DNA scission chemistry and EPR studies of four new bis(2,6-dimethoxyhydroquinone-3-mercaptoacetic acid)-peptide conjugates

Article abstract of DOI:10.1071/CH03111

In an effort to investigate the use of short peptide chains as carriers of new antitumour agents, four tripeptide cytotoxic agent conjugates, namely DMQ-MA-Lys(DMQ-MA)-Lys(Cbz)-Arg-OMe, DMQ-MA-Lys(DMQ-MA)-Phe-Arg-OMe, DMQ-MA-Lys(DMQ-MA)-Ile-Arg-OMe, and D

Full text of DOI:10.1071/CH03111

Full Paper
CSIRO PUBLISHING
www.publish.csiro.au/journals/ajc
Aust. J. Chem. 2004, 57, 243–251
DNA Scission Chemistry and EPR Studies of Four New
Bis(2,6-Dimethoxyhydroquinone–3-Mercaptoacetic
Acid)–Peptide Conjugates
Yu-Fei SongA,B
A School of Chemistry and Chemical Engineering, Shanxi University, Taiyuan 030006, P.R. China.
B Present address: CBAC/Leiden Institute of Chemistry, Leiden University, 2300 RA Leiden,
The Netherlands (e-mail: songyufei@hotmail.com).
Inanefforttoinvestigatetheuseofshortpeptidechainsascarriersofnewantitumouragents, fourtripeptidecytotoxic
agent conjugates, namely DMQ–MA–Lys(DMQ–MA)–Lys(Cbz)–Arg–OMe, DMQ–MA–Lys(DMQ–MA)–Phe–
Arg–OMe, DMQ–MA–Lys(DMQ–MA)–Ile–Arg–OMe, and DMQ–MA–Lys(DMQ–MA)–Val–Arg–OMe, were
synthesized. The cytotoxic agent conjugated to the N-terminal, and the ξ-amino group of lysine of the tripeptides
was 2,6-dimethoxyhydroquinone–3-mercaptoacetic acid (DMQ–MA).The tripeptides were synthesized by coupling
protected amino acid reꢀsidues in solution according to Pfp/DCC methods (where Pfp refers to pentafluorophenol
and DCC refers to N,N -dicyclohexylcarbodiimide). DNA scission chemistry showed that these compounds were
able to cleave supercoiled DNA into open-circular form in drug concentrations as low as 5–20 µM without addi-
tion of H2O2 or UV irradiation. DNA cleavage was clearly inhibited when hydroxyl radical scavengers such as
glycerol and sodium benzoate were added to the reaction system. When the drugs were added to rhodamine B,
the UV absorbance and fluorescence intensity of rhodamine B decreased quickly due to the degradation caused
by the hydroxyl radicals. Electron paramagnetic resonance (EPR) experiments employing the spin-trap agent 5,5-
dimethyl-1-pyrroline N-oxide (DMPO) showed that the signal intensity of the hydroxyl radicals generated from
these newly synthesized DMQ–MA–peptide compounds was a non-linear function of ascorbic acid concentration.
Deoxyribonucleic acid binding constants of these compounds were in the 104 M−1 range. It was concluded that
DNA cleavage was not related to the ability of these drugs to produce hydroxyl radicals exclusively; indeed, it was
related to their ability to produce various radicals and reactive species.
Manuscript received: 28 April 2003.
Final version: 25 September 2003.
OH
Introduction
OCH3
CH3O
Among the strategies for the chemotherapeutic treatment of
cancer is the use of naturally occurring antitumour antibi-
otics or their synthetic analogues.[1] These compounds cause
a variety of DNA lesions, ranging from DNA cross-linking
and alkylating to strand cleavage. There is accumulating
evidence that reactive oxygen species,[2–4] including the
superoxide anion radical hydrogen peroxide and the hydroxyl
radical, are generated in vivo during the metabolism and
molecular mechanics of the cytotoxic action of certain
antitumour antibiotics, especially those containing quinone
moieties.
SCH2COOH
OH
Scheme 1. DMQ–MA.
DMQ–MA congeners have been synthesized and most of
them displayed enhanced or retained in-vitro antitumour
activities compared to the parent compound DMQ–MA.[6,7]
The drug design is based several factors. Many acceptors
of amino acids, peptides, or amyloses, etc., exist on the sur-
face of tumour cells, and these cells can absorb nutrients to
propagate and proliferate.[8] Taking advantage of the rapid
phagocytosis of tumour cells, when peptide chains are com-
bined with tumour cells, DMQ–MA can access these cells
and exhibit its pharmacological effect.
