Thermodynamic quantities for different steps involved in the mechanism of the oxidation of 6-mercaptopurine by diperiodatocuprate(III) in aqueous alkaline medium

Article abstract of DOI:10.1080/17415993.2011.554836

The kinetics of oxidation of 6-mercaptopurine (6-MP) by diperiodatocuprate(III) (DPC) in aqueous alkaline medium at a constant ionic strength of 0.01mol/dm³ was studied spectrophotometrically. The reaction between DPC and 6-MP in an alkaline medium exhibits 1:4 stoichiometry (6-MP:DPC). The reaction is of first order in [DPC] and has less than unit order in both [6-MP] and [alkali]. However, the order of [6-MP] and [alkali] changes from first order to zero order as their concentration increases. Intervention of free radicals was observed in the reaction. An increase in periodate concentration decreases the rate. The oxidation reaction in an alkaline medium has been shown to proceed via a monoperiodatocuprate(III)-6-MP complex, which decomposes slowly in a rate-determining step followed by other fast steps to give the products. The main oxidative products were identified by spot test, IR, GC-MS and HPLC studies. The reaction constants involved in the different steps of the mechanism are calculated. The activation parameters with respect to a slow step of the mechanism are computed and discussed, and thermodynamic quantities are also determined. Figure Presented.

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Thermodynamic quantities for different
steps involved in the mechanism of
the oxidation of 6-mercaptopurine
by diperiodatocuprate(III) in aqueous
alkaline medium
a
a
Keerti M. Naik & Sharanappa T. Nandibewoor
a
P.G. Department of Studies in Chemistry , Karnatak University ,
Dharwad , 580003 , India
Published online: 17 Feb 2011.
To cite this article: Keerti M. Naik & Sharanappa T. Nandibewoor (2011) Thermodynamic
quantities for different steps involved in the mechanism of the oxidation of 6-mercaptopurine by
diperiodatocuprate(III) in aqueous alkaline medium, Journal of Sulfur Chemistry, 32:2, 123-136,
DOI: 10.1080/17415993.2011.554836
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Journal of Sulfur Chemistry
Vol. 32, No. 2, April 2011, 123–136
Thermodynamic quantities for different steps involved in
the mechanism of the oxidation of 6-mercaptopurine by
diperiodatocuprate(III) in aqueous alkaline medium
Keerti M. Naik and Sharanappa T. Nandibewoor*
P.G. Department of Studies in Chemistry, Karnatak University, Dharwad 580003, India
(Received 9 November 2010; final version received 11 January 2011)
The kinetics of oxidation of 6-mercaptopurine (6-MP) by diperiodatocuprate(III) (DPC) in aqueous alkaline
3
medium at a constant ionic strength of 0.01 mol/dm was studied spectrophotometrically. The reaction
between DPC and 6-MP in an alkaline medium exhibits 1:4 stoichiometry (6-MP:DPC). The reaction is
of first order in [DPC] and has less than unit order in both [6-MP] and [alkali]. However, the order of
[6-MP] and [alkali] changes from first order to zero order as their concentration increases. Intervention of
free radicals was observed in the reaction. An increase in periodate concentration decreases the rate. The
oxidation reaction in an alkaline medium has been shown to proceed via a monoperiodatocuprate(III)–
6-MP complex, which decomposes slowly in a rate-determining step followed by other fast steps to give
the products. The main oxidative products were identified by spot test, IR, GC-MS and HPLC studies.
The reaction constants involved in the different steps of the mechanism are calculated. The activation
parameters with respect to a slow step of the mechanism are computed and discussed, and thermodynamic
quantities are also determined.
S
SH
H
H
N
N
N
N
+
4 [Cu(H IO )(H O) ]
+ 4 Cu2+
2
6
2
2
O
N
N
H
N
H
N
O
H
+
4H IO62– + 6H O
3
2
Keywords: 6-mercaptopurine; diperiodatocuprate(III); oxidation; kinetics; mechanism
1
.
Introduction
6
-Mercaptopurine (6-MP) is a thiopurine drug in which carbon 6 of purine has been substi-
tuted with a sulfur-containing group. This drug with methotrexate is the drug of choice in
the treatment of childhood acute lymphoblastic leukemia, especially in those cases where pro-
longed duration of remission is required (1). 6-MP together with its prodrug azathioprine is used
*
Corresponding author. Email: stnandibewoor@yahoo.com
ISSN 1741-5993 print/ISSN 1741-6000 online
2011 Taylor & Francis
©
DOI: 10.1080/17415993.2011.554836
http://www.informaworld.com

