Synthesis and photophysical evaluations of fluorescent quaternary bipyridyl-1,8-naphthalimide conjugates as nucleic acid targeting agents

Article abstract of DOI:10.1080/10610278.2011.638381

A family of organic molecules containing the DNA intercalating chromophores, 4-nitro-and 4-amino 1,8-naphthalimide, conjugated to a diquat derivative by an orthogonal phenyl spacer have been prepared and characterised. Their binding interactions with double-stranded DNA were studied by a variety of spectroscopic techniques. These charged organic compounds are found to exhibit excellent binding affinities to DNA with binding constants comparable to those exhibited by metal complexes.

Full text of DOI:10.1080/10610278.2011.638381

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Synthesis and photophysical evaluations of fluorescent
quaternary bipyridyl-1,8-naphthalimide conjugates as
nucleic acid targeting agents
Gary J. Ryan a , Robert B.P. Elmes a , Susan J. Quinn a b & Thorfinnur Gunnlaugsson a
a School of Chemistry and Centre for Chemical Synthesis and Chemical Biology, Trinity
College, Dublin 2, Ireland
b School of Chemistry and Chemical Biology, Centre for Chemical Synthesis and Chemical
Biology, University College Dublin, Dublin 4, Ireland
Version of record first published: 16 Jan 2012.
To cite this article: Gary J. Ryan, Robert B.P. Elmes, Susan J. Quinn & Thorfinnur Gunnlaugsson (2012): Synthesis and
photophysical evaluations of fluorescent quaternary bipyridyl-1,8-naphthalimide conjugates as nucleic acid targeting agents,
Supramolecular Chemistry, 24:3, 175-188
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Supramolecular Chemistry
Vol. 24, No. 3, March 2012, 175–188
Synthesis and photophysical evaluations of fluorescent quaternary bipyridyl-1,8-naphthalimide
conjugates as nucleic acid targeting agents
Gary J. Ryana, Robert B.P. Elmesa, Susan J. Quinna,b* and Thorfinnur Gunnlaugssona*
aSchool of Chemistry and Centre for Chemical Synthesis and Chemical Biology, Trinity College, Dublin 2, Ireland; bSchool of Chemistry
and Chemical Biology, Centre for Chemical Synthesis and Chemical Biology, University College Dublin, Dublin 4, Ireland
(Received 21 September 2011; final version received 26 October 2011)
A family of organic molecules containing the DNA intercalating chromophores, 4-nitro- and 4-amino 1,8-naphthalimide,
conjugated to a diquat derivative by an ‘orthogonal’ phenyl spacer have been prepared and characterised. Their binding
interactions with double-stranded DNA were studied by a variety of spectroscopic techniques. These charged organic
compounds are found to exhibit excellent binding affinities to DNA with binding constants comparable to those exhibited by
metal complexes.
Keywords: DNA probes; naphthalimide; fluorescence; sensors; supramolecular systems
Introduction
(17–24). In a further development, Thomas et al. have
recently developed quaternarized dppz molecules and their
derivatives, as DNA binders, and shown that these exhibit
excellent binding affinity (25). In their work, the binding
of the extended form of the dppz ligand, dqdppn, to a
deformity in the double helix was noted. Inspired by this
work, we have recently developed the synthesis and
evaluated the binding of related dppz-functionalised
structures to DNA (26). We (14, 15) and others (27)
have also shown that the naphthalimide structure can
function as a strong DNA binder. Based on this work and
our general interest in the use of 1,8-naphthalimide-based
structures as probes and sensors (28), we set out to develop
new 1,8-naphthalimide-based ligands capable of partici-
pating in both DNA intercalative mode of action and
groove binding. Our systems comprise a cationic
bipyridine moiety conjugated to either a 4-nitro- or a 4-
amino-1,8-naphthalimide via a phenyl spacer, at the meta
or the para positions, yielding 1–3 (Figure 1). These bi-
functional structures should bind strongly to DNA through
synergetic action, where the quaternarized bipyridine
(diquat) moiety is expected to result in strong electrostatic
binding to the phosphate backbone of DNA, and hence
groove binding, while the naphthalimide moiety may
participate in DNA intercalation, or possible groove
binding. The relationship between the two DNA binding
motifs caused by the non-conjugated phenyl spacer can be
described as being linear, e.g. 1 or wedged, e.g. 2–3 and
would be expected to influence the binding in a unique
manner, the result of which would be expected to modulate
the photophysical properties of both moieties. Herein, the
synthesis of 1–4, their ability to bind to DNA and various
The rational design and synthesis of molecules capable of
binding DNA is a constantly evolving area of active
research which is driven by the need for new treatments
in diseases such as cancer, itself caused by malfunction
of DNA (1, 2). In particular, molecules that exhibit
multiple interactions through intercalation, groove bind-
ing and external electrostatic binding may allow for
stronger binding, increased selectivity and greater
specificity (3–11). Such systems have been the focus of
our research for a number of years now (10, 12). The
excellent binding properties of polypyridyl transition
metal ion complexes, such as Ru(II), Os(II), Rh(III) and
Cr(III) have led to the interest in the use of metal
complexes as anchors to locate a secondary external
binding group or facilitate an electrostatic interaction (13,
14). Indeed we have used such complexes to prepare a
range of new DNA binders. An example of our design is
the formation of several ‘rigid’ ruthenium tris bipyridyl
naphthalimide complexes that exhibit strong DNA
binding, and photoinduced DNA cleavage upon
irradiation (15). We have also developed mixed f–d
metal ion complexes, the emission of which is modulated
upon binding DNA, both in the visible and near infrared
(NIR) regions of the spectrum (15). Furthermore, in
related research we have also prepared purely cationic
organic naphthalimide cleft-like molecules that provide
shape recognition as DNA binders (16).
The intercalative properties of extended polypyridyl
ligands such as dipyrido[3,2-a:20,30-c]phenazine (dppz)
have been extensively exploited for DNA binding
particularly in Ru(II) and Rh(III) polypyridyl complexes
*Corresponding authors. Email: susan.quinn@ucd.ie; gunnlaut@tcd.ie
ISSN 1061-0278 print/ISSN 1029-0478 online
q 2012 Taylor & Francis
http://dx.doi.org/10.1080/10610278.2011.638381
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176
G.J. Ryan et al.
Figure 1. Family of quaternary bipyridine-conjugated naphthalimides 1–3 studied herein and the reference compound 4, all formed as
their Cl2 salts.
homopolymers are described. To the best of our knowl-
edge, these are the first examples of the use of the 1,8-
naphthalimide units in this manner. These fluorophores,
which absorb strongly in the visible region of the
spectrum (in the case of the 4-amino-1,8-naphthalimides
lmax , 450 nm) and emit at long wavelengths emitting
fluorophores are highly attractive for use as biologically,
and in particular DNA, targeting agents (29).
Results and discussion
Synthesis of compounds 1–4
The title compounds 1–3 were synthesised as outlined in
Scheme 1 (the synthesis of control diquat compound 4 is
presented in Supporting Information). The synthesis of
compound 5 has previously been reported by us (16). The
synthesis of the meta aniline functionalised bipyridine 10,
needed for the synthesis of 11, was easily achieved by
Scheme 1. Synthesis of 1,8-naphthalimide diquat compounds. Reagents and conditions: (a) excess dibromoethane, reflux 4.5 h,
(b) CH3COCO22 , EtOH, 2 M NaOH, 2 M HCl, (c) 2-pyridacyl pyridinium iodide, NH4OAc, H2O under reflux, (d) H2NNH2, Pd/C, EtOH,
(e) EtOH, argon atmosphere, 48 h, (f) H2NNH2, Pd/C, EtOH, (g) excess dibromoethane reflux 48 h.