2,6-Dimethoxyhydroquinone–3-mercaptoacetic
acid
(DMQ–MA; Scheme 1), one of derivatives of DMQ (2,6-
dimethoxybenzoquinone), is a natural product of fermented
wheatgerm. This compound was found to have in-vivo cyto-
toxicity to ehrlich ascites tumour cells (Table 1) under
the synergistic activation of l-ascorbate,[5] and has mod-
erate water solubility and high cytotoxicity. A number of
© CSIRO 2004
10.1071/CH03111
0004-9425/04/030243

244
Y.-F. Song
Using compounds with a high molecular weight as drug
The density of the negative charge on the surface of the
ehrlich kidney tumour cells is twice that on normal cells.[12]
The change in the charge density on the surface of can-
cer cells has a significant inhibitory effect on the growth
of such cells.[13] Incorporating arginine, which takes a posi-
tive charge under physiological conditions (pH 7.0), into the
peptide chains can possibly increase the affinity of the con-
jugates to the tumour cells. The lysyl residue was used to
anchor the cytotoxic agent DMQ–MA by the N-terminal and
the ξ-amino group. Two DMQ–MA molecules were incorpo-
rated into the conjugates in order to increase the likelihood
that the newly synthesized compounds would kill the tumour
cells.
carriers to target points is an active research field with some
success achieved in recent years,[9] but there are some lim-
itations with this method.[10] For example, when a drug is
linked to a polymer, it may lose activity because it cannot
interact with the drug target either due to steric constraints
or the inability of the drug to reach the target site.[10] Amino
acids are good carriers of cytotoxic drugs since they can carry
these across the cell membrane in some tumour cells with a
high rate or high affinity.[7,11] Herein, we attempt to use short
peptide chains as carriers of DMQ–MA (Scheme 2).
Table 1. In-vitro cytotoxicity of DMQ derivativesA[7]
ME: 3-mercaptoethanol;
It iswell known that reactiveoxygen speciesmediatemany
destructive mechanisms on cells,[14,15] and the hydroxyl
radical is regarded as one of the most powerful oxidants
in the biological system. The involvement of hydrogen per-
oxide in the cytotoxicity of DMQ–MA was demonstrated
by the drastic decrease in cytotoxicity when catalase was
administered.[8,14] The massive generation of hydroxyl rad-
icals was attributed to a radical cycling process in which
the semiquinone radical was regenerated whenever the ben-
zoquinone derivative interacted with ascorbic acid (AH2),
resulting in the production of an immense number of super-
oxide anions. The hydrogen peroxide then generated reacted
rapidly with Fe(ii) complexes to produce hydroxyl radicals
(Reactions (1)–(6 ); Q denotes benzoquinone derivatives,
QH• denotes semiquinone radicals, and CMX · Fe denotes
iron complexed to ATP or protein).
Ala: 3-mercaptoacetamido-α-methylacetamide;
Phe: 3-mercaptoacetamido-α-benzylacetamide
DMQ
derivative
KBB
PC-9C
−AHD2
+AHE2
AIF
−AH2
+AH2
AI
DMQ–MA
DMQ–ME
DMQ–Ala
DMQ–Phe
9.9
67.0
100.2
65.0
0.52
0.52
0.63
0.25
19
129
159
260
21.3
4.0
64.0
50.0
2.0
0.67
6.1
3.8
11
6
11
13
A Results are expressed as IC50 values (drug concentration required to
inhibit 50% of tumour cell growth).
B Oral epidermoid carcinoma cell line.
C Human pulmonary adenocarcinoma cell line.
D In the absence of l-ascorbic acid.
E In the presence of l-ascorbic acid.
F Activation index: IC50(−AH2)/IC50(+AH2).
OH
OH
H3CO
OCH3
H3CO
OCH3
SCH2CONH
CH COOCH3
(CH2)3
CH CONH CH CONH
(CH2)4
SCH2CONH
CH COOCH3
CH CONH CH CONH
(CH2)3
OH
(CH2)4
(CH2)4
SCH2CONH
CH2
OH
H3CO
NH
NH
C
NH
C
H3CO
SCH2CONH
OH
O
NH
NH
O
OH
HO
H3CO
HO
NH2
NH2
CH2
H3CO
DMQ–MA-Lys(DMQ–MA)-Phe-Arg-OMe 2c
DMQ–MA-Lys(DMQ–MA)-Lys(Cbz)-Arg-OMe 1c
OH
OH
H3CO
OCH3
H3CO
OCH3
CH COOCH3
CH CONH CH CONH
SCH2CONH
CH COOCH3
CH CONH CH CONH
SCH2CONH
(CH2)4
SCH2CONH
HC CH3
CH3
(CH2)3
NH
OH
OH
HC CH3
CH2
(CH2)3
(CH2)4
SCH2CONH
H3CO
NH
C
H3CO
C
NH
NH
CH3
OH
HO
NH2
HO
OH
NH2
H3CO
H3CO
DMQ–MA-Lys(DMQ–MA)-Ile-Arg-OMe 3c
DMQ–MA-Lys(DMQ–MA)-Val-Arg-OMe 4c
Scheme 2. Molecular structures of 1c, 2c, 3c, and 4c.

