A kinetic analysis of oxidation of the antioxidant N-acetyl-l-cysteine (NAC) by Pt(IV) complexes

Article abstract of DOI:10.1007/s11243-016-0021-z

N-acetyl-l-cysteine (NAC) is an antioxidant and a supplement and has been demonstrated to have protective effects for a variety of toxic effects of heavy metals. Although previous works have shown that NAC can ameliorate the severe toxic effects of cisplatin, there is a lack of understanding of the interactions between NAC and Pt(IV)-based prodrugs. In this work, the oxidation of NAC by a cisplatin prodrug (cis-[Pt(NH₃)₂Cl₄]), by a prototype of Pt(IV) anticancer drug ormaplatin ([Pt(dach)Cl₄]) and by a model compound (trans-[PtCl₂(CN)₄]^2-) was characterized in detail. NAC was oxidized to NAC-disulfide as identified by mass spectrometric analysis. Time-resolved spectral and stopped-flow kinetic measurements were carried out over a wide pH range, demonstrating that the oxidation followed overall second-order kinetics. The observed second-order rate constants k′ versus pH profiles were established. A reaction mechanism was deduced, involving three parallel rate-determining steps; conceivable transition states were also proposed for these steps. Rate constants of the rate-determining steps, obtained from the simulations of rate equation to the k′-pH profiles, were largely correlated with the electron density on the sulfur atom in NAC. The Pt(IV) prodrugs can execute oxidative stress in the biological systems of the human body by direct oxidation of relevant molecules, similar to HOCl/OCl^- and chloroamines. Instead, the oxidative stress involved in the severe toxic effects of cisplatin is produced via a different mode. NAC could be a chemoprotecting agent also for the Pt(IV) anticancer drugs if recent drug delivery technologies are used.

Full text of DOI:10.1007/s11243-016-0021-z

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Transition Met Chem (2016) 41:295–304
DOI 10.1007/s11243-016-0021-z
A kinetic analysis of oxidation of the antioxidant
N-acetyl-L-cysteine (NAC) by Pt(IV) complexes
Daofeng You1 Yanli Ren2 Shuying Huo2 Jingran Dong2 Shuguang Ren3
Tiesheng Shi2
•
•
•
•
•
Received: 21 December 2015 / Accepted: 8 January 2016 / Published online: 27 January 2016
Ó Springer International Publishing Switzerland 2016
Abstract N-acetyl-L-cysteine (NAC) is an antioxidant
and a supplement and has been demonstrated to have
protective effects for a variety of toxic effects of heavy
metals. Although previous works have shown that NAC
can ameliorate the severe toxic effects of cisplatin, there is
a lack of understanding of the interactions between NAC
and Pt(IV)-based prodrugs. In this work, the oxidation of
NAC by a cisplatin prodrug (cis-[Pt(NH3)2Cl4]), by a
prototype of Pt(IV) anticancer drug ormaplatin ([Pt
(dach)Cl4]) and by a model compound (trans-[PtCl2
(CN)4]2–) was characterized in detail. NAC was oxidized to
NAC-disulfide as identified by mass spectrometric analy-
sis. Time-resolved spectral and stopped-flow kinetic mea-
surements were carried out over a wide pH range,
demonstrating that the oxidation followed overall second-
order kinetics. The observed second-order rate constants k0
versus pH profiles were established. A reaction mechanism
was deduced, involving three parallel rate-determining
steps; conceivable transition states were also proposed for
these steps. Rate constants of the rate-determining steps,
obtained from the simulations of rate equation to the k0–pH
profiles, were largely correlated with the electron density
on the sulfur atom in NAC. The Pt(IV) prodrugs can exe-
cute oxidative stress in the biological systems of the human
body by direct oxidation of relevant molecules, similar to
HOCl/OCl- and chloroamines. Instead, the oxidative stress
involved in the severe toxic effects of cisplatin is produced
via a different mode. NAC could be a chemoprotecting
agent also for the Pt(IV) anticancer drugs if recent drug
delivery technologies are used.
Introduction
Electronic supplementary material The online version of this
article (doi:10.1007/s11243-016-0021-z) contains supplementary
material, which is available to authorized users.
N-Acetyl-L-cysteine (NAC) has been utilized as a supple-
ment for a long period of time as it can be bought over the
counter. It is one of the most frequently used antioxidant/
radical scavengers for experimental studies in tissue culture
and in vivo [1]. Moreover, NAC has been demonstrated to
have protective effects against a variety of toxic effects [1–
6], including a very high effectiveness in preventing toxic
effects of heavy metals [1–3]. As a matter of fact, it has
been employed as an antidote in cases of acute heavy metal
poisoning [7, 8]. Moreover, NAC has already been
approved for clinical use in humans, and it is essentially
safe when administrated via ingestion [1, 9].
