Bis-coenzyme Q0: Synthesis, characteristics, and application

Article abstract of DOI:10.1002/asia.201000477

A methylene-bridged bis-coenzyme Q₀, bis(2,3-dimethoxy-5-methyl- l,4-benzoquinone)methane (Bis-CoQ₀), that shows intramolecular electronic communications has been synthesized for the first time. By employing electrochemical, in situ UV/Vis, and electron paramagnetic resonance (EPR) spectroelectrochemical techniques, the unstable reduced intermediate speciesa-monoradicals, diamagnetic dianions and tetraanions of Bis-CoQ ₀a-have been observed. The electron-transfer process can be defined as a three-step reduction process with a total of four electrons in solution in CH₃CN. The chemical reaction in the third redox step process was confirmed by variable temperature cyclic voltammetry. In an aprotic CH ₃CN solution, the peak potential separation between electron-transfer steps diminished sequentially with increasing concentration of water. The hydrogen-bonding interactions between water and the electrochemically reduced intermediates of Bis-CoQ₀ can be estimated by peak potential shifts. The electronic communications of Bis-CoQ₀ may have been blocked when one reduction peak was observed with proper quantities of water in CH ₃CN solution. The antioxidant defense capacity of Bis-CoQ ₀-protected cells has also been assessed.

Full text of DOI:10.1002/asia.201000477

FULL PAPERS
DOI: 10.1002/asia.201000477
Bis-Coenzyme Q0: Synthesis, Characteristics, and Application
Xiuwen Wang, Wei Ma, Yilun Ying, Jie Liang, and Yi-Tao Long*[a]
In memory of Xiuwen Wang
Abstract:
A
methylene-bridged bis-
fined as a three-step reduction process
with a total of four electrons in solu-
tion in CH3CN. The chemical reaction
in the third redox step process was con-
firmed by variable temperature cyclic
voltammetry. In an aprotic CH3CN so-
lution, the peak potential separation
between electron-transfer steps dimin-
ished sequentially with increasing con-
centration of water. The hydrogen-
bonding interactions between water
and the electrochemically reduced in-
termediates of Bis-CoQ0 can be esti-
mated by peak potential shifts. The
electronic communications of Bis-CoQ0
may have been blocked when one re-
duction peak was observed with proper
quantities of water in CH3CN solution.
The antioxidant defense capacity of
Bis-CoQ0-protected cells has also been
assessed.
coenzyme Q0, bis(2,3-dimethoxy-5-
methyl-l,4-benzoquinone)methane
(Bis-CoQ0), that shows intramolecular
electronic communications has been
synthesized for the first time. By em-
ploying electrochemical, in situ UV/
Vis, and electron paramagnetic reso-
nance (EPR) spectroelectrochemical
techniques, the unstable reduced inter-
mediate species—monoradicals, dia-
magnetic dianions and tetraanions of
Bis-CoQ0—have been observed. The
electron-transfer process can be de-
Keywords: coenzymes
·
cyclic
voltammetry
electronic
·
electrochemistry
·
·
communication
hydrogen bonds
Introduction
Coenzyme Q0 (2,3-dimethoxy-5-methyl-l,4-benzoquinone;
CoQ0), shown in Scheme 1, is an intermediate in the synthe-
ses of coenzyme Qs, which are the most common redox-
active compounds that display the typical electronic proper-
ties of quinones. These naturally occurring compounds fulfill
several biological functions in a living cell. It participates in
electron and proton transport and adenosine triphosphate
(ATP) synthesis in the mitochondrial respiratory chain. It is
well documented that CoQ0 undergoes two sequential one-
electron reductions that correspond to the formation of the
radical anions and dianions in nonaqueous solvent sys-
tems.[13] Covalent coupling of two CoQ0 units may result in
favorable redox properties and widen its application
window.
In this study, the compound bis(2,3-dimethoxy-5-methyl-
l,4-benzoquinone)methane (Bis-CoQ0) has been synthesized
for the first time. We developed a practical synthetic route
to synthesize Bis-CoQ0 for possible applications in biocatal-
ysis, antioxidation, and antitumor activity. For most poten-
tial antioxidation agents, their biological functions are inti-
mately related to their electrochemical properties.[14] The
electron-transfer process is a fundamental issue for metabol-
ic reactions in living beings. Understanding the electron-
transfer process of a potential drug is therefore always a top
priority. For that reason, we have tried to understand the
Compounds with multiredox centers have received consider-
able attention because of their unique and tunable electro-
chemical properties.[1–4] By coupling two or more redox-
active sites in a molecule through suitable bridges, its elec-
trochemical properties could be changed due to intramolec-
ular electronic communication between redox-active
sites.[5–8] Typical electroactive compounds, such as ferrocenes
and quinones, have been widely investigated with a view
toward their potential optoelectronic and antioxidation ap-
plications, and much work has focused on coupling two fer-
rocenyl or quinonyl groups to construct bis-ferrocenes or
bis-quinones.[9–12] Coupled bis-ferrocenes or bis-quinones
have often exhibited different redox behaviors compared to
their original monomeric ferrocenes or quinones.
