Metal complexes of modified cyclen as catalysts for hydrolytic restriction of plasmid DNA

Article abstract of DOI:10.1016/j.jinorgbio.2010.04.007

Simple and novel nuclease models have been synthesized. These involve metal-binding ligand 1,4,7,10-tetraazlcyclododecane (cyclen) tethered to an acridine ring (a DNA-binding group) by amide linkers of various lengths. Binding of these probes to DNA was s

Full text of DOI:10.1016/j.jinorgbio.2010.04.007

Journal of Inorganic Biochemistry 104 (2010) 877–884
Contents lists available at ScienceDirect
Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
Metal complexes of modified cyclen as catalysts for hydrolytic restriction of plasmid DNA
⁎
Joel A. Krauser, Aarti L. Joshi, Ismail O. Kady
Department of Chemistry, East Tennessee State University, Box 70,695, Johnson City, TN 37614, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Simple and novel nuclease models have been synthesized. These involve metal-binding ligand 1,4,7,10-
tetraazlcyclododecane (cyclen) tethered to an acridine ring (a DNA-binding group) by amide linkers of
various lengths. Binding of these probes to DNA was studied by monitoring changes in their UV–visible
spectra affected by the presence of DNA. Titration of these compounds with increasing amounts of pBR322
DNA caused hypochromic effects and shifted the acridine absorption at 360 nm to a longer wavelength.
Under biologically relevant conditions (37 °C and pH 7.4), specific transition metal complexes of these
compounds are found to be highly effective catalysts toward the hydrolysis of plasmid DNA. This is
demonstrated by their ability to convert the super-coiled DNA (form I) to open-circular DNA (form II).
Structure-activity correlation studies show that hydrolytic activity depends on both the structure of ligand
(L1 NL2 NL3) and the nature of metal ion cofactor (Co3+ NZn2+ NCr2+ NNi2+ NCu2+ NFe3+).
Received 3 December 2009
Received in revised form 15 April 2010
Accepted 16 April 2010
Available online 24 April 2010
Keywords:
DNA
Acridine
Nuclease models
Metal complexes
Cyclen
© 2010 Elsevier Inc. All rights reserved.
Intercalation
1. Introduction
drugs is their ability to induce DNA strand breaks which result in
disruption of the topoisomerase enzyme function [14]. DNA topoi-
Molecular recognition and sequence-specific cleavage of DNA by
synthetic probes have been the subject of intensive studies for a
number of years [1–5]. Such restriction enzyme analogs, if proven
sufficiently efficient and specific, could have numerous applications,
such as DNA structure and sequence determination, recombinant DNA
manipulation, gene isolation and analysis, and development of
therapeutic agents [6–8]. Such applications have been confined to a
limited number of natural restriction endonucleases. Under physio-
logical conditions, phosphodiester bonds of DNA are found to be
remarkably stable and resistant to hydrolysis. Deoxyribonucleases, in
the presence of metal ion cofactors, are known to accelerate the
somerase enzymes, such as mammalian topo I and topo II, can untwist
the densely packed DNA by generating temporary breaks within the
DNA strands which result in topological changes in the tertiary
structure of the macromolecule. It is believed that molecules that can
stimulate permanent strand breaks in DNA would be of significantly
important in terms of developing new chemotherapeutic agents and
in elucidating the specific mechanisms involved in DNA-cleaving
processes [15]. However, mere intercalation of such drugs with DNA is
not sufficient for anti-neoplastic activity. Evaluation of a series of
substituted acridine derivatives showed that DNA intercalation,
coupled with the action of appropriately placed active side-groups,
is needed for antitumor and anti-neoplastic activities of such agents
[16,17]. Therefore, the central premise, underlying the design of
chemical agents that can mediate chemical reactions on nucleic acids
(and proteins), is that the juxtaposition of a target recognizing
domain with a catalytic moiety will result in molecules with
properties that are superior to the sum of the individual component
parts; this strategy provides a novel platform for drug design and
development.
Inspired by our previous work [18–21] and that of others [22–27]
which showed that certain metal complexes exhibit remarkable activity
toward hydrolysis of the phosphodiester bonds, and in an effort to
design relatively small synthetic molecules that can mimic deoxyribo-
nucleases, we synthesized a series of compounds L1–L3. These molecules
involve a DNA-binding group (acridine) tethered to a metal-binding
1,4,7,10-tetraazlcyclododecane cyclen ligand (Scheme 1). This work
demonstrates the effectiveness of some metal complexes of these
hydrolysis of DNA phosphodiester bonds by a factor of up to 1016
.
However, new tools for site-selective DNA hydrolysis are still needed
for further advancement in biotechnology and molecular biology,
such as manipulation of large DNA molecules of higher plants and
animals that cannot be manipulated by the natuarally occuring re-
striction endonucleases [9–12].
