Evaluation of the DNA-Binding and DNA-Photodamaging
Properties of Naphthoquinolizinium Derivatives
Heiko Ihmels,a,1 Milena Bressanini,b Giampietro Viola,b Daniela Vedaldib
aInstitut
für Organische Chemie, Universität Würzburg, Am Hubland, D-97074 Würzburg,
Germany, Telefax: +49 (0)931 888 4606; E-mail: ihmels@chemie.uni-wuerzburg.de;
bDepartment
of Pharmaceutical Sciences, University of Padova, Italy
The non-covalent binding of cationic aromatic compounds to DNA has attracted considerable
interest.1 The change of absorption or emission properties of such compounds on DNAbinding may be used to detect or characterize the nucleic acid.1 Moreover, irradition in the
chromophore absorption region often leads to photoinduced DNA damage, i.e. to
photonuclease activity,2 a property that may be applied in phototherapy.3
Among the compounds investigated along these lines are heterocycles with a quinolizinium
structure such as coralyne (1)4 and the related heterocyclic quinolinium derivative 2.5
Moreover, we have observed that the benzoquinolizinium derivatives (acridizinium) 3a,b and
indoloquinolizinium 4 exhibit DNA-binding and DNA-damaging properties.6 However, other
examples for DNA-binding quinolizinium derivatives with photonuclease activity are still
rare.5b During our studies of the influence of the substitution pattern on the DNA interaction
of quinolizinium derivatives, we became interested in the known naphthoquinolizinium
derivatives 5a,b7 The investigation of these compounds may allow to evaluate the influence
of the position of the postive charge on the DNA interactions. Moreoever, we anticipated, that
the presence of a fourth aromatic ring in quinolizinium drivatives 5, in comparison to the
tricyclic acridizinium salts 3, enhances the interaction between DNA base pairs and the
tetracyclic system. Herein, we present the preliminary investigations of the interactions of
naphthoquinolizinium bromides 5a and 5b with DNA.
2
OCH 3
H 3CO
OCH 3
N
HN
CH 3
N
OCH 3
N
CH 3
CH 3
1
2
H
N
R1
N
N
R2
1
Y
X
2
3a: R = NH 2 ; R = H
3b: R 1 = H; R 2 = NH 2
3c: R 1 = R 2 = H
4
Br
5a: X = N, Y = H
5b: X = H, Y = N
Interaction of quinolizinium salts 5a,5b with DNA followed by absorption and emission
spectroscopy. The naphthoquinolizinium bromides 5a and 5b were synthesized according to
literature procedure.7 They are yellow solids and their solutions exhibit a resolved absorption
band for the long-wavelength S0-S1 transition in the UV/vis region (Table 1). Both
compounds exhibit a broad fluorescence band on excitation with moderate fluorescence
quantum yields (Table 1). Neither absorption nor emission of naphthoquinolizinium salts
5a,5b are solvent dependent.
Table 1. Absorption and emission data of naphthoquinolizinium bromides 5a and 5b
5a
Solvent

(abs)a
5b
log 
[nm]

