Anilinic N-oxides support cytochrome P450-mediated N-dealkylation through hydrogen-atom transfer

Article abstract of DOI:10.1002/chem.201000185

The mechanism of N-dealkylation mediated by cytochrome P450 (P450) has long been studied and argued as either a single electron transfer (SET) or a hydrogen atom transfer (HAT) from the amine to the oxidant of the P450, the reputed iron-oxene. In our study, tertiary anilinic N-oxides were used as oxygen surrogates to directly generate a P450-mediated oxidant that is capable of N-dealkylating the dimethylaniline derived from oxygen donation. These surrogates were employed to probe the generated reactive oxygen species and the subsequent mechanism of N-dealkylation to distinguish between the HAT and SET mechanisms. In addition to the expected N-demethylation of the product aniline, 2,3,4,5,6-pentafluoro-N,N-dimethylaniline N-oxide (PFDMAO) was found to be capable of N-dealkylating both N,N-dimethylaniline (DMA) and N-cyclopropyl-N-methylaniline (CPMA). Rate comparisons of the N-demethylation of DMA supported by PFDMAO show a 27-fold faster rate than when supported by N,N-dimethylaniline N-oxide (DMAO). Whereas intermolecular kinetic isotope effects were masked, intramolecular measurements showed values reflective of those seen previously in DMAO- and the native NADPH/O₂-supported systems (2.33 and 2.8 for the N-demethylation of PFDMA and DMA from the PFDMAO system, respectively). PFDMAO-supported N-dealkylation of CPMA led to the ring-intact product N-cyclopropylaniline (CPA), similar to that seen with the native system. The formation of CPA argues against a SET mechanism in favor of a P450-like HAT mechanism. We suggest that the similarity of KIEs, in addition to the formation of the ring-intact CPA, argues for a similar mechanism of Compound I (Cpd I) formation followed by HAT for N-dealkylation by the native and N-oxide-supported systems and demonstrate the ability of the N-oxide-generated oxidant to act as an accurate mimic of the native P450 oxidant.

Full text of DOI:10.1002/chem.201000185

DOI: 10.1002/chem.201000185
Anilinic N-Oxides Support Cytochrome P450-Mediated N-Dealkylation
through Hydrogen-Atom Transfer
Kenneth M. Roberts and Jeffery P. Jones*[a]
Abstract: The mechanism of N-dealky-
lation mediated by cytochrome P450
(P450) has long been studied and
argued as either a single electron trans-
fer (SET) or a hydrogen atom transfer
(HAT) from the amine to the oxidant
of the P450, the reputed iron–oxene. In
our study, tertiary anilinic N-oxides
were used as oxygen surrogates to di-
rectly generate a P450-mediated oxi-
dant that is capable of N-dealkylating
the dimethylaniline derived from
oxygen donation. These surrogates
were employed to probe the generated
reactive oxygen species and the subse-
quent mechanism of N-dealkylation to
distinguish between the HAT and SET
mechanisms. In addition to the expect-
ed N-demethylation of the product ani-
line, 2,3,4,5,6-pentafluoro-N,N-dimeth-
G
aniline N-oxide (PFDMAO) was
lation of PFDMA and DMA from the
PFDMAO system, respectively).
PFDMAO-supported N-dealkylation of
CPMA led to the ring-intact product
N-cyclopropylaniline (CPA), similar to
that seen with the native system. The
formation of CPA argues against a
SET mechanism in favor of a P450-like
HAT mechanism. We suggest that the
similarity of KIEs, in addition to the
formation of the ring-intact CPA,
argues for
a similar mechanism of
Compound I (Cpd I) formation fol-
lowed by HAT for N-dealkylation by
the native and N-oxide-supported sys-
tems and demonstrate the ability of the
N-oxide-generated oxidant to act as an
accurate mimic of the native P450 oxi-
dant.
Introduction
Compound I (Cpd I) species of chloroperoxidase as the re-
active oxygen species (ROS) in these reactions.[1–4]
Cytochrome P450 (P450) enzymes are a ubiquitous super-
family of heme-containing monoxygenases capable of oxi-
dizing endogenous and exogenous substrates, including the
majority of clinically relevant pharmaceuticals.[1] Decades of
study have been spent understanding the mechanism of
P450-mediated oxidation. The consensus mechanism in-
volves a multistep activation of molecular oxygen.[1] Activa-
tion leads to a series of oxygen intermediates that culminate
in an iron(IV)–oxo porphyrin p-radical cation similar to the
N-Dealkylation has been studied as one of the many oxi-
dations performed by the proposed Cpd I. The pathway in-
À
volves oxygen insertion by Cpd I into the Ca H bond to
generate a carbinolamine that spontaneously dealkylates
forming the free amine and aldehyde (Scheme 1). Recent
Scheme 1. N-Dealkylation (N-demethylation) overview.
[a] Dr. K. M. Roberts, Prof. J. P. Jones
Department of Chemistry, Washington State University
PO BOX 644630, Pullman, WA 99164-4630 (USA)
Fax : (+1)509-335-8867
studies of this pathway have centered on two proposed
mechanisms.[5] One mechanism proposes a single electron
transfer (SET) from the target nitrogen to Cpd I forming an
iron(IV)–oxo porphyrin (Cpd II) and an aminium radical
cation (Scheme 2). Cation formation increases the acidity of
E-mail: jpj@wsu.edu
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201000185.
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pared propranolol oxidation in P4502D6 supported by
CuOOH and the native NADPH/O2 system and found dif-
ferent preferences for sites of oxidation for the two oxi-
dants.[18] Further, Guengerich et al. showed that the kinetic
isotope effects (KIEs) of the N-dealkylation of two substi-
tuted N,N-dimethylanilines (DMAs) by PhIO and CuOOH
were large (6.7–7.3 and 3.4–3.7, respectively) compared to
the small KIEs (1.7–2.3) seen for the native NADPH/O2
pathway of the enzyme.[16] This difference was supported by
theoretical calculations from Shaikꢁs group, who concluded
that PhIO and NADPH/O2 generated unique spin states of
Cpd I and that these spin states were responsible for the dif-
ferent isotope effects.[17]
Scheme 2. Pathways for hydrogen atom transfer (HAT) and single elec-
tron transfer (SET) (por=porphyrin).
Ca and drives a subsequent proton transfer to Cpd II. The
final step is the barrierless formation of the carbinolamine
by transfer of a hydroxyl radical to the resulting carbon rad-
ical.[6] In contrast, the second mechanism proposes that
Cpd I elicits a hydrogen atom transfer (HAT) from Ca to di-
rectly form the carbon radical and the protonated Cpd II
(Scheme 2). In attempts to distinguish between SET and
HAT mechanisms in metal-catalyzed N-dealkylations, stud-
ies with biomimetic systems have argued for SET mecha-
nisms in these systems.[7–10] Addressing this issue in enzymat-
ic systems, Shaffer et al. introduced N-cyclopropyl-N-meth-
ylaniline (CPMA) as a new probe for distinguishing between
the two mechanisms.[11,12] They found that P450-mediated
oxidation of CPMA led to products that left the cyclopropyl
ring intact.[12] This was in contrast to the solely ring-open
products found upon oxidation by horseradish peroxidase
(HRP),[11] an enzyme known to undergo a SET mecha-
nism.[13,14] They concluded that P450 follows a HAT mecha-
nism in the exclusion of the SET mechanism. Further,
Cerny and Hanzlik found that P450-mediated oxidation of
tertiary cyclopropylamines led to both ring-intact and ring-
open products.[15] However, ring-open products only ap-
peared in the second round of N-dealkylation. Simply, a
prior round of N-dealkylation was required and ring-opened
products derived solely from the ring-intact secondary
amine product of the first N-dealkylation. They concluded a
HAT mechanism for N-dealkylation that incorporated ab-
straction of the N hydrogen as explanation of the ring-open
products.
