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Unpublished research paper based on experiments done in 1998-1999.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 4-
hydroxybenzoic 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 .

INTRODUCTION (Coudray et al. 1995; Ste-Marie et al. 1996;

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 2,3-
dihydroxybenzoic 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
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 2-
hydroxybenzoic acid and 4-hydroxybenzoid acid.
Decarboxylation of 4-hydroxybenzoic acid
produced hydroquinone.
MATERIALS AND METHODS
Chemicals
All chemicals were obtained from Sigma-
Aldrich 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, 2,5-
dihydroxybenzoic acid, catechol, 3,4-

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dihydroxybenzoic acid and hydroquinone were The production of 3,4-DHBA and Hydroquinone
based upon the following assays: The assay were 76 % and 24 %, respectively, of the total
reaction mixture in glass tubes contained 2 mM hydroxylated products formed in the
2-hydroxybenzoic acid (or 4-hydroxybenzoic reaction.
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 Figure 1. Production of 3,4-dihydroxybenzoic
SCL-6A) equipped with an electrochemical acid () and hydroquinone (#) at different
detector ( Schimadzu L-ECD-6A) set at +0.7V concentrations of FeSO4 and at constant
versus a Ag/AgCl reference electrode. A concentration of 4-hydroxybenzoic acid (2 mM)
Brownlee Spheri-5 RP-18, 5: (220 x 4.6 mm) and Na2EDTA (2 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 The time course of the reaction was very fast and
detector was registred and the peak areas were finished in less than seconds. Iron in the form of
calculated as volt per second and quantified using ferritin or K4Fe(CN)6 , was ineffective in the
authentic standards of dihydroxybenzoic acids, reaction. Citric acid could replace EDTA in the
Catechol and Hydroquinone and corrected for the reaction, as could malic acid and oxalic acid
volumes applied. could to some extent, but higher concentrations
were necessary (data not shown). EDTA could
RESULTS not be replaced by 1,10-phenanthronline or the
calcium chelating agent EGTA. Standard
The results showed that FeSO4 and Na2-EDTA solutions of 2,4-dihydroxybenzoic acid, 2,6-
form stable adducts of 2,3-dihydroxybenzoic acid dihydroxybenzoic acid, resorcinol, and 2,4,6-
and 2,5-dihydroxybenzoic acid via an aromatic trihydroxybenzoic acid could not be measured
hydroxylation of 2-hydroxybenzoic acid. In when the detector response was set at 0.7 V.
addition decarboxylation of 2-hydroxybenzoic However, standard solutions of 3,4,5-
acid gave catechol (Figure 1, 2 & 3). The trihydroxybenzoic acid (gallic acid) and 2,3,4-
production of 2,3-dihydroxybenzoic acid, 2,5- trihydroxybenzoic acid could be detected.
dihydroxybenzoic 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.

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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 4-
hydroxybenzoic 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 Figure 3 Production of 3,4-dihydroxybenzoic
and Na2-EDTA or citric acid followed the same acid () and hydroquinone (#) at different
pattern. Also in this case saturation kinetics were concentrations of Na2EDTA and at
obtained with increasing concentrations of the constant concentration of FeSO4 (2 mM) and 4-
substrates in the reaction (see appendix). hydroxybenzoic 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).

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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 3,4-
dihydroxybenzoic 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 4-
hydroxybenzoic 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 2-
hydroxybenzoic 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

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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.; Fatme, 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 (1,2,4-
trimethylbenzene) 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 4-
hydroxybenzoate as an hydroxyl radical trap using gas chromatography-mass spectrometry and
high-performance liquid chromatography with electrochemical detection. Anal. Biochem. 241: 67-
74.

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Ste-Marie, L.; Vachon, L.; Bmeur, 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)-catechol-
H2O2 system. Mechanistic implication of the role of catechol. Bull. Chem. Soc. Jpn. 62:148-152.

Udenfriend, S.; Clark, C.T.; Axelrod, J.; Brodie, B.B. Ascorbic acid in aromatic hydroxylation.I. A
model system for aromatic hydroxylation. J.Biol. Chem. 208: 731-739; 1954.

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 4-
hydroxybenzoic acid (2 mM).

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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).

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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).

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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 2,5-
dihydroxybenzoic 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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