DNA Photocleavage and Binding Modes of Methylene Violet 3RAX and its Derivatives: Effect of Functional Groups

Article abstract of DOI:10.1071/CH16496

With 4′-amino-N,N-diethylaniline and aniline as starting materials, methylene violet 3RAX 1 and its derivatives 2-5 were synthesised. The five compounds were characterised by IR, UV-vis, and 1H NMR spectroscopy and mass spectrometry. The binding mode between the synthesised compounds and DNA were investigated. The results show that both compounds 1 and 5 bind to DNA by an intercalative mode, while compounds 2-4 interact with DNA through a mixed binding mode involving groove binding and electrostatic interactions. The photocleavage ability of the five compounds to DNA were calculated to be 38, 40, 30, 20, and 13%, respectively, when their concentration was adjusted to 400M. The singlet oxygen production of compounds measured by the 1,3-diphenylisobenzofuran method was consistent with the trend of DNA photocleavage ability. The DNA studies suggest that the binding mode between methylene violet 3RAX and DNA, the ability of methylene violet 3RAX to generate singlet oxygen, and the DNA photocleavage activity could be adjusted through modification of the amino group on methylene violet 3RAX.

Full text of DOI:10.1071/CH16496

CSIRO PUBLISHING
Aust. J. Chem.
Full Paper
http://dx.doi.org/10.1071/CH16496
DNA Photocleavage and Binding Modes of Methylene
Violet 3RAX and its Derivatives: Effect of Functional Groups
A
A
B
B
,
C
B
Ke Yang, Xiong Zhang, Fang Yang, Fengshou Wu, Xiulan Zhang,
, ,
A B C
and Kai Wang
A
Hubei Novel Reactor and Green Chemical Technology Key Laboratory, Wuhan
Institute of Technology, Wuhan 430073, China.
B
Key Laboratory for Green Chemical Process of Ministry of Education, Wuhan Institute of
Technology, Wuhan 430073, China.
C
Corresponding authors. Email: wfs42@126.com; kaiwang@wit.edu.cn
0
With 4 -amino-N,N-diethylaniline and aniline as starting materials, methylene violet 3RAX 1 and its derivatives 2–5 were
1
synthesised. The five compounds were characterised by IR, UV-vis, and H NMR spectroscopy and mass spectrometry.
The binding mode between the synthesised compounds and DNA were investigated. The results show that both
compounds 1 and 5 bind to DNA by an intercalative mode, while compounds 2–4 interact with DNA through a mixed
binding mode involving groove binding and electrostatic interactions. The photocleavage ability of the five compounds to
DNA were calculated to be 38, 40, 30, 20, and 13 %, respectively, when their concentration was adjusted to 400 mM. The
singlet oxygen production of compounds measured by the 1,3-diphenylisobenzofuran method was consistent with the
trend of DNA photocleavage ability. The DNA studies suggest that the binding mode between methylene violet 3RAX and
DNA, the ability of methylene violet 3RAX to generate singlet oxygen, and the DNA photocleavage activity could be
adjusted through modification of the amino group on methylene violet 3RAX.
Manuscript received: 7 September 2016.
Manuscript accepted: 25 January 2017.
Published online: 20 February 2017.
Introduction
3
RAX derivatives 2–4 (Scheme 1) are synthesised here through
1
Singlet oxygen ( O ) is the main cytotoxic agent in the process
transforming the amino group into hydroxy, 4-methyl-benzene-
sulfonimide, and benzene-1,2-dicarboxylic acid imide groups,
respectively. In addition, the conjugation of 1 with indometha-
cin yields compound 5. Indomethacin (INDO), as one of the first
non-steroidal anti-inflammatory drugs (NSAID), has been cho-
sen because it is widely used in the treatment of inflammatory
diseases, such as rheumatoid arthritis, osteoarthritis, gut arthri-
tis, burst, tendinitis, traumatic synovitis, and ankylosing spon-
[14]
dylitis.
indomethacin has antitumour activity.
ties of compounds 1–5 to calf thymus (CT)-DNA are studied by
means of various methods, such as UV-vis, fluorescence, and
circular dichroism (CD) spectroscopy. The singlet oxygen
generation ability of the compounds is determined using the
1,3-diphenylisobenzofuran (DPBF) method. Finally, we further
evaluate the DNA photocleavage activities of the compounds
through agarose gel electrophoresis.
