Penicillamine and captopril: mechanistic exploration of defensive actions of thiol drugs against a metal bound-superoxo complex

Article abstract of DOI:10.1080/00958972.2017.1303488

In acid-media ([H⁺]?=?0.01–0.06?M), each of the thiol compounds, D-penicillamine (PEN, L_PH₂) and captopril (CAP, L_CH₂) exist in several proton-dependent forms which can reduce the superoxo complex [(en)(dien)Co^III(O₂)Co^III(en)(dien)]⁵⁺ (1) to the corresponding peroxo [(en)(dien)Co^III(O₂)Co^III(en)(dien)]⁴⁺ (2) or the hydroperoxo complex [(en)(dien)Co^III(OOH)Co^III(en)(dien)]⁵⁺ (3). The observed first-order rate constants, k _o,P and k _o,C for PEN and CAP increase with the increase in [T_PEN] and [T_CAP] (which are the analytical concentrations of the respective thiols) but decrease with the increase in the media-acidity ([H⁺]) and the media ionic strength (I). The protolytic equilibria in aqueous solution allow several potentially reducing forms to coexist for both PEN (L_PH₃ ⁺, L_PH₂, L_PH^?, and L_P ^2?) and CAP (L_CH₂, L_CH^?, L_C ^2?) but the kinetic analyses reveal that the order of reactivity for the species are L_PH₃ ⁺ ~ L_PH₂?<<PH^? and L_CH₂?CH^??<<C ^2?, respectively. The predominance and higher reactivities of the anionic species, L_PH^? and L_C ^2? are supported by the negative slopes of the plots of k _o,P or k _o,C versus I. Moreover, a large value of k _H/k _D for PEN suggests an inner-sphere electroprotic reaction pathway while the absence of such effect for CAP strongly supports an outer-sphere electron transfer reaction. These propositions are supported by the structural features of L_PH^? and L_C ^2?.

Full text of DOI:10.1080/00958972.2017.1303488

Journal of Coordination Chemistry
ISSN: 0095-8972 (Print) 1029-0389 (Online) Journal homepage: http://www.tandfonline.com/loi/gcoo20
Penicillamine and captopril: Mechanistic
exploration of defensive actions of thiol drugs
against a metal bound-superoxo complex
Arabinda Mandal, Ranendu Sekhar Das, Bula Singh, Rupendranath Banerjee
& Subrata Mukhopadhyay
To cite this article: Arabinda Mandal, Ranendu Sekhar Das, Bula Singh, Rupendranath Banerjee
& Subrata Mukhopadhyay (2017): Penicillamine and captopril: Mechanistic exploration of defensive
actions of thiol drugs against a metal bound-superoxo complex, Journal of Coordination Chemistry,
DOI: 10.1080/00958972.2017.1303488
To link to this article: http://dx.doi.org/10.1080/00958972.2017.1303488
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Date: 20 March 2017, At: 14:49

Publisher: Taylor & Francis
Journal: Journal of Coordination Chemistry
DOI: http://dx.doi.org/10.1080/00958972.2017.1303488
Penicillamine and captopril: Mechanistic exploration of defensive actions of
thiol drugs against a metal bound-superoxo complex
ARABINDA MANDAL*†, RANENDU SEKHAR DAS*‡, BULA SINGH§, RUPENDRANATH BANERJEE‡
and SUBRATA MUKHOPADHYAY‡
†Department of Chemistry, Bidhannagar College, Kolkata 700 064, India
‡Department of Chemistry, Jadavpur University, Kolkata 700 032, India
§Department of Chemistry, Visva-Bharati, Santiniketan 731 235, India
In acid-media ([H+] = 0.01-0.06 M), each of the thiol compounds, D-penicillamine (PEN, LPH2)
and captopril (CAP, LCH2) exist in several proton-dependent forms which can reduce the
superoxo complex [(en)(dien)CoIII(O2)CoIII(en)(dien)]5+ (1) to the corresponding peroxo
[(en)(dien)CoIII(O2)CoIII(en)(dien)]4+ (2) or the hydroperoxo complex
[(en)(dien)CoIII(OOH)CoIII(en)(dien)]5+ (3). The observed first-order rate constants, ko,P and ko,C
for PEN and CAP increase with the increase in [TPEN] and [TCAP] (which are the analytical
concentrations of the respective thiols) but decrease with the increase in the media-acidity ([H+])
and the media ionic strength (I). The protolytic equilibria in aqueous solution allow several
+
2-
potentially reducing forms to coexist for both PEN (LPH3 , LPH2, LPH- and LP ) and CAP (LCH2,
LCH-, LC ) but the kinetic analyses reveal that the order of reactivity for the species are LPH3 ~
2-
+
2-
LPH2 <<< LPH- and LCH2 < LCH- <<< LC , respectively. The predominance and higher reactivities
of the anionic species, LPH- and LC2- are supported by the negative slopes of the plots of ko,P or
ko,C versus I. Moreover, a large value of kH/kD for PEN suggests an inner-sphere electroprotic
reaction pathway while the absence of such effect for CAP strongly supports an outer-sphere
electron transfer reaction. These propositions are supported by the structural features of LPH- and
2-
LC .
Keywords: Superoxide; Redox; Kinetics; Mechanism; PCET
*Corresponding authors. Email: arabindaju@gmail.com (A. Mandal); ranendu147@gmail.com (R.S. Das)
1

