Comptes Rendus Chimie

Parteek Kour

Anil Kumar

Vijai K. Rai

Aqueous microwave-assisted DMAP catalyzed synthesis of β-phosphonomalonates and 2-amino-4H-chromen-4-ylphosphonates via a domino Knoevenagel-phospha-Michael reaction

Rafael Estévez Brito

José Miguel Rodríguez Mellado

Mercedes Ruiz Montoya

Alberto Palma

Rafael Rodríguez-Amaro

Manuel Mayén

lable at ScienceDirect
C. R. Chimie 20 (2017) 365e369Contents lists avaiComptes Rendus Chimie
www.sciencedirect.comFull paper/MemoireSpectroscopic determination of the dissociation constants of
2,4- and 2,5-dihydroxybenzaldehydes and relationships to
their antioxidant activities
Rafael Estevez Brito a, Jose Miguel Rodríguez Mellado a, *,
Mercedes Ruiz Montoya b, Alberto Palma b, Rafael Rodríguez-Amaro a,
Manuel Mayen c
a Departamento de Química Física y Termodinamica Aplicada, Facultad de Ciencias, CeiA3, Campus Universitario Rabanales, edificio
Marie Curie, Universidad de Cordoba, 14014 Cordoba, Spain
b Departamento de Ingeniería Química, Química Física y Química Organica, Facultad de Ciencias Experimentales, Campus de “El
Carmen”, Universidad de Huelva, 21071 Huelva, Spain
c Departamento de Química Agrícola y Edafología, CeiA3, Campus Universitario Rabanales, edificio Marie Curie, Universidad de Cordoba,
14014 Cordoba, Spaina r t i c l e i n f o
Article history:
Received 21 March 2016
Accepted 2 May 2016
Available online 4 June 2016
Keywords:
2,4-Dihydroxybenzaldehyde
2,5-Dihydroxybenzaldehyde
Dissociation constants
UV-spectroscopy
Antioxidant activity* Corresponding author.
E-mail address: jmrodriguez@uco.es (J.M. Rodríg
http://dx.doi.org/10.1016/j.crci.2016.05.004
1631-0748/© 2016 Académie des sciences. Publishea b s t r a c t
UVevisible spectra of 2,4- and 2,5-dihydroxy-benzaldehydes (2,4DHB and 2,5DHB) are
recorded in a wide range of pH. The dissociation pK values obtained from these mea-
surements were 6.94 ± 0.03 and 9.28 ± 0.03 for 2,4DHB and 8.42 ± 0.03 and 10.93 ± 0.03
for 2,5DHB. The results indicate that the pH at which the assays for antioxidant capacity
measurements are made is very important in light of the hydroxyl group dissociation,
because of the different dissociation constants of the different isomers. The percentage of
dissociation of each group is essential, the positions of these groups in the ring appearing
as a secondary factor.
© 2016 Académie des sciences. Published by Elsevier Masson SAS. All rights reserved.1. Introduction (scavenging of a,a-diphenyl-b-picrylhydrazyl free radicals)2,5-Dihydroxybenzaldehyde (2,5DHB) is found in olive
processing effluents [1] and in Beta vulgaris seeds [2]. 2,4-
dihy-droxy-benzaldehyde (2,4DHB) is found in the heart-
wood of Toxicodendron vernicifluum [3], in red wines aged
in acacia barrels [4] or in the aerial parts of Cassia grandis
[5]. Both compounds showed bactericidal activities against
Campylobacter jejuni, Escherichia coli, Listeria mono-cyto-
genes, and Salmonella enteric [6]. They can also act as sen-
sitizers of antifungal agents [7,8] and as antioxidants
[3,9e11].
The antioxidant activity is typically measured far to the
physiological conditions. For example, in the DPPH methoduez Mellado).
d by Elsevier Masson SAS. Athe medium is methanol, and electrochemical measure-
ments using the H2O2 oxidation on mercury electrodes are
made in the pH range of 10e10.5 due to restrictions of the
electrode [9e11].
The dissociation state of phenolic benzaldehydes and
gallic acid derivatives [12] is crucial to understand their
chemical and bacteriological activities that take place
under physiological conditions. However, only one refer-
ence in the literature deals with the value of the higher
dissociation constant of gentisaldehyde [13]. Only the main
maximumwavelengths of the UV spectra in methanol have
been reported for both derivatives [14].
The aim of this paper was, first, to obtain the dissocia-
tion constants of the 2,4DHB and 2,5DHB to show the
relevance of the dissociation of the eOH groups in the
interpretation of the antioxidant activities. To reach thisll rights reserved.
R. Estevez Brito et al. / C. R. Chimie 20 (2017) 365e369366goal, the UV spectra of the aldehydes must be investigated
in order to obtain their dissociation constants and antiox-
idant activities are measured at two pH values.2. Experimental
2,5DHB and 2,4DHB were from Sigma and the rest were
from Merck (both analytical quality). Supporting electro-
lytes used for the determination of the antioxidant activity
were solutions of 0.1 M in both sodium bicarbonate, at
pH ¼ 10.5, and 0.1 M phosphoric acid at pH ¼ 12.5. The
ionic strength was adjusted to 0.5 M with solid KNO3 and
the pH was adjusted with solid NaOH. Antioxidants were
dissolved in ethanol and the stock solution concentrations
were 5  103 M. These solutions were stored in darkness
at 277 K to avoid decomposition.
UVevis absorption spectra (190e800 nm) were recor-
ded with a double beam PerkineElmer Lambda 750S
