Kinetics and mechanism of oxidation of isoleucine by n-bromophthalimide in aqueous perchloric acid medium

Article abstract of DOI:10.1155/2011/917657

The kinetics of oxidation of isoleucine with N-bromophthalimide has been studied in perchloric acid medium potentiometrically. The reaction is of first order each in [NBP] and [amino acid] and negative fractional order in [H ⁺]. The rate is decreased by the addition of phthalimide. A decrease in the dielectric constant of the medium increases the rate. Addition of halide ions or acrylonitrile has no effect on the kinetics. Similarly, variation of ionic strength of the medium does not affect the reaction rate. The reaction rate has been determined at different temperatures and activation parameters have been calculated. A suitable mechanism involving hypobromous acid as reactive species has been proposed. Copyright E-Journal of Chemistry 2004-2011.

Full text of DOI:10.1155/2011/917657

ISSN: 0973-4945; CODEN ECJHAO
E-Journal of Chemistry
2011, 8(4), 1728-1733
http://www.e-journals.net
Kinetics and Mechanism of Oxidation of
Isoleucine by N-Bromophthalimide in
Aqueous Perchloric Acid Medium
N.M.I. ALHAJI* and S. SOFIYA LAWRENCE MARY
P.G. and Research Department of Chemistry
Khadir Mohideen College, Adirampattinam - 614 701, India
alhajinmi@yahoo.in
Received 20 April 2011; Accepted 25 June 2011
Abstract: The kinetics of oxidation of isoleucine with N-bromophthalimide
has been studied in perchloric acid medium potentiometrically. The reaction is
of first order each in [NBP] and [amino acid] and negative fractional order in
[H+]. The rate is decreased by the addition of phthalimide. A decrease in the
dielectric constant of the medium increases the rate. Addition of halide ions or
acrylonitrile has no effect on the kinetics. Similarly, variation of ionic strength
of the medium does not affect the reaction rate. The reaction rate has been
determined at different temperatures and activation parameters have been
calculated. A suitable mechanism involving hypobromous acid as reactive
species has been proposed.
Keywords: Potentiometry, N-Bromophthalimide, Isoleucine, Oxidation, Mechanism
Introduction
Essential amino acids such as valine, leucine, isoleucine, methionine and phenylalanine, etc.
serve important functions in our biological system and play a significant role in metabolism.
These amino acids are used in biochemical, microbiological and nutrition investigation and
are employed as dietary supplements. Isoleucine also finds applications in medicine and
pharmaceuticals. The present investigation is a part of our broad program of studying
mechanistic aspects of the oxidation of amino acids by N-halo compounds. There are many
reports on the kinetics of oxidation of amino acids by various N-halo oxidants, such as
N-bromoacetamide1, benzyltrimethyl ammonium tribromide2, N-bromosuccinimide3,
chloramine-T4-6, bromamine-T7, chloramine-B8, bromamine-B9, N-bromonicotinimide10,
N-chlorosaccharin11, N-bromophthalimide12 etc. Here we report the results of the kinetic
studies of the oxidation of isoleucine(Ile) with N-bromophthalimide (NBP) in aqueous
perchloric acid medium.

