Synthesis, characterization, and measurement of antioxidant reactivity of salicylidene-D,L-tyrosine ethyl ester and copper(II)(salicylidene-D,L-tyrosine ethyl ester)2 in a linoleic acid peroxidation reaction system

Article abstract of DOI:10.1080/15533170600732718

Salicylidene-D,L-tyrosine ethyl ester and Cu(II)(SalTyrOEt) 2 were synthesized, characterized, and chain-breaking antioxidant and prooxidant reactivities determined in a 2,2′-azoisobutyronitrile initiated linoleic acid peroxidation reaction. Kinetics of oxygen consumption were found to be comparable to the antioxidant reactivity of 2,6-ditertiary-butyl-4- hydroxytoluene, due to efficient removal of linoleic acid peroxyl radical with the formation of linoleic acid hydroperoxide, LOOH, and correlate with the presence of tyrosyl phenolic OH groups. Copper in Cu(II)(SalTyrOEt) 2 accounts for prooxidant reactivity, catalyzing removal of LOOH, except at low concentrations when its antioxidant properties dominate. It is concluded that Cu(II)(SalTyrOEt) 2 is a unique antioxidant with prooxidant reactivity. Copyright

Full text of DOI:10.1080/15533170600732718

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Synthesis, Characterization, and Measurement of
Antioxidant Reactivity of Salicylidene‐D,L‐Tyrosine
Ethyl Ester and Copper(II)(Salicylidene‐D,L‐Tyrosine
Ethyl Ester)2 in a Linoleic Acid Peroxidation Reaction
System
S. H. Minasyan a , S. H. Ghazaryan b , V. J. Tonoyan c , S. A. Bajinyan c , K. P. Grigoryan b , J.
R. J. Sorenson d & F. T. Greenaway e
a Institute of Chemical Physics, Armenian National Academy of Sciences , Yerevan, Armenia
b Institute of Fine Organic Chemistry, Armenian National Academy of Sciences , Yerevan,
Armenia
c Research Center of Radiation Medicine and Burns, Ministry of Health , Yerevan, Armenia
d Division of Medicinal Chemistry, Department of Pharmaceutical Sciences, College of
Pharmacy , University of Arkansas, Medical Sciences Campus , Little Rock, Arkansas, USA
e Carlson School of Chemistry and Biochemistry , Clark University , Worcester, MA, USA
Published online: 15 Feb 2007.
To cite this article: S. H. Minasyan , S. H. Ghazaryan , V. J. Tonoyan , S. A. Bajinyan , K. P. Grigoryan , J. R. J. Sorenson &
F. T. Greenaway (2006) Synthesis, Characterization, and Measurement of Antioxidant Reactivity of Salicylidene‐D,L‐Tyrosine
Ethyl Ester and Copper(II)(Salicylidene‐D,L‐Tyrosine Ethyl Ester)2 in a Linoleic Acid Peroxidation Reaction System, Synthesis
and Reactivity in Inorganic, Metal-Organic, and Nano-Metal Chemistry, 36:5, 425-434
To link to this article: http://dx.doi.org/10.1080/15533170600732718
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Synthesis and Reactivity in Inorganic, Metal-Organic and Nano-Metal Chemistry, 36:425–434, 2006
Copyright # 2006 Taylor & Francis Group, LLC
ISSN: 0094-5714 print/1532-2440 online
DOI: 10.1080/15533170600732718
Synthesis, Characterization, and Measurement of
Antioxidant Reactivity of Salicylidene-D,L-Tyrosine Ethyl Ester
and Copper(II)(Salicylidene-D,L-Tyrosine Ethyl Ester)2 in a
Linoleic Acid Peroxidation Reaction System
S. H. Minasyan
Institute of Chemical Physics, Armenian National Academy of Sciences, Yerevan, Armenia
S. H. Ghazaryan
Institute of Fine Organic Chemistry, Armenian National Academy of Sciences, Yerevan, Armenia
V. J. Tonoyan and S. A. Bajinyan
Research Center of Radiation Medicine and Burns, Ministry of Health, Yerevan, Armenia
K. P. Grigoryan
Institute of Fine Organic Chemistry, Armenian National Academy of Sciences, Yerevan, Armenia
J. R. J. Sorenson
Division of Medicinal Chemistry, Department of Pharmaceutical Sciences, College of Pharmacy,
University of Arkansas, Medical Sciences Campus, Little Rock, Arkansas, USA
F. T. Greenaway
Carlson School of Chemistry and Biochemistry, Clark University, Worcester, MA, USA
Keywords Schiff base, salicylidene-tyrosine ethyl ester, copper(II)
(salicylideneTyrOEt)2, linoleic acid, peroxidation,
antioxidant
Salicylidene-D,L-tyrosine ethyl ester and Cu(II)(SalTyrOEt)2
were synthesized, characterized, and chain-breaking antioxidant
and prooxidant reactivities determined in a 2,20-azoisobutyroni-
trile initiated linoleic acid peroxidation reaction. Kinetics of
oxygen consumption were found to be comparable to the
antioxidant reactivity of 2,6-ditertiary-butyl-4-hydroxytoluene,
due to efficient removal of linoleic acid peroxyl radical with the for-
mation of linoleic acid hydroperoxide, LOOH, and correlate with
the presence of tyrosyl phenolic OH groups. Copper in
Cu(II)(SalTyrOEt)2 accounts for prooxidant reactivity, catalyzing
removal of LOOH, except at low concentrations when its antioxi-
dant properties dominate. It is concluded that Cu(II)(SalTyrOEt)2
is a unique antioxidant with prooxidant reactivity.
INTRODUCTION
Copper(II) complexes are the principal forms of copper
found in all tissues since the concentration of non-complexed
free copper(II) is ꢀ10218 M and not measurable with
existing equipment.[1,2] These copper complexes are small
molecular mass amino acid, carboxylic acid, and amino sugar
complexes, a copper albumin complex, and all copper-depen-
dent enzymes and storage proteins.[1,2] Thermodynamic stab-
ilities and coordination competitions account for ligand
exchange chemistry and the primary and ultimate existence
of the variation in copper complexes found in all biochemically
and physiologically relevant forms of copper in all biological
systems.
