Artificial transcription factors which mediate double-strand DNA Cleavage

Article abstract of DOI:10.1002/chem.201000552

A new family of artificial transcription factor (ATF)-based conjugates have been designed and synthesized as potent chemical nucleases. Polyamides as the important and efficient ATFs were used to modify and activate several anchor compounds. The results demonstrate that the resulting conjugates remarkably promote the rate accelerations and non-random double-strnd DNA cleavage activity. Interestingly, the cleavage activity of both the hydrolytic and oxidative agents was promoted efficiently through the modification of the ATFs.

Full text of DOI:10.1002/chem.201000552

FULL PAPER
DOI: 10.1002/chem.201000552
Artificial Transcription Factors which Mediate Double-Strand DNA Cleavage
Chao Li,[a] Chao Du,[a] Hua Tian,[a] Chao Jiang,[a] Min Du,[a] Yan Liu,[c]
Ren-Zhong Qiao,*[a, b] Yan-Xing Jia,*[b] and Yu-Fen Zhao*[c]
Abstract: A new family of artificial transcription factor (ATF)-based conjugates
have been designed and synthesized as potent chemical nucleases. Polyamides as
the important and efficient ATFs were used to modify and activate several anchor
compounds. The results demonstrate that the resulting conjugates remarkably pro-
mote the rate accelerations and non-random double-strand DNA cleavage activity.
Interestingly, the cleavage activity of both the hydrolytic and oxidative agents was
promoted efficiently through the modification of the ATFs.
Keywords: artificial transcription
factors
· DNA binding · DNA
cleavage · double-strand cleavage ·
polyamide
Introduction
tate dsDNA cleavage were reported, such as the bleomy-
cins,[3] the calicheamicin,[4] prodigiosin[5] and CeII-based com-
plexes.[6]
The recent impressive progress in the field of genomic and
biotechnology has stimulated the search for effective tools
to manipulate DNA.[1] There has been much interest in the
development of synthetic small molecules as nucleic acid
cleavage agents. Among them, double-strand break (dsb)
agents are thought to be more biologically significant than
single-strand breaks (ssb) as a source of cell lethality, be-
cause they apparently are less readily repaired by DNA
repair mechanisms.[2] In the past few years, agents that facili-
Particular interest has been paid to the field of artificial
transcription factors (ATFs), where the essential function of
some transcription factors is to recruit and promote the as-
sembly of a larger transcription complex, leading to the ex-
pression of a gene of interest.[7–10] In so-called artificial tran-
scription factors (ATFs), natural DNA-binding domains are
replaced by synthetic variants, such as triplex-forming pep-
tide nucleic acids (PNAs) and hairpin polyamides.[11] Poly-
amides[12] are an important class of artificial transcription
factors derived from nonproteinogenic amino acids, which
contain N-methylpyrrole (Py), N-methylimidazole (Im), or
N-methylhydroxypyrrole (Hp) groups that can bind in a
hairpin motif to the minor groove of double-stranded
DNA.[13] Because of their wide sequence recognition ability
and sequence specificity, polyamides have an enhanced ca-
pability to function as regulators of transcription in directed
applications.[14–17]
To further develop highly efficient dsbꢀs chemical nucleas-
es of practical value, we sought to explore new families of li-
gands that were readily available, of lower toxicity and
facile to activate under physiologically relevant conditions.
