998; 221–251.Google Scholar
  16. 15.(b)
    Rice-Evans, C. A.; Miller, N. J.; Paganga, G. Structure-Antioxidant Activity Relationships of Flavonoids and Phenolic Acids. Free Rad. Med. 1996, 20, 933–956.CrossRefGoogle Scholar
  17. 15.(c)
    Zhang, H. Y. Theoretical Methods Used in Elucidating Activity Differences of Phenolic Antioxidants. J. Am. Oil Chem. Soc. 1999, 76, 745–748.CrossRefGoogle Scholar
  18. 16.
    van Acker, S. A. B. E.; de Groot, M. J.; van den Berg, D. J.; Tromp, M. N. J. L.; den Kelder, D.-O. p. G.; Wim, J. F.; van der Vijgh, W. J. F.; Bast, A. A Quantum Chemical Explanation of the Antioxidant Activity of Flavonoids. Chem. Res. Toxicol. 1996, 9, 1305–1312.CrossRefGoogle Scholar
  19. 17.
    Meyer, M. Ab Initio Study of Flavonoids. Int. J. Quant. Chem. 2000, 76, 724–732.CrossRefGoogle Scholar
  20. 18.
    Alemán, C. Acid/Base Properties of Flavonoids Hydroxylated at Positions 2 and 3: A Novel Quantum Mechanical Study in the Gas-Phase and Solution. J. Mol. Struct. (Theochem.) 2000, 528, 65–73.CrossRefGoogle Scholar
  21. 19.
    Lau, K. S.; Mantas, A.; Chass, G. A.; Ferretti, F. H.; Estrada, M.; Zamarbide, G.; Csizmadia, I. G. Ab Initio and DFT Conformational Analysis of Selected Flavones: 5,7-Dihydroxyflavone and 7,8-Dihydroxyflavone. Can. J. Chem. 2002, 80, 845–855.CrossRefGoogle Scholar
  22. 20.
    Vasilescu, D.; Girma, R. Quantum Molecular Modeling of Quercetin-Simulation of the Interaction with the Free-Radical t-BuOO·. Int. J. Quant. Chem. 2002, 90, 888–902.CrossRefGoogle Scholar
  23. 21.
    Wright, J. S.; Johnson, E. R.; DiLabio, G. A. Predicting the Activity of Phenolic Antioxidants: Theoretical Method, Analysis of Substituent Effects, and Application to Major Families of Antioxidants. J. Am. Chem. Soc. 2001, 123, 1173–1183.CrossRefGoogle Scholar
  24. 22.
    Cren-Olivé, C.; Hapiot, P.; Pinson, J.; Rolando, C. Free Radical Chemistry of Flavan-3-ols: Determination of Thermodynamic Parameters and of Kinetic Reactivity from Short (ns) to Long (ns) Time Scale. J. Am. Chem. Soc. 2002, 124, 14027–14038.CrossRefGoogle Scholar
  25. 23.(a)
    Zhang, H. Y.; Sun, Y. M.; Wang, X. L. Substituent Effects on O-H Bond Dissociation Enthalpies and Ionization Potentials of Catechols: A DFT Study and Its Implications in the Rational Design of Phenolic Antioxidants and Elucidation of Structure-Activity Relationships for Flavonoid Antioxidants. Chem. Eur. J. 2003, 9, 502–508.CrossRefGoogle Scholar
  26. 23.(b)
    Zhang, H. Y.; Wang, L. F.; Sun, Y. M. Why B-Ring is the Active Center for Genistein to Scavenge Peroxyl Radical: A DFT Study. Bioorg. Med. Chem. Lett. 2003, 13, 909–911.CrossRefGoogle Scholar
  27. 24.
    Hussain, H. H.; Babic, G.; Durst, T.; Wright, J. S.; Flueraru, M.; Chichirau, A.; Chepelev, L. L. Development of Novel Antioxidants: Design, Synthesis, and Reactivity. J. Org. Chem. 2003, 68, 7023–7032.CrossRefGoogle Scholar
  28. 25.
    Gronet, S. Estimation of Effective Ion Temperatures in a Quadrupole Ion Trap. J. Am. Soc. Mass Spectrom. 1998, 9, 845–848.CrossRefGoogle Scholar
  29. 26.
    Bier, M. E.; Schwartz, J. C. Electrospray-Ionization Quadrupole Ion-Trap Mass Spectrometry. Electrospray Ionization Mass Spectrometry; Cole, R. B., Ed.; John Wiley & Sons: Chichester, 1997; 241.Google Scholar
  30. 27.
    Foresman, J. B.; Frisch, M. J. Exploring Chemistry with Electronic Structure Methods; Gaussian Inc: Pittsburgh, PA, 1995.Google Scholar
  31. 28.(a)
    Becke, A. D.. J. Chem. Phys. 1993, 98, 5648.CrossRefGoogle Scholar
  32. 28.(b)
    Lee, C.; Yang, W.; Parr, R. G.. Phys. Rev. B 1992, 45, 13244.CrossRefGoogle Scholar
  33. 29.
    DiLabio, G. A.; Pratt, D. A.; LoFaro, A. D.; Wright, J. S. Theoretical Study of X-H Bond Energetics (X = C, N, O, S): Application to Substituent Effects, Gas Phase Acidities, and Redox Potentials. J. Phys. Chem. A 1999, 103, 1653–1661.CrossRefGoogle Scholar
  34. 30.
    Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Zakreswki, V. G.; Montgomery, J. A., Jr.; Stratmann, R. E.; Burant, J. C.; Dapprich, S.; Millam, J. M.; Daniels, A. D.; Kudin, K. N.; Strain, M. C.; Farkas, O.; Tomasi, J.; Barone, V.; Cossi, M.; Cammi, R.; Mennucci, B.; Pomelli, C.; Adamo, C.; Clifford, S.; Ochterski, J.; Petersson, G. A.; Ayala, P. Y.; Cui, Q.; Morokuma, K.; Malick, D. K.; Rabuk, A. D.; Raghavachari, K.; Foresman, J. B.; Cioslowki, J.; Ortiz, J. V.; Stefanov, B. B.; Liu, G.; Liashenko, A.; Piskorz, P.; Komaromi, I.; Gomperts, R.; Martin, R. L.; Fox, D. J.; Keith, T.; Al-Laham, M. A.; Peng, C. Y.; Nanayakkara, A.; Gonzalez, C.; Challacombe, M.; Gill, P. M. W.; Johnson, B. G.; Chen, W.; Wong, M. W.; Andres, J. L.; Head-Gordon, M.; Replogle, E. S.; Pople, J. A. Gaussian 98, Revision A.3; Gaussian Inc: Pittsburgh, PA, 1998.Google Scholar
  35. 31.
    Cooks, R. G.; Koskien, J. T.; Thomas, P. D. The Kinetic Method of Making Thermochemical Determinations. J. Mass Spectrom. 1999, 34, 85–92.CrossRefGoogle Scholar
  36. 32.
    Wu, Z.; Fenselau, C. Gas-Phase Basicities and Proton Affinities of Lysine and Histidine Measured from the Dissociation of Proton-bound Dimers. Rapid Commun. Mass Spectrom. 1994, 8, 777–780.CrossRefGoogle Scholar
  37. 33.
    Armentrout, P. B. Entropy Measurements and the Kinetic Method: A Statistically Meaningful Approach. J. Am. Soc. Mass Spectrom. 2000, 11, 371–379.CrossRefGoogle Scholar
  38. 34.
    Drahos, L.; Vékey, K. How Closely Related are the Effective and the Real Temperature?. J. Mass Spectrom. 1999, 34, 79–84.CrossRefGoogle Scholar
  39. 35.
    Armentrout, P. B. Is the Kinetic Method a Thermodynamic Method?. J. Mass Spectrom. 1999, 34, 74–78.CrossRefGoogle Scholar
  40. 36.
    Afonso, C.; Modeste, F.; Breton, P.; Fournier, F.; Tabet, J. C. Proton Affinities of the Commonly L-aminoacids by Using Electrospray Ionization-Ion Trap Mass Spectrometry. Eur. J. Mass Spectrom. 2000, 6(443), 449.Google Scholar
  41. 37.
    Brodbelt-Lustig, J. S.; Cooks, R. G. Determination of Relative Gas-Phase Basicities by the Proton-Transfer Equilibrium Technique and the Kinetic Method in a Quadrupole Ion-Trap. Talanta 1989, 36, 255–260.CrossRefGoogle Scholar
  42. 38.
    Sykes, P. A Guide Book to Mechanism in Organic Chemistry 6th ed; Longman Scientific & Technical: Harlow, 1986.Google Scholar
  43. 39.
    Russo, N.; Toscano, M.; Uccella, N. Semiempirical Molecular Modeling into Quercetin Reactive Site: Structural, Conformational, and Electronic Features. J. Agric. Food Chem. 2000, 48, 3232–3237.CrossRefGoogle Scholar
  44. 40.
    Conrad, J. P.; Merlin, J. C. Spectroscopic and Structural Study of Complexes of Quercetin with Al(III). J. Inorg. Biochem. 2002, 92, 19–27.CrossRefGoogle Scholar
  45. 41.
    Cody, V.; Luft, J. Conformational Analysis of Flavonoids: Crystal and Molecular Structures of Morin Hydrate and Myricetin (1:2) Triphenylphosphine Oxide Complex. J. Mol. Struct. (Theochem.) 1994, 317, 89–97.Google Scholar
  46. 42.
    Jin, G. Z.; Yamagata, Y.; Tomita, K. I. Structure of Quercetin Dihydrate. Acta Crystallogr. 1990, C46(310), 313.Google Scholar
  47. 43.
    Tóth, J.; Remko, M.; Nagy, M. Structural Study of Flavonoids and Their Protonated Forms. Z Naturforsch C 1996, 51(784), 790.Google Scholar
  48. 44.
    Erkoç, F.; Erkoç, S. Theoretical Investigation of Flavonoids Naringenin and Genistein. J. Mol. Struct. (Theochem.) 2002, 583, 163–167.CrossRefGoogle Scholar
  49. 45.
    M. Preto, A. Melo, unpublished.Google Scholar
  50. 46.
    Agrawl, P. K.; Schneider, H. Deprotonation Induced 13 C NMR Shifts in Phenols and Flavonoids. Tetrahedron Lett. 1983, 24, 177–180.CrossRefGoogle Scholar
  51. 47.
    German, J. B. Nutritional Studies of Flavonoids in Wine. In Flavonoids in Health and Disease; Rice-Evans, C.; Packer, L., Eds.; Marcel Dekker: New York, 1998; p 343.Google Scholar

Copyright information

© American Society for Mass Spectrometry 2004

Authors and Affiliations

  • Hugo F. P. Martins
    • 1
  • J. Paulo Leal
    • 1
    • 2
  • M. Tereza Fernandez
    • 1
  • Victor H. C. Lopes
    • 3
  • M. Natália D. S. Cordeiro
    • 3
  1. 1.Departamento de Química e Bioquímica, Faculdade de CiênciasUniversidade de LisboaLisboaPortugal
  2. 2.Departamento de QuímicaInstituto Tecnológico e NuclearSacavémPortugal
  3. 3.CEQUP/Faculdade de CiênciasUniversidade do PortoPortoPortugal

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