Kinetics and mechanism for the scavenging reaction of the 2,2-diphenyl-1-picrylhydrazyl radical by synthetic artepillin c analogues

Article abstract of DOI:10.1246/bcsj.20120005

The kinetics for the reaction of the 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) with artepillin C, a prenylated phenylpropanoid found specifically in Brazilian propolis, and its analogues was examined in deaerated acetonitrile (MeCN) to shed light on the mechanism for the radical-scavenging reaction of phenolic antioxidants as well as on the structureactivity relationship. Among the examined analogues, a compound having a catechol moiety is found to have the largest second-order rate constant (k) for the DPPH-scavenging reaction. The deuterium kinetic isotope effect of 1.6 was observed for the DPPH-scavenging reaction of artepillin C in the presence of 0.13M CD ₃OD or CH₃OH in deaerated MeCN at 298 K. The log k values were found to be linearly correlated with calculated energy difference values (DHT, HT: hydrogen transfer) between the artepillin C analogues and the corresponding phenoxyl radicals, while such a linear correlation cannot be observed between the log k values and calculated ionization potentials (IP), DHT 1 IP, or experimental one-electron-oxidation potentials of the artepillin C analogues. These results together with a calculated structure of the transition state for the reaction between the artepillin C analogue and DPPH suggest that the DPPH- scavenging reaction of the artepillin C analogues in deaerated MeCN proceeds via a one-step hydrogen-atom transfer from the phenolic OH group to DPPH rather than an electron transfer followed by proton transfer.

Full text of DOI:10.1246/bcsj.20120005

© 2012 The Chemical Society of Japan
Bull. Chem. Soc. Jpn. Vol. 85, No. 8, 877-883 (2012)
877
Kinetics and Mechanism for the Scavenging Reaction of the 2,2-Diphenyl-
1-picrylhydrazyl Radical by Synthetic Artepillin C Analogues
Tomonori Kawashima,1 Sushma Manda,1 Yoshihiro Uto,2 Kei Ohkubo,3,4 Hitoshi Hori,2
Ken-ichiro Matsumoto,1 Kiyoshi Fukuhara,5 Nobuo Ikota,6 Shunichi Fukuzumi,*3,4,7
Toshihiko Ozawa,8 Kazunori Anzai,9 and Ikuo Nakanishi*1
1Radio-Redox-Response Research Team, Advanced Particle Radiation Biology Research Program, Research Center
for Charged Particle Therapy, National Institute of Radiological Sciences (NIRS), Inage-ku, Chiba 263-8555
2Department of Biological Science and Technology, Faculty of Engineering, The University of Tokushima,
Minamijosanjima-cho, Tokushima 770-8506
3Department of Material and Life Science, Graduate School of Engineering, Osaka University, Suita, Osaka 565-0871
4ALCA, Japan Science and Technology Agency (JST), Chiyoda-ku, Tokyo 102-0076
5Division of Organic Chemistry, National Institute of Health Sciences (NIHS), Setagaya-ku, Tokyo 158-8501
6School of Pharmacy, Shujitsu University, Okayama 703-8516
7Department of Bioinspired Science, Ewha Womans University, Seoul 120-750, Korea
8Department of Health Pharmacy, Yokohama College of Pharmacy, Yokohama, Kanagawa 245-0066
9School of Pharmaceutical Sciences, Nihon Pharmaceutical University, Kitaadachi-gun, Saitama 362-0806
Received January 4, 2012; E-mail: nakanis@nirs.go.jp
The kinetics for the reaction of the 2,2-diphenyl-1-picrylhydrazyl radical (DPPH•) with artepillin C, a prenylated
phenylpropanoid found specifically in Brazilian propolis, and its analogues was examined in deaerated acetonitrile
(MeCN) to shed light on the mechanism for the radical-scavenging reaction of phenolic antioxidants as well as on the
structure-activity relationship. Among the examined analogues, a compound having a catechol moiety is found to have
the largest second-order rate constant (k) for the DPPH•-scavenging reaction. The deuterium kinetic isotope effect of 1.6
was observed for the DPPH•-scavenging reaction of artepillin C in the presence of 0.13 M CD3OD or CH3OH in deaerated
MeCN at 298 K. The log k values were found to be linearly correlated with calculated energy difference values (DHT
,
HT: hydrogen transfer) between the artepillin C analogues and the corresponding phenoxyl radicals, while such a linear
correlation cannot be observed between the log k values and calculated ionization potentials (IP), DHT ¹ IP, or
experimental one-electron-oxidation potentials of the artepillin C analogues. These results together with a calculated
structure of the transition state for the reaction between the artepillin C analogue and DPPH• suggest that the DPPH•-
scavenging reaction of the artepillin C analogues in deaerated MeCN proceeds via a one-step hydrogen-atom transfer
from the phenolic OH group to DPPH• rather than an electron transfer followed by proton transfer.
