Photophysical and biological investigation of novel luminescent Ru(ii)-polypyridyl-1,8-naphthalimide Troeger's bases as cellular imaging agents

Article abstract of DOI:10.1039/c2cc17274g

The synthesis and photophysical properties of 1 and 2, two Ru(ii)-polypyridyl based-1,8-naphthalimide Troeger's bases, are described; these were found to stabilize double stranded DNA, undergo rapid cellular uptake, displaying good luminescence without affecting cell viability even after 24 hours of incubation. This journal is The Royal Society of Chemistry 2012.

Full text of DOI:10.1039/c2cc17274g

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Photophysical and biological investigation of novel luminescent
¨
Ru(II)-polypyridyl-1,8-naphthalimide Troger’s bases as cellular imaging agentsw
Robert B. P. Elmes,ac Marialuisa Erby,bc Sandra A. Bright,bc D. Clive Williams*bc and
Thorfinnur Gunnlaugsson*ac
Received 22nd November 2011, Accepted 13th January 2012
DOI: 10.1039/c2cc17274g
The synthesis and photophysical properties of 1 and 2, two
¨
Ru(II)-polypyridyl based-1,8-naphthalimide Troger’s bases, are
described; these were found to stabilize double stranded DNA,
undergo rapid cellular uptake, displaying good luminescence
without affecting cell viability even after 24 hours of incubation.
Troger’s base was first discovered in 1887, formed upon reaction
¨
of para-toluidine with formaldehyde under acidic conditions.1
This is a unique structural unit possessing a C2 axis of symmetry,
as well as being chiral due to two bridgehead stereogenic
nitrogen atoms. Formed as a racemic mixture, it is highly
strained, where the two aryl groups are close to being orthogonal
to each other.2,3 Because of this, Troger’s bases have a ‘cleft-like’4
¨
structure, which have found their use in supramolecular
chemistry;4–6 where they have been employed as a structural
scaffold for the formation of metallo-cages,7 -heterochiral rhombs,8
-aggregates,9 -organic-frameworks (MOFS)10 and -helicates.11 They
have also been used as building blocks in molecular tweezers,12
Scheme 1 The synthesis of the bis-Ru(II)polypyridyl naphthalimide
complexes and 2. (i) Paraformaldehyde, TFA. (ii) Ru(bpy)2Cl2,
1
calixarenes,13 and various other rigid scaffolds.14 Troger’s
¨
DMSO/H2O, microwave irradiation (See full experimental details in ESIw).
bases have also been used in the development of novel
luminescent materials,15 and for probing DNA structure, as
we16 and others17 have demonstrated, but recently, Kirsch-De
Mesmaeker et al.18 formed the first example of Ru(II) polypyridyl
to Ru(II)polypyridyl complexes.19d Herein we present the synthesis
and photophysical, cellular uptake and toxicity studies of two novel
bis-Ru(III)(bpy) Troger’s bases, 1 and 2, Scheme 1, derived from
¨
3
complexes of Troger’s bases as potential DNA binders.18
the 4-amino-1,8-naphthalimides 3 and 4, and we demonstrate their
application as MLCT luminescent cellular imaging agents.19a,21,22
The synthesis of 1 and 2 was achieved in two steps from
3 and 4, respectively, in an identical manner (the synthesis of
4 has previously been reported by us19d) and 3 was formed
similarly (see ESIw). After reacting 3 with 1.5 equivalents of
formaldehyde in neat TFA for 12 hours at room temperature,
the reaction mixture was treated with 6 M NaOH solution,
which resulted in the formation of a yellow precipitate. This
solid was further purified by precipitation from DMSO
¨
Many examples of Ru(II) complexes as DNA targeting binders
and as luminescent probes have been developed to date.19 These
often bind via an intercalation mode, as recently demonstrated by
Kelly, Cardin and co-workers, using X-ray crystallography of a
[Ru(TAP)2(dppz)]2+ complex intercalating into an oligonucleotide
sequence.20 While such complexes have often been shown to have
high binding affinity for DNA their potential in vivo has only
recently been accessed in any detail.21,22 Concomitantly, we have
developed many examples of naphthalimide based supramolecular
structures,23 which absorb and emit within the visible regions,
and we have shown that these can be conjugated as antennae
solution upon addition of MeOH, giving Troger’s base 5 in
¨
68% yield as a racemic mixture. In an identical manner, the
reaction of 4 gave 6 in 70% yield. Both compounds were fully
characterised (see full detail in ESIw). The reaction of 5 with
Ru(bpy)2Cl2, in a mixture of DMF/H2O (50 : 50) at 140 1C,
using a microwave-assisted synthesis, for 40 minutes, resulted in
the formation of an orange coloured solution, from which the PF6
salt of 1 was isolated by precipitation from H2O using NH4PF6.
