Voltammetric analysis of the possibility of derivatization of levodopa in the presence of aniline derivatives

Article abstract of DOI:10.1007/s13738-013-0263-z

Electrochemical oxidation of levodopa (LD) as one of the most well-known neurotransmitters has been studied in the presence of some aniline derivatives. The electron transfer of LD is followed by two competitive reactions in the presence of these amines.

Full text of DOI:10.1007/s13738-013-0263-z

J IRAN CHEM SOC
DOI 10.1007/s13738-013-0263-z
ORIGINAL PAPER
Voltammetric analysis of the possibility of derivatization
of levodopa in the presence of aniline derivatives
•
•
•
Lida Khalafi Mohammad Rafiee Mojgan Khoshnam
Seyed Mohammad Shoaei
Received: 27 December 2012 / Accepted: 12 April 2013
Ó Iranian Chemical Society 2013
Abstract Electrochemical oxidation of levodopa (LD) as
one of the most well-known neurotransmitters has been
studied in the presence of some aniline derivatives. The
electron transfer of LD is followed by two competitive
reactions in the presence of these amines. The reactions are
the Michael additions of side chain amine group of LD and/
or aromatic amines to electrochemically generated o-qui-
none. There are two ECE mechanisms for both pathways
and the competition between these inter and intramolecular
reactions drastically depends on the pH of the medium. The
pH dependence of reactions has been studied and the
observed homogeneous rate constants of the reactions were
estimated by digital simulation of cyclic voltammograms.
The effect of aniline substituents was also studied with
regard to their reactivities toward o-quinone of LD and the
competitive reactions. Based on the obtained results, the
products of intermolecular reactions are electroactive
diphenylamine derivatives and their half-wave potentials
depend on the nature of the aniline substituent.
Introduction
Levodopa (3,4-dihydroxyphenylalanine) is an unusual
amino acid which remains the gold standard drug for the
most effective treatment for symptomatic relief of Par-
kinson’s disease [1]. Parkinson’s disease is related to a
significant depletion of dopamine in the brain. LD, as an
immediate metabolic precursor of dopamine, is able to
cross the blood–brain barrier and is metabolized into the
central nervous system to dopamine [2]. Despite its unique
role, it has also been reported that the use of LD in high
doses, especially in patients with advanced disease, leads to
the development of motor complications very resistant to
therapy [3]. Due to this unique and important role, there are
many reported literature on the biological, chemical and
analytical study of this neurotransmitter and one of the
important concerns in these studies is the possibility of its
derivatization [4]. Among the various methods which have
been used in these studies; electrochemical methods are
more highlighted than others. LD and the other neuro-
transmitters have a well-known electron transfer reaction
and some interesting terms such as neuroelectrochemistry
and physcoanalytical electrochemistry have been used for
their electrochemical studies [5]. The well-defined elec-
trochemical behavior of neurotransmitters is related to the
presence of catechol in their structures. Similar to other
catechol containing neurotransmitters, LD undergoes
electrochemical oxidation and the product of this reaction
is its o-benzoquinone derivative. This electrochemically
oxidized o-quinone is a good electrophile and undergoes
Michael addition reaction in the presence of various
nucleophiles and its side chain amine group [6, 7]. Some
desired chemical reactions as well as Michael addition
have been employed successfully in derivatization and
determination of LD and other neurotransmitters [8–11].
Keywords Levodopa ꢀ Aniline derivatives ꢀ
Voltammetry ꢀ Digital simulation
L. Khalafi (&) ꢀ M. Khoshnam
Department of Chemistry, Shahr-e-Qods Branch,
Islamic Azad University, Tehran, Iran
e-mail: l_khalafi@yahoo.com
M. Rafiee
Department of Chemistry, Institute for Advanced
Studies in Basic Sciences, Zanjan, Iran
S. M. Shoaei
Department of Chemistry, Zanjan Branch,
Islamic Azad University, Zanjan, Iran
123