Many DNA-intercalating agents can bind reversibly to DNA by
intercalation of their planar, mostly aromatic, chromophores between
the nitrogen base pairs. One effect of clinically used DNA-intercalating
agents, such as the antitumor drug Amsacrine AMSA, is their ability to
selectively inhibit DNA synthesis, which is attributed to inhibition of
DNA polymerase I [13]. Yet another major effect of DNA-intercalating
⁎
Corresponding author. Tel.: +1 423 439 6910; fax: +1 423 439 5835.
E-mail address: kadyi@etsu.edu (I.O. Kady).
0162-0134/$ – see front matter © 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.jinorgbio.2010.04.007

878
J.A. Krauser et al. / Journal of Inorganic Biochemistry 104 (2010) 877–884
Scheme 1. Compounds synthesized and tested in this study.
probes in catalyzing the hydrolysis of DNA under biologically relevant
conditions.
(DMSO-d6): δ 8.26 (d, 2H, J=8.2 Hz), 8.15 (d, 2H, J=8.3 Hz), 7.89
(t, 2H), 7.77 (t, 2H), 3.83 (t, 2H), 3.42 (t, 2H). 13C NMR (DMSO-d6):
δ 173.45 (C O), 142.52, 133.48, 129.23, 127.42, 126.49, 125.28, 120.19,
63.27, 27.64. MS (m/z, %): 266 (M+, 9), 248 (78), 238 (27), 236 (32),
220 (31), 206 (36), 178 (100), 88 (15). Anal. calc. for C16H14N2O: C,
72.05; H, 5.31; N, 10.52. Found: C, 72.14; H, 5.21; N, 10.41.
2. Experimental
2.1. General information
All chemical reagents of puriss quality were purchase either from
Aldrich or Sigma company and were used without further purification.
Solvents were purchased from Fisher Scientific Company and used
untreated unless mentioned otherwise. Plasmid DNA pBR322 was
obtained from Sigma company, and concentrations of all DNA
solutions (base pairs per liter) were determined spectrophotometri-
cally. Ultra pure sterilized water was used in all experiment. The free
base ligands were synthesized as described below and purified by
crystallization of their HBr salts. Their purity was verified by 1H NMR
and mass spectroscopy to be N99%. UV–visible spectra were recorded
on Shimadzu UV-1700 spectrophotometer equipped with a temper-
ature control unit. NMR spectra were obtained on 400 MHz JEOL JNM-
ECS spectrometer, sodium 3-(trimethylsilyl)propionate-2,2,3,3-d4 in
D2O and tetramethylsilane (TMS) in CDCl3 were used as internal
references for 1H and 13C NMR measurements. The abbreviations used
to describe the peaks splitting patterns in the reported 1H NMR spectra
are as follows: d = doublet, t = triplet, m = multiplet, and brd =
broad. Thin-layer chromatography (TLC) was performed using silica
gel plates with fluorescent indicator UV-254 from Aldrich; column
chromatography were performed using silica gel (60–200) from TSI
Chemicals. Electrophoresis was performed using 1% agarose gels
submersed in TAE buffer (20 mM TRIS base, 20 mM acetic acid, 1 mM
EDTA) containing 1% SDS.
2.2.2. (3-hydroxyopropyl)-acridine-9-carboxamide (2)
A procedure similar to that of (2-hydroxyoethyl)-acridine-9-
carboxamide (1) was used, except using 3-aminopropanol in place
of 2-ethanolamine. Yield (54%); mp: 141 °C (dec). IR (KBr): 3220,
3028, 2949, 2931, 2863, 2826, 1725, 1603, 1518, 1442, 1435, 1300,
1038 cm−1 1H NMR (DMSO-d6): δ 8.31 (d, 2H, J=7.9 Hz), 8.17
.
(d, 2H, J=8.1 Hz), 7.85 (t, 2H), 7.73 (t, 2H), 3.92 (t, 2H), 3.38 (t, 2H),
2.05 (m, 2H). 13C NMR (DMSO-d6): δ 174.03 (C O), 141.25, 132.52,
128.95, 126.98, 125.23, 123.18, 120.11, 61.25, 50.95, 27.58. MS, m/z
(%): 281 (M+ +1, 12), 262 (80), 250 (23), 234 (17), 206 (35), 178
(100), 59 (8). Anal. calc. for C17H16N2O2: C, 72.83; H, 5.75; N, 9.99.
Found: C, 72.75; H, 5.64; N, 9.89.