[a] X. Wang, W. Ma, Y. Ying, J. Liang, Prof. Dr. Y.-T. Long
Key Laboratory for Advanced Materials and Department of Chemis-
try
East China University of Science and Technology
130 Meilong Road, Shanghai, 200237 (P. R. China)
Fax : (+086)21-642-50032
E-mail: ytlong@ecust.edu.cn
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/asia.201000477.
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teractions between water and the electrochemically reduced
intermediates of Bis-CoQ0 in an aprotic organic solvent
have been well studied. The electronic communications of
Bis-CoQ0 may be blocked in a solution of CH3CN with a
moderate concentration of water. The three-step reduction
process with a total of four electrons transferred gradually
converts into a two-step, two-electron one. In addition, the
protective effect of Bis-CoQ0 against hydrogen peroxide in-
duced oxidative damage to HeLa cells was also investigated.
Results and Discussion
Synthesis
Bis-CoQ0 was synthesized according to Scheme 1. CoQ0 was
reduced by using KBH4 in methanol at 08C for 10 min to
give 1. Compound 1 was treated with base and then methy-
lated by using dimethyl sulfate at room temperature to give
compound 2 in 69% yield. A mixture of 2, trioxane, and
InCl3 was stirred at 1208C for 6 h to give the methylene-
bridged 3 in 93% yield. Silver oxide in 6m HNO3 was used
to regenerate the oxidized quinone (Bis-CoQ0). After purifi-
cation by column chromatography, Bis-CoQ0 was obtained
in 53% overall yield. The structure of compound Bis-CoQ0
1
was confirmed by H and 13C NMR spectroscopy, MS, and
elemental analysis.
Scheme 1. Synthesis of methylene-bridged Bis-CoQ0.
Electrochemical Behavior of Bis-CoQ0, CoQ0, and CoQ0-
CH3 in Nonaqueous CH3CN
electrochemical properties and electron-transfer processes
as far as we could. The electrochemical experiments showed
that in nonaqueous solutions Bis-CoQ0 exhibited two pairs
of reversible redox peaks and one pair of irreversible redox
peaks, thus indicating intramolecular electronic communica-
tions. UV/Vis and EPR spectroelectrochemistry experiments
further supported the existence of intramolecular electronic
communications. The chemical reaction in the irreversible
redox process was also confirmed by variable-temperature
cyclic voltammetry (VT-CV). Due to the fact that most of
the quinones and their intermediates as well as reduced
forms exist inside hydrophobic cell membranes, it is to our
advantage to study their electrochemical properties in aprot-
ic organic solvents, which are useful in mimicking the non-
polar environment in the cell.[15] The hydrogen-bonding in-
To understand the electrochemical behavior of Bis-CoQ0,
CoQ0,
and
2,3-dimethoxy-4,5-methyl-l,4-benzoquinone
(CoQ0-CH3) they were compared by means of cyclic voltam-
metry. Figure 1a shows the CV results at a glassy carbon
(GC) working electrode in nonaqueous acetonitrile
(CH3CN) that contained 0.15m tetrabutylammonium per-
chlorate (TBAP) after complete removal of oxygen by purg-
ing with dry nitrogen for ten minutes. CoQ0 and CoQ0-CH3
displayed two pairs of typical reversible redox peaks of qui-
I
I
nones at ꢀ1.02 V (E = ), ꢀ1.65 V (EII = ), and ꢀ1.11 V (E = ),
1
1
1
2
2
2
ꢀ1.78 V (EII = ) versus ferrocene/ferrocinium (Fc/Fc+), re-
1
2
spectively. Bis-CoQ0 exhibited two sets of reversible redox
peaks at ꢀ1.02 V (EIa = ) and ꢀ1.20 V (EIb = ) and one set of
1
1
2
2
irreversible redox peaks at ꢀ1.73 V (EIIa,b = ). The two possi-
1
2
ble responses from tether-identical redox sites are attributed
to the presence and absence of electronic communica-
tions.[16] Tethered systems that do not display electronic
communication will exhibit the same behavior as nonteth-
ered redox probes. In the case of quinones, Figure 1a shows
a clear difference in the CV redox peaks of CoQ0 and Bis-
CoQ0, which is consistent with the different redox states of
Bis-CoQ0 that are in electronic communication with each
other.
Abstract in Chinese:
The first reduction peak of CoQ0 at ꢀ1.06 V is split into
two reduction peaks for Bis-CoQ0 at ꢀ1.06 and ꢀ1.24 V.
1
The large redox peak splitting (DE = =0.18 V) indicates that
2
strong intramolecular electronic communications exist be-
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Y.-T. Long et al.
