Kinetic and thermodynamic characterization of camptothecin hydrolysis at physiological pH in the absence and presence of human serum albumin

Article abstract of DOI:10.1002/kin.20449

To accurately derive the kinetic and thermodynamic parameters governing the hydrolysis of the lactone ring at physiological pH, a derivative spectrophotometric technique was used for the simultaneous estimation of lactone and carboxylate forms of camptoth

Full text of DOI:10.1002/kin.20449

Kinetic and Thermodynamic
Characterization of
Camptothecin Hydrolysis
at Physiological pH in the
Absence and Presence
of Human Serum Albumin
RISHI THAKUR, SASANK KUNADHARAJU, MICHALAKIS SAVVA
Division of Pharmaceutical Sciences, Arnold & Marie Schwartz College of Pharmacy & Health Sciences,
Long Island University, Brooklyn, NY 11201
Received 3 February 2009; revised 26 April 2009; accepted 22 May 2009
DOI 10.1002/kin.20449
Published online in Wiley InterScience (www.interscience.wiley.com).
ABSTRACT: To accurately derive the kinetic and thermodynamic parameters governing the
hydrolysis of the lactone ring at physiological pH, a derivative spectrophotometric technique
was used for the simultaneous estimation of lactone and carboxylate forms of camptothecin
(CPT). The hydrolysis of the CPT-lactone and the lactonization of CPT-carboxylate at 310.15 K
followed a first-order decay with apparent rate constants equal to 0.0279 0.0016 min−1 and
0.0282 0.0024 min−1, respectively. The activation energy associated with the hydrolysis of
the CPT-lactone and the lactonization of the CPT-carboxylate as calculated from the Arrhenius
equation was 89.18 0.84 and 86.49 2.7 kJ mol−1, respectively. The enthalpy and entropy
of the thermodynamically favored hydrolysis reaction were on average 10.49 kJ mol−1 and
48.00 J K−1 mol−1, respectively. The positive enthalpy and entropy values of the CPT-lactone
hydrolysis indicate that the reaction is endothermic and entropically driven. The stability of CPT-
lactone in the presence of human serum albumin (HSA) was also analyzed. Notwithstanding the
much faster hydrolysis of the CPT-lactone in the presence of HSA at various temperatures, the
energy of activation was determined to be similar to the one estimated in the absence of HSA,
suggesting that HSA does not catalyze the hydrolysis reaction, but it merely binds, sequesters,
and stabilizes the CPT-carboxylate species. ꢀ 2009 Wiley Periodicals, Inc. Int J Chem Kinet 41:
C
704–715, 2009
INTRODUCTION
Fifty years have past since the discovery of the topoiso-
merase I inhibitor, camptothecin (CPT) was publicly
announced, and still its fast hydrolysis in the blood
Correspondence to: Michalakis Savva; e-mail: msavva@liu.edu.
2009 Wiley Periodicals, Inc.
c
ꢀ

KINETIC AND THERMODYNAMIC STUDIES OF CPT HYDROLYSIS
705
(a)
H+
OH
O
+
O
O
H2O
+
O
OH
OH
CPT-lactone
CPT-carboxylate
(b)
Figure 1 (a) Chemical structure of CPT. (b) The reversible hydrolysis reaction of CPT-lactone (C20H16N2O4) to CPT-
carboxylate (C20H17N2O5). The forward direction of the reaction is shown from left to right. The reverse direction of the
reaction is the thermodynamically unfavorable esterification of the CPT-carboxylate species at pH 7.4.
precludes its efficient delivery [1–4]. The α-hydroxy
δ-lactone moiety of CPT (Fig. 1), which is structurally
important for the drug interaction with topoisomerase
I and passive diffusion of CPT into cancer cells, un-
dergoes a pH-dependent reversible hydrolysis at and
above pH 5 to a ring-opened carboxylate form. Thus,
the hydrolysis product of CPT, referred herein as CPT-
carboxylate, is quickly produced in the blood or in
aqueous media of neutral pH and is reported to be of
reduced activity, bioavailability, and the cause of toxic
side effects in human trials [5–8].
