A mechanistic comparison between cytochrome P450- and chloroperoxidase-catalyzed N-dealkylation of N,N-dialkyl anilines

Article abstract of DOI:10.1002/ejoc.200500333

Most peroxidases use histidine as an axial ligand for heme, while chloroperoxidase (CPO) uses a thiolate, which is similar to the ligand employed by cytochrome P₄₅₀ (P₄₅₀). Several studies have also shown that, unlike other peroxidases, CPO is capable of carrying out monooxygenation reactions in a similar manner to P₄₅₀ in addition to typical peroxidase-like reactions. These observations have been attributed to the similarities of the active-site architecture of the two enzymes. Both enzymes have been shown to efficiently catalyze the oxidative N-dealkylation of amines. The similar magnitudes of the kinetic isotope effects determined for P ₄₅₀- and CPO-catalyzed N-dealkylation of N,N-dimethylaniline have been used to propose that these reactions proceed through similar mechanisms. In this study, we have examined the mechanism of CPO-catalyzed N-dealkylation using a series of radical probes, 4-chloro-N-cyclopropyl-N-alkylanilines 1-3, which we have recently used in the mechanistic studies of P₄₅₀, and compared the results with those of P₄₅₀-catalyzed reactions. The results show that P₄₅₀- and CPO-catalyzed reactions proceed through distinctly different mechanisms. As previously reported, while P ₄₅₀-catalyzed reactions appear to proceed through a C _α-hydrogen abstraction mechanism, CPO-catalyzed reactions proceed through a single electron/proton transfer (SET/H⁺) mechanism, similar to reactions catalyzed by Horseradish peroxidase (HRP). Thus, CPO may not be a good mechanistic model for P₄₅₀-catalyzed N-dealkylations. Wiley-VCH Verlag GmbH & Co. KGaA, 2005.

Full text of DOI:10.1002/ejoc.200500333

FULL PAPER
A Mechanistic Comparison between Cytochrome P450- and Chloroperoxidase-
Catalyzed N-Dealkylation of N,N-Dialkyl Anilines
Mehul N. Bhakta[a] and Kandatege Wimalasena*[a]
Keywords: Cytochrome P450 / Chloroperoxidase / Horseradish peroxidase / Enzyme catalysis / Hydrogen abstraction /
Single electron abstraction / N-Dealkylation
Most peroxidases use histidine as an axial ligand for heme, of CPO-catalyzed N-dealkylation using a series of radical
while chloroperoxidase (CPO) uses a thiolate, which is sim- probes, 4-chloro-N-cyclopropyl-N-alkylanilines 1–3, which
ilar to the ligand employed by cytochrome P450 (P450). Several we have recently used in the mechanistic studies of P450, and
studies have also shown that, unlike other peroxidases, CPO compared the results with those of P450-catalyzed reactions.
is capable of carrying out monooxygenation reactions in a The results show that P450- and CPO-catalyzed reactions pro-
similar manner to P450 in addition to typical peroxidase-like ceed through distinctly different mechanisms. As previously
reactions. These observations have been attributed to the reported, while P450-catalyzed reactions appear to proceed
similarities of the active-site architecture of the two enzymes. through a Cα-hydrogen abstraction mechanism, CPO-cata-
Both enzymes have been shown to efficiently catalyze the lyzed reactions proceed through a single electron/proton
oxidative N-dealkylation of amines. The similar magnitudes transfer (SET/H+) mechanism, similar to reactions catalyzed
of the kinetic isotope effects determined for P450- and CPO- by Horseradish peroxidase (HRP). Thus, CPO may not be a
catalyzed N-dealkylation of N,N-dimethylaniline have been good mechanistic model for
P450-catalyzed N-dealky-
used to propose that these reactions proceed through similar lations.(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451
mechanisms. In this study, we have examined the mechanism Weinheim, Germany, 2005)
along with the convenient isolation and spectroscopic char-
acterization of a compound I type terminal active oxygen
Introduction
Chloroperoxidase (CPO) from Caldariomyces fumango is
an interesting heme peroxidase which uses cysteine as an
axial ligand,[1–3] while most other peroxidases use histidine
for this purpose. Thus, the active site of CPO is very similar
to those of the ubiquitous cytochrome P450 (P450) enzymes.