Recrystallization by diethyl-ether diffusion into a CH3CN
solution gave 1 as a deep-red solid in 68%, while 2 was formed
a School of Chemistry, Centre for Synthesis and Chemical Biology,
Trinity College Dublin, Dublin 2, Ireland. E-mail: gunnlaut@tcd.ie;
Fax: +353 1671 2826; Tel: +353 1896 3459
b School of Biochemistry and Immunology, Trinity College, Dublin 2,
Ireland. E-mail: clive.williams@tcd.ie
c Trinity College Biomedical Sciences Institute, Dublin 2, Ireland
w Electronic supplementary information (ESI) available: Synthesis,
22 figures and 2 tables. See DOI: 10.1039/c2cc17274g
c
2588 Chem. Commun., 2012, 48, 2588–2590
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in 63% yield using an identical procedure. Both complexes
were characterised using conventional methods (see ESIw).
Being synthesised as their chloride salts (stirring in Amberlite
Cl-form), both 1 and 2 were fully water-soluble, and as such all
photophysical analyses were carried out in pH 7.4 (phosphate)
buffered solutions (the photophysical analyses of the precursors
5 and 6 were carried out in CH2Cl2, see ESIw for full details).
The UV-Vis absorption spectrum of 1 is shown in Fig. 1, being
similar to 2 (ESIw), consisting of two bands at 240 nm and
290 nm while the absorption region between 350 nm and 500 nm
is composed of a single broad absorption band composed of
maxima at 350 nm, 400 nm, 430 nm and 460 nm. The bands at
240 nm and 290 nm are characteristic of p–p* intra-ligand
transitions within the bipyridine ligands while the less intense
bands between 350 nm and 500 nm are attributed to the
combination of ICT transitions within the naphthalimide
portion of 1 and 2, and the MLCT transitions associated with
the Ru(II) centres at 430 nm and 460 nm. Excitation into all of
the aforementioned absorption bands of 1 and 2 resulted in the
changes were observed, where in the UV/Vis absorption
spectra of 1, a small red shift coupled with 8% hypochromism
at 400 nm and 6% hypochromism at 460 nm was observed for
P/D 0 - 50 (see ESIw). For 2 a slightly larger change of 13%
hypochromism at 400 nm, and 10% hypochromism at 460 nm
was observed (see ESIw). Similarly, the effect on the luminescence
intensity upon the addition of stDNA to solutions of 1 and 2 over
the same range (P/D 0 - 50) was minor (see ESIw). In contrast
the Ru(II)(bpy)3 complex of 4 gave rise to large changes in their
absorption and the emission spectra. This clearly demonstrates
that the Troger’s base moiety in 1 and 2 had significant effect on
¨
the ability of these structures to bind to DNA in a classical groove
or intercalation fashion.