1. Introduction
D-penicillamine (PEN, 3,3-dimethyl-D-cysteine, HSC(CH3)2CH(NH2)CO2H) [1-4] and captopril
(CAP, 1-[(2S)-3-mercapto-2-methylpropionyl]-l-proline, HSCH2CH(CH3)CON(C4H7CO2H))
[5-7] are two of the renowned, highly potent, therapeutic thiol compounds (represented in
general as RSH) which are in frequent use for medicinal purposes. The former one, i.e., PEN is
administered to treat rheumatoid arthritis (RA) [8-10] as it acts as an immunosuppressant that
reduces inflammation and lowers pain and swelling [10]. PEN has also been found to be
effective as a chelating agent for the elimination of copper in patients suffering from Wilson's
disease [11, 12] and in treatment of cystinuria [13]. On the other hand, CAP is widely used as an
orally active antihypertensive drug [14-16] since CAP can function as an angiontensin-
converting enzyme (ACE) inhibitor that blocks the conversion of angiotensin I to angiotensin II
[16]. Despite the suspicion that prolonged use of PEN and CAP can cause proteinuria, renal
effects and other toxic effects [9, 16-19], these drugs are in wide use to fight against RA and
hypertension (HT).
The detailed causes behind the occurrence of RA are much less understood than HT but
recent studies have conclusively established that in both cases, the cellular oxidative stress serves
as an underlying mechanism behind the pathogenesis of these diseases [20-29]. Aerobic
organisms usually suffer from such oxidative stress when excess of the highly reactive oxygen
•-
species (ROS) like, O2 and H2O2 are produced due to partial reduction of molecular oxygen
[21, 22, 30]. It has been found that the ROS generated by NADPH oxidase can cause severe bone
and cartilage erosion [31, 32] as well as disruptions in intra-cellular signaling pathways [27].
To combat such stressful conditions, the living organisms are equipped with antioxidant
enzymes like, superoxide dismutase (SOD) or non-enzymatic molecules like, glutathione and
cysteine [33-35]. Antioxidant molecules like, glutathione, cysteine and thioglycolic acid which
•-
have reduced thiol (–SH) groups are well known and their efficacies in reducing O2 species are
well studied [36-38]. Two of the concerned drugs, PEN and CAP molecules, also possess the
thiol groups (figure 1) and are expected to react with the ROS species, but to our best of the
knowledge such studies with PEN and CAP are yet to be accomplished.
In our present study, we thus report the detailed kinetics of the redox reactions of PEN
and CAP with a superoxo complex in aqueous acidic media under laboratory condition. Since, in
2

•-
aqueous media, free O2 anions are unstable due to rapid disproportionation reactions of the
species [39], we have chosen a stable heteroleptic cobalt(III)-bound superoxide complex
[(dien)(en)CoIII(O2)CoIII(en)(dien)](ClO4)5 (1, en = NH2CH2CH2NH2 and dien =
NH2(CH2)2NH(CH2)2NH2) as the representative of the free superoxo species [40]. Detailed
kinetic observations and the outcome from our study are expected to shed light on the probable
mechanistic pathways through which thiol molecules like PEN and CAP react with the ROS
species.
2. Experimental
2.1. Materials and instrumentation
The perchlorate salt of the complex [(dien)(en)CoIII(O2)CoIII(en)(dien)]5+ (1, en =
ethylenediamine and dien = diethylenetriamime) was synthesized from Co(NO3)2·6H2O (SRL,
INDIA), en and dien (Aldrich) following the literature procedure [41]. Complex 1 is well
characterized [41, 42] and in aqueous acidic media it shows two characteristic UV-vis absorption
bands at 471 nm and 706 nm (figure S1). The purity of 1 synthesized for the present work is
more than 96% as the value of the molar extinction coefficient, ε at 706 nm was calculated as
1161 M-1cm-1 (reported 1210 M-1cm-1 [41]). The FTIR spectrum of 1 (figure S2) nicely matched
with the literature report [43, 44].
For the present work, the fresh stock solutions of penicillamine (Aldrich) and captopril
(Aldrich) were prepared by dissolving accurately weighed amount of the corresponding
compounds in aqueous acid media. The ionic strengths (I) of the reaction mixtures were held
constant with the addition of sodium perchlorate (Aldrich). All experimental solutions were
prepared with freshly fetched Milli Q water. Deuterium oxide (Aldrich) and perchloric acid
(Merck) were used without purification while the latter was standardized following standard
methods [45].
Kinetic runs were taken in situ in a thermostated cell housing (CPS-240A, 0.1 °C) of a
Shimadzu (UV-1700) spectrophotometer. Fourier Transform Infrared (FTIR) spectrum was
recorded on the Perkin Elmer (L1600300) FTIR spectrometer; EPR spectrum was recorded with
a Jeol (JES FA 200) spectrometer; mass spectrometry analysis was carried out with a micro-mass
Q-TOF spectrometer (Waters) with an electro-spray ionization source and 1H-NMR spectra were
recorded with a 300 MHz Bruker spectrometer using tetramethylsilane as an internal standard.
3

