Mechanistic insight from thermal activation parameters for oxygenation reactions of different substrates with biomimetic iron porphyrin models for compounds i and II

Article abstract of DOI:10.1007/s00775-011-0822-7

Compound I, an oxo-iron(IV) porphyrin p-cation radical species, and its one-electron-reduced form compound II are regarded as key intermediates in reactions catalyzed by cytochrome P450. Although both reactive intermediates can be easily produced from model systems such as iron(III) meso-tetra(2,4,6- trimethylphenyl)porphyrin hydroxide by selecting appropriate reaction conditions, there are only a few thermal activation parameters reported for the reactions of compound I analogues, whereas such parameters for the reactions of compound II analogues have not been investigated so far. Our study demonstrates that DH= and DS= are closely related to the chemical nature of the substrate and the reactive intermediate (viz., compounds I and II) in epoxidation and C-H abstraction reactions. Although most studied reactions appear to be enthalpy-controlled (i.e., DH=[-TDS=), different results were found for C-H abstractions catalyzed by compound I. Whereas the reaction with 9,10-dihydroanthracene as a substrate is also dominated by the activation enthalpy (DH= = 42 kJ/mol, DS= = 41 J/Kmol), the same reaction with xanthene shows a large contribution from the activation entropy (DH= = 24 kJ/mol, DS= = -100 J/kmol). This is of special interest since the activation barrier for entropy-controlled reactions shows a significant dependence on temperature, which can have an important impact on the relative reaction rates. As a consequence, a close correlation between bond strength and reaction rate- as commonly assumed for C-H abstraction reactions-no longer exists. In this way, this study can contribute to a proper evaluation of experimental and computational data, and to a deeper understanding of mechanistic aspects that account for differences in the reactivity of compounds I and II. SBIC 2011.

Full text of DOI:10.1007/s00775-011-0822-7

J Biol Inorg Chem (2012) 17:27–36
DOI 10.1007/s00775-011-0822-7
ORIGINAL PAPER
Mechanistic insight from thermal activation parameters
for oxygenation reactions of different substrates with biomimetic
iron porphyrin models for compounds I and II
•
Christoph Fertinger Alicja Franke
•
Rudi van Eldik
Received: 8 April 2011 / Accepted: 7 July 2011 / Published online: 30 July 2011
Ó SBIC 2011
Abstract Compound I, an oxo–iron(IV) porphyrin
p-cation radical species, and its one-electron-reduced form
compound II are regarded as key intermediates in reactions
catalyzed by cytochrome P450. Although both reactive
intermediates can be easily produced from model systems
such as iron(III) meso-tetra(2,4,6-trimethylphenyl)porphy-
rin hydroxide by selecting appropriate reaction conditions,
there are only a few thermal activation parameters reported
for the reactions of compound I analogues, whereas such
parameters for the reactions of compound II analogues
have not been investigated so far. Our study demonstrates
that DH= and DS= are closely related to the chemical
nature of the substrate and the reactive intermediate (viz.,
compounds I and II) in epoxidation and C–H abstraction
reactions. Although most studied reactions appear to be
enthalpy-controlled (i.e., DH= [ -TDS=), different
results were found for C–H abstractions catalyzed by
compound I. Whereas the reaction with 9,10-dihydroan-
thracene as a substrate is also dominated by the activation
enthalpy (DH= = 42 kJ/mol, DS= = 41 J/Kmol), the
same reaction with xanthene shows a large contribution
impact on the relative reaction rates. As a consequence, a
close correlation between bond strength and reaction rate—
as commonly assumed for C–H abstraction reactions—no
longer exists. In this way, this study can contribute to a
proper evaluation of experimental and computational data,
and to a deeper understanding of mechanistic aspects that
account for differences in the reactivity of compounds I
and II.
Keywords Iron porphyrin ꢀ Enzyme models ꢀ
Activation parameters ꢀ Peroxide ꢀ O–O bond activation
Introduction
The heme-containing cytochromes P450 form a super-
family of enzymes that are ubiquitous in aerobic organ-
isms. High-valence iron(IV)–oxo porphyrin species have
been identified as key intermediates in a vast number of
reactions catalyzed by these enzymes and their iron por-
phyrin biomimetics [1–4]. Throughout the past decades,
there was a vivid discussion among scientists to clarify the
different pathways for oxygenation reactions that lead to
different reaction products or product distributions. Com-
putational studies support a two-state reactivity [3–19] of
an iron(IV)–oxo porphyrin cation radical with closely lying
quartet (high-spin) and doublet (low-spin) states showing
different reactivity patterns. In comparison to this theo-
retical work, our own experimental work [20] ruled out an
involvement of an iron(III)–peroxo species, compound 0
[(Por)FeIII-OOR] (Por is porphyrin), as an oxidant in cat-
alytic reactions as proposed in the ‘‘multiple oxidant
hypothesis’’ [21–30].
from the activation entropy (DH= = 24 kJ/mol, DS=
=
-100 J/kmol). This is of special interest since the activation
barrier for entropy-controlled reactions shows a significant
dependence on temperature, which can have an important
Electronic supplementary material The online version of this
article (doi:10.1007/s00775-011-0822-7) contains supplementary
material, which is available to authorized users.