2.2.3. Acridine-tethered cyclen ligands (L1)
(2-Hydroxyoethyl)-acridine-9-carboxamide 1 (0.266 g, 1 mmol)
was dissolved in anhydrous tetrahydrofuran (THF, 10 mL) containing
anhydrous pyridine (0.316 g, 4 mmol). This solution was added grad-
ually to a solution of p-toluenesulfonyl chloride (Ts–SO2Cl, 0.285 g,
1.5 mmol) in THF (10 mL), cooled in ice bath under nitrogen atmo-
sphere. The solution was stirred at room temperature for 4 h, and the
white precipitate was removed by filtration. The filtrate was added
gradually to a solution of 1,4,7,10-tetraazlcyclododecane (cyclen
0.344 g, 2 mmol) in 10 mL of anhydrous THF. The reaction mixture
was stirred at room temperature for 8 h under nitrogen. The solvent
was removed under reduced pressure and the resulting oil was taken
up in 5 mL of 5% HCl, and the solution was washed three times with
methylene chloride (3×10 mL). The aqueous layer was treated with
2 mL of 20% NaOH and the product was extracted from the alkaline
solution with methylene chloride (3×10 mL). The combined organic
layers were dried over anhydrous Na2SO4 and solvent was evaporated
under diminished pressure. The resulting oil was dissolved in meth-
anol (3 mL) and treated with HBr solution (48%, 0.5 mL) to precipitate
the hydrobromide salt. This prodedure was repeated once more to
obtain pure L1·4HBr·4H2O, yield (0.35 g, 43%), m.p (102 °C, dec.). IR
(KBr): 3410, 2915, 2750, 2466, 2374, 1735, 1618, 1575, 1457, 1433,
2.2. Synthesis
2.2.1. (2-hydroxyoethyl)-acridine-9-carboxamide (1)
9-acridinecarbonyl chloride was prepared from 9-acridinecar-
boxylic acid using modified literature procedures [28]. Specifically,
9-acridinecarboxylic acid hydrate (0.48 g, 2 mmol) was refluxed in
freshly distilled thionyl chloride (5 mL) for 2 h under anhydrous
nitrogen atmosphere. The excess thionyl chloride was distilled under
reduced pressure and the remaining yellow solid product was further
dried under vacuum (0.1 mmHg) overnight and used without further
purification. The product (0.483 g, 2 mmol) was dissolved in anhy-
drous acetonitrile (10 mL) then added gradually, over a period of 2 h,
to a solution of 2-ethanolamine (0.3 g, 5 mmol) in acetonitrile (20 mL)
containing 0.5 mL of triethylamine. The reaction mixture was stirred at
room temperature (under nitrogen) for 12 h. The white precipitate
was separated by filtration and solvent was evaporated under reduced
pressure, the excess triethylamine and 2-ethanolamine were evapo-
rated in vacuum (0.1 mmHg) to leave a yellow solid. The crude product
was purified by silica gel column chromatography (methylene
chloride containing 10% methanol and 0.5% conc. NH4OH). Yield
(0.23, 48%); mp: 173 °C (dec). IR (KBr): 3210, 3035, 2953, 2941, 2857,
2835, 1732, 1605, 1523, 1458, 1445, 1305, 1163, 1012 cm−1. 1H NMR
1401, 1359 cm−1 1H NMR (D2O, pD 2): δ 8.73 (2H, brd), 8.28 (2H,
.
brd), 8.18 (2H), 8.12(2H), 3.96 (2H, t, –CONH–CH2), 2.80–3.52 (18H,
brd multiplet, –CH2N). 13C NMR (D2O, pD 2): δ 176.24 (C O), 149.57,
141.63, 133.89, 130.23, 126.27, 122.13, 119.91, 59.15, 53.45, 51.25,
49.34, 46.96, 26.89. Anal. calc. for C24H44N6O5Br4: C, 35.31; H, 5.44; N,
10.30. Found: C, 35.29; H, 5.32; N, 10.38.
2.2.4. Acridine-tethered cyclen ligands (L2)
The ligand L2·4HBr·4H2O was prepared from (3-hydroxyopropyl)-
acridine-9-carboxamide (2) following a procedure similar to the one
used to synthesize L1.4HBr.4H2O. Yield (3.98 g, 48%); m.p 95 °C, dec.).

J.A. Krauser et al. / Journal of Inorganic Biochemistry 104 (2010) 877–884
879
IR (KBr): 3408, 2935, 2745, 2474, 2354, 1739, 1628, 1567, 1437, 1428,
1405, 1336 cm−1 1H NMR (D2O, pD 2): δ 8.77 (2H, brd), 8.35 (2H,
3271, 2928, 2758, 2432, 2360, 1702, 1612, 1571, 1448, 1428, 1418,
1402, 1329, 1242, 1136, 1125, 958 cm−1 1H NMR (CDCl3 +10%
.
