2,5-Diamino-p-benzoquinone Derivatives as Photosystem I Electron Acceptors: Synthesis and Electrochemical and Physicochemical Properties

Article abstract of DOI:10.1021/jf970979g

A series of 2,5-diamino-p-benzoquinone derivatives have been prepared and their physicochemical properties studied. The sensitivity of their photoreduction potential to 3-(3,4-dichlorophenyl)-1,1-dimethylurea, 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone, and KCN, as well as the photosystem I (PSI) activity, suggests that the reduction of 2,5-diamino-p-benzoquinone derivatives in the illuminated thylakoid is at the primary electron acceptor of PSI and it is reversible. The half-wave potentials of these compounds to their corresponding radical anions in an aprotic medium such as acetonitrile were found to be comparable with the midpoint potential values of the electron transport carriers at the reducing site of PSI. The strong reductant produced by PSI is really more accessible to the strong lipophilic electron acceptor. These lipophilic p-benzoquinone derivatives can reach the carriers inside the thylakoid membrane more easily than the ionic electron acceptor. The accepting electron properties of these compounds at PSI are similar to those of the bipyridinium herbicides.

Full text of DOI:10.1021/jf970979g

724
J. Agric. Food Chem. 1998, 46, 724−730
2,5-Dia m in o-p-ben zoqu in on e Der iva tives a s P h otosystem I Electr on
Accep tor s: Syn th esis a n d Electr och em ica l a n d P h ysicoch em ica l
P r op er ties
Blas Lotina-Hennsen,*,† Lahoucine Achnine,† Norma Mac´ıas Ruvalcaba,‡ Aurelio Ortiz,§
J esu´s Herna´ndez,§ Norberto Farfa´n,§ and Martha Aguilar-Mart´ınez‡
Departamentos de Bioqu´ımica y Fisicoqu´ımica, Facultad de Qu´ımica, Universidad Nacional Autono´ma de
Me´xico, Ciudad Universitaria, Me´xico D.F. 04510, Mexico, and Departamento de Qu´ımica, Centro de
Investigacio´n y de Estudios Avanzados del IPN, Apartado Postal 14-740, 07000 Me´xico D.F., Mexico
A series of 2,5-diamino-p-benzoquinone derivatives have been prepared and their physicochemical
properties studied. The sensitivity of their photoreduction potential to 3-(3,4-dichlorophenyl)-1,1-
dimethylurea, 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone, and KCN, as well as the photo-
system I (PSI) activity, suggests that the reduction of 2,5-diamino-p-benzoquinone derivatives in
the illuminated thylakoid is at the primary electron acceptor of PSI and it is reversible. The half-
wave potentials of these compounds to their corresponding radical anions in an aprotic medium
such as acetonitrile were found to be comparable with the midpoint potential values of the electron
transport carriers at the reducing site of PSI. The strong reductant produced by PSI is really more
accessible to the strong lipophilic electron acceptor. These lipophilic p-benzoquinone derivatives
can reach the carriers inside the thylakoid membrane more easily than the ionic electron acceptor.
The accepting electron properties of these compounds at PSI are similar to those of the bipyridinium
herbicides.
Keyw or d s: 2,5-Diamino-p-benzoquinone derivatives; photosystem I; electron acceptors; cyclic
voltammetry; electrochemical reduction; half-wave potentials
INTRODUCTION
hydrophobicity of quinones is important for binding and
inhibition or diverting electrons away from the A1 site
(Itoh and Iwaki, 1989) and the bacterial QA site (Warncke
et al., 1987). Moreover, synthetic quinones with lower
midpoint potential values between those of Ao and the
iron-sulfur center Fx inhibit electron flow at the A1
level, in A1-depleted PSI particles (Itoh and Iwaki,
1989).
While searching for chemicals with potential herbicide
activity that affect the reducing site of PSII, we have
discovered a group of compounds, referred to as 2,5-
diamino-p-benzoquinone derivatives (compounds 2-7)
(Figure 1), which behave as electron acceptors at the
reducing site of PSI. Their analytical and spectroscopic
characterizations, as well as their redox potential de-
termination in aprotic medium, partition coefficients
reported as log P values (Fujita et al., 1964), and
extinction coefficients, have been investigated. The
study of the redox behavior of these compounds using
cyclic voltammetry has made possible the determination
of the one-electron reduction potential for the couple
Q/Q•- in a nonaqueous environment. The half-wave
potential for the reduction of this class of compounds
to the corresponding anion radical permitted us to locate
the site of electron acceptance at PSI.
Quinone redox systems play an important role as
electron and proton carriers in the photosynthetic
electron transport system of photosynthetic bacteria,
algae, and higher plants, as well as in mitochondria.
The electron transport chain of chloroplasts contains QA
and QB as the quinone cofactors in the reducing side of
photosystem II, bound by D2 and D1 proteins (Arntzen
and Pakrasi, 1986, and references cited therein), re-
spectively. On the other hand, PQH2 cofactors of the
cytochrome b6f complex are bound to b6 and/or the
Rieske iron sulfur protein (Trebst, 1985, and references
cited therein), and phylloquinone cofactor is part of the
core complex of PSI at the A1 site (Itoh and Iwaki, 1989).
In many cases, quinone derivatives (and/or analogues)
are used as electron donors, acceptors, redox mediators,
or inhibitors of natural reactions in the bioenergetics
field.