C. Fertinger ꢀ A. Franke ꢀ R. van Eldik (&)
Inorganic Chemistry,
Department of Chemistry and Pharmacy,
University of Erlangen-Nuremberg,
Egerlandstrasse 1, 91058 Erlangen, Germany
e-mail: vaneldik@chemie.uni-erlangen.de
Regardless of this intriguing controversy, it should be
kept in mind that even very fundamental effects coming
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J Biol Inorg Chem (2012) 17:27–36
from the solvent [22, 26] or the axial ligands [26, 31–35]
can have a dramatic impact on the observed reactivity or
even change the underlying reaction mechanism. There-
fore, in our recent work [36] we clarified the influence of
the nature of different substrates on their reaction with
different reactive intermediates that can participate in iron
porphyrin catalyzed reactions, i.e., compound 0, the iro-
n(IV)–oxo porphyrin cation radical species compound I
[(Porꢀ?)FeIV=O], and its one-electron-reduced form com-
pound II [(Por)FeIV=O]. We demonstrated a close rela-
tionship between the chemical nature of a substrate and its
reactivity towards different reactive intermediates and also
revealed changes in the reactivity order for compounds 0, I,
and II by investigating different types of reactions.
Materials and methods
Materials
All solutions were prepared in acetonitrile (99.9% AMD
CHROMASOLV from Sigma-Aldrich) m-Chloroperoxy-
benzoic acid was purchased from Acros Organics and
purified before use by recrystallization from hexane.
Iron(III) meso-tetra(2,4,6-trimethylphenyl)porphyrin hydrox-
ide [FeIII(TMP)OH; Scheme 1] was obtained from iron(III)
meso-tetra(2,4,6-trimethylphenyl)porphyrin chloride [FeIII
(TMP)Cl; Frontier Scientific] as described earlier [44]. The
resulting FeIII(TMP)OH was washed thoroughly with water
to remove trace impurities of NaOH, which could affect the
proper generation of compound I or compound II owing to
the pH sensitivity of these reactions. Iodosylbenzene was
synthesized according to a literature procedure [45]. 9,10-
Dihydroanthracene (DHA), 9H-xanthene, and cis-stilbene
(96%) were purchased from Aldrich (Scheme 2).
Especially changes in the regioselectivity in competitive
olefin oxygenation experiments leading to C=C epoxida-
tion and allylic C–H hydroxylation products are still the
subject of intense international research [21–26, 31–35]. In
this context, special attention was focused on the influence
of the meso substituents of the porphyrin system [37–40].
Recent studies [41, 42] demonstrated that a simple change
in the meso substituents can even change the regioselec-
tivity from C=C epoxidation to C–H hydroxylation in the
reaction of olefins with the cation radical species com-
pound I. Computational studies revealed that this change in
the reaction mechanism is caused by changes in the elec-
tronic nature of the porphyrin as well as by interactions of
the substrate with the ligand on the meso substituents of the
porphyrin system that affect the orientation of the substrate
towards the iron–oxo center.
Low-temperature rapid-scan measurements
Time-resolved UV/vis spectra were recorded with a quartz
glass dip-in detector (Spectralytics, Aalen, Germany)
coupled to a J&M TIDAS 500-3 (TSPEC-4) diode-array
spectrophotometer (J&M Analytik, Esslingen, Germany).
The optical dip-in detector had an optical path length of
1.0 cm and was connected to the spectrophotometer with
flexible light guides. A 20 ml double-wall reaction vessel
was used and thermostatted ( 0.1 °C) by a combination of
cold methanol circulation (WK 14-1 DS; Colora, Lorch,
Germany) and a 800 W heating unit. Complete spectra
were recorded between 350 and 726 nm with the integrated
software program J&M TIDAS-DAQ 2.3.7.4.
