Hydroxylation and decarboxylation of hydroxybenzoic acids by
Fe2+-chelates
Halvor Aarnes, Department of Biology, University of Oslo, POB 1066, Blindern, N-0316 Oslo,
NORWAY; E-mail: halvor.aarnes[at]bio.uio.no
Unpublished research paper based on experiments done in 1998-1999.
Abstract
Hydroxylation and decarboxylation of hydroxybenzoic acids occurs rapidly at pH 3 to 6.5 in a system
containing FeSO4 and Na2EDTA. EDTA could be replaced by citric acid. In this in vitro system 4hydroxybenzoic acid is hydroxylated to 3,4-dihydroxybenzoic acid (protocatechuic acid) and
decarboxylated to hydroquinone. In an analogous reaction 2-hydroxybenzoic acid (salicylic acid) is
hydroxylated to 2,3-dihydroxybenzoic acid and 2,5-dihydroxybenzoic acid (gentisic acid) and
decarboxylated to catechol. Surprisingly the reactions showed Micahelis-Menten saturation kinetics for
the products, and this paper is the first description of these reactions. From these data it is also
cautioned to use hydroxylation of hydroxybenzoic acids as an indicator of oxidative stress and hydroxyl
radicals in biological systems without proper controls.
Keywords- Catechol, decarboxylation, Fe 2+-chelates,
hydroxybenzoic acids, hydroquinone, hydroxylation .
(Coudray et al. 1995; Ste-Marie et al. 1996;
Myhre et al. 2000; Liu et al. 2002,). Stringent
tests for the experimental system in order to avoid
OH• -production from non-biological sources is
necessary (Montgomery et al. 1995; Myhre et al.
2000, ). During our work trying to detect OH• I
observed the possibility of artifactual production
of hydroxylated hydroxybenzoic acids. The
results presented here show that iron-chelates
without ascorbate can hydroxylate and
decarboxylate the hydroxybenzoic acids 2hydroxybenzoic acid and 4-hydroxybenzoid acid.
Decarboxylation of 4-hydroxybenzoic acid
produced hydroquinone.
INTRODUCTION
Since Udenfriend et al. (1954) and Brodie et al.
(1954) reported that ascorbic acid, Fe2+ , EDTA
and oxygen at pH 7 hydroxylated aromatic
compounds, a large number of model systems for
aromatic hydroxylation have been presented A
large number of model systems for aromatic
hydroxylation have been presented (Halliwell
1978; Grootveld.& Halliwell1986; Tamagaki
1989, Liu et al. 2002). In most cases the
hydroxylation is performed in a Fenton-like
reaction by hydrogenperoxide and ferrous ion
(Fe2+). In biological systems, the presence of
hydroxyl radicals (OH•) is regarded to be an
indicator of oxidative stress. Trapping by
hydroxybenzoic acid is a widely used sensitive
technique to detect OH• down to picomol
concentrations (Coudray et al. 1995; Ste-Marie et
al. 1996) using HPLC with electrochemical
detection. The salicylic acid method depends on
the formation of the two stable compounds 2,3dihydroxybenzoic acid and 2,5-dihydroxybenzoic
acid from 2-hydroxybenzoic acid (Coudray et al.
1995). Catechol is a also a product in the
salicylate assay (Grootveld.& Halliwell1986) .
The 4-hydroxybenzoic acid method is based
upon the formation of 3,4-dihydroxybenzoic acid
MATERIALS AND METHODS
Chemicals
All chemicals were obtained from SigmaAldrich Norway, except ethylacetate, FeSO4
•7H2O, and Titriplex III (Na2EDTA) which were
from Merck (Germany), and HPLC-grade
methanol from Rathburn (Scotland).