Urea and phenol type PSII herbicides are known to
inhibit photosynthetic electron transport at the reducing
site of PSII, at the level of the secondary two-electron
accepting plastoquinone QB. However, the herbicidal
action of some quinones is similar to that exhibited by
the bipyridinium herbicides (i.e. paraquat and diquat)
(Baker and Percival, 1990, and references cited therein)
due to their capacity to intercept electrons by competing
with the natural substrate ferredoxin. Furthermore,
MATERIALS AND METHODS
Ch em ica ls a n d Solven ts. All reagents used were com-
mercially available. p-Benzoquinone (compound 1), benzyl-
amine, o-aminophenol, (()-R-methylbenzylamine, (+)-R-me-
thylbenzylamine, (-)-norpseudoephedrine, and (+)-norephe-
drine were from Aldrich. Tricine, MV, sorbitol, sucrose, ADP,
DCPIP, DBMIB, DCMU, and diphenyl carbazide were from
* Author to whom correspondence should be addressed (e-
mail blas@servidor.unam.mx).
† Departamento de Bioqu´ımica.
‡ Departamento de Fisicoqu´ımica.
§ Departamento de Qu´ımica.
S0021-8561(97)00979-5 CCC: $15.00 © 1998 American Chemical Society
Published on Web 01/30/1998

2,5-Diamino-p-benzoquinone Derivatives as PSI Electron Acceptors
J. Agric. Food Chem., Vol. 46, No. 2, 1998 725
(DMSO-d6, 67.8 MHz) 180.0 (C-1,4), 148.63 (C-2,5), 146.78 (C-
8), 128.26 (C-7), 127.70 (C-11), 124.70 (C-12), 115.44 (C-10),
103.15 (C-9), 98.51 (C-3,6); IR (KBr) νmax 3226 (OH), 3220 (NH),
1565 (CdO), 1513, 1507, 1457 cm-1; UV (ethanol) λmax (log ꢀ)
434 (0.27); UV (octanol) λmax (log ꢀ) 342 (3.59) nm. Due to the
fact that compund 2 was insoluble in water and slightly soluble
in octanol, the partition coefficient could not be determined.
2,5-Di(benzylamine)-p-benzoquinone (3). A solution of p-
benzoquinone (5.00 g, 46.3 mmol) and benzylamine (3.29 g,
30.7 mmol) in 350 mL of ethanol was stirred at room temper-
ature for 4 h. The precipitate was filtered and washed with
ethanol/chloroform mixtures (7:3) to yield 3.65 g (74%) of 3 as
an orange solid: mp 253 °C; 1H NMR (DMSO-d6, 270 MHz) δ
4.36 (d, 2H, J ) 6 Hz, CH2-7), 5.10 (s, 1H, H-3,6), 7.22-7.35
(m, 5H, C6H5), 8.31 (t, 1H, J ) 6 Hz, NH); 13C NMR (DMSO-
d6, 67.8 MHz) δ 177.65 (C-1,4), 150.84 (C-2,5), 137.27 (Cipso),
128.38 (Cmeta), 127.08 (Cortho), 127.03 (Cpara), 93.1 (C-3,6), 44.98
(C-7); MS, m/z (%) 318 [M+, 48], 241 (2), 213 (11), 130 (5), 91
(100), 65 (29), 39 (11); IR (KBr) νmax 3278 (NH), 1552 (CdO),
1533, 1522, 1490, 1456, 1440, 1363, 1298, 1253 cm-1; UV
(ethanol) λmax (log ꢀ) 243 (4.42) nm; UV (octanol) λmax (log ꢀ)
347 (4.71) nm. Partition coefficient for compound 3 was not
determined due to its insolubility in both water and octanol.
2,5-Di-(R,S-(+)-R-methylbenzylamino)-p-benzoquinone (4).
To a solution of p-benzoquinone (12.97 g, 120 mmol) in 400
mL of ethanol was added (()-R-methylbenzylamine (9.69 g,
80 mmol), and the solution was stirred for 8 h at room
temperature. The solvent was evaporated to dryness, and the
resulting solid was chromatographed over silica gel eluting
with increasing polarities. Compound 4 was obtained from
the hexane/ethyl ether (3:7) fractions as a red solid: mp 224
°C in 10.8% yield (1.50 g); the polar fractions afforded
hydroquinone; 1H NMR (CDCl3, 270 MHz) δ 1.55 and 1.57 (d,
3H, J ) 7.26 Hz, CH3-8), 4.46 (quintet, 1H, J ) 6.6 Hz, CH-
7), 5.16 (s, 1H, H-3,6), 6.74 (d, broad, 1H, J ) 6.6 Hz, NH),
7.19-7.37 (m, 5H, C6H5); 13C NMR (CDCl3, 67.8 MHz) δ 178.70
(C-1,4), 149.60 (C-2,5), 141.66 (Cipso), 128.96 (Cmeta), 127.78
(Cpara), 127.80 (Cortho), 94.74 (C-3,6), 52.74 (C-7), 23.17 (C-8);
MS, m/z (%) 346 [M+, 65], 269 (2), 241 (22), 227 (23), 103 (21),
105 (100), 331 (24), 77 (32); IR (KBr) νmax 3330 (NH), 3280,
3271, 1635 (CdO), 1569, 1540, 1517, 1494, 1452, 1351, 1295,
1227 cm-1; UV (ethanol) λmax (log ꢀ) 341 (2.90) nm; UV (octanol)
λmax (log ꢀ) 342 (2.90) nm; log P 0.982, the concentration of 4
in the octanol phase, 0.58 mM.