Artepillin C [(E)-3-{4-hydroxy-3,5-bis(3-methyl-2-butenyl)-
phenyl}-2-propenoic acid] (1H) (Chart 1), a major component
(>5%) of Brazilian propolis,1 is a prenylated phenylpropanoid,
which is found specifically in Brazilian propolis and exhibits
important biological activities, such as antitumor,2 apoptosis-
inducing,3 immunomodulating,4 tumor anti-angiogenesis,5 po-
tential chemopreventive,6 anti-leukemic,7 and antioxidative
activities.8 It is known that there are two possible pathways
for antioxidative radical-scavenging reactions of phenolic
compounds.9-12 One is a one-step hydrogen-atom transfer
(Scheme 1a). The other is an electron transfer followed by
proton transfer (Scheme 1b).9-12 These mechanisms are also
known to strongly depend on solvent.10b,10d,13 However, very
little is known about the kinetics, mechanism, and structure-
activity relationship for the radical-scavenging reaction of 1H
and its analogues. Recently, we have reported that the scaveng-
ing reaction of the ¡,¡-dimethylbenzylperoxyl (cumylperoxyl)
radical (PhCMe2OO•) by 1H proceeds via the one-step hydro-
gen-atom transfer mechanism in propionitrile (EtCN) at a low
temperature (203 K).14 We have also reported the structure-
activity relationship by comparing the rates of PhCMe2OO•-
scavenging reaction by structurally modified artepillin C
derivatives.15 The energy difference values (DHT, HT: hydro-
gen transfer) between the artepillin C derivatives and the
corresponding phenoxyl radicals are linearly correlated with
the logarithm of the second-order rate constants (k) for
PhCMe2OO•-scavenging reaction.15 However, for the kinetic
investigation, stable radicals are advantageous, because their
concentrations are readily directly measurable at room temper-
ature. Among them, the 2,2-diphenyl-1-picrylhydrazyl radical
Published on the web August 3, 2012; doi:10.1246/bcsj.20120005

878
Bull. Chem. Soc. Jpn. Vol. 85, No. 8 (2012)
Radical-Scavenging by Artepillin C Analogues
Chart 1.
values as well as experimentally determined one-electron-
oxidation potentials (E0ox) of 1H-7H provides a valuable
insight into the mechanism and structure-activity relationship
of the radical-scavenging reactions of phenolic antioxidants.