Similarly, and in contrast to other peroxidases, CPO is
known to carry out both typical peroxidase-type oxidations
and more rare oxygen-transfer reactions including hydrox-
ylation of alkanes,[4] sulfoxidations of sulfides, N-dealky-
lation and N-oxygenation of alkylamines,[5–6] and epoxida-
tions of olefins[7–8] in a similar manner to P450. The oxygen
atom present in the products of these reactions has been
shown to originate exclusively from the oxidant, as in the
case of typical monooxygenation reactions carried out by
P450 enzymes.[3–8] One of the major differences between
CPO- and P450-enzyme catalysis is the source of oxygen in
their catalytic cycles. While CPO employs hydrogen perox-
ide as the source of oxygen, P450 enzymes employ molecular
oxygen and an ancillary reduction system that provides re-
ducing equivalents from NAD(P)H. These similarities,
species in the CPO catalytic cycle, make chloroperoxidase a
good model for the active oxygen species of P450 reac-
tions.[9–11]
P450-catalyzed N-dealkylations of N,N-dialkylamines
have been proposed to proceed through either an electron/
proton transfer (SET/H+) or a hydrogen-atom-abstraction
(HAT) mechanism, as shown in Scheme 1. Evidence for the
SET/H+ mechanism has been derived from studies with
chemically and mechanistically well-characterized perox-
idase model systems [e.g. horseradish peroxidase.
(HRP)],[12] kinetic isotope effects,[12] redox-potential corre-
lations,[13] and irreversible enzyme inactivation by various
radical probes.[14–15] However, recent mechanistic studies
[a] Wichita State University, Department of Chemistry
1845 Fairmount St., Wichita, Kansas 67260, USA
Fax: +1-316-978-3431
E-mail: kandatege.wimalasena@wichita.edu
Supporting information for this article is available on the
WWW under http://www.eurjoc.org or from the author.
Scheme 1.
Eur. J. Org. Chem. 2005, 4801–4805
DOI: 10.1002/ejoc.200500333
© 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
4801

M. N. Bhakta, K. Wimalasena
FULL PAPER
with sensitive radical probes[16] and kinetic isotope effect In contrast to the product profiles of the P450 reactions, the
studies[17] have indicated that a HAT mechanism could be CPO-catalyzed oxidations of 1–3 exclusively produced the
operative in P450-catalyzed N-dealkylation reactions. In N-cyclopropyl-cleaved products 1b–3b, with no detectable
contrast to P450-catalyzed N-dealkylations, relatively few N-methyl-, -ethyl-, or -isopropyl-cleaved products. In ad-
mechanistic studies have been carried out with CPO-cata- dition, in the presence of CN– in the incubation medium,
lyzed N-dealkylations, and thus the detailed mechanism has the cyclopropyl ring-opened, radical-cyclized CN– ad-
not been completely elucidated. However, a mechanism ducts[20] were detected as the major products in the CPO
similar to the SET mechanism proposed for P450 was reaction mixtures, similar to the products of parallel HRP
thought to be operative in CPO-catalyzed N-dealkylations, reactions (Table 1 and Scheme 3). These results demon-
primarily on the basis of the similar magnitudes of the kin- strate that while the HRP- and CPO-catalyzed oxidations of
etic isotope effects.[18–19]
1–3 exclusively proceed through a cyclopropyl ring-opening
We have recently shown that P450- and HRP-catalyzed followed by a radical cyclization pathway, P450 reactions
N-dealkylations of the radical probes 1–3 proceed through proceed through a pathway that does not involve the open-
distinct HAT and SET pathways, producing cyclopropyl ing of the cyclopropyl ring, but involves the isotope-sensi-
ring-unopened and -opened products, respectively tive removal of Cα-H from both N-alkyl and N-cyclopropyl
(Scheme 2).[16] In the present study, we have investigated the substituents (Scheme 3). On the basis of previous studies,[16]
product profiles of the CPO-catalyzed N-dealkylations of we conclude that the observed differences in the product
1–3 in order to test the above proposal that the mechanisms distributions between the P450 and CPO reactions, with re-
of the N-dealkylations catalyzed by P450 and CPO are spect to the substrates above, must be due to the fundamen-
mechanistically similar. The results show that the CPO-cat- tal differences in the chemistry of these two systems. Thus,
alyzed N-dealkylations of 1–3 give product profiles that are while P450-catalyzed monooxygenations of these substrates
diagnostic of a SET mechanism parallel to that of HRP. exclusively proceed through a HAT mechanism, CPO-cata-
These are clearly distinct from those of the P450-NADPH- lyzed oxidations exclusively proceed through an initial SET
mediated reactions. These results suggest that the mecha- from the benzylic nitrogen followed by fast opening of the
nism of the CPO-catalyzed N-dealkylation closely resembles cyclopropyl ring. These results further suggest that the rate
that of HRP rather than P450. Thus, the active-site con- of the cyclopropyl ring-opening is greater than that of the
straints, rather than the electronics of the axial thiolate li- Cα-deprotonation of the N-methyl, -ethyl, -isopropyl or
gand, play an important role in CPO-catalyzed reactions, -cyclopropyl groups of the nitrogen radical cation interme-
suggesting that CPO is not a good model for P450-catalyzed diate of these substrates under both CPO and HRP turn-
N-dealkylations.