J IRAN CHEM SOC
Recently, we have studied the electrochemical oxidation of
neurotransmitters in the presence of 4-aminobenzoic acid
and its competitive Michael addition with the side chain
amine group of neurotransmitters [12]. The condition for
domination of each reaction was obtained and the reaction
products of intermolecular reactions were claimed to be
diphenylamine (DPA) derivatives. DPA and its derivatives
are most commonly used as antioxidants. For example,
DPA is widely used to prevent post-harvest deterioration of
apple and pear crops. DPA is a parent compound of many
derivatives, which are used for the production of pharma-
ceuticals, dyes and further small-scale applications [13].
These interesting electrochemical aspects of reactants and
products encouraged us to extend our studies, and the
electrochemical oxidation of LD was performed in the
presence of various aniline derivatives.
Results and discussion
Figure 1 shows the CVs of 1.0 mM solution of LD in the
pH range of 3.0–7.0. As shown in curves (a) to (c), the CVs
show one anodic peak (A1) related to the conversion of the
catechol moiety of LD to o-quinone and one cathodic peak
(C1) related to the reduction of the produced o-quinone
under mild acidic conditions (pH 3–5). At pH values equal
and more than 6.0, the CVs show one anodic peak in the
positive-going scan and two cathodic peaks (C1 and C2) in
the negative-going scan (Fig. 1, curve d, e). In the second
cycle, the CVs show another anodic peak, A2, as the
counterpart of C2. The appearance of these new anodic and
cathodic peaks indicates the formation of an electroactive
species at this condition. The most probable reaction for
the formation of this product is the intramolecular Michael
addition of amine group to electrochemically generated o-
quinone. The pH dependence of the height of this new
redox couple (A2/C2) is also a good evidence for the pro-
posed reaction considering the basicity of the amine group
of LD. It is expected and observed that the heights of C2
and its counterpart, A2, increase by increasing the solution
pH due to deprotonation of the amine group of LD.
Experimental
Apparatus
Cyclic voltammetry was performed using a Behpajoh Model
BHP 2061-C potentiostat/galvanostat. In the voltammetry
experiments, a glassy carbon disc (2 mm diameter) and a
platinum wire were used as working and counter electrodes,
respectively. The working electrode potentials were mea-
sured versus Ag/AgCl (KCl 3.0 M). All electrodes were
purchased from AZAR electrode. Alumina powder (0.3 lM)
was used for mechanical polishing of the working electrode.
This treatment was followed by ultrasonic cleaning for 30 s.
More voltammetric studies were performed at various
scan rates and the normalized voltammograms of LD are
shown in Fig. 2. Normalized CVs are obtained by dividing
the current of CVs by the square root of the scan rate. They
can be simply overlaid for obtaining more useful infor-
mation [16]. The whole characteristics of a typical ECE
mechanism are observed for this electrode reaction in
which E represents an electron transfer at the electrode
surface and C represents a homogeneous chemical reaction.
The normalized heights of A1, A2 and C2 decrease by
Reagents
Levodopa, N-methylaniline, 4-bromoaniline, 4-nitroaniline,
3-nitroaniline and aniline were purchased from Fluka and
were used without further purification. Sodium acetate,
acetic acid, hydrochloric acid, sodium dihydrogen phos-
phate, disodium hydrogen phosphate and phosphoric acid
were reagent-grade materials from E. Merck. The stock
solutions of LD and aniline derivatives were prepared fresh
daily by dissolving the compounds in distilled water. Sam-
ples were prepared by taking the appropriate aliquots from
the stock solutions followed by dilution with buffer solu-
tions. The 0.15 M buffered solutions were prepared based on
Henderson–Hasselbalch equation and the suggested conju-
gate acid–bases in the Kolthoff tables considering their pKa
values [14]. For example, Na2HPO4/NaH2PO4 was selected
for pH values around 7; the pKa value of NaH2PO4 is 7.2.
The homogeneous rate constants were estimated by digital
simulation of cyclic voltammograms (CVs) using the
CVSIM simulation software and comparing the experi-
mental and simulated CVs [15].
15.0
e
a
A1
10.0
5.0
A2
0.0
-5.0
-10.0
C2
C1
-0.35
-0.15
0.05
0.25
0.45
0.65
E vs. Ag/AgCl (V)
Fig. 1 CVs of 1.0 mM LD at various pH values: a 3.0, b 4.0, c 5.0,
d 6.0 and e 7.0 at a glassy carbon electrode. Scan rate: 50 mV s-1
123