Experimental
Materials. Artepillin C (1H) and its analogues 2H, 4H,
and 6H were synthesized by regioselective C-prenylation of
the corresponding ortho-substituted phenols according to our
established procedure.15,19 Coniferyl alcohol (5H) was pur-
chased from Aldrich. Caffeyl alcohol (7H) was obtained
according to a literature procedure.20 The synthesis of 3H
was carried out with 4-iodo-2-methoxyphenol as a starting
material according to a literature procedure,21 although the
detailed procedure will be reported elsewhere. 1H NMR
(400 MHz, CDCl3): ¤ 1.60 (s, 3H, C(CH3)2), 1.68 (d, J =
0.90 Hz, 3H, C(CH3)2), 1.72 (s, 3H, ArCH2CHC(CH3)), 1.98-
2.12 (m, 4H, 2CH2), 3.36 (d, J = 7.3 Hz, 2H, ArCH2), 3.92
(s, 3H, ArOCH3), 5.11 (m, 1H, C(CH3)CH2CH2CH), 5.32
(m, 1H, ArCH2CH), 5.98 (s, 1H, OH), 6.28 (d, J = 15.8 Hz,
1H, CHCOOH), 6.93 (d, J = 1.7 Hz, 1H, H3-Ar), 6.98 (d, J =
1.2 Hz, 1H, H5-Ar), 7.65 (d, J = 15.9 Hz, 1H, ArCH); EI-MS
(m/z) 331 (M+). Anal. Calcd for C20H26O4: C, 72.79; H,
7.93%. Found: C, 72.97; H, 7.99%. The 2,2-diphenyl-1-
picrylhydrazyl radical (DPPH•) was commercially obtained
from Wako Pure Chemical Ind. Ltd., Japan and used as
received. MeCN used as a solvent in both spectrophotome-
tric and electrochemical measurements was purchased from
Nacalai Tesque, Inc. Japan, and used as received. CH3OH and
CD3OD used for measurements of the deuterium kinetic iso-
tope effect were obtained from Nacalai Tesque, Inc. Japan and
Wako Pure Chemical Ind. Ltd., Japan, respectively, and used as
received. Tetrabutylammonium perchlorate (Bu4NClO4) used
as a supporting electrolyte in electrochemical measurements
was purchased from Tokyo Chemical Industry Co., Ltd., Japan,
recrystallized from CH3CH2OH, and dried under vacuum at
313 K prior to use.22
Scheme 1. Two possible pathways for the DPPH•-scaveng-
ing reaction of 1H-7H (R1, R2, R3: See Chart 1).
(DPPH•) has been reported as a reactive hydrogen acceptor and
found to be useful to examine antioxidative activity.10a,13,16,17
In contrast to the case of PhCMe2OO•, DPPH• is also known to
act as a good electron acceptor in hydrogen-transfer reactions
with NADH (¢-nicotinamide adenine dinucleotide reduced
form) analogues.18 Thus, it is interesting to investigate the
mechanism of hydrogen-transfer reaction from artepillin C
analogues to DPPH• in terms of the one-step hydrogen-atom
transfer vs. electron transfer followed by proton transfer. We
report herein the kinetics and mechanism for the reaction of
1H and its analogues 2H-7H (Chart 1) with DPPH• in
deaerated acetonitrile (MeCN) at 298 K. The correlation
between the log k values determined by the stopped-flow
technique and calculated DHT and ionization potential (IP)
Spectral and Kinetic Measurements. To avoid the effect
of molecular oxygen (O2), the reactions were carried out under
strictly deaerated conditions. A continuous flow of argon (Ar)
gas was bubbled through an MeCN solution (3.0 mL) con-
taining DPPH• (4.0 © 10¹5 mol dm¹3) or an antioxidant (5.0 ©
10¹4-2.0 © 10¹3 mol dm¹3) in a reservoir of the spectropho-

T. Kawashima et al.
Bull. Chem. Soc. Jpn. Vol. 85, No. 8 (2012)
879
tometer for 7 min. The rates of DPPH•-scavenging reactions by
1H-7H in deaerated MeCN were determined by monitoring the
absorbance change at 516 nm due to DPPH• by a stopped-flow
technique on a UNISOKU RSP-1000-02NM spectrophotom-
eter at 298 K. The pseudo-first-order rate constants (kobs) were
determined by a least-squares curve fit. The first-order plots of
ln(A ¹ A¨) vs. time (A and A¨ are denoted as the absorbance
at the reaction time and the final absorbance, respectively)
were linear until three or more half-lives with the correlation
coefficient µ > 0.999.
Electrochemical Measurements. Second-harmonic alter-
nating current voltammetry (SHACV)23 was carried out with
three conventional electrodes on an ALS-630A electrochemical
analyzer with glassy carbon disk working electrode, platinum
wire counter electrode, and Ag/AgCl reference electrode.
Solution concentrations for the SHACV analysis were 1.0 ©
Figure 1. Time course of the absorbance change at 516 nm
in the reaction of 7H (2.0 © 10¹3 mol dm¹3) with DPPH•
(4.0 © 10¹5 mol dm¹3) in deaerated MeCN at 298 K. Inset:
The pseudo-first-order plot.