over conditions (Scheme 3). These findings are consistent
with the fast ring-opening rates predicted for the nitrogen
radical cation intermediates of N-cyclopropylamines
(Scheme 3).[21–22]
The proposed SET/H+ mechanism of hemo-protein-cata-
lyzed N-dealkylations assumes that a [P–FeO]2+ species,
rather than a protein-derived base, is responsible for the Cα-
deprotonation of the nitrogen radical cation intermedi-
ate.[19,23] Since the above results strongly suggest that CPO-
catalyzed N-dealkylations of 1–3 proceed through a SET
mechanism, the [P–FeO]2+ species of CPO could also be
capable of abstracting a proton from the SET intermediate,
the nitrogen radical cation, under normal circumstances.
Scheme 2.
Results and Discussion
Incubation of N-alkyl-N-cyclopropyl-p-chloroaniline de- However, the observed product profiles show that the rate
rivatives 1–3 with P450-2B1 gave two sets of products which of Cα-deprotonation of the nitrogen radical cation interme-
were identified as the N-cyclopropyl-p-chloroanilines 1a– diate of 1–3 is much slower than that of the cyclopropyl
3a, and the N-alkyl-p-chloroanilines 1b–3b, where the par- ring-opening in CPO-catalyzed reactions. Therefore, if the
tition ratios were dependent on the nature of the N-substit- [P–FeO]2+ species is in fact the putative base in CPO, which
uent (Scheme 2 and Table 1). The intramolecular isotope ef- is responsible for the deprotonation of Cα-H, then it must
fects (kH/kD) for the P450-2B1-catalyzed cleavage of the N- be a relatively weak base.[17,24] On the other hand, the pref-
ethyl and N-cyclopropyl groups of 2, which were deter- erential opening of the cyclopropyl ring in the CPO-cata-
mined from the partition ratios of deuterated derivatives, lyzed oxidations of 1–3 could also be due to the inaccessibl-
were 2.6 0.1 and 2.9 0.1, respectively. The kH/kD for the ity of the Cα-H to the [P–FeO]2+ species of the CPO active
cleavage of the N-isopropyl and N-cyclopropyl groups of 3 site because of steric constraints. Therefore, further studies
were 2.9 0.1 and 3.0 0.1, respectively. Furthermore, no are certainly necessary to distinguish between these pos-
detectable cyclopropyl ring-opened or ring-opened radical sibilities.
cyclized products 1c-3c, were observed in the reaction mix-
Taken together, the above results show that CPO- and
ture when the P450-2B1 reactions were carried out in the
P
450-catalyzed N-dealkylations of 1–3 are mechanistically
presence or absence of CN– in the incubation medium.[16] distinct. In contrast to previous proposals, while P450-cata-
4802
© 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
Eur. J. Org. Chem. 2005, 4801–4805

A Mechanistic Comparison between Cytochrome P450- and Chloroperoxidase
FULL PAPER
Table 1. Product distribution of P450-, HRP-, and CPO-catalyzed N-dealkylations of 1–3.
Product Distribution[a]
Reaction
1a
1b
1c
2a
2b
2c
3a
3b
3c
[b]
P450
90(2)
94(1)
ND
ND
ND
10(2)
6(1)
Ͼ98
5(1)
Ͼ98
4(1)
–
ND
–
95(1)
–
47(1)
48(1)
ND
ND
ND
53(1)
52(1)
Ͼ98
8(2)
Ͼ98
17(2)
–
ND
–
92(2)
–
30(1)
27(2)
ND
ND
ND
70(1)
73(2)
Ͼ98
32(1)
Ͼ98
–
ND
–
68(1)
–
P450/CN[c]
HRP[b]
HRP/CN[c]
CPO[b]
CPO/CN[c]
ND
96(1)
ND
83(2)
ND
35(2)
65(2)
[a] The product ratios were averages of at least three independent determinations. The standard deviations for the last significant figures
are given in parentheses. a. 4-chloro-N-cyclopropylaniline; b. 4-chloro-N-alkylaniline; c. CN adduct. [b] The% products were calculated
on the basis of the detectable products a and b (Scheme 3). [c] The% product ratios were calculated assuming 100% trapping of d
(Scheme 3) by CN–. ND: not detected.
