Hydrogen abstraction from neurotransmitters by active oxygen species facilitated by intramolecular hydrogen bonding in the radical intermediates

Article abstract of DOI:10.1039/b600111d

The reactivity of neurotransmitters toward hydrogen abstraction by an active oxygen species (the cumylperoxyl radical) is comparable to that of a strong antioxidant such as catechin due to the strong intramolecular hydrogen bonding, which has been successfully detected by ESR. The Royal Society of Chemistry 2006.

Full text of DOI:10.1039/b600111d

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Hydrogen abstraction from neurotransmitters by active oxygen species
facilitated by intramolecular hydrogen bonding in the radical intermediates
a
b
a
Kei Ohkubo, Yoshihiko Moro-oka* and Shunichi Fukuzumi*
Received 4th January 2006, Accepted 30th January 2006
First published as an Advance Article on the web 10th February 2006
DOI: 10.1039/b600111d
The reactivity of neurotransmitters toward hydrogen abstrac-
tion by an active oxygen species (the cumylperoxyl radical) is
comparable to that of a strong antioxidant such as catechin
due to the strong intramolecular hydrogen bonding, which
has been successfully detected by ESR.
for the first time. The radical intermediates, formed by hydrogen
abstraction from the neurotransmitters, have been successfully
detected by electron spin resonance (ESR) spectroscopy at low
temperature in solution.
The rates of the hydrogen transfer reactions of a series of
neurotransmitters with the cumylperoxyl radical to produce phe-
noxyl radical species were measured using ESR. The cumylperoxyl
radical is formed via a photoinduced radical chain process shown
in Scheme 1. Under photoirradiation of an oxygen-saturated
propionitrile (EtCN) solution of cumene and di-t-butyl peroxide,
the O–O bond of di-t-butyl peroxide is cleaved to produce the t-
Neurotransmitters are small molecules with pivotal biological im-
portance. Norepinephrine, epinephrine, serotonin, and dopamine
1,2
have neurotransmitter function in the human organism. Since
human beings consume more than 20% of their oxygen in the
brain, neurotransmitters are exposed to considerable amounts of
active oxygen species. Active oxygen species such as superoxide
or peroxide and their metabolic products may play a key role in
9,10
butoxyl radical, which abstracts a hydrogen atom from cumene
to produce the cumyl radical. The cumyl radical is readily trapped
by oxygen to produce the cumylperoxyl radical. The ESR signal
of the cumylperoxyl radical has the typical g value of a peroxyl
3–7
the etiology of certain disorders of the central nervous system.
The patients of schizophrenia are also known to be quite sensitive
to the mental and physiological stress that usually increases the
11
radical as shown in Fig. 1a. The cumylperoxyl radical can
abstract a hydrogen atom from cumene in the propagation step
to yield cumene hydroperoxide, accompanied by regeneration
8
concentration of active oxygen species. Under such conditions,
the catecholamine moiety of neurotransmitters may be easily
oxidized to the corresponding quinone, which might disturb
neurotransmission by sending abnormal signals to the receptors
or by behaving as an agonist like methamphetamine. Despite such
importance in understanding the possible roles of the active oxygen
species in disorders of the central nervous system, the reactivities
of neurotransmitters toward active oxygen species have yet to be
reported.
10
of the cumyl radical (Scheme 1). In the termination step,
cumylperoxyl radicals decay via a bimolecular reaction to yield
10
the corresponding peroxide and oxygen (Scheme 1). When the
illumination is cut off, the ESR signal intensity of the cumylperoxyl
radical (Fig. 1a) decays obeying second-order kinetics due to the
bimolecular reaction (Fig. 1b).
We report herein the determination of the rates of a series of neu-
rotransmitters (epinephrine, dopamine, L-DOPA, norepinephrine,
tyrosine and serotonin, which are shown in Chart 1) with an active
oxygen species (the cumylperoxyl radical), in order to evaluate
the reactivity of neurotransmitters toward active oxygen species
Scheme 1
Chart 1
In the presence of epinephrine, the decay rate of the cumylper-
oxyl radical after the light cutoff becomes much greater than
that in the absence of a neurotransmitter. The decay rate in the
presence of a neurotransmitter (e.g., epinephrine) obeys pseudo-
first-order kinetics rather than second-order kinetics. In such
a case, the decay of the ESR signal due to the cumylperoxyl
a
Department of Material and Life Science, Graduate School of Engineering,
Osaka University, SORST, Japan Science and Technology Agency (JST),
Suita, Osaka 565-0871, Japan. E-mail: fukuzumi@chem.eng.osaka-u.ac.jp;
Fax: +81-6-6879-7370; Tel: +81-6-6879-7368
b
College of Community Development, Tokiwa University, Miwa, Mito,
Ibaraki 310-8585, Japan
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because catechin has a much higher HOMO level than the
neurotransmitters (see Table 1 where the HOMO energies are
given) and the one-electron oxidation potential is more negative
12
than that of the cumylperoxyl radical. In contrast to the case
of catechin, electron transfer from the neurotransmitters, which
have much lower HOMO levels, to the cumylperoxyl radical is
energetically not feasible and thus, the hydrogen abstraction from
the neurotransmitters by the cumylperoxyl radical occurs by a one-
step hydrogen transfer, when the kobs value increases with decrease
of the calculated DHT value (Table 1).
