A bulge binding agent with novel wedge-shape topology for stimulation of DNA triplet repeat strand slippage synthesis

Article abstract of DOI:10.1016/j.bmcl.2008.10.038

Expansion of DNA repeat sequences is associated with many human genetic diseases. Bulged DNA structures have been implicated as intermediates in DNA slippage within the DNA repeat regions. Herein a bulge binding agent with novel wedge-shape topology of the aromatic moiety was designed and synthesized. The compound-bulge DNA interactions were characterized via UV melting experiments, circular dichroism and were quantitated by surface plasmon resonance with K_d of 41.5 μM. This compound showed remarkable stimulation for DNA triplet repeat strand slippage synthesis in vitro.

Full text of DOI:10.1016/j.bmcl.2008.10.038

Bioorganic & Medicinal Chemistry Letters 18 (2008) 6184–6188
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
A bulge binding agent with novel wedge-shape topology for stimulation
of DNA triplet repeat strand slippage synthesis
Liangliang Liu, Heyang Li, Long Yi, Xing Yang, Xin Wen, Zhen Xi *
State Key Laboratory of Elemento-Organic Chemistry, Department of Chemical Biology, Nankai University, 94 Weijin Road, Tianjin 300071, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Expansion of DNA repeat sequences is associated with many human genetic diseases. Bulged DNA struc-
tures have been implicated as intermediates in DNA slippage within the DNA repeat regions. Herein a
bulge binding agent with novel wedge-shape topology of the aromatic moiety was designed and synthe-
sized. The compound–bulge DNA interactions were characterized via UV melting experiments, circular
Received 29 August 2008
Revised 25 September 2008
Accepted 1 October 2008
Available online 11 October 2008
dichroism and were quantitated by surface plasmon resonance with K
showed remarkable stimulation for DNA triplet repeat strand slippage synthesis in vitro.
Ó 2008 Elsevier Ltd. All rights reserved.
d
of 41.5 lM. This compound
Keywords:
Phenothiazine
Bulge DNA
Wedge-shaped structure
Surface plasmon resonance
Slippage synthesis
Bulge structure in nucleic acid has been shown to play a signif-
a phenothiazine aminosugar 1 with large dihedron angle (ꢀ140°)
1
icant biological role in protein binding recognition, frameshift
between two benzene rings (Scheme 1). Aminosugar moiety in 1
was employed to enhance its aqueous solubility and binding to
the phosphate backbone at the bulged site. This phenothiazine
aminosugar was found to act as DNA bulge binders and stimulate
DNA slippage synthesis in vitro.
2
mutation, imperfect homologous recombination by repair en-
3
4
zymes, naturally occurring antisense RNA, and expansion of trip-
5
let repeats during DNA synthesis. Goldberg group have reported a
series of bulge binding molecules designed from the decomposed
6
product of NCS-chrom. NCS-chrom is a bicyclic enediyne antibi-
As shown in Scheme 2, compound 1 was prepared from pheno-
thiazine based on simple two-step organic synthesis. Phenothia-
7
otic. One of its decomposed products NCSi-gb shows bulge bind-
10
ing selectivity and its solution structure with bulge DNA has
zine 2 was firstly converted to 3 using NaH condition. The
8
11
been reported. Some other similar spirocyclic molecules such as
intermediate 3 was treated with trichloroacetimidate 4. After
DDI (double-decker intercalator) and ent-DDI have been designed
and synthesized, which are more stable compared with NCSi-gb
and show DNA slippage stimulation effect in vitro.6 The capacity
for DDI to recognize bulges stems specifically from the orientation
of the ring systems, transposed approximately 60° by the spirolac-
gel purification, all protecting groups were removed by sodium
methoxide in methanol to give 1. Compound 1 was structurally
characterized by 1- and 2-D NMR spectroscopy, including COSY,
HSQC, HMBC, and ROESY experiments, in conjunction with mass
spectrometry (see SI for NMR spectra).
Circular dichroism (CD) spectrum was used to test the bulge
binding selectivity of compound 1. The DNA sequences used in this
study contain B-formed double helix, hairpin-, and bugle-contain-
ing DNA structures. Compound 1 has nearly no CD signal in the
wavelength scope tested. The DNA concentration used in the
6a
tone, and the right-handed 35° twist of the molecules. On the
other hand, we recently reported binaphthol aminosugars as bulge
9
probe, which could stimulate DNA slippage synthesis in vitro. The
helical twist between two naphthyl rings in binaphthol aminosug-
ars is 90°. However, doxorubicin with nearly flat aromatic surface
was reported to inhibit the expansion. The topological structure of
the aromatic moiety seems to be important for its slippage prop-
erty. Whether an aromatic-containing aminosugar with relative
large dihedron angle h (90° < h < 180°) could also act as bulge
probe? In order to further study the topological affect of spirocyclic
moiety on bulge DNA binding, herein we designed and synthesized
experiment is 20 lM and [compound 1]/[DNA] ratio is 10 for each
sequence. Figure 1 shows the effect of compound 1 on the different
DNA sequences. When compound 1 was added, the change CD sig-
nal of bulge-containing DNA is distinctive, but little change was
observed for duplex and hairpin DNA. From the CD spectra, it could
be concluded that compound 1 can bind to DNA bulge structure
selectively. This result was consistent with the NMR research re-
ported on the selective interactions between phenothiazine deri-
*
Corresponding author. Tel./fax: +86 22 23504782.
1
2
E-mail address: zhenxi@nankai.edu.cn (Z. Xi).
vates and bulge-contained RNA.
0
960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.10.038