Four New Bis(2,6-Dimethoxyhydroquinone–3-Mercaptoacetic Acid)–Peptide Conjugates
245
•
QH2 + O2 → QH• + O− + H+
(1)
as shown in Fig. 1. It was shown that the product distribu-
tion clearly depended on the concentration of the conjugates,
a fact indicating that the DNA cleavage was drug-dose
dependent. With an increase in concentration, open-circular
DNA (OCC) was further cleaved into the linear form. The
low concentration of AH2 (5 µM) seems to have a syner-
gistic effect and it is selective. When other electron donors
such as glutathione were involved in the reaction system,
no obvious cleavage was observed. The conjugates began
to cleave supercoiled DNA (CCC) into the open circular
form in drug concentrations as low as 5–20 µM under the
synergistic activation of AH2 without addition of hydrogen
peroxideorUVirradiation.Additionof1 µMFe2+ ion, which
imparted no cleavage itself, improved the cleavage ability of
the DMQ–MA–tripeptide conjugates.
2
•
QH• + O2 → Q + O−2 + H+
(2)
(3)
(4)
(5)
Q + AH2 → QH• + AH•
•
2O−2 + 2H+ → H2O2 + O2
•
H2O2 + O−2 → OH• + OH− + O2
CMX · Fe2+ + H2O2 → CMX · Fe3+ + OH• + OH− (6)
The hydroxyl radical produced by DMQ–MA under the
activation of AH2 could be an important mediator of the
observed cytotoxicity.As we know, oxidants with free-radical
character are well known investigators of DNA damage. In
this paper, the DNA scission chemistry of the new DMQ–
MA–peptide conjugates and the reactive oxygen species are
investigated in detail.
Cleavage as a Function of Reaction Time
Results and Discussion
The computer software Gelworks 1D in GDS 8000 quantified
the intensity of the bands. Values for supercoiled plasmid
DNA were corrected by a factor of 1.47 owing to its lower
staining capacity by ethidium bromide.[16] The densitometric
quantitation of the bands produced by gel electrophoresis
of 1c is shown in Fig. 2. The supercoiled DNA was rapidly
DNA Scission Chemistry
Cleavage as a Function of Drug Concentration
DNA cleavage experiments were performed in the presence
of DMQ–MA–tripeptide conjugates andAH2 (ascorbic acid)
(a)
1
2
3
4
5
6
7
8
(b)
1
2
3
4
5
6
7
8
OCC
CCC
OCC
Linear
CCC
(c)
1
2
3
4
5
6
7
8
(d )
1
2
3
4
5
6
7
8
OCC
Linear
OCC
CCC
CCC
Fig. 1. DNA cleavage patterns of pBR322 DNA induced by (a) 1c, (b) 2c, (c) 3c, and (d) 4c on agarose electrophoresis
gel after incubation the drugs with pBR322 DNA in PBS at 37◦C for 10 min and at 65◦C for 10 min. The upper band
represents the open-circular form, the middle weak band represents the linear form, and the lower band, the supercoiled
form. Lane 1, DNA + 10 µM 1c (a), 2c (b), 3c (c), or 4c (d) +1 µM Fe2+ respectively; lanes 2–6, DNA + 100, 50, 20,
10, 5 µM 1c (a), 2c (b), 3c (c), or 4c (d) respectively; lane 7, DNA + 5 µM AH2; lane 8, DNA alone.

246
Y.-F. Song
converted into nicked DNA after incubating 1c and DNA for
30 min; form II (OCC) increased with increasing time, and
form I (CCC) completely disappeared after 60 min.
The observed distribution of DNA species in agarose gel
electrophoresis experiments provided a measure of the extent
of oxidative cleavage of DNA. The decrease in form I and the
increase in form II fitted well to a single exponential decay
curve.The oxidative rate constants for 1c at a concentration of
20 µM were estimated to be 0.0673 and 0.0859 min−1 respec-
tively for the decrease in form I and for the increase in form
II. Detailed information about the kinetic cleavage is under
investigation.
Addition of hydroxyl radical scavengers (Fig. 3), such as
glycerol (≥700 µM), sodium benzoate (≥700 µM), mannitol
(≥400 µM), methanol (≥700 µM), and dimethyl sulfoxide,
etc., inhibited markedly the cleavage of the pBR322 DNA
(Table 2); this indicated that hydroxyl radicals were involved
in the cleavage. (The effect of addition of the radical scav-
engers on the DNA cleavage chemistry of 2c, 3c, and 4c was
similar to that of 1c.) DNA scission chemistry of 1c in a glove
box filled with nitrogen was investigated, and it was estab-
lished that the DNA cleavage of 1c was clearly inhibited, a
result consistent with the mechanism.