F igu r e 1. Chemical structures of 2,5-diamino-p-benzoquin-
ones.
Sigma Chemical Co. Acetone, ethanol, and acetonitrile were
from Merck. Acetonitrile was distilled from phosphorus pen-
toxide prior to use. Tetraethylammonium tetrafluoroborate
(Et4NBF4) from Merck was used as supporting electrolyte and
dried for 24 h at 60 °C before being used.
Ap p a r a tu s. 1H and 13C NMR spectra were recorded using
J EOL FX90Q and J EOL GSX-270 (270 MHz) spectrometers.
Chemical shifts (ppm) are related to (CH3)4Si. DMSO-d6,
CDCl3, and acetone-d6 were used as solvents. Coupling
constants are quoted in hertz. The HETCOR standard pulse
sequence, which incorporates quadrature detection in both
domains, was used as well. Infrared spectra were determined
on a Perkin-Elmer 16 F spectrophotometer. Mass spectro-
metric analyses were carried out using a 5989A HP mass
spectrometer coupled to a gas chromatograph 5890 series II
(70 eV). UV spectra were determined on a Unicam SP 800
spectrophotometer. Melting points were obtained on a Gal-
lenkamp MFB-595 apparatus and remain uncorrected. Elec-
tron transport was determined with an oxygraph YSI model
5300. pH changes were measured in a Corning pH meter
model 12 attached to a Gilson recorder. Cyclic voltammetries
were carried out on a BAS 100 B/W electrochemical analyzer
with a 486 DX 33-MHz computer. Specific rotation was
determined with a Perkin-Elmer 241 polarimeter.
2,5-Di(R-(+)-R-methylbenzylamine)-p-benzoquinone (5). To
a solution of p-benzoquinone (4.00 g, 37.0 mmol) in 300 mL of
ethanol was added R-(+)-R-methylbenzylamine (3.00 g, 24.7
mmol), and the solution was stirred for 8 h at room temper-
ature. The solvent was evaporated to dryness, and the
resulting solid was chromatographed over silica gel eluting
with increasing polarities. Compound 5 was obtained from
the hexane/ethyl ether (3:7) fractions as a red solid: mp 190-
191 °C in 14.5% yield (600 mg); 1H NMR (CDCl3, 270 MHz) δ
1.57 (d, 3H, J ) 7.26 Hz, CH3-8), 4.46 (quintet, 1H, J ) 6.6
Hz, CH-7), 5.16 (s, 1H, H-3,6), 6.74 (d, broad, 1H, J ) 6.6 Hz,
NH), 7.19-7.37 (m, 5H, C6H5); 13C NMR (CDCl3, 67.8 MHz) δ
178.70 (C-1,4), 149.60 (C-2,5), 141.66 (Cipso), 128.96 (Cmeta),
127.78 (Cpara), 125.80 (Cortho), 94.74 (C-3,6), 52.74 (C-7), 23.17
(C-8); MS, m/z (%) 346 [M+, 65], 269 (2), 241(22), 227 (23), 103
(21), 105 (100), 331 (24), 77 (32); IR (KBr) νmax 3330 (NH) 3280,
3271, 1635 (CdO), 1569, 1540, 1517, 1494, 1452, 1351, 1295,
P r oced u r es for th e P r ep a r a tion of 2,5-Dia m in o-p-
ben zoqu in on es. p-Benzoquinone (1). UV (ethanol) λmax (log
ꢀ) 242 (3.27) nm; log P 0.176. The concentration of 1 in the
aqueous phase was 1.55 mM.
1227 cm-1; [R]25 -1164 (c 0.010, ethanol); UV (ethanol) λmax
D
(log ꢀ) 341 (2.90) nm; UV (octanol) λmax (log ꢀ) 342 (2.90) nm;
log P 0.982. Partition coefficients for compounds 4 and 5 are
identical, as expected, since they differ only in optical activity.
2,5-N,N-(1′R,2′R)-Norpseudoephedrine-p-benzoquinone (6).
A solution of (-)-norpseudoephedrine (5.00 g, 33 mmol) and
boric acid (681 mg, 11 mmol) dissolved in 75 mL of benzene
was heated to reflux for 2 h and cooled to room temperature,
and the solvent was evaporated to dryness. The resulting solid
was dissolved in 75 mL of ethanol, p-benzoquinone (5.34 g, 49
mmol) was added, and the solution was stirred overnight at
room temperature. The solvent was evaporated to dryness,
and the resulting solid was chromatographed over silica gel
using increasing solvent polarities. Compound 6 (5.5 g, 82%)
2,5-Di(o-aminophenol)-p-benzoquinone (2). p-Benzoquinone
(5 g, 46.3 mmol) was dissolved in 350 mL of ethanol, and
o-aminophenol (3.36 g, 30.7 mmol) was added. The solution
was stirred at room temperature for 8 h. The solvent was
evaporated to dryness, and the resulting solid was chromato-
graphed over silica gel eluting with increasing polarities. The
chloroform/ethanol (9:1) fractions afforded compound 2, which
was obtained as a red solid (2.02 g, 41%; mp 225 °C).