In this paper, our efforts focus on the modification of less
reactive and random dsbꢀs small compounds by Py/Im poly-
amide as artificial transcription factors. Through connection
of the two parts with simple linker, we obtained a new
family of ATFs-based DNA dsb chemical nucleases. In de-
tailed experiments, we will show that remarkable rate accel-
erations and non-random dsb activity of ATFs-based conju-
gates can be achieved based on the powerful affinity of the
[a] C. Li, C. Du, H. Tian, C. Jiang, M. Du, Prof. R.-Z. Qiao
State Key Laboratory of Chemical Resource Engineering
Beijing University of Chemical Technology
15 Beisanhuan East Road, 100029 Beijing (China)
Fax : (+86)010-82728926
E-mail: qrz@mail.tsinghua.edu.cn
[b] Prof. R.-Z. Qiao, Prof. Y.-X. Jia
State Key Laboratory of Natural and Biomimetic Drugs
School of Pharmaceutical Sciences
Peking University Health Science Center
38 Xueyuan Road, 100191 Beijing (China)
Fax : (+86)010-82805166
E-mail: yxjia@bjmu.edu.cn
[c] Y. Liu, Prof. Y.-F. Zhao
Department of Chemistry and
Key Laboratory for Chemical Biology of Fujian Province
College of Chemistry and Chemical Engineering
Xiamen University, 168 Daxue Road, 361005 Xiamen (China)
Fax : (+86)592-2185780
E-mail: yfzhao@xmu.edu.cn
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201000552.
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12935

ATFs moiety to double-stranded DNA compared with free
compounds.
reaction and as a consequence the minimum of cleavage
concentration lowered apparently. The appearance of a
well-defined electrophoresis band for the linear DNA by
both Vc and ImPyImPy-g-Vc suggests a double-strand cleav-
age process, although it is not clear whether single-strand
cleavage can be compared with double-strand cleavage.
In order to assess whether the observed linearization rep-
resents a non-random linearization process, the statistical
test of double-strand cleavage was applied.[21,22] Such analy-
sis assumes a Poisson distribution of the strands cuts and
calculates the average number of single (n1) and double (n2)
strands cuts from the fraction of supercoiled and linear
DNA present after the reaction. The Freifelder–Trumbo re-
Results and Discussion
Design and synthesis of ATFs-based conjugates: To confirm
whether the modification of ATFs moiety for improving the
ability of double strand cleavage is generally applicable, di-
verse small cleavage molecules were conjugated to ATFs, in-
cluding polyamines (cyclen) and bis(2-benzimidazolylme-
thyl)amine (IDB) for a hydrolytic and ascorbic acid (Vc) for
a oxidative mechanism. In addition, we attempted to conju-
gate these molecules to C-ter-
minus or N-terminus of oligo-
polyamide. The design and
structure of the ATFs-based
conjugates
are shown
in
Scheme 1. In all cases, the li-
gands were prepared starting
from a common precursor, that
is, the oligopolyamide back-
bone.
Py4-g-cyclen,
cyclen-
Py4Dp and Py4-g-IDB have
been prepared as previously de-
scribed.[18–20] A detailed synthe-
sis route of bis-cyclen-Py4Dp
and ImPyImPy-g-Vc is given in
the Supporting Information.
DNA cleavage with oxidative
agents: As for Vc, incubation of
supercoiled pUC18 DNA with
free Vc at 378C results in DNA
cleavage at lower concentra- Scheme 1. Structures of ATFs-based conjugates studied.
tions. Subsequently, all super-
coiled DNAs (form I) were
converted to nicked DNA
(Form II) by increasing the Vc
concentration while the linear
DNA (form III) was detected.
Further, linear DNA was gradu-
ally degraded into rogressively
smaller fragments when exces-
sive Vc was added to the reac-
tion mixture (Figure 1a). How-
ever, the catalytic activity was
enhanced remarkably due to
the introduction of the ATF
moiety. The linear DNA was
detected when the concentra-
tion of ImPyImPy-g-Vc was
about 0.1 mm. The strong bind-
Figure 1. Concentration dependence of pUC18 plasmid DNA cleavage by a) Vc and b) ImPyImPy-g-Vc in
ing affinity of polyamide to
DNA leads to an increase of
the effective molarity of the
40 mm pH 7.7 Tris-HCl buffer for 12 h at 378C. Lane 1, DNA control; lanes 2–12, 0.05, 0.1, 0.2, 0.4, 0.6, 0.8,
1.0, 1.2, 1.4, 1.6, 1.8, 2.0 mm cleavage agents in the reaction, respectively; lane 13, 500 bp DNA ladder (S: su-
percoiled DNA, N: nicked DNA, L: linear DNA). c) and d) Quantitation of % various DNA forms per lane
catalytic group in the cleavage by c) Vc and d) ImPyImPy-g-Vc.