The hydrolysis kinetics of the CPT and its analogues
at physiological pH and at ambient temperature have
been reported in many publications, but the thermo-
dynamic parameters governing this important hydrol-
ysis reaction have not yet been reported [6–10] except
for the 10-hydroxy camptothecin, which we have re-
cently completed [11]. This investigation is a continu-
ation of our recent study highlighting the superiority of
first derivative spectroscopy, a real-time nonperturbing
method developed in our laboratory for the simulta-
neous determination of nonequilibrium concentrations
of CPT, lactone (CPT-lactone) and carboxylate (CPT-
carboxylate) species at various temperatures. The tech-
nique was also very useful in assessing the stability of
CPT in the presence of human serum albumin (HSA),
thus demonstrating the potential of this technique in
CPTdeliverysystemselection. Thekineticand thermo-
dynamic analysis of the data presented herein has re-
vealed new information regarding the stability of CPT
in the presence and absence of HSA.
MATERIALS AND METHODS
Explanation of Terms
The terms CPT, CPT-lactone, and lactone are used
interchangeably throughout the article and denote
the native lactone form of the CPT. The term CPT-
carboxylate denotes the hydrolysis product of the
native CPT-lactone molecule. Moreover, the term for-
ward direction of the reversible hydrolysis reaction de-
notes the hydrolysis of the CPT-lactone and has as reac-
tants and products CPT-lactone and CPT-carboxylate,
respectively. The term reverse direction of the hydrol-
ysis reaction denotes the esterification or lactonization
of CPT-carboxylate and has as reactants and products
CPT-carboxylate and CPT-lactone, respectively. The
term lactonization reaction is used when the starting
material is CPT-carboxylate. The forward direction of
the lactonization reaction denotes the lactonization of
the CPT-carboxylate and has as reactants and prod-
ucts CPT-carboxylate and CPT-lactone, respectively,
whereas the term reverse direction of the lactoniza-
tion reaction denotes the hydrolysis of CPT-lactone.
International Journal of Chemical Kinetics DOI 10.1002/kin

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THAKUR, KUNADHARAJU, AND SAVVA
by diluting the respective stocks in 3 cm3 quartz cu-
vettes to give a final concentration of 8.612 × 10−7 M.
First-order derivatives of the spectral excitation scans
Finally, kinetic experiments performed with CPT-
lactone as the starting material are referred to as hydrol-
ysis reaction, whereas measurements collected with
CPT-carboxylate as the starting material are referred
to as lactonization or esterification reaction, albeit lac-
tone hydrolysis is the spontaneous or thermodynami-
cally favorable reaction.
were used to determine the zero-crossing points (λZCP
)
of pure CPT-lactone and CPT-carboxylate at 363 and
369 nm, respectively (Fig. 2). In the presence of HSA,
a bathochromic shift of the zero-crossing points was
observed at 369 and 379 nm, respectively (not shown).
Materials
Kinetic Investigation of the Hydrolysis
and Lactonization Reactions
The 20(S)-(+)-camptothecin (4-ethyl-4-hydroxy-
1H-pyrano-(3ꢁ,4ꢁ:6,7) indolizino (1,2-b)quinoline-
3,14-(4H,12H)-dione (CAS Number 7689-03-4),
C20H16N2O4, 348.352 g mol−1, stated mass fraction
purity ≥0.99 was purchased from LC Laboratories
(Woburn, MA). In close agreement with the vendor’s
stated purity, our elemental analysis of CPT-lactone
yielded a mass fraction purity of 0.99 (Calcd: C,
68.96; N, 8.04; H, 4.63. Found: C, 68.87; N, 8.20; H,
4.58; Robertson Microlit Laboratories, Madison, NJ).
In addition, HPLC analysis of the CPT using fluores-
cence detector did not show any presence of related
impurities (data not shown). All experiments were
performed using analytical-grade reagents without
further purification. Deionized water for preparation
of buffer solutions was obtained from Barnstead
NANO pure water system (Barnstead, Dubuque, IA).
Dimethyl sulfoxide (DMSO) and sodium hydroxide
(1 N) were purchased from Sigma-Aldrich (St. Louis,
MO). The CPT-lactone stock solutions were prepared
in DMSO at 2.871 × 10−4 M concentration. The
CPT-carboxylate stock solutions were prepared in
Kinetic measurements were performed by approaching
the equilibrium from both directions of the reaction, as
described elsewhere [11]. Briefly, to determine the re-
action kinetics of the hydrolysis reaction, the buffer
solution was preequilibrated to the desired tempera-
ture and an aliquot of CPT-lactone from the DMSO
stock solution was added to give the final concentra-
tion of 8.612 × 10−7 M. The excitation scans were
collected immediately and at different time intervals
(1.0–5.0 min), thereafter, depending on the rate of the
reaction. The percent CPT-lactone remaining at each
time interval was determined at the λZCP of the CPT-
carboxylate and vice versa. The stability and hydrol-
ysis kinetics of CPT-lactone in the presence of HSA
(3.008 × 10−4 M) in PBS (pH 7.4) at narrow tempera-
ture range (298.15–314.25 K) were analyzed similarly.