Hydroquinone was obtained as byproduct and separated from
the more polar fractions. 1H NMR (DMSO-d6, 270 MHz) δ 6.30
(s, 1H, H-3,6), 6.80 (s, 1H, OH), 7.34-7.42 (m, 3H, H-9,10,
11), 7.68 (d, 1H, J ) 7.3 Hz, H-12), 8.60 (s, 1H, NH); 13C NMR

726 J. Agric. Food Chem., Vol. 46, No. 2, 1998
Lotina-Hennsen et al.
was separated from the chloroform/hexane fractions (50:50)
as a red crystalline solid: mp 193-195 °C; 1H NMR (acetone-
d6, 90 MHz) δ 7.62-7.30 (m, 5H), 7.0 (d, 1H, J ) 8 Hz, NH)
5.33 (s, 1H, H-3), 5.00 (d, 1H, J ) 8 Hz, H-5), 4.95 (m, 1H,
H-4), 1.05 (d, 3H, J ) 6 Hz, CH3-4); 13C NMR (acetone-d6, 22.5
MHz) δ 178.8 (C-1), 151.8 (C-2), 143.6 (Cipso), 127.3 (Cortho),
129.2 (Cmeta), 128.5 (Cpara), 93.2 (C-3), 55.0 (C-4), 75.6 (C-5),
17.4 (CH3); MS, m/z (%) 406 (M+, 0.5), 300 (18), 193 (100),
177(32), 79 (54), 77 (43); IR (KBr) νmax 1563 (CdO), 3300 cm-1
;
[R]25 +849 (c 0.0155, ethanol); UV (ethanol) λmax (log ꢀ) 344
D
(2.86) nm; UV (octanol) λmax (log ꢀ) 346.5 (2.90) nm; log P 1.423,
concentration of compound 6 in the octanol phase, 0.521 mM.
2,5-N,N-(1′S,2′R)-(+)-Norephedrine-p-benzoquinone (7). Syn-
thesis was identical with that of 6 above. Compound 7 (6.00
g, 89%) was obtained from the chloroform/hexane (50:50)
1
F igu r e 2. Polarographic traces showing the electron acceptor
properties of compound 3 compared with MV and K3[Fe(CN)6].
Measurements were made by monitoring change of oxygen
concentration in the medium using a Clark electrode attached
to YSI oxygraph model 5300. The reaction mixture contained
0.1 M sorbitol, 5 mM MgCl2, 10 mM KCl, 15 mM tricine-KOH
buffer (pH 8.0), and thylakoids equivalent to 20 µg/mL
chlorophyll; reaction volume, 3.0 mL; temperature, 20 °C: (a)
Electron flow from water to 500 µM K3[Fe(CN)6] as oxygen
evolution; (b) electron transport from water to 50 µM MV; (c)
electron flow from water to 50 µM of compound 3. Numbers
on traces refer to mequiv e-h-1 (mg of Chl)-1. Arrows indicate
onset of illumination.
fractions as a red crystalline compound: mp 186-188 °C; H
NMR (acetone-d6, 90 MHz) δ 7.60-7.25 (m, 5H, C6H5), 7.00
(d, 1H, J ) 9 Hz, NH), 5.25 (s, 1H, H-3), 4.90 (d, 1H, J ) 4
Hz, H-5), 3.72 (m, 1H, H-4), 1.38 (d, 3H, J ) 6 Hz, CH3-4); 13
C
NMR (acetone-d6, 22.5 MHz) δ 177.1 (C-1), 146.6 (C-2), 142.1
(Cipso), 126.1 (Cortho), 127.7 (Cmeta), 126.9 (Cpara), 92.2 (C-3), 52.9
(C-4), 73.0 (C-5), 13.4 (CH3); MS, m/z (%) 406 [M+, 1.1], 300
(33), 193 (100), 79 (30), 77 (27); IR (KBr) νmax 1566 (CdO),
3330, 3263 (NH, OH) 2566 (CdO) cm-1; [R]25D -848.0 (c 0.0155,
ethanol); UV (ethanol) λmax (log ꢀ) 344 (2.86) nm; UV (octanol)
λmax (log ꢀ) 346.5 (2.90) nm; log P 1.423, concentration of
compound 7 in the octanol phase, 0.521 mM.
Biologica l Activity. Intact chloroplasts were prepared
from market spinach leaves (Spinacea oleracea L.) as reported
(Mills et al., 1980) and resuspended, unless indicated, in a
solution containing 400 mM sucrose, 5 mM MgCl2, and 10 mM
KCl buffered with 30 mM Na+-tricine at pH 8.0. KCN-
poisoned chloroplasts were incubated with 30 mM KCN (pH
7.8) at 0 °C for 30 min (Izawa, 1977). Because incubations
with cyanide are carried out with dilute thylakoid suspensions,
the thylakoids were collected by centrifugation and resus-
pended in a smaller volume of cyanide-free solution. Light-
induced, noncyclic electron transport in the presence of 2,5-
diamino-p-benzoquinone derivatives (either MV, K3[Fe(CN)],
or DCPIP) as electron acceptor (Saha et al., 1971; Calera et
al., 1995) was measured with an oxygen monitor using a Clark
electrode attached to a YSI oxygraph model 5300 at 20 °C.
KCN (0.1 mM) was added to inhibit any catalase activity. Stock
solutions of 2,5-diamino-p-benzoquinone derivatives were
prepared by dissolving these compounds in N′,N′-dimethyl-
formamide; the final concentration of the organic solvent in
the reaction mixture never exceeded 1%. Potassium ferricya-
nide is a useful electron acceptor that gives light-dependent
oxygen evolution during photolysis of water by thylakoids.