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Chem. Eur. J. 2010, 16, 12935 – 12940

Artificial Transcription Factors
FULL PAPER
lation suggests that approximately 100 ssb are required per
dsb if the process is completely random.[21] Thus, the smaller
the n1/n2 value, the higher the linearization activity of an
agent in question.[23] Table 1 shows the statistical results of
Table 1. Comparison of DNA linearization (n1/n2) activities of Vc and
ImPyImPy-g-Vc as function of reaction conditions.[a]
Complex
Vc
c [mm]
T [h]
n1
n2
n1/n2
1.0
0.3
0.2
1.0
2.0
0.3
0.3
0.3
0.3
12
20
12
12
12
0.5
7
4.0290
2.0103
0.3690
0.9933
1.6947
0.1391
0.4293
0.5781
0.8708
0.0420
0.0299
0.1485
0.3576
0.5126
0.0246
0.2208
0.3508
0.6710
95.9
67.2
2.5
2.8
3.3
5.7
1.9
1.6
1.3
ImPyImPy-g-Vc
12
20
[a] pUC18 DNA, 40 mm Tris-HCl, pH 7.4, 378C.
Figure 2. ATFs moiety mediate plasmid DNA dsbꢀs model.
DNA linearization experiments of Vc and ImPyImPy-g-Vc
performed under a variety of reaction conditions. Vc led to
DNA cleavage at 12 h (1.0 mm) and 20 h (0.3 mm) and
afford n1/n2 ratios 95.9 and 67.2, respectively. Linearization
derived from dsb, which is observed only when two cuts ran-
domly, occur sufficiently close on the two strands.[24] We can
thus demonstrate that Vc is not a true dsb agent in a sense
that a dsb is not formed as a result of a single excitation
event. However, the values of n1/n2 in the range 1–6, under
all reaction conditions for ImPyImPy-g-Vc, suggested a non-
random process for all combinations of reactions conditions.
In other words, dsb with ImPyImPy-g-Vc are caused by a
single event or interdependent events and not by a coinci-
dence of random ss breaks on opposite strands. We propose
that DNA affinity of the ATF moiety play an important role
in promoting activity of the true dsb. Due to the introduc-
tion of ATFs moiety, one DNA strand is cleaved, and then
the cleavage agent remains close to the first strand break to
carry out strand breaks elsewhere on the DNA. In addition,
the steady values under various conditions for ImPyImPy-g-
Vc suggest that the changes both in concentration and reac-
tion time hardly affect non-random cleavage path to effi-
ciently form linear DNA.
the double-strand. Further, since the cleavage agents were
restrained in its movements, a random cleavage was con-
trolled effectively. In this process, dsb in the presence of
ATFs moiety are caused by a single event or interdependent
events and not by the coincidence of random ssb on oppo-
site strands. In addition, there is a strong evidence of non-
random cleavage as no smaller fragments were obtained at
higher concentrations and longer reaction time (Figure 1b).
DNA cleavage with hydrolytic agents: The advantage of
ATF-based conjugates was demonstrated further through
the comparison of azacrown compounds. Figure 3a shows
the DNA cleavage behaviour of bis-cyclen-2ZnII under phys-
iological conditions. The cleavage agent converted the su-
percoiled plasmid to a mixture of supercoiled and nicked
DNA with non-selective ssb and random dsb reactions (see
Supporting Information for statistical data). As shown in
Figure 3b, however, the appearance of 1500–2000 bp linear
fragment provided strong evidence that the complex modi-
fied by ATFs (bis-cyclen-2ZnII-Py4Dp) works via a non-
random dsb event, since random cutting was expected to
produce fragments while increasing the cleavage agent con-
centration.[2] The similar results, using other ATFs-based
conjugates studied, were observed (see Supporting Informa-
tion for details).