Acid solutions were standardized by pH metric titration in Metrohm 736-GP Titrino instrument
and the pH of the reaction solution was determined with a Toshniwal pH-meter (CL-54, India).
2.2. Kinetic measurements
Since the title thiol drugs PEN and CAP combat with RA and HT under the physiological pH of
~ 7.0, we have chosen a lower pH range (1.2–2.0) to study the title reactions, mainly due to two
reasons. Firstly, 1 is stable only under pH ~ 6.5 and secondly at relatively higher pH condition,
the reaction between 1 with PEN and CAP proceeds too fast to study. We have thus studied the
oxidations of PEN and CAP by 1 in an acid perchlorate media ([H+] = 0.01–0.06 M) at constant
media ionic strength (I) (maintained by NaClO4). In acid perchlorate media, 1 is highly stable
against auto-decomposition and shows characteristic absorption at 706 nm where none of the
thiol (PEN and CAP) compounds absorb. Thus, when PEN or CAP reduces 1, from the
corresponding changes in the absorbance values at 706 nm with time, the progress of the reaction
can be followed. Such a time resolved spectral change for the reaction between 1 and PEN is
shown in figure 2 and a similar spectral profile for the reaction between 1 and CAP is shown in
figure S3. From the respective plots of the absorbance values versus time data, the values of the
corresponding observed rate constants for the reactions were determined. Since all the kinetic
experiments were performed in the presence of excess of thiol concentration over [1] ([TThiol]/[1]
≤ 10; [TThiol] denotes the analytical concentration of relevant thiol molecules, either PEN or
CAP) to maintain pseudo first-order condition, a non-linear least-square fit to the absorbance
versus time data (figures S4 and S5 for PEN and CAP, respectively) yields the observed pseudo
first-order rate constant, ko (represented as ko,P and ko,C for PEN and CAP, respectively). The
analytical software Origin 6.0 was used to plot the data and to extract the pseudo first-order rate
constant values.
All reaction mixtures for the kinetic studies were prepared by the addition of measured
volume of the solution of 1, NaClO4 and perchloric acid followed by addition of freshly prepared
-
aqueous solution of PEN or CAP. Here, it is worth remembering that, although ClO4 anions are
thermodynamically potent oxidants, the kinetic inertness in an aqueous solution renders them to
-
be non-oxidizing in nature [40, 46]. Thus the ClO4 anions do not interfere in the title redox
reaction. Earlier literature reports mention that the presence of ubiquitous metal ions like Cu2+
strongly catalyzes the oxidation of thiol molecules through the formation of Cu2+–thiol
4

complexes [38, 47]. To avoid such interferences from the metal ions, milli-Q water (18MΩ) was
used in preparation of the reagent solutions. Albeit independent kinetic runs were performed in
the presence of a well-known sequestering agent, dipicolinic acid (dpa, 2.0 mM), and the
calculated ko values were found within 95% of the ko values obtained from an independent
experiment with otherwise the same experimental conditions except dpa. Similar values of ko
from the independent experiments with or without dpa confirm that the reaction solutions are
free from any substantial Cu2+ ion impurities.
Each of the kinetic runs for this study was performed for at least three times. The
observed rate constant value reported here is the average of the best three independent kinetic
runs where the ko values lie close to each other. The errors associated with the ko values were
also provided in the corresponding data tables and the ordinates of the plots representing the ko
values also include the errors as the corresponding error bars.
2.3. Stoichiometry
The stoichiometries for the redox reactions between 1 and PEN or CAP were determined from
the estimation of excess [TPEN] or [TCAP] over [1] (here, [TPEN] and [TCAP] are the analytical
concentration of PEN and CAP, respectively). For this purpose, 1 was allowed to react with
excess amount of PEN and CAP and after completion of the reaction the remaining excess of the
thiol molecules was quantitatively determined by spectrophotometry.
Stoichiometric excess of PEN and CAP in the equilibrium reaction mixture was
determined with Ellman’s reagent (5,5´-dithio-bis-2-nitrobenzoic acid, DTNB) [48] which in
alkaline pH, reacts quantitatively with the free sulfhydryl group (–SH) of PEN and CAP to
produce yellow-colored 2-nitro-5-thiobenzoic acid (TNB) and mixed disulfides. TNB, in
aqueous alkaline media, shows characteristic absorbance at 412 nm (ε = 13.6×103 M-1 cm-1) [49]
and from the corresponding absorbance values the remaining excess of PEN and CAP was
determined (table S1).
The products resulting from the oxidation of the thiol compounds PEN and CAP were
characterized from the mass spectra of the corresponding product mixture. After completion of
the reaction, the reaction mixture was extracted with ethyl acetate and was evaporated under
vacuum at room temperature. The residue obtained thereby was dissolved in CH3CN for mass
5