Confirmation of the HAT mechanism, as well as other ox-
idations proposed to act via Cpd I, requires showing that
Cpd I elicits these reactions. However, the activation of mo-
lecular oxygen leading to Cpd I is complex and involves sev-
eral steps including proton and electron transfers.[1] Due to
this complicated process, alternate sources of generating this
species directly for use as mechanistic probes have been ex-
plored. These compounds, termed oxygen surrogates, in-
clude iodosylbenzene (PhIO), cumene hydroperoxide
(CuOOH), and substituted N,N-dimethylaniline N-oxides
(DMAOs). Yet, the ROSs resulting from surrogacy have re-
cently come into question as valid mimics of P450
Cpd I.[4,16,17] Studies by Dawson et al. observing Cpd I for-
mation by oxygen surrogates found no evidence for Cpd I
when P450BM3 was exposed to PhIO.[4] Bichara et al. com-
In contrast, anilinic N-oxides, such as N,N-dimethylaniline
N-oxide, have recently been shown to generate an oxidant
that strongly mimics the oxidant of the NADPH/O2 path-
way. Dowers et al., compared the KIEs of the N-demethyla-
tion of substituted DMAs by their respective N-oxides and
by NADPH/O2 and found them to be identical.[19] It was hy-
pothesized that DMAOs donate
a six-electron oxygen
(oxene) directly generating Cpd I, which in turn oxidizes the
resulting DMA (Scheme 3A).
Scheme 3. Proposed formations of Cpd I (A) and Cpd II (B) by oxygen
donation from an N-oxide (por=porphyrin).
Theoretical calculations by Shaikꢁs group supported this
conclusion finding that oxygen donation by DMAO gener-
ates Cpd I with
a
calculated energy barrier of
21 kcalmolÀ1.[17] In comparison, for the N-demethylation
step, they calculated Cpd I spin-state-dependent energy bar-
riers of 6.4 kcalmolÀ1 and 8.8 kcalmolÀ1 for the doublet and
quartet, respectively, demonstrating the rate-determining
nature of the oxygen donation.
DMAO-generated Cpd I would be expected to support
oxidation of secondary substrates. However, in early work
with a porphyrin mimetic system, Bruice and Nee found
DMAO unable to significantly support olefin epoxidation.[20]
They rationalized that the ease of oxidation of DMA out-
competed olefin epoxidation for the putative iron–oxene
(Cpd I) and that increasing the oxygen donation rate and
decreasing the rate of N-dealkylation would be required to
facilitate oxygen surrogacy. They proposed that electron-
withdrawing substituents on the aromatic ring would realize
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J. P. Jones and K. M. Roberts
both effects and, in support of this, they successfully tested
p-cyano-N,N-dimethylaniline N-oxide (CDMAO) as a surro-
gate oxygen donor able to support various olefin epoxida-
tions as well as cyclohexane hydroxylation.[20]
Scheme 4. Kinetic scheme for oxygen donation.
In a study with P4502B1, however, Seto and Guengerich
found that they were unable to significantly oxidize a
second distinct DMA with either DMAO or CDMAO.[21]
They concluded that oxygen donation resulted from homo-
and product formation. As described above, the calculations
of DMAO by Shaikꢁs group show a much higher barrier to
oxygen donation (k3) than oxidation of the aniline (k5),
21 kcalmolÀ1 and 6.4–8.8 kcalmolÀ1, respectively. This higher
barrier supports oxygen donation as the rate-determining
step in this pathway.[17] We examined the rate-determining
step experimentally by using KIEs for the N-demethylation
of an N-oxide by P450cam. Product formation rates for p-
cyano-N,N-dimethylaniline N-oxide (CDMAO) and p-
À
lytic cleavage of the N O bond generating Cpd II and the
aminium radical cation (Scheme 3B). While Cpd II is poised
to deprotonate the Ca of the aminium radical, the oxidant
would not be electrophilic enough to react with an unoxi-
dized DMA.
In this work, we investigated the nature of the ROS gen-
erated by anilinic N-oxides in P450 and its ability to oxidize
secondary substrates. With a P450 HAT mechanism for N-
dealkylation supported by the work of Shaffer et al. with
CPMA[12] and the identical KIE values found by Dowers et
al. for N-dealkylation by DMAOs and P450,[19] we hypothe-
sized that DMAOs donate an oxene to the P450 heme to
form a Cpd I poised to oxidize the resulting DMA through
a HAT mechanism. Further, similar to Bruice and Nee,[20]
we proposed that DMAO surrogacy is limited by the ease of
oxidation of the subsequent DMA and that increasing the
electron-withdrawing character on the aromatic ring would
increase the rate of oxygen donation and slow down the N-
demethylation of the subsequent aniline. To evaluate these
considerations, we employed a newly synthesized oxygen
surrogate, 2,3,4,5,6-pentafluoro-N,N-dimethylaniline N-oxide
(PFDMAO), with the expectation that the heavily electron-
withdrawing fluorines would increase the rate of Cpd I for-
mation and decrease the rate of N-dealkylation, unmasking
its ability to act as a surrogate. Experimental results demon-
strate that oxygen donation by PFDMAO is faster than by
DMAO with support by theoretical calculations that show a
lower barrier. We also show that the PFDMAO-derived oxi-
dant is capable of N-dealkylating DMA and CPMA, sub-
strates distinct from the generated aniline. In the surrogate
system, N-demethylation of DMA is faster than that of the
cyano-N,N-bis(trideuteriomethyl)aniline
N-oxide
([D6]CDMAO) were compared in intermolecular experi-
ments. Kinetic isotope effects were calculated from the
ratios of p-cyano-N-methylaniline and p-cyano-N-(trideuter-
iomethyl)aniline formed, both in separate incubations (non-
competitive) and when incubated together (competitive).
Given Scheme 4, three possibilities for isotope effects are
expected that depend on the rate-determining step: 1) On-
rates (k1H/k1D) would be expected to display no isotope
effect, so if binding is rate-determining, no isotope effect on
product formation would be observed; 2) a rate-determining
oxygen donation (k3H/k3D) would show a b-secondary iso-
tope effect on product formation; b-secondary isotope ef-
fects, however, can be small and may be indistinguishable
from 1. 3) If N-demethylation (k5H/k5D) is rate-determining,
a KIE of around 2.8 would be expected based on intrinsic
isotope effect values measured experimentally by Dower
et al.[19] Our intermolecular experiments show no significant
isotope effect (Table 1). This suggests that the isotope effect
Table 1. Kinetic isotope effects on N-demethylation by N-oxide-support-
ed P450cam.
kH/kD—[D0]/[D6]
kH/kD—[D0]/[D6]
kH/kD
intramolecular
noncompetitive[a,b]
competitive[a,b]
resulting
2,3,4,5,6-pentafluoro-N,N-dimethylaniline
CDMAO[c]
0.9Æ0.2
1.2Æ0.2
1.2Æ0.1
1.3Æ0.2
2.77Æ0.05[e]
PFDMAO[d]
2.33Æ0.07[f]
(PFDMA), supporting the greater ease of oxidation of
DMA relative to the more electron-withdrawn PFDMA.
Further, N-dealkylation of CPMA formed the ring-intact
product N-cyclopropylaniline (CPA). CPA formation, kinet-
ic values and theoretical calculations support a mechanism
of Cpd I formation followed by HAT.