spectrophotometer with Hanna quartz cuvettes of 1 cm
pathlength. Solutions of 0.1 M in both phosphoric and
acetic acids for pH < 8.5 and 0.1 M in both sodium bicar-
bonate and phosphoric acid for pH > 8.5 were used. pH was
measured using a Metrohm pH-meter. Alkali error was
corrected with the correction curves supplied by the
manufacturer. Ultrapure water was obtained with a
Millipore-Milli Q system.
A CHI650A electrochemical workstation from IJCambria
coupled to an EF-1400 controlled growth mercuryFig. 1. Left: DPV of 5  104 M H2O2. Numbers correspond to microliters of 5  103
the added amounts of 5  103 M antioxidant solution at pH: (up) 10.5, (down) 12electrode from BAS instruments was used in the HMDE
mode. The Hg drop area was 6.70  103 cm2. The tem-
perature was kept at 298 ± 0.1 K. All potentials were
measured against an AgjAgCljKClsat electrode (BAS MF-
2052). A platinum counter electrode BAS MW-1034 was
used. Differential pulse voltammetry (DPV) experiments
were made with a pulse amplitude of 0.05 V, a pulse width
of 0.05 s and a pulse period of 0.2 s. The antioxidant activity
was determined as reported [10,11].
To ensure the reproducibility of the measurements, the
experiments were made repeatedly, and the standard de-
viations of the data were less than 5%.3. Results and discussion
The antioxidant activity was obtained from the decrease
of the DPV signal corresponding to the H2O2 oxidation on a
mercury electrode in the presence of antioxidants [10,11].
This assesses the interaction with the radicals produced
during the oxidation of H2O2, being a measurement of the
scavenging ability. The measurement pH must be 10 or
higher, because at lower pH values the oxidation of the
mercury electrode prevents the appearance of the H2O2
signal. The volume that decreases the area of the H2O2
oxidation peak in 10% is related to the scavenging activity.
Its inverse was selected to express the antioxidant activity
because the higher the values of this parameter the higher
the scavenging activity [11].M 2,4DHB in 10 mL of final volume. Right: Decrease of the DPV peak area with
.5.
Fig. 2. UVevisible spectra. c ¼ 1  105 M.
R. Estevez Brito et al. / C. R. Chimie 20 (2017) 365e369 367Fig. 1 shows the DP voltammograms of the H2O2
oxidation after the addition of one of 2,4DHB at the two pH
values used. The experiments were made at a constant
ethanol content of 30%, both in the absence and in the
presence of antioxidants, to avoid the effect of this variable
on the DPV peak and to ensure the proportionality between
the peak area and the H2O2 concentration [10,11].
The oxidation peak decreases as the antioxidant con-
centration increases at both pH values but in the most
alkaline medium higher volumes of antioxidants areFig. 3. Fitting of the lognormal ploneeded to produce the decrease for the three compounds.
Fig. 1 also shows the percentages of the area diminution.
At pH 10.5, the antioxidant activity of 2,4DHB is ca.
double than that measured for the 2,5DHB, whereas at pH
12.5 the antioxidant activities of both compounds are
similar, though slightly lower in the case of the 2,5 isomer.
Proton dissociation is important to understand structure-
activity relationships of gallic acid derivatives with anti-
oxidant activity [12]. The above changes in the antioxidant
activities, both absolute and relative, could be discussed ints to experimental spectra.
Table 1
Characteristic parameters of the main individual bands.
Compound l/nm Dn1/2/kK b
2,4-DHB 276.5 3.8 0.1
313.3 3.8 0.15
248.0 3.0 0.0
327.0 3.3 0.16
357.0 3.0 0.0
2,5-DHB 258.0 3.3 0.15
361.0 3.4 0.05
420.0 3.4 0.1
452.0 4.4 0.22
R. Estevez Brito et al. / C. R. Chimie 20 (2017) 365e369368light of the dissociation of the hydroxyl groups. To assess
this, the knowledge of the pK values is essential. The
complete pK values of the dihydroxy-benzaldehydes are
not reported in the literature, as stated in the introduction,
and this will be the following task to do.
Fig. 2 shows the absorption spectra as functions of the
acidity for both dialdehydes. The curves showed several
main bands whose intensities depended on pH of the
solution.
The pH dependencies of the spectra indicate the
occurrence of acid-base reactions that must correspond to
the dissociation of the hydroxyl groups of the molecules:
The 2-hydroxyl group can form an intramolecular H-
bond with the adjacent carbonyl group [15] that stabilizes
the OH. For this reason, the more plausible dissociation
sequences are those shown in the above scheme.
The absorption spectra were analyzed by using a com-
puter program developed for Microsoft Visual Basic 2013,Fig. 4. Maximum absorbances obased on other programs from our laboratory and previ-
ously used in other pK determinations [16,17]. In this pro-
gram, it was assumed that the bands can be represented by
a log-normal band shape, which can be described by the
equation [18]:
ln A ¼ ln A0 þ ln2