spectroscopy in positive ion mode (figures S7 and S8 for PEN and CAP, respectively) and in
DMSO-d6 for 1H-NMR (figures S9 and S10, respectively).
3. Results and discussion
3.1. Stoichiometry of the reaction
The ratio Δ[1]/Δ[RSH] shown in table S1 indicates that each mole of PEN and CAP reacts with
one mole of 1. Both PEN and CAP possess one –SH group and thus are one-electron donors
[36, 38, 50]. On reaction with PEN and CAP, 1 is reduced by accepting the electron to produce
the corresponding peroxo complex [(dien)(en)CoIII(O2)CoIII(en)(dien)]4+ (2) [51, 52]. In acid
media, peroxo complex 2 quickly takes up a proton and isomerizes to form the corresponding
hydroperoxo complex [(dien)(en)CoIII(OOH)CoIII(en)(dien)]5+ (3) [53]. However, it should be
noted that the proton may also come from the reducing –SH group. The possibility of either a
proton coupled electron transfer (PCET) reaction pathway or the reaction pathway with an
electron transfer (ET) followed by the proton transfer (PT) was considered in detail in the
discussion section (vide supra). Unlike the hydroperoxo complexes with NH3 ligands, the
hydroperoxo complex with en and dien ligands (3) is much more stable under similar acidic
condition and 3 imparts a red coloration to the final product solution [53]. Here it is noteworthy
that 3 does not show any significant absorption at 706 nm and thus the formation of 3 from 1
does not interfere in the kinetic studies.
For superoxo complex 1 [(dien)(en)CoIII(O2)CoIII(en)(dien)]5+, the unpaired electron
-
resides on the dioxygen superoxo bridge (–O2 –) [2] and it is the prime center of the redox
activities [41, 42]. The presence of an unpaired electron results in an intense EPR response
-
(figure S6) [42] and when on reduction, the bridging superoxo group (–O2 –) of 1 is reduced to
2-
the corresponding bridging peroxo group (–O2 –) in 2, the final product mixture is completely
EPR silent. The Co(III) centers of 1 do take part in the redox interactions and they remain in +3
oxidation state in the hydroperoxo complex 3 [42, 52, 53]. The fact that the hydroperoxo
complex 3 is the end product is evident from the red color of the end product mixture since the
acidic solutions of similar hydroperoxo complexes also show absorption in the same wavelength
region (~500 nm) (figures 2 and S3) [52, 53].
Thiol molecules (PEN or CAP, in general RSH) usually on oxidation form the
corresponding disulfides, but in a handful of cases forms the thiol sulfinic acid (RSO2H), too
6

[36]. The mass spectra (figures S7 and S8) of the product mixtures though strongly support the
formation of disulfides from PEN and CAP; they also confirm that in these reactions no sulfinic
acid products were formed. While the product mixture from PEN (RSH, R =
SC(CH3)2CH(NH2)CO2H, M = 149.2 gM-1) shows a strong peak for the disulfide molecular ion
([M+H]+ in positive mode) at m/z = 297.1 (figure S6), peak for similar disulfide molecular ion
([M+H]+) from CAP (RSH, R = SCH2CH(CH3)CON(C4H7CO2H), M = 217.3 gM-1) appears at
m/z = 433.2 (figure S7). The end disulfide products were also characterized by 1H-NMR
analyses for which the end-products were extracted with ethyl acetate from the reaction mixture
and were then purified by column chromatography. The product obtained thereby was dissolved
in DMSO-d6 for 1H-NMR spectroscopy. The details of corresponding 1H-NMR spectrum for the
disulphides of PEN and CAP are shown in figures S9 and S10. Both the mass spectra and the
1H-NMR spectra confirm the formation of the corresponding disulfides on oxidation of PEN and
CAP.
Based on the above observation, we thus represent the redox reactions between 1 and the
thiols, PEN and CAP (RSH, R = SC(CH3)2CH(NH2)CO2H and
SCH2CH(CH3)CON(C4H7CO2H), respectively) by eq. 1, where 1 is reduced to 3 and on
oxidation, PEN and CAP formed the corresponding disulfide products represented as RSSR.
2[(en)(dien)CoIII(O2)CoIII(en)(dien)]5++ 2RSH
2[(en)(dien)CoIII(OOH)CoIII(en)(dien)]5+ + RSSR (1)
3.2. Variation of ko with [TPEN], [TCAP] and I
For PEN and CAP (table S2), the variation of the observed rate constants, ko,P values against
[TPEN] and variation of ko,C values against [TCAP] are shown in figures 3 and S11, respectively.
For both thiols, ko values increase linearly with increase in the analytical concentration of thiols
(r2 = 0.996 and r2 = 0.998 for figures 3 and S11, respectively). Moreover, the plots also pass
through the origin, which confirms that the auto-decomposition of 1 is insignificant under the
experimental condition.
The change of the media ionic strength, I was found to have effect on the ko values
(table S3) and the variations of ko,P and ko,C against I of the reaction media are shown in figures 4
7

and S12, respectively. It can be seen that with the increase in I, the ko values decrease linearly (r2
= 0.9970 and 0.9979 for figures 4 and S12, respectively). The negative slopes of the linear plots
indicate that at the rate steps of the redox reactions between 1 and PEN or CAP, two oppositely
charged species interact with each other. Since the oxidant, 1 is cationic the reactive species from
the reductants, PEN and CAP then must be anionic.
3.3. Variation of ko with [H+]
Our present study concerning the reactions between 1 and PEN or CAP were performed in acid
media where [H+] was varied over the range of 0.01–0.06 M. The variations of ko,P and ko,C
against 1/[H+] for PEN and CAP are shown in figures 5 and S13, respectively (table S4), which
show that with decrease in media [H+] the observed rate constant values increase linearly (r2 =
0.998 and 0.998 for figures 5 and S13, respectively). The reciprocal relationship between ko and
[H+] indicates the importance of the protolytic equilibria among the reactant species. As the
media acidity decreases the concentration of the deprotonated or anionic species increases, which
results in the increase in the observed rate constant values.
Among the reactants 1, PEN and CAP, 1 is very much stable over the entire experimental
-
range of acidity. While the superoxo moiety (O2 ) does not take up any proton due to its bridging
structure [51], the ligands en and dien also cannot participate in any protolytic-equilibria due to
their coordinative saturation. The reductant thiols PEN and CAP, on the other hand, participate
in several protolytic equilibria and result in several potential reactive species (schemes 1 and 2,
respectively).
8