Due to these relatively small spectral changes observed above,
we were unable to determine the binding constants from these
interactions accurately. Nevertheless, DNA denaturation (Tm)
studies using 1 and 2 clearly showed significant changes in the
melting behaviour of stDNA in the presence of both, see Fig. 2
for P/D = 10. Hence, while the binding of 1 and 2 to DNA did
not result in any major alternation in their photophysical
properties, both were able to stabilize the helical structure of
DNA to a large degree. Importantly, for both P/D ratios, 1
was observed to give rise to greater stabilisation over that seen
for 2, cf. Fig. 2 (and ESIw). This is most likely due to the
structural difference between the two systems, where orientation
3
appearance of MLCT based emission centred at ca. 615 nm;
the absence of any 1,8-naphthalimide associated emission
suggesting that sensitisation of the MLCT excited state occurs
through some mixing of low-lying isoenergetic triplet states,
3
3MLCT and Nap, as described by Castellano et al.24 as the
ICT emission centred at 505 nm overlaps with the 1MLCT
absorption of the Ru(II) centre. Similarly, the fluorescence
quantum yields measured for 1 and 2 were found to be identical
with FMLCT = 0.015 (Æ10%) but significantly lower than that
measured for the parent [Ru(bpy)3]2+ (FMLCT = 0.028) again
suggesting an interaction between the 1,8-naphthalimide and the
3MLCT triplet state of the Ru(II) centres. Excited state lifetime
measurements (tem) were also obtained in with tem = 187 ns and
174 ns for 1 and 2, respectively; indicating similar photophysical
behaviour.
of the two Ru(II) centres is with respect to the Troger’s base
¨
framework. In order to probe this interaction more thoroughly
and to evaluate the role of electrostatic interactions in the
binding of 1 and 2 to DNA, Tm studies were also undertaken
at varying concentrations of NaCl (25 mM and 50 mM). In the
absence of 1 and 2, the Tm for stDNA was determined as 76 1C
in the presence of 25 mM NaCl; but this value was increased
significantly in the presence of 1 and 2, where the melting
transition, once more, had not fully completed at 90 1C for 1
(see ESIw). Similar results were observed for 1, at 50 mM NaCl,
while Tm for 2 was shifted to 82 1C. It is clear from these
results that the interaction of 1 and 2 with DNA is highly
dependent on the ionic strength of the medium, and although
these interactions play a major role in the binding process, it
appears that there are other factors affecting the association of
these complexes with DNA.
As has been observed in the study reported by Veale et al.16
significant modulations of the photophysical characteristics of
Troger’s base analogues have been observed upon interaction
¨
with DNA suggesting a strong binding affinity of these
structures for the DNA biomolecule. With the inclusion of
the cationic Ru(II) polypyridyl centres we envisaged that both
1 and 2 may also bind to DNA and as such, UV/Vis absorption
and emission spectroscopy titrations with stDNA were initially
carried out to probe this interaction. Surprisingly, only minor
Because of the minor changes observed in the photophysical
properties of 1 and 2 upon binding to DNA, their potential to act
as luminescent cellular probes were next evaluated. Populations
of live HeLa cervical cancer cells (1 Â 105) were incubated
Fig. 1 The UV/Visible absorption, excitation and emission spectra of
1 in 10 mM phosphate buffer at pH 7.4. Inset: The UV/Vis absorption
region between 300 nm and 600 nm.
Fig. 2 Thermal denaturation curves of stDNA (150 mM) in 10 mM
phosphate buffer, pH 7.4, in the absence ( ) and presence of 1 ( ) and
2 ( ) at P/D = 10.
c
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Fig. 3 Confocal laser scanning microscopy live cell images of 1 and 2
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an imaging and potential therapeutic agent.
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In summary we have developed novel bis-Ru(II)(bpy) Troger’s
¨
3
bases 1 and 2, which have been found to bind to DNA and undergo
rapid cellular uptake, being internalised after just 2 hours and
highly fluorescent while displaying no effects on cellular viability.
We thank Science Foundation Ireland (SFI RFP 2009 and
SFI 2010 PI awards), HEA PRTLI Cycle 4, The Irish Research
Council for Science, Engineering and Technology (IRCSET Post-
graduate Studentship to RPBE) and PROGRAMMA MASTER
AND BACK—Regione Autonoma della Sardegna-2009—Alta
formazione (ME) for financial support. We also thank Dr Susan
J. Quinn and Dr Emma B. Veale for their helpful discussion.
Notes and references
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c
2590 Chem. Commun., 2012, 48, 2588–2590
This journal is The Royal Society of Chemistry 2012