2
of photodynamic therapy (PDT) to induce the apoptosis of
[
cancer cells through a type II mechanism.
can effectively damage the tiny blood vessels in the tumour
tissue through interaction with biological macromolecules,
such as DNA and proteins. Phenazine compounds and their
derivatives with a conjugated structure have been widely
1,2]
Singlet oxygen
[
3,4]
[5–8]
developed and applied in dyes, biomedicine,
9]
photoelectric materials, and analytical chemistry.
pesticides,
[10]
As one
[
Preliminary experiments have indicated that
The binding proper-
[15]
of the typical phenazine compounds, methylene violet 3RAX 1
displays a rigid planar phenazine structure. It has a potential
application in the PDT of cancers as a photosensitiser, as it might
change the molecular structure of DNA, undermine the spatial
configuration of DNA, and even induce the generation of
reactive singlet oxygen by its interaction with light to damage
DNA and cause cell death through interaction with DNA in
[
11,12]
tumour cells.
The functional groups of methylene violet 3RAX 1 allow
easy further modification of its structure. In 2013, Saha and
13]
Experimental
[
Kumar
prepared the methylene violet 3RAX derivative
Synthesis of Methylene Violet 3RAX 1 and its
Derivatives 2–5
indoine blue by transforming the diethylamino group at one
side of 1 into a (2-hydroxy-1-naphthalenyl) azo group and
introducing a methyl group. Studies showed that the affinity
of indoine blue to DNA was weaker than that of 1. However,
both of them still adopt an intercalation mode with DNA. To
further investigate the effect of functional groups at the other
side of 1 on its interaction with DNA, the methylene violet
Synthesis of 3-Amino-7-(diethylamino)-5-phenyl-
phenazinium (1)
0
Toa solution of 4 -amino-N,N-diethylaniline (5 g, 30.5 mmol),
aniline (5.8 g, 61.0 mmol), and concentrated hydrochloric acid
(8 mL) in acetic acid/sodium acetate buffer solution (200 mL)
Journal compilation � CSIRO 2017
www.publish.csiro.au/journals/ajc

B
K. Yang et al.
H
N
Ϫ
Cl
ϩ
N
N
N
N
OH
NaNO3
H SO
2
4
O
S
2
Cl
H
N
O
O
O
Ϫ
Cl
ϩ
N
S
TEA/CHCl3
N
H
H
N
Ϫ
Cl
O
O
3
N
ϩ
N
ϩ
N
NH2
H
N
O
O
ϩ
N
Ϫ
Cl
NH2
N
NH2
py
1
O
4
H
N
IMC/CHCl3
O
O
Ϫ
ϩ
N
Cl
DMAP/EDCl
N
N
N
H
Cl
5
O
Scheme 1. Synthetic routes of compounds 1–5.
was added to a large excess of potassium dichromate (19.7 g,
7.1 mmol). The reaction mixture was stirred for 3 h at 1058C,
(m, 2H), 7.60–7.64 (m, 2H), 7.74 (d, J 9.3, 1H). d (100 MHz,
C
CDCl ) 158.5, 149.5, 143.8, 132.8, 131.5, 130.8, 129.4, 125.7,
3
6
protected from light and atmospheric moisture. After cooling to
room temperature, the mixture was filtered, affording 2.3 g of
solid (yield 35 %). d (400 MHz, CDCl ) 1.09 (t, J 7.1, 6H), 3.33
121.1, 118.0, 113.2, 112.1, 47.2, 13.1. Anal. Calc. for
C H N ClO: C 69.56, H 5.84, N 11.06. Found: C 69.45,
H 5.77, N 11.12 %. nmax (KBr)/cm 3160 (OH), 1326, 1174
(C–O, OH), 1608, 1520, 1475 (C=C, C–N),1199 (C–N), 1060,
2
2 22 3
ꢀ
1
H
3
(
(
(
q, J 7.1, 4H), 5.63 (s, 1H), 5.99 (s, 1H), 7.13 (d, J 7.8, 1H), 7.32
d, J 7.8, 2H), 7.69–7.80 (m, 4H), 7.85 (d, J 8.9, 1H). dC
400 MHz, CDCl ) 149.3, 148.4, 143.8, 132.8, 130.2, 130.0,
1010, 997, 870, 844, 818, 778, 702 (C–H). l
530, 562. m/z (ESI) 344.1781 [M] .