The reaction kinetics of the lactonization reaction was
also monitored, starting with 100% CPT-carboxylate
at zero time.
0.001 N NaOH at concentration of 5.741 × 10−5
M
Kinetic Parameters
from the CPT-lactone stock solutions in DMSO
and equilibrated for 1 h at room temperature to
ensure complete hydrolysis (HPLC analysis, data
not shown). PBS (0.137 M NaCl, 0.0027 M KCl,
0.010 M Na2HPO4, 0.002 M KH2PO4) was used to
maintain the pH at 7.4. HSA, 66478 Da, CAS Number:
70024-90-7, fraction V, stated mass fraction purity
≥0.96 was purchased from Sigma-Aldrich.
The exponential decay of CPT-lactone versus time was
fitted using Eq. (1) by the method of nonlinear least
squares with [CPT-lactone]eq, [CPT-carboxylate]eq,
and kobs being the adjustable parameters.
[CPT-lactone] = [CPT-lactone]eq
obs·t
+ [CPT-carboxylate]eq · e−k
(1)
The [CPT-lactone], [CPT-lactone]eq, and [CPT-
carboxylate]eq are the concentrations of CPT-lactone
at time t, the CPT-lactone at equilibrium, and the CPT-
carboxylate at equilibrium, respectively, whereas kobs
is the overall or the observed first-order rate constant
of the hydrolysis reaction.
Fluorescence Measurements
Zero-time excitation scans of CPT-lactone and car-
boxylate were recorded within 20 s of stock dilu-
tion and used as reference spectra of the pure CPT-
lactone or pure CPT-carboxylate (Fig. 1b), on a Cary
Eclipse fluorescence spectrophotometer (Varian Inc.,
Victoria, Australia), as described elsewhere [11]. The
effect of DMSO on the spectral properties of lac-
tone was safely neglected as the volume fraction of
DMSO was less than 1%. Scans of either pure CPT-
lactone or CPT-carboxylate were collected at pH 7.4
It is to be noted that
kobs = kf + kr
(2)
where kf and kr are the forward or hydrolysis and the
reverse or lactonization rate constants, respectively, of
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KINETIC AND THERMODYNAMIC STUDIES OF CPT HYDROLYSIS
707
600
500
400
300
200
100
0
15
10
5
0
330
340
350
360
370
380
390
400
410
Wavelength / nm
Figure 2 Representative excitation scans of CPT-lactone (——) and CPT-carboxylate (——) and their respective first derivative
scans (thick and thin dashed lines) at temperature and pH of 304.15 K and 7.4, respectively. The primary y-axis represents
the intensity (I), and the secondary y-axis represents (dI/dλ) plotted against wavelength. The zero-crossing point λ(ZCP) is
defined as the wavelength at which the first derivative of the spectra is zero. The concentration of the CPT-lactone and the
CPT-carboxylate was kept constant at 8.612 × 10−7 M.
Recognize that both kf and krꢁ denote the true hy-
drolysis rate constant of the CPT-lactone calculated
by monitoring the kinetics of the reversible hydroly-
sis and lactonization reactions, respectively, whereas
kr and kfꢁ denote the true lactonization rate constant of
the CPT-carboxylate.