1
24 K.M. Naik and S.T. Nandibewoor
as immunomodulatory agent in the treatment of inflammatory bowel disease (2). It has been
suggested that in the cell, 6-MP is first converted to thioinosinic acid, by hypoxanthine–guanine
phosphoribosyl-transferase and then metabolized to thioguanine ribonucleotide and deoxyribonu-
cleotide; incorporation of these compounds into RNA and DNA results in the anti-tumour effect
of the drug (3). The first route is thought to involve the initial oxidation of 6-MP to 8-oxo-6-
mercaptopurine followed by the conversion to 6-thiouric acid (4). However, Zimm et al. (5) have
identified 6-thioxanthine in the urine samples of some patients who received 6-MP by intravenous
infusion, which may indicate that 6-MP is converted to 6-thiouric acid via 6-thioxanthine.
In recent years, the study of the highest oxidation state of transition metals has intrigued many
researchers. Transition metals in a higher oxidation state can be stabilized by chelation with
suitable polydentate ligands. Metal chelates such as diperiodatocuprate(III) (DPC) (6), diperi-
odatoargentate(III) (7), and diperiodatonickelate(IV) (8) are good oxidants in a medium with
an appropriate pH value. Copper(III) is shown to be an intermediate in the copper(II)-catalyzed
oxidation of amino acids by peroxydisulfate (9). The oxidation reaction usually involves the
copper(II)–copper(I) couple, and such aspects are detailed in different reviews (10). Copper
complexes have occupied a major place in oxidation chemistry because of their abundance and rel-
evance in biological chemistry (11). Copper(III) is involved in many biological electron-transfer
reactions (12). When the copper(III) periodate complex is oxidant and multiple equilibria between
different copper(III) species (13) are involved, it would be interesting to know which of the species
is the active oxidant.
To the best of our knowledge, oxidation of 6-MP by DPC has not been reported yet. In view
of the medicinal value and the potential pharmaceutical importance of 6-MP and lack of the
literature on the oxidation mechanism of this drug by DPC, there was a need for understanding
the oxidation mechanism of this bioactive compound. 6-MP contains different functional groups
and which group gets oxidized by the oxidant is of vital importance. Hence, we have undertaken
a careful study of the title reaction.
2
2
.
Results
.1. Stoichiometry and product analysis
Different sets of reaction mixtures containing different concentrations of DPC to 6-MP in the
−
◦
presence of constant amounts of OH and KNO3 were kept for 4 h in a closed vessel at 25 C
under an inert atmosphere (to avoid any potential reaction between oxygen and 6-MP). The
remaining concentration of DPC was estimated spectrophotometrically. The results indicated a
1
:4 stoichiometry as given in Equation 1.
S
SH
H
H
N
N
N
N
+
4 [Cu(H IO )(H O) ]
+ 4 Cu2+
2
6
2
2
O
N
N
H
(1)
N
N
O
H
H
+
4H IO62– + 6H O
3
2
The reaction product was extracted with ether and recrystallized from water and subjected to
HPLC and spectral studies. Only one product was obtained as evidenced by a single peak on the
HPLC spectrum and a single spot on thin layer chromatography, which was identified as 6-thiouric
acid by spot test (14) and was characterized by FT-IR and GC-MS spectral studies.

Journal of Sulfur Chemistry 125
−
1
6
-Thiouric acid was confirmed by its IR spectrum, which showed a band at 1654 cm due to
−
1
−1
>
C=O stretching, 3427 cm for −NH stretching and 2925 cm for −CH stretching of acid.
GC-MS data were obtained on a QP-2010S Shimadzu gas chromatograph mass spectrometer.
The mass spectral data showed a molecular ion peak at 185 m/z, confirming the presence of
6
-thiouric acid. All other peaks observed in GC-MS can be interpreted in accordance with the
observed structure of 6-thiouric acid. Another product Cu(II) was identified by UV–VIS spectra.
The reaction products do not undergo further oxidation under the present kinetic conditions.
Similar product was noticed (15) in in vitro study of 6-MP oxidation catalyzed by aldehyde
oxidase and xanthine oxidase.
2
.2. Reaction orders
The reaction orders were determined from the slope of log kobs versus log (concentration) plots
by varying the concentrations of 6-MP, alkali, and periodate in turn, while keeping all other
concentrations and conditions constant, except for DPC concentration.
2
.3. Effect of [DPC]
The oxidant, DPC, concentration was varied in the range 1.0 × 10− − 1.0 × 10 mol/dm3,
5
−4
and lack of variation in the kobs values indicate that order with respect to [DPC] was unity
(Table 1). This was also confirmed by linearity of the plots of log [absorbance] versus time
(r ≥ 0.9986, S ≤ 0.013) up to 90% completion of the reaction (Figure 1).
2
.4. Effect of [6-MP]
The effect of [6-MP] on the rate of reaction was studied at constant concentrations of alkali, DPC,
3
and periodate at a constant ionic strength of 0.01 mol/dm . The substrate, 6-MP, was varied in
−
−
◦
Table 1. Effect of [DPC], [6-MP], [IO ], and [OH ] on the oxidation of 6-MP by DPC in alkaline medium at 25 C,
4
3
I = 0.01 mol/dm .
5
4
−
4
−
3
2
2
[
DPC] × 10
[6-MP] × 10
[IO ] × 10
[OH ] × 10
kobs × 10
−1
kcal × 10
−1
4
3
3
3
3
(
mol/dm )
(mol/dm )
(mol/dm )
(mol/dm )
(s
)
(s
)
1
3
5
8
.0
.0
.0
.0
5.0
5.0
5.0
5.0
5.0
1.0
3.0
5.0
8.0
10.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
1.0
3.0
5.0
8.0
10.0
5.0
5.0
5.0
5.0
5.0
4.0
4.0
4.0
4.0
4.0
4.0
4.0
4.0
4.0
4.0
4.0
4.0
4.0
4.0
4.0
1.0
2.0
4.0
8.0
10.0
1.12
1.13
1.07
1.19
1.09
0.31
0.78
1.07
1.49
1.65
1.86
1.39
1.07
0.82
0.71
0.46
0.76
1.07
1.41
1.63
1.10
1.10
1.10
1.10
1.10
0.31
0.77
1.10
1.46
1.62
1.87
1.39
1.10
0.85
0.73
0.46
0.75
1.10
1.44
1.54
1
0.0
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
.0
.0
.0
.0
.0
.0
.0
.0
.0
.0
.0
.0
.0
.0
.0