Thus, experimental as well as computational studies
suggest large effects on the activation barrier of certain
types of reactions that can, for example, be accounted for in
terms of substrate orientation as mentioned above [43]. In
addition, the rates of hydrogen abstraction show a close
correlation with the energies of the C–H bonds in question,
which obviously should be reflected in the activation bar-
rier of the reaction. To clarify which effect dominates in
hydrogen abstraction and epoxidation reactions, we deter-
mined the thermal activation parameters DH= and DS= for
the reactions of some selected substrates with model por-
phyrin complexes for compound I, which is the most cru-
cial intermediate discussed as part of P450 catalysis, and
for its one-electron-reduced form compound II. Although
compound II is known to be a sluggish oxidant compared
with compound I [4], it is still a competent catalyst in a
number of different reactions [36]. As this is the first study
to investigate enthalpy and entropy effects for compound
II, and moreover the first to compare activation parameters
for both of the most discussed active species involved in
catalytic oxygenation reactions, it provides new insight into
mechanistic aspects that influence the reactivity of com-
pounds I and II.
Results
Iron(III) meso-tetramesitylporphyrin complexes are known
to be good biomimetics to study the catalytic reactions of
cytochrome P450 since the choice of an appropriate
oxidant in combination with carefully selected reaction
conditions allows a proper generation and sufficient sta-
bilization of different reactive intermediates, i.e., com-
pounds 0, I, and II, in solution for several minutes [20, 36].
The addition of an excess of m-chloroperoxybenzoic
acid to a solution of [FeIII(TMP)OH] or [FeIII(TMP)Cl] in
acetonitrile at low temperatures leads to the formation of
the cation radical species [(TMPꢀ?)FeIV=O] (compound I).
This is due to an acid-catalyzed heterolytic cleavage of the
O–O bond (two-electron oxidation), in which the excess of
m-chloroperoxybenzoic acid provides the necessary pro-
tons [20, 36, 46, 47].
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29
Scheme 1 Structure of
FeIII(TMP)X [TMP is meso-
tetra(2,4,6-
trimethylphenyl)porphyrin] and
its reaction with
m-chloroperoxybenzoic acid
(m-CPBA) or iodosylbenzene
(PhIO) to produce compound I
(Cpd I) or compound II
(Cpd II), respectively
Scheme 2 Structures of the substrates employed, viz., cis-stilbene,
9,10-dihydroanthracene (DHA), and 9H-xanthene
In aprotic solvents such as acetonitrile, the choice of a
nonacidic oxidizing agent—in our case iodosylbenzene—
converts [FeIII(TMP)OH] into compound II [(TMP)FeIV=O]
(see Scheme 1) [36, 48, 49].
Compounds I and II can be easily identified by careful
analysis of the spectral changes observed in the resulting
time-resolved UV/vis spectra. The formation of compound
I is associated with a large absorbance decrease and a shift
to a shorter wavelength of the Soret band combined with an
absorbance increase between 550 and 700 nm, which is
characteristic for a high-valence oxo–iron(IV) porphyrin
p-cation radical, [(TMPꢀ?)FeIV=O] (see Figs. 1, S1) [20,
36].
Fig. 1 Comparison of the UV/vis spectra for compound I (red line)
and compound II (green line) produced from [FeIII(TMP)OH] (TMP
is meso-tetra(2,4,6-trimethylphenyl)porphyrin) (black line). Inset:
Magnified view of the spectra between 475 and 625 nm
formed, and clean isosbestic points as well as clean one-
exponential kinetic fits support this observation.
In contrast to the cation radical compound I, the plain
iron(IV)–oxo species compound II shows a small shift to a
longer wavelength together with an absorbance increase of
the Soret band. Furthermore, a new broad band at 550 nm
occurs, which can be used to identify compound II spec-
troscopically (Figs. 1, S2) [36]. Unlike similar model
complexes, [FeIII(TMP)OH] is known to form compound II
as the sole stable product under these conditions [36, 49].
We found no evidence for the formation of a mixture of
reactive intermediates as reported for other systems [50],
i.e., compound II and traces of compound I formed via
disproportionation of compound II. In fact, the cation
radical band of compound I is absent when compound II is
Furthermore, an experiment that is commonly used to
prove the existence of a mixture of reactive intermediates
[50] was performed. The addition of various amounts of
[FeIII(TMP)OH], after the formation of compound II by a
minimum amount of iodosylbenzene and prior to the
addition of substrate, should suppress the fast dispropor-
tionation reaction of compound II and therefore the for-
mation of the very reactive compound I. Under such
conditions, the observed rate constants for the oxygenation
of the substrate (concentrations of the oxidant and the
substrate were kept constant in each experiment) should be
inversely proportional to the concentration of added
[FeIII(TMP)OH]. However, this experiment revealed only
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very small changes (within the experimental error limits) in
the resulting rate constants with increasing concentration of
added [FeIII(TMP)OH]. This is further strong evidence that
compound II is indeed the sole active oxidant in the system
studied.