Another electron acceptor used for measuring whole-chain
electrons utilizes the autoxidation of MV, which gives oxygen
evolution (Allen and Hollmes, 1886). Reaction time of il-
lumination was 1 min under aerobic conditions and saturating
white light. Kinetic parameters were obtained by nonlinear
regression analysis, using the Michaelis-Menten equation or
the substrate inhibition equation when pertinent. The Sigma
plot package was used to carry out the fitting procedure.
Extin ction a n d P a r tition Coefficien t Deter m in a tion s.
Extinction coefficients of p-benzoquinone and 2,5-diamino-p-
benzoquinone derivatives were determined using the spectro-
scopic absorption technique (Fujita et al., 1964; Hansch et al.,
1963). Partition coefficients were obtained by shaking the
solute between 50 mL of 1-octanol and 50 mL of water, followed
by spectrophotometric analyses of the octanol phases except
for compound 1.
RESULTS AND DISCUSSION
Syn th esis. Compounds 2-7 (Figure 1) were pre-
pared according to the method of Ross (1984) and were
assayed as electron acceptors. Physical and spectro-
scopic constants of 2,5-diamino-p-benzoquinone and its
derivatives are summarized under Materials and Meth-
ods. Compounds 2-5 were synthesized for the first time
in this work. Compounds 6 and 7 were synthesized as
described previously (Farfa´n and Contreras, 1985).
Bioch em ica l Stu d ies. If K3[Fe(CN)6] is used as
electron acceptor in illuminated thylakoids (Figure 2a),
oxygen is evolved from the photolysis of water (Allen
and Holmes, 1986). However, if MV is used as electron
acceptor at the whole electron transport chain of the
chloroplast (see Figure 2b), oxygen is consumed (Allen
and Holmes, 1986). Compound 3 stimulates oxygen
uptake in illuminated thylakoids in analogy to MV
(Figure 2c) and therefore accepts electrons from the
electron transport chain in thylakoids. As pointed out
by Hauska (1975), when chloroplasts are used in
combination with electron donors and acceptors, they
must not react with each other in a dark reaction. This
trivial criterion is completely met by 2,5-diamino-p-
benzoquinones, i.e., compound 3 (Figure 2). The data
suggest that reduced compound 3 reacts with the O2 of
•-
the medium producing O2 in the same way as shown
by reduced MV.
Photoreduction of compound 3 by illuminated thyla-
koids is given in Figure 3 as a function of increasing
concentration of 2,5-diamino-p-benzoquinone 3 and
measured as oxygen uptake. A concentration-response
curve of this shape was typical for each 2,5-diamino-p-
benzoquinone derivative. The photoreductions of K3-
[Fe(CN)6], MV, and DCPIP are included as positive
control.
Compounds 2 and 4-7 behave also as electron accep-
tors. The AC50 were obtained by kinetic analysis using
the Michaelis-Menten equation or the substrate inhibi-
tion equation, and the results for all compounds tested
are listed in Table 1. Compound 1 was used as control,
since it is known that p-benzoquinone accepts electrons
Electr och em ica l Stu d ies. The analyses were carried out
using cyclic voltammetry as previously reported (Aguilar-
Mart´ınez et al., 1996); the cyclic voltammograms of 3 mM
p-benzoquinone 3 and 2,5 diamino-p-benzoquinones 2-7 were
obtained using a polished platinum disk electrode (area 2 mm2)
as the working electrode in 0.1 M Et4NBF4/acetonitrile as the
electrolytic medium. The electroactivity range for this system
was 3.8 V (from -1.8 to 2.0 V vs SCE). All measurements
were performed at room temperature, and the scans were
made at 100 mV/s.

2,5-Diamino-p-benzoquinone Derivatives as PSI Electron Acceptors
J. Agric. Food Chem., Vol. 46, No. 2, 1998 727
at least after cytochrome b6f, since the photoreduction
of compounds 2-7 can also be inhibited by DBMIB
(Table 2).
To locate the site of electron acceptance of compounds
2-7, thylakoids were poisoned with KCN, an electron
transport inhibitor at the plastocyanin level (Izawa,
1977), since there is a stoichiometric release of plasto-
cyanin Cu from the lamellar membrane exposed to
KCN. Moreover, EPR spectroscopy confirmed the abil-
ity of reduced PMS to interact directly with P700 (Izawa
et al., 1973). Therefore, we used 500 µM PMS/1000 µM
ascorbate as the electron donor for P700, MV as PSI
electron acceptor, as well as 10 µM DCMU and 1 µM
DBMIB to fully inhibit any electron flow prior to PC.
Under these conditions, compounds 2-7 are still pho-
toreduced by the electron transport span from P700 to
the reducing side of PSI (Table 4), which suggests that
compounds 2-7 accept electrons at/or after P700, simi-
larly to bipyridinium herbicides or dioxathiadiazahet-
eropentalenes, which accept electrons at the FX level.
The log P values of these 2,5-diamino-p-benzoquinone
derivatives indicate that they have high hydrophobicity;
therefore, they accept electrons at the hydrophobic side
of the A1 site of PSI (Camillieri, 1984, and references
cited therein). It is noteworthy that the rate of electron
flow from reduced PMS to 2-7 is lower than expected,
due to the fact that PMS is a very good cyclic cofactor
of PSI and also because it reacts with the oxygen of the
medium.