1
26 K.M. Naik and S.T. Nandibewoor
2
2
1
1
0
0
.5
.0
.5
.0
.5
.0
0
1
2
3
4
5
Time (min)
5
3
Figure 1. First-order plots for the oxidation of 6-MP by DPC in aqueous alkaline medium at 298 K (10 DPC mol/dm ):
(1) 1.0; (2) 3.0; (3) 5.0; (4) 8.0; (5) 10.0.
6-MP]0.72 x 10 mol/dm
4
3
[
0
2
4 6 8
10
12
3.0
2.5
2.0
1.5
1.0
0.5
0.0
2.0
1.5
1.0
0.5
0.0
0
1
2
3
4
5
6
4
3
[
6-MP] x 10 mol/dm
Figure 2. Plots of kobs versus [6-MP]0.72 and kobs versus [6-MP] (from Table 1).
−
4
−3
3
the range 1.0 × 10 − 1.0 × 10 mol/dm . The kobs values increased with an increase in the
concentration of 6-MP. The order with respect to [6-MP] was found to be less than unity (Table 1)
(
r ≥ 0.9995, S ≤ 0.007) under the experimental concentrations. This less than unit order in the
0
.72
6
-MP was also confirmed by the linear plot of kobs versus [6-MP] . The plots of kobs versus
[6-MP] were nonlinear (Figure 2). However, at lower concentrations of 6-MP, the reaction was
first order in [6-MP] and at a high concentration of 6-MP, the reaction was independent of [6-MP].
The order of [6-MP] changes from first order to zero order as [6-MP] increases.
2
.5. Effect of [Alkali]
The effect of the increase in the concentration of alkali on the reaction was studied at constant
3
concentrations of 6-MP, DPC, and periodate at a constant ionic strength of 0.01 mol/dm at
◦
2
5 C. The rate constants increased with the increase in the alkali concentration (Table 1), indi-
cating apparent less than unit order dependence of rate on the alkali concentration (r ≥ 0.9994,
S ≤ 0.006). Similar to that in the case of 6-MP, the order of alkali changes from first order to zero
−
order as [OH ] increases.

Journal of Sulfur Chemistry 127
2
.6. Effect of [Periodate]
The effect of the increasing concentration of periodate was studied by varying the periodate
−
4
−3
3
concentration from 1.0 × 10 to 1.0 × 10 mol/dm keeping all other reactants’concentrations
constant. It was found that the added periodate had a retarding effect on the rate of reaction, the
order with respect to periodate concentration being negative less than unity (Table 1).
2
.7. Effect of ionic strength (I) and dielectric constant of the medium (D)
−
−
The addition of KNO3 at constant [DPC], [6-MP], [OH ], and [IO ] was found that with the
4
increasing ionic strength had no significant on the rate of the reaction.
Dielectric constant of the medium, D, was varied by varying t-butyl alcohol and water per-
centage. The D values were calculated from the equation D = DwVw + DBVB, where Dw and
DB are the dielectric constants of pure water and t-butyl alcohol, respectively, and Vw and VB are
the volume fractions of components water and t-butyl alcohol, respectively, in the total mixture.
There was no effect of dielectric constant on the rate of the reaction.
Thus, from the observed experimental results, the rate law for the reaction is given as follows:
Rate = kobs[6-MP] .72[DPC]1.0[OH ] [IO ]
0
− 0.52
− −0.41
4
2
.8. Effect of added products
−
4
The externally added products, 6-thiouric acid and copper(II) (CuSO4) in the range 1.0 × 10
−
.0 × 10− mol/dm did not have any significant effect on the rate of the reaction.
3
3
1
2
.9. Polymerization study (test for free radicals)
DPCisasingleequivalentoxidant. Hence, interventionofafreeradical, generatedfromtheorganic
compound, was expected. In view of this, for the reaction, the possibility of the formation of free
radicals was detected as follows. The reaction mixture, to which a known quantity of acrylonitrile
(scavenger) had been added initially, was kept in an inert atmosphere for 2 h. On diluting the
reaction mixture with methanol, a white precipitate was formed, indicating the intervention of free
radicals in the reaction. The blank experiments of either DPC or 6-MP alone with acrylonitrile did
not induce any polymerization under the same condition as those induced for a reaction mixture.
Initially, added acrylonitrile decreases the rate of reaction, indicating free radical intervention,
which is the case in earlier work (16).
2
.10. Effect of temperature
The kinetics was studied at five different temperatures under varying concentrations of 6-MP,
alkali, and periodate, keeping other conditions constant. The rate constants were found to increase
with the increase in temperature. The rate constants of the slow step (k) of Scheme 1 were obtained
2
6
−
from the slopes and intercepts of 1/kobs versus 1/[6-MP], 1/kobs versus [H3IO ], and 1/kobs
−
versus 1/[OH ] plots at five different temperatures and were used to calculate the activation
parameters. The activation parameters corresponding to these constants were evaluated from
ꢀ
the Eyring plot of (R ln k/T × h/kB) (r ≥ 0.9977, S ≤ 0.011) (h = Planck s constant, kB =
Boltzmann constant) versus 1/T and are tabulated in Tables 2 and 3.