FULL PAPERS
ꢀ
any radicals of quinonyl moieties, the other CH2-coupled
quinonyl radical moiety is electron donating, thereby sug-
gesting that a certain degree of stabilization is gained by the
methylene bridge. The dianions of CoQ0-CH3 at ꢀ1.82 V are
at a similar potential region to the Bis-CoQ0 tetranions. The
final reduction step to the tetranions of Bis-CoQ0 implies a
redox system; once at the diamagnetic dianions stage, it be-
haves as if it were a nontethered redox system. The elec-
tron-transfer process is totally different to bis-quinone with-
out electronic communications, for which only two reduction
peaks are observed. Each of two quinonyl groups of Bis-
CoQ0 accept one electron respectively, then the dianions of
Bis-CoQ0 convert to tetranions by accepting the other two
electrons almost simultaneously.
Square-wave voltammetry (SWV) was used to understand
the reduction process in detail. As shown in Figure 1b, the
half-peak width of the Bis-CoQ0 peaks Ia and Ib are equal
to that of peak I for CoQ0 and CoQ0-CH3, whereas the half-
peak width II for Bis-CoQ0 is larger than that of peak II for
both CoQ0 and CoQ0-CH3. Thus, the Bis-CoQ0 peak II
could be split into IIa and IIb. CoQ0 and CoQ0-CH3 exhibit-
ed the expected two peaks (I and II), attributed to two one-
electron redox reactions. Integration of peak I and II of
CoQ0 shows that the area of peak II is equal to 94% of the
area of peak I, and CoQ0-CH3 shows about 70%. The theo-
retical result should show peak area ratios of 1:1 for two
one-electron redox reactions. The deviation from 1:1 peak
areas is explained by invoking a chemical reaction of the
radical anions into an electrochemically inert species. The
assumption of a complexation reaction in which anion radi-
cals react to the dimer was in excellent agreement with the
experimental curves.[17]
Bis-CoQ0 displayed a more interesting result. The first re-
duction (peak I) of Bis-CoQ0 to the dianions is split into two
peaks (Ia and Ib) due to the electronic communication. The
integrated areas of the peak Ia and Ib are almost equal.
Peak II for Bis-CoQ0 was expected to be integrated to the
sum of peaks Ia and Ib; however, the area of peak II was
only 44% of the sum of Ia and Ib. This suggested that a sim-
ilar chemical reaction occurs for Bis-CoQ0 as for CoQ0.[15]
All half-widths of the peaks and their areas are shown in
the Supporting Information.
Figure 2 illustrates the influence of the potential scan rate
v on the cyclic voltammograms at a glassy carbon (GC) elec-
trode in a solution of 0.15m TBAP in nonaqueous CH3CN
that contains 0.2 mm of Bis-CoQ0 at a scan rate of 0.02, 0.04,
0.06, 0.08, 0.1, 0.15, and 0.20 Vsꢀ1. The scanning potential
range was ꢀ0.4 to ꢀ2.4 V. Inspection of the curves reveals
that an increase in the scan rates increases the current of all
the redox peaks of Bis-CoQ0. The first (Ia) and second (Ib)
redox peak potentials have no significant shifts. The third
(IIa,b) reduction peak shifted to more negative potentials,
and the oxidation peak shifted to more positive potentials.
The ratio of the reduction peak current to the oxidation
peak current for the first (Ia) and second (Ib) redox process
is close to unity (ꢁ1). However, the ratio of peak currents
(ipa/ipc) for the third (IIa,b) pair of peaks is found to be
Figure 1. a) CV values obtained at a GC electrode (3 mm in diameter) at
22ꢂ28C at 0.05 Vsꢀ1 for 0.5 mm CoQ0, CoQ0-CH3, and Bis-CoQ0 in non-
aqueous CH3CN that contained 0.15m TBAP. b) Experimental (black
line) and simulated (colored line) square-wave voltammetry (SWV)
values of CoQ0, CoQ0-CH3, and Bis-CoQ0. Increment E=0.004 V, fre-
quency=15 Hz.
tween the methylene-bridged quinonyl moieties of Bis-
CoQ0. The second reduction peak of Bis-CoQ0 is at a more
negative potential than the first reduction peak of CoQ0-
CH3 at ꢀ1.15 V. The CV results suggest that the two elec-
trons are captured successively by the two quinonyl moieties
and a more negative potential is needed for the second elec-
tron transfer. This phenomenon can be understood like this:
once Bis-CoQ0 accepts one electron on one quinonyl moiety
at ꢀ1.06 V, the electronic behavior of the other quinonyl
moiety is very similar to that of CoQ0-CH3. It is known that
the electron-acceptor capability is significantly enhanced
when the side chain of CoQ0 is substituted by an electron-
withdrawing moiety, but is decreased with an electron-do-
nating moiety. So the electron-acceptor capability of CoQ0-
CH2-R (R is a substituent) is significantly decreased when
ꢀ
ꢀ
the side chain of CH2 is coupled by an electron-donating
moiety of semiquinone radicals of CoQ0. Therefore the
second reduction peak potential of Bis-CoQ0 is more nega-
tive than that of CoQ0-CH3. Further reduction to the dia-
nions of CoQ0 occurs at ꢀ1.69 V, which is a more positive
potential than the tetranions of Bis-CoQ0 (ꢀ1.81 V). For
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Bis-Coenzyme Q0
Figure 3. Left: The UV/Vis spectra of CoQ0-CoQ0 (line a), CoQ0-CoQ0Cꢀ
2ꢀ
(line b), CoQ0Cꢀ-CoQ0Cꢀ (line c) and CoQ02ꢀ-CoQ0 (line d). Right: EPR
spectra of CoQ0-CoQ0Cꢀ at ꢀ1.10 V (line a) and ꢀ1.30 V (line b).