Received 23 February 2006; accepted 29 March 2006.
This work was supported by the International Science Technology
Center (A-361-00 Project). We thank Dr. Prof. L. Tavadyan, Institute
of Chemical Physics, NAS of Armenia, for his helpful discussions;
Dr. G. Sedrakyan and L. Haroutunyan for their technical assistance
and Dr. T. Stepanyan, Molecular Structure Research Center, Spec-
troscopy Laboratory, NAS of Armenia, for her assistance in obtaining
FTIR spectra.
Copper(II) complexes of amino acid Schiff bases have
become important due to their varied pharmacological
effects, which include anti-inflammatory, anti-ulcer, anti-
convulsant, anti-diarrheal, antimicrobial, and anticancer
Address correspondence to F. T. Greenaway, Carlson School of
Chemistry and Biochemistry, Clark University, Worcester, MA 01610-
1477, USA; Fax: 508-793-8861; E-mail: fgreenaway@clarku.edu
425

426
S. H. MINASYAN ET AL.
activities.[1–5] Schiff base Cu complexes are versatile models Schiff bases and in particular salicylidene-D,L-tyrosine ethyl
of metalloelement drugs and their structural and bonding ester [SalTyrOEt] (Figure 1a) were chosen for evaluation
modes provide a better understanding of protein-metalloele- with regard to their antioxidant and prooxidant reactivities
ment bonding in ligand exchange processes.[6–11] These com- using a biochemically relevant linoleic acid peroxidation
plexes can be used as catalysts for the decomposition of H2O2 reaction system.[24]
and organic hydroperoxides as catalase-mimetic and peroxi-
dase-mimetic agents respectively[1,2,12] and for the decompo-
sition of superoxide as superoxide dismutase (SOD)-mimetic
agents.[1–3,13]
The pharmacological effectiveness of copper complexes,
consistent with their involvement in various biochemical
process in normal tissues and in repair responses to
overcome diseases states associated with free radical pathol-
ogies, are suggested to be due, in part, to their antiradical
SOD-mimetic, catalase-mimetic, and peroxidase-mimetic
reactivities.[1,2,13] In these roles the reaction center is the
central metalloelement with redox reactivity having beneficial
effects in overcoming reactions associated with many disease
states. Since complexed forms of copper account for all mea-
surable amounts of copper in tissues and ligands forming
these complexes protect biochemical systems from the redox
reactivity of free Cu(II), indiscriminate coordination reactivity,
and chemical incompatibilities associated with physiological
pH values,[14–18] these complexed forms of copper are most
relevant for use as pharmacological agents.[1–5,11–13,19–23]
These complexes may also serve as Cu delivery agents and
facilitate de novo Cu-dependent enzyme syntheses need to
overcome disease pathology.[1,2] Copper complexes do have
prooxidant reactivity, which accounts for their peroxidase-
mimetic reactivity in removing or metabolizing lipid hydroper-
oxides in vivo, which always requires an oxidizable co-sub-
strate in biochemical systems.
The presence of antiradical centers in ligands bonded to
Cu, e.g., phenolic OH groups and the presence of redox
active Cu(II) capable of removing singlet oxygen (1O2),
superoxide, O22 †, hydroxyl radical (HO†), and hydroperoxyl
radical (HOO†), can play key roles in accounting for the
antioxidant reactivity of these complexes and directly take
part in preventing free radical processes associated with
the oxidation of biological and model substrates.[19–23]
copper(II) complexes of such ligands can produce beneficial
antioxidant effects in addition to the antioxidant reactivity
of the central metalloelement as an antiradical reaction
center. According to Berthon et al.[19,20] ligands that have
high antiradical reactivity decrease the manifestation of
prooxidant reactivity of the central metalloelement and may
be modified appropriately for utility in biological systems.
It is then reasonable to search for potential pharmacologically
effective Cu(II) complexes that possess a prooxidant to anti-
oxidant converting mechanism of action or a combination of
these reactivities in lipid media, taking into consideration the
great importance of preventing abnormal non-metabolic lipid
oxidation processes in biochemical systems. Accordingly,
Cu(II) complexes formed with lipophilic ligands such as
FIG. 1. (a) The salicylidene-DL-tyrosine ethyl ester Schiff base ligand and
(b) the Cu(II)(SalTyrOEt)2 complex.

ANTIOXIDANT REACTIVITY OF Cu(II)(SalTyrOEt)2
427
MATERIALS AND METHODS
After 20 h the solution was heated at 408C for 10 h to obtain
a light green solution, which when allowed to cool to room
temperature gave a green precipitate. The green solid was
filtered, washed with 5 ꢁ 20 ml diethyl ether and twice dis-
tilled water (5 ꢁ 20 ml), and after air-drying for 12 h gave a
green powder, the Cu(II) complex of salicylidene-D,L-
tyrosine ethyl ester. Yield: 1.31 g (63.7%). Melting point
with decomposition: 265 to 2708C (760 mm, Hg). Anal. Calc.
for CuC36H36N2O8 (%): C, 62.83; H, 5.27; N, 4.07; Cu 9.23.
Found: C, 63.19; H, 5.43; N, 4.06; Cu 8.73.A thin layer chro-
matogram gave a single spot, Rf ¼ 0.79, using a propanol:bu-
tanol:dimethylformamide:water (7:8:2:3) solvent system.
Reagents
Chlorobenzene (Aldrich) was dried over anhydrous CaCl2
and double distilled. Heptane, n-butanol, methanol, and aceto-
nitrile, all from Aldrich, were purified by double distillation.