1
28 K.M. Naik and S.T. Nandibewoor
2
-
O
I
HO
HO
O
O
O
OH
OH
I
Cu
HO
OH
O
HO
O
O
O
O
I
DPC
O
O
O
Cu
HO
HO
O
O
6-MP
H O
I
Cu
2
OH2
OH2
O
MPC
Complex
Scheme 1. Detailed mechanistic pathways for the oxidation of 6-MP by alkaline DPC.
Table 2. Effect of temperature on the rate constant of the
slow step of Scheme 1, k, for the oxidation of 6-MP by DPC
in aqueous alkaline medium.
2
−1
Temperature (K)
k × 10 s
2
2
2
3
3
88
93
98
03
08
1.19
1.91
3.03
4.45
6.69
Table 3. Thermodynamic activation parameter for the
oxidation of 6-MP by DPC in aqueous alkaline medium
with respect to slow step of Scheme 1.
Parameters
Values
Ea (kJ/mol)
63 ± 4
61 ± 4
#
ꢀ
ꢀ
ꢀ
H
S
G
(kJ/mol)
(J/K mol)
#
−70 ± 6
81 ± 1
#
2
(kJ/mol)
98 K
log A
9.6 ± 1.0
3
.
Discussion
5
−
The water-soluble copper(III) periodate complex is reported (17) to be [Cu(HIO6)2] . However,
in an aqueous alkaline medium and at a high pH range as employed in the study, periodate is
unlikely to exist as HIO4 (as present in the complex) as is evident from its involvement in the
−
6
multiple equilibria (18) (Equations (2)–(4)) depending on the pH of the solution.
−
+
+
+
K1 = 5.1 × 10−4
H5IO6 ꢀ H4IO + H
(2)
(3)
(4)
6
−
2−
−9
H4IO ꢀ H3IO + H
K2 = 4.9 × 10
6
6
H3IO2 ꢀ H2IO6 + H
−
3−
K3 = 2.5 × 10
−12
6
−
Periodic acid exists in an acid medium as H5IO6 and as H4IO around pH 7. Thus, under the
6
2
−
conditions employed in the alkaline medium, the main species are expected to be H3IO and
6