The above results showed that hydroxyl radicals might be
involved in the DNA cleavage of the DMQ–MA–tripeptide
conjugates. The deoxyribonucleic acid cleavage reaction was
concentration dependent. At high concentrations, the open
circular DNA was further cleaved into the linear form.All the
DNA cleavage experiments were carried out without addition
of hydrogen peroxide or UV irradiation.
100
80
CCC
OCC
linear
60
It was necessary to identify the reactive oxygen species
of these DMQ–MA–tripeptide conjugates since the forma-
tion of hydroxyl radicals, which are the main contributors to
DNA strand cleavage, was strongly implicated but not unam-
biguously proven. In the present work, evidence was provided
for the generation of hydroxyl radicals by the Rhodamine B
assay and ESR spectroscopy.
40
20
0
Table 2. The mass fraction of DNA species formed in the
cleavage reaction of pBR322 DNA
0
20
40
60
80
100
Time (min)
Fraction [%]
Cpd DNA
type 0 min 10 min 20 min 30 min 40 min 50 min 60 min
Fig. 2. The mass fraction of DNA species from the agarose gel elec-
trophoresis of products of the cleavage reaction of pBR322 DNA with
20 µM of 1c (ethidium bromide staining) as a function of the reac-
tion time. Cleavage conditions: 0.5 µg µL−1 pBR322 DNA (1 mL),
10 × PBS buffer (pH 7.1), at 37◦C for half the time and 65◦C for half the
time. The intensity of the bands was quantified by Gelworks 1D and
the results are displayed inTable 2. Fractions are calculated based on the
total intensity of the DNA control lane. The cleavage conditions for 2c,
3c, and 4c are the same as that for 1c.
2c CCC
OCC
3c CCC
OCC
80
20
81
19
63
37
60
40
54
46
49
51
44
56
43
57
36
64
32
68
27
73
21
79
25
75
17
76
7
linear
4c CCC
OCC
83
17
64
36
47
53
28
72
16
84
5
95
100
(a)
1
2
3
4
5
6
7
8
(b)
1
2
3
4
5
6
7
Fig. 3. (a) Effect of addition of radical scavengers on the DNA cleavage chemistry of 1c.The radical scavengers
includeglycerol(lane1), sodiumbenzoate(lane2), mannitol(lane3), methanol(lane4), DNA +AH2 (5 µM) + 1c
(40 µM) (lane 5), DNA alone (lane 6), DNA +AH2 (5 µM) (lane 7), and DMSO (lane 8). The effect of these
hydroxyl radical inhibitors on the DNA cleavage of 2c, 3c, or 4c is similar to that of 1c. (b) The DNA scission
chemistry was carried out under a glove box filled with nitrogen. DNA alone (lane 7), DNA +AH2 (5 µM)
(lane 6), DNA +AH2 (5 µM) + 1c (5 µM) (lane 5), DNA +AH2 (5 µM) + 1c (10 µM) (lane 4), DNA +AH2
(5 µM) + 1c (20 µM) (lane 3), DNA +AH2 (5 µM) + 1c (50 µM) (lane 2), DNA +AH2 (5 µM) + 1c (100 µM)
(lane 1).

Four New Bis(2,6-Dimethoxyhydroquinone–3-Mercaptoacetic Acid)–Peptide Conjugates
247
●
Investigation of the Hydroxyl Radicals by the
Rhodamine B (Rhb) Assay
of DMPO OH adducts. As the concentration of ascorbic
acid increased from 0 to 150 µM, a gradual increase in the
intensity of the characteristic 1 : 2 : 2 : 1 signals due to the
DMPO–hydroxyl radical was observed. When the concen-
tration of ascorbic acid was greater than 150 µM, a decrease
Hydroxyl radicals were quantified by following the degra-
dation of the dye Rhodamine B in the presence of 1c and
AH2.[17] The reaction was monitored at 554 nm in phosphate
buffer under aerobic conditions. The change in concentra-
tion of Rhb (ε 10.7 × 104 M−1 cm−1) at different times was a
direct measure of the concentration of hydroxyl radicals pro-
duced in the reaction mixture.[17] Similar experiments were
performed in the presence of 2c, 3c, and 4c.
●
in the signal intensity of DMPO OH appeared. As the cata-
lase was added to the reaction system, the signal due to the
hydroxyl radical almost disappeared, which indicated that
After each addition of 1c (20 µL) to Rhb (2 mL) (Fig. 4),
a sudden dip in the spectra was observed, indicating that the
hydroxylradicalwasproducedquickly(within10 s).Thetotal
volume of 1c added to the reaction system was 60 µL, and
the final concentration of 1c was 15 µM.
The effect of 1c on the fluorescence spectra of Rhodamine
B (Fig. 5) was investigated, which was evaluated by monitor-
ing the changes in the intensity of Rhodamine B at 584 nm.