L. Liu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6184–6188
6185
Scheme 1. Aminosugar compounds mentioned. The topological structures of three molecules are different from each other: the aromatic surface of doxorubicin is nearly
planar and is more flexible than the other two. The aromatic surfaces of DDI and 1 are fixed and wedge-shaped but they have different angle. The 3D structures were
1
6
17
generated using the InsightII molecular modeling program. The structures’ energy minimization was performed using the CVFF force field with a 0.001 kcal/mol energy
gradient convergence criterion by InsightII/Discover package. Graphics were generated using PyMOL (http://pymol.sourceforge.net).
Scheme 2. Synthesis of compound 1 from phenothiazine.
In order to test the ability of 1 to stabilize the possible bulge
intermediate, we measured the melting temperature change of
ATT and AAT bugle-contained DNA sequences by adding com-
pound 1 (Fig. S1). The melting temperature of ATT bugle-contain-
ing DNA changed from 35.8 to 36.6 °C by adding the compound
and that for AAT bugle-containing DNA from 35.7 to 37.5 °C, which
implying that compound 1 can stabilize both AAT and ATT bulge
structure.
ple, efficient, and rapid for bulged DNA–drug interactions,
which should facilitate the study on DNA slippage synthesis
mechanism and as a method for high-throughput screening of
bulge binders.
As bulge has been postulated as intermediate in DNA slip-
page synthesis involving templates with nucleotide repeats,
we examined the influence of compound 1 on the triplet repeat
expansion in vitro. The AATꢃATT trinucleotide repeats distribute
1
4
Surface plasmon resonance (SPR) measurements were further
made to quantitate DNA–drug interactions. As shown in Figure
in different types of genomic sequences, which is one of the
most instable triplet repeat sequences reported to slip in vi-
1
5
2, the steady state response for compound 1 binding to bulged
tro. DNA slippage synthesis was measured at 23 °C using the
primer/template oligomers of (ATT) and (AAT) in a reaction
catalyzed by the Klenow fragment of DNA polymerase I lacking
DNA surface was plotted versus the analyte concentration. The
3
5
data were fitted to a simple Langmuir isotherm for bimolecular
ꢁ
0
0
0
32
interactions: Req ¼ R ð½Aꢂ=ðK
d
þ ½AꢂÞÞ, wherein Req is the mea-
3 –5 exonuclease activity. The primer was 5 - P-end-labeled
max
3
2
sured response, [A] is the concentration of drug, Rmax is the
using [
dency experiment was carried out by changing the concentra-
tion of 1 from 4 to 400 M. The experimental result was
c- P] ATP and T4 polynucleotide kinase. Dose-depen-
maximum response at saturation of surface binding sites, and
K
d
is the dissociation constant.13 Compound 1 showed moderate
l
binding affinity (41.5 ± 5.0
ture, which was comparable with K
l
M) with ATT bulge-containing struc-
values reported for wedge-
shape bulge binding agents.6 However, the binding response
for hairpin (K = 140 ± 40 M) and double strand (K = 170 ±
M) structures are significantly smaller than that for ATT
bulge-containing structure. Herein SPR-based assay works sim-
analyzed by electrophoresis (Fig. 3). In the control reaction
(Fig. 3, lane 2), the primer was expanded to an average band
length of 17 bp (base pair), a little longer than 15 bp expected,
which indicated that the slippage occurred during DNA synthe-
sis. The slippage could be restrained by the addition of doxoru-
d
c
d
l
d
10 l
6
c
bicin (Fig. 3, lane 3). When compound 1 was hold at a relative

