Detection of aminium ion intermediates: N-cyclopropyl versus N-carboxymethyl groups as reporters [5]

Full text of DOI:10.1021/ja011648u

J. Am. Chem. Soc. 2001, 123, 10107-10108
10107
Scheme 1
Detection of Aminium Ion Intermediates:
N-Cyclopropyl versus N-Carboxymethyl Groups as
Reporters
Rheem A. Totah and Robert P. Hanzlik*
Department of Medicinal Chemistry
UniVersity of Kansas
Malott Hall 4048, 1251 Wescoe Hall DriVe
Lawrence, Kansas 66045-7582
ReceiVed July 9, 2001
Scheme 2
Oxidative N-dealkylation is the single most common reaction
in the biotransformation of alkylamines. This process is catalyzed
by a range of enzymes including the flavoproteins, monoamine
oxidase and sarcosine oxidase, the quinoproteins diamine oxidase,
lysyl oxidase, methylamine dehydrogenase, and plasma amine
oxidase, the non-heme iron oxidase ACC oxidase, and the heme-
thiolate cytochromes P450. N-Dealkylations catalyzed by cyto-
chrome P450 enzymes involve the enzymatic generation of a
carbinolamine, but the mechanism of this reaction is controversial.
One alternative is the hydrogen-atom transfer mechanism (HAT,
Scheme 1), but several important observations including low
kinetic deuterium isotope effects on N-dealkylation reactions, the
release of alkyl radicals during oxidation of 4-alkyl-1,4-dihydro-
pyridines, and the suicide substrate activity of cyclopropylamines
cannot be explained by this mechanism. To accommodate these
observations an alternative pathway involving single-electron
transfer (SET, Scheme 1) as an initiating step has been proposed.1
Considerable experimental effort has gone into differentiating
these two mechanisms.2-8 One approach has been to utilize
“radical clock” reporter groups which reveal cation radical inter-
mediates by undergoing unimolecular ring opening or fragmenta-
tion reactions. For this approach to be reliable, however, the probe
reaction must be fast enough to intercept and divert the postulated
aminium ion intermediate before it reacts to form the “normal”
products, and the probe must report unequiVocally, by giving
distinct products in model HAT versus SET reactions. The
cyclopropyl group has been widely applied to probe the mech-
anisms of amine-oxidizing enzymes.9-13 HAT oxidation of
cyclopropyl groups is known to lead to ring-intact products,14,15
but calculations16 and experimental results7,8,17,18 suggest that the
cyclopropylaminium ion undergoes rapid ring opening19 to a
distonic cation-radical intermediate. The latter has been postu-
lated, but never demonstrated, to be the species that inactivates
P450, and until recently,7,8 little was known about the fate of the
cyclopropyl moiety lost during N-dealkylation reactions (Scheme
2).
Thus, we turned to the N-carboxymethyl group as an alternative
to the cyclopropyl group. When attached to an electron-deficient
aminium cation radical center, this group undergoes rapid
decarboxylation20 and fragmentation21-23 to easily identifiable
products. To evaluate its potential as a reporter group for studying
enzymatic N-dealkylation mechanisms we first sought to compare
its reactivity to that of the cyclopropyl group by means of an
intramolecular competition using N-cyclopropyl-N-phenylglycine
(1a) as the oxidizable substrate and horseradish peroxidase (HRP)
as the SET oxidant (viz. Scheme 2). For context, we also studied
the related substrates 1b and 1c.
Since comparing the relative reactivity of the alkyl, cyclopropyl,
and carboxymethyl groups in 1a-1c upon SET oxidation depends
on quantitating the relative yields of monosubstituted anilines
(2a-2c) versus N-phenylglycine (1d, viz. Scheme 2) we first
investigated the stability of 2a-2c and 1d with HRP under
* Address correspondence to this author. Telephone: 785-864-3750. Fax
785-864-5326. E-mail: rhanzlik@ukans.edu.
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(19) Measured rates for the ring opening of cyclopropylaminium cation
radicals have not been reported, but the N-propyl-N-cyclopropylaminyl radical
undergoes ring opening much faster than the N-propyl-N-cyclobutylaminyl
radical (which fragments with a rate constant of 1.06 × 105 s-1at 25 °C; Maeda,
Y.; Ingold, K. U. J. Am. Chem. Soc. 1980, 102, 328-331), and the N-methyl-
N-(trans-2-phenylcyclopropyl)aminyl radical ring opens with a rate constant
of 7.2 × 1011 s-1 at 20 °C (Musa, O. M.; Horner, J. H.; Shahin, H.; Newcomb,
M. J. Am. Chem. Soc. 1996, 118, 8).
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P. S.; Falvey, D. E.; Yoon, U. C.; Oh, A. W. J. Am. Chem. Soc. 1998, 120,
10676-10686); the glycine cation radical decarboxylates at a rate g 108 sec-1
(Bonifacic, M.; Stefanic, I.; Hug, G. L.; Armstrong, D. A.; Asmus, K.-D. J.
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10.1021/ja011648u CCC: $20.00 © 2001 American Chemical Society
Published on Web 09/25/2001

