P450/NADPH/O2- and P450/PhlO-catalyzed N-dealkylations are mechanistically distinct

Article abstract of DOI:10.1021/ja0436143

A high-valent iron-oxo species analogous to the compound I of peroxidases has been thought to be the activated oxygen species in P₄₅₀-catalyzed reactions. Spectroscopic characterization of the catalytically competent iron-oxo species in iodosobenzene (PhIO)-supported model reactions and parallels between these model reactions and PhIO- and NADPH/O₂-supported P₄₅₀ reactions have been taken as strong evidence for this proposal. To support this proposal, subtle differences observed in regio- and chemoselectivities, isotope effects, and source of oxygen, etc., between NADPH/O₂- and PhIO-supported P₄₅₀ reactions have been generally attributed to reasons other than the mechanistic differences between the two systems. In the present study, we have used a series of sensitive mechanistic probes, 4-chloro-N-cyclopropyl-N-alkylanilines, to compare and contrast the chemistries of the NADPH/O₂- and PhIO-supported purified CYP2B1 N-dealkylation reactions. Herein we present the first experimental evidence to demonstrate that the NADPH/O₂- and PhIO-supported P₄₅₀ N-dealkylations are mechanistically distinct and, thus, the P₄₅₀/PhIO system may not be a good mechanistic model for P₄₅₀/NADPH/O₂-catalyzed N-dealkylations. Copyright

