Negative kinetic temperature effect on the hydride transfer from NADH analogue BNAH to the radical cation of N-benzylphenothiazine in acetonitrile

Article abstract of DOI:10.1021/jo061145c

The reaction rates of 1-(p-substituted benzyl)-1,4-dihydronicotinamide (G-BNAH) with N-benzylphenothiazine radical cation (PTZ^?+) in acetonitrile were determined. The results show that the reaction rates (k _obs) decreased from 2.80 × 10⁷ to 2.16 × 10⁷ M^-1 s^-1 for G = H as the reaction temperature increased from 298 to 318 K. The activation enthalpies of the reactions were estimated according to Eyring equation to give negative values (-3.4 to -2.9 kcal/mol). Investigation of the reaction intermediate shows that the charge-transfer complex (CT-complex) between G-BNAH and PTZ ^?+ was formed in front of the hydride transfer from G-BNAH to PTZ^?+. The formation enthalpy of the CT-complex was estimated by using the Benesi-Hildebrand equation to give the values from -6.4 to -6.0 kcal/mol when the substituent G in G-BNAH changes from CH₃O to Br. Detailed thermodynamic analyses on each elementary step in the possible reaction pathways suggest that the hydride transfer from G-BNAH to PTZ^?+ occurs by a concerted hydride transfer via a CT-complex. The effective charge distribution on the pyridine ring in G-BNAH at the various stages-the reactant G-BNAH, the charge-transfer complex, the transition-state, and the product G-BNA⁺-was estimated by using the method of Hammett-type linear free energy analysis, and the results show that the pyridine ring carries relative effective positive charges of 0.35 in the CT-complex and 0.45 in the transition state, respectively, which indicates that the concerted hydride transfer from G-BNAH to PTZ^?+ was practically performed by the initial charge (-0.35) transfer from G-BNAH to PTZ^?+ and then followed by the transfer of hydrogen atom with partial negative charge (-0.65). It is evident that the present work would be helpful in understanding the nature of the negative temperature effect, especially on the reaction of NADH coenzyme with the drug phenothiazine in vivo.

Full text of DOI:10.1021/jo061145c

Negative Kinetic Temperature Effect on the Hydride Transfer from
NADH Analogue BNAH to the Radical Cation of
N-Benzylphenothiazine in Acetonitrile
Xiao-Qing Zhu,* Jian-Yu Zhang, and Jin-Pei Cheng*
Department of Chemistry, The State Key Laboratory of Elemento-Organic Chemistry, Nankai UniVersity,
Tianjin 300071, China
xqzhu@nankai.edu.cn
ReceiVed June 5, 2006
The reaction rates of 1-(p-substituted benzyl)-1,4-dihydronicotinamide (G-BNAH) with N-benzylphe-
nothiazine radical cation (PTZ•+) in acetonitrile were determined. The results show that the reaction
rates (kobs) decreased from 2.80 × 107 to 2.16 × 107 M-1 s-1 for G ) H as the reaction temperature
increased from 298 to 318 K. The activation enthalpies of the reactions were estimated according to
Eyring equation to give negative values (-3.4 to -2.9 kcal/mol). Investigation of the reaction intermediate
shows that the charge-transfer complex (CT-complex) between G-BNAH and PTZ•+ was formed in front
of the hydride transfer from G-BNAH to PTZ•+. The formation enthalpy of the CT-complex was estimated
by using the Benesi-Hildebrand equation to give the values from -6.4 to -6.0 kcal/mol when the
substituent G in G-BNAH changes from CH3O to Br. Detailed thermodynamic analyses on each elementary
step in the possible reaction pathways suggest that the hydride transfer from G-BNAH to PTZ•+ occurs
by a concerted hydride transfer via a CT-complex. The effective charge distribution on the pyridine ring
in G-BNAH at the various stagessthe reactant G-BNAH, the charge-transfer complex, the transition-
state, and the product G-BNA+swas estimated by using the method of Hammett-type linear free energy
analysis, and the results show that the pyridine ring carries relative effective positive charges of 0.35 in
the CT-complex and 0.45 in the transition state, respectively, which indicates that the concerted hydride
transfer from G-BNAH to PTZ•+ was practically performed by the initial charge (-0.35) transfer from
G-BNAH to PTZ•+ and then followed by the transfer of hydrogen atom with partial negative charge
(-0.65). It is evident that the present work would be helpful in understanding the nature of the negative
temperature effect, especially on the reaction of NADH coenzyme with the drug phenothiazine in vivo.
Introduction
as the reaction temperature rises since an activation energy
barrier should be overcome in the reaction.11 But there is a case
Hydride transfer is one of the fundamental chemical and
biological reactions. The study of the hydride transfer from the
nicotinamide-adenine dinucleotide coenzyme (NADH) and its
analogues to the surrounding substrates1 has been a subject of
great interest and is increasingly drawing the attention of
researchers due to the biological importance of NADH in living
bodies.2-10 Generally, the rate of hydride transfer would increase
(1) (a) Walsh, C. Enzymatic Reaction Mechanism; W. H. Freeman: San
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Pyridine Nucleotide Coenzyme; Dolphin, D., Poulson, R., Avramovic, O.,
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10.1021/jo061145c CCC: $33.50 © 2006 American Chemical Society
Published on Web 08/10/2006
J. Org. Chem. 2006, 71, 7007-7015
7007

Zhu et al.
that the reaction rate could decrease as the reaction temperature
rises, i.e., the lower the temperature, the faster the reaction
rate.12-28 This phenomena used to go by the name of negative
kinetic temperature effect and was interpreted as follows: a
reaction intermediate, the energy of which is lower than the
reactant pair, lies in the course of reaction. Further, the energy
difference between the reactant pair and the reaction intermediate
is larger than the corresponding energy difference between the
intermediate and the transition state of the reaction. Kiselev and
Miller first reported the negative temperature dependence of
rates for the Diels-Alder reaction of tetracyanoethylene with
9,10-dimethylanthracene via the reaction intermediate of the
charge-transfer complex (CT-complex).12 Later, Fukuzumi and
co-worker observed the CT-complexes formed in the hydride
transfer from the Michler’s hydride to the quinone with the
negative temperature dependence of rates.27 Now, an interesting
question, whether the negative temperature effect exists also in
the hydride transfer from the nicotinamide-adenine dinucleotide
coenzyme NADH in vivo, is worthy of attention. This question,
in fact, has received our attention for a long time, since the
solution of this question evidently is very important to under-
standing the practical kinetics of the hydride transfer from
NADH in vivo.