Supramolecular Chemistry
177
11
14
12
13
N
N
N
10
9
8
7
6
O
O
11
5
1
4
2
3
NO2
10
9.4
9.2
9.0
8.8
8.6
8.4
8.2
8.0
7.8
7.6
(ppm)
Figure 2. The 1H NMR spectrum of 1 (D2O, 400 MHz) showing the aromatic region.
adapting a literature procedure previously reported for the
synthesis of the para analogue (29, 30). This involved the
synthesis of compound 7 (formed from 6, which was
synthesised from 3-nitrobenzaldehyde using an estab-
lished procedure (29)) (1 eq.), 2-pyridacyl pyridinium
iodide (1 eq.) and ammonium acetate (8 eq.) were added to
H2O and the mixture heated at reflux for 5 h. The solid was
filtered, and washed with water and acetone to give the
ammonium salt of the 4-(nitrophenyl)-6-carboxylate-2,20-
bipyridine, 8. This compound was not purified any further,
and immediately heated under vacuum until the evolution
of CO2 ceased. The resulting black solid was dissolved in
EtOAc (150 ml), activated charcoal was added and the
mixture was refluxed for 15 min. The mixture was filtered
through a pad of Celite and the solvent removed under
reduced pressure, to give 9 as a pale yellow solid in 37%
yield. This product was reduced to the corresponding
amino derivative 10 using 10% Pd/C in EtOH, under reflux
for 1 h, followed by the addition of hydrazine mono-
hydrate, and further reflux for 1 h. This gave the desired
product as an off-white solid in 97% yield. This aniline
derivative was reacted with 4-nitro-1,8-naphthalic anhy-
dride in refluxing ethanol solution under inert atmosphere
for 48 h giving 11 as a pale brown solid in 84% yield.
Reduction of 11 using hydrazine monohydride and Pd/C in
refluxing ethanol gave the 4-amino-1,8-naphthalimide
product 12 as an orange solid in 94% yield. The desired 4-
nitro-1,8-naphthalimide structures 1 and 2 were formed by
simply refluxing 5 and 11, respectively, in 1,2-dibro-
moethane for 48 h, followed by isolation of the resulting
precipitate by suction filtration. The collected solids were
washed repeatedly with acetone and ether, to remove any
unreacted starting material, giving 1 and 2 as a brown solid
in high 80% yield as dibromide salts.
In contrast, the synthesis of 3 by this method was
problematic and resulted in the formation of unidentified
decomposition products. However, by reducing the
reaction time to 4.5 h we were able to successfully obtain
3 as an orange solid, albeit in a much lower 1.6% yield.
A number of methods were subsequently explored with the
view of improving the yield of the formation of 3. These
included reduction of the corresponding quarternarized
nitro conjugate 2, by hydrogenation; however, this did not
furnish the desired product. Also the condensation of 10
with 4-amino-1,8-naphthalic anhydride to afford an
amino-substituted ligand precursor 12 directly proved
unfruitful. The bromide counter-ions for 1–3 were
exchanged for chloride using Amberlite ion exchange
resin (Cl2 form). These products were fully characterised,
see experimental. As an example Figure 2 shows the
1
partial H NMR for 1 clearly showing contributions from
the naphthalimide as well as the diquat moiety. For
1
comparison, the H NMR for 3 is shown in Figure S2c
(Supporting Information). For comparison studies, the
control molecule 4 was formed in analogous manner from
4-(4-methylphenyl)-2,20-bipyridne in 92% yield (See
Supporting Information).
Steady-state characterisation
Having successfully synthesised 1–3, their photophysical
properties were characterised. The absorption spectrum of

178
G.J. Ryan et al.
0.30
0.25
0.20
0.15
0.10
0.05
0.00
600
500
400
300
200
100
0
Absorbance
λex 320 nm
λem 530 nm
200
300
400
500
600
700
Wavelength (nm)
Figure 3. The UV/visible absorption spectrum, and steady state emission and excitation spectra of 1 (6.5 mM). All recorded in 10 mM
phosphate buffer at pH 7.
the linear derivative 1, recorded in 10 mM phosphate
buffered solution, is given in Figure 3. Three different
absorption maxima are present, a sharp band at 282 nm,
and two overlapping (but distinguishable) bands with lmax
at 320 and 338 nm, respectively. Comparison of this
spectrum with those obtained for the water soluble
controls, N-(propylammonium)-4-nitro-1,8-naphthalimide
13, and the diquat 4, revealed some differences (Figure S2
4-amino-1,8-naphthalimide which arises due to the
electron donating nature of the substituent (28). However,
other regions of the absorption spectrum were significantly
different than that expected. In particular, the band at
350 nm due to the diquat was found to essentially
disappear with diminished absorption also observed at ca.
319 nm. This suggests that the conjugation has a
significant effect on the absorption properties of the diquat
moiety, which is to be expected given the strong electron
accepting properties of diquat (31).
Supporting Information). The p–p interactions of 4-
*
nitro-1,8-naphthalimide in 1 (338 nm) are found to be blue
shifted from that observed in 13 (354 nm). The diquat
control 4 possesses a broad structured band at approxi-
mately 300 nm and second band at 350 nm. These bands
are also found to be significantly shifted in 1. The control
spectra indicate the higher energy band at 320 nm in 1 to
be diquat based, with the lower energy band (338 nm)
composed of contributions from both the diquat and
naphthalimide centres. This indicates a significant
electronic interaction between the two components. In
the case of the wedged structure 2 (Figure S3 Supporting
Information), the naphthalimide band is further blue
shifted from the position in 13 (See structure also in Figure
S3). The absorption spectrum for amino compound 3
displayed bands at 434 and 311 nm (Figure S4 Supporting
Information). An additive spectrum was also constructed
for 3, using the N-(propylammonium)-4-amino-1,8-
naphthalimide control 14 (16) (Figure S5 Supporting
Information as well as the structure). In this case the 1,3-
phenyl conjugation was found to have very little effect on
the 1,8-naphthalimide-based absorption with a small red-
shift for 3 (lmax ¼ 435 nm) compared to that of 14
(lmax ¼ 433 nm). Thus, this band was assigned to the
internal charge transfer (ICT) absorption character of the
The emission properties of 1–3 and the control
compound 4 were next recorded. Excitation of 1 at lmax
gave a single emission band centred at 530 nm, as shown in
Figure 3. This emission is not characteristic of a 4-nitro-
1,8-naphthalimides, which typically exhibit weak fluor-
escence (ca. 450 nm) due to the electron withdrawing nitro
group (32, 33). The excitation spectrum of 1 indicated the
emission at 530 nm originated primarily from the diquat
centre. Indeed, the excitation of 4 at 319 nm resulted in
emission with two maxima, at 415 and 530 nm (Figure S6
Supporting Information). In light of this the emission in 1
was attributed to the diquat portion of the molecule. The
absence of any emission from the nitro-1,8-naphthalimide
in 1 may be indicative of a single excited state, arising
from mixing of energy levels of the constituent
chromophores, or quenching of the 1,8-naphthalimide
excited state by the attached diquat moiety. The related
viologen moieties, covalently linked to 1,8-naphthali-
mides, have been shown to quench naphthalimide
emission through intramolecular electron transfer (28,
34). In contrast the wedged system 2 displayed quite
different, broad emission, upon excitation, more charac-
teristic of the 4-nitro1,8-naphthalimide emission. This