J IRAN CHEM SOC
increasing the scan rate, whereas the height of the C1 peak
considerably increases.
A1
pH=7
This behavior is in good agreement with the previously
reported results [7]: electrochemical oxidation of LD fol-
lowed by a cyclization reaction with an electroactive
product. The half-wave potential of the product is more
negative than the parent molecule. The main aim of this
paper is the designing of a competitive chemical reaction
for this intramolecular reaction and further derivatization
and it seems that aniline derivatives are good choices.
Figure 3 shows the CVs of LD in the presence of 1.0 mM
of 4-bromoaniline (4BA) as a desired nucleophile at the
same conditions and pH values for electrochemical oxi-
dation of LD.
10µA
A3
A2
C2
pH=6
C3
pH=5
pH=4
In the presence of 4BA, one new cathodic peak (C3) and
its anodic counterpart (A3) with half-wave potential more
negative than A1/C1 and more positive than A2/C2 redox
couples appear in CVs. For more details, the CVs of the
system were studied at pHs 3–5 in which A2/C2 couples did
not appear in the CVs (Fig. 4). The effects of CV scan rate
as a useful parameter were also studied on the normalized
CVs and the reaction mechanism.
pH=3
C3
C1
As clearly demonstrated in Fig. 4, all of the diagnostic
criteria are in good agreement with the ECE mechanism in
the presence of 4BA: oxidation of LD (E step) followed by
a chemical reaction (C step) in which its product is elec-
troactive and undergoes oxidation immediately (E step) at
the electrode surface. The same mechanism (ECE) is pro-
posed for the nucleophilic addition of 4BA to electro-
chemically generated o-quinone from oxidation of LD
(Scheme 1). The cathodic peak (C3) is due to the reduction
of the oxidized product of intermolecular addition of 4BA
on o-benzoquinone at the electrode surface. The reaction
-0.35
-0.15
0.05
0.25
0.45
0.65
E vs. Ag/AgCl (V)
Fig. 3 CVs of 1.0 mM LD in the presence of 1.0 mM 4BA at various
pH values. The solution pHs are shown on each curve. Scan rate:
100 mV s-1
1.7
A1
1.2
a
b
0.7
0.2
c
2.0
A3
1.5
1.0
A1
-0.3
C3
C1
-0.8
0.5
-0.2
0
0.2
0.4
0.6
A2
E vs. Ag/AgCl (V)
0.0
Fig. 4 Normalized CVs of 1.0 mM LD in the presence of 1.0 mM
4BA in acetate buffer solution (pH = 4.0). Scan rates from a to c are
50, 200 and 400 mV s-1
C2
-0.5
-1.0
C1
-0.4
-0.2
0
0.2
0.4
0.6
products are believed to be the new derivatives of diphe-
nylamine which have been confirmed in an earlier report
[12].
E vs. Ag/AgCl (V)
Fig. 2 Normalized CVs of 1.0 mM LD at a glassy carbon electrode
in phosphate buffer solution (pH 6.0). Scan rates from a to e are 50,
100, 200, 400 and 700 mV s-1, respectively
Based on the proposed mechanism, the above observed
changes in CVs can be explained as follows. At low scan
123