Hydroxylation and decarboxylation assay
Detection of 2,3-dihydroxybenzoic acid, 2,5dihydroxybenzoic acid, catechol, 3,41
dihydroxybenzoic acid and hydroquinone were
based upon the following assays: The assay
reaction mixture in glass tubes contained 2 mM
2-hydroxybenzoic acid (or 4-hydroxybenzoic
acid), 2 mM Titriplex III (Na2-EDTA), 2 mM
FeSO4@7H2O (freshly made), 20 mM Na-acetate
pH 4.0 (or pH5.0) and water in a final volume of
1 ml. Reactions were started by addition of
FeSO4 and whirlmixed for 5 s. The reaction
mixture was diluted 200 x with the mobile phase
(20 mM citrate, 30 mM Na-acetate, 0.1 mM
EDTA and 7 % (v/v) methanol (pH 4.0)) and
injected into the chromatograph. In some of the
experiments 2 ml ethylacetate was added to each
reaction mixture and immediately whirlmixing
for 20 s. When the layers were separated, 0.5 ml
of the supernatant was taken out, dried to dryness
overnight with circulating air in drams glass at
room temperature and dissolved in 1 ml of the
mobile phase. The dihydroxybenzoic acids were
measured by reversed-phase HPLC (Schimadzu
SCL-6A) equipped with an electrochemical
detector ( Schimadzu L-ECD-6A) set at +0.7V
versus a Ag/AgCl reference electrode. A
Brownlee Spheri-5 RP-18, 5: (220 x 4.6 mm)
with a RP-18 precolumn was used with the
mobile phase at a flowrate 0.2 - 0.8 ml min-1 at
ambient temperature. The output of the the
detector was registred and the peak areas were
calculated as volt per second and quantified using
authentic standards of dihydroxybenzoic acids,
Catechol and Hydroquinone and corrected for the
volumes applied.
The production of 3,4-DHBA and Hydroquinone
were 76 % and 24 %, respectively, of the total
hydroxylated products formed in the
reaction.
Figure 1. Production of 3,4-dihydroxybenzoic
acid (•) and hydroquinone (#) at different
concentrations of FeSO4 and at constant
concentration of 4-hydroxybenzoic acid (2 mM)
and Na2EDTA (2 mM).
The time course of the reaction was very fast and
finished in less than seconds. Iron in the form of
ferritin or K4Fe(CN)6 , was ineffective in the
reaction. Citric acid could replace EDTA in the
reaction, as could malic acid and oxalic acid
could to some extent, but higher concentrations
were necessary (data not shown). EDTA could
not be replaced by 1,10-phenanthronline or the
calcium chelating agent EGTA. Standard
solutions of 2,4-dihydroxybenzoic acid, 2,6dihydroxybenzoic acid, resorcinol, and 2,4,6trihydroxybenzoic acid could not be measured
when the detector response was set at 0.7 V.
However, standard solutions of 3,4,5trihydroxybenzoic acid (gallic acid) and 2,3,4trihydroxybenzoic acid could be detected.
RESULTS
The results showed that FeSO4 and Na2-EDTA
form stable adducts of 2,3-dihydroxybenzoic acid
and 2,5-dihydroxybenzoic acid via an aromatic
hydroxylation of 2-hydroxybenzoic acid. In
addition decarboxylation of 2-hydroxybenzoic
acid gave catechol (Figure 1, 2 & 3). The
production of 2,3-dihydroxybenzoic acid, 2,5dihydroxybenzoic acid and catechol were 50 %,
37 % and 13 %, respectively, of the total
hydroxylated products formed in the reaction.
Hydroxylation of 4-hydroxybenzoic acid gave
3,4-dihydroxybenzoic acid and decarboxylation
of 4-hydroxybenzoic acid gave hydroquinone.
2
The pH optimum of the hydroxylation and
decarboxylation reactions were between pH 3 to
pH 5, but with lower activity above pH 7.
Increasing the concentrations of the reactants 4hydroxybenzoic acid, and FeSO4 and citric acid
resulted in increasing amounts of products
showing saturation kinetics (Figure 1 & 2). The
same result was obtained varying the
concentrations EDTA, Fe2+-EDTA and citrate
(Figure 3). The results were confirmed with
separate experiments ex tracting the
hydroxybenzoic acids into etylacetate at pH 3.
Production of the products 2,3-dihydroxybenzoic
acid, 2,5-dihydroxybenzoic acid and catechol
from the reactants 2-hydroxybenzoic acid , FeSO4
and Na2-EDTA or citric acid followed the same
pattern. Also in this case saturation kinetics were
obtained with increasing concentrations of the
substrates in the reaction (see appendix).
Figure 3 Production of 3,4-dihydroxybenzoic
acid (•) and hydroquinone (#) at different
concentrations of Na2EDTA and at
constant concentration of FeSO4 (2 mM) and 4hydroxybenzoic acid (2 mM).
See appendix for additional data.