ln

1þ 2bðv v0Þ=Dv1=2

b
2
where A is the absorbance at the frequency n and A0 and n0
are the coordinates of the maximum; Dn1/2 is the band half-
width and b is a parameter which determines the degree of
asymmetry of the curve. As b tends to zero, the curve is
reduced to a Gaussian curve, i.e., the band is symmetrical.
The spectra were decomposed into sets of bands inte-
grated by the minimum number of bands possible, and
with the condition that the band parameters were inde-
pendent of both the pH and concentration (with the
obvious exception of the absorbance). Fig. 3 shows the
comparison between theoretical and experimental results
for both compounds at low and high pH values.
The intensities of some of the bands presented varia-
tions with the pH (either from zero to a maximum value,
either from a maximum value to zero), or present
maximum values at intermediate pH values and are zero at
very lower or higher values. The parameters of such bands
are shown in Table 1. These parameters remained virtually
unchanged in the pH range studied. Fig. 4 shows the
dependence of the maximum absorbances with the pH.
If the pH dependence of the maximum absorbance of
the individual bands is due to the abovementioned acid-
base equilibria, the following trivial equation must be
fulfilled:
pH ¼ pK þ log½ðAmax  AÞ=ðA AminÞ
where Amax and Amin are the maximum absorbances
measured at the maximum and minimum pH values,
respectively. The plots of log[(Amax  A)/(A  Amin)] versus
pH should give straight lines with the intercept equal to pK.
When the bands increase with pH, the slope of the straight
line obtained must be þ1, whereas in the opposite case the
slope must be 1. The pK values obtained from these plots
were 6.94 ± 0.03 and 9.28 ± 0.03 for 2,4DHB and
8.42 ± 0.03 and 10.93 ± 0.03 for 2,5DHB.f fitted bands versus pH.
R. Estevez Brito et al. / C. R. Chimie 20 (2017) 365e369 369The knowledge of the pK values permits us to calcu-
late the percentage of the different species at each pH. In
all cases, the lower the pH, the higher the antioxidant
activity found; that is, the occurrence of eOH groups is
related to high antioxidant activity. At pH 10.5, the hy-
droxyl group in position 4- of 2,4DHB is fully dissociated
and the hydroxyl group in position 2- is dissociated in ca.
94%. For 2,5DHB, the hydroxyl group in position 5- is fully
dissociated and the hydroxyl group in position 2- is
dissociated in ca. 37%. So, the dianionic form of 2,4DHB is
approximately 2.5 times more abundant than the dia-
nionic form of 2,5DHB at this pH value. At pH 10.5, the
antioxidant activity of the 2,4DHB is ca. double than that
of 2,5DHB. At the most alkaline pH (12.5), the two eOH
groups of 2,4DHB are fully dissociated and ca. the 97.5%
of 2,5DHB is dianionic. The antioxidant activity of this
last compound at pH 12.5 is only slightly lower than that
corresponding to 2,4DHB.
The above results show that the pH of the assays for
antioxidant capacity is very important in light of the eOH
group dissociation, because of the different dissociation
constants of the isomers. So to know the possible antioxi-
dant action of a given molecule in a living organism, the
assay must be made the closest possible to physiological