When 1c (50 µL) was added to Rhb (2 mL), the fluores-
cence intensity of Rhb at 584 nm declined very quickly due
to the production of hydroxyl radicals. The effect of the other
compounds such as 2c, 3c, and 4c on the degradation of Rhb
was similar to that of 1c.
Addition of 1c to Rhodamine B
240
235
230
225
220
215
210
205
EPR Experiments
EPR studies of 1c, 2c, 3c, and 4c employing the spin
trap5,5-dimethyl-1-pyrrolineN-oxide(DMPO)indicatedthe
0
10
20
30
40
50
60
Time (s)
●
presence of hyperfine splitting (HFS) of the DMPO OH
adduct. Fig. 6 shows the EPR spectra of 1c at various con-
centrations of ascorbic acid. The conjugate in the absence of
ascorbic acid gave a weak EPR signal due to the presence
Fig. 5. ThefluorescencespectraofRhbat584 nmwithincreasingtime
by addition of 1c to Rhb. Experimental conditions: Slit(Ex)/slit(Em)
10 nm/2.5 nm, λEx 554 nm, λEm 584 nm, scan speed 1500 nm min−1
.
(a)
(b)
1.68
1.60
1.66
1.64
1.62
1.60
1.58
1.56
1.54
1.58
1.56
1.54
1.52
1.50
1.48
1.46
0
100 200 300 400 500 600
Time (s)
0
100
200
Time (s)
300
400
500
(c)
(d )
1.68
1.66
1.64
1.62
1.60
1.58
1.56
1.54
1.68
1.66
1.64
1.62
1.60
1.58
1.56
1.54
1.52
1.50
0
100 200 300 400 500 600
Time (s)
0
100 200 300 400 500 600
Time (s)
Fig. 4. Quantitation of hydroxyl radicals by following the degradation of Rhb at 554 nm in 10 mM phosphate buffer (pH 7.1). The kinetics spectra
of Rhb (15 µM; 2 mL) at 554 nm in the presence of 10 µM of (a) 1c, (b) 2c, (c) 3c, and (d) 4c (three additions of 20 µL of drugs with the synergistic
activation of AH2 each time to Rhb (2 mL); 60 µL all together). The final concentration of 1c, 2c, 3c, and 4c is 15 µM respectively.

248
Y.-F. Song
(0)
(1)
(2)
(3)
(4)
b
a
a
a
a
(5)
(6)
(7)
(8)
●
Fig. 6. EPR spectra of the characteristic 1 : 2 : 2 : 1 signal of the DMPO OH adduct from DMQ–MA–Lys(DMQ–
MA)–Lys(Cbz)–Arg–OMe 1c (5 µM) with ascorbic acid (AH2) combinations. (0) The standard reaction solution contains
DMPO (0.1 mM) and FeSO4 (0.1 mM) in phosphate buffer saline (pH 7.1); (1) spectrum upon addition of 50 mM AH2;
(2) spectrum upon addition of 100 mM AH2; (3) spectrum upon addition of 140 mM AH2; (4) spectrum upon addition of
200 mM AH2; (5) spectrum upon addition of 250 mM AH2; (6) spectrum upon addition of 400 mM AH2; (7) spectrum
upon addition of 600 mM AH2; (8) spectrum upon addition of 800 mM AH2. aN = aH = 15.0 G; g 2.0068. In (5), peaks
N
a denote the DMPO OH signals, and peaks b denote the ascorbyl signal. For 2c, a = aH = 14.8 G, g 2.0065; for 3c,
●
aN = aH = 15.0 G, g 2.0058; for 4c, aN = aH = 14.9 G, g 2.0062.

Four New Bis(2,6-Dimethoxyhydroquinone–3-Mercaptoacetic Acid)–Peptide Conjugates
249
25
from high to low was as follows: Lys(Cbz), Ile,Val, Phe. DNA
cleavage experiments showed that the cleavage ability of the
DMQ–MA–peptide conjugates was weaker than that of the
parent compound DMQ–MA, and decreased in the following
order: DMQ–MA > 1c > 3c > 4c > 2c.
The agarose gel electrophoresis experiment showed that
no DNA cleavage by the peptide chains was observed, which
indicated that the cleavage behaviour of DMQ–MA–peptide
conjugates was attributed to DMQ–MA. Furthermore, addi-
tion of a catalytic amount Fe(ii), which caused no cleavage
by itself, had an obvious enhancement effect on the cleavage
behaviour of the DMQ–MA–peptide conjugates.