[a] [D0]=unlabeled. [b] [D6]=(CD3)2-labeled. [c] CDMAO=p-cyano-
N,N-dimethylaniline N-oxide. [d] PFDMAO=2,3,4,5,6-pentafluoro-N,N-
dimethylaniline N-oxide. [e] From reference [19]. [f] Measured by using
2,3,4,5,6-pentafluoro-N-methyl-N-trideuteriomethylaniline
for N-dealkylation is being masked by a prior, slower step:
either substrate binding or oxygen donation. Surface plas-
mon resonance measurements by Pearson et al. of the bind-
ing of the rod-like antifungals itraconazole and ketoconazole
with P450 3A4 measured substrate on-rates of 103–
104 mÀ1 sÀ1, much faster than either substrate or product off-
rates.[22] That the non-globular substrates itraconazole and
ketoconazole can still bind enzyme at this rate demonstrates
the rapidity of substrate binding, excluding binding as the
rate-determining step. The exclusion of the steps of sub-
Results and Discussion
Oxygen donation rates: A number of questions remain
about the mechanism of oxygen donation by anilinic N-
oxides, including the rate-determining step of the pathway,
the nature of the oxidant formed and the consequent
chemistry of N-dealkylation. We first address the rate-deter-
mining step in the N-oxide pathway. Scheme 4 demonstrates
a simple mechanism of N-oxide binding, oxygen transfer,
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strate binding and N-dealkylation supports oxygen donation
as rate-determining with CDMAO.
change of the rate-determining step, thus, unmasking the in-
trinsic isotope effect. Intramolecular isotope effects were de-
termined by using 2,3,4,5,6-pentafluoro-N-methyl-N-trideu-
teriomethylaniline N-oxide ([D3]PFDMAO) and measuring
the ratio of 2,3,4,5,6-pentafluoro-N-methylaniline (PFMA)
As a consequence of the electron-withdrawing character
of the p-cyano group, Bruice and Nee had used CDMAO to
increase the rate of oxygen donation relative to DMAO in
their experiments.[20] In an effort to further increase the rate
of oxygen donation, we turned to 2,3,4,5,6-pentafluoro-N,N-
dimethylaniline N-oxide (PFDMAO). The electron-with-
drawing character of the fluorines was expected to further
and
2,3,4,5,6-pentafluoro-N-trideuteriomethylaniline
([D3]PFMA). Intermolecular isotope effects were deter-
mined by using PFDMAO and 2,3,4,5,6-pentafluoro-N,N-
bis(trideuteriomethyl)aniline N-oxide ([D6]PFDMAO) and
measuring the ratios of the products PFMA and [D3]PFMA,
both in competitive and noncompetitive experiments.
Whereas a significant intramolecular isotope effect was
seen, no significant isotope effect for N-demethylation in
both the noncompetitive and competitive experiments was
observed (Table 1). As with CDMAO, the lack of an inter-
molecular isotope effect suggests that the intrinsic isotope
effect is masked by a prior step in the PFDMAO system
and that oxygen donation is still rate-determining. We
expect that the disagreement between the DFT calculations
and experimental results arises from the similarity of the
calculated barrier heights for oxygen donation and N-deal-
kylation (10.6 and 14.5 kcalmolÀ1). These values are not so
dissimilar as to be distinguishable by DFT calculations.
Though isotope effects suggest no change in the rate-de-
termining step for the PFDMAO system, it is still in ques-
tion whether the barrier for this step has been lowered.
PFDMAO and the unsubstituted DMAO were incubated
with P450cam and product formation rates were measured.
As shown in Table 2, the rate of N-demethylation from
PFDMAO was 27-fold faster than that of DMAO. Further,
À
weaken the N O bond and increase the rate of oxygen don-
ation. We performed DFT calculations for oxygen donation
by PFDMAO and found
a
barrier of 10.6 kcalmolÀ1
(Figure 1), significantly less than calculated by Cho et al. for
Table 2. Product formation rates by P450cam supported by DMAO or
PFDMAO.
Rate [nmolminÀ1 nmolP450À1
]
N-Oxide
Dimethyl product
Methyl product
Figure 1. Energies calculated by density functional theory for the reaction
of PFDMAO with P450. Values are uncorrected for zero-point energies
or solvation effects. TS1=transition state for oxygen donation; TS2=
transition state for hydrogen atom transfer; PFDMArad=PFDMA
carbon-centered radical (por=porphyrin).
DMAO[a]
0
1.2Æ0.2
32Æ2
PFDMAO[b]
1.5Æ0.3
[a] DMAO=N,N-dimethylaniline N-oxide. [b] PFDMAO=2,3,4,5,6-pen-
tafluoro-N,N-dimethylaniline N-oxide.
DMAO.[17] Further, the calculations support a barrier for N-
demethylation of the product aniline (PFDMA) of 14.5 kcal
molÀ1, several kcalmolÀ1 higher than that seen by Cho et al.
for DMAO[17] and 4 kcalmolÀ1 larger than that for oxygen
donation. This larger barrier implies that N-demethylation
should be rate-determining in the PFDMAO system. Our
calculations followed the reaction to the PFDMA carbon-
centered radical (Figure 1). Whereas this portrays an endo-
thermic reaction, Li et al. have demonstrated that oxygen
rebound is barrierless in the DMAO reaction leading to
very exothermic products (greater than 50 kcalmolÀ1).[23]
The products of oxygen rebound in the PFDMAO reaction
are expected to be similarly exothermic.
the unoxidized 2,3,4,5,6-pentafluoro-N,N-dimethylaniline
(PFDMA) was also isolated as a product. With N-demethy-
lation expected to require the prior step of oxygen donation,
combining the rates of both products in the PFDMAO
system gives a total rate of product formation of 34 nmol
minÀ1 nmolP450À1, a rate 28-fold faster than for DMAO.
Changes in the rate of product formation indicate a change
in the rate-determining step. With isotope effects supporting
oxygen donation as rate-determining, the increased rate of
product formation suggests a lowering of the barrier to
oxygen donation.
The faster rate of oxygen donation by PFDMAO also
lends insight to the mechanism of oxygen donation. The
electron-withdrawing nature of the aromatic fluorines offers
two outcomes for rate effects on oxygen donation, depen-
In light of our calculations, PFDMAO was synthesized
and kinetic isotope effects measured in P450cam to deter-
mine if this predicted faster oxygen donation resulted in a
À
dent on the nature of the N O bond scission. As discussed
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J. P. Jones and K. M. Roberts
above, two distinct oxygen donation mechanisms have been
proposed for N-oxide systems, a six-electron oxygen (oxene)
donation and a seven-electron oxygen donation. Donation
radical cations relative to their neutral anilines as calculated
by DFT: 282.32 and 154.76 kcalmolÀ1 for PFDMA and
DMA, respectively. It is important to note that the destabi-
lized aminium radical would be expected to show an in-
creased acidity of the methyl proton and a faster rate of
proton transfer. However, our isotope effect measurements
À
of an oxene results from heterolytic cleavage of the N O
bond, which returns both electrons to the nitrogen of the
aniline (Scheme 5). Electron-withdrawing groups would be
À
demonstrate that breaking the C H bond is not rate-deter-
mining and this increased rate would not be reflected in
product formation rates. Since homolytic cleavage is expect-
ed to be slower with PFDMAO, the finding that PFDMAO
donates its oxygen an order of magnitude faster than
DMAO excludes donation of a seven-electron oxygen and
the direct formation of Cpd II.
Further support for oxene donation is offered by spin
density calculations on the products of oxygen donation. We
used DFT calculations to observe the spin densities of the
product of oxygen donation from DMAO and compared
them to calculated spin densities of DMAO radical cation.
As shown in Figure 2, the calculations show large spin densi-
À
Scheme 5. Electronic effects on heterolytic cleavage of the N O bond of
DMAO and PFDMAO (por=porphyrin).
expected to pull the electrons away from the nitrogen and
À
the N O bond, weakening the latter. Oxygen donation and
Cpd I formation from heterolytic cleavage would be expect-
ed to be faster as a result of the weakened bond. This is sup-
ported by the over 10 kcalmolÀ1 lower activation energy for
oxygen donation from PFDMAO versus DMAO in DFT
calculations (Figure 1 and reference [13]). In contrast, dona-
tion of a seven-electron oxygen requires homolytic cleavage
À
of the N O bond, forming an aminium radical and Cpd II
Figure 2. Calculated spin densities of the products of DMAO oxygen
donation (left) and the DMA radical cation (right).
(Scheme 6). Electron-withdrawing substituents would fur-
ther increase the positive charge on the nitrogen in the ami-
nium radical. Destabilization of the product would be ex-
pected to be reflected in a slower rate of oxygen donation.