Scheme 3.
spectrometer or a Varian/Nicolet-400 spectrometer. All NMR spec-
tra were recorded in CDCl3 using TMS (δ =0.0 ppm) as an internal
standard. Proton chemical shifts (δ) are reported in parts per mil-
lion (ppm) downfield from TMS. Proton–proton coupling con-
stants are reported in Hertz (Hz) and reflect assumed first-order
behavior. Splitting patterns are designated as s, singlet; d, doublet;
t, triplet; q, quartet; sept, septet; m, multiplet; and br, broad. Carbon
chemical shifts are reported in ppm relative to CDCl3 (δ =
77.30 ppm) as an internal standard. GC analyses were performed
using a flame ionization detector with an Agilent 6890 series instru-
lyzed reactions proceed through a Cα-hydrogen-abstraction
mechanism, CPO-catalyzed reactions proceed through a
SET mechanism. Therefore, the magnitudes of the kinetic
isotope effects alone could clearly not be used to distinguish
between these two mechanisms. The mechanistic similarity
of N-dealkylation catalyzed by CPO to that catalyzed by
HRP rather than P450, suggests that the steric rather than
electronic constraints of the active site may play an impor-
tant role in CPO-catalyzed reactions. Therefore, although
the active-site architecture of CPO, especially with respect ment (30 m×0.320 mm carbon-packed capillary column) with a
temperature program of 100 °C for 3 min, 15 °C/min gradient from
100 °C to 280 °C and 280 °C for 10 min. GC–MS analyses were
performed with a Varian CP 3800 GC interfaced with a Varian-
Saturn Model 2200 GC/MS/MS (30 m×0.25 mm carbon-packed
capillary column) and the product mixtures were separated using a
temperature gradient similar to that above, and the spectra of de-
sired products were recorded and analyzed using standard software.
to the axial heme ligand, is similar to that of P450, it is not
a good model for P450-catalyzed N-dealkylations of N,N-
dialkylanilines.[27] Detailed isotope effect studies of the
P
450-, CPO-, and HRP-catalyzed N-dealkylations of N,N-
dialkylaniline derivatives are in progress to determine the
origin of the similarities and differences of the kinetic iso-
tope effects between the N-dealkylation reactions catalyzed
by these systems.
Enzymatic Incubation and Reactions
P
450-2B1 Reactions: Enzymatic reactions were carried out in a total
volume of 250 μL. The enzyme reaction mixtures containing (in
the order of addition) cytochrome P450 (2 μm), cytochrome P450
Experimental Section
-
Materials: Substrates and chromatographic standards were synthe-
sized according to literature procedures as described previously[25]
and purified by preparatory reverse-phase HPLC. Dilauryl-l-α-
phosphatidylcholine (DLPC), NADPH, chloroperoxidase (CPO;
EC 1.11.1.10), and horseradish peroxidase (HRP; EC 1.11.1.7, RZ
= 3.1) were purchased from Sigma–Aldrich. Catalase was pur-
chased from Roche Applied Sciences. Cytochrome P450-2B1 and
NADPH-P450-reductase were purified as reported previously.[26]
1H- and 13C NMR spectra were recorded with a Varian/Inova-300
reductase (4 μm), and DLPC (360 μm) were pre-incubated for
45 min on ice. After the pre-incubation, catalase (50 μL, 1 mg/mL),
potassium phosphate buffer (50 μL, 0.5 m, pH 7.4), H2O (28 μL),
and substrate (4 μL, 0.5 m) were added and incubated at 30 °C for
5 min. The reactions were initiated by adding NADPH to a final
concentration of 1.2 mm and incubated for 30 min at 30 °C. The
reactions were terminated by adding saturated K2CO3 (150 μL),
and the products were extracted with ethyl acetate (500 μL) with
N,N-diisopropylaniline as the internal standard, dried with a
Eur. J. Org. Chem. 2005, 4801–4805
www.eurjoc.org
© 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
4803

M. N. Bhakta, K. Wimalasena
FULL PAPER
stream of air, and dissolved in methanol. The products of this pro-
cedure were analyzed by GC and GC–MS.