Chlorobenzene and heptane were purified by passing them
over a column of activated Al2O3; 2,20-azoisobutyronitrile
(AIBN) (Polyscience, Inc.) was purified by recrystallization
from methanol; absolute ethanol was purified according to
known procedures,[25] and 2,6-di-tert-butyl-4-methylphenol
(BHT) (Aldrich) was repeatedly recrystallized from heptane
to obtain a constant melting point, 69 to 708C. Linoleic acid
(LH) (Aldrich), tetrabutylammonium perchlorate (TBAP),
D,L-tyrosine ethyl ester, salicylaldehyde, N,N0 bis(salicyli-
dene-ethylenediamine), Cu(II)2(CH3COO)4(H2O)2 (Fluka)
and Cu(II)2(stearate)4 (Chemreactiv, Russia) were reagent
grade and were used without further purification. All other
chemicals used were of highest available purity. Synthesis of
D,L-Tyrosine ethyl ester performed by a known method.[26]
Synthesis of salicylidene-tyrosine ethyl ester [SalTyrOEt]
(Figure 1a) and its Cu(II) complex (Figure 1b) were by
reported literature methods[27–30] as detailed below.
Spectroscopic Measurements
Fourier transform infrared (FTIR) spectra of the complex
and ligand were measured from 4000 to 400 cm21 using KBr
disks or nujol mulls respectively with a Nicolet FTIR Nexus
Spectrometer, using 32 scans and a resolution of 4 cm21
.
UV-Vis spectra were obtained for methanol solutions from
205 to 900 nm with a Specord M50 (Germany) spectropho-
tometer. Proton NMR spectra were obtained in dimethyl
sulphoxide-d6 using a 300 MHz Mercury, Varian (U.S.A.)
spectrometer. Copper analyses were performed with an AAS
1 atomic absorption spectrophotometer (Carl Zeiss, Jena,
Germany).
Synthesis of Salicylidene-D,L-Tyrosine Ethyl Ester
A 58C chloroform solution of the ethyl ester of D,L-tyrosine,
10 mmol (2.09 g), was added drop wise to a stirred 50 ml
chloroform solution of salicylaldehyde, 10 mmol (1.08 ml).
The reaction mixture was stirred for 2 h at 208C. Then
20 mmol (2.4 g) of anhydrous MgSO4 was added with stirring
to dry the chloroform solution. The mixture was stirred for
60 h, at 408C under solvent-saturated high purity nitrogen
gas. After the magnesium sulfate was removed by filtration
the chloroform solution was extracted with 5 ꢁ 10 ml of
twice distilled water, 5 ꢁ 10 ml of 0.5% solution of acetic
acid, and again with 5 ꢁ 10 ml of water. This chloroform
solution was dried over anhydrous sodium sulfate. The
yellow solution was concentrated by evaporation in vacuo at
508C to remove the chloroform and cooled to 308C to obtain
the yellow colored amorphous ethyl ester and dried in vacuo
over P2O5. Yield: 1.85 g (59.3%). Melting point: 132 to
1338C (760 mm, Hg). Anal. Calc. For C18H19NO4 (%): C,
69.07; H, 6.07; N, 4.47; Found: C, 69.10; H, 6.17; N, 4.55. A
thin layer chromatogram gave a single spot having an Rf of
0.79 using a propanol:water (7:3) solvent system.
Measurements of the Relative Antioxidant Efficiency (RAE)
using Oxygen Consumption Experiments
The method for determining antioxidant efficiencies was
based upon the decrease in rate of oxygen consumption follow-
ing the formation of linoleic acid radical (L†) in chloroben-
zene, by thermal cleavage of 2,20-azoisobutyronitrile (AIBN)
and initiation of the oxidation of linoleic acid, reactions 1–5.
Reaction 5, which is the rate limiting step, can be inhibited
with an antioxidant capable of reacting with LOO† and pre-
venting subsequent oxidation of LH via chain propagating
reactions, 4 and 5.
Kinetic Analysis
The mechanism of the free radical chain reactions leading to
the oxidation of linoleic acid in homogenous solution with a
required concentration of oxygen ꢂ 1024
M needed to
support the oxidation reaction (4) has been published.[31–36]
Initiation
Ox†idative chain†reactions were initiated with the formation
of (R0 ) and (R0OO ) by reactions (1) and (2); and the conver-
sion of these radicals to L† by reaction (3):
Synthesis of Copper(II) Salicylidene-D,L-Tyrosine Ethyl
Ester
Copper(II)2(acetate)4(H2O)2, 1.5 mmol (0.544 g), was vig-
orously stirred in a mixture of 50 ml absolute ethanol and
chloroform (1:1) to affect solution. Six mmol (1.88 g) of salicy-
lidene-D,L-tyrosine ethyl ester dissolved in 30 ml of chloroform
was added to the solution of copper acetate and gave a blue
solution. This reaction mixture was stirred for 30 h at 308C.
D
R0 ꢃ N ¼ N ꢃ R0 !½R0†N2R0†ꢄsolvent cage!2R0† þ N2
ð1Þ
very fast
0† þ O2 ! R0OO†
R
ð2Þ
ð3Þ
R0OO† þ LH!R0OOH þ L†

428
S. H. MINASYAN ET AL.
where R0 2 N ¼ N 2 R0 is the azo initiator, 2,20-azoisobutyr- of all tested compounds were prepared in butanol. The
†
†
onitrile, R0 is the cyano-iso-propyl radical, (CH3)2C C N, kinetics of oxygen consumption during the oxidation of
;;
and R0OO† is †the cyano-iso-propylperoxyl radical, linoleic acid was determined, at 760 torr O2, using a highly sen-
;;
(CH3)2(N C)COO :
sitive calibrated 1 mm diameter capillary gas micro-volume
meter to measure oxygen above the reaction mixture.[31,32]
This oxidation reaction was performed in a 10 ml glass
reaction vessel fitted with a water circulation jacket to facilitate
temperature control and mounted on a magnetic stirrer. The
reaction vessel was built to accommodate a membrane
designed for the injection of reagent solutions without
opening the cell. The vessel containing 4.6 ml 0.163 M LH in
chlorobenzene was attached to the micro-volume meter. The
stirred mixture was pre-equilibrated at the reaction tempera-
ture, 508C, for 25 to 30 min to allow thermal equilibration
and to stabilize the micro-volume meter reading prior to
reaction initiation. Then the oxidation reaction was initiated
as in the reaction sequence (1) to (3)[33–36] with the injection
of 0.3 ml of a solution of AIBN in chlorobenzene through the
membrane using a syringe. After 1 to 2 min, 0.1 ml of a
butanol solution of the test antioxidant compound was intro-
duced into the reaction mixture and, after 2 min, oxygen
uptake was recorded as a function of time. The final volume
of all reaction mixtures was 5 ml, the temperature was
50 + 0.18C, the final concentrations were 0.15 M LH, 0.01
M AIBN, and the concentration of the test compound varied
in the range from 1.1 ꢁ 1026 to 5.5 ꢁ 1024 M. The reaction
mixture was rapidly stirred at 600 rpm to ensure kinetic
rather than diffusion control of the oxidation reaction rate.