.
brd), 8.28 (2H), 8.10 (2H), 4.26 (2H, t,–CONH–CH2), 2.80–3.52 (18H,
brd multiplet,–CH2N), 1.81 (2H). 13C NMR (D2O, pD 2): δ 174.34 (C O),
150.12, 142.33, 134.02, 129.95, 125.17, 121.21, 118.95, 61.31, 54.35,
50.98, 48.31, 44.86, 39.96, 26.95. Anal. calc. for C25H46N6O5Br4: C,
36.16; H, 5.60; N, 10.12. Found: C, 36.25; H, 5.49; N, 10.18.
CD3OD): δ 8.36 (2H, brd, J=8.1), 8.20 (2H, J=9.0), 8.06 (2H), 7.86
(2H), 2.80–3.48 (16H, brd multiplet, cyclen CH2N). 13C NMR (CDCl3 +
10% CD3OD): δ 172.52 (C O, amide), 148.25, 140.19, 132.84, 129.10,
125.98, 121.12, 118.62, 56.72, 52.10, 51.66, 49.24. Anal. calc. for
C22H29Cl2N5O2Zn: C, 49.68; H, 5.51; N, 13.17. Found: C, 49.59; H, 5.49;
N, 13.03.
2.2.5. Acridine-tethered cyclen ligands (L3)
Asolution of 9-acridinecarbonyl chloride (0.241 g, 1 mmol) in
anhydrous THF (5 mL) was added gradually, over a period of 1 h, to a
solution of 1,4,7,10-tetraazlcyclododecane (cyclen, 0.344 g, 2 mmol) in
5 mL of anhydrous THF containing pyridine (0.158 g, 2 mmol). The
solution was stirred at room temperature for 4 h and the white precipitate
was filtered. The solvent was removed under reduced pressure, the
residual oil was dissolved in 5 mL of 5% HCl, and the solution was washed
three times with methylene chloride (3×10 mL). The aqueous layer was
treated with 2 mL of 20% NaOH and the product was extracted from the
alkaline solution with methylene chloride (3×10 mL). The combined
organic layers were dried over anhydrous Na2SO4 and solvent was
evaporated under diminished pressure. The resulting oil was dissolved in
methanol (3 mL) and treated with HBr solution (48%, 1 mL) to precipitate
the hydrobromide salt of the product. This prodedure was repeated once
more to obtain pure L3·3HBr·3H2O. Yield (0.487 g, 63%); m.p (125 °C,
dec.). IR (KBr): 3428, 2935, 2735, 2432, 1725, 1609, 1573, 1455, 1429,
1413 cm−1. 1H NMR (D2O, pD 2): δ 8.71 (2H), 8.19 (2H), 8.08 (2H), 7.95
(2H), 2.80–3.52 (16H, brd multiplet, –CH2N). 13C NMR (D2O, pD 2): δ
175.18 (C O), 148.25, 142.58, 133.19, 129.29, 125.97, 123.12, 118.89,
56.19, 52.25, 50.06, 48.394. Anal. calc. for C22H36N5O4Br3: C, 39.18; H, 5.39;
N, 10.38. Found: C, 39.15; H, 5.37; N, 10.40.
2.2.6. Acridine-tethered cyclen ZnII complex [ZnIIL1]Cl2.H2O
A solution of L1·4HBr·4H2O (0.408 g, 0.5 mmol) was dissolved in
5 mL of 10% NaOH. The alkaline solution was extracted with methylene
chloride (3×10 mL), the combined organic layers were washed with
distilled water (5 mL), dried over anhydrous Na2CO3, and evaporated
under reduced pressure. The oil residue was dissolved in 5 mL methanol
and was added to a solution of zinc(II) chloride (0.273 g, 2 mmol) in
10 mLof hot methanol. Thesolution wasrefluxed for 2 h under nitrogen,
the yellow solid were collected by filtration and recrystallized from
aqueous methanol. Yield of [ZnIIL1]Cl2·H2O (0.235 g, 82%). IR (KBr):
3418, 3285, 2925, 2795, 2486, 2335, 1739, 1609, 1579, 1477, 1453, 1425,
1402, 1369, 1278, 1163, 1109, 998, 923 cm−1 1H NMR (CDCl3+10%
.
CD3OD): δ 8.53 (2H, brd, J=7.9), 8.27 (2H, J=8.9), 8.03 (2H), 7.85(2H),
4.12 (2H, t, –CONH–CH2), 3.65 (2H, t, CH2–N), 2.98–3.48 (16H, brd
multiplet, cyclen CH2N). 13C NMR (CDCl3 + 10% CD3OD): δ 172.59 (C O,
amide), 170.9, 148.55, 142.65, 132.38, 129.51, 125.17, 121.19, 119.35,
55.19, 52.36, 50.26, 48.94, 45.89, 30.57. Anal. calc. for C24H34Cl2N6O2Zn:
C, 50.14; H, 5.97; N, 14.62. Found: C, 50.02; H, 5.95; N, 14.54.