(DMSO)/nm
max
þ
3
1
29.5, 125.6, 124.4, 118.5, 115.3, 113.3, 111.9, 47.4, 12.8. Anal.
Calc. for C H N Cl: C 69.74, H 6.12, N 14.79. Found: C
Synthesis of 3-(4-Methyl-benzenesulfonimide)-7-
(diethylamino)-5-phenyl-phenazinium (3)
2
2 23 4
ꢀ1
6
1
1
9.59, H 6.24, N 14.65 %. nmax (KBr)/cm 3058, 1649 (NH2),
339, 1198 (C–NH , N–H), 1601, 1524, 1484 (C=C, C–N),
069, 1006, 991, 874, 849, 812, 776, 704 (C–H), 1392 (–CH ).
2
To compound 1 (1 g, 2.63 mmol) was added 4-toluene
sulfonyl chloride (2.0 g, 10.52 mmol) and triethylamine
(3 mL) in dry chloroform (15 mL). The solution was heated to
3
þ
lmax (DMSO)/nm 560. m/z (ESI) 343.58 [M] .
6
mixture was poured into a saturated sodium chloride solution
08C for 2 h. After cooling to room temperature, the reaction
Synthesis of 3-Hydroxy-7-(diethylamino)-5-phenyl-
phenazinium (2)
(10 mL) and extracted with chloroform (3 ꢁ 5 mL). The organic
To compound 1 (1 g, 2.63 mmol), 5 mL of concentrated
sulfuric acid and 10 mL of water was added. The solution was
cooled to ꢀ108C. An aqueous solution (10 mL) of sodium nitrite
layers were combined, dried over anhydrous sodium sulfate, and
then concentrated under vacuum. The residue was purified on a
silica gel column eluted with chloroform/methanol (10 : 1) to
yield the desired product (1.1 g, yield 78 %). dH (400 MHz,
(
0.27 g, 3.95 mmol) was then added and stirred for 1.5 h. After
heating to room temperature, the reaction mixture was poured
into a solution of 1 g of copper sulfate, 5 mL of concentrated
sulfuric acid, and 5 mL of water. The mixture was stirred for
another 3 h at 1058C and then cooled to room temperature. The
pH of the solution was adjusted to 5 using sodium bicarbonate.
The crude product was precipitated with sodium chloride (5 g),
and then purified on a silica gel column with chloroform as
eluent. The first fraction was collected and evaporated to yield
CDCl ) 1.10 (t, J 7.1, 6H), 2.32 (s, 3H), 3.35 (q, J 7.1, 4H), 5.62
3
(s, 1H), 6.39 (s, 1H), 7.03 (d, J 8.9 , 1H), 7.11 (d, J 7.3, 2H), 7.31
(d, J 7.8, 2H), 7.46 (d, J 10.6, 1H), 7.57–7.62 (m, 3H), 7.80
(m, 4H) d (100 M, CDCl ) 149.7, 143.8, 137.6, 136.7, 133.0,
C
3
130.1, 129.4, 128.6, 125.4, 118.5, 116.5, 115.1, 113.8, 111.7,
47.3, 21.5, 13.1. Anal. Calc. for C H N ClSO : C 65.34,
2
9
29
4
2
H 5.48, N 10.51. Found: C 65.41, H 5.40, N 10.58 %. n
max
ꢀ1
(KBr)/cm 2959, 2924, 2853 (C–H), 1593, 1514, 1493 (C¼C,
0
.4 g of a solid product (yield 40 %). d (400 MHz, CDCl ) 1.03
H
C–N), 1342, 1121 (–SO NH–), 1200 (C–N), 1072, 1017, 957,
2
3
(
2
t, J 7.1, 6H), 3.24 (q, J 7.1, 4H), 5.55 (m, 2H), 6.81 (dd, J 2.3,
.5, 1H), 6.91 (d, J 9.6, 1H), 7.25 (d, J 7.4, 2H), 7.53–7.57
895, 852, 803, 769, 695 (C–H). l (DMSO)/nm 586. m/z (ESI)
498.29 [M þ H] .
max
þ

DNA Photocleavage and Binding Modes of Methylene Violet 3RAX
C
[
17]
Synthesis of 3-(Benzene-1,2-dicarboxylic acid imide)-
Measurement of the Production Rate of Singlet Oxygen
7
-(diethylamino)-5-phenyl-phenazinium (4)
1
DPBF was used as a selective singlet oxygen ( O ) acceptor,
2
1
which is bleached upon reaction with O .