Given a pKa of the carboxylate species ∼4, the
[CPT-carboxylate]eq is present almost exclusively in
its ionized form at pH 7.4 [12]. The concentration
of H2O was not used in the calculation of the appar-
ent equilibrium constant. The values of Khꢁ and kobs
were determined by direct nonlinear fitting of the ex-
perimental data by Eq. (1) using the Microsoft Excel
Solver through minimization of the sum of the squared
residuals at a 5% tolerance level. Subsequently, the rate
constants kf and kr were calculated by solving Eqs. (2)
and (3).
the hydrolysis reaction. Since the reaction being stud-
ied is a reversible reaction with both reactants and
products being present at equilibrium, the apparent
equilibrium constant of the hydrolysis reaction Khꢁ can
be defined as
kf
kr
[CPT-carboxylate]eq
[CPT-lactone]eq
Khꢁ =
=
(3)
where [CPT-lactone]eq and [CPT-carboxylate]eq are the
measured molar concentrations of CPT-lactone and
CPT-carboxylate, respectively, at equilibrium. Simi-
larly, the apparent chemical equilibrium constant of
the lactonization reaction Klꢁ is defined as
kfꢁ
1
Khꢁ
[CPT-lactone]eq
Klꢁ =
=
=
(4)
krꢁ
[CPT-carboxylate]eq
where kfꢁ and krꢁ are the forward or lactonization and
the reverse or hydrolysis rate constants, respectively, of
the lactonization reaction. The overall or observed rate
of the lactonization reaction koꢁ bs is equal to kfꢁ + krꢁ .
Thermodynamic Parameters
The temperature dependence of the rate constants
was employed to calculate the activation energy by
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THAKUR, KUNADHARAJU, AND SAVVA
using the integrated form of the Arrhenius equation,
grated fluorescence peak area against absorbance, and
refractive index. The refractive index of 0.1 M H2SO4
(η = 1.3335), PBS (η = 1.3340), and PBS + HSA
(η = 1.3370) were measured by a Bausch & Lomb
refractometer (type 33.44.68). Plots of integrated
fluorescence against the corresponding absorbance
were linear with a correlation coefficient greater
than 0.99.
k = A · e−(E /RT ), where A is the frequency factor
a
of the reaction. The rate constant k was determined
at five different temperatures ranging from 304.15 to
329.15 K. Plots of ln k against the reciprocal of the
corresponding absolute temperature were linear with
a correlation coefficient greater than 0.998. From the
slope and intercept of the linear fit, the activation en-
ergy and preexponential factor of CPT-lactone hydrol-
ysis in the absence and presence of HSA and esterifi-
cation of CPT-carboxylate were calculated.
RESULTS AND DISCUSSION
The standard Gibbs free energy of the reaction was
calculated from ꢀr Gꢁ◦(T ) = −RT · ln{Kꢁ(T )}, where
Kꢁ is the apparent chemical equilibrium constant of
either the hydrolysis or the lactonization reaction. To
verify the reliability of the thermodynamic parameters,
the absolute values of ꢀr Gꢁ◦(T ) for the hydrolysis and
lactonization reactions were compared and the differ-
ence was found to be statistically insignificant (95%
confidence interval). The standard enthalpy and en-
tropy of the reaction were determined from the slope
and intercept of the integrated form of the Van’t Hoff
equation, which was used to model the data:
The first derivative spectroscopy using the zero-
crossing technique was recently proved to be a
powerful tool to resolve the overlapping spectra
of 10-hydroxy-CPT-lactone and 10-hydroxy-CPT-
carboxylate for both qualitative and quantitative anal-
ysis [11]. In addition to the continuous monitoring ca-
pabilities of this method, unlike other chromatography
methods that require protein precipitation prior to anal-
ysis, the current method allowed real-time monitoring
of CPT hydrolysis in the presence of HSA.
The real-time monitoring of CPT-lactone hydroly-
sis of CPT (Figs. 3a and 3b) pointed out that, first,
the CPT-lactone hydrolysis rate increases exponen-
tially with temperature, second, the almost perfect fit
of the raw data using Eq. (1) signifies a unimolecular
reaction and, third and most importantly, being a symp-
tom of temperature-dependent lactone destabilization,
the apparent equilibrium constant Khꢁ of the hydrolysis
reaction increases with temperature.
The energy of activation Ea and the frequency factor
A were determined from the slope and intercept of the
corresponding linear fits of the Arrhenius plots shown
in Fig. 3b. As Table I indicates, the average Ea for the
hydrolysis reaction is 90.58 kJ mol−1 and is somewhat
higher than the energy barrier of the lactonization reac-
tion (81.12 kJ mol−1). The higher energy of activation
of the hydrolysis reaction is due to a much faster in-
crease of the kf with temperature as compared to the
kr . More specifically, kf is only 5.05 times larger than
kr at T = 304.15 K as compared to being 6.62 times
higher at T = 329.15 K (Table II).