Supramolecular Chemistry
179
contribution of naphthalimide emission to this spectrum
Investigation of DNA binding with 1–4 absorption
titrations
implies that the degree of interaction between the two
chromophores in this system is less than in 1 with the
wedged arrangement allowing for less effective overlap
but also showing sensitivity of emission to the phenyl ring
substitution. The excitation of the amino analogue 3 at
436 nm resulted in a single emission band centred at
550 nm. The band position is also characteristic of that
previously observed for 4-amino-1,8-naphthalimides (35).
The excitation spectrum also showed the origin of
emission to be from the naphthalimide centre. Excitation
in the region of the diquat moiety at 311 nm also resulted
in emission at 550 nm, though of reduced intensity, and
was accompanied by an additional band ca. 370 nm. Thus
the emission spectra indicate some mixing of the energy
levels of the two components. This is quite different to that
observed for the nitro analogue 2, where little or no
interaction was observed in the excited state. This is most
likely a consequence of ICT dynamics that are sensitive to
the conjugation at the imide position.
The binding of naphthalimides to DNA is typically
accompanied by significant changes in the absorbance
spectra (16, 29) and is known to be influenced by the ring
substituent (38, 39). The absorption spectra of molecules
1–4 were found to undergo significant changes in the
presence of increasing concentrations of DNA 10 mM
phosphate buffer. In all cases (each titration was repeated
several times, and were found to be fully reproducible), the
addition of DNA resulted in hypochroism. For molecule 1
a 25% hypochromic shift was observed (Figure S9
Supporting Information). A comparable change of 21%
was observed for the wedged compound 2 (Figure S10
Supporting Information). However, more dramatic hypo-
chromic changes were found for 3, see Figure 4. Here, a
42% decrease in the naphthalimide centred absorption
band at 450 nm was observed, with an 11% decrease found
in the diquat centred band at 311 nm. Furthermore, the
position of the 450 nm band was red-shifted shift by 11 nm
at high concentrations of DNA.
The redox behaviour of the 4-nitro-1,8-naphthalimide-
diquat conjugates 1 and 2 provided further evidence of the
interaction between the constituent chromophores result-
ing from the 1,3 arrangement of the phenyl spacer. Bridged
bipyridine compounds such as diquat display well-defined
reduction and oxidation processes (36). The 1,8-naphtha-
limides also display interesting electrochemistry depend-
ing on their nature and particular substitution pattern (31,
37). The cyclic voltammograms 1 and 2 were recorded in
aqueous solution (Ag/AgCl in 100 mM phosphate buffer,
at pH 7.0). Compound 1 possessed three reduction
processes (Figure S7 Supporting Information); the peaks at
20.28 and 20.78 V corresponding to sequential reduction
processes at the diquat moiety (36), while the peak at
20.49 V was attributed to the reduction of the 1,8-
naphthalimide. The redox behaviour of 2 was somewhat
different, where two reduction processes were observed
corresponding to reduction of the 1,8-naphthalimide and
the diquat moieties, respectively (Figure S8 Supporting
Information). In this case, the second reduction of the
diquat moiety was not observed. This we propose to be due
to the smaller degree of interaction between the 1,8-
naphthalimide and the diquat moiety, resulting from the
wedged arrangement of this system, in contrast to the
linear arrangement in 1 (which agrees with the results
obtained from the steady-state measurements above). The
oxidation process for 2 occurs at a similar potential
(20.38 V) to that of 1, as can be seen in Supporting
Information. Importantly, in the case of 1 and 2, the first
two reduction potentials demonstrate the ease with which
these structures can be reduced, which is a significant
factor in their ability to lead to redox damage of the DNA
polymer.
The changes in the absorbance spectra were analysed
using the Bard model (39, 40). In all three cases very
strong binding was found, see Table 1, with binding
constants in the range K ¼ 1.7 £ 107 (^0.5) M21 for 1 to
2.3 £ 106 (^0.5) M21 for 3. These binding constants
compare very well with known DNA binders and are
greater than those observed previously for naphthalimide-
based systems (41). Indeed, the binding constant for 1 is
comparable to that determined for the associated
ruthenium polypyridyl complex [Ru(bpy)2(5)]Cl2 which
was found to bind to DNA with a binding constant of
K ¼ 4.5 £ 106 (^0.7) M21 (12).
It is also interesting to note that the control diquat
compound 4 was found to bind strongly to DNA, with a 32%
decrease in the absorption intensity at 355 nm (Figure S11
Supporting Information) and a binding constant of
2.8 £ 106 (^0.5) M21. The binding site size, for 1–3 are
very similar ca. two base pairs (Bps), in contrast a binding
site of five Bps was determined for the control compound 4.
This clearly demonstrates the ability of compounds 1–3 to
intercalatewhile compound 4 is expected to bind externally.
This was confirmed by ionic strength studies. In the case of
4, the titration of concentrated salt solution resulted in
complete reversal of binding at 30 mM NaCl (Figure S12
Supporting Information), while partial displacement was
observed for 1–3 (Figure S13 Supporting Information).
Fluorescence emission quenching studies
All the molecules in the study were found to exhibit room
temperature emission. Excitation of 1 at 336 nm results in
emission centred at 530 nm, as discussed above. This
emission is found to be quenched by 90% in the presence