conditions, that is, in aqueous media with a medium-high
ionic strength and at pH 7e7.5, as is made in the
CUPRAC-AOA assay [19].
The antioxidant capacity of phenolic derivatives is
intrinsically related to the number of hydroxyl groups in
themolecule. When the number ofeOH in themolecules is
the same, the extension of the dissociation of each group is
essential. Undoubtedly, the radical scavenging capacity of
polyphenols depends on the interplay of other factors,
other than the acidity, such as redox potential of free rad-
icals, ionization potentials of phenol anions and ability of
the molecules to form a hydrogen bond. In addition, the
concentrations of hydroperoxide and superoxide anion
radicals generated in the oxidation of H2O2 on the mercury
electrode depend on pH. So, the measurement of scav-
enging ability of the DHB acid gives different absolutevalues at the pH 10.5 and 12.5, but the relative antioxidant
activities are related as discussed above.Acknowledgments
Financial support from Cordoba University within the
framework of the “Ayudas puente para el desarrollo de
proyectos de IþD precompetitivos 2015” is acknowledged.References
[1] P.P. Liebgott, M. Labat, A. Amouric, J.-L. Tholozan, J. Lorquin, J. Appl.
Microbiol. 105 (2008) 2084e2095.
[2] L. Gennari, M. Felletti, M. Blasa, D. Angelino, C. Celeghini, A. Corallini,
P. Ninfali, Phytochem. Anal. 22 (2011) 272e279.
[3] E.-M. Ahn, S.-J. Park, W.-C. Choi, S.-H. Choi, N.-I. Baek, J. Korean Soc.
Appl. Biol. Chem. 50 (2007) 358.
[4] B. Fernandez de Simon, E. Esteruelas, A.M. Mu~noz, E. Cadahía,
M. Sanz, J. Agric. Food Chem. 57 (2009) 3217e3227.
[5] A.G. Gonzalez, J. Bermejo, E. Valencia, Planta Medica 1996 (62)
(1996) 176e177.
[6] M. Friedman, P.R. Henika, R.E. Mandrell, J. Food Prot. 66 (2003)
1811e1821.
[7] N.C.G. Faria, J.H. Kim, L.A.P. Gonçalves, M.L. Martins, K.L. Chan,
B.C. Campbell, Lett. Appl. Microbiol. (2011) 506e513.
[8] S.W. Nowotarska, K.J. Nowotarski, M. Friedman, C. Situ, Molecules
19 (2014) 7497e7515.
[9] J.F. Arteaga, M. Ruiz Montoya, A. Palma, G. Alonso Garrido,
S. Pintado, J.M. Rodríguez Mellado, Molecules 17 (2012) 5126e5138.
[10] A. Palma, M. Ruiz Montoya, J.F. Arteaga, J.M. Rodríguez Mellado, J.
Electrochem. Soc. 160 (2013) H213eH218.
[11] A. Palma, M. Ruiz Montoya, J.F. Arteaga, J.M. Rodríguez Mellado, J.
Agric. Food Chem. 62 (2014) 582e589.
[12] H.-F. Ji, H.-Y. Zhang, L. Shen, Bioorg. Med. Chem. Lett. 16 (2006)
4095e4098.
[13] M. Xia, R. Dempski, R. Hille, J. Biol. Chem. 274 (1999) 3323.
[14] E.P. Crowell, W.A. Powell, C.J. Varsel, Anal. Chem. 35 (1963) 184e189.
[15] J.A. Valderrama, J. Benites, M. Cortes, D. Pessoa-Mahana, E. Prina,
A. Fourner, Tetrahedron (2002) 881e886.
[16] J.M. Rodríguez Mellado, M. Ruiz Montoya, Gazz. Chim. Ital. 120
(1990) 553e555.
[17] R. Perez, R. Marín Galvín, J.M. Rodríguez Mellado, Czech. Chem.
Commun. 67 (2002) 429e438.
[18] R.P. Fraser, E. Suzuki, The Use of Least Squares in Data Analysis,
Physical Principles of Protein Chemistry. Part C, Academic Press Inc,
New York, 1974.
[19] R. Apak, K.G. Güçlü, M. €Ozyürek, S.E. Çelik, Microchim. Acta 160
(2008) 413e419.

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