1
R ln{Kꢁ(T )} = ꢀr Sꢁ◦(T ) − ꢀr Hꢁ◦(T ) ·
(5)
T
Quantum Yield Study
Fluorescence quantum yield of CPT-lactone and CPT-
carboxylate in the presence and absence of 3.008 ×
10−4 M HSA in PBS buffer (pH 7.4) at 298.15 K was
determined using quinine sulfate in 0.1 M H2SO4 as
the reference with a quantum yield of φ = 0.545 [13].
UV–vis absorbance spectra of dilute solutions (ab-
sorbance <0.05) of the two molecules were collected
in reference to the corresponding solvents upon exci-
tation at 350 nm on a Cary 50 UV spectrophotometer
(Varian, Inc., Palo Alto, CA), whereas the corrected flu-
orescence spectra were recorded in the wavelength re-
gion of 371–600 nm using a Cary Eclipse fluorescence
spectrophotometer. The 350-nm absorbances and
the areas under the respective emission spectra of qui-
nine sulfate and CPT-lactone and carboxylate species
in the absence and presence of HSA were used to
calculate the quantum efficiency (φ) of CPT species
by Parker’s method [14]:
Although the energy of activation for the hydroly-
sis and lactonization reactions is similar, the frequency
factor A, which denotes the fraction of molecules col-
liding in the right orientation, is on average 225 times
higher for the forward direction when compared to
the reverse direction of the hydrolysis reaction and
∼360 times higher for the reverse direction of the lac-
tonization reaction. These results are very important
for the following reasons: First, the very high values of
the temperature-independent frequency factor are sup-
portive of the unimolecular nature of the CPT-lactone
ꢀ
ꢁ ꢀ
ꢁ
φs · mu
ηu2
ηs2
φu =
(6)
ms
where s and u represent the standard and unknown
samples, whereas φ, m, and η are, respectively, the
quantum yield, slope as obtained from the plots of inte-
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KINETIC AND THERMODYNAMIC STUDIES OF CPT HYDROLYSIS
709
100
80
60
40
20
0
0
50
100
150
200
(a)
t/min
0.003
0.00306
0.00312
0.00318
0.00324
0.0033
(b)
K/T
Figure 3 (a) Hydrolysis reaction kinetics of CPT-lactone at pH 7.4 and various temperatures: 304.15 K (ꢀ), 310.15 K (×|),
ꢁ
318.15 K ( ), 323.15 K (×), and 329.15 K ( ). Solid lines refer to the simulated hydrolysis kinetics profile with fitting
ꢀ
parameters derived as described in the methods section. (b) Linear fits of Arrhenius plots of ln kf (ꢂ) and ln k ( ) as a function
ꢃ
r
of the inverse of the temperature.
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THAKUR, KUNADHARAJU, AND SAVVA
Table I Activation Energy and Preexponential Factor for the Hydrolysis and Lactonization Reactions for CPT-Lactone
and CPT-Carboxylate
Hydrolysis Reaction
Ea (kJ mol−1 1011 A (min−1
89.18 0.84
90.58 0.96
81.12 0.37
Lactonization Reaction
Ea (kJ mol−1 1010 A (min−1
)
Reaction Direction
)
)
)
Overall
Forward
Reverse
320.5 107
471.1 179
2.10 0.2
86.49 2.7
76.61 3.7
88.09 2.6
149.9 135
0.58 0.4
232.9 207
Energy of activation (Ea) and frequency factor (A) are reported as the mean standard deviation of three independent experiments.
Overall, forward, and reverse directions are defined by the kobs and koꢁ bs, kf and kfꢁ and kr and krꢁ , respectively.
