EDTA bis-(ethyl phenylalaninate): A novel transition metal-ion chelating hydroxyl radical scavenger with a potential anti-inflammatory role

Article abstract of DOI:10.1016/S0960-894X(01)00496-6

Conjugation of ethylenediaminetetraacetic acid (EDTA) to ethyl phenylalaninate generates a novel radical scavenging metal-ion chelator EDTA bis-(ethyl phenylalaninate) (EBEP). The oxidation products o-, m- and p-tyrosine were isolated from hydrolysed, aqueous and aerated solutions containing EBEP, Fe(II) and H₂O₂. Data obtained demonstrate the potential of EBEP to act as a radical scavenging, iron-ion chelating antioxidant under physiologically relevant conditions.

Full text of DOI:10.1016/S0960-894X(01)00496-6

Bioorganic & Medicinal ChemistryLetters 11 (2001) 2573–2575
EDTA Bis-(ethyl phenylalaninate): A Novel Transition Metal-Ion
Chelating Hydroxyl Radical Scavenger with a Potential
Anti-inflammatory Role
Declan P. Naughtona,* and Martin Grootveldb
aSchool of Pharmacy and Biomolecular Sciences, University of Brighton, Cockcroft Building, Moulsecoomb, Brighton BN2 4GJ, UK
bThe Inflammation Research Group, Saint Bartholomew’s and the Royal London School of Medicine and Dentistry, London E1 2AD, UK
Received 30 March 2001; accepted 13 July2001
Abstract—Conjugation of ethylenediaminetetraacetic acid (EDTA) to ethyl phenylalaninate generates a novel radical scavenging
metal-ion chelator EDTA bis-(ethyl phenylalaninate) (EBEP). The oxidation products o-, m- and p-tyrosine were isolated from
hydrolysed, aqueous and aerated solutions containing EBEP, Fe(II) and H2O2. Data obtained demonstrate the potential of EBEP
to act as a radical scavenging, iron-ion chelating antioxidant under physiologically relevant conditions. # 2001 Elsevier Science
Ltd. All rights reserved.
The transition metal ion-dependent formation of
hydroxyl radical (ꢀOH) from hydrogen peroxide (H2O2)
in the presence of a reducing agent such as superoxide
rapidlywith ꢀOH radical to form a mixture of hydrox-
ylated products (for example, phenylalanine is attacked
by OH radical to produce three products: o-, m- and
ꢀ
ꢀ
ion (O2 À) or ascorbate (at low concentrations) is an
p-tyrosines). Indeed, aromatic hydroxylation has been
successfullyemployed as a method for measuring ꢀOH
radical production both in vivo and in vitro.7,8 Hence,
important mechanism of ‘oxidative stress’ leading to
irreversible cell and tissue damage.1,2 In particular,
metal-ion mediated oxidative stress has been attributed
a role in inflammation, atherosclerosis and Alzheimer’s
disease. Indeed, recent reports illustrate that amyloid
protein in Alzheimer’s plaques bind copper and iron
and produce H2O2 from oxygen via Udenfriend’s sys-
tem.3 Subsequent reaction between the generated H2O2
ꢀ
the design of an effective ‘site-specific’ [directed] OH
radical scavenger can in principle be achieved bycova-
lentlyattaching an aromatic moietyto a chelator that is
able to form a thermodynamically-stable complex with
iron ions. In this study, the ꢀOH radical-scavenging
potential of the powerful metal-ion chelator EDTA
bis-(ethyl phenylalaninate) (EBEP) has been investi-
ꢀ
and adjacent reduced metal ions generates OH radicals
ꢀ
in vitro. Iron chelators function as antioxidants to
diminish plaque and aggregate formation in neurode-
generative diseases and atherosclerosis.4,5 In addition to
disease states, much interest has been shown in the
contribution of oxidative damage to ageing. A recent
report outlined the extension of lifespan bya mean of
44% in one species bytreating with superoxide dis-
mutase/catalase mimetics.6 Clearly, new approaches to
the suppression of oxidative damage are a priorityin
both health and disease.
gated.9,10 The OH radical-mediated hydroxylation of
aromatic rings with the subsequent formation of rela-
tivelystable phenolate–iron(III) charge-transfer bonds
would favour a particular ligand for the role of an OH
ꢀ
radical-scavenging chelator. These properties maybe
useful in the development of novel dual-function anti-
oxidants to treat inflammation, atherosclerosis and
Alzheimer’s disease.
The ligand is accessible in good yield using standard
amide preparation following protection of the amino
acid carboxylic acid moiety. Using the method devel-
Aromatic compounds such as phenols, phenolic acids
and the amino acid phenylalanine react extremely
11
oped byGrootveld and Halliwell, the products o-, m-
and p-tyrosine were isolated from an aqueous, aerated
solution containing EBEP, Fe(II) and H2O2. No
detectable levels of the above hydroxylation products
were detected in the absence of added iron(II) ions. The
*Corresponding author. Tel.: +44-1273-642036; fax: +44-1273-
679333; e-mail: d.p.naughton@bton.ac.uk
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00496-6