Figure 2. CV values at a GC electrode (3 mm in diameter) of 0.2 mm Bis-
CoQ0 in nonaqueous CH3CN that contained 0.15m TBAP at 0.02, 0.04,
0.06, 0.08, 0.1, 0.15, and 0.2 Vsꢀ1. The inset shows the plots of peak cur-
transitions of the benzoquinonyl rings at 275 and 400 nm, as
shown in Figure 3left a. With an applied potential at
ꢀ1.10 V (one-electron reduction), the main absorption band
at 275 nm decreased and a new broad absorption band ap-
peared at 625 nm, as shown in Figure 3left b, which is as-
signed to the n–p* absorption the of radical anions (CoQ0-
CoQ0Cꢀ).[19] This absorption profile indicates that one of the
quinonyl groups is still in its oxidized form and the other
quinonyl ring is in its reduced radical anionic form. With
further reduction at ꢀ1.30 V, the absorption band at 275 nm
completely disappeared whereas the absorption band at
625 nm was retained. It signifies the absence of the quinonyl
group and the presence of one radical anion on each qui-
nonyl group. Other new bands appeared at 306 and 350–
450 nm, as shown in Figure 3left c. The radical anions con-
vert to diamagnetic dianions (CoQ0Cꢀ-CoQ0Cꢀ). At ꢀ1.90 V,
all the benzoquinonyl diamagnetic dianions bands disap-
peared and only the characteristic absorption band for the
tetranions of Bis-CoQ0 at 284 nm was left, as shown in Fig-
ure 3left d. If the diamagnetic dianions of Bis-CoQ0 just
accept one electron at this potential, at which one of the
quinonyl groups is in its reduced dianionic form and the
other quinonyl ring is still in its reduced radical anionic
form, the absorption of radicals of Bis-CoQ0 cannot disap-
pear completely. Therefore the absorption band at 284 nm
!
&
*
rents Ia ( ), Ib ( ), and IIa,b ( ) of Bis-CoQ0, ip, versus the square root
1
=
2
of the scan rate, v .
about 0.77. The ratio of peak currents and the peak poten-
tial shifts indicate that third redox process is irreversible.
All the redox peak currents increase linearly with the
1
=
2
square root of the scan rate, v ; the corresponding linear
1
equations are ipaIa (mA)=19.59v = (Vsꢀ1), (R=0.999); ipcIa
2
1
1
(mA)=ꢀ20.22v = (R=0.999); ipaIb (mA)=16.765v = (Vsꢀ1),
2
2
1
(R=0.999); ipcIb (mA)=ꢀ15.78v = (R=0.999); ipaIIa,b (mA)=
2
1
1
21.27v = (Vsꢀ1), (R=0.997); ipcIIa,b (mA)=ꢀ21.03v = (R=
0.996). The redox peak currents, ip, were enhanced with the
square root of the scan rate on the glassy carbon electrode.
The linear equation is inserted into Figure 2, which indicates
a diffusion-controlled electrode process. For a diffusion-con-
trolled electrode process, the peak current is expressed as a
function of scan rate and the concentration of redox species
in the following equation [Eq. (1)]:[18]
2
2
1
=
2
ð1Þ
ip ¼ 0:446nFcAðDvnF=RTÞ
in which D is the diffusion coefficient of the Bis-CoQ0 and
its intermediate species, c is its molar concentration, n is the
number of electrons that participated in the redox reaction,
A is the active area of the electrode, and the other variables
have conventional meanings. The value of diffusion coeffi-
cients of Ia, Ib, and IIa,b of Bis-CoQ0 are 2.625ꢁ10ꢀ5,
1.615ꢁ10ꢀ5, and 3.61ꢁ10ꢀ6 cm2 sꢀ1, respectively.
To confirm the electron-transfer mechanism, the molecu-
lar structures of the intermediates and final reduced species
were explored by using in situ UV/Vis spectroelectrochemi-
cal techniques. The UV/Vis spectra were recorded during
the reduction of 0.2 mm Bis-CoQ0 in nonaqueous CH3CN
that contained 0.15m TBAP in an optically transparent thin-
layer electrochemical cell (optical path length is (0.4ꢂ
0.1) mm). As shown in Figure 3left, before reduction Bis-
CoQ0 exhibited the characteristic p–p* and n–p* electronic
indicated that at ꢀ1.90 V, diamagnetic dianions of Bis-CoQ0
2ꢀ
accept two electrons and convert to tetranions (CoQ0
CoQ02ꢀ).