In order to better demonstrate whether the ATF-based
conjugates possess a better affinity to DNA compared with
free small compounds and free ATFs, the CD spectra of
Py4Dp, cyclen, cyclen-Py4Dp and Py4-g-cyclen were recorded
as shown in Figure 4. None of the ligands used showed CD
signals in the CT DNA absorption region. The absence of
any detectable ICD in the case of cyclen is indicative of the
absence of interaction between this small molecule and
DNA (Figure 4a). However, upon addition of cyclen-Py4Dp
and Py4-g-cyclen to CT DNA substantially induced CD sig-
nals (ICD) arise in 310–400 nm spectral region (Fig-
Possible process of non-random and random dsb: The differ-
ent cleavage models of plasmid DNA by cleavage agents in
the absence and presence of ATFs moiety are depicted in
Figure 2. Upon addition of free ligands to the cleavage reac-
tion, many sites of the supercoiled DNA were attacked non-
selectively to form ssb products (nicked DNA observed).
Subsequently, dsb (linear DNA observed) is observed only
when two cuts randomly occur sufficiently close on the two
strands. Thus, the observed linearization arose from a
random cleavage for free ligands. However, on account of
the strong affinity of the ATF to DNA, cleavage agents
modified were attached tightly to certain DNA sequences
and the effective molarity of the catalytic group might in-
crease to a sufficiently high level to allow facile cleavage of
Chem. Eur. J. 2010, 16, 12935 – 12940
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12937

R. Z. Qiao, Y. F. Zhao, Y. X. Jia et al.
ure 4c,d). The intensity of the
induced Contton effect ob-
served upon DNA binding of
ATF-based conjugates was
markedly higher than that ob-
served when the same number
of free Py4Dp (without linked
cyclen) was bound to CT DNA.
Furthermore, various impor-
tant characteristic interactions
between ATF-based conjugates
studied and duplex DNA were
studied, including CD spectra,
fluorescence quenching, Tm
measurements and UV spectro-
photometric titrations. All ex-
periments on the binding affini-
ty are given in Table 2 (see Sup-
porting Information for details).
The results were consistent with
DNA cleavage ability of small-
er molecules in the absence and
presence of ATF, which were
obtained by gel electrophoresis
experiments.
Figure 3. a) Concentration dependence of pUC18 plasmid DNA cleavage by Bis-Cyclen-2ZnII in 40 mm pH 7.7
Tris-HCl buffer for 12 h at 378C. Lane 1, DNA control; lanes 2–9, 0.1, 1.0, 10, 20, 40, 60, 80, 100 mm cleavage
agents in the reaction, respectively; lane 10, 500 bp DNA ladder (S: supercoiled DNA, N: nicked DNA, L:
linear DNA). b) pUC18 plasmid DNA cleavage by Bis-Cyclen-2ZnII-Py4Dp in the same conditions to Bis-
Cyclen-2ZnII. Lane 1, 500 bp DNA ladder; lane 2, DNA control; lanes 3–14, 0.05, 0.1, 0.5, 1.0, 10, 25, 30, 40,
50, 60, 80, 100 mm cleavage agents in the reaction, respectively (S: supercoiled DNA, N: nicked DNA, L: small
fragment). c) and d) Quantitation of % various DNA forms per lane by c) bis-cyclen-2ZnII and d) bis-cyclen-
2ZnII-Py4Dp.