O
C
CH3
SH
O
C
H3C
CH
CH3
SH
H3C
C
pKP1 = 1.8
pKP2 = 7.9
pKP3 = 10.5
C
+ H+
(2)
(3)
(4)
O
HO
CH
NH3
NH2
+
LPH3
LPH2
O
C
CH3
H3C
C
O
CH3
SH
H3C
C
C
O
SH + H+
O
CH
CH
NH2
NH3
LPH-
LPH2
O
O
CH3
H3C
CH3
H3C
C
C
+ H+
C
C
O
SH
O
S
CH
CH
NH2
2-
NH2
LPH-
LP
Scheme 1. pK values of successive deprotonation steps for PEN.
CH3
CH
CH3
CH
HS
O
HS
O
pKC1 = 3.7
C
H2
C
N
C
H2
+ H+
C
N
OH
(5)
(6)
O
C
C
O
O
LCH-
LCH2
CH3
CH
CH3
CH
S
HS
O
O
C
H2
C
+ H+
pKC2 = 9.8
C
N
C
N
O
O
H2
C
C
O
O
LCH-
2-
LC
Scheme 2. pK values of successive deprotonation steps for CAP.
+
According to scheme 1, a protonated molecule of PEN (LPH3 ) undergoes successive
+
deprotonations of the –CO2H group (eq. 2), –NH3 group (eq. 3) and finally –SH group (eq. 4) to
form the species LPH2, LPH- and LP , respectively [54]. The pKP1, pKP2 and pKP3 values for the
2-
+
deprotonation of the –CO2H, –NH3 and –SH groups of PEN were well known from both
experimental as well as theoretical works [55, 56] and values are 1.8, 7.9 and 10.5, respectively.
Similarly, according to scheme 2, a CAP molecule (LCH2) forms the species LCH- and
LC2- following deprotonation of the –CO2H group (eq. 5) and the –SH group (eq. 6), respectively
[55]. The reported pKa values for deprotonation of the –CO2H and –SH groups of CAP are pKC1
= 3.7 and pKC2 = 9.8, respectively [55, 56].
9

+
All the species, LPH3 , LPH2, LPH- and LP2- arising from PEN and the species, LCH2, LCH-
and LC2- arising from CAP molecules are the potential forms of the reductants and we can
assume that all of these species will react with 1. However, it is also important to note that only
the mercapto (–SH) groups of the PEN and CAP molecules can take part in the redox interaction
with 1. Since the Co-bound superoxo complexes are highly stable in acetic acid-sodium acetate
buffer [53], it can be inferred that the carboxylate (–COO-) groups of the PEN and CAP
molecules are redox inactive toward 1.
3.4. Effect of kinetic isotopic effect on ko
A decrease in the observed rate constant values was found when the reaction media was enriched
with D2O (table S5). From the linear plots of ko,P and ko,C vs. % D2O shown in figures 6 and S14
(r2 = 0.992 and 0.997, respectively), the ratio, kH/kD for PEN and CAP can be calculated as ~ 4.9
and ~ 1.4, respectively. From the ratio of kH/kD, it is very much evident that the isotopic
exchange has its profound effect on the reactions of PEN but not on the reactions of CAP. The
observed reaction rates for the simple reactions occurring through the electron-transfer
mechanism are hardly affected by the solvent isotopic exchanges and such significant fall in the
observed reaction rates for PEN indicates the involvement of the electroprotic reaction pathway
[58-61]. Moreover, the linearity of the plot shown in figure 6 also indicates the transfer of a
proton in the rate step [59].
3.5. Proposed mechanism for the reaction
+
It has been mentioned earlier that while for PEN, all the species, LPH3 , LPH2, LPH- and LP2- can
react with 1, for CAP the potential reacting species are LCH2, LCH- and LC . Considering all
2-
these species, the reactions between 1 and PEN are shown in the kinetic scheme 3 and the
reactions between 1 and CAP are shown in scheme 4.
10