quenched by adding saturated ammonium chloride solution, and
filtered. The supernatant liquid was extracted with ethyl acetate,
dried with Na2SO4, and concentrated. The crude reaction mixture
was purified using silica gel with hexane as the solvent to afford 1
(450 mg, 42%). 1H NMR (300 MHz, CDCl3): δ = 7.18 (dt, 1J =
3.3, 9.1 Hz, 2 H, 2 CH), 6.89 (dt, 1J = 3.3, 9.1 Hz, 2 H, 2 CH),
Horseradish Peroxidase (HRP) Reactions: Enzymatic oxidations
with HRP were carried out in potassium phosphate buffer (0.4 m,
pH 5.5) containing HRP (52 nm) and substrate (4 mm) in a total
volume of 250 μL. The reactions were initiated by adding hydrogen
peroxide to a final concentration of 4 mm. The reaction mixtures
were incubated for 10 min at room temperature and were quenched.
The products were extracted and analyzed as described above.
1
2.94 (s, 3 H, CH3), 2.35 (tt, J = 3.8, 6.6 Hz, 1 H, CH), 0.82 (m, 2
H, CH2), 0.60 (m, 2 H, CH2) ppm. 13C NMR (75.4 MHz, CDCl3):
δ = 149.6, 128.8, 122.4, 115.1, 39.3, 33.6, 9.3 ppm.
4-Chloro-N-cyclopropyl-N-ethylaniline (2): A mixture of 4-chloro-
N-ethylformanilide (1.0 g, 5.4 mmol) and Ti(OiPr)4 (1.7 g,
6.0 mmol) in THF (20 mL) was treated with ethylmagnesium chlo-
ride (4.8 mL, 25% in THF, 13.6 mmol) and stirred overnight. The
desired product was obtained as described above for 1 in 45% yield.
Chloroperoxidase (CPO) Reactions: Chloroperoxidase reactions
were carried out in a total volume of 250 μL of potassium phos-
phate buffer (0.1 m, pH, 6.0) containing CPO (20 units) and sub-
strate (4 mm). The reactions were initiated by adding hydrogen per-
oxide to a final concentration of 4 mm, and the reaction mixtures
were incubated at room temperature for 30 min. Products were ex-
tracted, identified, and quantified by GC–FID and GC–MS.
1
1H NMR (400 MHz, CDCl3): δ = 7.14 (td, J = 3.0, 9.1 Hz, 2 H,
1
2 CH), 6.87 (dt, 1J = 3.0, 9.1 Hz, 2 H, 2 CH), 3.42 (q, J = 7.2 Hz,
2 H, CH2), 2.37 (tt, 1J = 3.85, 6.6 Hz, 1 H, CH), 1.06 (t, 1J =
6.9 Hz, 3 H, CH3), 0.79 (m, 2 H, CH2), 0.56 (m, 2 H, CH2) ppm.
13C NMR (100 MHz, CDCl3): δ = 148.1, 128.9, 122.2, 115.7, 45.7,
31.2, 11.6, 9.2 ppm. MS: m/z = 195.
Determination of Intramolecular Kinetic Isotope Effects (kH/kD)
The intramolecular kinetic isotope effects (kH/kD) for the cleavages
of N-alkyl and N-cyclopropyl groups were determined from the
partition ratios of the corresponding proto- and deutero-substrates.
The substrates (1–3) used in this study primarily gave two partition
products, an N-alkyl-cleaved product, 4-chloro-N-cyclopropylani-
line, and an N-cyclopropyl-cleaved product, 4-chloro-N-alkylani-
line. Substitution of deuterium at the Cα-position of the substrate
alters the product partition ratios because of the kinetic deuterium
isotope effect. The intramolecular kH/kD values for the two path-
ways were obtained from the corresponding partition ratios. For
example, if specifically Cα-H and Cα-D cyclopropyl derivatives were
used:
4-Chloro-N-cyclopropyl-N-[1,1-D2]ethylaniline:
A mixture of 4-
chloro-N-[2,2-D2]ethylformanilide (1.0 g, 5.4 mmol) and Ti(OiPr)4
(1.7 g, 6.0 mmol) in THF (20 mL) was treated with ethylmagnesium
chloride (4.8 mL, 25% in THF, 13.6 mmol) and stirred overnight.