Full text of DOI:10.1021/ja0436143

Published on Web 01/12/2005
P
450/NADPH/O2- and P450/PhIO-Catalyzed N-Dealkylations Are Mechanistically
Distinct
Mehul N. Bhakta,† Paul F. Hollenberg,‡ and Kandatege Wimalasena*,†
Department of Chemistry, Wichita State UniVersity, Wichita, Kansas 67260-0051, and Department of
Pharmacology, UniVersity of Michigan Medical School, Ann Arbor, Michigan 48109-0632
Received October 20, 2004; E-mail: kandatege.wimalasena@wichita.edu
Scheme 1. Product Profiles of P450- and HRP-Catalyzed
N-Dealkylation of 1-3 under Various Conditions
Cytochrome P450 (P450) is a family of heme-monooxygenases
found in most organisms. Due to its central role, especially with
respect to drug metabolism and carcinogenesis, the chemical
mechanisms of P450 reactions have been extensively studied.1 Recent
cryocrystallographic and ENDOR techniques have unequivocally
identified the reaction intermediates Fe(II), Fe(III)O2, Fe(III)-OOH,
and Fe(III)‚ROH complexes of P450cam.2 Although numerous at-
tempts have been made, the proposed final active oxygen species,
the ferryl-oxo-π-radical species, has not been observed in the
catalytic cycle of any P450 so far. Spectroscopic evidence for the
formation of the ferryl-oxo species with m-chloroperbenzoic acid,
in the absence of substrate, has been recently reported with a
thermostable P450.3 On the other hand, catalytically competent ferryl-
oxo species have been characterized for chloroperoxidase,4 horse-
radish peroxidase (HRP),5 and oxygen surrogates such as iodosoben-
zene (PhIO)-activated, model metalloporphyrin systems.6 The
apparent parallels between the reactions catalyzed by P450/NADPH/
O2- and PhIO-supported P450 and metalloporphyrin model systems
have been taken as strong evidence for the involvement of a similar
ferryl-oxo species in P450/NADPH/O2 reactions.2 To support this
proposal, subtle differences observed in regio- and chemoselec-
tivities,7 isotope effects,8 and source of oxygen,9 etc., between
NADPH/O2- and PhIO-supported P450 reactions have been generally
attributed to reasons other than the mechanistic differences between
the two systems.6-8 Herein we present the first experimental
evidence to demonstrate that the NADPH/O2- and PhIO-supported
cleaved products, 1b-3b, and cyclopropyl ring-opened radical
cyclized CN9 adducts,12 1c-3c, in the presence of CN- in the
incubation medium (Table 1). These results demonstrate that while
HRP-catalyzed oxidations of 1-3 exclusively proceed through a
single electron transfer (SET) from the benzylic nitrogen followed
by facile cyclopropyl ring opening and radical cyclization pathway,
P450/NADPH/O2 reactions proceed through a distinct pathway that
does not involve the opening of the cyclopropyl ring, but involves
the isotope sensitive removal of hydrogen from the CR of both
N-alkyl and N-cyclopropyl substituents. These findings strongly
support our previous proposal that P450/NADPH/O2-mediated N-
dealkylations proceed through an initial CR-HAT rather than a SET
pathway.10
In contrast to the product profiles of P450/NADPH/O2/1-3
reactions, the P450/PhIO/1-3 reactions exclusively produce the
N-cyclopropyl cleaved products with little or no N-alkyl cleaved
products. In addition, P450/PhIO/1-3 reactions produce the N-
cyclopropyl group cleaved (1b-3b) and cyclopropyl ring-opened
radical cyclized products (1c-3c) in the presence of CN- in the
incubation medium, similar to the HRP-catalyzed reactions (Scheme
1 and Table 1). The control experiment revealed that the formation
of the products was strictly dependent on the presence of the active
P450 N-dealkylations are mechanistically distinct and, thus, the P450
PhIO system may not be a good mechanistic model for P450
NADPH/O2-catalyzed N-dealkylations.
/
/
We have previously shown10 that N-cyclopropyl-N-alkyl-p-
chloroaniline derivatives were good N-dealkylating substrates for
phenobarbital-treated rat liver microsomal preparations. The present
studies show that purified CYP2B1/NADPH/O2-catalyzed mo-
nooxygenation of 1-3 produces both N-cyclopropyl and N-alkyl
group cleaved products 1a-3a and 1b-3b, respectively, and the
partition ratios were dependent on the nature of the N-substituent
(Table 1; Scheme 1). The intramolecular isotope effects (kH/kD)
for the CYP2B1/NADPH/O2-catalyzed cleavage of N-isopropyl and
N-cyclopropyl groups of 1, which were determined from the
partition ratios of appropriately deuterated derivatives, were 2.9 (
0.1 and 3.0 ( 0.1, respectively. The kH/kD’s for the cleavages of
N-ethyl and N-cyclopropyl groups of 2 were 2.6 ( 0.1 and 2.9 (
0.1, respectively. Furthermore, no detectable cyclopropyl ring-
opened, radical cyclized products, 1c-3c, were observed in the
reaction mixtures when the CYP2B1/NADPH/O2/1-3 reactions
were carried out in the presence of CN- in the incubation medium.11
(Table 1 and Scheme 1). On the other hand, the HRP/H2O2-
catalyzed oxidation of 1-3 exclusively produces the N-cyclopropyl
enzyme and PhIO in all cases. The above results show that P450
/
PhIO and HRP/H2O2 systems favor the initial SET from the benzylic
nitrogen, whereas, P450/NADPH/O2 favors the HAT from the CR
of the substrate, under competitive conditions. These results are
also consistent with the previous observations that the chemistry
of P450/PhIO-catalyzed N-dealkylations is similar to that of HRP
rather than P450/NADPH/O2. For example, both HRP- and P450
/
PhIO-catalyzed N-dealkylations display high intramolecular isotope
effects in comparison to low isotope effects of similar P450/NADPH/
O2-supported reactions.8 In addition, the formation of nitrogen cation
radical intermediates has been observed for both P450/PhIO and HRP
systems,8 but similar species have not been detected in P450
NADPH/O2-mediated reactions.
/
Although the observed differences in product profiles of P450
/
PhIO reactions could simply be due to nonaccessibility of the CR-
H’s of the substrates to the active oxygen species due to the steric
† Wichita State University.
‡ University of Michigan Medical School.
9
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J. AM. CHEM. SOC. 2005, 127, 1376-1377
10.1021/ja0436143 CCC: $30.25 © 2005 American Chemical Society