KP1
KP2
+ H+
+ H+
+
(7)
(8)
LPH2
LPH-
LPH3
LPH2
KP3
2-
LP
+ H+
(9)
(10)
(11)
LPH-
1
kP1
kP2
+
Products
Products
LPH3
+
1
LPH2
LPH-
+
kP3
(12)
(13)
Products
Products
1
1
+
+
kP4
2-
LP
Scheme 3. Kinetic scheme for the reactions of PEN.
KC1
LCH-
+ H+
+ H+
(14)
LCH2
LCH-
KC2
2-
(15)
(16)
(17)
(18)
LC
kC1
kC2
kC3
Products
Products
Products
LCH2
LCH-
1
1
+
+
+
2-
LC
1
Scheme 4. Kinetic scheme for the reactions of CAP.
+
According to scheme 3, the rate constants for the reactions between 1 and LPH3 (eq. 10), LPH2
(eq. 11), LPH- (eq. 12) and LP2- (eq. 13) are kP1, kP2, kP3 and kP4, respectively, and the observed
rate constant for the reaction is given by the following eq. 19:
[
]
ꢀPꢅꢆPꢄ ꢀPꢈꢆPꢄꢆPꢅ ꢀPꢉꢆPꢄꢆPꢅꢆPꢈ TPEN
ꢀꢁ,ꢂ = ꢃꢀPꢄ
+
+
+
ꢊ
(19)
[Hꢇ]ꢅ
[Hꢇ]ꢈ
ꢋ
ꢇ
[
H
]
where
and
ꢆPꢄ
ꢆPꢄꢆPꢅ ꢆPꢄꢆPꢅꢆPꢈ
ꢇ
[
]
[ ]
ꢊ LPHꢈ (20)
TPEN = ꢃ1 +
+
+
[Hꢇ]ꢅ
[Hꢇ]ꢈ
ꢇ
[
H
]
ꢆPꢄ
ꢆPꢄꢆPꢅ ꢆPꢄꢆPꢅꢆPꢈ
ꢋ = ꢃ1 +
+
+
ꢊ (21)
[Hꢇ]ꢅ
[Hꢇ]ꢈ
ꢇ
[
H
]
11

Similarly, according to scheme 4, the rate constants for the reaction between 1 and LCH2 (eq. 16),
LCH- (eq. 17) and LC2- (eq. 18) are kC1, kC2 and kC3, respectively, and the observed rate constant
for the reaction is given by the following eq. 22:
[
]
ꢀCꢅꢆCꢄ ꢀCꢈꢆCꢄꢆCꢅ TCAP
ꢀꢁ,C = ꢃꢀCꢄ
+
+
ꢊ
(22)
[Hꢇ]ꢅ
ꢌ
ꢇ
[
H
]
where
and
ꢆCꢄ
ꢆCꢄꢆCꢅ
[Hꢇ]ꢅ
[
]
[ ]
ꢊ LCHꢅ (23)
TCAP = ꢃ1 +
+
ꢇ
[
H
]
ꢆCꢄ
ꢆCꢄꢆCꢅ
[Hꢇ]ꢅ
ꢌ = ꢃ1 +
+
ꢊ (24)
ꢇ
[
H
]
Eq. 19 and eq. 22 can be further rearranged as eq. 25 and eq. 26 as shown below:
ꢋꢀꢁ,P[Hꢇ]ꢈ
= (ꢀPꢄ[Hꢇ]ꢈ + ꢀPꢅꢆPꢄ[Hꢇ]ꢅ + ꢀPꢈꢆPꢄꢆPꢅ[Hꢇ] + ꢀPꢉꢆPꢄꢆPꢅꢆPꢈ) (25)
[
]
TPEN
and
ꢌꢀꢁ,C[Hꢇ]ꢅ
ꢇ ꢅ
ꢇ
[
[
H
]
)( )
+ ꢀCꢈꢆCꢄꢆCꢅ 26
= (ꢀCꢄ H ] + ꢀCꢅꢆCꢄ
[
]
TCAP
From the coefficient values of the non-linear least-square fit of order 3 of the right hand side of
eq. 25, i.e., (αko,P[H+]3/[TPEN]) against [H+] (figure S15) can yield the values of the composite
rate constants kP1, kP2KP1, kP3KP1KP2 and kP4KP1KP2KP3. Similarly, the values of the coefficients of
the non-linear least-square fit of order 2 of the right hand side of eq. 26, i.e., (βko,C[H+]2/[TCAP])
against [H+] (figure S16) can give the values of the constants kC1, kC2KC1 and kC3KC1KC2. Since
the values of the protolytic equilibria constants, KP1, KP2, KP3, KC1 and KC2 are known from the
literature [55, 56], values of rate constants kP1, kP2, kP3, kP4, kC1, kC2 and kC3 can be calculated.
The coefficient values yielded from the corresponding non-linear least-squares fits shown
in figures S15 and S16 are given in table 1. For PEN, though the values of kP1, kP2KP1 and
kP3KP1KP2 are positive, the value of kP4KP1KP2KP3 is negative with large error. Since a
12