-
For further validation of mechanism, an in situ EPR spec-
troelectrochemical study, a versatile technique for the de-
tecting paramagnetic species CoQ0-CoQ0Cꢀ, was conducted
under the same conditions. By applying a reductive voltage
at ꢀ1.10 V as shown in Figure 3right a, the three-line spectra
of CoQ0-CoQ0Cꢀ was obtained in nonaqueous CH3CN. The
values of hyperfine coupling constants, a, were obtained by
simulation. The main coupling constant for hydrogen atoms
at the methylene-bridged group (triple, a=3.10) suggested
that the radical anion is mainly located at the 4-position of
the quinonyl ring. When applying a further reduction at
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ꢀ1.30 V, the monoradical (CoQ0Cꢀ-CoQ0) obtained one
tively negative shifts with an increase in temperature. The
negative shifts are about 0.62 V from 7 to 578C. The striking
potential shifts of the third reduction can be explained by
complexation reactions. The anion radicals react to dimer
spontaneity. The dimer accepts electrons more easily than
diradical anions. As the temperature increases, the dimeriza-
tion equilibrium constant decreases,[17f] so the third peak po-
tential is most positive at low temperature and becomes less
positive as temperature increases.
·ꢀ
more electron to produce the diamagnetic dianion (CoQ0
-
CoQ0Cꢀ). The monoradical EPR signal gradually decreased
and vanished eventually. This phenomena indicated that the
radical anions convert to diamagnetic dianion (CoQ0Cꢀ-
CoQ0Cꢀ).
The temperature-dependent CV values of Bis-CoQ0 are
shown in Figure 4. Variable-temperature experiments were
performed with a nonisothermal electrochemical cell. The
reference electrode was kept at constant temperature ((20ꢂ
0.1)8C), whereas the half cell that contained the working
electrode and the junction to the reference electrode were
kept under thermostatic control with a water bath; its tem-
perature was varied from 7 to 578C, as shown in Figure 4.
Unlike the kinetics of a homogeneous reaction, the peak
potentials do not correspond to the thermodynamic formal
potentials (E8).[20] In a heterogeneous reaction, the redox
behaviors are remarkably influenced by temperature. The
first and second apparent standard potentials have no obvi-
ously negative shifts, whereas the third ones have distinc-
With this experimental configuration, the reaction entropy
for Bis-CoQ0 (DS8’(rc)) is given by Equation (2):[21]
DSꢃ0ðrcÞ ¼ Sꢃ0ðredÞꢀSꢃ0ðoxÞ ¼ nFðdEꢃ0=dTÞ
ð2Þ
Thus, DS8’(rc) was determined from the slope of the plot of
E8’ versus temperature, as shown in Figure 5, which turns
out to be linear under the assumption that DS8’(rc) is con-
Figure 5. The third (IIa,b) redox formal potentials of Bis-CoQ, E0’
IIa,b
versus T plots.
stant over the limited temperature range investigated. With
the same assumption, the enthalpy change (DH8’(rc)) was
obtained from the Gibbs–Helmholtz equation, namely, as
the negative slope of the E8’/T plotted versus 1/T. The E8’
values show a monotonic linear decrease with increasing
temperature from 280 to 330 K. DS8’(rc) and DH8’(rc) are
ꢀ1158 and 66670 Jmolꢀ1, respectively.
Reaction Mechanism
By taking into consideration the experimental data from
CV, SWV, UV/Vis, and EPR spectroelectrochemistry analy-
ses, the electron-transfer process of Bis-CoQ0 (CoQ0-CoQ0)
in nonaqueous CH3CN that contained 0.15m TBAP could
be suggested as follows:
Electrochemical Behavior of Bis-CoQ0 in CH3CN with
Varying Concentrations of Water
Figure 4. CV values at a GC electrode (3 mm in diameter) of 0.2 mm Bis-
CoQ0 in nonaqueous CH3CN that contained 0.15m TBAP at 280, 290,
300, 310, 320, and 330 K. The thermodynamic formal potentials EFꢃc=Fcþ
=
For a better understanding of the hydrogen-bonding interac-
tions between water and the electrochemically reduced in-
0 V.
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Bis-Coenzyme Q0
termediates of Bis-CoQ0 in an aprotic organic solvent, the
electrochemical properties of Bis-CoQ0 and its intermediate
species, monoradicals (CoQ0-CoQ0Cꢀ), diamagnetic dianions
(CoQ0Cꢀ-CoQ0Cꢀ), and tetraanions (CoQ02ꢀ-CoQ02ꢀ), were
studied in CH3CN that contained 0.15m TBAP with varying
concentrations of water. With the addition of water into the
CH3CN, which results in hydrogen-bonding interactions, the
three reduction peaks shifted positively. The third reduction
peak shifted most strongly and merged into the second one
(as shown in Figure 6); the new second one shifted more
strongly and finally merged into the first one. The represen-
tative cyclic voltammograms are given in Figure 8.