This is represented in the gas-phase energies of the aniline
ty on the iron-centered oxygen with very little spin density
on the anilinic nitrogen or the aromatic ring, indicative of a
neutral aniline paired with Cpd I. In contrast, spin density
calculations of an aminium radical cation show large spin
density on the nitrogen and throughout the aromatic ring.
We were unable to isolate Cpd II with the aminium radical.
À
Instead, lengthening of the N O bond generates Cpd I and
the aniline. The evident differences between the spin densi-
ties for the donation products and an aniline radical cation
further support the exclusion of homolytic cleavage of the
À
N O bond and direct Cpd II formation.
We have shown that the anilinic N-oxide-supported P450
pathway utilizes a mechanism that involves a fast N-dealky-
lation preceded by a rate-determining oxygen donation.
However, though perfluorination of the aromatic ring did
not result in a change in the rate-determining steps, product
formation rates of the PFDMAO system were significantly
increased compared to that of the unsubstituted DMAO
system. These faster donation rates that result from the elec-
tron-withdrawing fluorines with support from DFT calcula-
À
Scheme 6. Electronic effects on homolytic cleavage of the N O bond of
DMAO and PFDMAO (por=porphyrin).
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À
tions exclude homolytic cleavage of the N O bond to direct-
ly form Cpd II. In exclusion of the homolytic pathway, a
Oxidation of CPMA in the PFDMAO system was much
slower than either that of DMA or the system without sub-
strate. CPMA was N-dealkylated to MA and N-cyclopropyl-
À
mechanism that requires heterolytic cleavage of the N O
bond to form Cpd I is supported.
aniline with a total rate of 0.9 nmolminÀ1 nmolP450À1. N-
ACHTUNGTRENNUNG
Dealkylation of PFDMA was four times faster than that of
CPMA. Total oxygen donation was about four-fold slower
than the PFDMAO system without substrate and seven-fold
slower than the PFDMAO system with DMA present.
Having a methyl group replaced with a more electron-do-
nating cyclopropyl group, CPMA should be more suscepti-
ble to oxidation than DMA and a higher rate of oxidation
of CPMA would be expected. We expect the slower rates
seen in the CPMA–PFDMAO system to be an active site
binding effect, where CPMA inhibits PFDMAO from donat-
ing the oxygen. When PFDMAO is positioned for oxygen
donation, the resulting PFDMA outcompetes the CPMA by
a proximity effect. As with the DMA, total oxygen donation
in the PFDMAO system with CPMA present is not fully ac-
counted for by N-dealkylation products. In fact, approxi-
mately 80% of the PFDMA formation is unaccounted for.
We expect that, like DMA, CPMA elicits a third pathway of
N-oxygenation as the fate of the donated oxygen.
Oxygen surrogacy: Rate measurements and DFT calcula-
tions of the N-oxide-supported P450 system support a mech-
anism with
a rate-determining oxene donation. With
PFDMAO, this donation shows two fates for the product
aniline (PFDMA): it is either N-demethylated or released
from the enzyme (Table 2). The release of PFDMA requires
oxene donation without the subsequent N-demethylation.
With PFDMAO exhibiting faster rates for oxene donation
and a small portion of the donated oxenes not participating
in N-dealkylation, we tested the ability of PFDMAO to act
as an oxygen surrogate. PFDMAO and P450cam were incu-
bated in the presence of N,N-dimethylaniline (DMA) and
N-cyclopropyl-N-methylaniline (CPMA). Both substrates
were oxidized by P450cam when supported by PFDMAO
with product formation rates shown in Table 3.
The ability of PFDMAO to significantly support oxidation
of a second substrate is a feature previously unseen with
DMAOs and P450. In the experiments with DMA, not only
is the alternate substrate oxidized, but it outcompetes
PFDMA, the product of oxene donation, for oxidation. The
successful competition of DMA demonstrates the difficulty
of the N-dealkylation of PFDMA. In the PFDMAO system,
the more difficult oxidation of the product aniline
(PFDMA) permits competition between oxidation and sub-
strate exchange (Scheme 7). In contrast, the DMAO system
does not allow competition for the oxidant, expectedly the
result of the ease of oxidation of DMA. With the lifetime of
Cpd I permitting substrate exchange in the PFDMAO
system, the possibility of detection and characterization can
be entertained, similar to work with other various oxygen
surrogates by Dawson et al.[4] and Newcomb et al.[3,25] How-
Table 3. Product formation rates from alternate substrates by PFDMAO-
supported P450cam.
Rate [nmolminÀ1 nmolP450À1
]
PFDMA[a]
PFMA[b]
MA[c]
CPA[d]
DMA[e]
63Æ2
5Æ1
2.5Æ0.1
3.7Æ0.3
33.0Æ1
–
CPMA[f]
0.28Æ0.03
0.6Æ0.1
[a] PFDMA=2,3,4,5,6-pentafluoro-N,N-dimethylaniline.
[b] PFMA=
2,3,4,5,6-pentafluoro-N-methylaniline.
[d] CPA=N-cyclopropylaniline.
[c] MA=N-methylaniline.
[e] DMA=N,N-dimethylaniline.
[f] CPMA=N-cyclopropyl-N-methylaniline.
DMA was rapidly metabolized in the PFDMAO system
with a rate of N-methylaniline (MA) formation 27-fold
faster than the DMAO system. The rate of PFMA forma-
tion was decreased by a factor of ten, with the formation of
MA 11-fold faster than that of PFMA. This is expected be-
cause the donated oxygen is given a choice of substrates.
Any redirection of the oxygen towards DMA would result
in a decrease in PFMA formation. Further, because DMA
has a much lower oxidation potential, it is expected to out-
compete PFDMA for the donated oxygen, as is seen. Ex-
pectedly, the rate of formation of PFDMA was increased in
comparison to experiments without DMA. Redirection of
the donated oxygen to the DMA prevents oxidation of
PFDMA and, thus, permits its subsequent release. Total
oxygen donation in the presence of DMA was doubled in
comparison to experiments without DMA. Surprisingly, only
half of the PFDMA formation is accounted for by N-deme-
thylation products. The presence of DMA in the PFDMAO
system appears to elicit a third pathway for the donated
oxygen. We propose the formation of DMAO as this third
pathway, though oxidation of the P450 active site is also a
possibility.
Scheme 7. Competition of N,N-dimethylanilines for an N-oxide-generated
oxidant (por=porphyrin).
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J. P. Jones and K. M. Roberts
ever, no intermolecular isotope effects are observed with
PFDMAO demonstrating the rate-determining nature of
oxygen donation. There should be no significant buildup of
the Cpd I intermediate making detection difficult or impos-
sible.
oxidation than PFDMA. With its lower barrier to oxidation,
DMA would be expected to be preferentially oxidized over
PFDMA.
In the basic SET pathway, after oxene donation to form
Cpd I, a single electron transfer from the product aniline
occurs to form an aminium radical cation and Cpd II
(Scheme 8). Similar to the HAT mechanism, oxidation rates
of substrates depend on the rate of substrate exchange and
the ease of substrate oxidation. In the case of SET, the ease
of oxidation is determined by the single-electron oxidation
potential of competitive substrates. If substrate exchange is
fast relative to the SET event, the substrate with the lowest
oxidation potential will be oxidized first. With DMA having
a lower barrier to oxidation than PFDMA (Figure 2), we
would expect electron transfer from DMA to Cpd I to occur
faster. However, with P450 2B1, Seto and Guengerich found
only trace support of oxidation of [14C]DMA by three dis-
tinct DMAOs, including the electron-withdrawn CDMAO,
concluding that oxidation of a second substrate was prevent-
ed by a direct Cpd II formation from the N-oxide.[21] We
have excluded the direct formation of Cpd II by showing a
higher rate of oxygen donation
The N-oxide-generated oxidant: PFDMAO has been found
to be an oxene donor capable of acting as a surrogate for N-
dealkylation. Product formation rates and DFT calculations
support an oxene donation for anilinic N-oxides. However,
what occurs downstream of Cpd I formation is unclear. With
the observation that PFDMAO acts as an oxygen surrogate
for other substrates and DMAO does not, three possible
mechanisms for N-dealkylation in the N-oxide systems
become apparent. Two are the SET and HAT mechanisms
described above. The third mechanism is a SET-like path-
way that involves an outer-sphere electron transfer between
substrates.