2.2.7. Acridine-tethered cyclen ZnII complex [ZnIIL2]Cl2.H2O
This compound was prepared following a procedure similar to that
used in the synthesis of [ZnIIL1]Cl2.H2O, yield (0.247 g, 84%). IR (KBr):
3435, 3275, 2930, 2767, 2448, 2374, 1719, 1601, 1538, 1484, 1444, 1421,
1411, 1336, 1259, 1146, 1120, 987, 942 cm−1 1H NMR (CDCl3+10%
.
CD3OD): δ 8.48 (2H, brd, J=8.1), 8.23 (2H, J=9.0), 8.02 (2H), 7.77(2H),
4.32 (2H, t, –CONH–CH2), 3.64 (2H, t, CH2–N), 2.90–3.52 (16H, brd
multiplet, cyclen CH2N), 1.63 (2H, m). 13C NMR (CDCl3+10% CD3OD): δ
173.57 (C O, amide), 147.98, 141.59, 131.88, 128.91, 126.15, 120.18,
118.95, 56.12, 51.33, 49.86, 47.17, 44.88, 37.93, 29.97. Anal. calc. for
C25H36Cl2N6O2Zn: C, 50.98; H, 6.17; N, 14.27. Found: C, 50.86; H, 6.16;
N, 14.18.
Fig. 1. pH-Potentiometric titration curves of the ligands (L1–L3) at 25 °C, ionic strength
2.2.8. Acridine-tethered cyclen ZnII complex [ZnIIL3]Cl2.H2O
I=0.10 M NaNO3: (a) i. 1.0 mM L1.5H+
;
ii. 1.0 mM L1.5H+ +1.0 mM Zn2+
;
(b) i.
1.0 mM L2·5H+; ii. 1.0 mM L2 5H+ +1.0 mM Zn2+; (c) i. 1.0 mM L3 4H+; ii. 1.0 mM
L3 4H+ +1.0 mM Zn2+
This compound was prepared following a procedure similar to that
used to synthesize [ZnIIL1]Cl2·H2O, yield (0.200 g, 75%). IR (KBr): 3465,
.

880
J.A. Krauser et al. / Journal of Inorganic Biochemistry 104 (2010) 877–884
Table 1
2.3. pH measurments and potentiometric titration
Log of protonation constant (K=[LHn]/[LHn − 1][H+]), log stability constant of ZnII
complex (KZnL =[ZnIIL]/[ZnII][L]), and pKa of ZnII-bound water, at 25 °C and I=0.1 M
NaNO3.
2.3.1. Calibration of pH meter
All pH measurements were carried out using a Cole Parmer (model
5996-50) pre-calibrated electrode and pH meter. The pH meter was
regularly calibrated in the 2–12 pH range before measurements. The
calibration was done at 25 0.5 °C, using 100 ml of 4.00 mM HCl
solution (I=0.10 M NaNO3) to obtain the lower pH limit (pH1); then
8 mL of 0.10 M carbonate-free NaOH solution was added and the
upper pH limit (pH2) was measured. The corresponding theoretical
values of pH1 and pH2 were calculated to be 2.48 and 11.45,
respectively [29]. This method provided an experimental confidence
limit of 0.01 in the 2–12 pH range.
Equilibrium Reaction
logK (cyclen)a logK (L1) logK (L2) logK (L3)
L+H+ ⇌LH+
10.7
9.7
b2
b2
–
9.76
7.57
4.54
9.68
7.38
4.62
11.32
10.83
6.42
–
LH+ +H+ ⇌LH22+
LH22+ +H+ ⇌LH33+
LH33+ +H+ ⇌LH44+
LH44+ +H+ ⇌LH55+
L+ZnII ⇌ZnIIL(H2O)
b2b
b2c
11.42
7.39
b2b
b2c
11.36
7.46
–
16.2
8.84
7.25
ZnIIL(H2O)⇌ZnIIL(HO−)+H+ pKa =7.88
a
Based on reference [31].
Protonation constant of the tertiary amine nitrogen.
Protonation constant of the acridine nitrogen.
b
c
2.3.2. pH-potentiometric titrations
2.4. DNA experiments
The equilibrium constants for protonation of the free ligands and
deprotonation constants of the metal-bound water were determined
by potentiometric pH-titration. HYPERQUAD-2008 program was used
to calculate the protonation constants of the free ligands and the
stability constants of the metal complexes based the e.m.f. data [30–
32]. Titrations were carried out using carbonate-free NaOH solution,
at 25 °C and at a constant ionic strength (I=0.10 M NaNO3), in pH
range 2–10. The average of three independent titrations was
considered in the calculations. The value of Kw =[H+][OH−] used
under these conditions was 10−13.4. Fig. 1 shows pH-titration curves
for the protonated ligands (L·5H+) in the absence and in the presence
2.4.1. DNA-binding studies
Stock solutions (100 mM) of the ligands (free base) in tris–HCl
buffer (20 mM, pH 7.4, I=0.10 M NaCl), containing 10% by volume
dimethylsulfoxide DMSO were prepared. Spectroscopic titrations
were performed by adding DNA to the ligands' stock solutions; the
resulting solutions were then diluted with buffer to get the desired
concentrations of both the ligand and DNA. Concentration of ligands
in the final solutions were 50 μM and the molar ratio between ligand
and DNA (base pairs) ranged from 1:0 to 1:1. All UV–visible spec-
troscopic measurements were performed at 25 °C using 1 cm quartz
cuvettes.
of equimolar amount of Zn2+
.