Journal of Sulfur Chemistry 129
3
6
−
H2IO .At higher concentrations, periodate also tends to dimerize (19). However, the formation of
this species is negligible under conditions employed for kinetic study. Hence, at the pH employed
2
−
in this study, the soluble copper(III) periodate complex exists as DPC, [Cu(H2IO6)(H3IO6)] , a
conclusion also supported by earlier work (6).
The reaction between the DPC complex and 6-MP in the alkaline medium has the stoichiometry
1
:4 (6-MP:DPC) with a first-order dependence on [DPC] and an apparent order of less than unity
in [substrate], [alkali] and negative-fractional-order dependence on the periodate. Based on the
experimentalresults, amechanismisproposedforwhichalltheobservedordersineachconstituent
−
−
such as [oxidant], [reductant], [OH ], and [IO ] may be well accommodated. Lister (20) proposed
4
three forms of copper(III) periodate in the alkaline medium as DPC, monoperiodatocuprate(III)
(
MPC) and tetrahydroxocuprate(III). The tetrahydroxocuprate(III) is ruled out, as its equilibrium
−
11
◦
constant is 8.0 × 10
at 40 C. Hence, in the present study, DPC and MPC are considered to
be active forms of the copper(III) periodate complex. The result of the increase in the rate of the
reaction with an increase in alkalinity (Table 1) can be explained in terms of prevailing equilibrium
2
−
−
of the formation of [Cu(H3IO6)(H2IO6)] from [Cu(H3IO6)2)] as given in Equation (5).
K4
−
−
2−
[
Cu(H3IO6)2] + OH ꢀ[Cu(H2IO6)(H3IO6)] + H2O
(5)
Also the decrease in the rate with the increase in periodate concentration suggests that the
displacement of a ligand periodate takes place to give a free periodate and MPC species from
2
−
[
Cu(H2IO6)(H3IO6)] as given in Equation (6).
K5
Cu(H2IO6)(H3IO6)] − + 2H2O ꢀ[Cu(H2IO6)(H2O)2] + H3IO6
2
2−
(6)
[
Such types of equilibria have been well noticed in the literature (21). It may be expected that a
lower periodate complex such as MPC is more important in the reaction than the DPC in view of
the observed inverse fractional order in periodate. With the known equilibrium constants (21), the
individual concentrations of [DPC]f , DPC, and MPC were calculated. It was found that MPC was
−
in higher concentration and nearly paralleled the rate variation with different [OH ]. Furthermore,
−
the spectra of Cu(III) periodate complex was dependent on [OH ] and the absorption becomes
almost constant, indicating the predominance of one species, presumably [Cu(H2IO6)(H2O)2].
−
Becauseofthisandthefactthatrateisafunction of[OH ](lessthanunitorder), themainoxidation
species is likely to be [Cu(H2IO6)(H2O)2] and its formation equilibrium (6) is of importance in
the reaction.
The less than unit order in [6-MP] presumably results from the formation of a complex (C)
between the MPC species and 6-MP prior to the formation of the products. K6 is the composite
equilibrium constant comprising the equilibrium to bind active species MPC to 6-MP species
to form a complex (C). This complex (C) undergoes decomposition in a slow step to give the
free radical species of 6-MP, periodate, and Cu(II). This free radical species of 6-MP reacts
with one mole of MPC species in a fast step to form 6-thioxanthine, Cu(II), and periodate. This
6
-thioxanthine further reacts with one more mole of MPC in a fast step to form a free radical
species of 6-thioxanthine, Cu(II), and periodate. Further, this free radical reacts with one more
mole of MPC to form the final products, i.e. 6-thiouric acid, Cu(II), and periodate. So, the detailed
mechanistic pathways for the oxidation of 6-MP by DPC is presented in Scheme 1.
Since Scheme 1 is in accordance with the generally well-accepted principle of non-
complementary oxidations taking place in sequence of one-electron steps, the reaction between
the substrate and the oxidant would afford a radical intermediate. A free-radical scavenging
experiment revealed such a possibility. This type of radical intermediate has also been observed
in earlier work (22). Spectroscopic evidence for the complex formation between the oxidant
−
4
3
and the substrate was obtained from the UV–VIS spectra of 6-MP (5.0 × 10 mol/dm ), DPC

1
30 K.M. Naik and S.T. Nandibewoor
Figure 3. Spectroscopic evidence for the complex formation between DPC and 6-mercaptopurine: (a) UV–VIS spectra
of DPC complex (259 nm), (b) UV–VIS spectra of mixture of DPC and 6-MP (265 nm), and (c) UV–VIS spectra of 6-MP.
5
3
−
−3
3
(
5.0 × 10− mol/dm ), [OH ] (4.0 × 10 mol/dm ), and a mixture of both.A bathochromic shift
of about 6 nm from 259 to 265 nm in the spectra of DPC was observed (Figure 3). The Michaelis–
Menten plot also proved the complex formation between DPC and 6-MP, which explains the less
than unit order dependence on [6-MP] (Figure 4). Such a complex between a substrate and an
oxidant has been observed in other studies (23). The diamagnetic (dsp ) square planar structure
of Cu(III) periodate in the form of DPC and MPC and the paramagnetic (sp ) complex of Cu(III)
2
3
and 6-MP can be formulated as shown below. Similar structures have been reported in other
studies (24).
Scheme 1 leads to the rate law (7)
−
d[DPC]
Rate =
dt
−
kK4K5K6[DPC][6-MP][OH ]
=
,
(7)
(8)
2−
−
−
−
[
H3IO2−] + K4[H3IO ][OH ] + K4K5[OH ] + K4K5K6[OH ][6-MP]
6
6
which explains all the observed kinetic orders of different species.
Rate
kobs =
[
[
DPC]
−
kK4K5K6[6-MP][OH ]
=
.
H3IO2−] + K4[H3IO ][OH ] + K4K5[OH ] + K4K5K6[OH ][6-MP]
2−
−
−
−
6
6
The rate law (8) can be rearranged into the following form, which is suitable for verification:
2
−
2−
1
[H3IO6
]
[H3IO
]
1
1
6
=
+
+
+ k
.
(9)
−
kobs
kK4K5K6[6-MP][OH ]
kK5K6[6-MP]
kK6[6-MP]
−
According to Equation (9), other conditions being constant, plots of 1/kobs versus 1/[OH ]
r ≥ 0.9983, S ≤ 0.029), 1/kobs versus 1/[6-MP] (r ≥ 0.9995, S ≤ 0.016), and 1/kobs versus
(
2
6
−
[
H3IO ] (r ≥ 0.9994, S ≤ 0.011) should be linear and are found to be so (Figure 4). The slopes
3
and intercepts of such plots lead to the values of K4, K5, K6, and k as 24.6 dm /mol, 1.33 ×
−
3
3
3
3
−2 −1
1
0
dm /mol, 5.93 × 10 dm /mol, and 3.03 × 10
s , respectively. The magnitudes of K4
and K5 are in good agreement with those obtained by the earlier work (23), which supports the
proposed mechanism. Using these K4, K5, K6, and k values, the rate constants under different