In the HAT mechanism, after oxene donation by the N-
oxide, the generated Cpd I abstracts a hydrogen from the Ca
to form a carbon-centered radical (Scheme 8). The occur-
by PFDMAO over DMAO.
Failure of the DMAOs to act as
surrogates then suggests that
substrate exchange would need
to be slow relative to a fast
electron transfer step. In other
words, after Cpd I formation in
the DMAO system, the product
aniline (DMA) is oxidized by
one electron to form the amini-
um radical cation and Cpd II
before any substrate exchange
might occur. In contrast, the
higher barrier, and thus, slower
rate of electron transfer from
PFDMA in the PFDMAO
system could permit substrate
exchange. With substrates now
Scheme 8. Proposed N-oxide-supported HAT and SET mechanisms for N-dealkylation of PFDMAO (por=
porphyrin).
in competition for Cpd I, oxida-
tive preference determines
product formation. This system
would be expected to demon-
rence of competition for substrate oxidation is dependent
on the relative rates of substrate oxidation and substrate ex-
change. The inability of DMAO to support the oxygenation
of other compounds can be rationalized by its low barrier to
oxidation that permits its oxidation before exchange with an
alternate substrate can occur. Thus, when DMAO donates
its oxygen to form Cpd I, the resulting DMA immediately
transfers a hydrogen atom before any substrate exchange
occurs. In contrast, hydrogen atom transfer from PFDMA is
expected to be much slower. If substrate exchange is rapid
compared to the oxidation of PFDMA, then the relative
ease of oxidation presides over substrate preference. As de-
scribed, DMA has a much lower barrier to single-electron
strate surrogacy since DMA shows lower barriers to oxida-
tion than PFDMA (Figure 2).
Another possibility arises in the case that single electron
transfer from any anilinic N-oxide is faster than substrate
exchange. In this pathway, for a second substrate to be oxi-
dized by Cpd II, a second electron transfer must occur be-
tween the aminium radical cation and the substrate to be
oxidized (Scheme 9). Because the DMA radical cation has a
lower formation barrier, we would expect no electron trans-
fer from any substrates with a higher barrier to oxidation
(though transfer from the isotopically distinct [14C]DMA
would be expected.) We would, however, expect to observe
electron transfer from DMA to p-cyano-N,N-dimethylani-
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Cytochromes
FULL PAPER
were observed in these experi-
ments. Being a product of both
pathways, MA formation does
not distinguish between HAT
and SET, however, the observa-
tion of PFDMAO-supported
formation of CPA, a ring-intact
product, is revealing. Ring-
Scheme 9. Single electron transfer between two substrates of Cpd II (por=porphyrin).
line radical cation, since the electron-withdrawing nature of
the p-cyano group would decrease its single-electron oxida-
tion potential relative to DMA. In support of this rationale,
Seto and Guengerich did see an increased support of
[14C]DMA oxidation by CDMAO. In the PFDMAO system,
the highly electron-withdrawing nature of the aromatic fluo-
rines poises PFDMA radical cation for accepting an elec-
tron. With PFDMA having a much lower oxidation poten-
tial, any substrate with a higher oxidation potential would
be capable of transferring an electron to the PFDMA radi-
cal cation to generate a new substrate radical cation. This
new radical cation would then be susceptible to oxidation by
Cpd II.
Scheme 11. Product formations from CPMA directed by a HAT mecha-
nism.
The HAT and SET mechanisms predict similar outcomes
for the DMAO and PFDMAO systems. In each mechanism,
DMAO is not expected to support oxidation of other sub-
strates due to the ease of DMA oxidation. Further, each
mechanism also predicts the ability of PFDMAO to act as a
surrogate owing to a more difficult oxidation of the product
aniline (PFDMA). With DMAO seemingly unable to sup-
port oxidation of other substrates and PFDMAO supporting
oxidation of DMA and CPMA, none of these mechanisms is
excluded.
To distinguish between these mechanisms, we used the
chemical probes of Schaffer et al. and tested the metabolism
of CPMA in the PFDMAO system. Schaffer et al. found
that the metabolism of CPMA distinguished between SET
and HAT mechanisms.[11,12] With HRP, which follows a SET
mechanism, CPMA metabolism led exclusively to cyclo-
propyl ring-opened products (Scheme 10). In P450, now con-
intact product formation strongly argues against a SET
mechanism in the PFDMAO system, excluding both pro-
posed SET mechanisms. In the exclusion of SET, a HAT
mechanism for N-dealkylation in the N-oxide system is sup-
ported, similar to the native P450 system.
To further evaluate the similarity between the anilinic N-
oxide and native P450 systems, KIEs were measured for the
PFDMAO–P450cam system (Table 1). In the competitive
and noncompetitive intermolecular experiments, the intrin-
sic isotope effect is completely masked. However, the intra-
molecular experiment shows a significant isotope effect with
a value similar to those seen for N-dealkylation by both the
DMAO- and NADPH/O2-supported systems.
KIEs were also evaluated for the N-dealkylation of DMA
by the PFDMAO system (Table 4). In the noncompetitive
intermolecular experiment, the intrinsic isotope effect is
completely masked. However, partial unmasking of the in-
trinsic isotope effect is observed
in the competitive experiment.
This is likely due to a slow but
significant exchange of the
DMA prior to N-dealkylation.
This permits the heme-centered
oxidant to select between the
two isotopically-distinct DMAs
and, thus, unmask the isotope
Scheme 10. Product formations from N-cyclopropyl-N-methylaniline (CPMA) directed by a SET mechanism.
effect. That the intrinsic isotope
effect is not fully unmasked
suggests that this exchange is
sidered to follow a HAT mechanism, strictly ring-closed
products were formed (Scheme 11). We presented CPMA to
the PFDMAO system and successfully observed metabolites
of CPMA. Though metabolism of CPMA was much slower
than that of DMA, the two products, MA and N-cyclopro-
pylaniline (CPA), were observed. No ring-opened products
not exceedingly rapid relative to the rate of N-dealkylation.
The intramolecular isotope effect showed further unmasking
of the intrinsic isotope effect. As with the PFDMAO intra-
molecular isotope effect, this value is similar to those seen
for N-dealkylation by both the DMAO- and NADPH/O2-
supported systems.
Chem. Eur. J. 2010, 16, 8096 – 8107
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J. P. Jones and K. M. Roberts
Table 4. Product isotope effects for the N-demethylation of DMA[a] by
PFDMAO and P450cam.
Experimental Section
kH/kD—DMA/
[D6]DMA
kH/kD—DMA/
[D6]DMA
competitive
kH/kD—DMA/
[D3]DMA
Materials: Reagents or HPLC grade chemicals and solvents were sup-
plied by Alfa Aesar (Ward Hill, MA), Aldrich (St. Louis, MO), Fisher
Scientific (Pittsburgh, PA), EMD (Gibbstown, NJ), and Mallinckrodt
Baker (Phillipsburg, NJ). Isotopically-labeled compounds were supplied
by CDN Isotopes (Pointe-Claire, Quebec, CA). THF was distilled under
argon from sodium and benzophenone prior to use. 1H NMR spectra
were obtained at 300 MHz with a Varian Mercury 300 spectrometer
equipped with a quad-detection probe (1H, 13C, 31P, and 19F). 1H-decou-
pled 13C NMR spectra were obtained at 75 MHz. 19F NMR spectra were
obtained at 282 MHz. Gas chromatography/mass spectrometry was per-
formed on a ThermoQuest Voyager GC/MS (Thermo-Finnegan) coupled
to a CE Instruments GC8000Top affixed with a 30 m JW Scientific DB-1
GC column. Liquid chromatography/mass spectrometry was performed
on a ThermoQuest Surveyor LC affixed with an Agilent Eclipse Plus C-
18 column (5 mm, 2.1ꢂ150 mm) coupled to a Thermo-Finnegan LCQ Ad-
vantage ESI-MS.
noncompetitive[b]
intramolecular[c]
1.3Æ0.3
1.9Æ0.2
2.8Æ0.2
[a] DMA=N,N-dimethylaniline. [b] [D6]DMA=N,N-bis(trideuteriome-
thyl)aniline. [c] [D3]DMA=N-methyl-N-trideuteriomethylaniline.