Scheme 2. Synthetic scheme for preparation of ligands L1, L2, and L3.

J.A. Krauser et al. / Journal of Inorganic Biochemistry 104 (2010) 877–884
881
2.4.2. DNA-cleaving studies
Only zinc complexes were synthesized and characterized, since
these complexes (and that of cobalt) showed the highest DNA
hydrolytic activities, and because no signifcant difference in activity
was observed between pre-synthesized metal complexes and metal
complexes that were synthesized in situ. Thus we did not attempt to
isolate and characterize complexes of other metals, such complexes
were prepared in situ prior to reaction with DNA. The zinc complexes
of L1–L3 were synthesized by refluxing a methanolic solution con-
taining the free-base ligands and excess amount of zinc chloride.
All solvents and buffer solutions used in DNA-cleaving experi-
ments were sterilzed and free of oxygen; argon gas was bubbled
through the solutions for 30 min before reactions. In case when metal
complexes were prepared in situ, deoxygenated equimolar aqueous
solutions of both the ligand and the metal salt were mixed under
argon atmosphere for 2 h before they were added to the pre-
deoxygenated buffer solution of DNA. DNA cleavage experiments
were typically performed by incubating the DNA sample (100 μM,
base pairs) at 37 °C in 20 μL of a buffered solution (10 mM tris–HCl, pH
7.4, I=0.10 M NaCl) containing equimolar concentrations of either
the metal complex (ZnIIL), or the pre-mixed ligand/metal solution.
Reactions were allowed to proceed for 6 h under argon atmosphere.
DNA fragments were separated on a 1% agarose gel electrophoresed in
TAE buffer (20 mM TRIS base, 20 mM acetic acid, 1 mM EDTA)
containing 1% SDS. DNA bands were visualized by staining with
ethidium bromide solution and photographed. The relative intensities
of the DNA bands were quantified by densitometric analysis using a
Biorad 620 interfaced with a PC workstation. After adjusting for the
background and multiplying by a factor of 1.44 to account for the
reduced affinity of DNA for ethidium bromide, the results were
normalized and compared to the controls.
3.2. Potentiometric titration
Solution properties of the ligands and their ZnII complexes were
studied by pH-titration of the ligands in the absence and presence of
equimolar amounts of ZnII, at 25 °C and constant ionic strength
I=0.10 M NaNO3. The logarithm of protonation equilibrium constants
(logK) of the free ligands and the formation constants (logKZnL) of zinc
complexes were determined from the plot of pH versus the number of
sodium hydroxide equivalents added (Fig. 1). The results, summarized
in Table 1, show that the cyclen component of both ligands L1 and L2 has
one set of three relatively more basic amine groups (the primary amine
groups with logK=4.54, 7.57, 9.76 for L1, and logK=4.62, 7.38, 9.68 for
L2), and a second set of less basic tertiary amine group (with logKb2).
The protonation constant of the acridine nitrogen and that of the tertiary
nitrogen of the macrocycle were too low to measure by this method. The
pH-titration curves showed a buffer region (3bNaOH equivalentsb5)
corresponding to neutralization of the protonated nitrogen atoms of the
macrocycle, accompanied by the formation of the metal complex. The
titration curves of L1 and L2 show breaks when 5 equivalents of NaOH (4
equivalents in the case of L3) were added, after which deprotonation of
the metal-bound water becomes evident (Scheme 3). Interestingly, the
acidity of the ZnII-bound water (expressed as pKa values) of such
tetheredcyclencomplexes isenhanced(pKa=7.39, 7.46, and 7.25 for L1,
L2, L3, respectively) relative to that of un-tethered cyclen (pKa=7.88)
[31]. This acidity enhancement could be attributed to weakening of the
bond between Zn and the tertiary nitrogen of the macrocycle in L1, L2,
and more so between the metal and the amide nitrogen in L3 [33].