According to the experimental results, it can be concluded
that all the Bis-CoQ0 reduced species have hydrogen-bond-
ing interactions with water in CH3CN. Relative to the first
and second reduction peaks, a large positive potential shift
for the third reduction peak was observed. It would suggest
that the tetraanions have much stronger hydrogen-bonding
interactions with water.
Theoretical calculations found that the oxygen atoms of
monoquinones do have an increasing negative-charge densi-
ty during electrochemical reduction from the neutral qui-
nones to their radicals and then to their dianions.[22] Since
tetraanions of bis-quinones have large negative-charge den-
sities with two dianions of quinones, it would be expected
that the oxygen atoms with higher negative-charge densities
in the tetraanions will have a stronger interaction with water
molecules than those in the diradical anions. To treat the sit-
uation more quantitatively, the reaction formula can be ex-
pressed in the form of Equations (3)–(8):
Figure 6. CV values of Bis-CoQ0 at about 0.5 mm in CH3CN that con-
tained 0.15m TBAP with varying concentrations of water at a scan rate
of 0.1 Vsꢀ1
.
For the first redox process step [Eq. (9)]:[17a,23]
:
Eð11Þ ¼ E10 ð1ÞþRT=Fln ð1þKð1ÞCaH O
Þ
ð9Þ
=
2
eq
=
2
2
in which E01 (1) is the half-wave potential of the first redox
=
2
couple in the absence of water in solution, a is the number
of water molecules that are hydrogen bonded to CoQ0-
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Y.-T. Long et al.
FULL PAPERS
CoQ0Cꢀ, Keð1qÞ is the equilibrium constant of Equation (4), and
To further investigate hydrogen bonding in the monoradi-
cals, diamagnetic dianions, and tetraanions, in situ UV/Vis
spectroelectrochemical experiments were conducted on Bis-
CoQ0 in CH3CN in the presence of three different concen-
trations of water (0.22, 1.11, and 2.65m). All the band as-
signments of the UV/Vis spectra of Bis-CoQ0 and its re-
duced intermediate species at varying concentrations of
water are listed in Table 2.
As the concentration of water was increased to 0.22m, a
relatively small change in the UV/Vis spectrum of the singly
reduced species was observed, as shown in Figure 8A. Com-
pared with Bis-CoQ0 in CH3CN (as shown in Figure 3A),
the absorption band at 625 nm, which was given by the n–p*
electronic transition of monoradicals and diamagnetic dia-
nions, decreased with water in CH3CN but with no shifts.
Absorption bands of Bis-CoQ0 and its tetraanions at 274,
400, and 284 nm had no significant changes. This is indicated
that the n–p* electronic transition of benzoquinonyl rings is
sensitive to hydrogen-bonding interactions.
ð1Þ
CH O is the concentration of water. If K CaH O @1, a plot of
eq
2
2
Eð11Þ versus lnCH O gives a straight line with a slope of
=
2
2
2.3aRT/F, from which the value of a could be estimated. For
the sake of simplicity, only one-step, two-electron processes
have been considered for evaluating the degree of hydrogen
bonding for tetraanions. So for the second and third redox
process [Eqs. (10) and (11)], we have the reactions given in
Equations (5),(6) and (7),(8):
E1ð2Þ ¼ E10 ð2ÞþRT=Fln ð1þKð2ÞÞCbH O=ð1þKð1ÞÞCHa O
ð10Þ
ð11Þ
=
2
eq
eq
=
2
2
2
E1ð3Þ ¼ E10 ð3ÞþRT=Fln ð1þKð3ÞÞCcH O=ð1þKð2ÞÞCHb O
=
2
eq
eq
=
2
2
2
ð3Þ
in which b, c, and Kðe2qÞ, K are the number of water mole-
eq
2ꢀ
cules that are hydrogen bonded to CoQ0Cꢀ-CoQ0Cꢀ, CoQ0
-
2ꢀ
CoQ0 , and the equilibrium constant of Equations (6) and
(8), respectively. For a strong hydrogen bond, 1 in the de-
nominator and numerator in Equations (6) and (8) could be
neglected and thus the value of (bꢀa), (cꢀb) could be ob-
tained from the plot of Eð12 or 3Þ versus CH O as shown in
It is interesting that, at a high water concentration of
1.11m, only two reduction peaks were observed, as shown in
Figure 8B. The UV/Vis spectra of the species responsible for
the three-step, four-electron-transfer process could not be
controlled by adjusting the applied potential.
=
2
2
Figure 7. When more water is added, the equilibrium shifted
As shown in the CV results in Figure 8B, when the water
concentration was increased to 1.11m, the third reduction
peak shifted to more positive potentials and finally merged
with the second one. The new second peak currents in-
creased with loss of reversibility. The first redox peaks shift
positively, with no loss of reversibility. The n–p* electronic
transition band at 625 nm almost disappeared with an ap-
plied potential at the first reduction peak potential. With an
applied potential at the second reduction peak potential, the
characteristic band (at about 314 nm) of the diamagnetic
dianions exhibited a slight redshift and the hyperfine band
at about 350–450 nm was more clear compared to that in
CH3CN. This redshift is mainly caused by hyperconjugation
interactions.[24] The conformation of diamagnetic dianions
changed and is more conjugative. With an increase in the
time of electroanalysis, all absorption of the diamagnetic
dianions decayed and the tetraanion characteristic absorp-
tion band at 285 nm occurred. It is clearly a three-step re-
duction process.