Studies have shown large intramolecular isotope effects
(2.93–13.3) for N-dealkylation of DMAs by HRP[16,26] in
contrast to the smaller isotope effects (1.40–3.87) seen by
P450.[16,19,27] As HRP follows a SET mechanism, the small
magnitude of the intramolecular isotope effects in the
PFDMAO system further support a mechanism that does
not involve single electron transfer.
As we have shown, product formation rates of DMAO
and PFDMAO exclude direct Cpd II formation. Indirect for-
mation of Cpd II derived from electron transfer to Cpd I a
SET mechanism, is, in turn, excluded by the formation of
CPA. In the exclusion of Cpd II-derived mechanisms, HAT
is left to be the likely mechanism for N-dealkylation by the
anilinic N-oxide-supported P450. Further, the similarities in
KIEs for both the N-oxide and NADPH/O2 systems, togeth-
er with the observation of cyclopropyl ring-intact products
in both systems, strongly support a HAT mechanism for N-
dealkylation in these systems.
N-Oxide-supported P450cam—general procedure: P450cam was ex-
pressed and purified as described previously.[28] Incubations of P450cam
with 2,3,4,5,6-pentafluoro-N,N-dimethylaniline N-oxide (PFDMAO) or
N,N-dimethylaniline N-oxide (DMAO) were performed by using N-oxide
and P450cam in phosphate buffer (100 mm, pH 7.4). Samples were prein-
cubated at 308C prior to initiation with the N-oxide. For rate determina-
tions, reactions were incubated at 308C for discrete time points up to
60 min, dependent on the reaction. Reactions were quenched with ethyl
acetate containing 2,3,4,5,6-pentafluoroaniline (PFA) as an internal stan-
dard. The organic layer was collected and product further extracted twice
with ethyl acetate. Extracts were combined, dried with MgSO4, and con-
centrated in a 358C bath under a flow of N2(g) to <300 mL. Reaction
products were monitored by gas chromatography–mass spectrometry
(GC–MS) by using electron impact ionization. Except where indicated,
the GC method began at 708C for 5 min followed first by a 108C minÀ1
ramp to 1208C then by a 308C minÀ1 ramp to 2308C.
DMAO-supported P450cam procedure: Following the general procedure
above, incubations were performed by using DMAO hydrochloride
(20 mmol) and P450cam (1.0 nmol) in phosphate buffer (1.0 mL, 100 mm,
pH 7.4). Samples were preincubated at 308C for 10 min prior to initiation
with the N-oxide. For rate determinations, reactions were incubated at
308C and quenched at time points 0, 10, 20, 30, 45, and 60 min with ethyl
acetate (500 mL) containing PFA (50 nmol) as an internal standard. Addi-
tional ethyl acetate (500 mL) was added for product extraction. Product
was further extracted with ethyl acetate (2ꢂ1.0 mL). Extracts were com-
bined, dried with MgSO4, and concentrated in a 358C bath under a flow
of N2(g) to <300 mL. Reaction products were monitored by GC–MS using
electron impact ionization with the general method described above. Ions
with m/z 107.1 were monitored for quantization of N-methylaniline
(MA).
Conclusion
In conclusion, we have identified an N-oxide capable of sup-
porting N-demethylation of N,N-dimethylaniline (DMA) by
P450cam, implicating the generation of a porphyrin-directed
reactive oxygen species (ROS) as an intermediate to oxida-
tion. Kinetic isotope effects and product formation rates,
supported by DFT calculations, support a mechanism for
surrogacy that begins with a rate-determining oxene dona-
tion to form Compound I (Cpd I). Further, the formation of
PFDMAO-supported P450cam procedure: Following the general proce-
dure above, incubations were performed by using the trifluoroacetic acid
salt of PFDMAO (13 mmol) and P450cam (1.0 nmol) in phosphate buffer
(1.0 mL, 100 mm, pH 7.4). Samples were preincubated at 308C for 10 min
prior to initiation with the N-oxide. For rate determinations, reactions
were incubated at 308C for discrete time points up to and including
60 min. Reactions were quenched with ethyl acetate (500 mL) containing
PFA (50 nmol) as an internal standard. Additional ethyl acetate (500 mL)
was added for product extraction. Product was further extracted with
ethyl acetate (2ꢂ1.0 mL). Extracts were combined, dried with MgSO4,
and concentrated in a 358C bath under a flow of N2(g) to <300 mL. Reac-
tion products were monitored by GC–MS by using electron impact ioni-
zation with the general method described above. Ions with m/z 193.1 and
211.1 were monitored for quantization of 2,3,4,5,6-pentafluoro-N-methyl-
ring-intact products from N-cyclopropyl-N-methylACTHNUGTRNEUNGaniline
(CPMA) support a mechanism similar to that of native
P450, with the ring-intact products excluding a single elec-
tron transfer (SET) mechanism. The apparent similarities in
the native and N-oxide-supported systems together with
support for the formation of Cpd I argue for a Cpd I-direct-
ed hydrogen atom transfer (HAT) mechanism for P450-
mediated N-demethylation similar to that proposed for
P450-mediated alkyl hydroxylation. Further, these similari-
ties demonstrate the ability of the N-oxide-generated oxi-
dant to act as an accurate mimic of the native system, which
supports the use of N-oxides as mechanistic probes for other
P450-mediated oxidations. The application of anilinic N-
oxides as mechanistic probes as well as their potential as
oxygen surrogates for synthesis in porphyrin mimetic sys-
tems are currently under investigation by our group.
aniline
(PFMA)
and
2,3,4,5,6-pentafluoro-N,N-dimethylaniline
(PFDMA), respectively.
PFDMAO surrogacy procedure: Following the PFDMAO-supported in-
cubation procedure above, incubations were performed by using samples
consisting of the trifluoroacetic acid salt of PFDMAO (13 mmol),
P450cam (1.0 nmol), and N,N-dimethylaniline (DMA) or N-cyclopropyl-
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Cytochromes
FULL PAPER
N-methylaniline (CPMA) (20 mmol) in phosphate buffer (1.0 mL,
100 mm, pH 7.4). GC–MS was performed by using the general method
above monitoring ions with m/z 107.1, 132.1, 193.1, and 211.1 for MA, N-
cyclopropylaniline (CPA), PFMA, and PFDMA, respectively.
p-Cyano-N,N-dimethylaniline N-oxide hydrate: m-Chloroperbenzoic acid
(75%, 250 mg, 1.1 mmol) in chloroform (1.8 mL) was added dropwise to
a stirring solution of p-cyano-N,N-dimethylaniline (146 mg, 1.0 mmol) in
chloroform (2 mL). The reaction was allowed to proceed for 3.0 h on ice.
The product was purified by chromatography by using basic alumina.
The product mixture was loaded with chloroform and eluted with 25%
methanol in chloroform. Solvents were removed by rotary evaporation to
yield a white solid (178 mg, 99%). 1H NMR (300 MHz, CDCl3): d=2.83
(brs, 2H), 3.62 (s, 6H), 7.82 (d, J=7.2 Hz, 2H), 8.17 ppm (d, J=6.9 Hz,
2H); 13C{1H} NMR (75 MHz, CDCl3): d=63.49, 113.70, 117.70, 121.61,
133.58, 158.02 ppm.