polynomial-coefficient with negative sign usually is taken as an indication of the fact that the
particular reaction path is much less contributing or silent [50], we can infer that for PEN, the
species LP2- (eq. 13, scheme 3) either does not participate in reduction of 1 at all or its
contribution is almost negligible in comparison to other reducing species. With the known values
of pKP1, pKP2 and pKP3 (eqs. 2, 3 and 4, scheme 1), values for the second-order rate constants kP1,
kP2 and kP3 are calculated as ~ 0.042, ~ 1.2 and ~ 6.5×106 M-1 s-1, respectively.
For CAP, however, all the values of the rate constant composites kC1, kC2KC1 and
kC3KC1KC2 are positive (table 1) and with the known values of pKC1 and pKC2 (eqs. 5 and 6,
scheme 2), values of the second-order rate constants kC1, kC2 and kC3 are calculated as ~ 0.5,
~ 72.1 and ~ 5.4×109 M-1 s-1, respectively. The corresponding values of all these second-order
rate constants reproduced the observed ko values well within the range of 10% errors.
The calculated rate constant values for PEN and CAP bring out several interesting points.
From the results of the non-linear least-square fits, it can be seen that while for PEN kP1 ~ kP2 <<
kP3, for CAP, kC1 < kC2 << kC3. This suggests that the reductant species with negative charge(s)
(LPH- for PEN (eq. 12); LCH- and LC2- (eqs. 17 and 18) for CAP) play the dominant roles in
+
reducing 1 in comparison to the cationic (LPH3 (eq. 10)) or the neutral (LPH2 (eq. 11); LCH2
(eq. 16)) reductant species. The slow reactions of the cationic or the neutral reductant species are
quite expected and seem to be justified if we consider the electrostatic factors. Moreover, this
outcome strongly supports the observations made from the variations of ko vs. I (figures 4 and
S12), which also is suggestive for the interactions between the cationic complex 1 and anionic
reductants.
With the validation of the non-equalities (kP1 ~ kP2 <<< kP3 for PEN and kC1 < kC2 <<< kC3
for CAP), we now consider the structural features of the leading reducing species LPH-, LCH- and
2-
LC . As mentioned earlier that only the thiol end of the reductants are capable of reducing 1, we
notice that while LPH- and LCH- possess protonated thiol (–SH) group, LC2- possesses a
deprotonated thiolate (–S-) end. For PEN, the species LPH- thus can form a transient hydrogen
bond with the bridging superoxo moiety of 1 on close approach. In such a case, the reduction
reaction may proceed through an inner-sphere or electroprotic reaction mechanism [62, 63].
Support for such electroprotic reaction pathway comes from the kinetic isotope effect on the ko
values for PEN. Figure 6 shows that for PEN the kH/kD is ~ 4.9 and such large value of kH/kD
strongly favors an electroprotic reaction pathway where a proton transfer step is involved
13

[58-60]. However, an electroprotic reaction may proceed through either the concerted PCET
pathway or through the SPET pathway [61, 63]. The difference between PCET and SPET is very
subtle and the calculated rate constant data from our present study is not enough to show any
favoritism to any of these nearly close reaction pathways. We thus refer to these processes
(PCET or SPET) as an inner-sphere electroprotic reaction in general.
For CAP, the kinetic isotope effect is however absent and the value of kH/kD is only ~ 1.4
(figure S14). This suggests a simple outer-sphere or electron-transfer reaction for CAP. The
reason for the absence of the kinetic isotope effect for CAP is easily conceivable from the
structural consideration of the species LC2- and its much larger contribution over LCH-
(kC3 >>> kC2, table 1) in terms of the observed rate constant values. Though the species LCH- can
form hydrogen bond with 1, the predominant reactive species LC2- which possesses the
deprotonated thiolate (–S-) end cannot form such hydrogen bond and the value of kC3
(~ 109 M-1 s-1) indicates a diffusion-controlled outer-sphere ET reaction.
Based on this discussion, the mechanistic schemes for the reactions between 1 and PEN
or CAP can be described through the interaction of the superoxo complex and RSH and RS-
(schemes 5 and 6, respectively). In scheme 5, reaction occurs through an inner-sphere
electroprotic mechanism, for PEN, RSH = -O2CHC(NH3)C(CH3)2SH (LPH-). Similarly, in
scheme 6 where the outer-sphere electron transfer reaction is controlled by the rate of diffusion,
-
2-
for CAP, RS- = -SCH(CH3)CON(C4H7CO2 ) (LC ). In both schemes, the disulfide products from
PEN or CAP are identical with RSSR shown in eq. 1.
III
5+
III
O
5+
III
III
O
Co(en)(dien)
(en)(dien)Co
Co(en)(dien)
(en)(dien)Co
O
O
H
K
1
+
(27)
RSH
SR
H
O
O
5+
5+
III
III
O
III
III
Co(en)(dien)
Co(en)(dien)
(en)(dien)Co
(en)(dien)Co
O
k
slow
3
+
RS
H
(28)
(29)
SR
fast
2 RS
RSSR
Scheme 5. Mechanistic scheme for the reactions of PEN.
14

K
RS- + H+
RSH
(30)
(31)
[(en)(dien)CoIII(O2)CoIII(en)(dien)]5+
[(en)(dien)CoIII(O2)CoIII(en)(dien)]4+
k
1
2
+
slow
+
RS
RS-
H+
fast
[(en)(dien)CoIII(O2)CoIII(en)(dien)]4+
[(en)(dien)CoIII(HO2)CoIII(en)(dien)]5+
(32)
(29)
3
2
fast
RS
2
RSSR
Scheme 6. Mechanistic scheme for the reactions of CAP.
3.6. Activation energies of the reactions
We can get an idea for the activation energies of the redox interactions between 1 and PEN or
CAP from the corresponding slopes of the Arrhenius plots which show the changes of lnko
values against T-1 for PEN and CAP, respectively (figures S17 and S18; table S6). It is however
very interesting to find that both of the Arrhenius plots shown in figures S17 and S18 are convex
in nature and thus the activation energies of the respective reactions are not constant over the
experimental range of temperatures (288-313 K). We can divide the experimental temperature
range into a lower and a higher zone and from the corresponding slopes of the linear plots of lnko
vs. T-1 we can calculate the activation energies as Ea,L and Ea,H. While for PEN the activation
energy changes from ~43.9 (Ea,L) to 23.2 kJ M-1 (Ea,H), for CAP the change is from ~75.5 (Ea,L)
to 31.5 kJ M-1 (Ea,H) (table S7). The inversion temperatures Tinv for PEN and CAP are
approximately 299.7 and 298.5 K, respectively. Here, although the experimental temperature
range is narrow and the calculated activation energy values are imprecise but they serve an
important purpose to highlight the fact that for both PEN and CAP, the activation energies in the
higher temperature zone became almost half of the energy in the lower temperature zone. The
convex nature of the Arrhenius plot has been a subject of much discussion [64-66] and
undoubtedly it indicates towards the intricate relationships between the molecular associations in
the rate steps with the temperature. Detailed investigations in this area are beyond the scope of
15