Journal of Sulfur Chemistry 131
experimental conditions were calculated by Equation (8) and compared with experimental data.
There is a good agreement between them (Table 1), which fortifies Scheme 1.
The negligible effect of ionic strength and dielectric constant of the medium on the rate
qualitatively explains the reaction between neutral charged species, as seen in Scheme 1.
The thermodynamic quantities for the first, second, and third equilibrium steps of Scheme 1
2
−
−
can be evaluated as follows. The [H3IO6 ], [6-MP], and [OH ] (as in Table 1) were varied at five
−
different temperatures. The plots of 1/kobs versus 1/[OH ], 1/kobs versus 1/[6-MP], and 1/kobs
2
−
versus [H3IO ] should be linear. From the slopes and intercepts, the values of K4 were calculated
6
at different temperatures and these values are given in Tables 4 and 5. A van’t Hoff’s plot was
made for the variation of K4 with temperature (log K4 versus 1/T (r ≥ 0.9811, S ≤ 0.006)) and
the values of the enthalpy of reaction ꢀH, entropy of reaction ꢀS, and free energy of reaction
ꢀG were calculated for the first equilibrium step (Tables 4 and 5). A comparison of the ꢀH value
3
-
4
3
[
H
2
IO
4
6
] x 10 mol/dm (2)
6
(a)
0
2
8
10
1
0
.0
.5
0.20
0
0
0
0
0
0
.6
.5
.4
.3
.2
.1
0
0.15
(2)
0.10
(3)
0.05
(1)
0.0
0.00
0
2
4
6
8
10
-3
3
1
1
/[6-MP] x 10 dm /mol (1)
0
2
4
6
8
10
-
-2
3
1
1
/[OH ] x 10 dm /mol (3)
3
-
4
3
[
H
2
IO
6
] x 10 mol/dm (2)
6
(b)
0
2
4
8
10
5
4
4
3
3
2
2
1
1
0
.0
.5
.0
.5
.0
.5
.0
.5
.0
.5
1.0
0.8
0.6
0.4
0.2
0.0
2
2
1
1
0
0
.5
.0
.5
.0
.5
.0
(
2)
3)
(
(
1)
0.0
0
2
4
6
8
10
-3
3
1
1
/[6-MP] x 10 dm /mol (1)
0
2
4
6
8
10
-
-2
3
1
1
/[OH ] x 10 dm /mol (3)
◦
Figure 4. Verification of rate law (8) in the form of Equation (9) for the oxidation of 6-MP by DPC. (a) At 15 C, (b) at
2
◦ ◦ ◦ ◦
0 C, (c) at 25 C, (d) at 30 C, and (e) at 35 C.

1
32 K.M. Naik and S.T. Nandibewoor
3
-] x 104 dm-3 mol (2)
1
2 6
[H IO
(
c)
0
2
4
6
8
10
4
.0
.5
1
.4
2.5
2.0
1.5
1.0
0.5
0.0
3
(
2)
1.2
3.0
(3)
1.0
0.8
0.6
0.4
2
.5
.0
2
(1)
1.5
1.0
0.5
0.0
0.2
0.0
0
2
4
6
8
10
/[6-MP] x 10- dm mol1 (1)
3
3
1
0
2
4
6
8
10
/[OH ] x 10 dm mol1 (3)
-
-2
3
1
3
-
4
3
[
4
H
2
IO
6
] x 10 mol/dm (2)
6
(
d)
0
2
8
10
1
0
0
.0
.5
.0
0.3
0.3
0
0
0
0
0
0
0
.6
(
2)
3)
.5
.4
.3
.2
.1
.0
0
.2
(
0.2
(
1)
0
0
.1
.1
0.0
0
2
4
1
6
8
10
-
3
3
1
/[6-MP] x 10 dm /mol (1)
0
2
4
6
8
10
-
-2
3
1
1
/[OH ] x 10 dm /mol (3)
3
-
4
3
[
4
H
2
IO
6
] x 10 mol/dm (2)
6 8
(
e)
0
2
10
1
0
0
.0
.5
.0
0.20
0.15
0.10
0
0
0
0
0
0
0
.6
.5
.4
.3
.2
.1
.0
(
(
2)
3)
0.05
(1)
0.00
0
2
4
1
6
8
10
-
3
3
1
/[6-MP] x 10 dm /mol (1)
0
2
4
6
8
10
-
-2
3
1
1
/[OH ] x 10 dm /mol (3)
Figure 4. Continued.