Compounds 2-7 did not inhibit the Hill reaction at
concentrations lower than those needed to reach AC50.
However, at higher concentrations (>AC50) some of the
quinones (2, 3, and 7) studied inhibited the Hill reaction.
The basal electron transport rate in the presence of
compounds 2-7 was accelerated by ADP plus Pi, or an
uncoupler, and the phosphorylating electron flow was
also accelerated by an uncoupler, as occurs with ferri-
cyanide or MV (Table 2) in analogy to previous studies
(Saha et al., 1971). Thylakoids used in this work were
coupled, since control experiments using ammonium
chloride (5 mM) induced 2- or 3-fold increases in the
electron transport rate from water to MV. To find out
whether electron transport from water to 2,5-diamino-
p-benzoquinone derivatives is coupled to ATP formation,
ATP synthesis was measured using compound 3 as
electron acceptor. Figure 4 shows that the rate of ATP
synthesis is directly proportional to the concentration
of compound 3 up to 5 µM. Saturation of the system is
reached at ≈20 µM of compound 3. These data indicate
that ATP formation is coupled to electron transport. The
P/2e- ratio calculated from the rate of ATP synthesis
and reduction of compound 3 was >1.00, which indicates
that both photosystems are coupled to ATP synthesis,
at low concentrations of the compound. However, as
concentrations of compound 3 increase, the P/e2 de-
creases (see the inset in Figure 4), which may indicate
that the compound 3 has more than one action; that is,
it inhibits H+-ATPase, redox enzyme, or both. In Figure
4 is also included the P/e2 of MV as control; the values
obtained are similar to that reported by Saha et al.
(1971).
F igu r e 3. Rate of electron transport associated with the
photoreduction of electron acceptors, compound 3 (b), methyl
viologen (9), K3[Fe(CN)6] (2), and DCPIP (1), by illuminated
thylakoids. In each case a cuvette contained 20 µg of chlorophyll/
mL. The reaction contained the following mixture and condi-
tions: intact thylakoids (20 µg of chlorophyll/mL) were incu-
bated in 3.0 mL of the following electron transport medium:
0.1 M sorbitol, 0.01 M KCl, 5 mM MgCl2, 10 mM K-tricine
(pH 8.0). Each point represents the mean of four determina-
tions. Vertical bars on each curve represent the maximal value
for standard deviation.
at the reducing side of PSII (Berg et al., 1980). More-
over, p-benzoquinone (1) allowed us to evaluate the
effect of amine substitution at the 2- and 5-positions,
thus, providing information on the redox potentials and
biochemical studies. MV was also used as control for
electron acceptor at the reducing side of PSI, and its
sensitivity to inhibitors is compared with that of 2,5-
diamino-p-benzoquinone derivatives. According to the
AC50 (Table 1), the order of electron acceptance was 3
> 2 > 6 > 7 > 4 > 5, compound 3 being the most
powerful electron acceptor, due to the lower AC50
displayed. The AC50 values of compounds 4 and 5 were
reached at higher concentrations than the other p-
benzoquinone derivatives tested (Table 1). The satura-
tion curve of compounds 2-7 was reached at different
concentrations. These maximum concentrations were
used in the experiments to locate the site(s) of action of
compounds 2-7 in the electron transport chain of
thylakoids (Table 2).
Further experiments were performed to determine the
site where compounds 2-7 accept electrons using
specific inhibitors of the electron transport chain, and
adequate electron transport donors were included.
DCMU and DBMIB (Izawa, 1977) inhibit uncoupled
electron transport rates from water to compounds 2-7
(see Table 2); however, compound 1 was sensitive to
DCMU and insensitive to DBMIB, indicating that it
accepts electrons after QA and before cyt b6f complex.
These results indicate that compounds 2-7 accept
electrons after PQ.
To further clarify the zone of electron acceptance for
compounds 2-7, PSI activity was measured with DCPIP/
ascorbate as electron donors and MV as electron accep-
tors, and the results are shown in Table 3. It is observed
that photoreduction of compounds 2-7 by thylakoids
is inhibited by DCMU (Table 2), and it is reestablished
after the addition of DCPIPH2. The results are similar
to those obtained when DCPIPH2 was added to DCMU-
inhibited thylakoids in the presence of MV. These data
suggest that compounds 2-7 accept electrons in PSI or
Electr och em ica l Stu d ies. To corroborate the site
of electron acceptance for compounds 1-7, their elec-
trochemical properties were evaluated by cyclic volta-
mmetry (scanned at 100 mV/s) of deoxygenated solu-
tions in 0.1 M Et4NBF4 in acetonitrile. Half-wave
reduction potentials [E1/2 ) (Epa + Epc)/2 (Heinze, 1984)]

728 J. Agric. Food Chem., Vol. 46, No. 2, 1998
Lotina-Hennsen et al.