The equilibrium constants for the formation of ZnII complexes
were determined from the pH-titration data. The calculated logKZnL
for L1, L2, and L3 are 11.42, 11.36, 8.84 mol−1 L−1, respectively. The
3. Results and discussion
3.1. Synthesis
Compounds L1 and L2 were synthesized from 9-acridinecarbonyl
chloride [28], upon treatment first with at least two equivalents of
H2N–(CH2)xOH, followed by tosylation. The tosylate derivatives were
treated with excess cyclen (1,4,7,10-tetraazacyclododecane) to give
the desired products (Scheme 2). Compound L3 was prepared directly
from 9-acridinecarbonyl chloride upon treatment with two equiva-
lents of cyclen in THF. After the reactions were complete, solvent was
evaporated and excess unreacted cyclen was recovered by sublima-
tion under reduced pressure. Products were obtained as yellow oil.
Purification of crude products was achieved by converting the free
base ligands into their hydrobromide salts, followed by recrystalliza-
tion from methanol. All analytical and spectral data of the products
were consistent with the assigned structures.
Scheme 3. Protonation of ligands and formation of Zn(II) complexes.

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J.A. Krauser et al. / Journal of Inorganic Biochemistry 104 (2010) 877–884
3.3. DNA experiments
3.3.1. DNA-binding studies
The ability of these compounds to bind to plasmid DNA was
demonstrated by monitoring the changes in their absorption spectra
when DNA is added. UV–visible absorption spectrum of a buffered
solution of L1 (in 10 mM tris–HCl, pH 7.4, at 25 °C and I=0.10 M NaCl),
in the absence of DNA, shows four absorption bands: 340 nm
(ε=1.5×104 M−1 cm−1), 360 nm (ε=1.8×104 M−1 cm−1), 405 nm
(ε=5.5×103 M−1 cm−1), and 429 nm (ε=3.2×103 M−1 cm−1). The
360 nm band is believed to be due to the extended π-bonds conjugation
of the acridine ring [34]. Only the binding of L1 to DNA was studied since
complexes of this ligand exhibited the highest hydrolytic activity. The
extent of binding was determined by monitoring the change in the
360 nm absorption band upon increasing the concentration of DNA
(Fig. 2). Comparative studies of the features of the absorption spectrum
of L1, in the absence and presence of DNA showed that the extent of the
red-shift of the 360 nm band depends on DNA concentration. Such a
shift can be attributed to the strong DNA-ligand interaction [24,35]. In
addition, DNA-ligand interaction resulted in a significant hypochromic
effect on the absorption spectrum of the ligand. These results indicate
clearly that the acridine group in these compounds interacts strongly
with the double-stranded DNA, mostly through intercalation. Interac-
tions of this nature between acridine derivatives and DNA have been
previously observed in other related studies [23,36,37].
Fig. 2. Changes in the UV–visible absorption spectrum of L1 (50.0 μM) upon titration
with plasmid pBR322 DNA in tris–HCl buffer, at pH 7 and 25 °C. The bathochromic shift
and hypochromocity of the 360 nm band is dependent on the concentration of DNA.
relatively small stability constant for L3, compared to that of L1 and L2,
may be attributed to the weaker bond between Zn and the amide
nitrogen compared to the Zn-tertiary amine bond.
3.3.2. DNA hydrolysis
Hydrolytic reactions between selected metal complexes of these
ligands and DNA were studied by monitoring the conversion of closed-
Fig. 3. (a) 1% agarose gel showing cleavage of plasmid pBR322 DNA by metal complexes of compounds L1, L2, and L3, at 37 °C for 6 h. All reactions were conducted under argon
atmosphere in 20 μL of degassed buffer (20 mM Tris–HCl, pH 7.4, I=0.10 M NaCl) containing 100 μM DNA (base-pair). Lane 1: 100 μM of each of cyclen and acridine; lane 2: 100 μM
of each of cyclen, acridine, and Co3+; lane 3: 100 μM of each L3 and Co3+; lane 4: 100 μM of each L2 and Co3+; lane 5: 100 μM of each L1 and Co3+; lane 6: 100 μM of each L1 and Fe3+
;
lane 7: 100 μM of each L1 and Co3+, in presence of 200 μM ethidium bromide; lane 8: 100 μM of each L1 and Zn2+; lane 9: 100 μM of each L1 and Cr2+; lane 10: 100 μM of each L1 and
Cu2+; lane 11: 100 μM of each L1 and Ni2+; lane 12: DNA only (control). (b) Densitometeric quantitative analysis of the relaxation of plasmid DNA by metal complexes of L1–L3, based
on lanes analysis in part (a). % Relative relaxation=[(form II)/(form I+form II)]×100%.