Journal of Sulfur Chemistry 133
Table 4. Effect of temperature on equilibrium constants of Scheme 1 for the oxidation of 6-MP by DPC in
aqueous alkaline medium.
3
3
3
K6 × 10−3 (dm /mol)
3
Temperature (K)
K4 (dm /mol)
K5 × 10 (mol/dm )
2
2
2
3
3
88
93
98
03
08
10.3
17.2
24.6
32.5
41.4
3.67
2.27
1.33
0.92
0.51
1.48
2.51
5.93
6.59
8.76
Table 5. Thermodynamic quantities with respect to K4, K5, and K6.
Thermodynamic quantities
Values from K4
Values from K5
Values from K6
ꢀ
ꢀ
ꢀ
H (kJ/mol)
S (J/K mol)
G298 K (kJ/mol)
51 ± 3
196 ± 10
−8.0 ± 0.4
−71 ± 3
−293 ± 15
16 ± 1
67 ± 3
294 ± 15
−21 ± 2
#
(
51 ± 3 kJ/mol) from K4 with that of ꢀH (61 ± 4 kJ/mol) of the rate-limiting step supports the
fact that the first step of Scheme 1 is fairly fast since it involves low activation energy compared
with that obtained for the slow step of the reaction (25). In the same manner, K5 and K6 values
were calculated at different temperatures and their corresponding values of the thermodynamic
quantities are given in Tables 4 and 5.
The values of ꢀH and ꢀS were both favorable for electron-transfer processes. The negative
value of ꢀS indicates that the complex (C) is more ordered than the reactants (26). The value of
#
#
#
#
ꢀ
S within the range for radical reaction has been ascribed (27) to the nature of electron pairing
and unpairing processes and to the loss of degrees of freedom formerly available to the reactants
upon the formation of a rigid transition state. The observed modest enthalpy of activation and a
relatively low value of the entropy of activation as well as a higher rate constant of the slow step
indicate that the oxidation presumably occurs via an inner-sphere mechanism. This conclusion is
supported by an earlier observation (28).
4
. Conclusions
Among the various species of DPC in an aqueous alkaline medium, [Cu(H2IO6)(H2O)2] is con-
sidered as an active species for the title reaction. The results indicated that in carrying out this
reaction, the role of pH in the reaction medium is crucial. The rate constant of the slow step and
other equilibrium constants involved in the mechanism are evaluated. The activation parameters
with respect to the slow step of the mechanism were computed. The overall mechanistic sequence
described here is consistent with all the experimental evidences including the product, spectral,
mechanistic, and kinetic studies.
5
5
.
Experimental
.1. Materials and reagents
All chemicals used were of reagent grade, and doubly distilled water was used throughout the
work. A solution of 6-MP (Sigma Aldrich) was prepared by dissolving an appropriate amount
of 6-MP in doubly distilled water. The required concentration of 6-MP was prepared from its

