tert-butoxyl as a model for radicals in biological systems: Caveat emptor [2]

Full text of DOI:10.1021/ja005730l

5808
J. Am. Chem. Soc. 2001, 123, 5808-5809
tert-Butoxyl as a Model for Radicals in Biological
Systems: Caveat Emptor
Scheme 1
§
J. M. Tanko,* Robert Friedline, N. Kamrudin Suleman, and
Neal Castagnoli, Jr.
Department of Chemistry
Virginia Polytechnic Institute and State UniVersity
Blacksburg, Virginia 24061-0212
ReceiVed October 25, 2000
Table 1. Absolute Rate Constants and H/D Isotope Effects for
Reaction of BuO with MPTP (and Deuterated Derivatives)
ReVised Manuscript ReceiVed March 28, 2001
t
•
t
•
The tert-butoxyl radical ( BuO ) has been used as a chemical
model for C-H bond cleavage in enzyme-catalyzed oxidations
1-3
such as those catalyzed by cytochrome P450, methane mono-
oxygenase,3 and monoamine oxidase. The general idea in such
,4
5,6
t
•
studies is to probe the “similarity” between the chemistry of BuO
and the enzyme, “similarity” being assessed in terms of regio-
chemistry (i.e., which C-H bond is cleaved) and kinetic isotope
effects (the effect on rate associated with the replacement of
hydrogen by deuterium). A common (radical) mechanism is
1
substrate
k (M- s-1
)
H D
k /k
8
8
8
8
MPTP
MPTP-d2
2.27 ((0.06) × 10
1.60 ((0.14) × 10
1.21 ((0.20) × 10
1.07 ((0.15) × 10
a
b
c
t
•
1.41 ((0.13)
1.31 ((0.25)
1.13 ((0.25)
assumed to be operating if the enzyme and BuO behave
similarly”.
-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP, 1) is a
MPTP-d
MPTP-d
4
7
“
1
a
b
c
tertiary amine of enormous biological significance. Oxidation
products arising from MPTP lead to a parkinsonian syndrome in
humans. Monoamine oxidase B (MAO-B) is the principal brain
enzyme that catalyzes the oxidation of MPTP.7 The initial events
in the oxidation of amines by MAO are suspected to involve either
homolytic10 or electron-transfer pathways, illustrated in Scheme
d vs d . d vs d . d vs d .
2
0
4
2
7
4
t
•
substituted-MPTP by BuO were determined. The results are
summarized in Table 1.
The magnitude of the observed isotope effect (k /k , Table 1)
H D
is consistent with hydrogen abstraction by BuO at C-6 (the allylic
position). The observation of a small primary isotope effect at
C-6 was expected because this R-C-H bond is the weakest bond
in the molecule (ca. 80 kcal/mol). However, the isotope effects
at C-2 and C-7 (the N-methyl group, Table 1) suggest that BuO
abstracts hydrogen from all R-carbons of MPTP, and not only
the allylic position. These results were unanticipated because the
-9
11
t
•
1
for MPTP.
Initially, the objective of this study was to measure the absolute
t
•
rate constant for hydrogen abstraction from MPTP (by BuO ),
to provide insight into the mechanism of MAO-B catalysis. Our
initial results led to a more general study of the rates and activation
t
•
t
•
parameters associated with the reaction of BuO with tertiary
amines. Experimentally, absolute rate constants for the reaction
13,14
C-2 and C-7 C-H bonds are at least 10 kcal/mol stronger.
t
•
of BuO with amines were determined by laser flash photolysis
The above findings are in contrast to the MAO-B- catalyzed
pathway, where only allylic R-C-H bond cleavage is observed;
no isotope effect is observed at either the C-2 or N-methyl
t
•
(
LFP). In this approach, BuO was generated by irradiating di-
tert-butyl peroxide (DTBPO) with a Nd:YAG laser (2 ns pulse)
12
in the presence of an amine.
15
positions.
Photolysis of DTBPO/MPTP (benzene solvent) gave rise to a
t
•
Thus, despite large differences in bond strengths, BuO exhibits
little or no selectivity in hydrogen abstractions from MPTP. In
fact, BuO seems to exhibit little or no selectivity in hydrogen
transient species (λmax 385 nm) assigned to the MPTP-derived
radical 3. Similar spectra were obtained for MPTP-d
2
, -d
4
, and
t
•
7
-d .) By measuring the observed rate constant for formation of
this species as a function of amine concentration, absolute rate
constants for hydrogen abstraction from MPTP and deuterium-
abstractions from amines, in general. For example, the rate
t
•
12
constant for reaction of BuO with triethylamine and MPTP
are nearly identical, despite the fact that the R-C-H bond in
MPTP is weaker by at least 10 kcal/mol.14
§
On sabbatical leave from the Division of Natural Sciences, University of
Guam, Mangilao, Guam 96923, U.S.A.