C O M M U N I C A T I O N S
Table 1. Product Distributions of CYP2B1/NADPH/O2-, CYP2B1/PhIO-, and HRP/H2O2-Catalyzed N-Dealkylation of 1-3
% product distributiona
reaction
1a
1b
1c
2a
2b
2c
3ac
3bc
3cc
b
CYP2B1/NADPH/O2
30(1)
27(2)
ND
ND
ND
70(1)
73(2)
>98
47(1)
48(1)
ND
ND
ND
53(1)
52(1)
>98
20(1)
22(1)
ND
ND
ND
80(1)
78(1)
>98
CYP2B1/NADPH/O2/CNd
CYP2B1/PhIOb
ND
ND
ND
CYP2B1/PhIO/CNd
50(2)
>98
50(2)
68(1)
15(2)
>98
85(2)
92(2)
40(2)
>98
60(2)
75(2)
b
HRP/H2O2
HRP/H2O2/CNd
ND
32(1)
ND
8 (2)
ND
25(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, CN9, adduct. b The intermediates d and e (Scheme 1) are not organic soluble
and could not be detected under the experimental conditions, and % products were calculated based on the detectable products, a and b. c In CYP2B1/
NADPH/O2/3 reactions a significant amount of an unidentified aromatic ring hydroxylated product was also detected; % products were calculated based on
total N-dealkylated products. d The % product ratios were calculated assuming that trapping of e by CN- was 100%. ND, not detected.
effects of the remnant PhI, the previous literature evidence13 and
the following experimental observations argue against such a
possibility: (a) The product distribution patterns of N-cyclopro-
pylaniline derivatives were not altered when the steric bulk at the
benzylic nitrogen of the substrate was significantly altered by
substituting N-Me, N-Et, N-iPr, and N-benzyl for both systems;
(b) structure optimization of these substrates shows that the nitrogen
atoms are sterically relatively more shielded than the CR-H’s; (c)
the product profiles of all the above substrates were not altered
when the P450/NADPH/O2 or P450/PhIO reactions were carried out
in the presence of excess PhI (up to 2 mM) in the reaction mixtures;
(d) product profiles were not changed when the wild-type CYP2B1
is replaced with the active site double mutant I114A/F297G, which
has an enlarged active site and has been shown to produce multiple
hydroxylation products from progesterone including 16R, 15R, 21,
and two other unknown hydroxylated products14 (data not shown);
(e) no ring hydroxylated or any other products of PhI or PhIO were
detected in any of the reaction mixtures. Therefore, the differences
of the product distribution patterns of the PhIO- and NADPH-
supported P450 reactions of the above substrates are most likely
associated with the differences of the chemistries of the two
systems.15
As mentioned above, PhIO-supported model metalloporphyrin-
catalyzed hydroxylations and epoxidations have been commonly
used as models for P450-catalyzed reactions, and the active oxygen
species of these reactions is widely believed to be the ferryl-oxo
species.2 However, numerous recent model studies suggest that the
catalytically relevant active oxygen species in these reactions may
not be the ferryl-oxo species, but could be a complex between the
iron(III)-porphyrin and the oxidant.16-18 Therefore, a parallel
attractive possibility for the above observations is that the catalyti-
cally relevant oxygen species of P450/PhIO reactions could also be
a similar complex between the PhIO and the heme center of the
thank Prof. James R. Halpert for providing the CYP2B1 active site
double mutant I114A/F297G.
Supporting Information Available: Experimental methods and GC
and GS-MS traces of the reaction products (PDF). This material is
available free of charge via the Internet at http://pubs.acs.org.
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Acknowledgment. This work was supported by the National
Institutes of Health, GM 45026 (K.W.) and CA16954 (P.F.H). We
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