¹3
10¹3 mol dm for all artepillin C derivatives. The working
electrode was polished with BAS polishing alumina suspension
and a polishing cloth and rinsed with Millipore Milli-Q water
followed by CH3OH prior to each measurement. The electrode
¹3
was cycled several times in MeCN containing 0.1 mol dm
Bu4NClO4 within the range of 0 to +1.9 V to reassure the
absence of voltammetric signal due to the supporting electro-
lyte. All electrochemical measurements were performed at
298 K. To minimize the effect of O2 on the electrochemical
behavior of these antioxidants, the inert atmosphere in the
electrochemical cell was maintained throughout the experi-
ments by bubbling Ar gas in the solution before the experi-
ments and a continuous Ar gas flow over the solution during
the measurements. Addition of 45 mV to the E0 values vs.
ox
Ag/AgCl were carried out, to report the E0 values vs. the
ox
saturated calomel electrode (SCE).24
Theoretical Calculations.
Density functional theory
(DFT) calculations were performed on an 8CPU workstation
(PQS, Quantum Cube QS8-2400C-064). Geometry optimiza-
tions were carried out using the Becke3LYP and 6-31G(d) basis
set for the phenoxyl radicals with the unrestricted Hartree-Fock
(UHF) formalism as implemented in the Gaussian 09 program
Revision A.02.25 The DHT values were determined by the single
point energy calculations at the B3LYP/6-31G(d) basis set with
the restricted open shell Hartree-Fock (ROHF) formalism.
Transition state search on the geometry of DPPH•-7H was
obtained from the highest energy point from scanning the bond
length (1.2-2.5 ¡) between the N atom of DPPH• and OH
group of 7H. The transition state was further optimized at the
DFT using the option “opt = (ts, calcfc, noeigentest)” applying
B3LYP/6-31G(d) basis set. To verify the character of the
stationary points, they were subjected to frequency analyses.
The optimized transition structure was characterized as pos-
sessing a single imaginary frequency corresponding to the
transition vector (or reaction coordinate mode) for a particular
chemical transformation, in contrast to energy minima with all-
real vibrational frequencies.
Figure 2. Plots of kobs vs. [1H-7H] for the reaction of 1H-
7H with DPPH• in deaerated MeCN at 298 K.
sentative example). These spectral changes suggest that hydro-
gen transfer from the phenolic OH group in 1H-7H to DPPH•
may take place to scavenge DPPH•. The rate of the DPPH•-
scavenging reaction of 1H-7H was measured by monitoring
the decrease in the absorbance at 516 nm due to DPPH• using
a stopped-flow technique. The decay of the absorbance at
516 nm obeyed pseudo-first-order kinetics when the concen-
tration of 1H-7H ([1H-7H]) was maintained at more than a
10-fold excess of DPPH• concentration (inset of Figure 1). The
kobs values increase with increasing [1H-7H], exhibiting first-
order dependence on [1H-7H], as shown in Figure 2. From the
slopes of the linear plots of kobs vs. [1H-7H], the second-order
rate constants (k) were determined and are listed in Table 1.
Structurally modified artepillin C analogues 2H-7H could
afford significantly larger k values than the parent artepillin
C (1H). The k value for 7H (3.1 © 10 mol¹1 dm3 s¹1) is the
largest among the examined artepillin C analogues in this
study. It is also found that the DPPH•-scavenging reactivity
of the artepillin C derivatives in deaerated MeCN at 298 K
Results and Discussion
DPPH•-Scavenging Rates of Artepillin C and Its Ana-
logues.
Upon addition of 1H-7H to a deaerated MeCN
solution of DPPH•, the absorbance at 516 nm due to DPPH•
immediately decreased, as shown in Figure 1 (7H as a repre-

880
Bull. Chem. Soc. Jpn. Vol. 85, No. 8 (2012)
Radical-Scavenging by Artepillin C Analogues
Table 1. Second-Order Rate Constants (k) for DPPH•-Scavenging Reaction, Energy Difference Values (DHT),
Ionization Potentials (IP), DHT ¹ IP, and One-Electron-Oxidation Potentials (E0ox) of 1H-7H
b)
ka)
DHT
IPb)
/kcal mol
DHT ¹ IPb)
/kcal mol
E0 (vs. SCE)c)
ox
Compound
¹1
¹1
¹1
¹1
/mol¹1 dm3 s
/kcal mol
/V
1H
2H
3H
4H
5H
6H
7H
3.4
7.2
398.3
396.2
394.2
394.1
393.8
392.3
390.2
162.4
154.3
162.6
162.3
158.2
153.2
161.0
235.9
241.9
231.6
231.8
235.6
239.1
229.2
+0.93
+0.83
+0.94
+1.10
+0.85
+0.84
+0.91
1.1 © 10
1.3 © 10
1.3 © 10
1.7 © 10
3.1 © 10
a) Determined in deaerated MeCN at 298 K. b) Calculated by DFT. c) Determined by SHACV with a glassy
¹3
carbon working electrode in deaerated MeCN (0.1 mol dm Bu4NClO4).