& Shuguang Ren
rsg9801@126.com
& Tiesheng Shi
rock@hbu.edu.cn
1
The First Hospital of Hebei Medical University,
Shijiazhuang 050031, Hebei Province, People’s Republic of
China
2
College of Chemistry and Environmental Science, and the
MOE Key Laboratory of Medicinal Chemistry and Molecular
Diagnostics, Hebei University,
Baoding 071002, Hebei Province, People’s Republic of
China
On the other hand, platinum-based anticancer drugs are
still playing a central role among the chemotherapeutic
pharmaceuticals as exemplified by the fact that cisplatin
and carboplatin have been used worldwide [10, 11].
3
The Fourth Hospital of Hebei Medical University,
Shijiazhuang 050031, Hebei Province, People’s Republic of
China
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Transition Met Chem (2016) 41:295–304
Cisplatin was the first platinum anticancer drug and is still
the most used one. Despite its tremendous success, cis-
platin has several severe side effects including nephrotox-
icity, neurotoxicity, ototoxicity, myelotoxicity and
hemolytic anemia [12–17], with nephrotoxicity as the most
severe. Carboplatin has a similar curing spectrum as cis-
platin, but shows slightly less severe toxicities. Not sur-
prisingly, the design and synthesis of a new generation of
platinum-based anticancer agents including numerous
Pt(IV) compounds have been of a great interest with a goal
of minimizing the side effects [10, 11, 17–19]. Although
great efforts have been made, progress appears to be slow
due to a poor understanding of the anticancer and toxico-
logical mechanisms [10, 11, 17–19].
(trans-[PtCl2(CN)4]2–); their structures are given in
Scheme 1. In general, Pt(IV) compounds have an octahedral
configuration; substitution reactions on these compounds are
usually very slow. Comparatively, reductions of the Pt(IV)
compounds are fast; thus, Pt(IV) anticancer active com-
pounds are often regarded as prodrugs due to their ease of
reduction [11, 18, 19]. Ormaplatin was a prototype of the
Pt(IV) anticancer drugs developed earlier and was very
promising due to its property of oral administration. After
clinical trials, ormaplatin was abandoned because of its
severe nephrotoxicity [23]. cis-[Pt(NH3)2Cl4] is the prodrug
of cisplatin, while trans-[PtCl2(CN)4]2– has been utilized as a
model compound for the Pt(IV) prodrugs [24]. We herein
report our results.
Concurrent to the designing of a new generation of
platinum drugs is the development of some chemopro-
tecting agents which are used to maintain cisplatin/carbo-
platin anticancer efficacy while protecting against
chemotherapy side effects. Indeed, a few chemoprotecting
agents have been discovered including sodium thiosulfate,
amifostine, N-acetyl-L-cysteine (NAC), D-methionine,
L-selenomethionine, and ebselen [16, 20]. These chemo-
protecting agents are all antioxidants, showing some degree
of relief for the toxic effects of cisplatin and carboplatin
[16, 20–22].
Experimental
Materials
K2[Pt(CN)4]Á3H2O, cis-[Pt(NH3)2Cl4], and NAC were
obtained from Sigma-Aldrich (St. Louis, MO). Potassium
tetrachloroplatinate(II), (1R,2R)-(-)-1,2-diaminocyclohex-
ane (dach), acetic acid, sodium acetate, sodium chloride,
hydrochloric acid, sodium dihydrogenphosphate, disodium
hydrogenphosphate, sodium perchlorate were, all in ana-
lytical grade, purchased either from Fisher Scientific (in
Beijing, China) or from Alfa Aesar (in Tianjin, China) and
were used without further purification. K2[PtCl2(CN)4] was
synthesized using the method reported [25]; the UV–Vis
In view of the protective role of NAC for the Pt-based
drugs, a detailed kinetic analysis was undertaken in this work
on oxidation of NAC by three Pt(IV) compounds: ormaplatin
([Pt(dach)Cl4]), cis-diamminetetrachloroplatinum(IV) (cis-
[Pt(NH3)2Cl4]) and trans-dichlorotetracyanoplatinate(IV)
Scheme 1 Structures of the
Pt(IV) complexes, the fully
protonated form of N-acetyl-L-
cysteine (NAC) and of NAC-
disulfide
_
2
Cl
Cl
Cl
Cl
Cl
CN
CN
Cl
H3N
H3N
NH2
NC
NC
Pt
Pt
Pt
NH2
Cl
Cl
Cl
Cl
_
[Pt(dach)Cl4] (ormaplatin)
CH3COHN
cis-[Pt(NH3)2Cl4]
trans-[PtCl2(CN)4]2
CH3COHN
COOH
H
CH2S SCH2
COOH
CH2 SH
H
H
NHCOCH3
COOH
N-Acetyl-L-cysteine (NAC)
NAC-disulfide (RSSR, fully protonated form)
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Transition Met Chem (2016) 41:295–304
297
spectra of aqueous solutions prepared from K2[PtCl2(CN)4]
were in excellent agreement with that reported earlier for
trans-[PtCl2(CN)4]2– [26]. Ormaplatin (also called tetra-
platin, [Pt(dach)Cl4]) was from the same batch, synthesized
and characterized in a recently work [27].