Conclusion
In summary, we have shown herein that a new family of
ATFs-based conjugates as effective double-strand DNA
cleavage agents were designed and evaluated. Polyamide, a
prevalent kind of ATFs, was chosen to modify some lower
capable and random dsb small molecule agents. The prelimi-
nary gel electrophoresis suggested that the conjugates pro-
moted remarkably rate accelerations and non-random dsb
activity. Due to the introduction of ATFs, small molecules
were effectively fixed on the certain site in double-strand
DNA and it resulted in effective molarity of the catalytic
group was enhanced. The probability of both strands cleav-
age is higher than single strand of that, since cleavage
agents are restricted within narrow limits. Owing to the sig-
nificant sequence selectivity of hairpin polyamides, small
cleavage agents conjugated to hairpin polyamide have been
an ongoing area of study in our group, and they could regu-
late efficiently double-strand cleavage with well-defined se-
quence selectivity.
Figure 4. CD spectra of CT DNA upon addition of increasing amount of
ligands. a) Cyclen. b) Py4Dp. c) Cyclen-Py4Dp. d) Py4-g-Cyclen. All ex-
periments were carried out at pH 8.0 (10 mm Tris-HCl buffer) with
10 mm NaCl. Spectra were recorded at the following r values: 0, 0.001,
0.002 to 0.01 (from bottom to top at 339 nm).
Table 2. Summary of melting temperature measurements and apparent
binding constant measurement.
Compound
DTm [8C]
c50 [mm]
Kapp [mÀ1
]
distamycin[a]
Py4Dp
cyclen
Py4-cyclen
cyclen-Py4Dp
bis-cyclen
bis-cyclen-Py4Dp
IDB
7.7ꢂ106
1.4ꢂ108
3.4ꢂ103
1.7ꢂ108
2.6ꢂ108
6.4ꢂ106
3.6ꢂ107
7.8ꢂ103
5.0ꢂ104
1.3ꢂ105
8.4
2.2
11.5
13.8
9.1ꢂ10À3
3700
Experimental Section
8.8ꢂ10À3
7.2ꢂ10À3
2500
General information: Details MS (ESI) mass spectral data were recorded
on a Finnigan LCQDECA mass spectrometer. 1H NMR spectra were
measured on a Bruker AV600 spectrometer and chemical shifts in ppm
are reported relative to internal Me4Si (CDCl3) or (D2O). Electrophore-
sis apparatus was using a Biomeans Stack II-Electrophoresis system,
PPSV-010. Bands were visualized by UV light and photographed, record-
ed on an Olympus Grab-IT2.0 Annotating Image Computer System. All
8.2ꢂ10À2
Py4-IDB
1ꢂ10À1
9ꢂ10À2
Py4-IDB-ZnII
[a] See refs. [25,26].
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Artificial Transcription Factors
FULL PAPER
other chemicals and reagents were obtained commercially and used with-
out further purification. Electrophoresis grade agarose, plasmid DNA
(pUC18), polyACHTUNGTRENNUNG(dA–dT)·polyACHTUTNGRENNUG(dA–dT) and polyACHNUTGERTG(NNUN dC–dG)·polyACHTNUGRTEN(NUGN dC–dG)
were purchased from Promega Corporation. Calf thymus DNA (CT
DNA) was purchased from Sigma (USA) Company and used as received.