Table II First-Order Rate Constants Reported as the Mean SD of Three Experiments for Hydrolysis and
Lactonization Reactions
Hydrolysis Reaction
kf (min−1
Lactonization Reaction
krꢁ (min−1 kfꢁ (min−1
)
T (K)
kobs (min−1
)
)
kr (min−1
)
koꢁ bs (min−1
)
)
304.15 0.0151 0.0005 0.0126 0.0004 0.00250 0.0001 0.0157 0.0003 0.0132 0.0003 0.00250 0.0001
310.15 0.0279 0.0016 0.0235 0.0013 0.00440 0.0003 0.0282 0.0024 0.0239 0.0021 0.00430 0.0005
318.15 0.0745 0.0073 0.0640 0.0067
0.0105 0.0007 0.0650 0.0026 0.0558 0.0020 0.00920 0.0006
323.15
329.15
0.117 0.0044
0.213 0.013
0.101 0.0042
0.185 0.012
0.0160 0.0005
0.0280 0.0014
0.117 0.0060
0.204 0.012
0.102 0.0056
0.179 0.0089
0.0150 0.0013
0.0246 0.0032
hydrolysis since orientation of the colliding molecules
does not play a critical role. Second, the much higher
frequency factor of the hydrolysis as compared to that
of the lactonization reaction, coupled to the similar
energetic barrier characterizing both reactions, sug-
gests that the greater flexibility of the E-ring of the
CPT-carboxylate is the reason for the less efficient lac-
tonization of the hydrolyzed CPT.
cates that hydrolysis of CPT-lactone is favorable at
higher temperatures. The enthalpy, ꢀr Hꢁ◦(T ), and en-
tropy, ꢀr Sꢁ◦(T ), associated with the hydrolysis reac-
tion of CPT-lactone (9.90 0.82 kJ mol−1 and 43.30
1.9 J K−1 mol−1, respectively) and the esterification
of CPT-carboxylate (−11.55
1.4 kJ mol−1 and
−51.56
4.9 J K−1 mol−1, respectively) were de-
termined from the slope and intercept of the linear
fits of the plots of ln Kꢁ versus 1/T (Fig. 5). The de-
tailed thermodynamic parameters are summarized in
Table III. The positive value of enthalpy signified the
endothermic nature of the hydrolysis. Interestingly, the
positive entropy factor (T ꢀr Sꢁ◦(T )) overcompensated
the unfavorable enthalpic part for the hydrolysis re-
action, yielding a negative ꢀr Gꢁ◦(T ). In contrast, the
chemical equilibrium constant Klꢁ for the lactoniza-
tion reaction decreased with increasing temperatures,
indicative of an exothermic reaction, but yet not spon-
taneous due to a large negative ꢀr Sꢁ◦(T ), as reflected
in the positive values of ꢀr Gꢁ◦(T ). The similarity of
these results with the published hydrolysis kinetics re-
sults of 10-hydroxy camptothecin strongly advocates
that A-ring substitutions do not affect the stability of
the E-ring [11,12].
These conclusions are also supported by Fig. 4,
which depicts the kinetics of the esterification reac-
tion with the CPT-carboxylate as the starting material.
As expected, at lower temperatures the esterification
reaction slows down, while in agreement with Fig. 3a,
lactonization is favored at lower temperatures. Fur-
thermore, as indicated in Table I, the Ea for the reverse
direction of the hydrolysis reaction is slightly higher
than that of the forward direction of the esterification
reaction (81.12
0.37 vs. 76.61
3.7 kJ mol−1).
The small differences in the values calculated from the
two experiments could be due to the small volume of
DMSO used only during the study of the hydrolysis
reaction as compared to the pure buffer solution used
during the study of the lactonization reaction.
Values of the standard Gibbs free energy associ-
ated with the hydrolysis of CPT-lactone, calculated
from ꢀr Gꢁ◦(T ) = −RT · ln {Kꢁ(T )}, are tabulated in
Table III. The negative ꢀr Gꢁ◦(T ) values indicate that
the hydrolysis reaction is the thermodynamically sta-
ble reaction at pH 7.4. In addition, the decrease in
ꢀr Gꢁ◦(T ) values with increasing temperatures indi-
The stability of CPT in the presence of 3.008 ×
10−4 M HSA deteriorates as indicated by the shorter
half-life (t1/2) and reduced levels of equilibrium CPT-
lactone concentration. More specifically, the t1/2 and
percent [CPT-lactone]eq were found to be 13.1 min
and 1.4%, respectively, as compared to 24.9 min and
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98
95
92
89
86
83
80
0
50
100
150
200
(a)
t/min
0.003
0.00306
0.00312
0.00318
0.00324
0.0033
(b)
K/T
Figure 4 (a) Lactonization reaction kinetics of CPT-carboxylate at pH 7.4 and temperatures: 304.15 K (ꢀ), 310.15 K (×|),
ꢁ
318.15 K ( ), 323.15 K (×), and 329.15 K ( ). Solid lines refer to the simulated lactonization kinetics profile with fitting
ꢀ
parameters derived as described in the methods section. (b) Linear fits of Arrhenius plots of ln kfꢁ (ꢂ) and ln kꢁ ( ) as a function
ꢃ
r
of the inverse of the temperature.