employed for kinetic measurements; the kinetic traces were
simulated by the software provided by Applied Photo-
physics. Stock solutions of NAC (20–30 mM) were prepared
just before the kinetic measurements by dissolving a certain
amount NAC in buffers; the stock solutions were flushed
with nitrogen for 10 min and were only used for a couple of
hours. Solutions of NAC and of the Pt(IV) complexes for
kinetic measurements were prepared, respectively, by add-
ing an appropriate amount of the Pt(IV) stock solution and
NAC to a specific buffer. Those solutions were flushed for
10 min with nitrogen before loading onto the stopped-flow
machine. Reactions were started by mixing equal volumes of
NAC and platinum(IV) solutions directly in the stopped-flow
machine. Pseudo first-order conditions were fulfilled by
keeping NAC in at least tenfold excess.
Solutions
Stock solutions of 1.0 mM of [Pt(dach)Cl4], cis-[Pt(NH3)2-
Cl4] and trans-[PtCl2(CN)4]2– were prepared by dissolving
appropriate amounts of each of the Pt(IV) complexes in
solutions containing 0.90 M NaClO4, 0.09 M NaCl and
0.01 M HCl. These solutions were wrapped with aluminum
foil and were put in a refrigerator; the stock solutions of cis-
[Pt(NH3)2Cl4] and [Pt(dach)Cl4] were freshly prepared each
day. Buffers with combinations of acetic acid/sodium acetate,
NaH2PO4/Na2HPO4, and NaHCO3/Na2CO3 (0.2–0.3 M),
were prepared to cover a wide pH range (for pH[ 3). All the
buffer solutions contained 0.10 M of NaCl and 2.0 mM of
EDTA; sodium perchlorate was used to adjust the ion strength
(l) of buffers to 1.0 M. The addition of NaCl was to suppress
the hydrolysis of the platinum(IV) complexes. EDTA in the
buffers was to eliminate the catalytic effects in the autooxi-
dation of NAC caused by trace amounts of metal ions such as
Cu(II) and Fe(III) [28–30]. The pH values of buffer solutions
were measured with an Accumet Basic AB15 Plus pH meter
equipped with an Accumet combination pH electrode (Fisher
Scientific, Pittsburgh, PA). Standard buffers of pH 4.00, 7.00
and 10.00, also from Fisher Scientific, were used to calibrate
the electrode. For solutions with pH\ 3, hydrogen-ion con-
centrations [H?] were maintained by perchloric acid, while
the pH values were calculated by: pH = -log[H?] ? 0.11,
which is based on a mean activity coefficient of 0.77 for
1.00 MÁNaClO4 [31]. Doubly distilled water was used to
prepare all the solutions.
Mass spectra
For each of three Pt(IV) complexes, two reaction mixtures
were prepared and flushed by nitrogen gas: (A) 1 mM Pt(IV)
and 8 mM NAC in 10 mM HCl; and (B) 1 mM Pt(IV) and
2 mM NAC in 10 mM HCl. Low resolution mass spectra
were recorded on an Agilent 1200/6310 ion-trap mass
spectrometer with electrospray ionization (ESI); both posi-
tive and negative modes were employed for the reaction
mixtures prepared above from trans-[PtCl2(CN)4]2– and cis-
[Pt(NH3)2Cl4], respectively. High-resolution mass spectra
were recorded on an Apex Ultra 7.0T FT-ICR mass spec-
trometer (Bruker Daltonik, Germany) with an ESI of positive
mode for the reaction mixtures made from [Pt(dach)Cl4].
Results and discussion
Acquisition of kinetic data
Electronic and time-resolved spectra
In order to gain some general information about the oxi-
dation processes, time-resolved spectra were recorded for
each of the Pt(IV) compounds. Figure 1A shows the
spectra for the oxidation of NAC by [Pt(dach)Cl4] under a
set of reaction conditions; apparently, the weak absorption
band around 265 nm did not shift during the reaction
process. Further, the absorbance at 265 nm as a function of
time was simulated by a single exponential, namely
Eq. (1), where kobsd pertains to the observed first-order rate
constants, and At, A0, and A? stand for the absorbance at
time t, zero and infinity, respectively.