[18]
Phthalic anhydride (0.39 g, 2.65 mmol) and compound 1 (1 g,
.63 mmol) were dissolved in pyridine (20 mL).The solution
Five sample
2
2
solutions containing DPBF in DMSO (50 mM) were prepared in
the dark. Each sample container was covered with aluminium
foil with a yellow filter (with cut-off wavelength , 500 nm) on
one side. The samples were then exposed to light (50 W) through
the filter. After irradiation, the visible spectra of the sample was
measured. The normalised absorbance of DPBF at 418 nm in
was heated to 1208C for 12 h under nitrogen atmosphere. After
cooling to room temperature, the reaction mixture was poured
into a saturated sodium chloride solution (100 mL). The precip-
itate was purified on a silica gel column with chloroform/
methanol (100 : 1) as eluent. The first fraction was collected as
the desired product (0.8 g, yield 60 %). d (400 MHz, CDCl )
.10 (t, J 7.1, 6H), 3.35 (q, J 7.1, 4H), 5.65 (s, 1H), 6.40 (s, 1H),
.01 (d, J 8.9, 1H), 7.31 (d, J 7.8, 2H), 7.40 (d, J 10.6, 1H), 7.55–
.75 (m, 5H), 8.05 (m, 4H). d (100 MHz, CDCl ) 167.3, 150.4,
the presence of 1–5 was reported as a function of the photo-
H
3
1
irradiation time. From this plot, the production rates of O were
determined.
1
7
7
1
1
2
C
3
43.3, 133.6, 132.2, 130.1, 129.4, 128.4, 125.1, 123.0, 121.7,
20.5, 118.5, 113.8, 112.4, 47.3, 13.1. Anal. Calc. for
DNA Photocleavage Assay
The DNA photocleavage activities of compounds 1–5 were
measured using the plasmid DNA relaxation assay. Briefly,
plasmid DNA (0.5 mg), enriched with the covalently closed
circular or supercoiled conformer (Form I), and the one-phor-all
plus buffer (pH 7.5) was vortexed. Aliquots of the DNA were
pipetted into different Eppendorf tubes. Various amounts of
autoclaved water (control sample) or solutions of compounds
C H N ClO : C 70.79, H 4.95, N 11.01. Found: C 70.82,
3
H 4.90, N 11.08 %. nmax (KBr)/cm : 2935, 2856 (C–H), 1590,
0
25
4
2
ꢀ1
1
9
528, 1472 (C¼C, C–N), 1678 (–CONH–), 1197 (C–N), 1073,
55, 877, 856, 801, 751, 682 (C–H). l (DMSO)/nm 553, 588.
max
þ
m/z (ESI) 474.41 [M þ H] .
Synthesis of Methylene Violet 3RAX-INDO 5
1–5 (test sample) were added into the Eppendorf tubes to give a
A mixture of compound 1 (1 g, 2.63 mmol), indomethacin
final volume of 20 mL in each sample tube. The sample mixtures
were then photo-irradiated at 400–500 nm for 60 min using a
transilluminator (Vilber Lourmat) containing 4 ꢁ 15 W light
tubes (Aqua Lux) with maximum emission at 435 nm. After
photo-irradiation, 2 mL of the 6x sample dye solution was added
to each Eppendorf tube and mixed well by centrifugation. The
sample mixtures were loaded onto a 0.8 % (v/v) agarose gel
(
2
1.4 g, 3.95 mmol), dimethylaminopyridine (DMAP, 0.3 g,
.63 mmol), and 1-ethyl-3-(3-dimethylaminopropyl)carbodii-
mide (EDCI) (1 g, 5.26 mmol) in dry trichloromethane
10 mL) was heated to 608C for 12 h. After cooling to room
temperature, the reaction mixture was poured into a saturated
sodium chloride solution (10 mL) and extracted with chloroform
(
(
3 ꢁ 5 mL). The organic layers were combined and dried over
(
1
pH 8) used as supporting electrolyte, and electrophoresed at
ꢀ
1
.3 V cm for 3 h using a mini gel set. After electrophoresis, the
anhydrous sodium sulfate, and then concentrated under vacuum.