(em)b

(em)c
[nm]
 (abs)a
log 
[nm]
 (em)b
 (em)c
[nm]
Bufferd
402
3.84
418
0.13
391
4.34
422
0.26
H2O
402
3.80
419
0.16
392
4.31
422
0.32
MeOH
403
3.90
420
0.18
393
4.33
422
0.48
CH3CN
402
3.87
420
0.20
392
4.36
421
0.39
a
c = 10–4 mol/L, S0-S1 transition, maximum at longest wavelength. – b ex (5a)= 380 nm; ex (5b) = 370 nm;
c = 10–5 M. – c Relative to quinine sulfate in 1 N H2SO4; error: ±0.02. – d 10 mM phosphate buffer (pH = 7).
3
The addition of calf thymus (ct) DNA to an aqueous solution of salts 5a,5b was monitored by
absorption and emission spectroscopy. The spectrophotometric titration revealed a profound
interaction of the quinolizinium salts 5a and 5b. With an increasing DNA concentration a
decrease of the absorbance was observed, along with a significant red shift of 10 nm (5a) and
11 nm (5b) and a partial loss of the fine structure (Figure 1 and 2). Such a perturbation of the
4
4
B
A
Abs. 2
Abs. 2
0
0
280
330
380
Wavelength / nm
430
280
330
380
430
Wavelength / nm
Figure 1. Spectrophotometric titration of naphthoquinolizinium 5a (A) and 5b (B) with ct
DNA in aqueous buffer solution ([dye] = 10–4 M, V = 2 mL; titration interval: 2 x 10–7 moles
of DNA (in bases); 10 mM phosphate buffer)
absorption spectra on DNA addition usually indicates the strong association of a cationic dye
with DNA.1,8 Moreover, isosbestic points were detected (5a: 411 and 345 nm; 5b: 398 and
340 nm) in each case which indicate that one type of quinolizinium-DNA complex is formed
almost exclusively. Mostly remarkable is the observation, that on addition of DNA to
quinolizinium salts 5 a significant red shift appears, whereas these compounds do not exhibit
any solvatochromic behaviour. By contrast, the benzoquinolizinium bromide 2c does not
exhibit such a significant change of the absorption bands on DNA addition.6a Thus, it may be
proposed that the significant perturbation of the electronic structure of the chromophore
results from a strong binding interaction between DNA and the dye.
The fluorescence intensity of naphthoquinolizinium salts 5a,5b is significantly quenched by
addition of DNA (Figure 2), however, essentially no shift of the emission maximum was
4
4
3
I0 /I
2
1
0
60
120
180
[DNA] / M
Figure 2. Stern-Volmer plot from fluorometric titration of naphthoquinolizinium salts 5a and
5b with ct DNA in aqueous buffer solution (5a ( ): ex = 345 nm; 5b ( ): : ex = 340 nm;
c(5a) = c(5a) =10–5 M, 10 mM phosphate buffer)
observed. Since a significant red shift in the absorption spectrum of salts 5 on DNA addition
had been observed, presumably due to a stabilization of the excited state by  stacking with
the nucleic bases, a similar bathochromic shift may be expected in the emission spectrum.
However, since no such effect has been observed on fluorometric titration, it may be
concluded that the emission of the bound naphthoquinolizinium molecule is totally quenched
and the observed reduced fluorescence results solely from the free dye molecule. A SternVolmer plot derived from the fluorometric titration exhibits linear behaviour of the titration
curve of salt 5b, whereas the one of isomer 5a is linear at DNA concentrations up to 60 M
(Figure 3). From these data, quenching efficiencies were estimated by the Stern-Volmer
costant KSV, which is also a rough estimate of the binding constant at static quenching. The
quenching constants (5a: KSV = 8600 M–1; 5b: 5700 M–1) reveal that the
naphthoquinolizinium derivatives 5a and 5b have an affinity to DNA which comparable to
those of known anthracene derivatives.9
DNA photocleavage. Preliminary experiments showed that irradiation of supercoiled plasmid
DNA (pBR 322) in the presence of the naphthoquinolizinium salts 5 at  = 350 nm under
5
aerobic conditions resulted in single strand breaks, i.e. formation of the open-circular form.
After 15 min of irradiation, 90% and 65% DNA damage were detected by gel electrophoresis
(Figure 3). Under anaerobic conditions, essentially the same extend of strand breaks was
observed, so that it may be concluded that at least under these conditions, an oxygenindepedent DNA-cleavage mechanism takes place.
100
5a
5a
80
5b
60
5b
5a
40
5b
20
0
5 min
10 min
15 min
Figure 3. Photocleavage of plasmid DNA pBR 322 with naphthoquinolizinium salts 5a
(white column) and 5b (black column) under aerobic conditions; data derived from gel
electrophoresis of the photolysate; (irradiation time: 5, 10, and 15 min; max = 350 nm, [pBR
322] = 10 g/mL; [dye] = 0.1 mM)
In summary, we have established with the present work that the naphthoquinolizinium salts
5a,b exhibit pronounced DNA-binding and DNA-photodamaging properties, which offer a
promising basis for extended studies of these readily available heterocycles.
Acknowledgements
This work was generously financed by the Bundesministerium für Bildung und Forschung, the
Deutsche Forschungsgemeinschaft, the Deutscher Akademischer Austauschdienst (Vigoni
programm) and the Fonds der Chemischen Industrie. Constant support and encouragement by
Prof. Waldemar Adam is gratefully appreciated.
6
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