By examining the past publications, it is found that except
for the paper where Fukuzumi used 9,10-dihydroacridine as the
NADH model and found the negative kinetic temperature effect
on the reaction of 10-methyl-9,10-dihydroacridine with quino-
ne,28 no paper has reported the negative kinetic temperature
effect on the hydride transfer from NADH or its analogues until
now. Since 10-methyl-9,10-dihydroacridine is quite different
from NADH in structure, it is evident that the observation of
the negative kinetic temperature effect on the hydride transfer
from 10-methyl-9,10-dihydroacridine to the quinone is not an
efficient indicator that NADH could also have the same property
of negative kinetic temperature effect on the hydride transfer.
1-Benzyl-1,4-dihydronicotinamide (BNAH), due to having
a very similar structure as NADH, is extensively used as the
model of NADH,4,9,29 and many experiments have shown that
BNAH can form a charge-transfer complex with some suitable
substrates before the complete hydride transfer,30 which indicates
that the negative kinetic temperature effect on the hydride
transfer from NADH and its close analogue BNAH could occur.
In this paper, we used BNAH as the NADH model and the
radical cation of N-benzylphenothiazine (PTZ•+)31 as the hydride
acceptor to examine the kinetic temperature effect on the hydride
transfer from BNAH to PTZ•+. As a result, a significant negative
kinetic temperature effect on the hydride transfer from BNAH
to PTZ•+ was observed.
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Results and Discussion
Reaction of G-BNAH with PTZ•+ and the Negative Kinetic
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7008 J. Org. Chem., Vol. 71, No. 18, 2006

NegatiVe Kinetic Temperature Effect on HydrideTransfer
TABLE 1. Second-Order Observed Rate Constants (kobs) for the
Reaction of G-BNAH with PTZ•+ in Deaerated Acetonitrile at
Various Temperatures
a
kobs × 10 -7 (M-1 s-1
)
G-BNAH
298 K
303 K
308 K
313 K
318 K
p-OCH3
p-CH3
p-H
p-Cl
p-Br
3.49
3.16
2.80
2.10
2.13
3.19
2.87
2.60
1.99
2.01
3.01
2.72
2.50
1.89
1.90
2.73
2.58
2.27
1.73
1.75
2.60
2.33
2.16
1.66
1.68
a The experimental errors are within (5%.
FIGURE 1. Stack plot of spectra for the reaction of BNAH (1.0 ×
10-4 M) with the radical cation of N-benzylphenothiazine (PTZ•+) (1.0
× 10-4 M) in acetonitrile at 298 K.
azine (PTZ•+) and 1-(p-substituted benzyl)-1,4-dihydronicoti-
namide (G-BNAH; G ) CH3O, CH3, H, Cl, Br) were synthe-
sized according to literature methods.32,33 When G-BNAH was
treated by the radical cation of N-benzylphenothiazine (PTZ•+)
in acetonitrile at room temperature, the corresponding com-
poundssG-BNA+, N-benylphenothiazine (PTZ), and PTZH+s
were obtained as the final products, which clearly shows that
during the reaction process, G-BNAH released a hydride ion.
The spectrophotometric titration experiments suggest that 2 mol
of PTZ•+ were required to consume 1 mol of G-BNAH
completely (eq 1, Figure S1). The reaction rate was determined
FIGURE 2. The plots of ln(kobs/T) against the reaction temperature
(K) for the reaction of PTZ•+ with CH3O-BNAH (9), CH3-BNAH
(b), BNAH (2), and Cl-BNAH (1) in acetonitrile.
substituent (G) on the benzyl ring of G-BNAH was going from
the electron-withdrawing group (G ) Br) to the electron-
donating group (G ) OCH3) (Figure 2). By examining the
activation parameters of the reactions, it is found that the
activation enthalpies (∆Hqobs) all were negative, and the negative
values increased as the substituent was going from the electron-
withdrawing group to the electron-donating group (from -2.9
kcal/mol for p-Br to -3.4 kcal/mol for p-OCH3). To our best
knowledge, this is the first time the negative kinetic temperature
dependence of rate in the hydride transfer mediated by the
NADH model BNAH has been observed. Since the Hammett
reaction constant F for the reaction is a negative value (F )
-0.436) (see Figure 3) and the logarithmic values of kobs are
strongly dependent on the standard oxidation potentials of
G-BNAH (Figure 4), it is conceived that charge transfer, electron
transfer, or hydride transfer from G-BNAH to PTZ•+ would be
in the rate-determining step of the reaction.
with an Applied Photophysics SX.18MV-R stopped-flow spec-
trometer by monitoring the spectra change of PTZ•+ (Figure
1). The second order observed rate constants (kobs) for the
reaction at different temperatures between 298 and 318K are
listed in Table 1. The activation parameters of the reactions
activation enthalpy (∆Hqobs) and activation entropy (∆Sqobs)s
were derived from the Eyring plots of ln(kobs/T) versus the
reciprocal of the absolute temperature (1/T),34 which were listed
in Table 3.
According to the negative activation enthalpies (∆Hqobs) for
the reactions of G-BNAH with PTZ•+, it is conceivable that
between the reactant pair (G-BNAH and PTZ•+) and the
transition state of the reactions would exist a CT-complex as
the reaction intermediate,12-28,35 and the state energy of the CT-
complex should be not only smaller than that of the reactant
pair, but also smaller than that of the transition state of the
reactions. So, to determine the origin resulting in the negative
kinetic temperature effect on the reaction of G-BNAH with
PTZ•+, it is necessary to examine and determine the CT-complex
formed between G-BNAH and PTZ•+ during the reaction
process.
From Table 1, it is clear that the most striking feature of the
reaction of G-BNAH with PTZ•+ is that as the reaction
temperature increases from 298 to 318 K, the reaction rates (kobs
)
decreased rather than increased and the negative kinetic tem-
perature effect on the reaction rates (meaning “the lower the
temperature, the faster the reaction rate”28) increased as the
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Foces-Foces, C.; Lo´pez-Rupe´rez, F.; Jose, C. J. Chem. Soc., Perkin Trans.