6
186
L. Liu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6184–6188
Figure 1. Circular dichroism spectra of DNA (20
l
M) alone (solid line) and 1–DNA complex after addition of 1 (200
l
M) (dashed line). The CD spectra were performed on a
. Data were recorded at a
M phosphate, pH = 7.0. All oligonucleotides
Jasco-715 spectropolarimeter, using quartz cylindrical cell of 1 mm path length. The cell compartment was continuously purged with dry N
bandwidth of 1.0 nm and measured at every 0.2 nm over of 230–330 nm at 20 °C in phosphate buffer containing 50 M NaCl,10
20 M) were heated to 95 °C for 1 min and then cooled to the 15 °C slowly before use.
2
l
l
(
l
low concentration, little affect was detected (Fig. 3, lanes 4 and
). The stimulating effect of 1 became distinctive at higher con-
centration. The average band length of slippage increased with
that the trinucleotide repeats DNA slippage synthesis stimulated
by small molecule could be a dynamic equilibrium, so molecule
with moderate bulge-binding affinity can give well slippage re-
5
15
9
the concentrations of 1, such as 18, 20, 32, 71 bp for 50, 100,
sult. Compared with the stimulation effect of DNA strand slip-
6
c
9
2
00, 400
l
M, respectively. The enhancement of DNA slippage
page for DDI or binaphthol aminosugars, the title compound
exhibits relative smaller effect at low concentration
(<100 M). However, with enhancement of the concentration
to 400 M, compound 1 has similar effect with that of DDI,
synthesis by compound 1 could also be restrained by the addi-
tion of doxorubicin (Fig. 3, lane 10). Phenothiazine or amino-
sugar alone did not show any detectable DNA bulge selective
binding, nor stimulate DNA slippage (data not shown). The
experimental result demonstrated that compound 1 having dif-
ferent aromatic topology against DDI could also stimulate the
DNA slippage synthesis in vitro. Comparing the electrophoresis
result with the binding affinity revealed by SPR, we propose
1
l
l
where the triplet expansion nearly reaches saturation. Further
work is also needed to clearly elucidate the stimulation ability
of DNA stand slippage for compound 1.
In summary, we have synthesized and characterized a pheno-
thiazine aminosugar compound with novel wedge-shaped aro-

L. Liu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6184–6188
6187
Figure 3. (A) Dose effects of compound 1 on the expansion of the ATTꢃAAT trinucleotide repeat. A standard reaction (23 °C, 12 h) containing 5 -32P-end-labeled (ATT)
0
3
5
and unlabeled template (AAT) was catalyzed by the Klenow fragment of Escherichia coli. DNA polymerase. The products were analyzed on 15% denaturing PAGE gel.
Lane 11 is marker, 42 bp (top) and 26 bp (bottom); lane 1 is the reaction without Klenow fragment; lane 2 is the reaction with 2% DMSO as control; lane 3 is the
control reaction mixed with doxorubicin 40
l
M; lanes 4–9 is reaction in the presence of 1 from 4, 20, 50, 100, 200 to 400 M, respectively, and lane 10 is the reaction
*
M) and doxorubicin (40 lM). (B) The average slippage length is calculated as the total (slippage length its intensity)/the total band
l
containing both compound 1 (100
l
intensity of each lane.
Acknowledgments
This work was supported by the National Key Project for Basic
Research of China (2003CB114403), National Natural Science Foun-
dation of China (20272029, 20572053, 20421202, 20432010), Min-
istry of Education of China (104189), and Nankai University ISC.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2008.10.038.
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