J.A. Krauser et al. / Journal of Inorganic Biochemistry 104 (2010) 877–884
883
circular super-coiled cc-DNA (Form I) to the nicked (Form II) and the
linear (Form III). The extent of strand scission of DNA was assessed by
agarose gel electrophoresis. Typically, pBR322 plasmid DNA was
incubated in a buffer solution containing either the pre-synthesized
metal complex (in case of ZnL), or the ligand and equimolar amount of
the metal salt. No significant differences in hydrolytic activities were
observed between using pre-synthesized metal complexes versus using
metal complexes that were synthesized in situ; therefore all the
comparative DNA-cleaving studies that were performed in this study
involved in situ syntheses of the metal complexes. DNA hydrolysis
reactions were carefully carried out under oxygen-free sterilized
conditions tominimizeoxidative or enzymic damage to DNA. Fragments
of the cleaved DNA were separated by electrophoresis (Fig. 3a). After
staining the gel with ethidium bromide solution, the photograph of the
gel was scanned and the bands were quantified. After adjusting for the
background, the results were normalized relative to controls (Fig. 3b).
Generally, metal complexes of L1 are more active toward hydro-
lysis of the phosphodiester bonds of DNA than those of L2 and L3,
however the activity trend based on the metal ion cofactors was
similar in all three ligands. Results show that the hydrolytic activities
of these compounds depend on both the structure of the ligand
(activity order: L1 NL2 NL3) and the nature of metal ion cofactor
(activity order: Co3+ NZn2+ NCr2+ NNi2+ NCu2+ NFe3). This was
demonstarted by the conversion of cc-DNA (form I) to open circular
oc-DNA (form II), a process that normally requires one nicking in cc-DNA.
The high activity of L1 could be justified on the bases of having the
optimum length of linker between the metal-binding and DNA-binding
groups. This structural advantage allows proper positioning of the metal
complex relative to the phosphodiester bonds. A longer or shorter linking
arm decreased the restriction activity. Although the highest hydrolytic
activities were observed in the case of cobalt and zinc complexes,
complexes of other metals such as chromium, nickel, and copper showed
good activities; iron complexes on the other hand had the least
restriction activity. Interestingly, control tests involving incubation of
DNA in a solution containing the metal ion, acridine, or cyclen failed to
elicit any activity beyond the background. This substantiates the
necessity of close cooperation between all these three components.
The extent of DNA hydrolysis by these metal complexes is not
significantly affected by the presence of catalase enzyme, dithiothreitol
(DTT), or when excess amounts of radical scavengers such as potassium
iodide or potassium formate were used. This indicates that the mode of
the observed DNA cleavage is not of an oxidative nature. However, the
presence of excess amounts of ethidium bromide, a known powerful
intercalator, profoundly suppresses DNA cleavage, indicating that
intercalation of the acridine moiety in the double-stranded DNA is
required to achieve a good hydrolytic activity. Formation of linear DNA
(form III), a process that normally requires at least two nearby nickings
in the double-helix, was not observed except with CoIIIL1 and only under
prolonged reaction time, more than 24 h (data is not shown).
Fig. 4. (a) Agarose gel showing the effect of pH on DNA hydrolysis by ZnIIL1 at 37 °C,
I=0.10 M (NaCl), reaction time 6 h. (b) The pH-profile of DNA hydrolysis in part a.
arms of various lengths. DNA-binding experiments showed that the
acridine group of these compounds can bind to double-stranded DNA by
intercalating between its nitrogen base pairs. This was evident from
both the strong hypochromic effect and the bathochromic shift in the
absorption spectra of theligands upon titration with increasingamounts
of DNA.
Metal complexes of these compounds are able to hydrolyze plasmid
DNA effectively and under biologically relevant conditions (pH 7.4, and at
37 °C). Comparative studies based on the ligand showed that hydrolytic
activity generally follows the order: L1NL2NL3. Results show that
activity also depends on the nature of the metal (Co3+ NZn2+ NCr2+
N
Ni2+ NCu2+ NFe3+). This study also clearly shows that the mode of DNA
cleavage by these metal complexes is hydrolytic and not oxidative.
The higher hydrolytic efficiency of L1, relative to that of L2 and L3,
indicates that it has the optimum length of spacing arm between the
metal-binding and the DNA-binding subgroups. This allows position-
ing of the metal complex in the proper location relative to the
phosphodiester bonds. A longer linking arm (in L2) as well as a shorter
one (in L3) resulted in a decrease in restriction activities.
The optimum pH for DNA hydrolysis is found to be around the pKa
values of the metal complexes, which suggests possible involvement of
metal-hydroxo intermediates in the reaction. Such intermediates are
usually formed by deprotonation of at least one water molecule that is
coordinated to the metal center [38–40]. To further understand the
mechanism of DNA cleavage, we compared the hydrolytic activities of
metal complexes at different pH values. The results show that the
activity is largely dependent on the pH (Fig. 4). In the case of Zn(II)L1
complex, the highest activity was observed at pH 7.4. Reactions at
higher pH values resulted in a decrease in hydrolytic activity, possibly
due to destabilization of the metal complex under alkaline conditions.
Acknowledgment
This work was supported in part by institutional support from East
Tennessee State University, and by the Schering-Plough Industrial
Award (No. 3403).
4. Conclusions
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