tration and evaporation of the mixture yield yellow solid. Subsequently,
the Boc groups were removed with TFA (0.4 mL) in CH2Cl2 (2 mL). The
crude product was purified by a silica gel column with CHCl3/CH3OH/
NH3·H2O (66:33:1) as eluent to obtain the title compound (347.5 mg,
35%). 1H NMR (600 MHz, [D6]DMSO): d=1.85 (m, 2H), 2.73 (m, 8H),
2.79, 2.80 (s, 6H), 2.84 (m, 8H), 3.05–3.12 (br, 18H), 3.25 (q, 2H, J=6.0),
3.74 (s, 4H), 3.82–3.86 (br, 12H), 6.79 (s, 1H), 6.93 (s, 2H), 6.96–7.10 (br,
4H), 7.15–7.20 (br, 4H), 8.14 (s, 1H), 9.46–9.93 (br, 4H), 10.16 ppm (s,
1H); 13C NMR (100 MHz, [D6]DMSO): d=165.56, 162.24, 159.34, 159.11,
158.45, 136.84, 125.16, 123.18, 122.92, 122.25, 121.29, 119.16, 118.04,
115.98, 105.30, 105.08, 67.49, 55.62, 55.23, 47.41, 44.81, 42.69, 42.35, 42.22,
39.99, 39.86, 39.72, 39.59, 39.45, 39.31, 39.18, 36.44, 35.82, 25.01 ppm; MS
(ESI+): m/z: 1094 [M+HÀ7TFA]+; HRMS (ESI+): m/z: calcd for
Synthesis of bis-cyclen-Py4Dp: Detailed compound structures and synthe-
sis route of Bis-Cyclen-Py4Dp are given in the Supporting Information
(Scheme S1).
Compound 2: Compound 1 was dissolved in DMF (30 mL) containing
anhydrous K2CO3 (1.38 g, 10.0 mmol) with stirring at room temperature.
Then, BnBr (1.78 mL, 15 mmol) was dropwise to the solution above for
30 min. After 4 h, the reaction was quenched by cold water (100 mL) and
solution was extracted with CHCl3 (50 mLꢂ6). The combined organic
phases were washed with saturated aqueous NaCl and dried over
Na2SO4. Filtration and evaporation of the solvent yield compound 2
(2.34 g, 78%). MS (ESI+): m/z: 323 [M+Na]+.
C57H87N18O8F3: 604.3454; found: 604.3453 [M+2H]2+
.
ImPyImPy-g-Vc: The detailed structure and synthesis route of ImPyIm-
Py-g-Vc are given in the Supporting Information (Scheme S1). Treatment
of l-ascorbic acid with acetyl chloride in acetone afforded the 5,6-ketal
of l-ascorbic acid. Benzylation of the C-2’ and C-3’ hydroxy groups of
the lactone ring in compound 7 was accomplished by using K2CO3 and
benzyl bromide in DMF to provide compound 8. Deblocking of the 5,6-
O,O-protected derivative of l-ascorbic acid 8 with acetic acid in metha-
nol gave 2,3-O,O-dibenzyl-l-ascorbic acid (9). The flexible g-aminobuty-
ric acid linker was conjugated to compound 9 by BOP reagent and
DIEA to obtain compound 10. Then the polyamide backbone ImPyIm-
PyCOOH and compound 10 were conjugated by BOP reagent and
DIEA in DMF/dichloromethane. Finally, the hydrogenation reaction cat-
alyzed by Pd/C removed the benzyl group. The desired product was puri-
fied by silica gel column chromatography with CHCl3/CH3OH 60:40 as
eluent.1H NMR (600 MHz, [D4]CD3OD): d=7.46 (s, 2H), 7.39 (d, 1H),
7.28 (s, 2H), 7.04–7.08 (d, 1H), 6.85 (s, 1H), 4.75 (d, 1H), 4.26 (m, 1H),
4.25 (m, 1H), 4.24 (s, 1H), 4.23 (s, 6H), 4.08 (s, 3H), 3.97 (s, 3H), 2.49 (t,
2H), 1.94 (t, 2H), 1.55 ppm (t, 2H); 13C NMR (100 MHz, [D6]DMSO):
d=174.0, 173.1, 162.6, 160.7, 157.9, 157.7, 156.3, 154.4, 154.2, 146.0, 134.7,
131.4, 131.2, 130.8, 130.5, 126.0, 125.9, 123.5, 120.3, 120.1, 118.5, 77.3,
65.7, 65.6, 39.8, 37.3, 36.9, 34.4, 34.7, 31.0, 23.7 ppm; MS (ESIÀ): m/z:
734.5 [M]À; HRMS (ESI+): m/z: calcd for C32H36O11N10: 737.2643; found:
737.2638 [M+H]+.