180
G.J. Ryan et al.
0.10
0.08
0.06
0.04
0.02
0.00
1.0
0.8
0.6
0.4
0.2
0.0
0
1
2
3
Bp/D ratio
300
400
500
600
Wavelength (nm)
Figure 4. Changes in the UV/Visible absorption spectrum of 3 (6.5 mM) upon addition of DNA (0221.45 mM Bps) in 10 mM phosphate
buffer, at pH 7.
of increasing concentrations of DNA, with a small blue
shift being observed, see Figure 5. A similar behaviour
was observed for 4 (Figure S14 Supporting Information†)
indicating that the changes observed for 1 are dominated
by the interaction of the diquat moiety and DNA. The
oxidising nature of the diquat moiety strongly suggests
that quenching of emission is due to electron transfer from
the DNA bases to the diquat centre (42). The efficiency of
this process is improved when the oxidising agent is held
in close proximity to the bases. Hence the extent of
quenching can be taken as a further indication of tight
binding. In contrast to the significant changes seen in
Figure 5, the wedged compound 2 exhibited much smaller
changes in emission upon addition of DNA with 31%
quenching observed (Figure S15 Supporting Information).
Following this, the 4-amino compound 3 was
considered. Titration with DNA resulted in significant
quenching of the naphthalimide emission by 55%, see
utilised models. Nevertheless, the above results show the
potential of our design for probing the binding interaction
of such molecules with DNA. The difference in quenching
behaviour for 2 and 3 also arises due to steric effects of the
ring substituent at the 4th position. This illustrates, that in
addition to the nature of the spacer, the nature of the ring
substituent on the naphthalimide greatly influence the
binding and behaviour of these molecules.
The interaction of 1 and 2 with DNA was evaluated by
using ethidium bromide displacement assay (43) and the
binding constants were determined to be 1.7 £ 107 M21
and 2.4 £ 107 M21, respectively. In the case of the
ethidium bromide titration, the binding constant for 1 is
in agreement with that calculated from UV/visible
titration. However, the value for 2 is considerably removed
from that determined by UV/visible study, being almost an
order of magnitude different. Compound 1 was determined
to bind DNA with higher affinity, as the 1,8-naphthalimide
may intercalate in a manner that is not restricted by the
diquat, which points in the opposite direction, while
binding of 2 to DNA might involve both intercalative
interaction of the 1,8-naphthalimide, but also a consider-
able degree of groove association of the diquat part of the
molecule, permitted by the wedged arrangement. In such a
mode both parts of the molecule may effectively displace
Figure 6, again with only very minor changes in the lmax
.
The changes in the relative emission intensity for the lmax
were plotted against the Bp to DNA-binding conjugate, D
ratio (Bp/D) and are shown as insets in Figures 5 and 6,
respectively. These plots clearly demonstrate similar
binding behaviour. However, we were unable to determine
accurate binding constants from this data, regardless of the
Table 1. Various parameters obtained from the changes in the UV/Visible absorption spectra upon binding to DNA.
No
Hypochroism (%)
Bathochroism
Binding constant K (M21
)
Binding site size n (BPs)
R 2
1
2
3
4
25
21
42
32
–
–
1.7 £ 107 (^0.5)
6.5 £ 106 (^1.8)
2.3 £ 106 (^0.5)
2.8 £ 106 (^0.5)
2.26 (^0.04)
1.95 (^0.06)
1.98 (^0.05)
5.84 (^0.21)
0.99
0.99
0.99
0.99
11 nm
8 nm

Supramolecular Chemistry
181
500
400
300
200
100
0
1.0
0.8
0.6
0.4
0.2
0.0
0
1
2
3
4
5
Bp/D ratio
400
450
500
550
600
650
700
750
Wavelength (nm)
Figure 5. Changes in the emission spectrum of 1 (6.5 mM) (lex 336 nm) upon addition of DNA (0–29.25 mM) in 10 mM phosphate
buffer at pH 7.
DNA bound ethidium bromide and thus the apparent
affinity from the displacement assay is higher.
AT or GC rich DNA. The titration profiles resulting from
the addition of [poly(dG–dC)]2 and [poly(dA–dT)]2 to
these compounds are shown in Figure S16 (Supportive
Information).
Studies of 1–4, in the presence of different DNA
alternating co-polymers, were also carried out with the aim
of establishing whether these compounds displayed any
sequence selectivity in their binding. However, none of the
systems displayed a marked preference in affinity for
either [poly(dA–dT)]2 or [poly(dG–dC)]2. Furthermore,
these compounds in general did not display any marked
differences in their fluorescence spectra upon binding to
Thermal denaturation studies
Having determined that 1–3 bind to DNA with significant
changes in their spectroscopic properties we next
400
300
200
100
0
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0
1
2
3
Bp/D ratio
450
500
550
600
650
700
750
800
Wavelength (nm)
Figure 6. Changes in the emission spectrum of 3 (6.5 mM) (lex 436 nm) upon addition of DNA (0–21.45 mM) in 10 mM phosphate
buffer, at pH 7.

182
G.J. Ryan et al.
Table 2. Thermal denaturation data for compounds 1–4 (all
reported with an error or ^0.58C).
1.0
0.8
Calf-thymus [poly(dA–dT)]2 [poly(dA–dT)]2
buffer
Sample
(Bp/D ¼ 5)
Absence
buffer
(Tm/8C)
buffer þ 50 mM
(,Tm/8C)
NaCl (,Tm/8C)
0.6
,69
.75
.75
,72
,75
47
64
70
58
–
59
66
70
67
–
0.4
1
2
3
4
0.2
0.0
–0.2
expected to play an important role in any external binding;
for this reason, measurements under conditions of
increased ionic strength are desired. These were facilitated
by the low temperature melting transition of [poly(dA–
dT)]2 which had 598C in the presence of 50 mM NaCl
(in 10 mM phosphate buffer). Significant changes in the
melting profile were still observed for 1–3 probed under
these conditions, see Figure 8(b). The greatest changes
were again observed for the wedged nitro compound 2
(DTm ¼ 108C). In this case, a single phase was observed in
the melting profiles implying the loss of one of the binding
modes. Thus, the increased ionic strength is expected to
have inhibited an electrostatic mode of interaction.
The melting studies of 1–4 illustrated the high affinity
of all the conjugate systems for DNA, with significant
shifts in Tm values.
50
60
70
80
90
Temperature °C
Figure 7. Thermal denaturation curves of DNA (150 mM) in
10 mM phosphate buffer, at pH 7, in the absence (B) and
presence of 1 at a Bp/D ratio of 10 (X) and 5 (O), and 2 at a Bp/D
ratio of 10 (P) and 5 (V).
evaluated these interactions using denaturation studies, as
these can provide important information regarding the
influence of binding on the stability of the DNA secondary
structure. The presence of both nitro compounds 1 (linear)
and 2 (wedged) were found to cause a dramatic change to
the melting profile, with neither profile reaching a plateau
at 908C, see Figure 7. In these cases a Tm of greater than
75.08C was assigned. The Tm value for DNA in the absence
of the compounds was found to be 698C. The reference 4
was also found to stabilise the double helix DNA, with Tm
values of 73.4 and 75.48C being obtained at a Bp/D ratio of
10 and 5, respectively. However, a very unusual melting
profile was observed for 3 (Figure S17 Supporting
Information). Between 30 and 608C a decrease in
absorbance was observed with an increase observed after
668C. This negative melting curve structure has previously
been associated with increasing DNA structure with
increasing temperature, such as the formation of a duplex.
It is possible that the presence of 3 results in disruption of
regions of the duplex and these undergo change in the 30–
608C temperature range after which the expected influence
of bound 3 is observed (44). These results are summarised
in Table 2. Thermal denaturation studies on 1–3 were also
carried out in the presence of [poly(dA–dT)]2, which has a
Tm of 478C under buffered conditions. All three
compounds were found to cause very large changes in
the observed Tm, see Figure 8(a). While a 16.58C
difference in Tm was found for 1 even greater changes
were found for the wedged compounds, which points to
greater stabilisation in the case of 2 (DTm ¼ 238C).
Furthermore, the melting profile of 2 revealed two regions
of change, which was also observed for 3.
Circular dichroism (CD) and linear dichroism (LD)
studies
To shed further light on the binding mode of these
molecules their interactions with DNA were studied using
both CD and LD. For the former, the titrations were carried
out by varying the concentration of 1–4 in the presence of a
constant concentration of DNA and considering the induced
circular dichroism (ICD) signal, using normal experimental
procedures. Such ICD signals are a very useful tool for the
determination of groove versus intercalative binding (45,
46). In the case of the linear nitro compound 1, increasing
concentration resulted in the appearance of a weak
(,2.5 mdeg) positive ICD signal at 352 nm (Figure S19).
This signal was found to persist at higher salt concentrations
(100 mM NaCl). The greatest changes were observed for
the wedged compound 2, see Figure 9. A larger positive
signal (,5 mdeg) was observed at 350 nm with greater
changes also observed at lower wavenumbers. Again this
signal persisted in the presence of 100 mM NaCl. While the
assignment of a binding mode from changes in the CD
spectrum is not definitive, generally small changes imply
intercalation, whereas larger signals are indicative of
groove binding and proximity to the chiral sugar residues.
The origin of the induced signal at 350 nm for both 1 and 2
could arise due to either the naphthalimide component or
The first-order derivative of the data allowed these
melting transitions to be readily distinguished (Figure S18
Supportive Information). Electrostatic interactions are