Figure 2. Production of 3,4-dihydroxybenzoic
acid (•) and hydroquinone (#) at different
concentrations of 4-hydroxybenzoic acid and at
constant concentration of FeSO4 (2 mM) and
Na2EDTA (2 mM).
3
Figure 4. Summary of the Fe2+-EDTA induced hydroxylation and decarboxylation of 2-hydroxybenzoic
acid giving 2,5-dihydroxybenzoid acid , 2,3-dihydroxybenzoic acid and catachol. The products from
Fe2+-EDTA induced hydroxylation and decarboxylation of 4-hydroxybenzoic acid are 3,4dihydroxybenzoic acid and hydroquinone.
DISCUSSION
This work show that Fe2+-EDTA or Fe2+-citrate at pH 3-6.5, without hydrogenperoxide or ascorbate,
can induce hydroxylation and decarboxylation of 2-hydroxybenzoic acid and 4-hydroxybenzoic acid.
This work also suggests that 4-hydroxybenzoic acid can be hydroxylated and decarboxylated to give
3,4-DHBA and HQ, respectively, in an analogous reaction. Formation hydroquinone from 4hydroxybenzoic acid by Fe2+-chelates has not been reported earlier. Not only EDTA, but also citric acid
can be used as a iron-chelate in the reaction.
It is well known that Fe2+ can react with hydroxybenzoic acid in siderophore-complex (Montgomery
et al. 1995). Thus, it is likely that no free intermediates occur in the reaction. The time course of the
reaction indicate a fast reaction kinetics as reported earlier for the ascorbate-dependent hydroxylation
(Ste-Marie et al. 1999 ). Ascorbate, in addition to be an antioxidant, can work as an enzyme cofactor
of hydroxylase enzymes.
In biological systems enzymes are known to catalyze the same type of reactions presented in this
paper e.g. salicylate 1-monooxygenase (EC 1.14.13.1) catalyzing oxidative decarboxylation of 2hydroxybenzoic acid and 4-hydroxybenzoate 3-monooxygenase (EC 1.14.13.2) catalyzing
hydroxylation of 4-hydroxybenzoic acid, but in these cases NAD(P)H are used as an electron donor
4
instead of Fe2+. In addition, the cytochrome P450 system can hydroxylate hydroxybenzoic acids (14).
Because only a limited number of hydroxybenzoic acids can give response when the detector was set
to the potential +0.7 V, there is a possibility that also other products can be produced in the reactions
investigated here.
The biological relevance of addition of Fe2+-chelate to a biological system in order to produce OH• can
be questioned. It is cautioned to use hydroxylation of hydroxybenzoic acids as an indicator of oxidative
stress in biological systems without proper controls. Data from such measurements can be at least
partially artifactual and should be interpreted with caution.
It is suggested that the oxidative hydroxylation and decarboxylation of hydroxybenzoic acids can
happen without involvement of hydroxyl radicals.
REFERENCES
Brodie, B.B.; Axelrod, J.; Shore, P.A.; Udenfriend, S. Ascorbic acid in aromatic hydroxylation. II.
Products formed by reaction and substrates with ascorbic acid, ferrous ion, and oxygen. J. Biol. Chem.
208: 741-750; 1954.
Coudray, C.; Talla; M.; Martin, S.; Fatôme, M. & Favier, A. 1995. High-performance liquid
chromatography-electrochemical determination of salicylate hydroxylation products as an in vivo
marker of oxidative stress. Anal. Biochem. 227:101-111.
Grootveld, M.& Halliwell.1986. Aromatic hydroxylation as a potential measure of hydroxyl-radical
formation in vivo. Identification of hydroxylated derivatives of salicylate in human body fluids.
Biochem. J. 237:499-504.
Halliwell, B. 1978. Superoxide-dependent formation of hydroxyl radicals in the presence of iron
chelates. FEBS Letters 92:321-326.
Liu, M., Liu, S., Peterson, S.L., Miyake, M. & Liu, K.J.: On the application of 4-hydroxybenzoic acid
as a trapping agent to study hydroxyl radical generation during cerebral ischemia and reperfusion. 2002.
Mol.Cell.Biochem. 234/235:379-385.
Montgomery, J.; Ste-Marie, L.; Boismenu, D.& Vachon, L. 1995. Hydroxylation of aromatic
compounds as indices of hydroxyl radical production: a cautionary note revisited. Free Radic Biol.
Med. 19:927-933.