J IRAN CHEM SOC
+
Scheme 1
O
O
NH3
+
−2e- −2H+
HO
HO
NH3
CO2H
CO2H
+
HO
HO
NH3
Br
+
O
O
NH3
CO2H
NH
+
H2N
CO2H
Br
+
+
O
O
NH3
HO
HO
NH3
CO2H
−2e- −2H+
CO2H
NH
NH
Br
Br
12.0
rates, the time scale and extent of reaction increase and
oxidation of this produced intermediate at the electrode
surface causes an increase in the normalized height of A1
anodic peak. In other words, the current function for the A1
peak, (IpA1/v1/2), decreases by increasing the scan rate. The
height of C1 in the negative-going scan is proportional to
the remaining o-quinone and the height of C3 is propor-
tional to the oxidized product. Therefore the height of C1
decreases whereas the normalized heights of C3 and A3
increase at low scan rates and there is a greater extent of
the chemical step.
A1
9.0
6.0
3.0
0.0
d
c
b
a
A3
C1
-3.0
-6.0
C3
Based on the proposed mechanism and considering the
observation that A2/C2 couple did not appear at pH values
less than 6.0, it can be concluded that the main coupled
chemical reaction at this conditions is the addition of 4BA
to o-quinone. At pH values more than 6, both inter and
intermolecular reactions take place and both the related
redox peaks (A2/C2 and A3/C3) of the products are
observed. The A2/C2 redox peaks have the same half-wave
potential as those that appear in the absence of 4BA. At the
pH value equal to 7, the ratio of C2 over C3 is more than
unity. This is due to the predominance of the intramolec-
ular over intermolecular Michael addition at this pH value.
Voltammetric studies of LD have been extended in the
presence of N-methylaniline (MA), 4-nitroaniline (4NA),
3-nitroaniline (3NA) and aniline (AN). Figure 5 shows the
CVs of LD in the presence of the above-mentioned aro-
matic amines.
0
0.1
0.2
0.3
0.4
0.5
E vs. Ag/AgCl (V)
Fig. 5 CVs of LD in the presence of 1.0 mM of a N-methylaniline,
b 3-nitroaniline, c 4-bromoaniline and d aniline in acetate buffer
solution (pH = 4.0). Scan rate: 50 mV s-1
observed for the desired nucleophiles. It can be explained
based on the difference in nucleophilicity and basicity of
these aromatic amines with the aliphatic amine group of
LD. The amine group of LD is a stronger base and
nucleophile than aromatic amines, but it is fully protonated
with good estimation. Therefore, intramolecular reaction
did not take place at all under mild acidic conditions.
However, the aniline derivatives, especially those that have
electron-withdrawing substituent, have weak basic char-
acter. For example, the pKb value of 4BA is 10.04 [17].
With good estimation, more than 50 % of 4BA molecules
The same and interesting pH-dependent interplay
between inter and intramolecular reactions are also
123