Compound 3: Compound
2 (1.20 g) dissolved in anhydrous THF
(70 mL), which was dropwise to suspension THF (20 mL) containing
LiAlH4 (0.77 g, 20 mmol) for 30 min at 08C under an N2 atmosphere.
After 2 h, CH3OH (5 mL), H2O (5 mL) and NaOH (10 mL, 10%) were
added in the solution above, respectively, and the mixture was filtrated
with celatom. Evaporation of solution gave compound 3 (0.97 g, 99%) as
yellow solid. MS (ESI+): m/z: 251 [M+Li]+.
Compound 4: Compound 3 (0.73 g, 3.0 mmol) was dissolved in anhydrous
CH2Cl2. PBr3 (0.66 mL, 6.9 mmol) was added in the solution with stirring
3 h at 08C under an N2 atmosphere. After warming to room temperature,
the mixture was poured in cold water (70 mL), and its pH was adjusted
to pH 7.0–7.2 using saturated aqueous NaHCO3. The solution was ex-
tracted with Et2O (50 mLꢂ5). The combined organic phases were dried
over Na2SO4. Filtration and evaporation of the solvent yield compound 4
(0.68 g, 61%). MS (ESIÀ): m/z: 372 [M]À.
Compound 5: Under an N2 atmosphere, 3Boc-Cyclen (1.39 g, 2.95 mmol)
was dissolved in CH3CN (70 mL) and compound 4 (0.55 g, 1.48 mmol)
and anhydrous Na2CO3 (0.38 g, 3.57 mmol) was added in the solution.
The remaining reaction was refluxed at 858C for 20 h. Filtration and
evaporation of the solvent yield yellow solid. Crude product was purified
by a silica gel column with ethyl acetate/petroleum ether (50:50) as
Circular dichroism measurements: All experiments were performed
under a continuous flow of nitrogen using a Jasco-720 spectropolarimeter.
A path length cell of 1 cm was used and all experiments were performed
at room temperature. A 0.5 mm solution of cyclen, Py4Dp and cyclen-
Py4Dp was titrated into the DNA solutions (at pH 8.0, 10 mm Tris-HCl
buffer with 10 mm NaCl). The standard scan parameters for all experi-
ments were as follows: The wavelengths were scanned from 400 to
220 nm. The sensitivity was set at 100 mdeg and the scan speed was set at
200 nm per minute. Three scans were accumulated and averaged by the
computer.
eluent. 1H NMR (600 MHz, CDCl3): d=1.40, 1.44 (br, 54H, OC
ACTHNUGTRNEUGN(CH3)3),
2.60 (m, 8H, CH2NCH2), 3.2–3.29 (m, 16H, NCH2CH2), 3.56 (m, 8H,
NCH2CH2), 3.68 (s, 4H, NCH2Ar), 4.97 (s, 2H, OCH2Ar), 6.61 (s, 1H,
OArH4), 6.75 (s, 2H, OArH2, OArH6), 7.25–7.28 (br, 1H, ArH4), 7.33–
7.36 ppm (br, 4H, ArH2, ArH3, ArH5, ArH6); 13C NMR (100 MHz,
CDCl3): d=155.73, 137.45, 128.53, 127.93, 127.43, 115.94, 79.41, 69.92,
56.04, 55.47, 54.15, 49.86, 47.95, 28.47 ppm; MS (ESI+): m/z: 1176
[M+Na]+.