1729
N.M.I. ALHAJI et al.
Experimental
N-Bromophthalimide (Aldrich), purity 99% was used as received. The standard solution in
distilled water was prepared afresh every week and standardized against thio.
Chromatographically pure isoleucine (SRL, India) was further assayed by acetous perchloric
method13. All other chemicals were of Analar grade. Ionic strength of the medium was kept
constant by using sodium perchlorate.
Kinetic measurements
The reactions were carried out under pseudo-first order conditions by keeping an excess
(x10 or greater) of the amino acid over NBP. The kinetics has been followed
potentiometrically by setting up a cell made up of the reaction mixture in which platinum
electrode and a standard calomel electrode were dipped. The EMF of the cell was measured
periodically using an Equip-tronics potentiometer, while the reaction mixture was
continuously stirred using a magnetic stirrer. The pseudo first-order rate constants (kobs)
were calculated from the slopes of ln (Et - E∞) versus time plots (r > 0.990) and the results
were reproducible to an accuracy of 5%. The second-order rate constants (k2) were
obtained as kobs/[Ile].
Stoichiometry and product analysis
The reaction between N-bromophthalimide (0.05 M) and isoleucine (0.01 M) was conducted
in 100 mL of water in the presence of perchloric acid (0.1 M). The reaction was allowed to
proceed for 24 hours to ensure the completion of the reaction. The residual
N-bromophthalimide was determined iodometrically. With varying amounts of isoleucine
the analysis showed that 2 mol of N-bromophthalimide were consumed for 1 mol of the
amino acid. This occurs because the aldehyde, initial product of the oxidation, was further
oxidized to the corresponding carboxylic acid in the presence of an excess of the oxidant.
O
+
2
Br
H2NCH(R)COOH
N
O
+ 2H2O
O
+
2H+ + 2Br−
RCOOH + NH3 + CO2
+
2
HN
O
(R = -CH(CH3)CH2CH3)
The formation of carboxylic acid product was confirmed by spot tests14 and ammonium ions
by Nessler’s reagent. The liberated CO2 was predicted by lime ware test.
Results and Discussion
Kinetics of oxidation of isoleucine by N-bromophthalimide was investigated at several
initial concentrations of the reactants in HClO4 medium under pseudo-first order conditions
of [substrate] >> [NBP]. At constant [Ile]o, [HClO4], [NaClO4] and temperature, plots of
ln(Et - E∞) vs. time were linear (r > 0.993) indicating first-order dependence of rate on
[NBP]o. Further, the rate constants remained constant with increase in the concentration of
NBP (Table 1), confirming first-order dependence on [NBP]o. Also, the rate data in Table 1
point out that kobs value increases linearly with increase in the initial concentration of
isoleucine. A plot of log kobs vs. log [Ile]o was linear (r = 0.999) with a slope value of 0.98.
Also the plot of kobs vs. [Ile]o passed through the origin (Figure 1; r = 0.999), establishing the

Kinetics and Mechanism of Oxidation of Isoleucine
1730
first order dependence of rate on [Ile]o. The rate decreased with increase in [HClO4] (Table 1)
and the plot of log kobs vs. log [H+]o was found to be linear ((Figure 2; r = 0.993; slope = −0.52)
showing a negative fractional-order dependence on [H+]o. Further, the plot of kobs vs. 1/[H+]o
was linear and passed through the origin, indicating that the oxidation occurred only through
the acid dependent path under these conditions.
Table 1. Pseudo first-order and second-order rate constants for the oxidation of isoleucine
by NBP in aqueous perchloric acid at 303 Ka,b
103[Ile]o, M
2.0
104[NBP]o, M
102[H+]o, M
4.0
104 kobsc, s-1
2.95 0.27
5.76 0.51
11.7 1.1
102k2 , M-1 s-1
14.6 1.4
14.4 1.3
14.7 1.4
14.6 1.4
13.9 1.4
14.2 1.3
14.3 1.3
14.6 1.4
18.4 1.7
10.4 1.0
7.71 0.78
5.67 0.53
d
2.0
2.0
2.0
2.0
2.0
0.5
1.0
3.0
2.0
2.0
2.0
2.0
4.0
8.0
4.0
4.0
12.0
20.0
4.0
4.0
4.0
17.5 1.6
27.8 2.8
0.4
4.0
5.68 0.52
5.71 0.53
5.82 0.54
7.35 0.70
4.16 0.39
3.08 0.31
2.26 0.21
4.0
4.0
4.0
2.0
8.0
12.0
4.0
4.0
4.0
4.0
20.0
aAs determined by a potentiometric technique following the disappearance of NBP; the error quoted in
k values is the 95% confidence limit of ‘Student t test’15. bGeneral conditions: [I] = 0.75 M; cEstimated
from pseudo first-order plots over 60% reaction. dIndividual k2 values estimated as kobs/[Ile]o
[lle,] M
Figure 1. Direct plot of kobs versus [amino acid] for the oxidation of isoleucine with NBP