Supramolecular Chemistry
183
(a)
(b)
1.0
0.8
0.6
0.4
0.2
0.0
1.0
0.8
0.6
0.4
0.2
0.0
30
40
50
60
70
80
90
40
50
60
70
80
90
Temperature °C
Temperature °C
Figure 8. Thermal denaturation curves of [poly(dA–dT)]2 (150 mM), in the absence (B) and presence of 1(X), 2 (O) and 3 (P) all at a
Bp/D ratio of 5. (a) 10 mM phosphate buffer, at pH 7, (b) 10 mM phosphate buffer þ50 mM NaCl, at pH 7.
the diquat. However, the increase in magnitude may imply
more favourable groove binding interaction of the diquat
component in the case of 2. Significantly, no ICD signal was
observed for the diquat control 4, which has a strong
absorbance in the region 300–350 nm (Figure S20). This
indicates that the presence of the naphthalimide is required
to allow strong associative bonding of the diquat moiety
with the groove.
of the compound. This suggests that structural differences in
groove size in both homopolymers allows for additional
interactions between the sugar environment and chromo-
phores of 1 and 2. Thus, while the strength of binding may
not be very different between the homopolymers the nature
of the binding interactions are somewhat modified.
Interestingly, in the case of 3 an additional ICD band
was observed to grow in at ca. 311 nm, and a less intense
band at ca. 440 nm (Figure S22). Furthermore, in contrast
to the signals observed for 1 and 2, both ICD bands were
negative. From the absorption spectrum (Figure S5), the
band at 311 nm can be seen to originate from the diquat
moiety, whereas the band at 440 nm is assigned to the 1,8-
naphthalimide group. The relative intensities of these
bands imply that the naphthalimide is intercalating and
that the diquat is held close to the sugar groups with some
partial intercalation of the linker group. The negative-
induced signals have previously been observed for
intercalating naphthalimide bi-functionalised spermine
compounds (44) and may arise due to the better
intercalating nature of the amino-substituted naphthali-
mide, as noted previously (38).
Having previously studied the binding of 1–4 to
homopolymers, the CD titrations of 1 and 2 with synthetic
polymers [poly(dA–dT)]2 and [poly(dG–dC)]2 were also
performed (Figure S21 Supporting Information). Large ICD
was observed for both molecules in the presence of the
polymers, particularly in the region above 300 nm, with
ICD greater than that seen upon binding to DNA. The
structure of the ICD band in the visible region was broader
and more closely in agreement with the absorption spectrum
20
DNA
Bp/D 25
Bp/D 15
15
Bp/D 10
Bp/D 5
10
To further investigate this we carried out LD
investigations where compounds 1 and 2 were studied in
the presence of DNA. The changes in the LD spectrum for
both, when recorded in 10 mM phosphate buffer solution
with P/D ratio of 3, are shown in Figure 10. For the linear
structure 1, Figure 10(a), it can be seen that both negative
and positive signals are observed, suggesting that
contribution from both groove binding and intercalation
to DNA for 1. These results support our finding above that
both chromophores are contributing to the overall binding,
though through different modes of interactions. Similarly,
compound 2, the wedged derivative also showed
significant changes in the LD upon binding to DNA,
Bp/D 2.5
5
0
–5
–10
200
250
300
350
400
450
Wavelength (nm)
Figure 9. Circular dichroism curves of DNA (150 mM) in
10 mM phosphate buffer, at pH 7, in the absence and presence of
2 at varying ratios.

