Intramolecular base-accelerated radical-scavenging reaction of a planar catechin derivative bearing a lysine moiety

Article abstract of DOI:10.1039/b913714a

A planar catechin derivative incorporating a lysine moiety was synthesized and showed ～400-fold increased radical-scavenging activity relative to naturally-occurring (+)-catechin.

Full text of DOI:10.1039/b913714a

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Intramolecular base-accelerated radical-scavenging reaction of a planar
catechin derivative bearing a lysine moietyw
Kiyoshi Fukuhara,*a Ikuo Nakanishi,*bc Kei Ohkubo,c Yoshinori Obara,d Ayako Tada,e
Kohei Imai,e Akiko Ohno,a Asao Nakamura,e Toshihiko Ozawa,bf Shiro Urano,e
Shinichi Saito,d Shunichi Fukuzumi,c Kazunori Anzai,b Naoki Miyataag and Haruhiro Okudaa
Received (in Cambridge, UK) 9th July 2009, Accepted 28th August 2009
First published as an Advance Article on the web 14th September 2009
DOI: 10.1039/b913714a
A planar catechin derivative incorporating a lysine moiety was
synthesized and showed B400-fold increased radical-scavenging
activity relative to naturally-occurring (+)-catechin.
catechin derivatives having various alkyl side chain lengths.19
It was found that increasing the number of carbon atoms
included in the alkyl chain resulted in increasing radical
scavenging abilities and oxidative protection.
Herein, we describe a new type of planar catechin derivative
with a lysine moiety as a basic substituent with the aim to
improve antioxidant and radical-scavenging activities. This
idea is based on our previous report that the radical-scavenging
reaction of a vitamin E model is significantly accelerated by
the presence of a base, such as pyridine.20 As vitamin E is
oxidized to form the corresponding radical cation by a radical-
scavenging reaction, the acceleration is attributed to the
stabilization of the radical cation by base. Therefore, if a basic
functional group were introduced into 2, the radical-scavenging
activities of 2 would be increased even in neutral conditions
without addition of base, thereby leading to the development
of a super antioxidant effective for the prevention and treatment
of oxidative stress-related diseases.
Oxidative stress contributes to the incidence of brain dysfunction,
cancer, cardiovascular diseases, and inflammation.1–4 The
prevention of such diseases by using phenolic antioxidants
such as flavonoids and vitamin E is receiving increasing
attention.5–8 The radical-scavenging activity of phenolic anti-
oxidants, essential for prevention of such oxidative stress
related diseases, is based on their redox properties which are
dominated by the rate of hydrogen transfer reactions between
the phenolic antioxidant and reactive oxygen species.9 Among
the different antioxidants, particular attention has focused on
(+)-catechin (1) which naturally occurs in green tea.10–12 The
structural feature responsible for the radical-scavenging activity
of (+)-catechin is the ortho-dihydroxy functionality in the
catechol ring.13–15 Introduction of electron-donating or with-
drawing substituents into this catechol ring might regulate the
radical-scavenging activities by altering the redox property of
catechin. In addition, other chemical modifications affecting
O–H bond dissociation enthalpy or stabilization of the phenoxyl
radical might also be effective for improving antioxidative
activities. In this context, we previously synthesized a planar
catechin analogue (2), in which the catechol ring and chromane
structure in 1 are constrained to be planar. This analogue
showed an approximate 5-fold increase in radical-scavenging
activity compared with 1.16,17 The predominant radical-
scavenging activity of 2 is attributed to a more negative
oxidation potential than 1.18 We also synthesized planar
In the design of the planar catechin derivative, the orientation
of the amino substituent with respect to the catechol structure
of planar catechin was especially taken into account. The
amino substituent should have a similar conformation to the
catechol structure in 2 so as to stabilize the radical cation of
catechol formed by reaction with various types of reactive
oxygen species. The planar catechin derivative (3) designed in
this study is shown in Fig. 1. The lysine moiety in 3 is
incorporated into the planar catechin through a hydroxy
propyl group as an appropriate linker that defines a precise
geometry between the planar catechin and lysine. The
conformational space of 3 was explored by DFT (B3LYP/
6-31G*) calculations. As shown in Fig. 2, the design provides a
favorable relationship between the amino functional group
and the hydroxyl group of the catechol structure. The distance
between the 14-OH group of the catechol and N of the amino
group is 1.78 A, indicating that formation of an intra-
molecular O–HÁ Á ÁN hydrogen bond is conformationally
favorable which would enhance radical-scavenging activities.
In addition, the ionization potential of 3, calculated by DFT,
is 6.14 eV which is much lower than that of 2 (7.38 eV),
suggesting a higher potential for the one-electron oxidation
reaction responsible for radical-scavenging activities.
a Division of Organic Chemistry, National Institute of Health
Sciences, Setagaya-ku, Tokyo 158-8501, Japan.
E-mail: fukuhara@nihs.go.jp; Fax: +81 3-3707-6950;
Tel: +81 3-3700-1141
b Research Center for Charged Particle Therapy, National Institute
of Radiological Sciences, Inage-ku, Chiba 263-8555, Japan
c Graduate School of Engineering, Osaka University, SORST, Japan
Science and Technology Agency, Suita, Osaka 565-0871, Japan