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J. Org. Chem, Vol. 71, No. 18, 2006 7009

Zhu et al.
TABLE 2. Equilibrium Constants for the Formation of the
CT-Complexes of G-BNAH with PTZ•+ in Deaerated Acetonitrile at
Various Temperatures
a
KCT × 10 -3 (M-1
)
λ
max (nm) of
G-BNAH CT-complex 298 K 303 K 308 K 313 K 318 K
p-OCH3
p-CH3
p-H
p-Cl
p-Br
722
722
720
719
718
3.9
3.7
3.4
3.3
3.2
3.4
3.2
2.9
2.7
2.8
2.7
2.6
2.5
2.3
2.3
2.4
2.3
2.0
2.0
2.0
2.0
1.9
1.8
1.7
1.7
a The uncertainties are not larger than (7%.
TABLE 3. Standard Formation Enthalpy and Entropy Changes of
CT-Complex Formed between G-BNAH and PTZ•+, Activation
Parameters of the CT-Complex, and the Eyring Activation
Parameters of the Reaction of G-BNAH and PTZ•+ in Acetonitrile
FIGURE 3. Correlation of log kobs (at 298 K) versus Hammett
substituent constants σ for the reaction of G-BNAH with PTZ•+
.
a
b
a
b
a
b
G-BNAH ∆H°CT
∆S°CT
∆Hq
-
∆Sq
-
∆Hq
∆Sq
H
T
H
T
obs
obs
p-OCH3
p-CH3
p-H
p-Cl
p-Br
-6.4
-6.3
-6.2
-6.0
-6.0
-5.1
-4.7
-4.6
-4.1
-4.1
3.0
3.0
3.1
3.1
3.1
-30.5
-30.8
-30.3
-30.8
-30.7
-3.4
-3.3
-3.1
-2.9
-2.9
-35.6
-35.5
-34.9
-34.9
-34.8
a Units of kcal‚mol-1 b Units of cal K-1 mol-1
. .
absorbance of the CT-complex by changing the concentrations
of G-BNAH according to the Benesi-Hildebrand equation (eq
2).36 The values of the equilibrium constant (KCT) at different
temperatures from 298 to 318K are summarized in Table 2.
The corresponding thermodynamic parameters for the CT-
complex formation were obtained from the plots of ln KCT vs
1/T according to the van’t Hoff equation,37 which are listed in
Table 3.
FIGURE 4. Correlation of log kobs (at 298 K) versus the standard
oxidation potentials of G-BNAH.
[PTZ•+]/A ) 1/(KCTꢀ[G-BNAH]) + 1/ꢀ
(2)38
From Figure 5 and Table 2, it is clear that before the hydride
transfer from G-BNAH to PTZ•+, a CT-complex was formed
between G-BNAH and PTZ•+. The value scale of the standard
formation enthalpy of the CT-complex ranges from -6.4 kcal/
mol for G-BNAH (G ) CH3O) to -6.0 kcal/mol for G-BNAH
(G ) Br). Since the negative values of the standard formation
enthalpy of the CT-complex are increased from the electron-
withdrawing group (G ) Br) to the electron-donating group (G
) OCH3), the CT-complex could be constructed by charge
transfer from G-BNAH to PTZ•+.26-28 In terms of the relation-
ship that the observed activation enthalpy ∆Hqobs for the reaction
of G-BNAH with PTZ•+ is equal to the combination of the
formation enthalpy of the CT-complex (∆H°CT) (always negative
values) and the activation enthalpy of the CT-complex to reach
the transition state (∆HqH-T) (eq 3),15,18,26,27 the activation
FIGURE 5. Visible-UV spectra of the CT-complex of G-BNAH (3.0
× 10-4 M) with the radical cation of N-benzylphenothiazine (PTZ•+
)
enthalpy of the CT-complex to reach the transition state (∆Hq -
(3.0 × 10-4 M) in acetonitrile at 298 K.
H
T) can be easily derived from eq 3, and the results were
summarized in Table 3.
CT-Complex Formed between G-BNAH and PTZ•+. When
BNAH and PTZ•+ were mixed in acetonitrile, a new broad
absorption band at λmax ) 720 nm, which is the characteristic
of a CT-complex, is immediately observed (Figure 5). Since
neither BNAH nor PTZ•+ has absorption at this absorption range
and BNAH can form a CT-complex with many suitable
substrates before the hydride complete transfer, the new broad
absorption band should originate from the CT-complex formed
between G-BNAH and PTZ•+ (Figure 5).30 The equilibrium
constant (KCT) of the CT-complex was estimated from the initial
∆Hqobs ) ∆H°CT + ∆Hq
(3)
-
H T
From Table 3, it is clear that ∆Hq T is positive, but ∆H°CT
H-
is negative. The increase of the reaction temperature is favorable
(36) Benesi, H. A.; Hildebrand, J. H. J. Am. Chem. Soc. 1949, 71, 2703.
(37) Song, H.-C. J. Zhengzhou Institute Light Ind. 1995, 10, 1.
(38) A ) absorbance at λmax of the CT-complex; KCT ) equilibrium
constants for CT-complex formation; ꢀ ) extinction coefficient.
7010 J. Org. Chem., Vol. 71, No. 18, 2006

NegatiVe Kinetic Temperature Effect on HydrideTransfer
SCHEME 1. Possible Mechanisms for the Reaction of G-BNAH with PTZ•+ in Acetonitrile
to the activation of the formed CT-complex, but unfavorable
to the formation of the CT-complex at the same time. Since
another PTZ•+ to yield the final products; the second reaction
step for the path b-f is hydrogen atom transfer from G-BNAH•+
to another PTZ•+ to form the final reaction products; and the
second reaction step for the path b-e-g is proton transfer from
G-BNAH•+ to PTZ to become PTZH+ and G-BNA•, and the
latter was then oxidized by another PTZ•+ to yield the final
products. In the path c-g, the hydride transfer was initiated by
neutral hydrogen atom transfer from G-BNAH to PTZ•+ to form
PTZH+ and G-BNA•, and the latter was then oxidized by another
PTZ•+ to yield the final products. It is clear that all five pathways
give the same final reaction products. It follows that an
interesting but difficult question is produced: which one is the
practical pathway of the hydride transfer from G-BNAH to
PTZ•+. Obviously, it is necessary to access the thermodynamic
data of each mechanistic step for the hydride transfer from
G-BNAH to PTZ•+ to determine the practical pathway of the
hydride transfer from G-BNAH to PTZ•+.
the absolute value of ∆H°CT is larger than the value of ∆Hq
,
T
H-
the temperature factor has a larger effect on the formation of
the CT-complex than on the activation of the formed CT-
complex, which resulted in the abnormal experimental situation
for the reaction of G-BNAH with PTZ•+ in acetonitrile that the
higher the temperature, the slower the reaction.