184
G.J. Ryan et al.
(a) 0.003
(b) 0.003
0.002
Absorbance
DNA Alone
1 (P/D = 3)
Absorbance
DNA Alone
2 (P/D = 3)
0.002
0.001
0.001
0.000
0.000
–0.001
–0.002
–0.003
–0.001
–0.002
–0.003
200
250
300
350
400
450
500
200
250
300
350
400
450
500
Wavelength (nm)
Wavelength (nm)
Figure 10. Linear dichroism curves of DNA (150 mM) in 10 mM phosphate buffer, in the presence of 1 (a) and 2 (b) shown in blue.
The LD spectra of DNA (in red) and the UV–Vis absorption spectra of both 1 and 2 (in black) are included for reference.
where the contribution from groove binding seems to be
increased in comparison to compound 1, suggesting that
groove binding is the dominant mode of interaction,
confirming the results seen in the CD spectra of 2 above.
Experimental
Materials and instrumentation
All NMR spectra were recorded using 400.13 MHz for 1H
NMR and 100.6 MHz for 13C NMR or 600.1 MHz for H
1
NMR and 150.2 MHz for 13C NMR. Shifts are referenced
relative to the internal solvent signals. High-resolution
mass spectra were determined by a peak matching method,
using leucine Enkephalin (Tyr–Gly–Gly–Phe–Leu), as
the standard reference (m/z ¼ 556.2771). The lumines-
cence quantum yields were calculated by comparison with
[Ru(bpy)3]2þ. CD spectra were recorded at a concentration
corresponding to an optical density of approximately 1.0,
in buffered solutions. All reagents and solvents were
purchased commercially and used without further
purification. Anhydrous solvents were prepared using
standard procedures. Calf thymus and salmon testes DNA
were used in this study, both of which have 33% G:C
solutions of DNA in 10 mM phosphate buffer (pH 7) gave
a ratio of UV absorbance at 260 and 280 nm of 1.86:1,
indicating that the DNAwas sufficiently free of protein. Its
concentration was determined spectrophotometrically
using the molar absortivity of 6600 M21 cm21 (260 nm).
Poly(dA–dT)poly(dA–dT) and poly(dG–dC)poly(dG–
dC) concentrations determined spectrophotometrically
using the molar absortivities of 6600 M21 cm21 (262 nm)
and 8400 M21 cm21 (254 nm), respectively.
Conclusion
In conclusion, we have demonstrated the high binding
affinity of a family of new bipyridyl-derivatised
naphthalimide molecules that exhibit binding affinities
greater than that previously reported for naphthalimide
systems and, in the case of 2, comparable to that of the
parent metal complex. The dramatic impact on the melting
temperature and ICD signals indicate that molecules such
as 1 and 2 interact through intercalation, while LD
measurements support stronger groove binding inter-
actions in the case of 2. This is further supported by the
persistence of behaviour under conditions of increased
ionic strength. Denaturation experiments also reveal
multiple modes of interaction including unusual phenom-
enon that may be due to changes in the secondary structure
of DNA due to the cationic nature of the diquat molecule.
The fluorescence titrations also reveal the different
response of charge transfer sensitive and electron-
mediated, quenching mechanisms to the DNA binding
event. The binding is found to be sensitive to the ring
substituent and the nature of connectivity. Overall the
DNA titrations reveal that both the diquat and naphtha-
limide moieties contribute to the enhanced binding. These
results clearly demonstrate how simple modification (in
this case through quaternarization) of ligands designed to
be employed in DNA binding metal complexes; can
transform them into effective DNA binding ligands in their
own right. These molecules are currently the focus of an
in-depth biological study to evaluate their therapeutic
potential this will be the subject of a future publication.
Synthesis 4-[N-(p-phenyl)-4-nitro-1,8-naphthalimide]-
2,20-ethylenebipyridyldiylium dichloride (1)
4-[N-(p-phenyl)-4-nitro-1,8-napthalimide]-2,20-bipyridine
(0.33 g, 0.70 mmol, 1 eq.) was suspended into 30 ml of
dibromoethane and heated under reflux for 48 h. The
reaction mixture was cooled to room temperature, the
resulting precipitate collected by filtration and washed
with acetone (5 ml) and hexane (5 ml). The product was

Supramolecular Chemistry
185
dried under high vacuum. The product was dissolved in
MeOH and stirred for 1 h with Amberlite ion exchange
resin (Cl2). The suspension was filtered and the
solvent removed under reduced pressure giving the
product as a brown solid (0.32 g, 80%). Calculated for
C30H20F12N4O4P2: C, 45.59; H, 2.55; N, 7.09. Found: C,
45.37; H, 2.46; N, 6.88; accurate MS (m/z) calculated for
C30H20N4O4 (M2þ): 500.1485. Found 500.1478; 1H NMR
dH (D2O, 400 MHz): 9.23 (1H, s, Ar-H), 9.17 (2H, m,
2 £ Ar-H), 9.06 (1H, d, J ¼ 8.0 Hz, Ar-H), 8.87 (1H, m,
Ar-H), 8.63 (1H, d, J ¼ 6.0 Hz, Ar-H), 8.53 (1H, d,