The desired product was obtained as described above for 1 in 35%
1
1
yield. H NMR (400 MHz, CDCl3): δ = 7.14 (td, J = 3.5, 9.1 Hz,
1
1
2 H, 2 CH), 6.87 (dt, J = 3.5, 9.1 Hz, 2 H, 2 CH), 2.37 (tt, J =
3.85, 6.6 Hz, 1 H, CH), 1.05 (s, 3 H, CH3), 0.79 (m, 2 H, CH2),
0.56 (m, 2 H, CH2) ppm. 13C NMR (100 MHz, CDCl3): δ = 148.14,
128.94, 122.24, 115.60, 31.04, 11.31, 9.04 ppm. MS: m/z = 197, deu-
terium content Ͼ98%.
4-Chloro-N-[1-D]cyclopropyl-N-ethylaniline: A mixture of 4-chloro-
N-ethyl[D]formanilide (1.0 g, 5.4 mmol) and Ti(OiPr)4 (1.7 g,
6.0 mmol) in THF (20 mL) was treated with ethylmagnesium chlo-
ride (4.8 mL, 25% in THF, 13.6 mmol) and stirred overnight. The
desired product was obtained as described above for 1 in 37% yield.
where PRC–H/R–H and PRC–D/R–H are the partition product ratios
for the proteo- and deutero-derivatives, and kC–H, kR–H, and kC–D
are the rates of the cyclopropyl Cα-H, cyclopropyl Cα-D, and alkyl
Cα-H abstraction steps, respectively.
1
1H NMR (400 MHz, CDCl3): δ = 7.14 (td, J = 3.3, 9.1 Hz, 2 H,
1
2 CH), 6.87 (dt, 1J = 3.3, 9.1 Hz, 2 H, 2 CH), 3.44 (q, J = 6.9 Hz,
2 H, CH2), 1.06 (t1J = 6.9 Hz, 3 H, CH3), 0.82 (m, 2 H, CH2), 0.58
(m, 2 H, CH2) ppm. 13C NMR (100 MHz, CDCl3): δ = 148.1,
128.9, 122.2, 115.7, 45.6, 11.6, 9.0 ppm. MS: m/z = 196, deuterium
content Ͼ98%.
Syntheses of 4-Chloro-N-isopropyl-N-cyclopropylanilines (General
Procedures): The starting material, 4-chloro-N-isopropylaniline,
was prepared by refluxing 4-chloroaniline with 2-bromopropane
(1.2 equiv.) for about 1 h. The N-isopropyl Cα-deuterated derivative
was obtained by the same procedure, except that 2-bromopropane
was replaced with 2-deutero-2-bromopropane. 4-Chloro-N-ethyl-
aniline was obtained by treating the corresponding acetanilide with
borane–methyl sulfide complex (2.0 equiv.) in THF at 0 °C fol-
lowed by refluxing for 2 h. Deuterium was incorporated at the Cα-
position of the ethyl group by treating the desired acetanilide with
LiAlD4 (Ͼ98% deuterium content) in THF (25 mL) at 0 °C, fol-
lowed by refluxing overnight. The corresponding 4-chloro-N-alkyl-
formanilides of these N-alkyl derivatives were obtained by treating
with formic acid (1.5 equiv.). The corresponding Cα-deuterofo-
rmanilides were obtained using the same procedure, except that
formic acid was replaced with Cα-D formic acid. The N-alkyl-N-
formanilide derivatives were converted into the corresponding N-
cyclopropyl derivatives by using the method of Chaplinski and de
Mejeire.[25]
4-Chloro-N-cyclopropyl-N-isopropylaniline (3): A mixture of 4-
chloro-N-isopropylformanilide (0.98 g, 5 mmol) and Ti(OiPr)4
(1.5 mL, 5.4 mmol) in THF (25 mL) was treated with ethylmagne-
sium chloride (4.3 mL, 25% in THF, 12.4 mmol) and stirred over-
night. The desired product was obtained as described above for 1
1
1
in 40% yield. H NMR (400 MHz, CDCl3): δ = 7.16 (td, J = 3.3,
8.9 Hz, 2 H, CH2), 6.93 (td, 1J = 3.3, 8.9 Hz, 2 H, CH2), 3.84
(septet, 1J = 6.7 Hz, 1 H, CH), 2.25 (tt, 1J = 3.8, 6.4 Hz, 1 H, CH),
1
1.24 (d, J = 6.7 Hz, 6 H, 2 CH3), 0.81 (m, 2 H, CH2), 0.48 (m, 2
H, CH2) ppm. 13C NMR (100 MHz, CDCl3): δ = 150.2, 128.4,
123.5, 118.8, 54.8, 26.1, 21.0, 9.2 ppm. MS: m/z = 210.