Ta ble 1. Ha lf-Wa ve P oten tia ls E1/2 for Ben zoqu in on e 1 a n d 2,5-Dia m in o-p-ben zoqu in on es 2-7 Mea su r ed by Cyclic
Volta m m etr y (100 m V/s vs SCE), 0.1 M Et4NBF 4/Aceton itr ile, a n d AC50 Da ta a
b
b
c
b
compd E1/2 (mV) vs SCE ipa/ipc
E1/2Ec vs SCE ipa/ipc
E1/2 (mV) vs NHE E1/2c (mV) vs NHE AC50 (µM) Ki (mM) Vmax
1
2
3
4
5
6
7
MV
-483
-898
-1049
-999
-1001
-991
-974
1.01
1.07
1.02
0.95
1.03
0.91
0.93
-1155
-1372
-1536
-1457
-1442
-1234
-1331
1.03
0.83
0.97
0.87
0.94
0.79
0.92
-239
-654
-805
-755
-757
-747
-730
-911
-1128
-1292
-1213
-1198
-990
55.00
0.65
0.23
2.90
15.40
0.68
0.84
1520
309
221
270
0.101
1.75
290
382
0
-1087
a
AC50 is the concentration of the compound needed to accept 50% of electrons from the chloroplast electron transport chain. Vmax in
equiv e- h-1 (mg of Chl)-1
.
Quinone/semiquinone redox couple. c Semiquinone/dianion redox couple.
b
Ta ble 2. In h ibition of Electr on Tr a n sp or t by DCMU a n d
DBMIB w ith Com p ou n d s 1-7 a n d MV a s Electr on
Accep tor s
Ta ble 4. Un cou p led Electr on Tr a n sp or t fr om P 700 to MV
or 2,5-Dia m in o-p-ben zoqu in on e Der iva tives in
KCN-Tr ea ted Th yla k oid sa
exptl
conditions
UET +
UET +
electron
donor
electron
acceptor
rate of electron
transport (% of control)
BET
PET
UET
DCMU
DBMIB
condition
none
control
MV
1
2
3
4
5
6
7
238
880
190
242
290
241
227
265
319
800
288
281
319
269
273
358
950
1520
796
788
826
418
819
881
0
0
0
0
0
0
0
0
0
H2O
MV
100
1500
0
0
0
0
0
0
KCN treated H2O
KCN treated PMS/ASC
MV
MV
3
8
82
100
none
control
H2O
KCN treated H2O
3
3
2
4
5
6
7
9
85
84
80
85
84
82
KCN treated PMS/ASC
KCN treated PMS/ASC
KCN treated PMS/ASC
KCN treated PMS/ASC
KCN treated PMS/ASC
KCN treated PMS/ASC
a
Reaction mixture contained tricine buffer (pH 8.0), 10 mM;
thylakoids with 20 µg of chlorophyll/mL; MgCl2, 5 mM; KCl, 10
mM; sorbitol, 100 mM for basal electron transport (BET). For
measurement of phosphorylating electron transport (PET) ADP,
1 mM and Pi, 3 mM, were added. for uncoupled electron transport
(UET), NH4Cl, 5 mM, was added. if used, DCMU, 10 µM; DBMIB,
1 µM. Rates are in µequiv e- h-1 (mg of Chl)-1. Saturation
concentration of MV was 150 µM. Saturation concentrations of
a
Reaction mixture as described in Table 2. Concentrations of
MV and compounds 2-7 were 50, 5, 5, 100, 40, 30, and 20 µM,
respectively. Control values for electron transport from 500 µM
PMS/1000 µM ASC to 2,5-diamino-p-benzoquinone derivatives
were 2, 469; 3, 438; 4, 419; 5; 438; 6, 427; 7, 519 µequiv e- h-1
(mg of Chl-1). Control value for electron flow from H2O to MV (in
KCN-treated thylakoids) was 96 µequiv e- h-1 (mg of Chl-1).
Uncoupled electron transport rate from H2O to 3. H2O to 3 (KCN
treated), PMS/ASC to 3 (KCN treated) were 850, 76.5, 438 µequiv
e- h-1 (mg of Chl-1), respectively. Other conditions as in Tables 2
and 3.
compounds 1-7 were 400, 10, 10, 150, 50, 60, and
respectively.
3 µM,
Ta ble 3. Un cou p led P h otosystem I Electr on Tr a n sp or t of
2,5-Dia m in o-p-ben zoqu in on e Der iva tivesa
electron
donor
electron
acceptor
UET +
DCMU
UET + DCMU
+ DCPIPH2
UET
H2O
H2O
H2O
H2O
H2O
H2O
H2O
MV
2
3
4
5
910
796
788
826
418
819
881
0
0
0
0
0
0
0
900
790
790
820
420
820
880
1 and 2. The cyclic voltammogram for compound 3
showed two successive cathodic and anodic peaks (Fig-
ure 5). The first cathodic peak at -1100 mV corre-
sponds to the reduction of Q to Q•-, and the anodic peak
at -998 mV is consistent with the oxidation of Q•- to Q
(E1/2 ) -1049 mV). The reduction of Q•- to Q2- is
observed at -1577 mV, while the oxidation of Q2- to
Q•- is at -1494 mV (E1/2 ) -1536 mV) (Table 1).
Table 1 also shows that the introduction of aromatic
amines and alkylamines as substituents on the p-
benzoquinone system causes a shift in the half-wave
potential to a more negative value compared to that of
p-benzoquinone 1. This can be explained by considering
two separate electronic effects of the amino substituents.
First, the inductive electron-withdrawing effect of the
nitrogen atom, which always operates, regardless of the
steric hindrance, i.e., if operated alone, would render
reduction of 2,5-diaminobenzoquinones easier than
reduction of 1,4-benzoquinone itself. Second, the reso-
nance effect is generally predominant and results in
delocalization of the lone pair of electrons of the nitrogen
atom over the enone system of the quinone nucleus (as
Scheme 1). This delocalization requires that the amino
substituents can adopt a conformation in which the
π-orbitals carrying the lone pair of electrons are parallel
to the π system of the ring.