~
&
1
*
Figure 7. Plots of DE = of the first ( ), second ( ), and third ( ) redox
2
peaks of Bis-CoQ0 against lnCH O
.
2
When the concentration of water was increased to 2.65m,
as shown in Figure 8C, only one reduction peak was ob-
served. The spectra of the reduced forms change dramatical-
ly. With an applied potential at ꢀ1.15 V, the absorption
band at 275 nm decreased and then disappeared; the new
bands clearly appeared at 314, 365, 420, and 440 nm, where-
as the absorption band at 625 nm completely disappeared.
towards the hydrogen-bonded forms, but because of the ln
relationship with the equilibrium expression in the Nernst
equation, the potential shifts are greatest at the lowest
ð1Þ
ð2Þ
water concentrations. The parameters of a, b, c, K , K
,
eq
eq
ð3Þ
and K for Bis-CoQ0 are summarized in Table 1.
eq
Table 1. Electrochemical parameters: reduction of Bis-CoQ0 in the presence of water in CH3CN.
ðnÞ
n
K
Linear regression
R
eq
Bis-CoQ
Bis-CoQ0
Bis-CoQ0
N
0.610
1.57
4.15
30.7
953
DEð11Þ =0.016lnCH O +0.054
0.997
0.998
0.999
=
2
2
DEð12Þ =0.025lnCH O +0.084
=
2
2
6.48ꢁ104
DEð13Þ =0.066lnCH O +0.280
=
2
2
1070
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Chem. Asian J. 2011, 6, 1064 – 1073

Bis-Coenzyme Q0
Table 2. Band assignment of UV/Vis spectra of Bis-CoQ0 and its reduced intermediates in CH3CN with varying concentrations of water.
Water
Characteristic absorption band [nm]
-
2-
4-
concentration in CH3CN [m]
Bis-CoQ0
275, 400
Bis-CoQ0
Bis-CoQ0
Bis-CoQ0
284
0
625
625
625
306, 625
350, 450
306, 400
625
314, 365
420, 440
313, 365
420, 440
0.22
1.11
2.65
275, 400
275, 400
275, 400
284
285
306
The absorption bands of monoradicals were not observed,
or perhaps the absorption bands of monoradicals and dia-
magnetic dianions are too similar to separate them. It may
be caused by the fact that the first two electron-transfer pro-
cesses were too close to be distinguished separately. With an
increase in the time of electroanalysis, all absorption of the
radicals had decayed and the characteristic p–p* electronic
transition absorption band for tetraanions at 310 nm oc-
curred. The absorption band of the tetraanion experienced a
redshift of about 20 nm relative to that in CH3CN with low
water concentration. The obvious redshift cannot be due to
the change in bulk polarity, since even in water the absorb-
ance band of tetraanions of Bis-CoQ0 is at 288 nm. To our
knowledge, electron-transfer reactions and the electrochemi-
cal behavior of compounds that contain several identical
redox centers without electronic communications should be
the same as those of monomeric ones. The absorption bands
of diamagnetic dianions and tetraanions of Bis-CoQ0 and
their electrochemical behavior are very similar to the ab-
sorption bands of radical anions and dianions of CoQ0
under the same solution conditions. The three-step reduc-
tion process with a total transfer of four electrons gradually
converts into a two-step, two-electron reduction process for
Bis-CoQ0 with increasing water concentrations in water/
CH3CN solutions. The process is that the two quinonyl
groups of Bis-CoQ0 accept one electron respectively, and
then the diamagnetic dianions of Bis-CoQ0 convert in tet-
raanions by accepting the other two electrons. The absorp-
tion bands of Bis-CoQ0 intermediates exhibit redshifts. The
electronic communications gradually decreased with en-
hanced hyperconjugative interactions. When only one reduc-
tion peak was observed, the electronic communications of
Bis-CoQ0 may have been blocked.
Assessment of Antioxidant Capacity
To estimate the antioxidation efficacy of Bis-CoQ0, the pro-
tective effect of Bis-CoQ0 against oxidative damage induced
by hydrogen peroxide to HeLa cells was investigated. The
capability of Bis-CoQ0 to attenuate the damage induced by
hydrogen peroxide toward HeLa cells was much improved
compared to that of coenzyme Q10 (CoQ10). CoQ10 is an es-
sential natural cofactor found in the mitochondria of every
cell in the body and has an antioxidation biological function.