Regardless of the particular oxygenation products, the
rate constants determined in dehydrogenation/hydroxyla-
tion and epoxidation reaction sequences concern the rate-
determining initial reaction steps. These steps are identical
for compounds I and II, i.e., hydrogen abstraction and
formation of the intermediate with a radical residue in
dehydrogenation/hydroxylation reactions, and the forma-
tion of a C–O bond to form the radical intermediate in
epoxidation reactions, respectively (see Schemes 3, 4).
Therefore, we can directly compare the activation param-
eters determined for compounds I and II for the particular
type of oxygenation reactions, as they can be associated
with the same rate-limiting steps.
Since compounds I and II can be selectively produced
and unambiguously identified by their characteristic UV/
vis spectra, temperature-dependent measurements can be
performed as these intermediates show a remarkable sta-
bility for many minutes in acetonitrile over a sufficiently
wide temperature range.
Compounds I and II were produced in solution as
described already, and their formation and stability in
solution were continuously monitored by UV/vis spec-
troscopy before the addition of the substrate. In the course
of this study, three different substrates were used (see
Scheme 2). cis-Stilbene is a common substrate used to
investigate epoxidation of olefins by compound I or com-
pound II [20, 36, 51, 52], whereas DHA and 9H-xanthene
were used to study C–H abstraction reactions (see
Schemes 3, 4) [36, 52, 53]. The latter are known to be the
rate-determining initial steps in dehydrogenation and
hydroxylation reactions [10, 41, 43], operating in compli-
ance with the ‘‘rebound mechanism,’’ which was initially
postulated by Groves et al. [54] more than 30 years ago and
which is still gaining growing support by current research
[55]. Important to note, there are two possible pathways
following the initial C–H abstraction step [41]. In the case
of DHA, a dehydrogenation rather than a hydroxylation
step is expected to operate, leading to anthracene as a very
stable aromatic product [53].
Upon injection of a substrate into the reaction mixture,
the decomposition reaction of the generated intermediate
was observed, which is directly related to the reaction with
the substrate. In all cases the reactive intermediates
reverted to iron(III) porphyrin species, giving clean isos-
bestic points in the resulting rapid-scan spectra recorded
during the overall reaction sequence (see Figs. 2, 3, S3–S5).
In all reactions compound I or compound II was remarkably
stable even at high temperatures, and the spontaneous
decomposition of these reactive intermediates was extremely
slow compared with the observed reaction with the substrate
(see Fig. S6). Moreover, the single-exponential fits of the
kinetic traces obtained indicate that only primary oxidation
reactions have to be taken into account since secondary
reactions are not involved.
Two reaction steps operate in the hydroxylation or
dehydrogenation reactions of compound I, viz., an initial
reduction of the cation radical species [(TMPꢀ?)FeIV=O] to
[(TMP)FeIV–OH], which can also be regarded as a com-
pound II intermediate [57–59], and a second, very fast
‘‘rebound’’ step ending with the reformation of an iron(III)
porphyrin (see Fig. S3) [10, 41, 43]. Since the lifetimes of
the radical intermediates are known to be ultrashort
because the rebound step is extremely fast [2, 4, 10], only
the rate-determining initial step is reflected in the kobs
values determined. In contrast, in the case of C–H
abstraction reactions with compound II, a single reaction
step operates, which is also equivalent to C–H abstraction
(see Figs. 2, 3). Therefore, this C–H abstraction step can be
In the same way, epoxidation reactions can be investi-
gated, where the rate-determining step is the initial C–O
bond formation (see Scheme 4) [10, 51, 52]. Competing
hydroxylation/epoxidation reactions [41] do not play a role
when these substrates are used, as on the one hand cis-
stilbene does not bear any saturated hydrocarbons neces-
sary for C–H abstraction, and on the other hand DHA and
xanthene lack double bonds accessible for epoxidation. In
general, benzylic hydrogen is known to be a rather sluggish
reaction partner in hydroxylation reactions [56], so these
possible side reactions have no impact on our studies.
Scheme 3 The ‘‘rebound
mechanism’’ in hydroxylation
reactions with compound I
including the transition state
(TS) for the rate-limiting step
[5, 10]
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J Biol Inorg Chem (2012) 17:27–36
31
Scheme 4 The mechanism for
epoxidation reactions with
compound I including the
transition state (TS) for the
rate-limiting step [10]
investigated for the reaction of DHA or xanthene with each
of the two reactive intermediates. In the same manner, this
can also be accomplished for the rate-determining C–O
bond formation step in epoxidation reactions (see Figs. S4,
S5).
For both oxidizing species, kobs showed a linear
dependence on the substrate concentration with no signif-
icant intercept even at higher temperatures (see Fig. S7).