4-Chloro-N-cyclopropyl-N-[1-D]isopropylaniline: A mixture of 4-
chloro-N-[2-D]isopropylformanilide (0.57 g, 2.9 mmol) and
Ti(OiPr)4 (0.91 g, 3.2 mmol) in THF (25 mL) was treated with eth-
ylmagnesium chloride (2.5 mL, 25% in THF, 7.2 mmol) and stirred
overnight. The desired product was obtained as described above
4-Chloro-N-cyclopropyl-N-methylaniline (1): A mixture of 4-chloro-
N-methylformanilide (1.0 g, 5.90 mmol) and Ti(OiPr)4 (1.86 g,
1
1
for 1 in 38% yield. H NMR (300 MHz, CDCl3): δ = 7.14 (td, J
6.49 mmol) was dissolved in THF (20 mL) and treated with ethyl- = 3.0, 9.0 Hz, 2 H, CH2), 6.92 (dt, 1J = 3.0, 9.0 Hz, 2 H, CH2),
magnesium chloride (5.19 mL, 25% in THF, 14.75 mmol) and
stirred overnight at room temperature. The reaction mixture was
2.52 (tt, 1J = 3.85, 6.6 Hz, 1 H, CH), 1.23 (s, 6 H, 2 CH3), 0.78 (m,
2 H, CH2), 0.48 (m, 2 H, CH2) ppm. 13C NMR (75.4 MHz,
4804
© 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
Eur. J. Org. Chem. 2005, 4801–4805

A Mechanistic Comparison between Cytochrome P450- and Chloroperoxidase
FULL PAPER
[9] M. T. Green, J. H. Dawson, H. B. Gray, Science 2004, 304,
1653–1656.
[10] H. B. Dunford in Peroxidases in Chemistry and Biology (Eds.:
J. Everse, K. E. Everse, M. B. Brisham), CRC, Boca Raton,
1991, 1–24.
[11] a) J. H. Dawson, Science 1988, 240, 433–439; b) J. H. Dawson,
M. Sono, Chem. Rev. 1987, 87, 1255–1276.
[12] a) F. P. Guengerich, Chem. Res. Toxicol. 2001, 14, 611–650; b)
Cytochrome P450: Structure, Mechanism, and Biochemistry (Ed.:
P. R. Ortiz de Montellano), Plenum Press, New York, 1995.
[13] T. L. Macdonald, W. G. Gutheim, R. B. Martin, F. P. Guenger-
ich, Biochemistry 1989, 28, 2071–2077.
[14] O. Agusto, H. S. Beilan, P. R. Ortiz de Montellano, J. Biol.
Chem. 1982, 257, 11288–11295.
[15] R. P. Hanzlik, R. H. Tullman, J. Am. Chem. Soc. 1982, 104,
2048–2050.
[16] a) M. N. Bhakta, K. Wimalasena, J. Am. Chem. Soc. 2002, 124,
1844–1845; b) M. N. Bhakta, P. F. Hollenberg, K. Wimalasena,
Chem. Commun. 2005, 265–267; c) M. N. Bhakta, P. F. Hollen-
berg, K. Wimalasena, J. Am. Chem. Soc. 2005, 127, 1376–1377.
[17] a) S. B. Karki, J. P. Dinnocenzo, J. P. Jones, K. R. Korzekwa,
J. Am. Chem. Soc. 1995, 117, 3657–3664; b) J. I. Manchester,
J. P. Dinnocenzo, L. Higgins, J. P. Jones, J. Am. Chem. Soc.
1997, 119, 5069–5070.
CDCl3): δ = 150.3, 128.5, 123.4, 119.0, 54.4 (t), 26.5, 20.9, 9.3 ppm.
MS: m/z = 211, deuterium content Ͼ98%.
4-Chloro-N-[1-D]cyclopropyl-N-isopropylaniline: A mixture of 4-
chloro-N-isopropyl-N-[D]formanilide (0.48 g, 2.4 mmol) and
Ti(OiPr)4 (0.76 g, 2.6 mmol) in THF (20 mL) was treated with eth-
ylmagnesium chloride (2.1 mL, 25% in THF, 6.1 mmol) and stirred
overnight. The desired product was obtained as described above
1
1
for 1 in 39% yield. H NMR (300 MHz, CDCl3): δ = 7.14 (td, J
= 3.5, 9.0 Hz, 2 H, CH2), 6.92 (dt, 1J = 3.3, 9.0 Hz, 2 H, CH2),
3.84 (septet, 1J = 6.6 Hz, 1 H, CH), 1.24 (d, 1J = 6.6 Hz, 6 H, 2
CH3), 0.77 (m, 2 H, CH2), 0.48 (m, 2 H, CH2) ppm. 13C NMR
(75.4 MHz, CDCl3): δ = 150.4, 128.5, 123.4, 119.1, 54.8, 26.1 (t),
21.0, 9.2 ppm. MS: m/z = 211, deuterium content Ͼ98%.