The DNA scission ability of DMQ–MA and its conju-
gates was equivalent to or greater than some of the metal
chelates, since these chelates necessitated addition of hydro-
gen peroxide or UV irradiation for DNA scission. Herein, our
newly synthesized compounds were able to generate super-
oxide anions, which gave rise to hydrogen peroxide and then
hydroxyl radicals via the slow Haber–Weiss reaction or via
the fast Fe(ii) complex route in situ. It needs to be pointed out
that the addition of H2O2 or UV irradiation was not required
for DNA cleavage of these compounds. The role of the 5 µM
AH2 appeared to be a synergistic one. In the absence of AH2,
DMQ–MA at low concentrations was not cytotoxic, and was
able to cleave DNA.[14]
2c
4c
DMQ–MA
1c
3c
20
15
10
5
0
0
200
400
600
800
Ascorbic acid (10Ϫ6 mol/L)
Fig. 7. Effect of ascorbic acid concentration on the generation of
hydroxyl radicals from 1c, 2c, 3c, 4c, and DMQ–MA (5 µM).
hydrogen peroxide was involved in the DMQ–MA–peptide
conjugates under the synergistic activation of AH2.
It was found that ascorbic acid promoted the generation
of hydroxyl radicals from dimethoxyhydroquinone deriva-
tives within certain concentrations.[7,14,18] However, ascorbic
acid appeared to have scavenging effect on hydroxyl rad-
icals over certain concentrations. The interpretation of the
observed scavenging process was as follows: the rate con-
stants of ascorbic acid and DMPO in the reaction competing
for the hydroxyl radicals were comparable, and an increase in
the concentration of ascorbic acid would favour its reaction
with hydroxyl radicals and yield more ascorbyl radicals at the
Hydroxyl radical generation was further confirmed by
the Rhodamine B assay. The hydroxyl radical was produced
very quickly (within 10 s), which led to a decrease in UV
absorbance at 554 nm and fluorescence intensity at 584 nm
of Rhodamine B.
●
expense of the DMPO OH adduct.
Ascorbic acid may promote the generation of the
semiquinone radical from the dimethoxybenzoquinone
derivative Q, which recycled to provide substrate in reaction
(2). The result was a rapid generation of O−2 , and conse-
quently an immense production of hydroxyl radicals. The
DMQ–MA–peptide conjugates, activated by AH2, gave rise
to a semiquinone radical species.[15,19] ESR spectra showed
the superimposition of the signals of the semiquinone radicals
EPR experiments showed that many hydroxyl radicals
were trapped by DMPO, and the characteristic 1 : 2 : 2 : 1
●
intensity of the DMPO OH signal increased within certain
concentrations of ascorbic acid. However, over some concen-
trations, ascorbic acid seemed to have a scavenging effect,
and the signal intensity of ascorbyl increased at the expense
●
●
of DMPO OH. The signal intensity of the DMPO OH
descended in the order 4c > 1c > 3c > DMQ–MA ≈ 2c. The
inconsistent results of the DNA cleavage ability and the
hydroxyl radical intensity can be explained by the presence
of other radicals, such as semiquinone radicals, involved in
the DNA damage.
●
and the characteristic 1 : 2 : 2 : 1 DMPO OH pattern (peaks a
in Fig. 6).[19] The strong hyperfine splitting signals in the cen-
tre of the ESR spectra were identified as the ascorbyl radical
signal (peaks b in Fig. 6).[14,18]
●
The intensity of the EPR signal of DMPO OH for the four
Binding constants of the drugs to DNA can be estimated
by measuring the loss of ethidium fluorescence as a func-
tion of the added drug (Table 3). The drug concentration,
which produced 50% inhibition of fluorescence, was approx-
imately inversely proportional to the binding constant. (The
relationship takes no account of differences in binding site
size or cooperative effects. Moreover, for drugs with bind-
ing constants that are orders of magnitude larger than that
of ethidium, it invariably leads to a minimum value for
the binding constant.) Since the binding constants of these
conjugates were quite weak (10−4 M−1),[7,20] the degree of
DNA cleavage was not related to the binding constant of
the drugs to DNA, but rather related to the relative ability
of the drugs to generate various free radicals and reactive
species.
DMQ–MA derivatives as a function of ascorbic acid concen-
tration is shown in Fig. 7. As can be seen, the maximum
signal intensity for DMPO OH with 1c, 4c, and DMQ–MA
occurred at an ascorbic acid concentration of 250 µM, while
for 2c and 3c, the maximum signal intensity occurred at an
ascorbic acid concentration of approximately 140 µM. The
derivatives had quite similar spectroscopic profiles.
●
Conclusions
In this paper, four new bis(dimethoxyhydroquinone–3-
mercaptoacetic acid)–peptide conjugates were synthesized in
a stepwise fashion in solution by Pfp/DCC methods.