Mechanism and Thermodynamics of the Reaction of
G-BNAH with PTZ•+. From the previous studies,26-28 it
appeared that the negative temperature effects on the hydride
transfer from organic hydrides (such as Michler’s hydride and
9-substituted 10-methyl-9,10-dihydroacridine) all were thought
to occur in the initial single-electron-transfer step. No paper
was found to report the negative temperature effect that occurred
in the concerted hydride transfer process. As to the present
reaction that also has a negative temperature effect, whether or
not the hydride transfer from G-BNAH to PTZ•+ also was
initiated by single electron transfer is the question. To thoroughly
elucidate the practice pathway of the reaction, the detailed
thermodynamics of each mechanistic step for the hydride
transfer from G-BNAH to PTZ•+ needs to be obtained. Ac-
cording to the typical chemical properties of G-BNAH and
PTZ•+ as well as the reaction final products (eq 1), five possible
pathways can be proposed for the practice mechanism of the
hydride transfer from G-BNAH to PTZ•+ as shown in Scheme
1 (i.e., the paths of a-h; b-d-h; b-f; b-e-g; and c-g). In
the path a-h, the hydride transfer from G-BNAH to PTZ•+ was
completed by direct one-step transfer mechanism to form
G-BNA+ and PTZH•, and the latter was then oxidized by another
PTZ•+ to yield the final products (PTZH+ and PTZ). In the paths
b-d-h, b-f, and b-e-g, the hydride transfer was initiated
by single electron transfer from G-BNAH to PTZ•+ to form
G-BNAH•+ and PTZ, but the second reaction step among the
three pathways is different: the second reaction step for the
path b-d-h is hydrogen atom transfer from G-BNAH•+ to PTZ
to become G-BNA+ to PTZH•, the latter was then oxidized by
To estimate the standard state enthalpy change of each
mechanistic step, eight equations (4-11)39 were developed, in
which eqs 4, 6, 7, 8, and 9 were derived from five suitable
thermodynamic cycles (Schemes S1-S5 in the Supporting
Information) according to Hess’ law (where F ) 23.06 kcal
mol-1 V-1 is the Faraday constant), from which the change of
the standard state enthalpy or standard state free energy for each
elementary step shown in Scheme 1 can be easily derived only
if the enthalpy change of the reaction (eq 1) and the standard
redox potentials of the related species are available. Evidently,
the enthalpy changes of reaction 1 (∆Hrxn) can be obtained from
the corresponding reaction heat, which can be directly deter-
mined by titration calorimetery (Figure 6). The standard redox
(39) It should be pointed out herein that we used the term free energy
change ∆GeT to replace the enthalpy change ∆HeT for the electron-transfer
processes. The validation of using free energy change ∆GeT instead of
enthalpy change ∆HeT for electron-transfer processes is that entropies
associated with electron transfer are negligible, and ∆GeT can be combined
directly with ∆HeT, which has been verified by Arnett’s work: Arnett, E.
M.; Amarnath, K.; Harvey, N. G.; Cheng, J.-P. J. Am. Chem. Soc. 1990,
112, 2, 344.
J. Org. Chem, Vol. 71, No. 18, 2006 7011

Zhu et al.
TABLE 5. Energetics of Each Mechanistic Step Shown in Scheme
1 (kcal/mol)a
∆H° (or ∆G°)
G-BNAH step a step b step c step d step e step f step g step h
CH3O
CH3
H
Cl
Br
-7.3 -3.3 -7.4 -4.0 -4.1 -39.6 -35.6 -35.7
-7.0 -3.1 -8.1 -3.9 -5.0 -39.6 -34.6 -35.7
-6.7 -2.8 -8.0 -3.9 -5.3 -39.6 -34.4 -35.7
-6.2 -2.2 -8.0 -4.0 -5.8 -39.7 -33.9 -35.7
-6.1 -2.2 -7.9 -3.9 -5.7 -39.6 -33.9 -35.7
a The enthalpy changes calculated from eqs 4 and 6-9 for step a and
c-f are the combinations of enthalpy and free energy terms. The free energy
for the electron transfer ∆G° to replace the enthalpies changes ∆H° is
reasonable. See ref 39.
FIGURE 6. Isothermal titration calorimettry (ITC) for the reaction of
BNAH with PTZ•+ in acetonitrile at 298 K. Titration was conducted
by adding 10 µL of BNAH (1.89 mM) every 500 s into the acetonitrile
solution containing PTZ•+ClO4 (5.01 mM).
-
TABLE 4. Reaction Enthalpy Changes of the Reactions of
G-BNAH with PTZ•+ in Acetonitrile (kcal/mol), along with the
Redox Potentials of Relative Species in Acetonitrile (V vs Fc+/0
)
E1/2
E°
E°
E°
a
b
c
c
c
G-BNAH ∆Hrxn (PTZ+/0
)
(G-BNAH+/0
)
(G-BNA+/0
)
(PTZH+/0
)
p-OMe -43.0
0.334
0.334
0.334
0.334
0.334
0.189
0.201
0.214
0.237
0.239
-1.209
-1.168
-1.156
-1.136
-1.135
-1.214
-1.214
-1.214
-1.214
-1.214
FIGURE 7. Coordinate diagram of the electron transfer from BNAH
to PTZ+• in acetonitrile.
p-CH3
p-H
p-Cl
-42.7
-42.4
-41.9
-41.8
transfer (step c). Since the state energy change for the hydrogen
atom transfer is more negative than that for the hydride transfer
and for the electron transfer, respectively, step c should be more
favorable than the other two possible initial steps to take place.