A radical intermediate, the phenoxyl radical species in Scheme 1,
was detected by ESR in the hydrogen abstraction from epinephrine
with the t-butoxyl radical. Photoirradiation of an EtCN solution
containing di-t-butyl peroxide and epinephrine with a 1000 W
mercury lamp at 193 K affords the corresponding phenoxyl
radical. Under photoirradiation, the O–O bond of di-t-butyl
peroxide is cleaved to produce the t-butoxyl radical which can
abstract hydrogen from epinephrine to produce the phenoxyl
radical. The ESR spectrum of the phenoxyl radical thus produced
under photoirradiation is shown in Fig. 2a. The well-resolved
◦
Fig. 1 (a) ESR spectrum of the cumylperoxyl radical in EtCN at −70 C
2
generated by the photoirradiation of an O -saturated EtCN solution
−
3
−3
containing di-t-butyl peroxide (1.0 mol dm ) and cumene (1.0 mol dm
)
with a 1000 W high-pressure mercury lamp. (b) Time dependence of the
ESR signal intensity of the cumylperoxyl radical in the presence of the
2
various concentrations of epinephrine in O -saturated EtCN at 193 K.
radical is ascribed to the hydrogen atom transfer from epinephrine
to the cumylperoxyl radical (Scheme 1). The pseudo-first-order
rate constant (k) exhibits first-order dependence with respect to
the concentration of epinephrine. The slope gives the second-
order rate constant (kobs) for the hydrogen transfer process of
epinephrine. The rate constant of the hydrogen transfer from
epinephrine to the cumylperoxyl radical was determined as 2.9 ×
2
−1
3
−1
1
0 mol dm s . This value is the largest among the examined rate
2
−1
3
−1
constants and is comparable to the value (6.0 × 10 mol dm s )
1
2,13
of catechin, which is one of the strongest antioxidants.
The rate constants of the hydrogen transfer reactions of other
neurotransmitters have also been determined and the results are
summarized in Table 1, together with the energy difference values
(DHT) between the phenoxyl radicals and the phenols determined
14
by the density functional theory (DFT) calculations. All the
neurotransmitters afford significantly larger hydrogen transfer rate
−
1
3
−1 15,16
constants than tyrosine (<5 × 10 mol dm s ),
precursor of neurotransmitters. The kobs values of serotonin and
-methylphenol were also small, and this is consistent with the
which is a
4
large DHT values. The kobs value increases with the decrease of
the calculated DHT value. In the case of catechin, the hydrogen
abstraction from catechin by the cumylperoxyl radical occurs
via electron transfer from catechin to the cumylperoxyl radical,
Table 1 Rate constants (kobs) for hydrogen abstraction from neurotrans-
mitters by the cumylperoxyl radical in O -saturated EtCN at 193 K, energy
2
difference values (DHT) between phenoxyl radicals and phenols in reference
to epinephrine and energies of the HOMO level determined by DFT
calculations
−
1
3
−1
a
−1 d
Neurotransmitter
k
obs/mol dm s
D
HT/kJ mol
HOMO/eV
2
2
2
2
2
b
Catechin
Dopamine
L-DOPA
Epinephrine
Norepinephrine
6.0 × 10
2.0 × 10
3.5 × 10
2.9 × 10
1.8 × 10
1.1 × 10
<5 × 10
<5 × 10
−25
−5.73
−8.53
−8.61
−8.68
−8.76
−5.74
−8.09
−8.99
−2.9
−2.1
Fig. 2 (a) ESR spectrum of a phenoxyl radical observed under photo-
e
0
irradiation of
a deaerated EtCN solution containing epinephrine
1.7
7.9
7.9
46
−
3
t
t
−3
c
(0.1 mol dm ) and Bu OOBu (0.1 mol dm ) at 193 K. (b) Com-
puter simulation spectrum; the maximum slope linewidth (DHmsl) =
0.033 mT. (c) hfc values (in mT) together with the calculated val-
ues given in parentheses with the chemical structure of the phenoxyl
radical. (d) DFT optimized structure with spin distribution of the
phenoxyl radical, calculated using the UB3LYP/6-311+G(d,p) basis set.