PFDMAO intermolecular kinetic isotope effect (KIE) determinations:
Samples consisted of the trifluoroacetic acid salt of PFDMAO (1.3 mmol)
and/or 2,3,4,5,6-pentafluoro-N,N-bis(trideuteriomethyl)aniline N-oxide
([D6]PFDMA) (1.3 mmol), and P450cam (1.0 nmol) in phosphate buffer
(1.0 mL, 100 mm, pH 7.4). Samples were preincubated at 308C for
5.0 min. Reactions were initiated by addition of N-oxide and incubated at
308C for 60 min. Reactions were quenched with dichloromethane
(1.0 mL) and anisole (20 nmol) in dichloromethane (100 mL) was added
as an internal standard. The organic layer was collected and product fur-
ther extracted with dichloromethane (2ꢂ1.0 mL). Extracts were com-
bined, dried with MgSO4, and concentrated in a 358C bath under a flow
of N2(g) to <300 mL. Reaction products were monitored by GC–MS by
using electron impact ionization with the following method. The GC
method began at 358C for 5 min followed by a 208CminÀ1 ramp to
2308C. Ions with m/z 197.1 and 200.1 were monitored for quantization of
N,N-Dimethylaniline N-oxide hydrochloride: DMA (1.27 mL, 10 mmol)
was added dropwise to a mixture of m-chloroperbenzoic acid (75%,
3.45 g, 15 mmol) in dichloromethane (35 mL). Reaction was run for 1.5 h
at room temperature and the solvent was removed by rotary evaporation.
The product mixture was purified by column chromatographed by using
basic alumina. The product was loaded with chloroform and eluted with
25% methanol in chloroform. Fractions containing product were com-
bined and the solvent was removed by rotary evaporation. The product
was reconstituted with water (10 mL) and the solution was rinsed with di-
ethyl ether (2ꢂ10 mL). Water was removed by rotary evaporation to give
oil. Concentrated hydrochloric acid (1 mL) was added to the oil and the
mixture was concentrated in vacuum to give a solid. The product was re-
crystallized from acetone to yield long, colorless crystals (350 mg, 20%).
1H NMR (300 MHz, CDCl3): d=4.09 (s, 6H), 7.55 (m, 3H), 7.91 (d, J=
8.4 Hz, 2H), 13.86 ppm (brs, 1H); 13C{1H} NMR (75 MHz, CDCl3): d=
61.06, 119.56, 130.48, 131.20, 149.03 ppm.
PFMA
and
2,3,4,5,6-pentafluoro-N-trideuteriomethylaniline
([D3]PFMA), respectively.
PFDMAO intramolecular KIE determination: Following the general in-
cubation procedure above, samples consisted of the trifluoroacetic acid
salt of 2,3,4,5,6-pentafluoro-N-methyl-N-(trideuteriomethyl)aniline N-
oxide ([D3]PFDMA) (670 nmol) and P450cam (1.0 nmol). Samples were
preincubated at 308C for 5.0 min. Reactions were initiated by addition of
N-oxide and incubated at 308C for 20 min. Reactions were quenched
with PFA (50 nmol) in ethyl acetate (500 mL). Additional ethyl acetate
(500 mL) was added for product extraction. Product was further extracted
with ethyl acetate (2ꢂ1.0 mL). Extracts were combined, dried with
MgSO4, and concentrated in a 358C bath under a flow of N2(g) to
<300 mL. Reaction products were monitored by GC–MS by using elec-
tron impact ionization with the general method described above. 3 Da
spans of ions with m/z 194.6–197.6 and 197.6–200.6 were monitored for
quantization of PFMA and [D3]PFMA, respectively, correcting for iso-
topic overlap.
2,3,4,5,6-Pentafluoro-N-methylaniline: Potassium tert-butoxide (tBuOK)
(2.0 g, 18 mmol) was added at room temperature over 2 min to a vigo-
rously stirring solution of 2,3,4,5,6-pentafluoroaniline (2.8 g, 15 mmol)
and iodomethane (0.94 mL, 15 mmol) in dry THF (100 mL). TLC after
addition of potassium tert-butoxide showed incomplete conversion of the
starting material with a trace of doubly methylated product present. Pre-
cipitates (potassium iodide and unreacted tBuOK) were removed by fil-
tration through Celite. THF was removed from the filtrate by rotary
evaporation to a volume of 5 mL. The mixture was then purified by
column chromatography over Silica 60, eluting the product with 2.5%
ethyl acetate in hexane to give 2,3,4,5,6-pentafluoro-N-methyl aniline
(1.27 g, 43%) as a pale yellow oil. 1H NMR (300 MHz, CDCl3): d=3.05
(m, 3H), 3.58 ppm (brs, 1H); 13C{1H} NMR (75 MHz, CDCl3): d=
33.60 ppm; 19F NMR (282 MHz, CDCl3): d=À172.84 (m, 1F), À165.13
(m, 2F), À161.13 ppm (d, J=23.7 Hz, 2F).
DMA KIE determinations: Following the general incubation procedure,
samples consisted of the trifluoroacetate salt of PFDMAO (670 nmol),
P450cam (1.0 nmol), and the appropriate DMA. Noncompetitive reac-
tions used either DMA (1.0 mmol) or N,N-bis(trideuteriomethyl)aniline
([D6]DMA) (1.0 mmol). Competitive reactions contained both DMA
(1.0 mmol) and [D6]DMA (1.0 mmol). Intramolecular experiments con-
tained N-methyl-N-trideuteriomethylaniline ([D3]DMA) (1.0 mmol). Sam-
ples were preincubated at 308C for 8.0 min. Reactions were initiated by
addition of PFDMAO and incubated for 20 min at 308C. Reactions were
quenched with PFA (20 nmol) in ethyl acetate (500 mL). Additional ethyl
acetate (500 mL) was added for product extraction. Product was further
extracted with ethyl acetate (2ꢂ1.0 mL). Extracts were combined, dried
with MgSO4. Reaction products were monitored without concentration
by GC–MS by using electron impact ionization with the general method
described above. For the noncompetitive assays, 4 Da spans of ions with
m/z 105.6–109.6 and 107.6–111.6 were monitored for quantization of MA
and N-trideuteriomethylaniline ([D3]MA), respectively. For the competi-
tive and intramolecular assays, 3 Da spans of ions with m/z 104.6–107.6,
107.6–110.6 were monitored correcting for isotopic overlap.
2,3,4,5,6-Pentafluoro-N,N-dimethylaniline: tBuOK (5.0 g, 45 mmol) was
added at room temperature over 10 min to a vigorously stirring solution
of 2,3,4,5,6-pentafluoroaniline (2.8 g, 15 mmol) and iodomethane
(2.3 mL, 38 mmol) in dry THF (100 mL). TLC after 10 min showed com-
plete conversion of the starting material to 2,3,4,5,6-pentafluoro-N,N-di-
methylaniline. Precipitates (potassium iodide and unreacted tBuOK)
were removed by filtration through Celite. THF was removed from the
filtrate by rotary evaporation to a volume of 5 mL. The product was puri-
fied by flash chromatography (15% chloroform in hexane) to yield
2,3,4,5,6-pentafluoro-N,N-dimethylaniline (1.68 g, 53%) as a clear oil.
1H NMR (300 MHz, CDCl3): d=2.90 ppm (t, J=1.8 Hz, 6H);
13C{1H} NMR (75 MHz, CDCl3): d=43.71 ppm (t, J=3.6 Hz); 19F NMR
(282 MHz, CDCl3): d=À165.19 (t, J=21.7 Hz, 1F), À164.53 (m, 2F),
À151.25 ppm (d, J=21.7 Hz, 2F).
p-Cyano-N,N-dimethylaniline N-oxide (CDMAO) intermolecular KIE
determinations: Samples containing CDMAO (1.0 mmol) and/or p-cyano-
N,N-bis(trideuteriomethyl)aniline N-oxide ([D6]CDMAO) (1.0 mmol) and
P450cam (5 nmol) in phosphate buffer (500 mL, 100 mm, pH 7.4) were in-
cubated for 60 min at 308C. Reactions were quenched with acetonitrile
(1.0 mL) containing aniline (30 nmol) as an internal standard followed by
vortexing and centrifugation to pellet the protein. Supernatant was col-
lected and reaction products monitored by LC–ESIMS. The LC method
began at 5% methanol, 0.1% acetic acid in water for 2.0 min followed by
a 5% methanolminÀ1 ramp to 95% methanol, 0.1% acetic acid in water
(2.0–20.0 min). Ions with m/z 133.1 and 136.1 were monitored for p-
cyano-N-methylaniline (CMA) and p-cyano-N-trideuteriomethylaniline
([D3]CMA), respectively.
