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ences in pH dependent affinity as well as in the fluorimetric
response. However, fluorimetric titrations showed a very
complex pattern of interactions between 5 and DNA/RNA, with
no straightforward correlations with the structure–activity
relationship. Thus for a more detailed structural analysis of
the complexes formed, CD spectroscopy was applied.8,9 It
should be noted that due to its weak intensity the CD spec-
trum of 5 (ESI†) could be accurately subtracted from CD
spectra of 5/DNA complexes. The CD spectrum of ds-RNA
upon titration with 5 did not change significantly (ESI†). The
absence of any ICD bands was attributed to the lack of one
dominant and well-structured binding mode of 5 with respect
to the chiral axis of the RNA double helix.
In contrast to ds-RNA, the addition of 5 to any of the ds-
DNA studied at either pH 5 or pH 7 resulted in pronounced
changes in the CD spectra. Common for all studied DNA was a
moderate decrease of the CD band of the DNA itself at λ =
245–250 nm, which is characteristic for a distortion of the
double stranded helix. For both GC- and AT-DNA isoelliptic
points λ < 265 nm supports one dominant type of DNA struc-
ture in the complex. However, at λ > 265 nm the observed
changes in the CD spectra were strongly influenced by the
Fig. 1 Normalised fluorescence changes of 5 upon titration at pH 7.0
with poly(dG-dC)2 ( , c = 4.7 × 10−6 M), poly A–poly U ( , c = 4.0 × 10−6
■
M), poly(dA-dT)2
(
, c = 1.0 × 10−7 M), Na cacodylate buffer, I = 0.05 M.
DNA basepair composition. Spectral changes at
λ
=
265–300 nm arise from several spectroscopically active species
(free and bound DNA, ICD effects of bound 5). The individual
contributions of each species could not be differentiated from
one another.
A more detailed analysis revealed that the CD spectrum of
poly dGdC–poly dGdC significantly changed at λ > 275 nm
upon addition of 5 (Fig. 3, up) due to the appearance of a
strong ICD band at 290–313 nm. As this is the absorption
band of the GCP moiety, this ICD band can be attributed to
the positioning of the GCP chromophore along one of the
DNA grooves.9,10,12 Furthermore, the absence of any ICD band
> 330 nm suggests that the pyrene chromophore is not bound
in the minor groove but rather outside of the GC-DNA. Most
likely steric hindrance between the amino groups of guanine
in the minor groove and the sterically demanding linker pre-
vents alignment of the pyrene within the groove. A strongly
non-linear dependence of the ICD intensity at 290 nm pointed
to a saturation of the dominant binding site of the GCP moiety
at r[5]/[dGdC–dGdC] = 0.3.
In contrast to GC-DNA, the addition of 5 to poly dAdT–poly
dAdT (Fig. 3, down) resulted in a decrease and a bathochromic
shift of the CD band of the DNA at λ = 263 nm. Two positive
ICD bands appeared at 308 and 350 nm, which agreed nicely
with UV/vis maxima of 5 bound to the poly dAdT–poly dAdT
complex (ESI,† Table S2). The ICD band at 350 nm is specific
for the 5/AT-DNA complex as this band is not observed in the
5/GT-DNA complex. This band can be attributed to the
uniform positioning of pyrene within the DNA double
helix.10,12 Furthermore, the positive sign of this ICD band
suggests that the pyrene is either positioned along the minor
groove9 or partially intercalated with its long axis oriented per-
pendicularly to the long axes of the adjacent DNA basepairs.9
For the accurate differentiation between these two binding
Fig. 2 Normalised fluorescence changes of 5 upon titration at pH 5.0
−7
with poly(dG-dC)2 ( , c = 1 × 10 M), poly A–poly U ( , c = 1 × 10−7 M),
■
poly(dA-dT)2
(
, c = 1.0 × 10−7 M), Na cacodylate buffer, I = 0.05 M.
Table 1 Binding constants (log Ks), ratios n = [bound compound]/[poly-
nucleotide] of and with ds-polynucleotides calculated from
5
2
fluorimetric titrations at pH 7 and pH 5 (buffer sodium cacodylate,
I = 0.05 M).a
dAdT–dAdT
log Ks (n)
dGdC–dGdC
log Ks (n)
rA-rU
log Ks (n)
pH 7
pH 5
5
7.3 (0.3)
6.6 (0.2)
5.3 (0.2)
6.5 (0.3)
5.4 (0.9)
5.7 (0.2)
6.4 (0.1)
6.3 (0.2)
5.0 (0.9)
b
2c
5
d
2c
5.4 (0.2)
a Titration data were processed using the Scatchard equation,14
accuracy of the obtained n 10 − 30%, consequently log Ks values vary
in the same order of magnitude. b Aggregation occurred. c Published
results.11,12 d Too small changes for accurate data calculation.
emission response is not proportionally related to the DNA/
RNA binding affinity. Most intriguingly, at pH 5 the selectivity
of 5 for a different ds-DNA is reversed (Table 1). For ds-RNA no
quantitative binding data could be obtained as negligible
fluorimetric changes hampered data processing.
Comparison of the binding parameters with those of the
previously studied analogue 2 revealed several distinct differ-
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Org. Biomol. Chem.