Experiments to determine prooxidant reactivity of the test
compound in the presence of LOOH were performed after
allowing LOOH to accumulate in the absence of the test
compound. Only after the accumulation of ꢀ0.001 M LOOH,
based upon the amount of consumed oxygen as determined
earlier under the same reaction conditions, was a solution of
the studied compound injected into the reaction mixture.
Propagation
Propagation involves a cyclic process converting L† to
LOO† in the presence of oxygen:
v:fast
L† þ O2 ! LOO†
ð4Þ
ð5Þ
kp;slow
LOO† þ LH ! LOOH þ L†
where kp is the rate constant for propagation.
Termination
Chain termination reaction (6) limits the extent of lipid per-
oxidation in the absence of inhibitors, destroying two peroxyl
radicals and yielding non-radical products.
kt
LOO† þ LOO† ! non-radical products
ð6Þ
where kt is the rate constant for termination. In the absence of a
chain-breaking antioxidant and under a long-chain approxi-
mation, where the number of R† ! ROO† cycles is large,
the overall rate of uninhibited oxidation (Runinh) is described
by the kinetic equation:[32–34]
d½O2ꢄuninh
kp
Runinh ¼ ꢃ
¼
½LHꢄR1i =2
ð7Þ
1=2
dt
ð2 ktÞ
where Ri is the rate of initiation, and 2d[O2]uninh/dt is the rate
of oxygen uptake for the uninhibited oxidation.
In the presence of a peroxyl radical trapping antioxidant,
AH, the rate-controlling step for inhibition by the removal of
LOO† produced in reaction (4) is:
kinh
LOO† þ AH !LOOH þ A†
ð8Þ
ð9Þ
Calculation of Oxygen Consumption Rates
fast
LOO† þ A† ! non-radical products
The kinetics of oxygen consumption measured in the
presence of various concentrations of the studied compounds
based upon a kinetic plot of oxygen concentration change
with time are shown in Figure 2. Initial rates of oxygen con-
sumption were determined from these kinetic plots. The
ð10Þ initial slope for the plot of to to t for these kinetic plots was cal-
Under conditions of inhibition, the rate of oxidation is given
by the kinetic equation;
d½O2ꢄinh
kp
Rinh ¼ ꢃ
¼
½LHꢄRi
dt
nkinh½AHꢄ
culated to obtain the initial rate of LH oxidation, d[O2]inh
/
where 2 d[O2]inh/dt is the rate of oxygen uptake for the inhib- dt(to).
ited oxidation, Rinh is the rate of inhibited oxidation of linoleic
Measurements of the Antioxidant Efficiency (AE), Relative
Antioxidant Efficiency (RAE), and Antiperoxyl Radical
Capacity (ARC)
acid, kinh is the rate constant for the inhibition of the reaction of
linoleic acid peroxyl radical with an antioxidant, and n is a stoi-
chiometric factor for the removal of linoleic acid peroxyl
radical by an antioxidant.[31–36]
Calculations of AE and RAE have been described in
detail.[33] The plot of the initial rate of oxygen consumption,
d[O2]inh/dt (to), is dependent upon the inverse concentration
Oxygen Consumption Experiments
Stock solutions of 0.163 M LH and 0.1667 M AIBN were of antioxidant, [AH]2o 1, added to the oxidation reaction
prepared in nitrogen-saturated chlorobenzene. Stock solutions system. The concentration range was chosen to obtain a

ANTIOXIDANT REACTIVITY OF Cu(II)(SalTyrOEt)2
429
FIG 2. Kinetic plots of oxygen consumption during thermally initiated oxi-
dation of 0.15 M linoleic acid by 1022 M AIBN in chlorobenzene at 508C
(Ri ¼ 4 ꢁ 1028 Ms21): without antioxidant (1); 1.1 ꢁ 1024 M SalTyrOEt
(2); 0.55 ꢁ 1024 M Cu(II)(SalTyrOEt)2 (3); 1.1 ꢁ 1024 M BHT (4); in the pre-
sence of 0.275 ꢁ 1024 M Cu(II)(stearate)2 (5); or 1.1 ꢁ 1024 M SalTyrOEt
and 0.275 ꢁ 1024 M Cu(II)(stearate)2 mixture (6).