2574
D. P. Naughton, M. Grootveld / Bioorg. Med. Chem. Lett. 11 (2001) 2573–2575
selectivity of the hydroxylation process afforded by the
proximityof the intramolecular aromatic rings is
apparent on comparison of the hydroxylation pattern
detected for the intact phenylalanine containing ligand
EBEP relative to phenylalanine itself (Fig. 1). In the
case where the aromatic rings were not constrained in a
close localityto the iron (i.e., where the metal ion was
complexed by EDTA), hydroxylation sites were in the
order o-ꢁm-ꢂp-. However, for EBEP, hydroxylation
occurred in the order p->o->m-. Furthermore, expo-
studies show that hydroxylation does not occur in the
absence of ascorbate.
The abilityof EBEP to trap ꢀOH radicals was assessed
by the deoxyribose assay.12 As expected, addition of
EBEP resulted in a marked decrease in OH radical
ꢀ
attack on ribose. A reduction of 20% in hydroxyl radi-
cal attack on deoxyribose in the presence of EBEP
(0.104 mM) contrasts to a reduction of only2% on
addition of EDTA and phenylalanine. The inhibition of
ꢀ
ꢀ
sure of phenylalanine to Fenton-derived OH radical
greater than 50% of OH-mediated damage at 5.2 mM
reflects the abilityof this metal ion chelating scavenger
to limit Fe3+ mediated ROS damage in vitro (Table 1).
generated a similar pattern of isomeric tyrosines irre-
spective of whether EDTA was present in solution.
Thus, site-specific hydroxylation occurs for EBEP.
1H NMR provided further evidence for hydroxylation
of the phenylalanine aromatic rings by Udenfriend’s
system. The influence of added iron(III) chloride (1.00
molar equivalent in aqueous solution) and ascorbate
(5.00 mol equiv) on the 1H NMR spectrum of a solution
of EBEP in H2O was investigated. For this purpose, the
reaction mixture was incubated at room temperature for
a 30 min period. The electronic absorption spectrum of
this solution exhibits a broad shoulder at ca. 550 nm indi-
cative of the presence of phenolate–Fe(III) binding (data
not shown). Removal of iron ions as hydrated iron(IꢃII)
oxide was achieved byraising the pH, incubating at 60 C
for 2.5 h and centrifugation. The resulting solution was
lyophilised and the 1H NMR spectrum exhibits character-
istic splitting patterns of the aromatic proton resonances
corresponding to p- and o-tyrosine isomers, demonstrating
that ꢀOH radical attack on the aromatic ring has occurred
in two or more positions (data not shown). Control
Figure 1. Products derived from the hydroxylation of EDTA bis(ethyl
phenylalaninate): (A) contains molar equivalents of EBEP and Fe(II)
and three molar equivalents of H2O2; (B) contains molar equivalents
of PA, Fe(II) and EDTA, and three equivalents of H2O2; and (C)
contains molar equivalents of PA, Fe(II) and three equivalents of
H2O2.
Table 1. Antioxidant efficacyof EBEP as assessed bythe deoxyribose
assay
Compound
Concentration (mM)
Absorbance
% Inhibition
—
EBEP
EBEP
EBEP
EDTA+
Phenylanine
0.000
0.104
1.040
5.200
0.104
0.208
2.13
1.71
1.23
1.04
2.09
0.0
20
42
51
0.2
Scheme 1.