1
34 K.M. Naik and S.T. Nandibewoor
stock solution. The copper(III) periodate complex was prepared by the standard procedure (29).
The existence of copper(III) complex was verified by its UV–VIS spectrum, which showed an
absorption band with the maximum absorption at 415 nm. The aqueous solution of copper(III)
was standardized by iodometric titration and gravimetrically by the thiocyanate (30) method. The
copper(II) solution was prepared by dissolving the known amount of copper sulfate (BDH) in
distilled water. Periodate solution was prepared by weighing the required amount of sample in hot
water and used after keeping it for 24 h. Its concentration was ascertained iodometrically (31) at
neutral pH by phosphate buffer.Aqueous solutions of KOH and KNO3 (BDH,AR) were employed
to maintain the required alkalinity and ionic strength, respectively, in reaction solutions.
5
.2. Instruments used
(i) For kinetic measurements, a Peltier Accessory (temperature control) attached Varian CARY
5
0 Bio UV–VIS spectrophotometer (Varian, Victoria-3170, Australia) was used.
(
ii) For product analysis, a QP-2010S Shimadzu gas chromatograph mass spectrometer and a
Nicolet 5700-FT-IR spectrometer (Thermo, USA) and for pH measurements, an Elico pH
meter model LI120 were used.
5
.3. Kinetic measurements
The kinetics was followed under pseudo-first-order condition, where [6-MP] > [DPC] at 25 ±
◦
0
.1 C, unless specified. The reaction was initiated by mixing the DPC with 6-MP solution, which
also contained the required concentration of KNO3, KOH, and KIO4. The progress of the reaction
was followed spectrophotometrically by measuring the decrease in the absorbance of DPC in
the reaction mixture as a function of time, in a 1 cm quartz cell in a thermostated compartment
of Hitachi 150-20 UV–VIS spectrophotometer, at its λmax = 415 nm. Previously, it was verified
that there is negligible interference from other species present in the reaction mixture at this
wavelength. That the absorbance by DPC obeys Beer’s law at its λmax = 415 nm was verified
3
earlier, and the molar absorbency index, ε was found to be 6230 ± 100 dm /mol cm.
The pseudo-first-order rate constants, kobs, were determined from the log (absorbance) versus
time plots and were the average of duplicate runs (Table 1). The plots were linear up to 90%
−
completion of reaction under the range of [OH ] used and the rate constants were reproducible to
within ± 5%. The orders for various species were determined from the slopes of plots of log kobs
versus log respective concentration of species except for [DPC] in which no variation of kobs
was observed as expected to the reaction condition. During the kinetics, a constant concentration
−
4
3
viz. 5.0 × 10 mol/dm of KIO4 was used throughout the study unless otherwise stated. Since
periodate is present in excess in DPC, the possibility of the oxidation of 6-MP by periodate in
alkaline medium at 25 C was tested. The progress of the reaction was followed iodometrically.
◦
However, it was found that there was no significant reaction under the experimental conditions
employed compared with the DPC oxidation of 6-MP. The total concentrations of periodate and
−
OH was calculated by considering the amount present in the DPC solution and that additionally
added. Kinetic runs were also carried out in N2 atmosphere in order to understand the effect of
dissolved oxygen on the rate of reaction. No significant difference in the results was obtained
under a N2 atmosphere and in the presence of air. In view of the ubiquitous contamination of
carbonate in the basic medium, the effect of carbonate was also studied. Added carbonate had no
effect on the reaction rates. The spectral changes during the reaction are shown in Figure 5. It is
evident from the figure that the concentration of DPC decreases by observing the absorbance at
4
15 nm.

Journal of Sulfur Chemistry 135
Figure 5. UV–VIS spectra changes during the oxidation of 6-mercaptopurine by alkaline DPC at 298 K,
−
5
−4
−
−3
3
[
DPC] = 5.0 × 10 , [6-MP] = 5.0 × 10 , [OH ] = 4 × 10 , and I = 0.01 mol/dm with scanning time of (1)
0
.5 min, (2) 1.0 min, (3) 1.5 min, (4) 2.0 min, and (5) 2.5 min.
Regression analysis of experimental data to obtain the regression coefficient r and standard
deviation S of points from the regression line was performed using Microsoft 2003 Excel program.
References
(
(
(
(
(
(
(
(
(
1) Van Scoik, K.G.; Johnson, C.A.; Porter, W.R. Drug Metab. Rev. 1985, 16, 157–174.
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(
(
(
(
(
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(
(
20) Lister, M.W. Can. J. Chem. 1953, 31, 638–652.
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1
36 K.M. Naik and S.T. Nandibewoor
Appendix
Derivation of rate law for the reaction
According to Scheme 1:
d[DPC]
Rate = −
= k[Complex]
dt
Rate = kK6[6-MP][Cu]
kK5K6[6-MP][Cu2−]
Rate =
Rate =
2
6
−
[
H3IO
]
−
kK4K5K6[6-MP][DPC][OH ]
(A1)
(A2)
2
6
−
[H3IO
]
The total concentration of DPC, [DPC]T, is given by
DPC]T = [DPC]f + [Cu −] + [Cu] + Complex
2
[
where T and f refer to total and free concentrations.
2
6
−
[
DPC]T[H3IO
]
[
DPC]f =
2
6
−
−
2−
6
−
−
[
H3IO ] + K4[OH ][H3IO ] + K4K5[OH ] + K4K5K6[OH ][6-MP]
Similarly, the concentration of 6-MP is
[
6-MP]T = [6-MP]f + [Complex]
2
6
−
[6-MP]T[H3IO
]
[
6-MP]f =
2
−
−
−
[
H3IO ] + K4K5K6[OH ][Cu ]
6
−
−
2−
In view of the low concentrations of DPC used, the term in K4K5K6[OH ][Cu ] compared with [H3IO ] can be
6
neglected. Hence,
[
6-MP]f = [6-MP]T
(A3)
Similarly,
−
−
[
OH ]f = [OH ]T
(A4)
Substituting Equations (A2)–(A4) into Equation (A1), we get
−
−
d[DPC]
kK4K5K6[DPC][6-MP][OH ]
6
Rate =
=
2
6
−
2−
−
−
−
dt
[H IO ] + K [H IO ][OH ] + K K [OH ] + K K K [OH ][6-MP]
3
4
3
4
5
4
5
6