FIG. 3. The effect of various concentrations of Cu(II)(SalTyrOEt)2 on oxygen
consumption during thermally initiated oxidation of 0.15 M linoleic acid by
1022 M AIBN in chlorobenzene at 508C. Without antioxidant (1), or in the pre-
sence of 5.5 ꢁ 1026 M (2), 11 ꢁ 1026 M (3), 22 ꢁ 1026 M (4), or 55 ꢁ 1026
M
(5) Cu(II)(SalTyrOEt)2, or in the presence of both 22 ꢁ 1026
M
Cu(II)(SalTyrOEt)2 and 1 ꢁ 1023 M LOOH (40) (Ri ¼ 4 ꢁ 1028 Ms21). The
induction period, t, is indicated by an arrow.
rectilinear plot. From equation (10), a plot was calculated with
the slope of S where S was defined in equation (11):
molecule.[33] For SalTyrOEt and Cu(II)(SalTyrOEt)2 these
values were calculated by measuring the induction period in
the presence of the reference BHT antioxidant, tBHT, and the
test compound, tAH, according to equation (15):
kp½LHꢄRi
S ¼
ð11Þ
nkinh
The ratio of kind to kp shown in equation (12) is defined as
AE,
ARCAH ¼ ð2½BHTꢄotAHÞ=ð½AHꢄotBHT
Þ
ð15Þ
kinhib ½LHꢄRi
AE ¼
¼
ð12Þ
where [AH]o is the initial concentration of AH.
kp
nS
These values can be calculated from S, Ri, and n, which
usually equals 2 for a monophenolic antioxidant.[31–36] The
initiation rate, Ri, was calculated by measuring the time of
RESULTS AND DISCUSSION
the induction period, t (see Figure 3), in the presence of a refer- Spectral Characterization of SalTyrOEt and
ence antioxidant, BHT, according to equation (13):
Cu(II)(SalTyrOEt)2 Complex
The SalTyrOEt ligand was characterized by 1H NMR,
FTIR, and UV-Visible spectra. Proton NMR spectra were
obtained in DMSO-d6 and contained absorbances (d, ppm) at
1.26 t (3H, J ¼ 7.1 Hz, C10H3); 2.97 dd (1H, J ¼ 13.6,
8.3 Hz, C8Ha); 3.18 dd (1H, J ¼ 13.6, 5.4 Hz, C8Hb); 4.16 dd
(1H, J ¼ 8.3, 5.4 Hz, NC7H), 4.16 q (2H, J ¼ 7.1, Hz,
OC9H2); 6.61 m (2H, C12H); 6.81 td (1H, J ¼ 7.4, 1.3 Hz,
C3H); 6.84 br.d (1H, J ¼ 8.5, Hz, C1H); 6.91 m (2H, C11H);
7.22 dd (1H, J ¼ 7.6, 1.8 Hz, C4H), 7.27 ddd (1H, J ¼ 8.5,
7.2, 1.8 Hz, C2H); 8.15 s (1H, N ¼ C5H); 8.83 br.(1H,
Ri ¼ 2½BHTꢄot
where [BHT]o is the initial concentration of BHT.
ð13Þ
The RAE was obtained directly from the ratio of the plot
slopes by dividing the value obtained for the test antioxidant,
AH, by the value obtained by the reference antioxidant,
BHT, as shown in equation (14):
AEAH
kinh
SBHT
SAH
RAE ¼ ꢃ
¼
¼
ð14Þ
AEBHT kinh;BHT
The RAE can also be determined by dividing the value of salicyl-O6H); 12.84 br. (1H, tyrosyl-O13H). The salicyl OH
AE for the antioxidant by the value of AE for BHT. The AE evidences the greatest shift due to intramolecular hydrogen
and RAE values are the quantitative kinetic parameters for bonding shown in Figure 1a. The tyrosine phenolic group is
antioxidant reactivity. Using experimental values for AE also shifted due to intermolecular hydrogen bonding with
and RAE as well as literature data for BHT kinh
3 ꢁ 104 M21 21 [32]
,
kinh was determined for a test antioxidant
DMSO.
The UV-Visible spectrum of SalTyrOEt contained three
maxima, lmax (nm) and 1 (M21cm21), at 214.6 (25640) for
s
from equation (14).
,
Antiperoxyl radical capacity (ARC), which is the measure C55O, 258.8 (11340) for the salicyl ring, and 317.2 (4060)
of the number of radicals that react per antioxidant for the tyrosyl ring.

430
S. H. MINASYAN ET AL.
The FTIR spectrum of the Schiff base contains absorptions
Results of elemental analyses, FTIR, and UV-Vis spectra
at 1735 and 1643 cm21 assignable to the ester carbonyl group show that SalTyrOEt coordinates with the Cu(II) atom in a tri-
and the azomethyne, CH55N, group vibrations, respectively. dentate manner via the carbonyl oxygen, azomethine nitrogen,
The salicyl phenolic OH group exhibits a very broad absorption and the salicyl-phenolate O2 groups to form a mononuclear
through the range of 2200 to 3200 cm21 due to overlapping complex with proton loss from the salicyl phenolic OH group
aromatic absorptions centered at 2520, 2600, 2683, and consistent with the structure shown in Figure 1b.
2806 cm21. A broad absorption through the range of 2800 to
3200 cm21 is assigned to aromatic and aliphatic CH absor-
bances, which overlap with the 2806 cm21 absorption. The
Antioxidant Efficiencies
As shown in Figure 2, plot 1, there was a rapid linear con-
tyrosyl-phenolic OH group gave a broad absorption through
the range of 3200 to 3600 cm21, centered at 3454 cm21 and
overlapping with absorptions for the CH and salicyl-phenolic
OH absorptions. The broad absorption for the salicyl OH
group was attributed to intramolecular hydrogen bonding
between the phenolic OH hydrogen atom and the nitrogen of
the azomethyne group as shown in Figure 1a. The absorption
observed at 1242 cm21 is attributed to salicyl phenolic C–O
stretching and the absorption observed at 1258 cm21, is attrib-
uted to tyrosyl phenolic C–O stretching
sumption of oxygen with time in the absence of antioxidant
for the uninhibited reaction: LOO† þ LH ! LOOH þ L†
(Reaction 5) leading to the regeneration of LOO† in the sub-
sequent reaction: L† þ O2 ! LOO† (Reaction 4). Addition of
1.1 ꢁ 1024 M SalTyrOEt, 5.5 ꢁ 1025 M Cu(II)(SalTyrOEt)2
or 1.1 ꢁ 1024 M BHT, representing equal concentrations of
the phenolic OH group, caused a marked decrease in rate of
oxygen uptake (plots 2, 3, and 4, respectively) compared to
the uninhibited reaction rate (plot 1). This shows that the
studied compounds have chain-breaking antioxidant reactivity,
which may be due to the antioxidant reactivity of SalTyrOEt
and the removal of LOO† (Reactions 16–18 in the case of SalT-
yrOEt). The decrease in the oxygen consumption rate is also
consistent with antioxidant reactivity in the case of TyrOEt.