The residue was purified on a silica gel column with chloroform/
methanol (10 : 1) as eluent. The second fraction was collected as
ꢀ1
gel was stained with 0.5 mg mL ethidium bromide solution for
30 min and then washed with deionised water for 10 min. The
resulting gel image was viewed under 365 nm and captured
the desired product (1.5 g, yield 80 %). d (400 M, CDCl ) 1.15
H
3
[
19]
(t, J 7.1, 6H), 2.31 (s, 3H), 3.49 (q, J 7.1, 4H), 3.80 (s, 3H), 4.10
(s, 2H), 5.76 (s, 1H), 5.56 (d, J 8.1, 1H), 6.86 (d, J 8.1, 2H), 7.43
(m, 5H), 7.59 (d, J 8.6, 2H), 7.79 (m, 3H), 8.01–8.10 (m, 3H),
digitally using a gel documentation system (BioRad).
Results and Discussion
8
1
1
1
6
.58 (d, J 8.8, 2H). d (100 MHz, CDCl ) 167.2, 164.4, 154.4,
C 3
Synthesis of Compounds
49.6, 143.6, 130.4, 135.7, 134.6, 132.2, 131.9, 129.3, 128.6,
27.6, 125.4, 124.1, 121.3, 120.4, 118.1, 116.3, 113.1, 111.7,
04.6, 55.9, 47.3, 12.8, 9.8. Anal. Calc. for C H N Cl O : C
The synthesis of compounds 1–5 was carried out as shown in
0
Scheme 1. With 4 -amino-N,N-diethylaniline and aniline as
starting materials, methylene violet 3RAX 1 was synthesised in
35 % yield following the procedure described in the litera-
4
0
35
5
2 3
8.18, H 5.01, N 9.94. Found: C 68.09, H 5.08, N 9.88 % n
max
ꢀ
1
(KBr)/cm 2975 (C–H), 1662 (C=O), 1577, 1551, 1512, 1460
[
20]
(C=C, C–N), 1259 (Ph–O–CH ), 1199 (C–N), 1071, 1013, 925,
3
ture. The derivatives 2–5 were synthesised through a routine
coupling reaction in moderate yield. The amino group in com-
pound 1 was oxidised by an acid-catalysed reaction, affording 3-
hydroxy-7-(diethylamino)-5-phenyl-phenazinium (2) in 40 %
yield. In addition, the amino group in compound 1 was coupled
with 4-toluene sulfonyl chloride, phthalic anhydride, or indo-
methacin in dry solvent, affording compounds 3, 4, and 5,
respectively, with moderate yields. The structures of 1–5 were
confirmed by UV, NMR, and MS spectra.
8
6
71, 829, 755, 699 (C–H). l (DMSO)/nm 546, 578. m/z (ESI)
max
þ
83.92 [M þ H] .
Determining the Mode of Interaction Between
Compounds 1–5 and CT-DNA
The spectroscopic measurements were performed at room
temperature in buffer (pH 7.4, 0.05 M TRIS-HCl, 0.1 M NaCl).
1
An extinction coefficient of 6600 M cm at 260 nm was
ꢀ
ꢀ1
[16]
used to determine the concentration of CT-DNA.
UV-Vis
UV-Vis Absorption Spectroscopy
spectra were measured on a Shimadzu 1901 spectrometer.
Fluorescence spectra were measured on a Perkin Elmer LS-55
spectrometer. Circular dichroism was run on a JASCO J-810
spectrometer. The UV-vis absorption spectra of compounds
were recorded within a range of 450–740 nm. Fluorescence
spectra of compounds were recorded at 557, 551, 574, 562, and
The effect of the stoichiometric addition of CT-DNA on the
UV-vis absorption spectra of compounds 1 to 5 is shown in
Fig. 1. As shown in Table 1, with the increase of DNA con-
centration, moderate hypochromism for compounds 1–5
(H ¼ 23–49 %) was found, together with a significant red or
blue-shift for compound 1 and 5 (Dl ¼ 12, ꢀ14, respectively),
indicating that compound 1 and 5 may intercalate DNA at high
5
51 nm with excitation at 633, 621, 640, 674, and 675 nm,
respectively. Induced CD spectra were recorded within a range
of 500–700 nm.
[
15]
concentration.