This finding unambiguously rules out the participation of
side reactions such as competing spontaneous decomposi-
tion of the reactive intermediate, the reformation of the
latter with an excess of oxidant present in the reaction
medium, and the contribution of secondary oxidations of
the substrate in the measured processes.
To ensure that the reformation of compound I or com-
pound II is not reflected in the resulting rate constants, the
observed rate constants for the formation of the oxidizing
species and for its decomposition in the reaction with the
substrate were tuned in such way (based on the known
second-order rate constants for these reactions and the
relative concentration of the substrate and oxidant used)
that the reformation of the respective intermediate did not
have a notable effect on the resulting data.
Fig. 2 Rapid-scan spectra for the conversion of [FeIII(TMP)OH] into
compound II upon addition of iodosylbenzene (PhIO) with the
subsequent reformation of [FeIII(TMP)OH] upon addition of xan-
thene. Experimental conditions: 2.7 9 10-6
M
FeIII(TMP)OH,
6.0 9 10-6 M PhIO, 4.5 9 10-3 M xanthene in acetonitrile at
- 15 °C
To compare the particular reactivity of each oxidizing
intermediate at different temperatures (usually between
-25 and 5 °C), we examined their reactions with various
substrates under pseudo-first-order conditions. As demon-
strated by Fig. 4, the reaction rate shows a significant depen-
dence on the selected temperature (see also Figs. S8–S10).
According to the logarithmic form of the Eyring equation, the
enthalpy of activation can be calculated from the slope and
the entropy of activation can be calculated from the intercept
of the resulting linear plot of ln(kcompound I/T) versus 1/T or
ln(kcompound II/T) versus 1/T for the oxygenation reactions by
compounds I and II, respectively (see Figs. 5, 6). A summary
of the activation parameters determined is presented in
Table 1.
Fig. 3 Kinetic trace for the reaction shown in Fig. 2 recorded at the
Soret band (417 nm). Starting with [FeIII(TMP)OH] (1), the addition
of PhIO leads to the formation of compound II (2). Upon addition
of xanthene, the reduction back to [FeIII(TMP)OH] (3) can be
monitored. Experimental conditions: 2.7 9 10-6 M FeIII(TMP)OH,
6.0 9 10-6 M PhIO, 4.59 10-3 M xanthene in acetonitrile at –15 °C
The second-order rate constants at -15 °C (see Table 1)
calculated from the activation parameters are in close
agreement with those determined from the concentration
dependence measurements in our earlier study [36], which
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Fig. 6 Determination of the thermal activation parameters by
temperature-dependent measurements of the reaction of compound
II with various substrates
Fig. 4 The temperature-dependent reduction of compound II back to
[FeIII(TMP)OH] can be monitored upon addition of xanthene.
Experimental conditions: 2.7 9 10-6 M FeIII(TMP)OH, 6.0 9 10-6
M
PhIO, 4.5 9 10-3 M xanthene in acetonitrile. Kinetic traces recorded at
the Soret band (417 nm)
complexes for compounds I and II. For most of the reac-
tions studied, the activation enthalpy contributes most to
the overall activation barrier. Only in a few cases does the
activation entropy make a considerable contribution or
contribute even more than the activation enthalpy. These
general trends form the basis for the further discussion.
If C–H abstraction reactions are enthalpy-controlled, the
respective rate constants should be closely related to the
strength of the bond to be activated. Since the formation of
the transition state is connected with partial electron
transfer from the carbon atom, accompanied by the partial
dissociation of the C–H bond with a concomitant formation
of an O–H bond, it can be expected that the activation
barriers for the formation of the transition state should
correlate with the dissociation energies of C–H and O–H
bonds, i.e., BDEC–H and BDEO–H, respectively. If we
consider substrate oxygenation reactions by one oxidant
(compound I or compound II), the BDEO–H value remains
constant throughout the series of hydrogen abstraction
reactions with various substrates. Therefore, only the cor-
relation of the activation barrier, DH=, with BDEC–H has to
be considered. As a result, this bond strength should be
reflected by the activation enthalpy, i.e., a stronger C–H
bond requires a higher activation enthalpy, which causes a
higher activation barrier (DG=) and consequently a lower
rate constant for this particular reaction. This trend can be
observed when comparing the activation parameters
determined for DHA and xanthene (i.e., a lower BDEC–H
causes a lower DH=, which leads to a lower DG= and a
higher rate constant).
Fig. 5 Determination of the thermal activation parameters by
temperature-dependent measurements of the reaction of compound I
with various substrates
further supports the proposal that the resulting activation
parameters can be assigned to the reactions investigated.