But, according to the strong dependence of the reaction rate of
G-BNAH with PTZ•+ on Hammett substituent constant σ (F )
- 0.436) (see Figure 3) and on the standard oxidation potentials
of G-BNAH (the line slope is -4.5, see Figure 4), it is
reasonable to conclude that the initial hydrogen atom transfer
from G-BNAH to PTZ•+ (step c) should be ruled out as the
initial step for the reaction of G-BNAH with PTZ•+.
Concerning steps a and b, a comparison of the state energy
changes of the two initial reaction steps (step a and b) with the
corresponding observed activation enthalpy changes of the
reactions (∆Hqobs) clearly shows that the observed activation
enthalpy changes (∆Hqobs) (-3.8 to -2.7 kcal/mol) are more
negative than the corresponding standard state energy changes
of the electron transfer in step b (-3.3 to -2.2 kcal/mol), but
less negative than the concerted hydride transfer process in step
a (-7.3 to -6.1 kcal/mol). On the basis of a general reaction
law that the activation energy change is always larger than or
at least equal to the corresponding standard state free energy
change for any elemental reaction,41 it is evident that step b
should be ruled out as the initial reaction, and the only remaining
step a should be suitable for the reaction law, i.e., the reaction
of G-BNAH with PTZ•+ was initiated by the concerted hydride
transfer rather than by one-electron or hydrogen atom transfer,
even though PTZ•+ was generally regarded as a typical one-
electron oxidant (Figure 7). These results unambiguously support
that the practical mechanism of the hydride transfer from
G-BNAH to PTZ•+ is the path of steps a-h, i.e., the hydride
transfer from G-BNAH to PTZ•+ was completed by a direct
p-Br
a Measured in acetonitrile solution at 298 K in kcal/mol by titration
calorimetry. The data given were the average values of at least two
independent runs, each of which was again an average value of at least 7
consecutive titrations. The reproducibility was e1.0 kcal/mol. b Measured
in acetonitrile solution at 298 K in volts by the CV method vs ferrocenium/
ferrocene redox couple. Reproducible to 5 mV or better. c Measured by
OSWV and SHACV methods in acetonitrile solution at 298 K in volts vs
ferrocenium/ferrocene redox couple. See ref 40.
potentials can be measured by cyclic voltammetry (CV),
Osteryoung square wave voltammetry (OSWV), and second-
harmonic ac voltammetry (SHACV)40 (Figure S2-S4). The
detailed experimental results were summarized in Table 4,
respectively. The thermodynamic data of each mechanistic step
shown in Scheme 1 are summarized in Table 5.
From Table 5, it is easy to find that the value scales of the
state energy changes for the three initial steps (steps a, b, and
c) in the five possible pathways (see Scheme 1) range from
-7.3 to -6.1 kcal/mol for the concerted hydride transfer process
(step a), from -3.3 to -2.2 kcal/mol for the electron transfer
(step b), and from -8.1 to -7.4 kcal/mol for the hydrogen
(40) The SHACV and OSWV methods provide superior approaches to
directly evaluating the one-electron redox potentials in the presence of a
follow-up chemical reaction and it is regarded that both phase selective ac
and square wave techniques are powerful in studying irreversible or nearly
irreversible systems. See: (a) Bard, A. J.; Faulkner, L. R. Electrochemical
Methods, Fundamental and Applications; John Wiley & Sons: New York,
2001; Chapter 10, pp 368-416. (b) McCord, T. G.; Smith, D. E. Anal.
Chem. 1969, 41, 1423. (c) Bond, A. M.; Smith, D. E. Anal. Chem. 1974,
46, 1946. (d) Wasielewski, M. R.; Breslow, R. J. Am. Chem. Soc. 1976,
98, 4222. (e) Arnett, E. M.; Amarnath, K.; Harvey, N. G.; Cheng, J.-P. J.
Am. Chem. Soc. 1990, 112, 344. (f) Fukuzumi, S.; Nishimine, M.; Ohkubo,
K.; Tkachenko, N. V.; Lemmetyinen, H. J. Phys. Chem. B 2003, 107, 12511.
(g) Ofial, A. R.; Ohkubo, K.; Fukuzumi, S.; Lucius, R.; Mayr, H. J. Am.
Chem. Soc. 2003, 125, 10906. (h) Osteryoung, J. G.; Osteryoung. R. A.
Anal. Chem. 1985, 57, 101. (i) Ivaska, A. U.; Smith, E. E. Anal. Chem.
1985, 57, 1910. (j) O’Dea, J.; Wojciechowski, M.; Osteryoung, J. Anal.
Chem. 1985, 57, 954-955.
(41) (a) Hine, J. AdV. Phys. Org. Chem. 1977, 15, 1. (b) Page, M.;
Williams, A. Organic and Bio-organic Mechanism; Addison-Wesley
Longman Limited 1997; Chapter 1, pp 1-22.
7012 J. Org. Chem., Vol. 71, No. 18, 2006

NegatiVe Kinetic Temperature Effect on HydrideTransfer
constant σ. From Figure 8, three excellent straight lines with
the line slopes of 0.78 ( 0.03 (equivalent to F of -0.14)43 for
the formation process of the CT-complex, 1.00 ( 0.03
(equivalent to F of -0.18)43 for the process from the reactants
to the transition state, and 2.24 ( 0.12 (equivalent to F of
-0.39)43 for the hydride transfer from G-BNAH to PTZ•+ are
observed, which means that the Hammett linear free energy
relationship holds in the three chemical processes. According
to the intrinsic property of the Hammett substituent effect, it is
conceived that the sign of the line slope values reflects an
increase or decrease of the effective charge on the nitrogen (N1)
atom in the pyridine ring and the magnitude of the line slope
values is a measurement of the effective charge change on the
N1 atom during the corresponding reaction processes.44 To
quantitatively evaluate the relative effective charge changes on
the N1 atom for the three reaction processes, we defined that
the effective charge on the N1 atom in G-BNAH is zero, and
the effective charge on the N1 atom in G-BNA+ is a positive
one (+1), which is from our basic knowledge of the electronic
structures of the neutral G-BNAH and the corresponding
quaternary ammonium salts G-BNA+. This definition indicates
that the positive line slope of 2.24 for the hydride transfer from
G-BNAH to PTZ•+ is equivalent to the positive effective charge
increase of one unit (+1) on the N1 atom from G-BNAH to
G-BNA+. According to this relationship, it is easy to deduce
that the line slope of 0.78 for the formation of the CT-complex
is equivalent to the positive effective charge increase of 0.35
on the N1 atom from the free G-BNAH as the reactant to the
complex state of G-BNAH in the CT-complex.9b,c,42,45 Since
the effective charge on the N1 atom in free G-BNAH has been
defined as zero, the effective charge on the N1 atom in the CT-
complex should be +0.35 (Scheme 2). Similarly, the slope of
1.00 for the chemical reaction process from the reactants to the
transition state should be equivalent to the positive effective
charge increase of 0.45 on the N1 atom in going from the free
G-BNAH as the reactant to the G-BNAH moiety in the transition
state, which indicates that the N1 atom on the pyridine ring in
the transition state should possess an effective charge of +0.45.