6
7
a
Basal electron transport medium was used as reaction mixture
as described in Table 2. Concentrations of electron acceptors as
indicated in Table 2. If used, DCPIP/ascorbate, 100 µM/300 µM.
Rates are in µequiv e- h-1 (mg of Chl-1).
are shown in Table 1. The E1/2 value is a good ap-
proximation of the formal reduction potential E′o for the
quasi-reversible one electron reduction process of non-
polar p-benzoquinone and 2,5-diamino-p-benzoquinones.
Under these conditions, all compounds were reversible
(Fray, 1972) and stable radical anions were formed. The
ratio of the anodic and cathodic peak currents was
nearly equal to 1. It is well-known that in aprotic
solvents the reduction reaction of quinones generally
involves the following two succesive steps (Mann and
Barnes, 1970):
Q + e- f Q•-
•- + e- f Q2-
(1)
(2)
Q
The behavior of the seven quinones studied here is
consistent with the redox pathways outlined in reactions
In quinone 2 with arylamino substituents at the C2
and C5 positions, the E1/2 cathodic peak values for the

2,5-Diamino-p-benzoquinone Derivatives as PSI Electron Acceptors
J. Agric. Food Chem., Vol. 46, No. 2, 1998 729
This steric hindrance is probably the major factor
responsible for the appreciably higher half-wave poten-
tials for compound 3 compared with those of compounds
4-7. It is noteworthy that compounds 6 and 7, having
the same substituent chain but different stereochemis-
tries, showed different E1/2 values.
It is well-known (Iwaki and Itoh, 1989) that the
midpoint potential values for the electron transport
carriers at the reducing side of PSI, from A0 to FX, vary
from -1.0 to -0.7 V vs NHE. Our results show that
the capacity of 2,5-diamino-p-benzoquinone derivatives
to accept electrons at the reducing side of PSI seem to
be dependent on the first half-wave values for the Q/Q•-
couple (E1/2) listed in Table 1, which vary between
-0.805 and -0.654 V vs NHE (column 6). These redox
potential values suggest that the p-benzoquinone de-
rivatives accept electrons at the FX site of PSI, which is
in agreement with their activity in the electron trans-
port chain of thylakoids. The low solubility of these
compounds in water (refer to log P values under
Materials and Methods) suggests that they accept
electrons at the lipophilic site of the PSI where the
phylloquinone A1 to FX are located.
Con clu sion s. The synthesized 2,5-diamino-p-ben-
zoquinone derivatives 2-7 had appropriate E1/2 poten-
tials for the first wave to intercept electron transport
at A1 due to some degree of structural similarity or FX
level. Reduction of these 2,5-diamino-p-benzoquinones
leads to formation of the anion radicals Q•-, which react
rapidly with oxygen to yield superoxide O2•- and gener-
ate Q, in analogy to bipyridinium herbicides (Camilleri
et al., 1984). The environment where p-benzoquinone
derivatives accept electrons is in the hydrophobic do-
main of PSI, since the partition coefficients of these
compounds showed that they are soluble in the octanol
phase.
F igu r e 4. Photophosphorylation from water to MV (b) and
to compound 3 (9) as electron acceptors in thylakoids isolated
from spinach leaves. Photophosphorylation was measured in
the presence of 1.0 mM ADP and 3.0 mM K2HPO4. The
reaction contained the following mixture and conditions:
thylakoids (20 µg of chlorophyll/mL) were incubated in 3.0 mL
of the following electron transport medium: 0.1 M sorbitol,
0.01 M KCl, 5 mM MgCl2, 10 mM K-tricine (pH 8.0). (Inset)
P/e2 represents the ratio of the number of molecules of ATP
formed to the number of pairs of electrons transferred to the
acceptors methyl viologen (O) and compound 3 (0).
ABBREVIATIONS USED
DBMIB, 2,5-dibromo-3-methyl-6-isopropyl-p-benzo-
quinone; DCPIP, dichlorophenolindophenol; DCMU (di-
uron), 3-(3,4-dichlorophenyl)-1,1-dimethylurea; MV, meth-
yl viologen or paraquat; PC, plastocyanine; PMS,
phenazine methosulfate (N-methylphenazonium metho-
sulfate); PSI, photosystem I; PSII, photosystem II; Q,
quinone; Q•-, semiquinone radical anion; Q2- quinone
dianion; QA, primary quinone electron acceptor of pho-
tosystem II; QB, secondary quinone electron acceptor;
PQH2, reduced plastoquinone pool; SCE, saturated
calomel reference electrode.
F igu r e 5. Cyclic voltammogram of 3 mM 2,5-di(R-methyl-
benzylamine)-p-benzoquinone containing 0.1 M Et4NBF4 in
CH3CN (working electrode Pt, reference SCE; scan rate 100
mV/s).
Sch em e 1
LITERATURE CITED
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alkylamine substituents. The ortho steric effect in
compound 2 could indeed be resposible for the loss of
coplanarity of the amine group with the quinone ring,
as shown in Scheme 1.
It was observed that p-benzoquinones having methyl
groups in R-positions to nitrogen (compounds 4-7)
showed higher E1/2 than 3 (Table 1). This may be due
to the fact that the methyl groups hinder the molecular
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J F970979G