1731
N.M.I. ALHAJI et al.
2 + log [H+]
Figure 2. Double logarithmic plot for the oxidation of isoleucine with NBP
The rate of the reaction is not significantly affected by the change in the ionic strength
of the medium (Table 2) brought about by the addition of sodium perchlorate, pointing out
the participation of a neutral species as a reactant in the rate-determining step. Addition of
phthalimide causes a decrease in the rate of reaction (Table 2) suggesting that the step in
which phthalimide is formed as one of the products is reversible.
Table 2. Influence of ionic strength and phthalimide (NHP) on reaction rate for the
oxidation of isoleucine with NBP at 303 Ka
[I], M
0.50
0.75
1.00
104kobs, s-1
5.79 0.54
5.76 0.51
5.81 0.55
104[NHP], M
104kobs, s-1
5.76 0.51
5.28 0.49
4.59 0.46
0
1.0
3.0
aGeneral conditions: [NBP] = 0.0002 M; [Ile] = 0.004 M; [H+] = 0.04 M; [I] = 0.75 M unless
otherwise stated
The involvement of free-radical intermediates during the reaction can be excluded as
the rate constant is not affected by the addition of acrylonitrile (Table 3). The added KBr has
no effect on the rate of oxidation (Table 3), establishing that the course of the oxidation does
not involve Br+ ion or Br2 as active species. Addition of acetonitrile to the reaction mixture
increased the rate (Table 3) and a plot of log kobs vs. 1/D is linear with a positive slope.
Table 3. Influence of added acrylonitrile (AN), KBr and solvent polarity on reaction rate for
the oxidation of isoleucine with NBP at 303 Ka
[AN], M
0
104kobs, s-1
5.76 0.51
5.78 0.52
5.74 0.52
[KBr], M 104kobs, s-1
% CH3CN, v/vb
104kobs, s-1
5.76 0.51
6.92 0.59
8.84 0.72
10.8 1.0
0
5.76 0.51
5.72 0.52
5.69 0.55
0
0.003
0.006
0.001
0.004
10
20
40
aGeneral conditions: [NBP] = 0.0004 M; [Ile] = 0.004 M; [H+] = 0.04 M; [I] = 0.75 M

Kinetics and Mechanism of Oxidation of Isoleucine
1732
The reaction was studied at three other temperatures (298, 308 and 318 K) keeping
other experimental conditions constant. From the Arrehenius and Eyring plots, the
thermodynamic parameters for the oxidation of isoleucine were found to be Ea = 49.42
kJ/mol, ∆H ≠ = 46.86 kJ/mol, ∆G≠ (298 K) = 78.56 kJ/mol and ∆S≠ =106.39 JK1 mol1.
Mechanism
The possible oxidation species in acidified NBP solution are NBP, NBPH+, HOBr and
H2OBr+. In the present study the involvement of NBPH+ can be ruled out on the basis that
the reaction shows a negative dependence on [H+]. Molecular bromine or bromonium ion
the reactive species may not be the reactive species here because the addedBr- has no
effect on the rate of the reaction in present study. The retarding effect of phthalimide
suggest that the pre-equilibrium step involves a process in which phthalimide is one of the
products.
O
O
Br
N
+ H2O
HN
+ HOBr
O
O
Therefore, it can be assumed that HOBr is the most likely oxidizing species in the
present reaction. In aqueous solution amino acids exist in the following equilibrium.
RCH(NH2)COOH
RCH(NH3)COO
RCH(NH3)COOH
(A)
(C)
(B)
The linear decrease in the rate with an increase in the concentration of H+ in the reaction
mixture suggests that the amino acid is being removed progressively as kinetically inactive
forms (B) and (C), thus establishing that the amino acid involves in the reaction in the form (A).
Based on the foregoing discussions, the following mechanism (Scheme 1) involving an
electrophilic attack of HOBr on the amino group of the amino acid has been proposed. A
similar mechanism has been postulated in the oxidation of amino acids by chloramine-T16
and N-bromoacetamide1.
NH2
NHBr
slow
RCH
+
HOBr
RCH
H2O
+
COOH
NH
COOH
Br
fast
+ CO2 + H+ + Br−
RCH NH
RCH
C
O H
O
fast
+ H2O
+ NH3
RCH NH
RCHO
Scheme 1
The proposed mechanism is also supported by the moderate value of energy of
activation and other thermodynamic parameters. The large negative ∆S≠ indicates the
formation of an arranged complex in the rate determining step.

1733
N.M.I. ALHAJI et al.
Conclusion
In the present kinetic study of the oxidation of isoleucine by NBP in HClO4 medium, a
mechanism involving an electrophilic attack of HOBr on the amino group of isoleucine has
been proposed. This mechanism is supported by the experimental data such as the reaction
stoichiometry, the oxidation products and the activation parameters.
Acknowledgment
The authors thank the college management for providing the necessary facilities.
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