Since the CT-complex was formed by the charge transfer of
-0.35 from G-BNAH to PTZ•+, the reaction process from the
CT-complex to the complete formation of G-BNA+ was
performed only by hydrogen atom transfer with partial negative
charge (-0.65). The details of the effective charge changes on
the N1 atom during the hydride transfer from G-BNAH to PTZ•+
is shown in Scheme 2.
FIGURE 8. Hammett plots of ∆H°CT (9), ∆Hq (b), and ∆H°H
-
obs
T
(1) vs Hammett substituent constants. The values of ∆H°CT and ∆Hq
obs
-
are listed in Table 3; the values of ∆H°H (energies changes of step
T
a) are listed in Table 5, respectively.
one-step mechanism to form G-BNA+ and PTZH•, the latter
was then oxidized by another PTZ•+ to yield the final products
(PTZH+ and PTZ). Considering that the state energy change
for the second step (step h) (-35.7 kcal/mol) is much more
negative than that for the corresponding initial reaction step (step
a), it is conceivable that the hydride transfer in the initial step
is the rate-determining step. The coordinate diagram of the
concerted hydride transfer from BNAH to PTZ•+ via a CT-
complex in acetonitrile is shown in Figure 7. Since the structure
of G-BNAH is quite closed to the redox active center of NADH
and PTZ•+ can be generated in the metabolism of phenothiazine-
based drug in vivo,31 the negative kinetic temperature effect
observed here in the hydride transfer from G-BNAH to PTZ•+
may suggest that a similar phenomenon could also occur in the
mammalian body, i.e., when the temperature of the mammalian
body increases, the reaction rate of NADH with the radical
cation of phenothiazine drug could decrease.
Evaluation of the Effective Charge Distribution on the
Pyridine Ring in the Hydride Transfer Process. Although
CT-complexes in the NADH mimicing reductions have been
observed for a long time, no quantitative estimation about the
charge transfer from the pyridine ring in the CT-complexes has
been available until now. To quantitatively examine the process
details of the hydride transfer from G-BNAH to PTZ•+ and
further elucidate the electrostatic character of the charge-transfer
complex and the transition state of the reaction, the effective
charge distribution on the pyridine ring in G-BNAH at the
various stages (the reactant G-BNAH, the charge-transfer
complex, the transition state, and the product G-BNA+) was
estimated by using the method of Hammett-type linear free
energy analysis, since the Hammett linear free-energy relation-
ship analysis can provide a very efficient access to estimate the
effective charge distribution.42 Figure 8 shows the plots of the
formation enthalpy of the CT-complex (∆H°CT), the observed
activation enthalpy of the reaction of G-BNAH with PTZ•+
(∆Hqobs), and the energy change of hydride transfer from
From Scheme 2, it is clear that relative to the free G-BNAH,
the G-BNAH moiety in the CT-complex carries the effective
positive charge of 0.35, which means that the CT-complex was
formed by the charge (-0.35) transfer from G-BNAH to PTZ•+.
Similarly, relative to the free G-BNAH, the G-BNAH moiety
in the transition state of the reaction of G-BNAH with PTZ•+
carries the effective positive charge of 0.45, which means that
the formation of the transition state from the reaction of
G-BNAH with PTZ•+ was completed by the transfer of 45%
hydride ion. To our best knowledge, this is the first time the
charge distribution at the stage of CT-complex and the transition
(43) According to Hammett equation log K ) Fσ and ∆H ≈ -RT log
K, the line slope of ∆H against σ should be approximately equal to -5.7F.
See more in refs 9b, 9c and 45.
G-BNAH to PTZ•+ (∆H°H T) against the Hammett substituent
-
(44) Isaacs, N. S. Physical Organic Chemistry, 2nd ed.; John Wiley &
Sons: New York, 1995; Chapter 4, p 161.
(45) (a) Zhu, X.-Q.; Hao, W.-F.; Tang, H.; Wang, C.-H.; Cheng, J.-P. J.
Am. Chem. Soc. 2005, 127, 2696. (b) Zhu, X.-Q.; Li, Q.; Hao, W.-F.; Cheng,
J.-P. J. Am. Chem. Soc. 2002, 124, 9887.
(42) (a) Page, M.; Williams, A. Organic and Bio-organic Mechanism;
Addison-Wesley Longman Limited: Reading, MA, 1997; Chapter 3, pp52-
79. (b) Williams, A. Acc. Chem. Res. 1989, 22, 387. (c) Williams, A. J.
Am. Chem. Soc. 1985, 107, 6335.
J. Org. Chem, Vol. 71, No. 18, 2006 7013

Zhu et al.