Electronic spectra for the Pt(IV) complexes in aqueous
solution and the time-resolved spectra for some of the
reaction courses between the Pt(IV) compounds and NAC
were recorded on a TU-1900 spectrophotometer (Beijing
Puxi, Inc., Beijing, China) using 1.00 cm quartz cells. The
spectrophotometer had a cell compartment, which were kept
at 25.0 0.2 °C by circulating water from a thermostat
(BG-chiller E10, Beijing Biotech Inc., Beijing). For the time-
resolved spectra, both the Pt(IV) and NAC solutions in the
same pH buffer were pre-thermostated at 25.0 0.2 °C.
At ¼ ðA0ÀA1Þ expðÀkobsdtÞ þ A1
At ¼ A0 þ ðA1ÀA0Þ½1 À expðÀkobsdtފ
ð1Þ
ð2Þ
Kinetic experiments
The simulation generated a good fit shown in Fig. 1B.
An Applied Photophysics SX-20 stopped-flow spectrometer
(Applied Photophysics Ltd., Leatherhead, U.K.) was
Time-resolved spectra for the oxidation of NAC by cis-
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Transition Met Chem (2016) 41:295–304
and 285.7 nm are discernible in Fig. 2; an enlarged scale at
the latter point is given in Fig. 2 as an insert. Moreover, the
kinetic trace obtained at 255 nm could also be well simu-
lated by a single exponential equation, i.e. Eq. (2) [32],
where the absorbance increased. Hence, the NAC oxidation
processes by all the Pt(IV) compounds were indeed first-
order in [Pt(IV)]. The values of kobsd reported in this work
were the averages from 3 to 7 duplicate runs; standard
deviations were usually \5 %.
Rate equation
The impact of varying [NAC] on the oxidation rate was
studied in the region 0.20 mM B [NAC] B 6.00 mM
depending on the buffer capacity. The experiments were
designed to warrant that the [NAC] variations did not cause
any pH changes of the reaction medium. The kobsd versus
[NAC] relations are shown in Figs. 3, 4 and 5 for reactions
of trans-[PtCl2(CN)4]2–, cis-[Pt(NH3)2Cl4], and [Pt(dach)Cl4],
respectively. Clearly, the plots ofkobsd versus [NAC] are linear
without any significant intercepts, indicating that the oxidation
reactions are also first order in [NAC]. Hence, the oxidation
reactions all follow an overall second-order rate law expressed
by Eq. (3a, b), where k0 denotes observed second-order rate
constants and [Pt(IV)] = [Pt(dach)Cl4]/cis-[Pt(NH3)2Cl4].
Àd½PtðIVފ=dt ¼ kobsd½PtðIVފ ¼ k0½NACŠ½PtðIVފ
ð3aÞ
or
Fig. 1 A Time-resolved spectra for reaction between [Pt(dach)Cl4]
and NAC under the reaction conditions: [Pt(IV)] = 0.08 mM,
[NAC] = 2.00 mM, [Cl-] = 0.10 M, pH = 4.51 acetic acid/acetate
buffer, l = 1.0 M and 25.0 °C. The first spectrum was obtained ca.
10 s after reaction and time between scans was 30 s. B Kinetic trace
at 265 nm; the data points are from the absorbance readings in
Fig. 1A. The solid curve was obtained by fitting Eq. (1) to the
experimental data by use of a nonlinear least-squares method
[Pt(NH3)2Cl4] are given in Figure S1A in the Supporting
Information. The absorbance changes were simulated
according to Eq. (1) at the two absorption bands 225 and
270 nm and are shown in Figure S1B in the Supporting
Information. Clearly, the simulations afforded good fits and
moreover, the values of kobsd derived at these two wave-
lengths are identical within the experimental errors. These
attributes suggested that the overall spectral changes
observed in Figure S1A originated from a single reaction
process.
Time-resolved spectra for the NAC oxidation by trans-
[PtCl2(CN)4]2– are presented in Fig. 2; the emergence of an
absorption band around 255 nm and of a weak band around
279 nm was attributed to the formation of [Pt(CN)4]2– as a
reaction product. Two clear-cut isosbestic points at 242.7
Fig. 2 Time-resolved spectra for reaction between trans-[PtCl2
(CN)4]2- and NAC under the reaction conditions: [Pt(IV)] =
0.10 mM, [NAC] = 1.00 mM, [H?] = 0.010 M, [Cl-] = 0.10 M,
l = 1.0 M and 25.0 °C. The first spectrum was obtained ca. 10 s
after reaction and time between scans was 30 s. Insert an expended
scale around the isosbestic point of 285.7 nm
123