Quaternarized pdppz: Synthesis, DNA-binding and biological studies of a novel dppz derivative that causes cellular death upon light irradiation

Article abstract of DOI:10.1039/c0cc04303f

The quaternarized pdppz derivative 1 was shown to bind strongly to DNA with concomitant changes in its ground and excited state photophysical properties. Furthermore, the compound also showed rapid cellular uptake, and induced apoptosis upon light irradiation in various cancer cell lines after 24 hours of incubation. The Royal Society of Chemistry 2011.

Full text of DOI:10.1039/c0cc04303f

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Quaternarized pdppz: synthesis, DNA-binding and biological studies of
a novel dppz derivative that causes cellular death upon light irradiationw
Robert B. P. Elmes,a Marialuisa Erby,b Suzanne M. Cloonan,b Susan J. Quinn,*c
D. Clive Williams*b and Thorfinnur Gunnlaugsson*a
Received 8th October 2010, Accepted 2nd November 2010
DOI: 10.1039/c0cc04303f
The quaternarized pdppz derivative 1 was shown to bind strongly
to DNA with concomitant changes in its ground and excited state
photophysical properties. Furthermore, the compound also showed
rapid cellular uptake, and induced apoptosis upon light irradiation
in various cancer cell lines after 24 hours of incubation.
The development of novel low molecular weight cancer
Scheme 1 Synthesis of 2 (free ligand) and corresponding dicationic
therapeutics is a highly topical area of research. Such
organic derivative 1. (i) EtOH, reflux; (ii) dibromoethane, reflux.
molecules should ideally possess good water solubility, possess
The synthetic pathway of 1 and 2 is shown in Scheme 1.
The synthesis of 2 was achieved by condensation of 5,6-
diaminoquinoxaline15 4 with 1,10-phenanthroline-5,6-dione16
3 by reflux in EtOH yielding 2 as a grey solid in 95% yield.
Subsequent reflux in dibromoethane over 48 h and precipitation
from methanol with diethyl ether yielded 1 as a pure beige solid
in 78% yield. Synthesised as its chloride salt, 1 was water-
soluble, and its photophysical properties were investigated in
10 mM phosphate-buffered aqueous solutions at pH 7.4.
The characteristic absorption spectrum together with the
excitation and emission spectra of 1 are shown in Fig. 1. The
absorption spectrum of 1 shows absorbance maxima at ca.
285 nm, 310 nm, 370 nm, 390 nm along with a broad shoulder
rapid cellular uptake, undergo selective localisation and be able
to induce programmed cell death of the cancer cells.1–4 In recent
work, we have demonstrated that pyrrolo-1,5-benzoxazepine,5
sulfur-substituted a-alkyl phenethyl-amine,6 naphthalimide,7
and polyamide8 structures show potential as anticancer agents
in a variety of cancer cell lines, as well as in solid tumours.9 In
other related work, we have developed new polypyridyl ligands
possessing various organic fluorophores, either directly
attached, or conjugated via short spacers to the Ru(II)-
polypyridyl unit, for use as DNA targeting ligands and
imaging agents.10 Herein, we present the synthesis of 1, a
novel quaternarized polypyridyl molecule formed from 2,
pyrazino[2,3-h]dipyrido[3,2-a:20,30-c]phenazine, or pdppz. This
new species is based on combining, in a single structure, two well
known polypyridyl ligands, dipyrido[3,2-a:20,30-c]phenazine
(dppz) and 1,4,5,8-tetraazaphenanthrene (TAP), that are
commonly used as DNA binding ligands in Ru(II) polypyridyl
complexes,11–13 and has not, to the best of our knowledge, been
synthesised before. The quaternarisation of polypyridyl ligands
has recently been shown by Thomas et al.14 to be an effective
way of increasing the DNA binding affinity of such organic
structures. Hence, we embarked on investigating the ability of 1,
to bind to DNA, as due to its extended aromatic surface, in
concert with its cationic character, we foresaw that 1 would be
an ideal candidate as a DNA intercalator. Moreover, its cationic
nature also furnishes 1 with good aqueous solubility and should
facilitate its cellular uptake in various cancer cell lines, where its
ability to initiate apoptosis could potentially be controlled by
light irradiation.
centred at 460 nm. The band at 285 nm (e = 31 400 cmꢀ1 Mꢀ1
)
is characteristic of p–p* transitions of the phen moiety while
the intense band at 310 nm (e = 44 100 cmꢀ1 Mꢀ1) is attributed
to p–p* transitions with less intense bands at 370 nm and
390 nm (e = 6900 cmꢀ1
M
ꢀ1) attributed to n–p* transitions
within the phenazine part of the ligand.17 These transitions are
typical of the related compound dppz. Upon excitation into
the intense band at 310 nm 1 gave intense emission with lmax
at 515 nm (FF = 0.053). Similarly, the excitation of 1 (lem
515 nm) yielded a spectrum structurally identical to the
=
absorption spectrum.