Moreover, the bathochromic shift after

D
K. Yang et al.
CDNA [ϫ10Ϫ6 mol LϪ1
]
0
.14
.12
CDNA [ϫ10Ϫ6 mol LϪ1
]
0.16
0.14
0.12
0.10
0.08
0.06
0.04
0.02
0
0
Compound 1
Compound 2
9
2
5
7
9
1
1
1
1
1
1
.23
7.70
5.40
3.87
0
0
5
9
1
1
7.40
5.67
14.80
33.93
0.10
2.33
10.80
29.27
47.73
56.97
66.20
75.43
0.08
0.06
0.04
0.02
0
153.07
172.20
181.77
191.33
200.90
460 480 500 520 540 560 580 600 620 640 660 680 700
480
500
520
540
560
580
600
CDNA [ϫ10Ϫ6 mol LϪ1
]
CDNA [ϫ10Ϫ6 mol LϪ1
]
0.16
0.14
0.12
0.10
0.08
0.06
0.04
0.02
0
0.18
Compound 4
0
0
6.87
0.16
0.14
0.12
0.10
0.08
0.06
0.04
0.02
0
19.03
Compound 3
4
7.58
76.13
04.68
123.72
42.75
20.60
41.20
1
5
6
8
9
1
1
1
1
4.93
8.67
2.40
6.14
09.87
16.73
23.60
30.47
1
152.27
171.30
180.82
190.33
137.33
460 480 500 520 540 560 580 600 620 640 660 680 700
480 500 520 540 560 580 600 620 640 660 680 700 720 740
CDNA [ϫ10Ϫ6 mol LϪ1
]
0
0.09
0.08
0.07
0.06
0.05
0.04
0.03
5
3
5
7
8
8
.27
1.60
2.67
3.73
4.27
9.53
Compound 5
94.80
100.07
105.33
110.60
500
520
540
560
580
600
Wavelength [nm]
ꢀ1
ꢀ1
Fig. 1. UV-Vis absorption spectra of compounds 1–5 with the titration of CT-DNA, [compound 1–5] ¼ 4 mmol L , buffer solution (pH 7.4, 0.05 mol L
ꢀ1
TRIS-HCl þ 0.1 mol L NaCl).
interaction with DNA suggests compound 1 adopts an end-to-
end interaction with DNA (J aggregate). On the contrary,
compound 5, with hypsochromic shift, tends to exhibit a parallel
stacking arrangement (H aggregate) probably due to the high
hypochromicity (. 20 %) without a significant spectroscopic
shift. These weak spectroscopic variations suggest that
compounds 2–4 may interact with DNA through a groove
binding mode.
[
21]
steric hindrance, as reported previously. Generally speaking,
for a given DNA, intercalation will result in a large change of its
absorption spectrum, while small changes occur upon the
The binding affinities between compounds 1–5 and DNA
were expressed by the binding constant (K), which was calcu-
[22,23]
lated using Eqn 1:
[
18]
groove binding. At a high concentration of CT-DNA, the
absorption bands of compounds 2–4 exhibited substantial
D=jeA ꢀ eFj ¼ D=jeB ꢀ eFj þ 1=ðjeB ꢀ eFjKÞ
ð1Þ

DNA Photocleavage and Binding Modes of Methylene Violet 3RAX
E
Table 1. Spectroscopic characterisation of compounds 1–5 in the presence of CT-DNA
Compd
UV-vis hypochromicity (H)/
Fluorescence emission hypochromicity (H)/
wavelength shift (Dl)
[%]/[nm]
CD r ¼ 0.01
bathochromic shift (Dl)
Positive band
Negative band
[
%]/[nm]
[
nm]
[nm]
1
2
3
4
5
30.1/12
22.8/2
25.9/ꢀ14
ꢀ14.3/ꢀ5
ꢀ26.5/ꢀ2
13.9/ꢀ8
—
598
584
573
606
591
604
608
597
—
49.3/1
25.2/1
22.2/ꢀ14
58.3/ꢀ12
2
1
1
2
2
0
1
0
Ϫ1
Ϫ2
Ϫ3
Ϫ1
500
550
600
650
700
500
550
600
650
700
3
2
3
4
2
1
1
0
0
Ϫ1
Ϫ2
Ϫ1
500
550
600
650
700
500
550
600
650
700
2
1
5
0
Ϫ1
Ϫ2
500
550
600
650
700
Wavelength [nm]
Fig. 2. Induced CD spectra of compounds 1–5.