J ¼ 9.0 Hz, Ar-H), 8.49 (1H, d, J ¼ 7.5 Hz, Ar-H), 8.44
(1H, d, J ¼ 8.0 Hz, Ar-H), 8.32 (1H, t, J ¼ 7.0 Hz, Ar-H),
8.23 (3H, m, 3 £ Ar-H), 7.83 (1H, t, J ¼ 7.6 Hz, Ar-H),
7.65 (2H, d, J ¼ 8.0 Hz, Ar-H), 5.30 (4H, d, J ¼ 7.0 Hz,
2 £ CH2); 13C NMR dC (D2O, 100 MHz): 165.4, 164.4,
157.5, 149.8, 148.2, 147.1, 146.9, 139.6, 135.8, 134.8,
133.0, 132.7, 131.4, 130.7, 130.3, 130.0, 129.6, 128.6,
128.4, 128.2, 127.1, 125.7, 125.4, 124.3, 122.9, 121.5,
52.8, 51.7; IR nmax (cm21): 1712 (m, ZCOZNZCOZ),
1523 (m, CZNO2), 1347 (m, CZNO2).
heated at reflux for 1 h. Hydrazine monohydrate (98%)
(2.01 g, 1.95ml, 62.75 mmol, 20 eq.) was added and the
mixture heated at reflux for 1 h. The mixture was filtered
through a pad of celite and the solid washed with CH2Cl2
(40 ml). The filtrate was washed with water, dried over
MgSO4, and the solvent removed under reduced pressure
giving the product as an off-white solid (0.75 g, 97%).
m.p. 131–133 8C; accurate MS (m/z) calculated for
1
C16H14N3 (M þ H): 248.1188. Found 248.1193; H NMR
dH (CDCl3, 400 MHz): 8.70 (1H, d, J ¼ 3.5 Hz, Ar-H), 8.67
(1H, d, J ¼ 15.1Hz, Ar-H), 8.46 (1H, d, J ¼ 8.0 Hz, Ar-H),
7.82 (1H, m, Ar-H), 7.48 (1H, dd, J ¼ 1.5, 5.0 Hz, Ar-H),
7.31 (1H, m, Ar-H), 7.25 (1H, m, Ar-H), 7.13 (1H, d,
J ¼ 7.5 Hz, Ar-H), 7.05 (1H, d, J ¼ 2.0 Hz, Ar-H), 6.74
(1H, dd, J ¼ 1.5, 8.0 Hz, Ar-H), 3.89 (2H, br s, NH2); 13
C
NMR dC (CDCl3, 100 MHz): 156.0, 155.7, 149.1, 148.7,
146.7, 138.9, 136.5, 129.5, 123.4, 121.2, 120.8, 118.5,
116.9, 115.3, 113.1; IR nmax (cm21): 3422 (w, ZNH2
stretch), 1626 (w, ZNH2 bonding).
4-[N-(m-phenyl)-4-nitro-1,8-napthalimide]-2,20-
bipyridine (11)
4-(3-Nitrophenyl)-2,20-bipyridine (9)
4-(3-Aminophenyl)-2,20-bipyridine (0.50 g, 2.04 mmol, 1
eq.) and 4-nitro-1,8-naphthalic anhydride (0.50 g,
2.04 mmol, 1 eq.) were suspended in high performance
liquid chromatography (HPLC) grade ethanol and the
mixture refluxed under an argon atmosphere for 48 h. The
reaction mixture was cooled to room temperature and ether
added to further precipitate product, which was collected by
suction filtration giving the product as a pale brown solid
(0.81 g, 84%). Calculated for C28H16N4O4.0.4H2O: C,
70.11; H, 3.53; N, 11.68. Found: C, 70.27; H, 3.35; N,
11.40; accurate MS (m/z) calculated for C28H17N4O4
(M þ H): 473.1250. Found 473.1268; 1H NMR dH (CDCl3,
400 MHz): 8.81 (1H, s, Ar-H), 8.79 (1H, d, J ¼ 4.5 Hz, Ar-
H), 8.69 (4H, m, 4 £ Ar-H), 8.62 (1H, d, J ¼ 8.0 Hz, Ar-H),
8.46 (1H, d, J ¼ 7.5 Hz, Ar-H), 8.17 (1H, m, Ar-H), 8.07
(1H, s, Ar-H), 8.04 (1H, d, J ¼ 8.0 HZ, Ar-H), 7.98 (1H, dt,
J ¼ 1.5, 8.0 Hz, Ar-H), 7.86 (1H, m, Ar-H), 7.76 (1H, m,
Ar-H), 7.59 (1H, d, J ¼ 8.0 Hz, Ar-H), 7.49 (1H, m, Ar-H);
13C NMR dC (CDCl3, 100 MHz): 163.3, 162.5, 156.1,
155.0, 150.2, 149.3, 149.2, 147.2, 138.0, 137.4, 136.6,
131.7, 130.1, 130.0, 129.9, 129.6, 128.9, 128.8, 127.4,
127.3, 126.8, 124.4, 124.2, 123.4, 122.9, 121.4, 120.6,
117.4; IR nmax (cm21): 1718 (w, ZCOZNZCOZ), 1524
(m, CZNO2), 1350 (m, CZNO2).
(E)-4-(3-Nitrophenyl)-2-oxo-3-butenoic acid (2.13 g,
9.64 mmol, 1 eq.), 2-pyridacyl pyridinium iodide (3.14 g,
9.64 mmol, 1 eq.) and ammonium acetate (5.95 g,
77.12 mmol, 8 eq.) were added to H2O and the mixture
heated at reflux for 5 h. The solid was filtered and washed
with water (2 £ 10 ml) and acetone (2 £ 5 ml) to give the
ammonium salt of the 4-(nitrophenyl)-6-carboxylate-2,20-
bipyridine. The ammonium salt was heated under vacuum
with a heat gun until evolution of CO2 ceased. The
resulting black solid was dissolved in EtOAc (150 ml),
activated charcoal added and the mixture refluxed for
15 min. The mixture was filtered through a pad of celite
and the solvent removed under reduced pressure, giving
the product as a pale yellow solid (0.92 g, 37%). m.p. 107–
1098C; accurate MS (m/z) calculated for C16H12N3O2
(M þ H): 278.0930. Found 278.0925; 1H NMR dH
(CDCl3, 400 MHz): 8.78 (1H, d, J ¼ 5.2 Hz, Ar-H), 8.71
(2H, m, 2 £ Ar-H), 8.59 (1H, m, Ar-H), 8.47 (1H, d,
J ¼ 7.6 Hz, Ar-H), 8.29 (1H, dd, J ¼ 1.2, 8.2 Hz, Ar-H),
7.86 (1H, dt, J ¼ 1.8, 8.2 Hz, Ar-H), 7.68 (1H, t,
J ¼ 7.6 Hz, Ar-H), 7.56 (1H, dd, J ¼ 1.7, 5.3 Hz, Ar-H),
7.36 (1H, m, Ar-H); 13C NMR dC (CDCl3, 100 MHz):
156.6, 155.1, 149.6, 148.8, 148.3, 146.3, 139.6, 136.6,
132.6, 129.7, 123.7, 121.6, 121.0, 120.9, 118.4; IR nmax
(cm21): 1525 (s, CZNO2), 1343 (s, CZNO2).
4-[N-(m-phenyl)-4-nitro-1,8-naphthalimide]-2,20-
ethylenebipyridyldiylium dichloride (2)
4-(3-Aminophenyl)-2,20-bipyridine (10)
4-(3-Nitrophenyl)-2,20-bipyridine (0.87 g, 3.14mmol, 1 eq.)
and 10% Pd/C were added to EtOH (30 ml) and the mixture
Compound 11 (0.25 g, 0.53 mmol, 1 eq.) was suspended
into 30 ml of dibromoethane and heated under reflux for
48 h. The reaction mixture was cooled to room