a School of Chemistry, Centre for Synthesis and Chemical Biology,
Trinity College Dublin, Dublin 2, Ireland. E-mail: gunnlaut@tcd.ie;
Fax: +353 1 671 2826; Tel: +353 1 896 3459
b School of Biochemistry and Immunology, Trinity College, Dublin 2,
Ireland. E-mail: clive.williams@tcd.ie; Fax: +353 1 8963130;
Tel: +353 1 8962596
c School of Chemistry and Chemical Biology, Centre for Chemical
Synthesis and Chemical Biology, University College Dublin, Belfield,
Dublin 4, Ireland
w Electronic supplementary information (ESI) available: Synthesis, 22
figures and 2 tables. See DOI: 10.1039/c0cc04303f
Fig. 1 The UV/Visible absorption spectra, the excitation and the
fluorescence emission spectra of 1 (10 mM) in aerated 10 mM
phosphate buffer, at pH 7.4.
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We next investigated the ability of 1 to bind to salmon testes
DNA (stDNA) at pH 7.4 (10 mM phosphate buffer), using the
absorption and emission spectroscopy of 1, Fig. 2. As can be
seen from these results, the absorption spectrum was
significantly affected over the entire wavelength range. From
these changes, we were able to determine the affinity binding
constant Kb as 6.5 ꢁ 105 Mꢀ1 (ꢂ0.5) (and a binding site size of
2.6 (ꢂ0.03)) using the method of Bard et al.18 This indicates
a strong binding affinity for DNA, comparable to that seen
for many transition metal based intercalators, and of similar
magnitude to that reported by Thomas et al.14 Concomitantly,
the changes in the emission spectra were also dramatic, where
the emission was almost completely ‘switched off’, as
demonstrated by an inset in Fig. 2. Similar effects were seen
in the fluorescence emission spectra upon excitation at other
wavelengths such as 390 nm. Again, analysis of these changes
(of which 80% occurred over 0 - 10 P/D, see ESIw) by fitting
data using the model of McGhee and von Hippel,19 gave Kb =
3.55 ꢁ 105 Mꢀ1 (ꢂ0.02), consistent with that seen in the
absorption spectra. We also investigated the effect of ionic
strength on these changes by carrying out a back titration in
the presence of increasing concentrations of NaCl (see ESIw).
This showed that the binding of 1 to DNA was only marginally
affected; indicating that electrostatic interactions are not the
main form of interaction with DNA. Furthermore, while no
induced circular dichroism (CD) was observed, significant
changes were evident in the linear dichroism (LD) spectrum
(see ESIw), which resulted in the formation of a strong negative
band at 310 nm, resulting from the p–p* transition of the
phenazine part of the ligand; strongly supporting an
intercalative mode of DNA interaction for 1.20
(and a binding site size of 1.7), while for poly(dA–dT) a
significantly lower Kb of 4.85 ꢁ 105 Mꢀ1 (ꢂ0.33) was
determined. Thermal denaturation studies also showed
that these compounds greatly stabilized the stDNA structure
(DTm 4 5–10 1C, see ESIw).
Having established strong affinity of 1 for DNA, we next
wished to evaluate its DNA photocleavage efficiency. Agarose
gel electrophoresis of pBR322 plasmid DNA was undertaken,
Fig. 3. When incubated in the dark 1 showed no DNA
cleavage. However, after 30 minutes of irradiation at a low
intensity (2 J cmꢀ1), under aerobic conditions at a P/D ratio of
10, 1 showed extremely efficient photocleavage, resulting in
essentially complete conversion of the supercoiled DNA into
nicked circular form (Fig. 3, lane 8). Efficient photocleavage
was also observed when 320 nm and 400 nm cut off filters were
employed (Fig. 3, lanes 8 and 6, respectively). Interestingly,
when irradiated in the presence of the known singlet oxygen
scavenger, NaN3,
1 showed diminished photocleavage
efficiency suggesting that reactive oxygen species could be the
primary mechanism aiding in the photocleavage of the DNA
after treatment with 1.
We anticipated that 1 would be taken up by cells due to its
cationic nature, possibly through receptor mediated transport,
or by diffusion driven by plasma membrane potential. Fig. 4
shows the fluorescence confocal laser scanning microscopy
picture of live HeLa (cervical cancer) cells upon excitation at
310 nm after incubation with 1 (10 mM) 24 h prior to imaging
(Fig. 4A). The image clearly demonstrates the localisation of 1
in all cells, with apparent localisation in the cytoplasm and to a
lesser extent in the nucleus, as demonstrated in Fig. 4B, a
bright field image showing co-localisation of 1 (green) with the
nucleic acid stain DAPI (blue). To assess the anticancer
Similar spectroscopic titrations were also carried out using
the homo-polymers poly(dG–dC) and poly(dA–dT) (see
binding isotherms in ESIw); the results showed that the
emission of 1 was again significantly quenched in both cases.