D is the concentration of DNA during the process of titration. eA
is the ratio of Amax/[1–5] during the process of titration. e is the
Fluorescence Spectroscopy
B
A fluorescence titration was performed to further probe the
binding mode between compounds 1–5 and CT-DNA. With the
titration of CT-DNA, an increase in intensity of fluorescence
emission of compounds 1, 4, and 5 is observed (Fig. S1,
Supplementary Material). This might be ascribed to the
ratio of Amax/[1–5] after saturating with DNA, and e is the ratio
F
of Amax/[1–5] without DNA. The values of K obtained for
3
3
4
compounds 1–5 are 7.48 ꢁ 10 , 2.72 ꢁ 10 , 3.88 ꢁ 10 ,
3
ꢀ1
2
.82 ꢁ 10 , and 1.56 ꢁ 104 mol L , respectively.

F
K. Yang et al.
conjugated plane structures of compounds 1 and 5, which could
insert between the base pairs of the DNA double-helix and are
protected by the strong hydrophobic core of the DNA so will
Circular Dichroism Spectroscopy
To further clarify the binding mode between the compounds and
DNA, the induced CD spectra of compounds 1–5 were recorded
in the presence of CT-DNA (Fig. 2). In the case of duplex DNA,
[24]
avoid the quenching effect of water molecules.
As for com-
pound 4, it could insert into the grooves of the DNA double-helix.
The hydrogen bonding of base pairs and the layer stacking of the
bases providesa completely hydrophobic environment. However,
compound 2 and 3 exhibited a decrease in fluorescence emission
intensity upon titration with DNA, due to the insertion into the
structure of DNA, which will change the electronic excited
state. At the same time, there were electrostatic interactions
between the positive charges of compounds 1–5 and the negative
charges of the phosphate groups of DNA. The electrostatic
[25]
interaction will strengthen the heterozygosity.
a positive induced CD band indicates groove binding, and a
[23]
negative induced CD band indicates intercalation.
In solu-
tions of DNA and compounds 1–5 (100 : 1, v/v), compounds 1
and 5 show a strong negative peak (centred at 598 and 591 nm,
respectively), which further indicates they interact with DNA
through intercalation. For compounds 2, 3, and 4, a strong
positive peak (centred at 604, 608, and 597 nm, respectively)
and a weak negative peak (centred at 584, 573, and 606 nm,
respectively) was obtained, suggesting they adopt a combina-
tion of groove binding and intercalation with DNA. The results
suggest that the modification of the amino group could change
the binding properties of these compounds to DNA.
[22]
1.00
0.95
0.90
0.85
0.80
0.75
0.70
0.65
1
Photogeneration of O
2
1
DPBF was able to capture the O photogenerated by com-
2
[
26]
pounds 1–5, which reduces its own light activity.
value acquired from DPBF v. illumination time could indirectly
1
reflect the O yield of compounds 1–5. As shown in Fig. 3, the
The A/A0
2
absorbance of DPBF at 418 nm decreased gradually with the
increase of illumination time in the presence of compounds 1–5.
DPBF control
DPBFϩ5
DPBFϩ4
DPBFϩ3
DPBFϩ1
DPBFϩ2
1
From the slope of the line, the relative production rates of O for
2
1
compounds 1–5 could be obtained. The order of O yield was
2
1
2 . 1 . 3 . 4 . 5. The yield of O by compound 2 was more
2
than the other compounds owing to the hydroxy group on the
methylene violet 3RAX, which could increase the electronic
effect and intermolecular hydrogen bond. In addition, com-
1
0
10
20
30
40
50
pound 1 generated O much faster than compounds 3, 4, and 5,
2
lllumination time [s]
as the acyl group could reduce the electron density of the
1
compounds. Moreover, different yields of O photogeneration
2
Fig. 3. Relation between irradiation time and DPBF at 418 nm ultraviolet
absorption ratio (A/A ).
indicated that modification of the amino group could affect the
ability of the compounds to generate singlet oxygen.
0
(a)
(b)
Conc [µM]
Conc [µM]
0
100
200
300
400
0
100
200
300
400
Form II
Form I
0
100
0
100
11
89
16
84
20 %
80 %
Form II
Form I
0
100
0
100
22
78
32
68
38 %
62 %
(
c)
(d)
Conc [µM]
Conc [µM]
0
100
200
300
400
0
100
200
300
400
Form II
Form I
0
100
0
100
0
100
8
92
30 %
70 %
Form II
Form I
0
100
0
100
0
100
0
100
13 %
87 %
(
e) Conc [µM]
0
100
200
300
400
Form II
Form I
0
100
20
80
26
74
38
62
40 %
60 %
Fig. 4. Agarose gel electrophoresis images of DNA photocleavage assay of (a) 1, (b) 2, (c) 3, (d) 4, and (e) 5 at different concentrations.