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THAKUR, KUNADHARAJU, AND SAVVA
Table III Thermodynamic Parameters for the CPT-Lactone Hydrolysis, CPT-Carboxylate Lactonization, and Combined
Average Values of the Thermodynamically Favored Hydrolysis Reaction
Hydrolysis reaction
T (K)
% [CPT-lactone]eq
% [CPT-carboxylate]eq
Khꢁ
ꢀr Gꢁ◦(T ) (kJ mol−1
)
304.15
310.15
318.15
323.15
329.15
16.53
15.73
14.14
13.61
13.13
83.47
84.27
85.86
86.39
86.87
5.052 0.039
5.367 0.24
6.090 0.44
6.355 0.25
6.618 0.15
−4.096 0.024
−4.330 0.14
−4.774 0.20
−4.966 0.13
−5.171 0.075
ꢀr Hꢁ◦ = 9.900 0.82 kJ mol−1
ꢀr Sꢁ◦ = 43.30 1.9 J K−1 mol−1
Lactonization reaction
% [CPT-carboxylate]eq
T (K)
% [CPT-lactone]eq
Klꢁ
ꢀr Gꢁ◦(T )/(kJ mol−1
)
304.15
310.15
318.15
323.15
329.15
16.03
15.32
14.18
12.81
12.04
83.97
84.68
85.82
87.19
87.96
0.191 0.008
0.181 0.017
0.165 0.006
0.147 0.014
0.137 0.013
4.188 0.11
4.413 0.24
4.762 0.094
5.159 0.26
5.450 0.26
ꢀr Hꢁ◦ = −11.55 1.4 kJ mol−1
ꢀr Sꢁ◦ = −51.56 4.9 J K−1 mol−1
Combined average parameters for the thermodynamically favored hydrolysis reaction
T (K)
Khꢁ
ꢀr Gꢁ◦ (T ) (kJ mol−1
)
304.15
310.15
318.15
323.15
329.15
5.147 0.18
5.460 0.38
6.072 0.31
6.599 0.52
6.983 0.61
−4.142 0.08
−4.372 0.18
−4.768 0.14
−5.063 0.21
−5.310 0.23
ꢀr Hꢁ◦ = 10.49 1.6 kJ mol−1
ꢀr Sꢁ◦ = 48.00 5.4 J K−1 mol−1
Average apparent equilibrium constant at the corresponding temperature calculated at the mean of Kꢁ (forward direction) and 1/Kꢁ (reverse
direction).
15.7% in the absence of HSA, at pH 7.4 and 310.15 K
To further investigate the mechanism of hydrolysis
of CPT in the presence of HSA, we have carried out
quantum yield measurements of the two species in the
presence and absence of HSA under physiological pH
(Fig. 6). Furthermore, unlike the hydrolysis of CPT-
lactone alone, in the presence of HSA the equilibrium
lactone concentration does not change with tempera-
ture (Fig. 7a). Theactivationenergy(Ea) andfrequency
factor (A) of CPT-lactone hydrolysis in the presence
of HSA were determined from the dependence of rate
constants (k) on temperature using the Arrhenius equa-
tion (Fig. 7b and Table IV). Interestingly, the Ea of
CPT-lactone hydrolysis in the presence of HSA was
found to be similar to that of the hydrolysis reaction
of CPT-lactone alone at pH 7.4. The equilibrium of
the hydrolysis reaction completely shifted toward the
CPT-carboxylate with a loss of the reversibility of the
reaction. The kobs and equilibrium CPT concentrations
at 310.15 K are in close agreement with previously
reported kinetic analysis using HPLC as the analytical
method [7–8,15–17].
Table IV First-Order Rate Constants and Energy of
Activation for Hydrolysis of CPT in the Presence of HSA,
Reported as the Mean SD of Two Independent
Experiments
T (K)
kobs (min−1
)
% [CPT-carboxylate]eq
298.15
302.15
306.15
310.15
314.15
0.0135 0.0001
0.0209 0.0013
0.0311 0.0004
0.0526 0.0010
0.0673 0.0027
99.8 0.23
99.7 0.46
98.3 0.20
98.6 0.12
98.9 0.54
Ea = 80.66 0.39 kJ mol−1
.