4
-Methylphenol
Serotonin
Tyrosine
a
b
c
d
Determined in MeCN. ref. 12. ref. 15. Values were determined by
ROHF formalism with the B3LYP/6-31G* basis set. 1665.5 kJ mol
e
−1
.
1
000 | Org. Biomol. Chem., 2006, 4, 999–1001
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ESR spectrum in Fig. 2a allows us to determine the hyperfine
coupling constants (hfc) from the simulation spectrum as shown in
Fig. 2b. The computer simulation spectrum using these hfc values
5 K. Ishige, D. Schubert and Y. Sagara, Free Radical Biol. Med., 2001,
3
0, 433.
6
(a) D. A. Butterfield, Curr. Med. Chem., 2003, 10, 2651; (b) A. I. Bush,
Trends Neurosci., 2003, 26, 207.
(
Fig. 2b) agrees well with the observed ESR spectrum (Fig. 2a).
7 K. Murakami, K. Irie, H. Ohigashi, H. Hara, M. Nagao, T. Shimizu
The hfc assignment is also supported by the DFT calculation
using B3LYP/6-311+G(d,p) (see the calculated hfc values in
and T. Shirasawa, J. Am. Chem. Soc., 2005, 127, 15168.
8
(a) P. Janicak, J. Davis, S. Preskorn and F. Ayd, Jr., Principles and
Practice of Psychopharmacotherapy, Lippincott Williams & Wilkins,
Maryland, USA, 2nd edn, 1997; (b) B. J. Sadock and V. A. Sadock,
Kaplan & Sacick’s Synopsis of Psychiatry, Lippincott Williams &
Wilkins, Maryland, USA, 9th edn, 2003.
14,17
parentheses in Fig. 2c).
The ESR spectrum of the hydrogen
bonded radical in Fig. 2a is quite different from that reported for
18
the free epinephrine semiquinone radical anion. The existence of
strong intramolecular hydrogen bonding in the phenoxyl radical is
clearly indicated by the a(H) value (0.220 mT) at the phenol proton
9
J. K. Kochi, P. J. Krusic and D. R. Eaton, J. Am. Chem. Soc., 1969, 91,
1
877.
1
0 S. Fukuzumi and Y. Ono, J. Chem. Soc., Perkin Trans. 2, 1977, 622.
19
(
Fig. 2c). This is supported by the DFT calculation (Fig. 2d).
11 S. Fukuzumi, K. Shimoosako, T. Suenobu and Y. Watanabe, J. Am.
Chem. Soc., 2003, 125, 9074.
The high hydrogen abstraction reactivity of epinephrine may be
ascribed to the strong intramolecular hydrogen bonding, which
1
2 I. Nakanishi, K. Miyazaki, T. Shimada, K. Ohkubo, S. Urano, N. Ikota,
T. Ozawa, S. Fukuzumi and K. Fukuhara, J. Phys. Chem. A, 2002, 106,
11123.
20
stabilizes the hydrogen abstracted radical.
In conclusion, neurotransmitters are susceptible to hydrogen
abstraction by an active oxygen species (the cumylperoxyl radical).
In particular, the hydrogen abstraction reactivity of catechol
amines is found to be comparable to that of a strong antioxidant
such as catechin due to the strong intramolecular hydrogen
bonding in the phenoxyl radical, which has been successfully
detected by ESR.
13 Protective effects of antioxidants against oxidative stress-induced
neurodegeneration have been reported, see: H. J. Heo and C. Y. Lee,
J. Agric. Food Chem., 2004, 52, 7514.
4 Density functional theory (DFT) calculations were performed on a
COMPAQ DS20E computer. Geometry optimizations were carried out
using the Becke3LYP and 6-31G* basis set for the phenoxyl radical with
the unrestricted Hartree–Fock (UHF) formalism as implemented in the
Gaussian 03 program. The DHT values were determined by the restricted
open shell Hartree–Fock (ROHF) formalism with the B3LYP/6-31G*
basis set.
1
1
1
1
5 The kobs value of tyrosine could not be determined accurately in the
present experimental conditions because of the low solubility in MeCN
at low temperature.
6 The kobs values of phenol derivatives have been reported, see: T. Osako,
K. Ohkubo, M. Taki, Y. Tachi, S. Fukuzumi and S. Itoh, J. Am. Chem.
Soc., 2003, 125, 11027.
7 The geometry optimization of other isomers of hydrogen abstracted
radicals of epinephrine has also been carried out to compare the
energies of the isomers using DFT calculations. The structure drawn in
Fig. 2c is the most stable one among all the isomers.
Acknowledgements
This work was partially supported by a Grant-in-Aid (nos.
1
6205020 and 17750039) from the Ministry of Education, Culture,
Sports, Science and Technology, Japan.
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Org. Biomol. Chem., 2006, 4, 999–1001 | 1001