Discussion
The results summarized in Table 1 allow us to distinguish
between contributions arising from the activation enthalpy
(DH=) and the activation entropy (expressed as TDS=)
towards the overall activation barrier (DG= = DH=-
TDS=). In this way it is possible to draw conclusions
concerning the importance of enthalpy and entropy effects
during the oxygenation of different substrates by the model
Comparison of the activation enthalpies for compound I
with those for compound II measured for the same sub-
strate reveals that the activation enthalpies for the reaction
of compound I are substantially lower than those for
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33
Table 1 Activation parameters, C–H bond energies [70], and rate constants calculated from DG= at -15 °C for the reaction of compounds I and
II with various substrates
Â
Ã
ꢂ
¼
kCpdIðꢁ15 CÞ Mꢁ1 Sꢁ1
Substrate
DH= (kJ/mol) DS= (J/K mol) -T-15 °CDS= (kJ/mol)
BDEC–H (kJ/mol)
DGꢁ15 ꢂC(kJ/mol)
Compound I
cis-Stilbene
DHA
46
42
1
1
-43
-41
4
5
4
11
11
25
1
1
1
57
53
49
2
2
5
–
15.8 0.6
(1.01 0.04) 9 102
(6.5 0.7) 9 102
319.45
310.66
Xanthene
24.4 0.9
-100
Compound II
cis-Stilbene 52.9 0.5
-57
-41
-85
2
5
5
14.7 0.5
68
62
59
1
5
3
–
0.094 0.001
1.5 0.1
DHA
51
37
3
1
11
22
2
2
319.45
310.66
Xanthene
6.2 0.3
DHA 9,10-dihydroanthracene, BDE bond dissociation energy
compound II. This finding is in agreement with the
experimentally observed oxygenation strength of these two
oxidants. Significantly higher activation barriers for com-
pound II indicate that the hydrogen abstraction by com-
pound II is sluggish compared with that for compound I.
Theoretical calculations of activation barriers, viz., quan-
tum mechanical/molecular mechanical studies, demon-
strated that hydrogen abstraction barriers for compound II
are approximately 16 kJ/mol higher than those for com-
pound I [60, 61]. In this work we found the experimentally
determined differences between the activation enthalpies
for compounds I and II measured for various substrates to
range between 9 and 13 kJ/mol depending on the substrate
used.
difference of the alkyl residue in the free substrate and in
the fully relaxed geometry of the alkyl radical (REAlk), i.e.,
DC–H = BDEC–H ? REAlk. Consistent with this finding is
the possibility that the substrates characterized by higher
BDEC–H can display a similar or even lower energy barrier
for the transition state than a substrate with lower BDEC–H
if the value of REAlk is high enough. This is the case when
the formation of the alkyl radical residue in the transition
state is energetically favorable. When we take into account
that the REAlk values for the substrates studied can differ
(especially in the case of xanthene, owing to the stabilizing
resonance energy), the combination of a low activation
enthalpy with a relatively high BDEC–H observed in our
study appears to be reasonable and in line with computa-
tional predictions.
Moreover, DH= might not even play a decisive role for
the height of the activation barrier and thus for the resulting
rate constants. In the case of DHA, the overall process is
clearly enthalpy-controlled, whereas for xanthene the
activation entropy also contributes significantly, especially
in the case of compound I, where the contribution of TDS=
is very similar to that of DH=.
As mentioned before, hydroxylation reactions follow the
oxygen rebound mechanism that involves a rate-deter-
mining C-H abstraction as the first reaction step [10, 41, 54,
55]. Takahashi et al. [43] found that this type of reaction
can be entropy-controlled when the cation radical com-
pound I is used as oxidizing species. This finding implies
that the initial hydrogen abstraction step requires a highly
ordered transition state characterized by a loss of transla-
tional and rotational degrees of freedom that results in a
significantly negative DS=. On the basis of the data given
in Table 1, one can calculate that in the reaction of xan-
thene with compound I the entropy term TDS= shows a
higher contribution than DH= to DG= at temperatures
above -29.2 °C. In contrast, the reactions with DHA
show a significantly less negative activation entropy
and are therefore enthalpy-controlled. To understand this
It seems that the low barrier for C–H activation for
xanthene is the result of other factors. According to the
computational calculations, the barrier for the C–H
abstraction reaction can be lowered by an increase in res-
onance energy of the transition state. It can be expected
that in the case of xanthene an increase in resonance energy
caused by an enhanced conjugation of the oxygen lone pair
results in a decrease of the activation barrier [62]. In the
same way, the activation entropy is also affected, since this
stabilization suggests energetically more stable intermedi-
ates and thus a closer interaction between the reaction
partners. Consequently, their translational and rotational
degrees of freedom are limited, which is reflected by a
more negative DS= compared with that for DHA.