186
G.J. Ryan et al.
temperature, the resulting precipitate collected by filtration
and washed with acetone (5 ml) and hexane (5 ml). The
product was dried under high vacuum. The product was
dissolved in MeOH and stirred for 1 h with Amberlite ion
exchange resin (Cl2). The suspension was filtered and the
solvent removed under reduced pressure, giving the
product as a brown solid (0.25 g, 82%). Calculated for
C30H20F12N4O4P2: C, 45.59; H, 2.55; N, 7.09. Found: C,
45.82; H, 2.42; N, 7.08; accurate MS (m/z) calculated for
C30H19N4O4 (M2þ): 499.1406. Found 499.1411; 1H NMR
dH (D2O, 400 MHz): 9.14 (1H, d, J ¼ 6.0 Hz, Ar-H), 9.11
(1H, d, J ¼ 6.5 Hz, Ar-H), 9.07 (1H, s, Ar-H), 8.95 (1H, d,
J ¼ 8.0 Hz, Ar-H), 8.79 (1H, t, J ¼ 8.0 Hz, Ar-H), 8.54
(1H, d, J ¼ 8.6 Hz, Ar-H), 8.46 (3H, m, 3 £ Ar-H), 8.28
(1H, m, Ar-H), 8.23 (1H, d, J ¼ 8.0 Hz, Ar-H), 8.11 (1H,
d, J ¼ 8.0 Hz, Ar-H), 8.02 (1H, s, Ar-H), 7.76 (2H, m,
2 £ Ar-H), 7.62 (1H, d, J ¼ 7.5 Hz, Ar-H), 5.26 (4H, d,
J ¼ 8.0 Hz, 2 £ CH2); 13C NMR dC (D2O, 100 MHz):
165.4, 164.4, 157.5, 149.7, 148.2, 147.1, 146.9, 139.6,
139.5, 135.8, 134.8, 133.0, 132.7, 131.4, 130.7, 130.3,
130.0, 129.5, 128.5, 128.4, 128.2, 127.1, 125.8, 125.7,
125.4, 122.9, 121.5, 52.5, 51.6; IR nmax (cm21): 1715 (m,
ZCOZNZCOZ), 1523 (m, CZNO2), 1345 (m, CZNO2).
moethane (30 ml) and the mixture heated at reflux for 4.5 h.
The reaction mixture was cooled to room temperature, the
resulting precipitate collected by filtration and washed with
acetone (5ml) and hexane (5ml). The product was adsorbed
onto a short pad of silica and purified using MeOHZNH3(-
sat). A concentrated solution of NH4PF6 was added which
gave a precipitate that could not be isolated by filtration. The
product was extracted into DMF/CH2Cl2 and the solution
concentrated under vacuum. The product was precipitated
out by dropping into toluene and isolated by filtration. After
drying under vacuum the product was obtained as an orange
solid (0.003g, 1.6%). Accurate MS (m/z) calculated for
1
C30H23N4O2 (M2þ): 471.1821. Found 471.1806; H NMR
dH (D2O, 400 MHz): 9.11 (1H, d, J ¼ 6.0 Hz, Ar-H), 9.00
(1H, d, J ¼ 6.5, Ar-H), 8.93 (1H, s, Ar-H), 8.81 (1H, d,
J ¼ 8.5, Ar-H), 8.69 (1H, m, Ar-H), 8.35 (1H, d, J ¼ 5.5 Hz,
Ar-H), 8.23 (1H, m, Ar-H), 8.17 (1H, d, J ¼ 8.0 Hz, Ar-H),
8.07 (1H, d, J ¼ 8.0 Hz, Ar-H), 8.03 (1H, s, Ar-H), 7.96 (1H,
d, J ¼ 8.6 Hz, Ar-H), 7.90 (1H, d, J ¼ 8.5 Hz, At-H), 7.58
(2H, m, Ar-H), 7.35 (1H, m, Ar-H), 6.54 (1H, d, J ¼ 8.5 Hz,
Ar-H), 5.21 (4H, m, 2CH2); 13C NMR dC (D20, 150 MHz):
166.3, 165.2, 157.3, 153.6, 148.1, 147.2, 146.8, 139.45,
139.38, 137.1, 135.0, 134.3, 133.2, 123.4, 131.2, 130.7,
130.2, 129.7, 128.8, 128.7, 128.2, 126.7, 125.0, 124.4, 120.0,
118.9, 109.4, 106.7, 52.5, 51.5.
4-[N-(m-phenyl)-4-amino-1,8-napthalimide]-2,20-
bipyridine (12)
4-(4-Methylphenyl)-2,20-ethylenebipyridyldiylium
dibromide (4)
Compound 11 (0.30 g, 0.64 mmol, 1.eq.) was dissolved in
dimethyl formamide (DMF) (30 ml) and MeOH (30 ml)
added. To this was added 10% Pd/C (0.05 g) and the mixture
heated at 658C for 1 h. Hydrazine monohydrate (0.41 g,
0.40 ml, 12.74 mmol, 20 eq.) was added and the mixture
heated at 658C for further 16 h. The reaction mixture was
filtered through celite and the solvent removed under
vacuum. The resulting residue was tritrated with water to
remove unreacted hydrazine and the solid isolated by
filtration to give the product as an orange solid (0.27 g,
94%). Accurate MS (m/z) calculated for C28H19N4O2
(M2þ): 443.1508. Found 443.1495; 1H NMR dH
(DMSO[D6], 400 MHz): 8.78 (1H, d, J ¼ 5.5 Hz, Ar-H),
8.69 (3H, m, Ar-H), 8.45 (2H, m, Ar-H), 8.23 (1H, d,
J ¼ 8.5 Hz, Ar-H), 8.00–7.92 (2H, m, Ar-H), 7.84 (1H, d,
J ¼ 5.0 Hz, Ar-H), 7.71 (2H, m, Ar-H), 7.53–7.47 (4H, m,
Ar-H þ NH2), 6.90 (1H, d, J ¼ 8.5 Hz, Ar-H); 13C NMR
dC (DMSO[D6], 100 MHz): 164.5, 163.6, 156.4, 155.4,
153.2, 150.5, 149.6, 147.8, 138.2, 138.0, 137.7, 134.3,
131.5, 130.7, 130.6, 130.1, 129.9, 128.1, 126.6, 124.7,
124.3, 122.7, 121.9, 121.0, 119.9, 117.9, 108.6, 108.2.
4-(4-Methylphenyl)-2,20-bipyridne (0.13 g, 0.53 mmol,
1.eq.) was suspended in dibromoethane (5 ml) and the
mixture heated at reflux for 4 h. The reaction mixture was
cooled to room temperature, the resulting precipitate
collected by filtration and washed with acetone (5 ml) and
ether (5 ml). After drying under high vacuum, the product
was obtained as a yellow solid (0.21 g, 92%). Calculated
for C19H18Br2N2.1.25H2O: C, 49.81; H, 4.55; N, 6.11.
Found: C, 49.94; H, 4.22; N, 6.07; accurate MS (m/z)
calculated for C19H19N2 (M2þ): 275.1548. Found
1
275.1555; H NMR dH (D2O, 400 MHz): 9.13 (1H, dd,
J ¼ 0.8, 6.0 Hz, Ar-H), 9.05 (1H, d, J ¼ 2.3 Hz, Ar-H),
8.99 (1H, dd, J ¼ 1.0, 8.2 Hz, Ar-H), 8.98 (1H, d,
J ¼ 6.52 Hz, Ar-H), 8.83 (1H, dt, J ¼ 1.3, 8.0 Hz, Ar-H),
8.46 (1H, dd, J ¼ 2.0, 6.5 Hz, Ar-H), 8.8 (1H, m, Ar-H),
7.91 (2H, d, J ¼ 8.5 Hz, Ar-H), 7.22 (2H, d, J ¼ 8.5 Hz,
Ar-H), 5.25 (2H, m, CH2), 5.18 (2H, m, CH2), 2.37 (3H, s,
CH3); 13C NMR dC (D2O, 100 MHz): 159.0, 148.2, 147.0,
146.3, 145.0, 140.1, 139.3, 130.6, 130.5, 130.1, 128.4,
128.2, 126.2, 124.8, 52.7, 51.3, 20.7.
4-[N-(m-phenyl)-4-amino-1,8-napthalimide]-2,20-
ethylenebipyridyldiylium dichloride (3)
Absorption and fuorescence titrations
4-[N-(m-Phenyl)-4-amino-1,8-napthalimide]-2,20-bipyri-
dine (0.15 g, 0.34mmol, 1.eq.) was suspended in dibro-
Solutions of calf-thymus DNA in 10 mM phosphate buffer
(pH 7) were found to give a ratio of UV absorbance at 260

Supramolecular Chemistry
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temperature. The intrinsic binding constant K and the
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Thermal denaturation experiments were performed on a
thermoelectrically coupled Varian Cary 50 Spectrometer.
The temperature in the cell was ramped from 20 to 908C, at
a rate of 18C min21 and the absorbance at 260 nm of DNA
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0.28C.
Supporting Information Available
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Acknowledgements
We are grateful to Trinity College Dublin, Science Foundation
Ireland (SFI for RF 2008 and 2009 Programmes) and IRCSET
(postgraduate studentships to G. J. Ryan and R. P. B. Elmes) for
financial support. We also wish to thank Dr Michael E. G. Lyons
and Dr Gareth Keeley for assistance with the electrochemistry.
We particularly would like to thank Prof. John M. Kelly for
valuable discussions and his continuous support. This article is
dedicated to his 65th birthday.
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