It is tempting to explain these results on the basis of the
reactivity/selectivity principle. The strength of the O-H bond in
(
(
1) Karki, S. B.; Dinnocenzo, J. P. Xenobiotica 1995, 25, 711-724.
2) Manchester, J. I.; Dinnocenzo, J. P.; Higgins, L.; Jones, J. P. J. Am.
Chem. Soc. 1997, 119, 5069-5070.
t-BuOH is 105 kcal/mol, while the R-C-H bonds of most amines
lie in the range ca. 80-90 kcal/mol.1
3,14
Consequently, the
(
3) Choi, S.-Y.; Eaton, P. E.; Hollenberg, P. F.; Liu, K. E.; Lippard, S. J.;
Newcomb, M.; Putt, D. A.; Upadhyaya, S. P.; Xiong, Y. J. Am. Chem. Soc.
996, 118, 6547-6555.
4) MacFaul, P. A.; Arends, I. W. C. E.; Ingold, K. U.; Wayner, D. D. M.
J. Chem. Soc., Perkin Trans. 2 1997, 135-145.
5) Franot, C.; Mabic, S.; Castagnoli, N., Jr. Bioorg. Med. Chem. 1997, 5,
519-1529.
6) Franot, C.; Mabic, S.; Castagnoli, N., Jr. Bioorg. Med. Chem. 1998, 6,
hydrogen abstraction process is exothermic by at least 15 kcal/
1
t
•
(
mol, and because of its high reactivity, BuO is expected to exhibit
low selectivity. However, given the extreme exothermicity of these
reactions, several questions arise: Why are these reactions so
slow? Why are they not diffusion-controlled?
In an attempt to answer these questions, rate constants for
reaction of BuO with several amines were measured over a
(
1
2
1
1
(
83-291.
(
7) Heikkila, R. E.; Manzino, L.; Duvoisin, R. C.; Cabbat, F. S. Nature
t
•
984, 311, 467-469.
(
8) Langston, J. W.; Irwin, I.; Langston, E. B. Science 1984, 225, 1480-
temperature range of 10-70 °C. Because in most cases, the
482.
9) Chiba, K.; Peterson, L. A.; Castagnoli, K. P.; Trevor, A. J.; Castagnoli,
(
(13) Wayner, D. D. M.; Clark, K. B.; Rauk, A.; Yu, D.; Armstrong, D. A.
J. Am. Chem. Soc. 1997, 119, 8925-8932.
N., Jr. Drug Metab. Dispos. 1985, 13, 342-347.
(
(
(
10) Edmondson, D. E. Xenobiotica 1995, 25, 735-753.
(14) Dombrowski, G. W.; Dinnocenzo, J. P.; Farid, S.; Goodman, J. L.;
Gould, I. R. J. Org. Chem. 1999, 64, 427-431.
11) Silverman, R. B. Acc. Chem. Res. 1995, 28, 335-342.
12) Griller, D.; Howard, J. A.; Marriott, P. R.; Scaiano, J. C. J. Am. Chem.
(15) Ottoboni, S.; Caldera, P.; Trevor, A.; Castagnoli, N., Jr. J. Biol. Chem.
1989, 264, 13684-13688.
Soc. 1981, 103, 619-623.
1
0.1021/ja005730l CCC: $20.00 © 2001 American Chemical Society
Published on Web 05/24/2001

Communications to the Editor
J. Am. Chem. Soc., Vol. 123, No. 24, 2001 5809
Table 2. Rate Constants and Activation Parameters for Reaction of
t
•
BuO with Several Amines
2
2
BDE
k
E
a
H
1
amine
(kcal/mol) (M- s-1)a
(kcal/mol)
log A
b
b
b
b
b
7
1
(CH
PhN(CH
(PhCH
2
dCHCH
2
)
3
N
82.6
85.4
89.1
90.7
91.7
4.42 × 10 0.076 ((0.43) 7.70 ((0.31)
7
2
3
4
5
6
2
Ph)
2
2.78 × 10 1.12 ((0.62) 8.27 ((0.44)
7
2
)
3
2
N
4.91 × 10 0.22 ((0.30) 7.85 ((0.21)
8
(CH CH
3
)
3
N
1.59 × 10 2.45 ((0.34) 10.0 ((0.24)
8
PhN(CH
quinuclidine
3
)
2
1.00 × 10 2.41 ((0.27) 9.78 ((0.19)
95.8c
9.26 × 10 2.43 ((0.46) 8.76 ((0.32)
6
a
Rate constant at 22 °C, calculated from activation parameters.
b
Literature, ref 14. c Calculated, using energies obtained via density
functional theory (B3LYP/cc-pVTZ).
q
q
Figure 2. T∆S vs ∆H for hydrogen abstractions from amines by
t
•
BuO .