d Faculty of Science, Tokyo University of Science, Shinjuku-ku, Tokyo
158-8501, Japan
e Department of Applied Chemistry, Shibaura Institute of Technology,
307 Fukasaku, Minuma-ku, Saitama-shi, Saitama 337-8570, Japan
f Yokohama College of Pharmacy, Tozuka-ku, Yokohama, Kanagama
245-0066, Japan
The overall strategy for the synthesis of 3 is outlined in
Fig. 3. Treatment of 1 with 4-oxovaleric acid ethyl ester
resulted in the formation of a planar catechin structure with
an alkyl substituent (4). After protection of phenolic OH with
benzyl bromide, the ethyl ester of 4 was reduced with LAH to
g Graduate School of Pharmaceutical Sciences, Nagoya City
University, Mizuho-ku, Nagoya, Aichi 467-8603, Japan
w Electronic supplementary information (ESI) available: Experimental
details. See DOI: 10.1039/b913714a
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Fig. 1 Structures of 1, 2 and 3.
Fig. 3 Synthesis of 3: (a) ethyl 4-oxovalerate, TMSOTf, THF (76%);
(b) benzyl bromide, K2CO3, DMF (90%); (c) LiAlH4, THF (92%);
(d) Na-Boc-Ne-Z-Lys, DCC, DMAP, CH2Cl2 (62%); (e) H2, 10% Pd/C,
THF (81%).
that the introduction of the lysine moiety increases the k value
compared to 2 by 73 times, or a 420-fold increase over the
k value of 1. The k value of 3 is remarkably high and to our
knowledge is indeed the strongest antioxidant ever observed.
There are two possibilities for the mechanism of the radical-
scavenging reaction toward the oxyl radical: either a one-step
hydrogen atom transfer or an electron transfer followed by a
proton transfer.20,21 Previously, 1 and 2 were found to scavenge
the oxyl radical by an electron transfer mechanism via the
formation of a radical cation intermediate.18 In analogy with
planar catechin 2, the GOꢁ-scavenging reaction of 3 is thought
to proceed by a mechanism involving an electron transfer from
3 to GOꢁ rather than a one-step hydrogen atom transfer.
Therefore, the amino group in lysine, which is located close to
the catechol moiety and participates in intramolecular hydrogen
bonding, is capable of stabilizing the radical cation intermediate
Fig. 2 DFT (density functional theory) optimized structure of 3
calculated using the B3LYP/6-31G* basis set.
afford the alcohol derivative (5), which was then reacted with
Na-Boc-Ne-Z-Lys using DCC, followed by deprotection with
hydrogenation (Pd/C, H2) to furnish 3. In the NOESY
spectrum, cross-peaks were observed for H15/H27 and H15/H29
(data not shown), revealing that the amino group was spatially
close to the catechol structure and formed interactions via
hydrogen bonding. The radical-scavenging properties of 3
were tested in a non-aqueous system using the galvinoxyl
radical (GOꢁ) as an oxyl radical species. When 3 was added
to a deaerated acetonitrile solution of GOꢁ, the visible absorption
band at 428 nm due to GOꢁ disappeared immediately, indicating
that the GOꢁ-scavenging reaction of 3 was taking place. As the
spectrum change is very fast, the rate of the radical-scavenging
reaction of 3 was measured by monitoring the decrease in
absorbance at 428 nm due to GOꢁ using the stopped-flow
technique. The decay of the absorbance of 428 nm obeyed
+
3ꢁ generated in the electron-transfer oxidation of 3 by GOꢁ,
resulting in the enhanced radical-scavenging activity. In
pseudo-first-order kinetics when the concentration of
was maintained at a more than 10-fold excess of the GOꢁ
concentration. The pseudo-first-order rate constant (kobs
3
)
increased linearly with an increase in concentration of 3 as
shown in Fig. 4. From the slope of the linear plot of kobs vs. [3],
the second-order rate constant (k) for the radical-scavenging
reaction was determined as 1.1 Â 104 MÀ1
s
À1. The k values
Fig.
4
Plot of the pseudo-first-order rate constants (kobs) vs.
[catechin] for the reaction of 2 (’) or 3 (K) with GOꢁ (2.4 Â 10À5 M)
for 1 and 2 were determined in the same manner to be 2.6 Â 10
and 1.5 Â 102 MÀ1
s
À1, respectively. These results indicated
in deaerated MeCN at 298 K.
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stronger radical-scavenging activity compared to 2. The
intramolecular hydrogen bond O–HÁ Á ÁN between the amino
moiety in lysine and 14-OH in catechol was validated by a
DFT calculation of 3. Proton transfer from 14-OH to the
amino group, thereby displacing the hydrogen bonding to
+
OÁ Á ÁH–N, was also shown in the optimized geometry of 3ꢁ
.
Stabilization of the radical cation arising from this hydrogen
bond by the amino group of lysine results in the enhancement
of radical-scavenging activity. This study improves our
understanding of the radical-scavenging mechanism of
phenolic antioxidants, and contributes to the further
development of synthetic antioxidants with strong radical-
scavenging activities.
Fig. 5 DFT optimized structure of 3ꢁ+ calculated using the B3LYP/
6-31G* basis set.
Notes and references
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formation of 3(-H)ꢁ by a one-step hydrogen atom transfer
mechanism, no interaction between the amino group and
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had been transferred to the amino group of the lysine and the
length of the resultant OÁ Á ÁH–N hydrogen bond was 1.68 A.
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bonding into OÁ Á ÁH–N significantly contributes to the
stabilization of 3ꢁ+, resulting in an enhancement in the
radical-scavenging activity of 3 by the electron transfer
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Fig. 6 Proposed radical-scavenging mechanism of 3.
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This journal is The Royal Society of Chemistry 2009
6182 | Chem. Commun., 2009, 6180–6182