J IRAN CHEM SOC
are in the deprotonated form at pH 4 and act as nucleophile.
Therefore, only the nucleophilic additions of these aniline
derivatives take place and the relative peaks of their
product are observed. All of the desired aniline derivatives
undergo nucleophilic addition with electrochemically
generated o-quinone except 4-nitroaniline. Under neutral
conditions, considering partial deprotonation of amines of
LD, both reactions and their related peak were observed.
Finally at the more basic conditions in which both amines,
side chain amine group and anilines are in their reactive
form, no competition was observed and the intramolecular
reaction consumed all of the electrochemically produced o-
quinone. This is due to the predominance of intramolecular
reactions and more nucleophilic character of alkyl amines.
There are also some interesting findings which can be
concluded by comparison of the CVs of the desired reac-
tions. A relatively large negative shift in the redox poten-
tials of the products of both inter and intramolecular
reactions is clearly observed in all CVs compared to the
parent LD molecule. This can be explained in terms of
strong resonance electron-donating property of the substi-
tuted amine groups. But the half-wave potentials of the
products of intermolecular reactions are more positive than
the half-wave potential of the cyclization product. It can be
also described by the fact that the electron pair of aromatic
amines are not free despite of aliphatic ones, due to reso-
nance with the benzene ring, to take part in resonance with
the catechol ring.
The other explanation is that in the resonance form of the
product the nitrogen of N-methylaniline should be posi-
tively charged, which lessens its tendency for the resonance
of its electron pair.
Finally, to obtain more information and quantitative
explanation, the rate constants of homogeneous coupled
chemical reactions were obtained by digital simulation of
CVs and comparison of the digitally simulated and
experimental CVs. The simulation was carried out
assuming semi-infinite one-dimensional diffusion at a disk
electrode geometry based on the proposed mechanism.
E
start, Efinal, scan rate and electrode area were entered as
experimental parameters. The formal potentials were
obtained experimentally as the midpoint potential between
the anodic and cathodic peaks (Emid). The transfer coeffi-
cients (a) were assumed to be 0.5. The heterogeneous rate
constants (k0) of electron transfer at different potentials
were calculated from Koutecky–Levich equation and con-
sisted of the extrapolation of the reciprocal of the current
density versus the square root of rotation of the electrode in
hydrodynamic voltammetry. Standard heterogeneous rate
constant, k0, was also obtained (0.005 cm s-1) considering
the over-potentials and Emid for each k0 value [19].
All of the above-mentioned parameters were kept con-
stant and the rate constants (kobs) of the desired homoge-
neous reactions were allowed to change through the fitting
process. A good agreement between the simulated CVs and
those obtained experimentally was observed. The observed
rate constant of the coupling reaction of oxidized CAs with
various aniline derivatives is presented in Table 1. Each
reported rate constant is the average of four independent
simulations at various scan rates and the relative standard
deviations (RSD) of all reported values are less than 9 %.
The same trend in the rate constant of reactions can be
derived considering the electron-withdrawing or donating
characters of aniline derivatives. The table simply shows
that the presences of electron-withdrawing substituents and
steric hinderance decrease the reactivity of the nucleophiles
as discussed based on the diagnostic criteria of CVs.
The other finding achieved by comparing the CVs in
Fig. 5 is the difference in reactivities of various aniline
derivatives. Based on the above discussion, in an ECE
mechanism the ratio of A1 to C1 can be considered as the
reactivity of nucleophiles toward o-quinone. The order of
A1 to C1 peak current ratio is: aniline [ 4-bromoani-
line [ 3-nitroaniline [ N-methylaniline. It suggests that
the presence of electron-withdrawing groups diminishes
the nucleophilicity of aniline derivatives to some extent.
The only exception to this order is N-methylaniline, which
can be explained by the higher steric hindrance of this
secondary amine. There is also a trend in the half-wave
potential of products and the electron-donating or with-
drawing character of aniline derivatives. The order of half-
wave potentials of products is: N-methylaniline [ 3-nitro-
aniline [ 4-bromoaniline [ aniline. The presence of elec-
tron-withdrawing groups on aniline rings decreases their
tendency for resonance of the electron pair of nitrogen with
the catechol ring and thus shifts the half-wave potential of
products to more positive values. There is again an
exception with N-methylaniline: the presence of the methyl
group in this derivative and its steric hindrance with the
chain group of LD in the ortho position force it to the out
of plane conformation that decreases the possibility of it
resonance less than other primary aniline derivatives [18].
Table 1 The observed homogeneous rate constant (kobs) of Michael
addition of aniline derivative and electrogenerated o-quinone from the
oxidation of LD and half-wave potential of products
kaobs
Eb1/2
Aniline
0.44
0.38
0.35
0.27
0.155
0.172
0.188
0.257
4-Bromoaniline
3-Nitroaniline
N-methylaniline
a
Homogeneous rate constant at pH = 5 (M-1 s-1
)
b
Half-wave potential of the reaction product at pH = 5
123

J IRAN CHEM SOC
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Based on these results, the o-benzoquinone produced from
the oxidation of LD is attacked by its side chain amine
group or aniline derivatives in mild acidic solutions. There
is close interplay between the inter and intramolecular
reactions in the presence of aniline derivatives. The solu-
tion pH is an influential parameter that affect the superior
of each nucleophilic addition toward o-quinones. It can be
described taking into account the difference in nucleophi-
licity and basicity of side chain amine group as aliphatic
amines and aniline derivatives as aromatic amines. This
essay has argued that all of the desired aniline derivatives
have suitable reactivity toward o-quinone except 4-nitro-
aniline, which is expected due to its strong resonance
electron-withdrawing character of nitro in the para position
of aniline. It is shown that the rate constant of Michael
addition of anilines decreases by increasing the electron-
withdrawing character of their substituent. There is also a
significant correlation between half-wave potential of
products and the nature of aniline substituents. The reaction
products are claimed to be diphenylamine derivatives and
are known as antioxidants. The tuning of their half-wave
potential and their electrochemical measurement may be
interesting from the synthetic and biological aspects of
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