2,3,4,5,6-Pentafluoro-N,N-dimethylaniline N-oxide trifluoroacetic acid
salt: tBuOK (16.8 g, 150 mmol) was added at room temperature over
5 min to
a vigorously stirring solution of 2,3,4,5,6-pentafluoroaniline
(9.16 g, 50 mmol) and iodomethane (7.8 mL, 125 mmol) in dry THF
(200 mL). TLC after 10 min showed complete conversion of the starting
material to 2,3,4,5,6-pentafluoro-N,N-dimethylaniline. Precipitates (potas-
sium iodide and unreacted tBuOK) were removed by filtration through
Celite. THF was distilled from the filtered product mixture and the prod-
uct was dissolved in dichloromethane (50 mL) and stirred on ice. Trifluo-
ACHUTNGRENUrNG oACTHUNGRTENNUpG eracetic acid (TFPA) was generated in situ by using a method adapted
from Emmons and Lucas.[29] Hydrogen peroxide (50%, 7.1 mL,
125 mmol) was added dropwise to dichloromethane (12 mL) stirring on
Chem. Eur. J. 2010, 16, 8096 – 8107
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J. P. Jones and K. M. Roberts
ice. To this mixture, trifluoroacetic anhydride (21.1 mL, 150 mmol) was
added dropwise over 20 min. After stirring the reaction for 20 min, the
mixture was warmed to room temperature. The TFPA mixture was
added dropwise over 15 min to the stirring PFDMA mixture on ice. TLC
after 120 min showed only a trace of PFDMA in the reaction mixture.
The product was extracted from the reaction mixture with distilled water
(7ꢂ50 mL). Extracts were combined and rinsed with diethyl ether
(100 mL). Water was removed by rotary evaporation and the product
was precipitated with diethyl ether. The product was then recrystallized
from diethyl ether/THF (75:25) to yield 2,3,4,5,6-pentafluoro-N,N-dime-
thylaniline N-oxide trifluoroacetic acid salt (6.36 g, 37%) as large pale
yellow crystals. 1H NMR (300 MHz, CDCl3): d=4.14 (t, J=1.8 Hz, 6H),
11.20 ppm (brs, 1H); 13C{1H} NMR (75 MHz, CDCl3): d=62.6 ppm (t,
J=5.7 Hz,); 19F NMR (282 MHz, CDCl3): d=À156.95 (m, 2F), À146.99
(m, 1F), À138.32 (d, J=17.8 Hz, 2F), À76.46 ppm (s, 3F).
N-Cyclopropyl-N-methylaniline: N-Cyclopropyl-N-methylaniline was
synthesized by using the method of Shaffer et al.[11] Ethylmagnesium bro-
mide (3.0m in Et2O, 10 mL, 30 mmol) was added dropwise to a stirring
solution of N-methylformanilide (1.3 mL, 10 mmol) and titanium isoprop-
oxide (3.8 mL, 14 mmol) in dry THF (50 mL). The reaction was heated
at 658C for 30 min followed by stirring for 24 h at room temperature.
The reaction was quenched with a saturated aqueous solution of NH4Cl
(40 mL) and THF was removed by rotary evaporation. The product mix-
ture was diluted with 1N HCl (36 mL) and the product was extracted
with diethyl ether (7ꢂ13 mL). The combined extracts were dried with
MgSO4, filtered through Celite, and the solvent was removed by rotary
evaporation to give oil. The product was purified by flash chromatogra-
The reaction mixture was removed from ice and stirred for 12 h allowing
to warm to room temperature. The reaction was carefully quenched with
distilled water (10 drops) and brought up in diethyl ether (10 mL). The
mixture was washed with distilled water (3ꢂ5 mL) and with brine
(5 mL). The organic layer was dried with Na2SO4, filtered and, the sol-
vent was removed by rotary evaporation. The product was purified by
flash chromatography (4% ethyl acetate in hexane) to yield N-cyclopro-
pylaniline (125 mg, 61%) as a pale yellow oil. 1H NMR (300 MHz,
CDCl3): d=0.44 (m, 2H), 0.65 (m, 2H), 2.35 (m, 1H), 4.09 (brs, 1H),
6.67 (t, J=7.2 Hz, 1H), 6.72 (d, J=7.5 Hz, 2H), 7.12 ppm (t, J=7.2 Hz,
2H); 13C{1H} NMR (75 MHz, CDCl3): d=7.63, 25.45, 113.37, 117.95,
129.34, 148.90 ppm.
Computational methods: Density functional calculations were performed
by using Gaussian 03[33] or Jaguar[34]. The B3LYP[24] functional was used
with the LACVP basis set with effective core potential on iron and the 6-
31G on sulfur, nitrogen, carbon, and hydrogen. Energies are reported as
enthalpies of gas-phase reactions without correction for zero-point ener-
gies or solvation effects. The optimized geometries are available in the
Supporting Information. The heme model was the abbreviated heme
[17]
À
with an S H as fifth ligand used by Shaik and co-workers. The spin
density was calculated for the ground state radical cation by using stan-
dard Mulliken population analysis.
Acknowledgements
phy (15% CH2Cl2 in hexane) to yield N-cyclopropyl-N-methylACHTNUTGRNEUNGaniline
1
This work was supported by NIH Grant GM84546.
(590 mg, 40%) as a pale yellow oil. H NMR (300 MHz, CDCl3): d=0.64
(m, 2H), 0.84 (m, 2H), 2.39 (m, 1H), 3.00 (s, 3H), 6.79 (t, J=7.5 Hz,
1H), 7.01 (d, J=7.5 Hz, 2H), 7.27 ppm (t, J=7.5 Hz, 2H); 13C{1H} NMR
(75 MHz, CDCl3): d=9.31, 33.52, 39.34, 114.01, 117.62, 129.07,
151.10 ppm.
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a saturated aqueous solution of Na2CO3
(2.5 mL) was added dropwise over 3 min and the organic layer was col-
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bined organic layers were dried with Na2SO4 and filtered under vacuum
to a final volume of 1 mL. The filtrate was added dropwise to a stirring
solution of aniline (370 mL, 4.0 mmol) and triethylamine (700 mL,
5.0 mmol) in pentane (1.5 mL) and the reaction mixture was heated to
reflux for 72 h. The reaction mixture was brought up in dichloromethane
(15 mL) and washed with distilled water (2ꢂ10 mL) and with saturated
brine (10 mL). The aqueous layers were combined and back-extracted
with dichloromethane (15 mL). The two organic layers were combined
and the solvent was removed by rotary evaporation. The product was pu-
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a pale yellow oil.
1H NMR (300 MHz, CDCl3): d=0.89 (q, J=4.5 Hz, 2H), 1.13 (m, 5H),
3.57 (q, J=7.2 Hz, 2H), 4.79 (brs, 1H), 6.77 (t, J=7.5 Hz, 1H), 6.88 (d,
J=7.2 Hz, 2H), 7.20 ppm (t, J=7.5 Hz, 2H); 13C{1H} NMR (75 MHz,
CDCl3): d=15.23, 15.58, 61.11, 68.77, 114.36, 118.57, 129.27, 145.71 ppm.
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Received: January 23, 2010
Published online: June 2, 2010
Chem. Eur. J. 2010, 16, 8096 – 8107
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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8107