As seen for the titration of stDNA, the major changes occurred
with the addition of 5 P/D for poly(dG–dC) (95% quenched);
while 25 P/D was needed to achieve 70% quenching for
Fig. 3 Agarose gel electrophoresis of pBR322 DNA (1 mg mlꢀ1) after
poly(dA–dT), demonstrating
a clear preference for the
30 min irradiation (2 J cmꢀ2) in 10 mM phosphate buffer, pH 7.4; lane
2+
1: plasmid DNA control; lane 2: Ru(bpy)3 (P/D 20); lane 3: NaN3
binding of 1 to poly(dG–dC). This was further confirmed by
fitting the changes in the absorption and the emission spectrum,
which for the former resulted in Kb = 1.35 ꢁ 107 Mꢀ1 (ꢂ0.55)
control; lane 4: 1 in the dark (P/D 10); lanes 5 and 6: 1 (P/D 20, 10)
(l 4 400 nm); lanes 7 and 8: 1 (P/D 20, 10) (l 4 320 nm); lanes 9 and
10: 1 + 10 mM NaN3 (P/D 20, 10). See further information in ESIw.
Fig. 2 Changes in the absorption spectrum of 1 (10 mM) with
increasing concentration of stDNA (0–172 mM). Inset: the
corresponding changes in the fluorescence emission spectrum of 1
(10 mM) (lex 310 nm). The blue and the red spectra indicate the
beginning and the end point of the titrations.
Fig. 4 Confocal laser scanning microscopy live cell images of 1
(10 mM) with HeLa cells. Shown is the image obtained with cells (A)
stained with 1 (green) and (B) overlay of 1 (green), nuclear co-stain
DAPI (blue) and the bright field view after 24 h of incubation.
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potential of 1, its antiproliferative effects were assessed in a
number of malignant cell lines, including HeLa cells, the
mesothelioma cell lines ONE58 and CRL5915 and the
Burkitt’s lymphoma cell lines, DG-75 and MUTU-I. Fig. 5A
illustrates that 1 (100 mM) was found to reduce the viability of
these malignant cells by approximately 50–60% in the dark
and by 80–90% in the light indicating both a dark and a light-
readily taken up by various cancer cell lines, and that 1 induces
cell death, which is enhanced upon photoactivation.
We thank Science Foundation Ireland (SFI RFP 2009),
HEA PRTLI Cycle 4, The Irish Research Council for
Science, Engineering and Technology (IRCSET Postgraduate
Studentship to RPBE) and TCD (Postgraduate Award to ME)
for financial support.
dependent cytotoxic death effect (IC50 values for
1
were determined to be between 52–200 mM in the dark, and
34–47 mM under light irradiation in these cell lines). This
cytotoxicity occurs at a similar potency to the well known
platinum-based chemotherapeutic cisplatin (shown as C in
Fig. 5A).7b Further studies carried out using propidium iodide
FACS (fluorescent activated cell sorter) analysis, which detects
the proportion of apoptotic bodies in the pre-G1 phase of the
cell cycle,21 also suggest that 1 induces light-dependent
apoptosis in HeLa cells (Fig. 5B). This indicates that
cytotoxicity may involve a programmed cell death mechanism.
These results clearly demonstrate that these small molecular
weight agents have the potential to be used as novel analogues to
classical photodynamic therapeutic (PDT) drugs, as has also
recently been demonstrated by O’Shea et al.3
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Fig. 5 (A) The antiproliferative effects of 1 (100 mM) on the malignant
cell lines CRL5915, HeLa, ONE58, MUTU-I and DG-75. 1–5 ꢁ 104
cells 200 mlꢀ1 were seeded and treated with cisplatin (C) (10 mM) or 1
for 24 h, irradiated with 4 J cmꢀ2 light and incubated for 24 h. Alamar
Blue reagent (10 ml) was added to measure cell viability at 590 nm
(excitation 544 nm). Values represent the mean ꢂ SEM of six data
points and untreated cells represented 100% cell viability. (B)
Compound 1 (100 mM) induces light-dependent apoptosis in HeLa
cells. 0.25 ꢁ 106 cells were treated with 1 (100 mM) for 24 h, irradiated
with 4 J cmꢀ2 light, incubated for 24 h, harvested by centrifugation and
fixed in 100% ethanol. FACS analysis was carried out upon incubation
with propidium iodide and RNAse A. 10 000 cells were counted using
appropriate gates. Values represent the mean ꢂ SEM of three
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1 were statistically
compared to control cells by unpaired T-test using GraphPad prism
Apoptosis: A User’s Guide, ed. Z. Darzynkiewicz, X. Li,
T. G. Cotter and S. J. Martin, Cytometry, London, U.K., 1996.
software. * = p o 0.05.
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688 Chem. Commun., 2011, 47, 686–688
This journal is The Royal Society of Chemistry 2011