Different amino acids were combined in the side chains of
the DMQ–MA–peptide conjugates, and the polarity sequence

250
Y.-F. Song
The syntheses of Boc–Phe–Arg–OMe 2a [(77%) m/z [MH]+ 436
(Found: C 58.2, H 7.7, N 16.5. C21H33N5O5 requires C 57.9, H 7.6,
N 16.1%)], Boc–Ile–Arg–OMe 3a [(78%) m/z [MH]+ 402 (Found: C
54.0, H 8.9, N 17.1. C18H35N5O5 requires C 53.9, H 8.8, N 17.4%)],
and Boc–Val–Arg–OMe 4a [(75%) m/z [MH]+ 388 (Found: C 52.9,
H 8.1, N 18.4. C17H33N5O5 requires C 52.7, H 8.6, N 18.1%)] were
similar to that of 1a.
Table 3. DNA-binding constants of 1c, 2c, 3c, and 4c determined
by the ethidium bromide assay[20]
Scan speed 1500 nm min−1, λEx 525 nm, λEm 600 nm,
slit(Ex)/slit(Em) 15 nm/15 nm. [EB] 1.27 µM, [DNA] 1.55 µM. The
assays were carried out with pH 8.0 buffer (5 mM Tris-HCl, 0.5 mM
EDTA, 0.5 µg mL−1 ethidium bromide buffer). The fluorimeter cell
compartment and circulation water bath were thermostatted at 25◦C
Drug
Binding constant [M−1
]
Synthesis of Boc–Lys(Boc)–Lys(Cbz)–Arg–OMe 1b
1c
2c
3c
4c
1.24 × 104
1.27 × 104
1.76 × 104
1.81 × 104
1.84 × 104
Compound 1a (0.22 g, 0.40 mmol) in CH2Cl2 (3 mL) was stirred with
trifluoroacetic acid (TFA; 2 mL) for 1 h. The solvents were removed
under vacuum, and the TFA salt of 1a was obtained. Boc–Lys(Boc)
(0.14 g, 0.40 mmol) in CH2Cl2 (5 mL) was treated with Pfp (0.11 g,
0.60 mmol) in an ice bath for 15 min, DCC (0.10 g, 0.40 mmol) was
added, and the reaction mixture was kept at 0◦C for 15 min and then
at room temperature for 90 min. The reaction mixture was added to
the TFA salt of 1a, and DIEA was added to adjust the pH to 7.0.
After 120 min, the reaction mixture was filtered, and the filtrate was
diluted with CH2Cl2 and washed successively with citric acid (10%,
30 mL), saturated NaHCO3 (30 mL), and water (20 mL), then dried
over MgSO4 and evaporated under vacuum to give the crude solid.
Purification using silica gel chromatography and stepwise elution with
1% CH2Cl2 in 2–15% CH3OH/CH2Cl2 afforded the tripeptide as a
white solid (71%), RF 0.57 (20 : 80 CH3OH/CH2Cl2). m/z [MH]+ 779
(Found: C 57.3, H 7.6, N 14.6. C37H62N8O10 requires C 57.1, H 8.0,
N 14.4%).
DMQ–MA
Experimental
General Experimental
The protected amino acids were purchased from Sigma. Medium-
pressure column chromatography was performed using Merck 230–400
mesh silica gel.TLC was performed on Merck silica gel 60 on aluminum
sheets. Mass spectra were recorded on a JEOL JM-HX 110 instrument
and MA SPEC system operating in the FAB (fast-atom bombardment)
mode. Elemental analyses were performed with a Perkin–Elmer 240c
instrument. Calf thymus DNA, pBR322 DNA, and Tris buffer were
purchased from Beijing Sino-American Biotechnology Company. The
DNA solution was prepared by dissolving DNA in aqueous solution and
dialyzing this against a buffer until the UV absorbance ratio A260/A280
was greater than 1.90. The concentration of the prepared stock solu-
tion was expressed as DNA(p), and was determined according to its
absorbance at 260 nm using ε260 6600 M−1 cm−1. The electrophoresis
experiments were carried out on a set of electrophoresis systems (made
by Beijing Liuyi Instruments) by usingTAE buffer (pH 7.2).The gel was
stained with ethidium bromide for 20 min after electrophoresis. Images
were analyzed on a Complete Gel Documentation and Analysis system
GDS 8000. UV-vis spectra were carried out on a Hewlett Packard 8453
spectrophotometer. Fluorescence measurements were determined with
a Perkin–Elmer LS50B fluorescence spectrophotometer. Spin trapping
of radical species by ESR spectroscopy was carried out with a Bruker
ER 200D ESR spectrometer. Experimental conditions were as follows:
microwave power 63 mW, modulation amplitude 2.0 G, modulation
frequency 100 kHz, sweep time 30 G min−1, receiver gain 6.3 × 105,
time constant 500 ms. The standard reaction solution contained 0.1 mM
DMPO and 0.1 mM FeSO4 in phosphate buffer saline (pH 7.1). Vari-
ous concentrations of ascorbic acid were added to the standard reaction
solution followed by the addition of the synthesized compounds (4 µM)
in a total volume of 0.35 mL.