Scheme 2
Thus, intrinsic reactivities are masked because the rate constants
t
•
for hydrogen abstractions from amines by BuO are governed
by entropy considerations. Entropy requirements also explain why
these reactions are not diffusion-controlled. This issue is likely
not to be restricted simply to amines, but is likely to be important
for substrates whose C-H bond strengths are less than ca. 95
Figure 1. Activation energy for hydrogen abstraction from amines by
BuO as a function of R-C-H bond strength.
t
•
t
•
kcal/mol. BuO is not “peculiar” per se, although its bulk and
asymmetry will require a more ordered transition state than
smaller, more symmetric radicals (e.g., HO•, Cl•), the entropy
requirements are not unusually stringent.
radicals resulting from hydrogen abstraction from these amines
do not give a strong UV/vis absorption, diphenylmethanol (DPM)
12
t
•
was used as a probe. Activation parameters were determined
by fitting to the Arrhenius equation.16 The results of these
experiments are summarized in Table 2. (Rate constants at 22
Is BuO a good model for radicals in biological systems? In
terms of regiochemistry, the answer is unequivocally no, at least
for reactive substrates (i.e., C-H bonds weaker than 95 kcal/
mol). For such substrates, the reaction rate and regiochemistry
are dominated by entropy considerationssintrinsic reactivity
patterns are masked. (It is unlikely the entropy requirements
°
C, calculated from the activation parameters, compare well to
-
1
-1
values in the literature (units M s ): (CH
3
CH
, 1.4 × 10 ; quinuclidine, 6.00 × 10 ). The
rate constants do not reveal any sensible structure/reactivity
2
)
3
N, 1.80 ×
8
12
8 17
6 12
1
3 2
0 ; PhN(CH )
t
•
associated with BuO would in any way serve as an effective
model for entropy requirements in an enzyme active site). Many,
and perhaps most, substrates of biological significance contain
heteroatoms (N, O, S) or sites of unsaturation which stabilize
radicals, resulting in weak C-H bonds. Hence, these consider-
ations are not simply restricted to amines. In terms of H/D isotope
effects (or trends in isotope effects with a series of substrates),
t
•
trend: Substrates with the weakest C-H bond react with BuO
at a lower rate! However, as illustrated in Figure 1, the activation
energies do parallel the strength of the C-H bond.
a
Clearly, the intrinsic reactivity trend observed in the E ’s is
offset by the preexponential (A-factor) term in the Arrhenius
equation. It is in fact the magnitude of the A-factor which is
preventing these rate constants from being diffusion controlled.
This situation is easy to understand if rather than Arrhenius
t
•
BuO may be a reasonable model (for an extremely reactive
radical center). It is likely that the entropy requirements and
transition state structure will not change with the substitution of
parameters (E
and entropy of activation. A plot of T∆S (T ) 298 K) vs ∆H
a
, A), the discussion is recast in terms of the enthalpy
q
q
D for H in a substrate. Thus, the magnitude of the isotope (k
) should be a reasonable measure of the differences in activation
energy for the hydrido and deutero analogues.
H
/
q
q
(
Figure 2) reveals that for all these amines (a) T∆S > ∆H (i.e.,
k
D
at room temperature, the entropy of activation contributes more
to the barrier than does the enthalpy of activation), and (b)
substrates which have a low enthalpy of activation (because of
stabilization of the resulting radical) have a higher cost to “pay”
in terms of entropy (reflecting a more highly ordered transition
state). There are two considerations pertaining to the entropy of
the transition state for these reactions: (1) In the transition state
for hydrogen abstraction, the C-H bond must align with the
nitrogen lone pair (and the orbitals of an adjacent π-system, when
present) for maximum resonance stabilization of the developing
t
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Inevitably, as with any model, the usefulness of BuO requires
a thorough understanding of its chemical properties. In the proper
t
•
context, BuO can be an effective model for reactive radicals in
biological systems. Improperly used, it can lead to confusing and
perhaps meaningless results.
Warning: MPTP is a known nigrostriatal neurotoxin and
should be handled using disposable gloves in a properly ventilated
hood. Detailed procedures for the safe handling of MPTP have
been reported.18
t
•
radical center (Scheme 2). (2) Most of the surface of BuO is
unreactive, aliphatic bulk. Hence, there are stringent requirements
Acknowledgment. Financial support from the National Science
Foundation (CHE-9732490) is gratefully acknowledged. Also, we thank
Kay Castagnoli for her efforts in preparation of a sample of MPTP-d .
7
t
•
associated with the trajectory of the approach of BuO to the
amine.
JA005730L
(
16) Benson, S. W. The Foundations of Chemical Kinetics; McGraw-Hill:
New York, 1960.
17) Dinnocenzo, J. P.; Karki, S. B.; Jones, J. P. J. Am. Chem. Soc. 1993,
15, 7111-7116.
(18) Pitts, S. M.; Markey, S. P.; Murphy, D. L.; Weisz, A.; Lunn, G.
Recommended Practices for the Safe Handling of MPTP In MPTP-A
Neurotoxin Producing a Parkinsonian Syndrome; Academic: New York, 1986;
pp 703-716.
(
1