10108 J. Am. Chem. Soc., Vol. 123, No. 41, 2001
Communications to the Editor
addition of fresh HRP causes a rapid resumption of oxidation of
1a for a brief period, followed again by inactivity that can only
be reversed by addition of fresh HRP. Furthermore, HRP exposed
to 1a and H2O2 for even a few minutes is incapable of oxidizing
other standard substrates including N,N-dimethylaniline and
pyrogallol. Thus, compound 1a is an apparent suicide substrate
for HRP (partition ratio ca. 800 turnovers/inactivation). Cyclo-
propanone hydrate25 and trans-2-phenylcyclopropylamine12 are
also suicide substrates for HRP; however, the action of these
compounds and 1a toward HRP contrasts with that of 1d, 2a,
and 4,7,8 none of which detectably inactivates HRP.
To investigate the role of the ionized carboxyl group in
substrates 1a-1c we also submitted esters 3a and 3b to oxidation
with HRP and monitored product formation by HPLC. The
disappearance of 3b follows zero-order kinetics (45 µM‚min-1
)
standard peroxidatic conditions.24 Under these conditions the
disappearance of 2a-2c and 1d follows first-order kinetics to
g95% completion with apparent rate constants of 0.053, 0.035,
0.48, and 0.015 min-1, respectively; aniline is the product in all
cases. Under similar conditions the disappearance of 1b and 1c
follows strictly zero-order kinetics through at least 95% consump-
through g95% consumption, and the only products are 2b and
methyl glyoxylate (detected as its DNP derivative), both formed
in g95% yield. In this respect the HRP oxidation of 3b is
analogous to the HRP oxidation of other N,N-dialkylanilines, with
the highly selective loss of the ester side chain attributable to the
enhanced acidity of its R-hydrogens. The disappearance of 3a
also follows zero-order kinetics (16 µM‚min-1) through g95%
consumption, but in contrast to 1a, 3a does not detectably
inactivate HRP. The products formed from 3a include N-
phenylglycine methyl ester (3d, trace), aniline (2d, 10 mol %,
formed by rapid secondary oxidation of 3d; 146 µM‚min-1),
methyl glyoxylate (18 mol %), and quinolinium compound 5 (81
mol %, detected, isolated, and identified by HPLC and FAB-
MS). The formation of 5 is analogous to the HRP-induced
conversion of N-cyclopropyl-N-methylaniline (4) to 1-methyl-
quinolinium ion.8 Thus, the relative rates of reaction of substituents
on a tertiary aminium ion (N+•) center are: decarboxylation of
N-carboxymethyl > cyclopropyl ring opening > R-deprotonation
of -CH2COOMe group > R-deprotonation of N-alkyl group.
In conclusion, we have found that the carboxymethyl group
fragments at least an order of magnitude more rapidly than the
cyclopropyl group in response to the one-electron oxidation of a
directly attached nitrogen center. Since N-carboxymethyl groups
are synthetically much easier to introduce into amine substrates
than are N-cyclopropyl groups, this finding should enable a new
approach to investigating mechanisms of amine-oxidizing en-
zymes, notably the cytochrome P450s. The inactivation of HRP
by compound 1a, which contains both probe groups, is enigmatic
in that it is the only compound among those studied that shows
this activity; efforts to elucidate its mechanism of action are
currently underway.
tion. The apparent rate constants are 41 and 102 µM‚min-1
,
respectively, and in both cases the product consists almost entirely
of the corresponding N-alkylaniline (i.e., 2b and 2c) along with
an equivalent amount of formaldehyde and traces of aniline;
N-phenylglycine (1d) is not detected.
When 1a was incubated with HRP, a single major metabolite
(>66% yield) and three minor metabolites were formed. By means
of GC/MS7 and HPLC comparisons to authentic standards the
major metabolite was identified as N-cyclopropylaniline (2a), and
one of the minor metabolites, as aniline (formed by secondary
oxidation of 2a). N-Phenylglycine (1d) was not observed among
the products at any time throughout the reaction. From the relative
rates of oxidation of 2a versus 1d given above, it is clear that if
1d had formed it would have accumulated and been detected.
Since controls showed that as little as a 5 mol % yield of 1d
would have been readily detectable, 1a+• must react preferentially
if not exclusively by decarboxylation rather than by cyclopropyl
group fragmentation.
The kinetics of HRP oxidation of 1a proved to be very different
from the oxidation of 1b and 1c. The disappearance of 1a is quite
rapid up to about 25-30% consumption (within ca. 10 min),
whereupon reaction ceases. Neither addition of H2O2, 30-fold
dilution, nor waiting 24 h restores enzyme activity, whereas
(24) Incubations were conducted at room temperature under air and
contained (in order of addition) 830 µL of potassium phosphate buffer (0.4
M, pH 5.5), 1.0 µmol of substrate (40 µL of a 25 mM solution in MeCN), 3.0
µmol of H2O2 (30 µL of 0.1 M) and 78 pmol of HRP (Sigma, RZ ) 3.0,
added in 100 µL of buffer). To determine rates of substrate disappearance
and product formation, 100 µL aliquots of incubation mixture were removed
at different times and quenched with 100 µL of MeCN. In some cases 2,4-
dinitrophenylhydrazine reagent was also added (see refs 7 and 8). Aliquots
of quenched incubation mixtures (20 µL) were injected onto a Vydac C-18
column (5 µm, 4.6 mm × 150 mm) eluted at 1.0 mL/min with the following
rapid two-step gradient: 0-5 min, 10-60% solvent B (MeCN) in solvent A
(5% MeCN in 50 mM NH4OAc, pH 7.2); 5-10 min, 60-10% B in A. Peaks
were detected at 240 nm and integrated electronically.
Acknowledgment. R.T. is a NIH Predoctoral Trainee (GM-08545).
Supporting Information Available: Syntheses and characterizations
of compounds 1a-c, 3a-c, and 5 (PDF). This material is available free
of charge via the Internet at http://pubs.acs.org.
JA011648U
(25) Wiseman, J. S.; Nichols, J. S.; Kolpak, M. X. J. Biol. Chem. 1982,
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