When TyrOEt, SalTyrOEt or BHT were introduced into the oxi-
dation system at concentrations ranging from 5.5 ꢁ 1026 to
5.5 ꢁ 1024 M, the initial rate of oxygen consumption decreased
linearly with increasing concentration. As shown in Figure 3,
addition of increasing concentrations of Cu(II)(SalTyrOEt)2
ranging from 5.5 ꢁ 1026 to 2.2 ꢁ 1025 M (plots 2 to 4) also
decreased the initial rate of oxygen consumption in a concen-
tration related manner when compared with the uninhibited
reaction (plot 1), with a nearly constant decrease in the initial
rate of oxygen uptake over the first 2000 sec for plots 2, 3
and 4 when compared to plot 1.
The Cu(II) complex is soluble in DMF, DMSO, methanol,
butanol, and other polar solvents and is quite stable in
air under dry and dark conditions, giving reproducible
kinetics as an antioxidant over a 6 month period. Elemental
analysis data support the formation of a 1:2 complex,
Cu(II)(SalTyrOEt)2. The UV-Visible spectrum obtained
in methanol had absorptions at lmax nm with
1
(M21cm21) ¼ 223.4 (55160), 242 (34950) shoulder, 269.4
(23000), 370 (8260), and 632 (120). The broad absorption
centered at 632 nm was assigned to a d-d transition and indi-
cates a distorted octahedral geometry around the Cu(II) atom
with two nitrogen ligand atoms. The band at 370 nm is due
to a phenolate ! Cu(II) charge transfer transition, which indi-
cates bonding of the salicyl-phenolate oxygen atom to
Cu(II).[37,38] The FTIR spectrum of the Cu(II) complex con-
tained absorptions at 3419, 1714, and 1619 cm21 assignable
to the tyrosyl-phenolic OH group, the ester carbonyl group,
and the azomethyne group vibrations, respectively. The
21 cm21 shift to lower frequency for the carbonyl group and
a 24 cm21 shift for the azomethyne group as well as the
decrease in absorption intensity for the carbonyl oxygen
suggest bonding of the carbonyl oxygen and azomethyne N
atoms to copper, consistent with published literature for com-
Quantitative kinetic antioxidant parameters obtained using
experimental curves for oxygen consumption in the presence
of various concentrations of the studied compounds and
equations (12–15) for the addition of TyrOEt, SalTyrOEt,
Cu(II)(SalTyrOEt)2, or BHT to the linoleic acid oxidation
reaction mixture are presented in Table 1.
Based upon the RAE, AE, kinh, and ARC data shown in
Table 1, Cu(II)(SalTyrOEt)2 and SalTyrOEt have antioxidant,
antiperoxyl radical reactivity, similar to that of BHT in inhibit-
ing oxidations caused by linoleic acid peroxyl radical in a lipid
reaction medium. Dividing the RAE and ARC values obtained
for Cu(II)(SalTyrOEt)2 by 2 to obtain an equivalent number of
tyrosyl-phenolic OH groups (Table 1) shows that the apparent
greater antioxidant reactivity of this complex is due to its
greater number of tyrosyl-phenolic OH groups. Thus, these
results indicate a BHT-like chain-breaking antioxidant mech-
anism[32] implying that mechanisms of antiperoxyl radical
and antioxidant reactivity of Cu(II)(SalTyrOEt)2, SalTyrOEt,
and TyrOEt involve abstraction of the hydrogen atom from
the tyrosyl-phenolic OH group by linoleic acid peroxyl
plexes with similar ligands[37] [40]
. The salicyl phenolic C–O
-
absorbance at 1242 cm21 is lost and new broad absorptions
occur at 1258 cm21 and 756 cm21 upon complex formation,
indicating bonding of the salicyl phenolic oxygen atom
and the Cu(II) atom, which is also consistent with the published
literature.[37–40] The very broad 2200 to 3200 cm21 absorption
of the free salicyl phenolic OH group of the ligand is not
present in the copper complex, which supports deprotonation
of the salicyl-phenolic OH group with Cu(II) coordination.
This is also supported by the appearance of a new band at
460 cm21 which is attributed to Cu(II)–O salicyl-phenolate
vibrations.[37–40]

ANTIOXIDANT REACTIVITY OF Cu(II)(SalTyrOEt)2
431
TABLE 1
Values of RAE, AE, kinh and ARC
Antioxidant
RAE
AE ¼ kinh/kp
kinh
ARC
BHT
TyrOEt
1
a300
57
b3 ꢁ 104
5.66 ꢁ 103
9.35 ꢁ 103
5.78 ꢁ 104
2.89 ꢁ 104
2
c
0.189
0.316
1.92
0.96
SalTyrOEt
Cu(II)(SalTyrOEt)2
Cu(II)(SalTyrOEt)2 4 2
94
2.55 + 0.20
3.58 + 0.20
1.79 +0.10
578
289
aThe kinh/kp value for BHT agrees with the literature,[32] bkp ¼ 100 M21 21 [41] c
s
,
not determined.