SCHEME 2
TABLE 6. Comparison of the Related Thermodynamic Data for the Various Reaction Systems with the Negative Kinetic Temperature Effecta
d
e
d
e
d
e
reaction systemb
reaction type c
∆Hq
∆Sq
∆H°inter
∆S°inter
∆Hq
∆Sq
ref
obs
obs
inter
inter
TM-3
AcrH2/DDQ
RR
ET
ET
ET
ET
ET
H-T
ET
DT
HT
ET
ET
ET
AD
HT
ET
DA
ET
ET
-7.8
-7.7
-4.2
-3.5
-3.3
-3.2
-3.1
-3.1
-2.7
-2.7
-2.6
-2.4
-2.3
-2.3
-1.5
-44.7
-40.7
-42.3
-44.2
-
-44.2
-34.9
-32
-50
-57
21a
28
20a
26
*Ru(bpy)32+/methyl m-nitrobenzoate
MH2/DDQ
-7.0
-8.8
-10.3
-
3.5
-34.2
MH2/DDQ
5.7f
7.9
3.1
27
MGH/DDQ
-11.0
-6.2
-26.6
-4.6
-17.7
-30.3
26
BNAH/PTZ•+
twg
17
2+
MAQ/*Ru(bpy)3
FeII(D2bip)/FeIII(Dbip)
FeII(H2bip)/TEMPO
15a
15b
20a
26
-9.4
-30
6.7h
9.0
-27i
*Ru(bpy)32+/p-nitrobenzaldehyde
MHOMe/DDQ
-26.2
-43.3
-52
-11.4
-28.2
-15.1
2,6-(MeO)2Pyridine/[(DH)2Co(Me)(Py)]+
PhCCl/TME
<-6.9j
18
-28
23a
15a
13
12
13
FeII(H2bip)/FeIII(Hbip)
-45
Fe(H2O)62+/Ru(phen)3
-1.35
-1.19
-0.6
-36
3+
DMA/TCNE
-41
Fe(H2O)62+/Fe(terpy)2
-38
3+
ZnT(t-Bu)PP/ZnT(t-Bu)PP•+
-0.24
-4
16
a To facilitate the comparison, all units are converted to kcal‚mol-1 or cal K-1 mol-1 b Abbreviations: TM-3 ) 3,5,5-trimethyl-2-oxomorpholin-3-yl
.
radical; AcrH2 ) 10-methyl-9,10-dihydroacridine; DDQ ) 2,3-dichloro-5,6-dicyano-p-benzoquinone; *Ru(bpy)32+ ) excited tris(2,2′-bipyridine)ruthenium(II);
MH2 ) bis[4-(dimethylamino)phenyl]methane; MGH ) Leuco Malachite Green; BNAH ) benzyl-1,4-dihydronicotinamide; PTZ•+ ) N-benzylphenothiazine
radical cation; MAQ ) 2-methylanthraquinone; FeII(H2bip) ) iron 2,2′-bi(tetrahydro)pyrimidine; FeII(D2bip) ) deuterated iron 2,2′-bi(tetrahydro)pyrimidine;
FeIII(Hbip) ) iron bi(tetrahydro)pyrimidine; FeIII(Dbip) ) deuterated iron bi(tetrahydro)pyrimidine; TEMPO ) 2,2,6,6-tetramethylpiperidine-N-nitroxyl
radical; MHOMe ) bis[4-(dimethylamino)phenyl]methoxymethane; DH ) dimethylglyoxime; Py ) pyridine; PhCCl ) phenylchlorocarbene; TME )
tetramethylethylene; Ru(phen)33+ ) tris(1,10-phenanthroline)ruthenium(III); DMA ) 9,10-dimethylanthracene; TCNE ) tetracyanoethylene. c RR ) radical
combination; ET ) electron transfer; DT ) deuterium-atom transfer; H-T ) hydride transfer; HT ) hydrogen-atom transfer; AD ) carbine/alkene addition;
DA ) Diels-Alder reaction. d Units of kcal‚mol-1
.
e Units of cal K-1 mol-1
. .
f The original data take the value of 24 kJ‚mol-1 g This work. h Calculated
with eq 3. i Calculated with the equation ∆Sq ) ∆S°CT + ∆Sq
.
CT
j The value is estimated by the author. See ref 18.
obs
state of the hydride transfer with negative temperature effect
have been estimated. It is believed that this information would
be valuable to understand the nature of the hydride transfer from
NADH to the drug phenothiazine in vivo.
effect. The reason is that in the electron transfer process, the
charge-transfer complex as reaction intermediate is easily
formed, and the formation heat of the charge-transfer complex
is frequently larger than the activation heat of the charge-transfer
complex for the following reaction. From Table 6, we also find
that the formation enthalpy (∆H°inter) of the charge-transfer
complex generally is not too negative (generally larger than -12
kcal/mol). It is conceived that if the negative temperature effect
can be observed on a reaction, the activation enthalpy of the
intermediates (∆Hqinter) should be quite small (generally smaller
than 12 kcal/mol), which means that it is impossible to observe
this unusual negative kinetic temperature effect in a very slow
reaction. In fact, up until now, no paper has been released to
report the negative kinetic temperature effect in a slow chemical
reaction. As is well-known, electron transfer is universal in
living bodies and the transfer rate generally is very fast, and it
is reasonable to deduce that the reactions with negative kinetic
temperature effects in vivo could not be few.
To search for the common origin resulting in the negative
kinetic temperature effect on the reaction rate, the present
reaction system and some reaction systems with negative kinetic
temperature effect reported previously were summarized to-
gether in Table 6 for comparison. From Table 6, it is clear that
most of the negative kinetic temperature effects were found to
occur in the electron-transfer processes.13,14,16-18,20,26-28 Al-
though in some formal non-electron-transfer reactions, such as
hydrogen atom transfer, hydride transfer, Diels-Alder reac-
tion,12 and carbine/alkene addition,23a the negative kinetic
temperature effects can also be observed, in fact, these reactions
are generally believed to be initiated by electron transfer or by
concerted electron transfer, and it is reasonable to suggest that
the electron transfer reaction should be the most common
reaction type, which can result in negative kinetic temperature
7014 J. Org. Chem., Vol. 71, No. 18, 2006

NegatiVe Kinetic Temperature Effect on HydrideTransfer
the kinetic traces were recorded on an Acorn computer and analyzed
by Pro-K Global analysis/simulation software, or translated to PC
for further analysis. The well data fitting used the equation of Abs
) P1/(P1/k/t +1) + P2, which integrated in the Pro-K Global
analysis/simulation software or Origin Software. The plot of 1/Abs
vs time t would also give the same value of the rate constants. The
corresponding observed rate constants was obtained by using the
equation kobs ) k × ꢀb. In each case, it was confirmed that the rate
constants derived from five to seven independent measurements
agreed within an experimental error of (5%. The equilibrium
constant (KCT) of the CT-complex was estimated from the initial
absorbance of the CT-complex by changing the concentrations of
G-BNAH according to the Benesi-Hildebrand equation (eq 2).36
The corresponding thermodynamic parameters for the CT-complex
formation were obtained from the plots of ln KCT vs 1/T according
to the van’t Hoff equation (Supporting Information). The spectra
of the CT complex were obtained by plotting the initial absorbance
against the wavelength with the stopped-flow spectrophotometer.