DNA Photocleavage and Binding Modes of Methylene Violet 3RAX
G
DNA Photocleavage
[4] P. Juzenas, J. Moan, J. Environ. Pathol. Toxicol. Oncol. 2006, 25, 29.
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The DNA photocleavage ability of compounds 1–5 were
detected by monitoring the conversion of supercoiled form
(
Fig. 4, only compounds 1, 2, and 5 showed photocleavage
activities towards DNA when their concentration was 200 mM.
When the concentration of the compounds was elevated to
[
A. Monge, H. Cerecetto, M. Gonzalez, Eur. J. Med. Chem. 2010, 45,
[
form I) into the nicked circular form (form II). As shown in
27]
5
362. doi:10.1016/J.EJMECH.2010.08.061
[
6] M. L. Lavaggi, G. Aguirre, L. Boiani, L. Orelli, B. Garci, H. Cerecetto,
M. Gonzalez, Eur. J. Med. Chem. 2008, 43, 1737. doi:10.1016/J.
EJMECH.2007.10.031
4
00 mM, the DNA photocleavage activities of compounds 1–5
[7] H. Cerecetto, M. Gonzalez, M. L. Lavaggi, A. Azqueta, A. L. Cerain,
were calculated to be 38, 40, 30, 20, and 13 %, respectively, with
the order 2 . 1 . 3 . 4 . 5, which was consistent with their
ability to generate singlet oxygen. The DNA photocleavage
activities of the five compounds were all less than 40 % due to
their molecular structures and binding affinities with DNA.
A. Monge, J. Med. Chem. 2005, 48, 21. doi:10.1021/JM0492150
[
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Conclusions
[
[
[
2
We prepared methylene violet 3RAX 1 and four methylene
violet 3RAX derivatives 2–5. The five compounds were char-
1
acterised by IR, UV-Vis, H NMR, and MS analysis. The
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K. Kaminski, J. Phys. Chem. B 2009, 113, 12536. doi:10.1021/
JP905162R
binding mode between the synthesised compounds and DNA
and their DNA photocleavage ability were investigated. The
results show that both 1 and 5 bind to DNA by intercalation,
while compounds 2–4 interacted with DNA through a mixed
binding mode involving groove binding and electrostatic
interactions. The DNA photocleavage ability of compounds 1–5
were calculated to be 38, 40, 30, 20, and 13 %, respectively. The
singlet oxygen production of compounds 1–5 measured by the
DPBF method was consistent with the trend of DNA photo-
cleavage ability, suggesting they have potential as PDT agents.
These results suggest that the binding mode between methylene
violet 3RAX and DNA, and its ability to generate singlet oxygen
and DNA photocleavage activity could be adjusted through
modification of the amino group on methylene violet 3RAX.
[
[
[
[
[
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1
6, 3488. doi:10.3390/MOLECULES16053488
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Yu, H. Zhang, Z. Y. Li, J. Porphyr. Phthalocyanines 2012, 16, 85.
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Supplementary Material
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H. Zhang, Z. Y. Li, Eur. J. Inorg. Chem. 2009, 922. doi:10.1002/EJIC.
Fluorescence spectra of compounds 1–5 with the titration of CT
DNA are available on the Journal’s website.
2
00801047
[
[
20] Wang K., Song S. H., Chen Y., Yu F., Chi R., Huang T., Synthesizing
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Acknowledgements
The authors are grateful for the financial support of the Wuhan Science and
Technology Talent Training Program of Chenguang Project (grant no.
[22] K. Wang, X. Lin, X. Wan, T. Jia, X. L. Zhang, H. Zhang, X. L. Ju,
2015070404010190), Key Project of Scientific Research Project of Hubei
Chem. J. Chin. Univ. 2012, 33, 2663 (in Chinese).
Provincial Department of Education (grant no. D20141506), National
Natural Science Foundation of China (grant no. 21601142), and Scientific
Research Project of Hubei Provincial Department of Education (grant no.
Q20161507).
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