A = (1.85 0.33) 1012 min−1
.
International Journal of Chemical Kinetics DOI 10.1002/kin

KINETIC AND THERMODYNAMIC STUDIES OF CPT HYDROLYSIS
713
2
1
1
0.00300
0.00305
0.00310
0.00315
0.00320
0.00325
0.00330
K/T
Figure 5 Van’t Hoff plots depicting the temperature dependence of the apparent equilibrium hydrolysis constant Kꢁ ( ) and
◦
apparent equilibrium lactonization constant Klꢁ (ꢀ). The relationship between ln Kꢁ and (T /K)−1 was linear with r2h> 0.985.
ꢀr Hꢁ◦(T ) and ꢀr Sꢁ◦(T ) were calculated from the slope and intercept values of Eq. (5) as described in the methods section.
[Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]
100
80
60
40
20
0
0
50
100
150
200
t/min
Figure 6 Hydrolysis kinetics of CPT-lactone at pH 7.4 and temperature 310.15 K in the presence (—) or absence (∗) of HSA.
conditions. To avert species interconversion during the
study, all readings were obtained within 10–20 s, and to
ensure rapid binding of CPT molecules on the protein,
HSA was used in great excess at a molar ratio of HSA
to CPT ranging from 30/1 to 240/1.
As shown in Table V, fluorescence quantum yields
of the CPT-lactone and CPT-carboxylate in PBS were
found to be almost identical, indicating that light ab-
sorption and emission capacities of CPT in PBS are
not affected by the status of the E-ring of the molecule.
International Journal of Chemical Kinetics DOI 10.1002/kin

714
THAKUR, KUNADHARAJU, AND SAVVA
100
80
60
40
20
0
0
50
100
150
200
250
300
t/min
(a)
0.00315
0.0032
0.00325
0.0033
0.00335
0.0034
(b)
K/T
Figure 7 (a) Hydrolysis kinetics of CPT-lactone in the presence of HSA at pH 7.4 and various temperatures: 298.15 K (ꢀ),
ꢁ
302.15 K (×|), 306.15 K ( ), 310.15 K (×), and 314.15 K ( ). Solid lines are the fitted data. (b) Arrhenius plot of the apparent
ꢀ
hydrolysis rate constant kobs of the CPT-lactone in the presence of HSA.
Contrary to that, due to the close proximity of CPT
chromophore groups to HSA, significant quenching
in quantum yield for both the species was observed
in the presence of HSA. Interestingly, the reduction
of quantum yield was approximately three times for
CPT-carboxylate but only ∼2 times for CPT-lactone.
More specifically, upon excitation at 350 nm, absorp-
tion of CPT-lactone in the presence of HSA was on
average reduced by 23.4% as compared to a 29.7% re-
duction of the CPT-carboxylate. Similarly, integrated
International Journal of Chemical Kinetics DOI 10.1002/kin

KINETIC AND THERMODYNAMIC STUDIES OF CPT HYDROLYSIS
715
Table V Fluorescence Quantum Yield of CPT-Lactone
BIBLIOGRAPHY
and CPT-Carboxylate in Presence and Absence of 0.020
g cm−3 HSA in PBS Buffer at pH 7.4 and 298.15 K,
Reported as the Mean SD of Two Independent
Experiments
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fluorescence intensity of the CPT-lactone was reduced
by 59.9% as compared to a 77.1% reduction of CPT-
carboxylate. When the study was conducted using an
excitation wavelength of 370 nm, the absorption inten-
sity of both species in the presence of HSA was simi-
larly reduced by 5.5% most probably because 370 nm
is closer to the λmax, of both CPT species. However,
the integrated fluorescence of CPT-carboxylate was
disproportionally reduced by 73.4% as compared to
a 63.1% reduction of the CPT-lactone. Nonetheless,
the calculated quantum yield of both species from
both studies was practically identical. The find that
CPT-carboxylate undergoes nonradiative relaxation to
a higher extent, coupled with the higher affinity of CPT-
carboxylate for HSA [8,15,16], supports the mech-
anistic explanation that HSA does not catalyze the
hydrolysis reaction of CPT-lactone, but it rather binds,
sequesters, and stabilizes the CPT-carboxylate species
thus shifting the lactone-carboxylate equilibrium
to the right.
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International Journal of Chemical Kinetics DOI 10.1002/kin