Figure 3. Plots of log k vs. (a) DHT, (b) IP, (c) E0ox, and (d) DHT ¹ IP.
follows a similar trend to their PhCMe2OO•-scavenging
reactivity in EtCN at 203 K in our recent report.15 Thus,
DPPH• is a useful tool to evaluated the scavenging reactivity
of phenolic antioxidants against peroxyl radicals.
DPPH•-Scavenging Mechanism of Artepillin C and Its
Analogues. If the radical-scavenging reaction of phenolic
compounds proceeds via one-step hydrogen-atom transfer
(Scheme 1a) as the rate-determining step, the O-H bond
dissociation energy of phenolic OH group is an important
parameter to control the reactivity of the radical-scavenging
reaction.9,10d,26 Thus, the O-H bond dissociation energies of
phenolic OH group in 1H-7H, which equal the DHT values
between 1H-7H and the corresponding phenoxyl radicals
1•-7•, were determined by DFT calculations at the B3LYP/
6-31G(d) level (see the Experimental Section). The results are
listed in Table 1. The lowest DHT value of 7H results from
the electron-donating (ED) property of the ortho OH group
and the intramolecular hydrogen bonding, which significantly
stabilizes 7•. Comparison of the DHT values of the examined
artepillin C analogues with their log k values revealed the
linear correlation between these two parameters, as shown in
Figure 3a. The smaller the DHT value is, the larger the k value
becomes. Thus, the DPPH•-scavenging reaction of 1H-7H may
proceed via the one-step hydrogen-atom transfer mechanism

T. Kawashima et al.
Bull. Chem. Soc. Jpn. Vol. 85, No. 8 (2012)
881
Figure 5. Plots of kobs vs. [1H] in the presence of CH3OH
(3.5 © 10¹1 mol dm¹3) (closed circles) and CD3OD (3.5 ©
10¹1 mol dm¹3) (open circles) for the reaction between
1H and DPPH• (6.6 © 10¹5 mol dm¹3) in deaerated MeCN
at 298 K.
Figure 4. Second-harmonic alternating current voltammo-
¹3
gram for the one-electron oxidation of 4H (1.0 © 10
¹3
mol dm¹3) in deaerated MeCN (0.1 mol dm Bu4NClO4)
at 298 K. Scan rate: 4 mV s¹1; amplitude: 25 mV.
(Scheme 1a). On the other hand, the reactivity of the electron-
transfer reaction (Scheme 1b) of antioxidants is related to their
IP or E0 values.9,10d,26 Thus, the IP values for 1H-7H were
ox
also calculated by DFT and the results are shown in Table 1.
No linear correlation was observed in the plot of log k vs. IP,
as shown in Figure 3b, suggesting that the electron-transfer
process may not be involved as the rate-determining step in the
DPPH•-scavenging reaction of 1H-7H. In addition, the E0
ox
values of 1H-7H were directly determined by the SHACV.23
A representative SHACV wave for 4H using a glassy carbon
working electrode is shown in Figure 4. The E0 value (vs.
ox
¹3
SCE) of 4H in MeCN (0.1 mol dm Bu4NClO4) was deter-
mined from the intersection of the SHACV wave (Figure 4) to
be +1.10 V. The E0 values of other artepillin C derivatives
ox
were also determined in a similar manner (Table 1). No linear
Figure 6. The optimized transition structure of 7H-DPPH•
calculated by DFT (B3LYP/6-31G(d) level).