D. P. Naughton, M. Grootveld / Bioorg. Med. Chem. Lett. 11 (2001) 2573–2575
2575
The ‘site-specific’ hydroxylation of the o-position of
tyrosine is evident from examination of space filling
models. However, hydroxylation of the p-position is
accounted for bythe relative position of the aromatic
rings. In the case where the aromatic rings interact with
the metal orbitals the proximityof the p-position to the
metal orbital would be expected to cause an enhanced
degree of hydroxylation at this site.13 The results from
visible absorption spectroscopystudies indicate that
hydroxylation of the aromatic ring followed by phe-
nolate binding to the ferric ion occurs. From exam-
ination of space-filling models, o-tyrosine is the only
8. Hermes-Lima, M.; Santos, N. C.; Yan, J.; Andrews, M.;
Schulman, H. M.; Ponka, P. Biochim. Biophys. Acta 1999,
1426, 475.
9. Experimental: l-Phenylalanine (50 g, 0.3026 mol) was
refluxed in dryethanol (350 mL, 80 ꢃC, 3 h) with a slow stream
of dry HCl gas bubbling continuously. The hydrochloride salt
was recovered byevaporating the excess ethanol. The free
ester was then obtained byneutralisation with aqueous NaOH
solution (1.0Â10À1 mol dmÀ3) and then extracted with ethyl
acetate. EDTA bis-anhydride (3.306 g, 0.0129 mol), phenyl-
alanine ethyl ester (4.26 mL, 0.0258 mol) and pyridine (2.0 mL)
were refluxed in tetrahydrofuran (THF) for 3.5 h. The white
product was filtered and recrystallised from hot ethanol (mp
170 ꢃC). Elemental analysis: C, 59.99% (59.96), H, 6.27%
(6.62), N, 8.74% (8.58). 1H NMR analysis (D2O: 200 MHz,
298 K): d 1.23 [t, 3H, 3J=7 Hz, CH3], 2.5 [s, 4H, CH2CH2],
ꢀ
phenolate species, resulting from OH attack on the
phenylalanine moiety of EBET, which can participate
in intramolecular phenolate–iron(III) bonding (Scheme
1).
3
3.15 [m, 4H, J=11 Hz, CH2], 3.05 [s, 4H, CH2CONH], 3.18
[s, 4H, CH2COO], 4.19 [q, 3J=7 Hz, OCH2CH3], 4.65 [dt, 1H,
3J=11 Hz, CH], 7.30 [m, 8H, Ar].
These results show the potential of EBEP as a radical
scavenging chelator in biological systems. The stabilis-
ing effect and steric crowding caused byphenolate
binding to the ferric ion mayprevent the occurrence of
potentiallydeleterious redox chemistryat the active
metal ion centre.
10. HPLC studies: To an aqueous solution of the ligand
(EBEP, 2.20Â10À2 mol dmÀ3), one molar equivalent of iron(II)
sulphate (in an aqueous, N2-purged solution) was added fol-
lowed by3.00 mol equiv of H 2O2 solution. Subsequent to
incubation at 40 ꢃC for a 40 min period, the samples were
treated with 1.0 mol dmÀ3 NaOH solution at 60 ꢃC for a per-
iod of 2.5 h in order to hydrolyse the amide bond. This proce-
dure was followed bycentrifugation to remove the hydrated
iron(III) oxide precipitate. The resulting supernatants were
then subjected to HPLC analysis for separation and measure-
ment of aromatic hydroxylation products. Detection of these
products was byelectrochemical detection with the detector
potential set in the +0.70 to +0.85 V range. Authentic o-, m-
and p-tyrosine standards were subjected to HPLC analysis to
pre-calibrate the system.
Acknowledgements
We are grateful to Professor Peter Sadler, Universityof
Edinburgh and Dr. Christine Cardin, Universityof
Reading for helpful discussions.
In order to ensure that the proximityof the ligand to the
metal ion’s coordination sphere demonstrated an enhanced
potential with respect to the scavenging of OH radicals gen-
References and Notes
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