Although the bond energies can be regarded as the
thermodynamic driving force, they do not necessarily
correlate with the level of the activation barrier for the
transition state. According to recent findings from com-
putational studies [62], the calculated activation barriers
can show a poor correlation with BDEC–H, but a very good
correlation with the bond strength quantity (DC–H). The
latter is a more reliable measure of the interaction strength
between the bound alkane and hydrogen moieties, and is
defined as the combination of BDEC–H and the energy
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difference, one should consider that DHA is a symmetrical
molecule, whereas xanthene has a notable dipole moment
that originates from the partially negatively charged oxy-
gen bridge and a more positive CH2 bridge on the opposite
side of the molecule [63]. If we consider that both reactive
intermediates (compounds I and II) bear a very positive
iron center, a possible explanation is that xanthene tends to
approach the reaction partner in a ‘‘wrong’’ orientation in
contrast to DHA, i.e., with the oxygen bridge pointing to
the iron center. If we keep in mind that compound II is an
iron(IV) species, whereas compound I is an iron(IV) cation
radical [and therefore a formal iron(V) species], it is rea-
sonable that this effect might have a larger impact on
reactions with compound I owing to its higher charge,
which is in line with the data given in Table 1.
agreement with the results from previous studies on com-
pound I mimics [43]. As a consequence, the difference in
the reactivity between compounds I and II for the epoxi-
dation of cis-stilbene clearly arises from effects that control
the activation enthalpy, i.e., factors such as bond strength
rather than the order of the transition state. This is in sharp
contrast to the results for C–H abstraction reactions, where
different entropic factors induce different reactivities for
compounds I and II, as this reaction proceeds via another
pathway and transition state.
The large impact of the activation entropy, in general,
and the entropic control of the hydrogen abstraction reac-
tion of xanthene with compound I, in particular, have
important consequences for practical work as well as for
theoretical considerations. For such a scenario the Gibbs
free energy relationship DG= = DH=-TDS= demands a
close relation with the reaction temperature. As shown in
Table 1, at -15 °C the reaction of compound I with DHA
has a contribution from the activation entropy of 11 kJ/mol
In contrast, the reaction with the symmetrical DHA
molecule shows no difference in the activation entropies
and gives almost identical values for the reaction with
compounds I and II. In this case the difference in the
reactivity clearly arises from different activation enthalpies,
which reflect a higher ability of compound I to induce C–H
abstraction processes. For this reason, the reaction with
DHA is enthalpy-controlled, no matter whether the reaction
partner is compound I or compound II. In contrast, the
corresponding reaction of xanthene with the cation radical
species (compound I) is entropy-controlled, whereas the
reaction with compound II is still enthalpy-controlled as
here the activation enthalpy is generally higher.
¼
to the overall activation barrier of DGꢁ15ꢂC ¼ 53 kJ=mol;
whereas the reaction of xanthene has a much larger entropy
contribution of -TDS= = 25.7 kJ/mol and DH=
=
24.4 kJ/mol to the free energy activation barrier. Conse-
quently, the reaction of compound I with xanthene
becomes slower compared with the reaction with DHA on
increasing the temperature. On the basis of the data given,
an isokinetic temperature of 25.2 °C can be calculated
where both reactions have the same activation barrier.
Upon a further increase in temperature, the reaction rate for
the C–H abstraction of compound I with DHA even
exceeds that for the reaction with xanthene. Even though
the respective reactions with compound II are in general
enthalpy-controlled, the isokinetic temperature describing
this change in the reactivity order is only 45.0 °C, as in the
case of xanthene again a lower activation enthalpy is
combined with an about twice as high activation entropy
in comparison with DHA (see the example in Table 1 at
-15 °C). Although the isokinetic temperatures given
should be evaluated with caution since they include large
error limits due to the calculation method, they at least
suggest that these switchover points can play a role in a
biologically relevant temperature range.
In the same way, a change in regioselectivity by the
variation of the temperature in the competitive C=C bond
epoxidation versus C–H hydroxylation of cyclohexene
has been reported in some cases [42]. Again, this can be
accounted for in terms of a large contribution of the
activation entropy term to the activation barrier in
hydroxylation reactions that increases with temperature,
whereas the epoxidation reaction is clearly dominated by
the activation enthalpy. Therefore, the latter becomes
favorable over the hydroxylation reaction at higher
temperatures.
To further clarify this impact, we studied another com-
mon type of reaction, viz., the epoxidation of cis-stilbene.