correlation was also observed in the plot of log k vs. E0
,
ox
as shown in Figure 3c, supporting the ruining of the possi-
bility of the electron transfer as the rate-determining step in
the DPPH•-scavenging reaction of 1H-7H. Furthermore, the
reactivity of the proton-transfer process following the initial
electron transfer (Scheme 1b) can be evaluated by the
calculated deprotonation energies of the radical cation inter-
mediates 1•+-7•+, which correspond to DHT ¹ IP values in
Table 1. As shown in Figure 3d, no linear correlation was
observed between log k and DHT ¹ IP values, suggesting that
the proton-transfer process is also not the rate-determining step
of the DPPH•-scavenging reaction of 1H-7H. These results
strongly suggest that the DPPH•-scavenging reaction of 1H-7H
proceeds via the one-step hydrogen-atom transfer as the case of
the PhCMe2OO•-scavenging reaction by artepillin C analogues
(Scheme 1a).15 In fact, the deuterium kinetic isotope effect
(KIE, kH/kD) of 1.6 (kH = 7.6 © 10 mol¹1 dm3 s¹1, kD = 4.7 ©
10 mol¹1 dm3 s¹1) was observed for the DPPH•-scavenging
reaction of 1H in the presence of 0.13 M CD3OD or CH3OH in
deaerated MeCN at 298 K (Figure 5),27 although the acceler-
ation by the presence of CD3OD or CH3OH was observed as
compared to its absence. Such a KIE value strongly supports
the one-step hydrogen atom transfer mechanism. We also
evaluated the transition state structure of the reaction between
7H and DPPH• by DFT calculation (see the Experimental
Section). The optimized transition structure of 7H-DPPH•
is shown in Figure 6. The distance between the hydrogen
atom of the phenolic OH group in 7H and the nitrogen atom
in DPPH• is 1.283 ¡, which is close enough to occur the
hydrogen-atom transfer.
Structure-Activity Relationship. The enhancement in the
radical-scavenging activity of 2H-7H as compared to 1H can
be explained by the fact that the ED groups reduce the DHT
values and electron-withdrawing (EW) groups have a reverse
effect on it.15,28,29 The presence of ED groups and absence of
EW groups in synthetic artepillin C analogues lowers their DHT
values and increases their k values. For example, the replace-
ment of a methyl group in 2H with a methoxy group to become
6H resulted in more than 2-fold enhancement in the DPPH•-
scavenging activity. A slight increase in the k values was
observed by replacing a carboxy group in 4H with a hydroxy-

882
Bull. Chem. Soc. Jpn. Vol. 85, No. 8 (2012)
Radical-Scavenging by Artepillin C Analogues
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K. Anzai, Chem. Commun. 2008, 626.
Conclusion
Artepillin C (1H) and its analogues 2H-7H showed an
efficient DPPH•-scavenging activity in deaerated MeCN at
298 K. Among them, 7H having a catechol structure showed
the highest DPPH•-scavenging activity due to the stabilization
of the corresponding phenoxyl radical 7• via the intramolecular
hydrogen bonding. The linear correlation between the log k
and DHT values as well as the deuterium kinetic isotope effect
and the calculated transition state structure strongly suggests
that artepillin C and its analogues may follow the one-
step hydrogen-atom transfer rather than the electron transfer
followed by proton transfer in the DPPH•-scavenging reac-
tion in deaerated MeCN. Furthermore, the DPPH•-scavenging
activity of artepillin C analogues can be predicted by the
DHT values calculated by DFT. It is clear that the electron
richer environment the compound has, the lower its DHT value
is and the higher its radical-scavenging activity becomes. The
structure-activity relationship obtained in this study provides
valuable and fundamental information about the radical-
scavenging mechanism of phenolic antioxidants in biological
systems as well as about the development of novel antioxidants
with higher activity than natural occurring ones.
16 S. Goldschmidt, K. Renn, Ber. Dtsch. Chem. Ges. A/B
1922, 55, 628.
This work was partially supported by Grant-in-Aid (Nos.
23590064, 23750014, and 20108010) from the Ministry of
Education, Culture, Sports, Science and Technology, Japan, the
Global COE (center of excellence) program “Global Education
and Research Center for Bio-Environmental Chemistry” of
Osaka University from MEXT, Japan, and NRF/MEST of
Korea through WCU (No. R31-2008-000-10010-0) and GRL
(No. 2010-00353) programs.
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