Therefore, Bis-CoQ0 may have potential as a therapeutic
Figure 8. The UV/Vis spectra of CoQ0-CoQ0 (line a), CoQ0-CoQ0Cꢀ
2ꢀ
(line b), CoQ0Cꢀ-CoQ0Cꢀ (line c), and CoQ02ꢀ-CoQ0 (line d) in CH3CN
with varying concentrations of water.
Chem. Asian J. 2011, 6, 1064 – 1073
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemasianj.org
1071

Y.-T. Long et al.
FULL PAPERS
trolled from 280 to 330 K with a Jinghong DKB-501A (Jinghong Co. Ltd,
Shanghai, China) variable-temperature water-circulating bath.
agent in the treatment of oxidative stress-related tissue
injury and thereby minimize the oxidative damage suffered
by mitochondrial DNA.
In Situ Electron Paramagnetic Resonance (EPR) Spectroelectrochemistry.
The home-built spectroelectrochemical cell used in EPR experiments
was as described earlier.[25] Silver wire was used as the working electrode
and the length of it matched the height of the EPR cavity. An alloy wire
electrode and a silver electrode served as the counterelectrode and the
reference electrode, respectively. EPR measurements were performed in
the X-band region with a Bruker EMX-8/2.7 spectrometer at room tem-
perature. The potential during the EPR measurements was controlled
with an electrochemical workstation. EPR scan parameters used the fol-
lowing: microwave frequency 9.87 GHz, modulated frequency 100 KHz,
modulated amplitude 0.15 G, time constant 81.92 s, conversion time
163.84 s.
Conclusion
In summary, we have synthesized a methylene-bridged bis-
coenzyme Q0 and investigated its electrochemical, UV/Vis,
and EPR spectroelectrochemical properties under different
redox states in organic solvents. The chemical reaction in
the third redox step process was confirmed by variable tem-
perature cyclic voltammetry. Strong intramolecular electron-
ic communications between two methylene-bridged quinon-
yl moieties of Bis-CoQ0 were found. Hydrogen-bonding in-
teractions between water and the electrochemically reduced
intermediates of Bis-CoQ0 in an aqueous organic solvent
have also been studied. The electronic communications of
Bis-CoQ0 may have been blocked when one reduction peak
was observed with proper quantities of water in water/
CH3CN solution. The presence of anion radicals and dia-
magnetic dianions during the redox processes of Bis-CoQ0
may strengthen its capacity as an antioxidant. We are cur-
rently investigating this capability. In addition, this study
gave us an indication that it might be possible to modify and
control the electrochemical properties of Bis-CoQ0 by
tuning the coupling groups and therefore widen the poten-
tial application window of CoQs.
In Situ UV/Vis Spectroelectrochemistry.
In situ UV/Vis spectroelectrochemistry was carried out with a PC-con-
trolled Oceanoptics DT-Mini-2-GS situ spectrometer at a resolution of
1 nm and with an optically transparent thin-layer electrode (OTTLE) cell
at room temperature. The OTTLE cell was constructed from quartz, and
the OTTLE is a platinum gauze situated in a (0.4ꢂ0.1) mm optical-path-
length section of the cell with a platinum wire counterelectrode, Ag/
AgCl wire pseudoreference electrode, and a Teflon tube for purging with
dry nitrogen.
Assessment of Antioxidant Capacity
The antioxidation capability of Bis-CoQ0 against oxidative damage in-
duced by hydrogen peroxide to HeLa cells was determined by the con-
ventional 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) assay.[26]
Acknowledgements
We thank the financial support of the National High Technology Re-
search and Development Program of China (863 Project:
2008AAO6A406), and the Ministry of Health (grant no. 2009 ZX 10004-
301). Y.-T.L. is supported by the Program for Professor of Special Ap-
pointment (Eastern Scholar) at the Shanghai Institutions of Higher
Learning.
Experimental Section
Chemicals
Tetrabutylammonium perchlorate (TBAP Fluka, >99.0%) was dried
under vacuum at 358 K for 24 h and stored under vacuum. HPLC-grade
CH3CN (Sigma) was initially dried by distillation over CaH2 before use.
All chemicals were used as received without further purification unless
specified.
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HPLC-grade CH3CN (Sigma) was initially dried by distillation over
CaH2 before use. All solutions for electrochemistry were dried by placing
the solvent, electrolyte, and Bis-COQ0 inside a 25 mL vacuum syringe
(Yikang Medical Instrument Group Co. Ltd, Jiangxi, China) that con-
tained 3 ꢂ molecular sieves (dried under vacuum at 513 K for 12 h) and
storing the syringe under a dry nitrogen atmosphere for at least 48 h.
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Apparatus and Electrochemical Measurements
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controlled CHI 660 electrochemical station (Chenhua Co. Ltd, Shanghai,
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using ferrocenium/ferrocene as an internal standard. Variable-tempera-
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cell. The cell was jacketed in a glass sleeve and the temperature was con-
1072
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ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Asian J. 2011, 6, 1064 – 1073

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Received: July 7, 2010
Published online: November 16, 2010
Chem. Asian J. 2011, 6, 1064 – 1073
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemasianj.org
1073