Obtained for test compounds in the thermally initiated oxidation of 0.15 M linoleic acid with 1022 M AIBN
(Ri ¼ 4 ꢁ 1028 Ms21) in chlorobenzene at 508C, equations (12) to (14).
radical to form a tyrosyl-phenoxyl radical, TyrO†, as shown in
reaction (16), which rapidly reacts to form non-radical the Cu(II) atom cannot be involved in antiperoxyl radical reac-
Since the ARC value for Cu(II)(SalTyrOEt)2 is close to 2,
products, as in reactions (17) and (18):
tions of Cu(II)(SalTyrOEt)2. Direct participation of Cu(II) in
the removal of peroxyl radicals would result in a larger ARC
value for the complex, close to 2.5. The high oxidation potential
of Cu(II), E8Cu(II)/Cu(III) ¼ –2.6 V at pH ꢅ 7,[45] indicates that
the reaction Cu(II) þ LOO† ! Cu(III) is much less likely to
occur than oxidation reactions involving peroxyl radicals such
as tert-butylOO†, E8t–ert-butylOO†/tert-butylOO ¼ 20.7 V.[46] The
lack of a measurable reaction between Cu(II) and tert-butyl
peroxyl radical has been previously reported.[23]
kinh
LOO† þ TyrOH !LOOH þ TyrO†
ð16Þ
ð17Þ
ð18Þ
fast
LOO† þ TyrO† ! non-radical products
fast
TyrO† þ TyrO† ! non-radical products
Additional evidence that the antioxidant or antiperoxyl
radical reactivities of SalTyrOEt and Cu(II)(SalTyrOEt)2 are
due to the presence of the tyrosyl-phenolic OH group in these
molecules comes from the fact that Schiff bases that do not
have an equivalent tyrosyl-phenolic OH group such as N,N0-
bis (salicylidene)ethylenediamine and salicylidene Schiff bases
of ethyl esters of g-aminobutyric acid or tryptophan do not
possess measurable antiperoxyl radical reactivity (our unpub-
lished observation). The phenolic OH of the salicyl group
present in N,N0-bis(salicylidene)ethylenediamine, in salicylidene
Schiff bases of g-aminobutyric acid ethyl ester and tryptophan,
as well as in SalTyrOEt, does not contribute to antiperoxyradical
reactivity because of intramolecular hydrogen bonding between
the hydrogen atom of the salicyl-phenolic OH group and the
nitrogen of azomethyne group as shown in Figure 1a for SalTyr-
OEt. This intramolecular hydrogen bonding prevents phenoxyl
radical formation and markedly reduces the antioxidant or anti-
peroxyl radical reactivity of these phenolic hydroxyl groups,
consistent with literature reports.[23,42–44]
Prooxidant Reactivities
TyrOEt, SalTyrOEt, and BHT have no prooxidant reactivity
in our experimental conditions. Copper(II) complexes possess
prooxidant reactivities including peroxidase-mimetic reactiv-
ity, which is due to the ability of Cu(II) complexes to
catalyze the removal of accumulated LOOH formed in the
chain oxidation of linoleic acid as well as in the antioxidation
reaction of LOO† with tyrosyl phenolic OH groups. Products
of prooxidation, LOO† and LO†, formed in these reaction pro-
cesses then co-initiate the chain peroxidation of linoleic
acid.[14–21] The prooxidant reactivity of Cu(II) complexes,
which involves the oxidation of LOOH (19), the homolysis
of LOOH (20) by Cu(I) formed in reaction (19) and the
peroxidase-mimetic reactivity (21), was evaluated for
Cu(II)(SalTyrOEt)2 at times long after the peroxidation
reaction induction period, t, to allow for accumulation of
LOOH. The increased oxidation rate in the presence of
Cu(II)(SalTyrOEt)2, in comparison with the uninhibited oxi-
dation reaction rate, indicates that this prooxidant reactivity,
reactions (19) and (20), is observable once linoleic acid hydro-
peroxide accumulates as a reaction product.
The ARC value for SalTyrOEt and Cu(II)(SalTyrOEt)2, cal-
culated per tyrosine ligand, is close to 2, see Table 1, which
indicates that the ligand has one phenolic antiperoxyl radical
reaction center. The somewhat larger ARC value for SalTyr-
OEt than for BHT or Cu(II)(SalTyrOEt)2, suggests that the sal-
icylic phenolic OH group may play some role in reactions with
peroxyl radicals, although this role is probably small since the
difference in ARC values is not large. Conversely, the value of
kinh is larger for Cu(II)(SalTyrOEt)2 than for compounds
having salicylic phenolic OH groups.
CuðIIÞ þ LOOH!CuðIÞ þ LOO† þ Hþ
CuðIÞ þ LOOH!CuðIIÞ þ LO† þ OHꢃ
CuðIÞ þ LOOH!LO ꢃ CuðIIIÞ ꢃ OH
ð19Þ
ð20Þ
ð21Þ
An alternative explanation for the ability of Cu(II) to induce
lipid peroxidation in the presence of a lipid hydroperoxide but

432
S. H. MINASYAN ET AL.
not a reducing agent, involves the oxidation of Cu(II) by the reactivity is also consistent with the observation the oxygen
hydroperoxide, forming Cu(III) and the lipoxyl radical, consumption rate decreased as the complex concentration
reaction 22:[18]
increased up to 2.2 ꢁ 1025 M Cu(II)(SalTyrOEt)2 (Figure 3,
plots 2, 3 and 4). Upon a further increase in concentration to
5.5 ꢁ 1025 M, the antioxidant reactivity begins to exceed the
prooxidant reactivity (Figure 3, plot 5), as expected when
CuðIIÞ þ LOOH!CuðIIIÞ þ LO† þ OH
ð22Þ
Both the lipoxyl radical LO† and Cu(III) generated
according to reaction (22) are capable of initiating lipid per-
oxidation, reaction (23), leading to the regeneration of
LOO†, reaction (4):
compared with the decrease shown for 2.2 ꢁ 1025
M
Cu(II)(SalTyrOEt)2. In addition, when the concentration of
Cu(II)(SalTyrOEt)2 is increased, the decrease in the rate of
oxygen consumption persists longer. These results indicate
that Cu(II)(SalTyrOEt)2 possesses low prooxidant reactivity
at concentrations ꢀ 5.5 ꢁ 1025 M.
CuðIIIÞ þ LH!CuðIIÞ þ L† þ Hþ
ð23Þ
v:fast
L† þ O2 ! LOO†
Comparison of Antioxidant and Prooxidant Reactivities
The marked chain-breaking antioxidant reactivities of
Cu(II)(SalTyrOEt)2 are kinetically dominant and Cu(II)(Sal
TyrOEt)2 acts as a phenolic antioxidant as does the free
ligand, SalTyrOEt, which does not possess any measurable
prooxidant properties under conditions of these experiments.