Myhre, O.; Vestad, T.A., Sagstuen, E.; Aarnes, H.& Fonnum; F. 2000.
The Effects of Aliphatic (n-nonane), Naphtenic (1,2,4-trimethylcyclohexane) and Aromatic (1,2,4trimethylbenzene) Hydrocarbons on Respiratory Burst in Human Neutrophil Granulocytes. A
Fluorescence and Electron Paramagnetic Resonance (EPR) Spectroscopy Study. Toxicol. Appl.
Pharmacol. 167: 222-230.
Ste-Marie, L.; Boismenu, D.; Vachon, L.& Montgomery,J. 1996. Evaluation of sodium 4hydroxybenzoate as an hydroxyl radical trap using gas chromatography-mass spectrometry and
high-performance liquid chromatography with electrochemical detection. Anal. Biochem. 241: 6774.
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Ste-Marie, L.; Vachon, L.; Bémeur, C.; Lambert, J.; Montgomery, J. 1999. Local striatal infusion of
MPP+ does not result in increased hydroxylation after systemic administration of 4-hydroxybenzoate
Free Radic. Biol. & Med. 27: 997-1007.
Tamagaki, S.; Suzuki, K.& Tagaki, W. 1989. Aromatic hydroxylation with an iron(III)-catecholH2O2 system. Mechanistic implication of the role of catechol. Bull. Chem. Soc. Jpn. 62:148-152.
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Appendix
Appendix 1. Production of 3,4-dihydroxybenzoic acid (•,34DHB) and hydroquinone (#,HQ) at
different concentrations of citrate (0-5 mM) and at constant concentration of FeSO4 (2 mM) and 4hydroxybenzoic acid (2 mM).
6
Appendix 2: Production of 2,3-dihydroxybenzoic acid (•, 2,3-DHBA) and 2,5 dihydroxybenzoic
acid (B, 2,5-DHBA) at different concentrations of Na2EDTA (0-4 mM) and at constant
concentration of FeSO4 (2 mM) and 2-hydroxybenzoic acid (2 mM).
Appendix 3: Production of 2,3-dihydroxybenzoic acid (•, 2,3-DHBA) and 2,5 dihydroxybenzoic
acid (B, 2,5-DHBA) at different concentrations of 2-hydroxybenzoic acid (2-HBA) and at constant
concentration of FeSO4 (2 mM) and Na2EDTA (2 mM).
7
Appendix 4: Standard curve for 2,5-dihydroxybenzoic acid (2,5-DHBA) measured by HPLC
equipped with electrochemical detector set at 0.7V. The concentration of 2,5-DHBA was measured
as volt@second (Vs). The same kind of standard curve was made for all the compounds used in this
paper..
Appendix 5: Standard curve for 3,4-dihydroxybenzoic acid (3,4-DHBA) measured by HPLC
equipped with electrochemical detector set at 0.7V. The concentration of 3,4-DHBA was measured
as volt@second (Vs).
8
Appendix 6: Production of 2,3-dihydroxybenzoic acid (•, 2,3-DHBA) and 2,5 dihydroxybenzoic
acid (B, 2,5-DHBA) at different concentrations of FeSO4- Na2EDTA 0-4 (mM) and at constant
concentration of 2-hydroxybenzoic acid ( mM).
9
Appendix 7: A. Detection of 2,3-dihydroxybenzoic acid (23DHB); 2,5-dihydroxybenzoic acid
(25DHB) and catechol (CC) from 2-hydroxybenzoic acid diluted 100x with the mobile phase.
Detection with HPLC equipped with electrochemical detector (ECD) at 0.7V and the amount of
products is measured as millivolt (mV). B. Elution of a standard mixture containing 1 :M 2,5dihydroxybenzoic acid (25DHB); 2,3-dihydroxybenzoic acid (23DHB); 3,4-dihydroxybenzoic acid
(34DHB) and catechol (CC). C. Chromatogram of reaction mixture with 4-hydroxybenzoic acid and
Fe2+-EDTA diluted 100x with the mobile phase. D. Elution of a standard mixture containing 1:M
of 2,3,4-trihydroxybenzoic acid (234THB); 3,4,5-trihydroxybenzoic acid (345THB), hydroquinone
(HQ); 2,3-dihydroxybenzoic acid (23DHB); 3,4-dihydroxybenzoic acid (34DHB) and catechol
(CC).
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