Other UV-vis spectra were performed on a HITACHI UV-3000
spectrometer.
Conclusions
According to the examination of the kinetics, thermodynam-
ics, and the reaction intermediate for the reaction of NADH
analogue G-BNAH with the radical cation of N-benzylphe-
nothiazine (PTZ•+) in acetonitrile, the following conclusions can
be drawn:
(1) The rate of hydride transfer from G-BNAH to PTZ•+
decreased as the reaction temperature increased. The activation
enthalpies of the hydride transfer all are negative values (-3.4
to -2.9 kcal/mol). The reason is that G-BNAH and PTZ•+
formed a charge-transfer complex prior to the hydride transfer
from G-BNAH to PTZ•+ and the absolute value of the formation
enthalpy for the CT-complex is larger than the activation
enthalpy of the CT-complex, which results in negative values
of the activation enthalpies for the hydride transfer from
G-BNAH to PTZ•+.
(2) The reaction of G-BNAH with N-benzylphenothiazine
radical cation (PTZ•+) in acetonitrile was initiated by the
concerned hydride transfer via CT complex, rather than one-
electron transfer, even though PTZ•+ was generally regarded
as a typical one-electron oxidant.
(3) The effective charge distribution on the pyridine ring in
G-BNAH at the stages of the CT-complex and the transition
state was estimated to be 0.35 and 0.45 by using the method of
Hammett-type linear free energy analysis.
Titrated Calibration Experiments. Titrated calibration experi-
ments were performed in acetonitrile solution at 298 K on a CSC
4200 isothermal titration calorimeter. Prior to use, the instrument
was calibrated against an internal heat pulse. Data points were
-
collected every 2 s. The reaction heat of G-BNAH with PTZ•+ClO4
was determined following nine automatic injections from a 250
µL injection syringe containing 2 mM G-BNAH into the reaction
cell (1.00 mL) (containing 5 mM PTZ•+ClO4-). Injection volumes
(10 µL) were delivered at 0.5 s intervals with 500 s between every
two injections. The reaction heat was obtained by area integration
of each peak except the first one.
(4) Since the structure of G-BNAH is quite close to the redox
active center of NADH, it is reasonable to suggest that negative
temperature effect could occur on the reactions of NADH
coenzyme with the drug phenothiazine in vivo.
To our best knowledge, this paper not only reports the
negative kinetic temperature effect on the reactions mediated
by the NADH close analogue G-BNAH for the first time, but
also provides detailed thermodynamic data (such as, state energy
change) for each elementary step of the hydride transfer from
the NADH analogue G-BNAH to PTZ•+ and the detailed charge
distribution on G-BNAH at the various stages during the hydride
transfer process, especially at the stage of CT-complex, and also
examines the common origin resulting in the negative kinetic
temperature effect on the chemical reactions, which, in fact, is
never found in previous reports on this subject. It is evident
that all this hard-to-get and very important information would
be very valuable in understanding the nature of negative kinetic
temperature effect, especially on the reactions mediated by
NADH coenzyme in vivo.
Measurement of Redox Potentials. The electrochemical experi-
ments were carried out by cyclic voltammetry (CV), Osteryoung
square wave voltammetry (OSWV), and second-harmonic ac
voltammetry (SHACV), using a BAS-100B electrochemical ap-
paratus in deaerated acetonitrile solution under an argon atmosphere
at 298 K as described previously.9,40,46 n-Bu4NPF6 (0.1 M) was
employed as the supporting electrolyte. A standard three-electrode
cell consists of a glassy carbon disk as working electrode, a platinum
wire as counter electrode, and 0.1 M AgNO3/Ag (in 0.1 M Bu4-
NPF6-CH3CN) as reference electrode. All sample solutions were
about 1.5 mM. The ferrocenium/ferrocene redox couple (Fc+/0) was
taken as the internal standard.
Acknowledgment. Financial support from the Ministry of
Science and Technology of China (Grant No. 2004CB719905),
the National Natural Science Foundation of China (Grant Nos.
20125206, 20272027, 20332020, 20472038, and 20421202),
Ministry of Education of China (Grant No. 20020055004), and
Natural Science Fund of Tianjin (Grant No. 033804311) is
gratefully acknowledged.
Experimental Section
Materials. G-BNAH (G ) p-OCH3, p-CH3, H, p-Cl, p-Br) and
-
PTZ•+ClO4 were synthesized according to the literature meth-
ods.32,33 Reagent grade acetonitrile was refluxed over KMnO4 and
K2CO3 for several hours and was distilled over P2O5 under argon
before use.
Kinetic and Spectral Measurement. The kinetic measurement
of the reactions was carried out on an Applied Photophysics
SX.18MV-R stopped-flow spectrometer, which was thermostated
((0.1 °C) by circulating water, by mixing the same concentration
(1.0 × 10-4 M) of the two reactants. The SX.18MV-R uses a novel
cell cartridge system that has a dead time of around 1 ms with
very high sensitivity. The 5 µL cell with 1 mm path length light
guide with a dead-time around 500 µs was applied in the
experiments. The G-BNAH and PTZ•+ were weighed accurately
by using an analytic balance (d ) 0.01 mg) to ensure the some
concentration of the two reactants. The spectra changes of PTZ•+
(λmax ) 512 nm, ꢀ ) 6.0 × 103 M-1 cm-1) were monitored and
Supporting Information Available: Experimental details,
thermodynamic cycles for calculating the change of the standard
state enthalpy or free energy shown in Scheme 1, the electrochemi-
cal graphs for BNAH with three different electrochemical methods,
and characterization spectra of BNAH and PTZ•+. This material is
available free of charge via the Internet at http://pubs.acs.org.
JO061145C
(46) Arnett, E. M.; Venimadhavan, S. J. Am. Chem. Soc. 1991, 113,
6967.
J. Org. Chem, Vol. 71, No. 18, 2006 7015