The antiradical tyrosyl-phenol OH reaction center of
Cu(II)(SalTyrOEt)2 removes LOO† formed in reaction (5), as
well as LOO† and LO† formed in the removal of LOOH by
reactions (19), (20), and (22), and thus suppresses the free
radical oxidative reactions initiated by these species as well
as the chain propagation reactions (4), and (5). During the
induction period, when the concentration of LOOH is still
low and the concentration of the tyrosine centers is still
appreciable, the formation of non-radical products occurs as
in reaction (21). Therefore this ligand converts potential proox-
idant reactivity of Cu(II) to a lipid hydroperoxide detoxifying
and utilizing antioxidant compound in a lipid medium. Under
these conditions the resultant complex manifests properties
of a sterically non-hindered phenolic antioxidant without
direct involvement of the central metalloelement in antiperoxyl
radical reactions, without demonstrating prooxidant reactivity.
It should be noted that as the oxidation reaction proceeds,
the antioxidant copper complex becomes depleted and other
copper complexes accumulate in the system, the ligands of
which are the products of peroxyl radical reactions with
tyrosine OH groups. These ligands, which do not have antiper-
oxyl radical centers, can influence the Cu(I)/Cu(II), Cu(II)/
Cu(III), Cu(I)/Cu(III) redox potentials and strengthen the
prooxidant reactivity of the metabolites of Cu(II)(SalTyrOEt)2.
This could explain the observed increase in rate of oxygen con-
sumption at the end of uninhibited oxidations in the presence of
Cu(II)(SalTyrOEt)2 (Figure 3, plots 2 and/or 4).
Copper chelation by the SalTyrOEt ligand suppresses the
prooxidant reactivity of the central metalloelement. This is evi-
denced by the fact that Cu(II)(SalTyrOEt)2 more effectively
suppresses the rate of oxygen consumption during the oxi-
dation of linoleic acid than does an equimolar mixture of
Cu(II)2(stearate)4 and SalTyrOEt (see Figure 2, plots 3 and
5). Additional evidence for this comes from the observation
that Cu(II)2(stearate)4 does not show antioxidant reactivity,
but instead has prooxidant reactivity as shown in Figure 2,
plot 6. Addition of 2.75 ꢁ1025 M Cu(II)2(stearate)4 to the
reaction mixture caused a rapid consumption of oxygen at a
level which was slightly greater than that observed for the unin-
hibited reaction.
SUMMARY AND CONCLUSIONS
We report here the synthesis, characterization, and evalu-
One possible mechanism explaining the prooxidant reactiv-
ity of Cu(II) complexes could be oxygen activation. However, ation of a new Schiff base, salicylidene-D,L-tyrosine ethyl
when the oxidation reaction of linoleic acid is performed in air ester ligand, SalTyrOEt (Figure 1a), and the Cu(II) complex,
(at an oxygen partial pressure of 150 mm Hg, no change in Cu(II)(SalTyrOEt)2 (Figure 1b) for antioxidant and prooxidant
kinetics or initial rate was observed for the oxidation reactivities. Kinetic parameters for chain-breaking antioxidant
reaction. This result suggests that oxygen activation involving efficiencies were determined in an in vitro linoleic acid
coordination to Cu(II) does not occur under our experimental initiated peroxidation reaction system selected as a biochemi-
conditions.
However, Cu(II)(SalTyrOEt)2 has prooxidant reactivity. The
cally relevant lipid model system.
Results of this research show that SalTyrOEt coordinates
initial reaction kinetics for the reactivity of Cu(II)(SalTyrOEt)2 with Cu(II) in a monobasic tridentate manner via a carbonyl
prooxidation were studied in the presence of 0.001M hydroper- oxygen, the azomethine nitrogen, and the salicyl-phenolate
oxide, LOOH. As shown in Figure 3, plot 40, the rate of oxygen oxygen produced by loss of the proton from the salicyl-
consumption markedly increased, demonstrating prooxidant phenolic OH group. Cu(II)(SalTyrOEt)2 and its ligand were
reactivity for Cu(II)(SalTyrOEt)2, with an ARC value for found to possess antiperoxyl radical antioxidant reactivity as a
Cu(II)(SalTyrOEt)2 being smaller than 4 (Table 1). These phenolic antioxidant in a lipid medium. This complex and its
experimental data directly show that reactions (19), (20), ligands react as sterically unhindered phenols to remove
and/or (22) occur in the oxidation system. This prooxidant peroxyl radicals, which are responsible for the chain

ANTIOXIDANT REACTIVITY OF Cu(II)(SalTyrOEt)2
433
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LIST OF ABBREVIATIONS
LH
L†
Linoleic acid
Linoleic acid alkyl radical
Linoleic acid alkyl peroxyl radical
Cyanoisopropyl radical
Cyanoisopropylperoxyl radical
Rate of peroxidation in the absence of anti-oxidant
Rate of initiation
LOO†
†
R0
R0OO†
Ro
Ri
BHT
kinh
2,6-di-tert-butyl-4-methylphenol
Rate constant of inhibition
Stoichiometric factor
copper(II)3.5H2O
and
aqua(50-phosphopyridoxylidene-DL-
phenylalaninato) copper(II)2.5H2O. Polyhedron 1991, 10,
2163–2169.
N
R
Rate of peroxidation
Squared correlation factor
Linoleic acid
R2
LH
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chemistry of copper(II) complexes of N-salicylidene-aminoaci-
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catalase. Ind. J. Chem. 1993, 32A, 108–113.
LOOH
LO†
ARC
ArO†
FTIR
T
Linoleic acid hydroperoxide
Linoleic acid oxyl radical
Antiperoxyl radical capacity
Phenoxyl radical
Fourier Transform Infrared
Temperature
2,20-Azobis(isobutyronitrile)
Antioxidant
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AIBN
AH
NMR
UV
Nuclear Magnetic Resonance
Ultraviolet
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