Supporting Information (see footnote on the first page of this arti-
cle): Selected GC traces of the products of P450-, HRP-, and CPO-
catalyzed N-dealkylation of 4-chloro-N-cyclopropyl-N-alkylani-
lines.
Acknowledgments
[18] G. T. Miwa, J. S. Walsh, G. L. Kedderis, P. F. Hollenberg, J.
Biol. Chem. 1983, 258, 14445–14449.
[19] O. Okazaki, F. P. Guengerich, J. Biol. Chem. 1993, 268, 1546–
1552.
[20] a) C. L. Shaffer, S. Harriman, M. Y. Koen, R. P. Hanzlik, J.
Am. Chem. Soc. 2002, 124, 8268–8274; b) C. L. Shaffer, M. D.
Morton, R. P. Hanzlik, J. Am. Chem. Soc. 2001, 123, 8502–
8508.
This work was supported by the National Institutes of Health, GM
45026. We are grateful to Dr. Vincent Storhaug at the Department
of Chemistry at this institution for his assistance with the GC–MS
analyses of enzymatic products and Dr. Paul F. Hollenberg at The
University of Michigan for providing us with the purified P450 and
reductase.
[21] T. M. Nguyen, S. Creve, T. K. Ha, Chem. Phys. Lett. 1998, 293,
[1] M. Sundaramoorthy, J. Terner, T. L. Poulos, Structure 1995, 3,
1367–1377.
[2] a) P. G. Debrunner, A. F. Dexter, C. E. Schulz, Y.-M. Xia, L. P.
Hager, Proc. Natl. Acad. Sci. USA 1996, 93, 12791–12798.
[3] a) P. R. Ortiz de Montellano, Annu. Rev. Pharmacol. Toxicol.
1992, 32, 89–107; b) P. R. Ortiz de Montellano, Acc. Chem.
Res. 1987, 20, 289–294.
90–96.
[22] L. A. Coosky, H. F. King, W. H. Richardson, J. Org. Chem.
2003, 68, 9441–9452.
[23] E. Baciocchi, F. Gerini, O. Lanzalunga, A. Lapi, M. G. Pipar-
aro, S. Mancinelli, Eur. J. Org. Chem. 2001, 2305–2310.
[24] a) J. P. Dinnocenzo, S. B. Karki, J. P. Jones, J. Am. Chem. Soc.
1993, 115, 7111–7116; b) S. B. Karki, J. P. Dinnocenzo, Xenobi-
otica 1995, 25, 711–724.
[4] a) P. H. Toy, M. Newcomb, L. P. Hager, Chem. Res. Toxicol.
1998, 11, 816–823; b) M. B. McCarthy, R. E. White, J. Biol.
Chem. 1983, 258, 9153–9158.
[25] V. Chaplinski, A. de Mejeire, Angew. Chem. Int. Ed. Engl. 1996,
35, 413–414.
[5] M. A. Ator, P. R. Ortiz de Montellano, J. Biol. Chem. 1987,
[26] I. H. Hanna, J. F. Teiber, K. L. Kokones, P. F. Hollenberg,
Arch. Biochem. Biophys. 1998, 350, 324–332.
262, 1542–1551.
[6] S. R. Blanke, L. P. Hager, J. Biol. Chem. 1988, 263, 18739–
18743.
[7] P. R. Ortiz de Montellano, Y. S. Choe, G. DePillis, C. E. Catal-
ano, J. Biol. Chem. 1987, 262, 11641–11646.
[8] L. Casella, M. Gullotti, R. Ghezzi, S. Poli, T. Beringhelli, S.
Colonna, G. Carrea, Biochemistry 1992, 31, 9451–9459.
[27] Our studies were exclusively carried out with N-substituted ani-
line derivatives, and the results may or may not be extended to
aliphatic amines. For example, see M. A. Cerny, R. P. Hanzlik,
Arch. Biochem. Biophys. 2005, 436, 265–275.
Received: May 09, 2005
Published Online: September 28, 2005
Eur. J. Org. Chem. 2005, 4801–4805
www.eurjoc.org
© 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
4805