this work and we thus debar ourselves from correlating these energy values to the proposed
mechanistic schemes to justify them.
4. Conclusion
In acid-media the superoxo complex, [(en)(dien)CoIII(O2)CoIII(dien)(en)]5+ (1), is quantitatively
reduced by two thiol drugs, D-penicillamine (PEN, LPH2) and captopril (CAP, LCH2). The
observed rate constant (ko) values of the reactions though increase with increase in thiol
concentrations but linearly decrease with the increase in media [H+] and ionic strength, I. In
+
solution both PEN and CAP undergo several protolytic equilibria and exist in cationic (LPH3 ),
2-
2-
neutral (LPH2, LCH2) and anionic (LPH-, LP , LCH-, LC ) reactive forms. Detailed kinetic
+
analyses indicate that while for PEN, the order of reactivities run as LPH3 ~ LPH2 <<< LPH-, for
2-
CAP the order is LCH2 < LCH- <<< LC . While, for PEN, the predominant reactive species, LPH-
which possesses the reduced thiol (–SH) end preferably reacts through the concerted
electroprotic reaction pathway, for CAP, the species, LC2- with the deprotonated thiolate (–S-)
end reacts through the diffusion-controlled electron-transfer mechanism. The large kinetic
isotopic effect for PEN supports the electroprotic inner-sphere reaction mechanism. Overall, the
present study on the redox reactions of the thiol molecules, PEN and CAP offers us an insight
into the interplay of the protonic equilibrium between the potential reactive species in the way to
the determination of their overall redox abilities against the ROS species.
Acknowledgement
Authors thankfully acknowledge UGC UPE-II program of Jadavpur University for financial
support and Department of Chemistry, Bidhannagar College for allowing A. M. for
infrastructural facilities as when it was necessary.
Disclosure statement
No potential conflict of interest was reported by the authors.
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19

CH3
CH
O
C
CH3
SH
H3C
HS
O
C
C
H2
C
N
OH
O
HO
CH
NH2
D-Penicillamine (PEN)
C
Captopril (CAP)
Figure 1. Both PEN and CAP possess reduced thiol (–SH) group.
20

0.6
0.5
0.4
0.3
0.2
0.1
0.0
1
14
400
500
600
700
800
900
wavelength (nm)
Figure 2. Time resolved spectra of the reaction between 1 and PEN recorded at an interval of
30 s. Condition: [1] = 0.5 mM; [TPEN] = 10.0 mM; [H+] = 0.05 M; I = 0.5 M; T = 298.0 0.1 K.
21

20
15
10
5
0
0
5
10
15
20
25
30
103[TPEN] (M)
Figure 3. Variation of ko,P vs. [TPEN]. Condition: [1] = 0.5 mM; [H+] = 0.05 M; I = 0.5 M; T =
298.0 0.1 K.
22

8
7
6
5
4
3
2
K
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6
I (M)
Figure 4. Variation of ko,P vs. I. Condition: [1] = 0.5 mM; [TPEN] = 10.0 mM; [H+] = 0.05 M; T =
298.0 0.1 K.
23

40
35
30
25
20
15
10
5
0
0
20
40
60
80
100
[H+]-1 (M-1)
Figure 5. Variation of ko,P with [H+]-1. Condition: [1] = 0.5 mM; [TPEN] = 10.0 mM; I = 0.5 M; T
= 298.0 0.1 K.
24

8
7
6
5
4
3
2
k
0
20
40
60
80
100
%D2O (v/v)
Figure 6. Variation of ko,P with D2O content of the reaction media. Condition: [1] = 0.5 mM;
[TPEN] = 10.0 mM; [H+] = 0.05 M; I = 0.5 M; T = 298.0 0.1 K.
25

Table 1. Coefficients values derived from the plots of eq. 25 and eq. 26.
PEN
CAP
Rate constants and its
composites
Rate constants and its
composites
Value
Value
kP1 (M-1 s-1)
(4.2 0.7) × 10-2 kC1 (M-1 s-1)
(1.9 0.6) × 10-2 kC2KC1 (s-1)
(1.3 0.4) × 10-3 kC3KC1KC2 (M s-1)
–
(5.2 1.1) × 10-2
(1.4 0.2) × 10-2
(2.0 0.8) × 10-4
kP2KP1 (s-1)
kP3KP1KP2 (M s-1)
kP4KP1KP2KP3 (M2 s-1)
26

Graphical abstract
O
O
O
O
HO
SH
O
SH
O
SH
O
S
NH3
NH3
NH2
NH2
2-
pKP3
pKP2
pKP1
LPH-
+
LPH2
LP
LPH3
kP2
kP3
PEN
CAP
kP1
kP4
[(dien)2(en)2Co2(O2)]5+
Products
kC1
kC3
kC2
HS
O
HS
O
S
O
O
O
O
pKC2
N
pKC1
N
N
OH
O
O
LCH-
2-
LCH2
LC
27