Boc–Lys(Boc)–Phe–Arg–OMe 2b [(76%) m/z [MH]+ 664 (Found:
C 58.3, H 8.2, N 14.5. C32H53N7O8 requires C 57.9, H 8.1, N 14.8%)],
Boc–Lys(Boc)–Ile–Arg–OMe 3b [(78%) m/z [MH]+ 630 (Found: C
55.7, H 8.3, N 15.7. C29H55N7O8 requires C 55.3, H 8.8, N 15.6%)],
and Boc–Lys(Boc)–Val–Arg–OMe 4b [(74%) m/z [MH]+ 616 (Found:
C 54.3, H 8.2, N 16.3. C28H53N7O8 requires C 54.6, H 8.7, N 15.9%)]
were synthesized in a similar way to 1b.
Synthesis of DMQ–MA–Lys(DMQ–MA)–Lys(Cbz)–Arg–OMe 1c
Compound 1b (0.44 g, 0.56 mmol) in CH2Cl2 (2 mL) was stirred with
TFA (2 mL) for 1 h, and the solvents were removed under vacuum.
DMQ–MA (0.44 g, 1.68 mmol) in CHCl3 (10 mL) was stirred with Pfp
(0.47 g, 2.40 mmol) in an ice bath for 15 min, and then DCC (0.35 g,
1.69 mmol) was added. The reaction mixture was stirred at 0◦C for
another 20 min and at room temperature for 2 h, and then was filtered.
The filtrate was dried under vacuum for 30 min. The TFA salt of 1b
in DMF (3 mL) was added, and the pH was adjusted with DIEA to
7.0. The reaction mixture was filtered after 2 h. The filtrate was diluted
with CHCl3 and washed with citric acid, saturated NaHCO3, and water
successively, then dried over MgSO4, and evaporated to give a solid.
This solid was purified by silica gel chromatography and eluted step-
wise with 1% CH2Cl2 in 2–15% CH3OH/CHCl3 to afford a purple
solid (67%), RF 0.53 (20 : 80 CH3OH/CH2Cl2). m/z [MH]+ 1063
(Found: C 52.9, H 6.0, N 10.3. C47H66N8O16S2 requires C 53.1, H 6.3,
N 10.5%).
Synthesis
DMQ,[5] Boc–Lys(Boc),[21] and DMQ–MA[6] were synthesized accord-
ing to literature procedures.
DMQ–MA–Lys(DMQ–MA)–Phe–Arg–OMe2c[(65%)m/z[MH]+
948 (Found: C 53.5, H 6.3, N 10.17. C42H57N7O14S2 requires C 53.2, H
6.1, N 10.3%)], DMQ–MA–Lys(DMQ–MA)–Ile–Arg–OMe 3c [(63%)
m/z [MH]+ 914 (Found: C 51.5, H 6.2, N 11.1. C39H59N7O14S2
requires C 51.3, H 6.5, N 10.7%)], and DMQ–MA–Lys(DMQ–MA)–
Val–Arg–OMe 4c [(67%) m/z [MH]+ 900 (Found: C 51.0, H 6.2,
H 11.17%. C38H57N7O14S2 requires C 50.7, H 6.4, N 10.9%)] were
synthesized in a similar way to 1c.
Synthesis of Boc–Lys(Cbz)–Arg–OMe 1a
Nα-Boc–Nξ-Cbz–lysine hydrochloride (0.50 g, 1.31 mmol) in CH2Cl2
(10 mL) was stirred with Pfp (0.36 g, 1.97 mmol) in an ice bath for
15 min, and then DCC (0.27 g, 1.31 mmol) was added. The mixture was
stirred at 0◦C for a further 15 min, and at room temperature for 2 h. It
was then filtered and the solvent was removed under vacuum.A solution
of Arg–OMe (0.33 g, 1.31 mmol) in dimethylformamide (DMF; 2 mL)
was added, and the reaction mixture was then adjusted to pH 7.0 with
DIEA (N,Nꢀ-diisopropylethylamine). The reaction mixture was kept
for 3.5 h at room temperature. The mixture was distilled under reduced
pressure to produce a white solid, which was purified by column chro-
matography with 100 mL of 1% CH2Cl2 in 2–11% CH3OH/CH2Cl2.
The solvents were evaporated to give a white solid (76%), RF 0.54
(15 : 85 CH3OH/CH2Cl2). m/z [MH]+ 551 (Found: C 56.9, H 7.5,
N 15.2. C26H42N6O7 requires C 56.7, H 7.7, N 15.3%).
Acknowledgments
The author thanks Dr L. Sheh at Tunghai University, Taiwan,
for experimental assistance and valuable discussion. The
author acknowledges the support of the National Natural Sci-
ence Foundation of China and Provincial Natural Foundation
of Shanxi.

Four New Bis(2,6-Dimethoxyhydroquinone–3-Mercaptoacetic Acid)–Peptide Conjugates
251
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