Compound 6: Compound
5 (230.8 mg, 0.2 mmol) and Pd/C (5%)
(22.7 mg) were added to anhydrous CH3CH2OH. The solution was stirred
at 1 atm under an H2 atmosphere. After 3 h, filtration and evaporation of
the residue gave brown solid (161.7 mg, 76%). 1H NMR (600 MHz,
Plasmid DNA cleavage: Electrophoresis experiments were performed
with plasmid DNA (pUC18). In a typical experiment, supercoiled pUC18
DNA (5 mL, 0.08 mgmLÀ1) in Tris-HCl buffer (40 mm) was treated with
different concentration ligands (dissolved in dimethyl sulfoxide, Vc
system in water) at different pH value, followed by dilution with the Tris-
HCl buffer to a total volume of 80 mL. The samples were then incubated
at different temperature and time intervals, and loaded on a 1% agarose
gel containing ethidium bromide (1.0 mgmLÀ1). Electrophoresis was car-
ried out at 85 V for 1 h in TAE buffer. Bands were visualized by UV
light and photographed followed by the estimation of the intensity of the
DNA bands using a Gel Documentation System.
CDCl3): d=1.24–1.71 (br, 54H, OCACHTNUTRGNEUNG(CH3)3), 2.11–2.61 (m, 8H,
CH2NCH2), 3.35–3.70 (m, 24H, NCH2CH2), 4.11–4.17 (s, 4H, NCH2Ar),
6.85 (s, 1H, ArH4), 7.38, 7.73 ppm (s, 2H, ArH2, ArH6); MS (ESI+): m/z:
1064 [M+H]+.
The above-mentioned product (424.8 mg, 0.40 mmol), anhydrous K2CO3
(55.4 mg, 0.40 mmol) and BrCH2CH2COOEt (134 mL, 1.20 mmol) were
added to acetone (25 mL). The solution was extracted with ethyl acetate
(40 mLꢂ4) and dried over Na2SO4 after refluxed for 10 h. Filtration and
evaporation of the solution yield yellow solid. Subsequently, it was hydro-
lyzed to obtain the corresponding acid compound 6 (391.6 mg, 95%).
Fluorescence quenching assay: Fluorescence spectra were recorded on
Hitachi model F-4500 spectrofluorimeter, with excitation and emission
band bass: 10 nm (lex =520, lem =620 nm). 0.5 mm solution of Py4Dp, bis-
cyclen and bis-cyclen-Py4Dp were titrated into the DNA-EB solution at
pH 8.0 10 mm Tris-HCl buffer with 10 mm NaCl. Apparent binding con-
stants (Kapp) of the ligands with CT DNA were estimated and compared
by measuring the loss of EtBr fluorescence as a function of added ligand.
The Kapp values were calculated from: KEtBr·ACTHNUGRTENNU[G EtBr]=Kapp·ACHTGNUTREN[NUNG ligand], where
[EtBr] and KEtBr are the concentrations and binding constants of EtBr,
respectively, and [ligand] is the concentration of ligand at 50% of maxi-
1H NMR (300 MHz, CDCl3): d=1.23–1.47 (br, 54H, OC
ACTHUNGTRNEUNG(CH3)3), 2.86 (br,
8H, CH2NCH2), 3.40–3.89 (m, 28H, NCH2CH2, NCH2Ar), 4.62 (br, 2H,
ArOCH2), 6.96 ppm (br, 3H, ArH2, ArH4, ArH6); MS (ESI+): m/z: 1122
[M+H]+.
Bis-cyclen-Py4Dp: Compound 6 (657.2 mg, 0.59 mmol) was converted
into activated esters with HOBt (135.1 mg, 1 mmol) and DCC (206.5 mg,
1 mmol) in CH2Cl2 (20 mL). NH2Py4Dp was added to the stirred solution
in room temperature and the reaction mixture was stirred overnight. Fil-
Chem. Eur. J. 2010, 16, 12935 – 12940
ꢁ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
12939

R. Z. Qiao, Y. F. Zhao, Y. X. Jia et al.
mal EtBr fluorescence. The binding constant of EtBr was taken to be 1ꢂ
107.
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Received: March 3, 2010
Published online: September 28, 2010
12940
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Chem. Eur. J. 2010, 16, 12935 – 12940