In this case we are dealing with a partially negatively
charged double bond, and it is therefore likely that its
attraction to a more positively charged iron center bearing
a more electrophilic oxygen promotes the reaction with
compound I more than the reaction with compound II,
since different contributions from aspects such as orienta-
tion, trajectory, and accessibility can be expected. Conse-
quently, DS= is less negative for compound I than for
compound II in epoxidation reactions, whereas the opposite
effect is observed when C–H abstraction reactions are
studied.
In summary, the differences in the activation enthalpies
can largely account for the higher reactivity of compound I
compared with compound II in epoxidation as well as in
C–H abstraction reactions. This result reflects the higher
oxidative power of compound I and is in line with a
stronger Fe=O bond and stronger stabilization of oxygen in
compound II models as reported in the literature [64]. This
effect might be smaller than expected since similar bond
lengths were found for the Fe=O moiety in some com-
pound I and compound II species [65].
The epoxidation reaction is clearly enthalpy-controlled
for both compound I and compound II models, which is in
123

J Biol Inorg Chem (2012) 17:27–36
35
In this respect, also some theoretical approaches that are
based on the application of the activation energy Ea should be
reconsidered, since Ea neither reflects entropic effects nor
shows a dependence on the temperature. As a consequence,
the use of Ea instead of DG= in computational studies can be
misleading when the activation entropy (and in this respect
also the temperature) plays a dominant role [43].
models for compounds I and II in epoxidation and C–H
abstraction reactions. This comparison reveals an important
impact of the chemical nature of the substrate and the
reactive intermediate (viz., compound I or compound II) on
the resulting reaction rate in different types of reaction,
which goes beyond the common concepts that relate the
activation barrier to bond energies in C–H abstraction
reactions. In fact, the crucial question is to what extent the
activation entropy contributes to the overall activation
barrier and thus to the resulting rate constants.
Furthermore, a common method to visualize a direct
relationship between the C–H bond strength and the rate
constants for the rate-determining hydrogen abstraction
reaction is to plot log(k/n) versus BDEC–H (where n is the
number of abstractable substrate hydrogen atoms). This
plot is expected to result in a linear relationship showing
that log(k/n) decreases with increasing bond strength
according to the Bell–Evans–Polanyi relationship [66, 67].
Remarkably, such a close connection between BDEC–H and
DH= is not necessarily given, as discussed above. In
addition, this correlation demands enthalpy-controlled
reactions, as only in that case can bond strengths be liable
for the resulting rate constants by affecting the dominant
activation enthalpy and in this way the activation barrier.
As soon as the activation entropy plays a major role in the
activation process, such a close relation no longer exists,
although the logarithmic expression of the rate constants as
well as compensation effects might cover the decreasing
impact of the bond strength if the data set is limited to only
a few substrates. Nevertheless, especially weak C–H bonds
suggest a relatively large influence of the activation
entropy. Consequently, a plot of the low BDE values ver-
sus log(k/n) should result in a horizontal line. This fact has
been intensively discussed for different hydrogen-
abstracting agents such as the t-butoxyl radical [68], but
has been widely ignored when considering reactions of
P450 biomimetics.
As demonstrated in this study, epoxidation reactions are
clearly enthalpy-controlled, whereas C–H abstraction
reactions can in many cases be dominated by factors con-
tributing to the activation entropy. Since an important
impact of the activation entropy contribution leads to a
significant dependence of the activation barrier on the
temperature, a close correlation between bond strength and
reaction rate—as commonly assumed for C–H abstraction
reactions—no longer exists. In this respect, also theoretical
calculations using the activation energy (Ea) instead of
DG= as a measure for the activation barrier have to be
interpreted with caution, because Ea is independent of
temperature.
Therefore, comparative studies in which rate constants
for different substrates, different reaction types, or different
reactive intermediates (viz., compounds I and II) are
determined can in some cases be misleading. As a conse-
quence, the particular impact of the entropic contribution
and a possible influence of the reaction temperature should
be considered when evaluating experimental as well as
theoretical results, as these effects can cause substantial
changes in the reactivity order of different catalytic
species.
Acknowledgment The authors gratefully acknowledge continued
financial support from the Deutsche Forschungsgemeinschaft.
Transferring the results of biomimetic studies at low
temperatures to biological conditions even amplifies
the relevance of the entropic contribution. Furthermore,
enzymes appear to have sterically more demanding super-
structures. On the other hand, the entropic impact may be
compensated by the fact that water molecules leave the
active pocket when substrates enter. Therefore, these mole-
cules already lose a large part of their rotational and trans-
lational degrees of freedom prior to the actual reaction with
the iron center. As a consequence, the activation entropy,
and for this reason the free energy barrier, for enzyme
reactions is usually lower than for bimolecular model system
reactions [5, 69].
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