Chapter III: Stereoselective interaction of cationic [4]helicenes with DNA
III- 4.3.Time resolved fluorescence studies
74 | P a g e Figure III-11: Fluorescence spectra of (-)-M-nPr2-DMQA with different ratios of base pairs DNA (upon
the addition of DNA, the volume increases by 3%) after excitation at 400 nm.
Upon raising the DNA concentration, the ratio of DNA-bound DMQA vs free DMQA in solution increases, which explains the increase in the fluorescence. And since the aggregated forms do not permit the fluorescence of the molecules, the water solution is less photon emitting.
In summary, as we can see from this study, the interaction of the nPr2-DMQA and Me2 -DMQA chromophores with the double-stranded helical polynucleotide results in several changes in the absorption and fluorescence spectra along with increasing fluorescence quantum yields.
III-4.3.Time resolved fluorescence studies
Excited-state dynamics of the M, the P and the racemic form of each of the studied chromophores were recorded at the maximum of fluorescent band after 400 nm excitation in different solutions (Figure III-12). The fluorescence profiles in the phosphate buffer solution (PBS/EDTA) were reproduced by a biexponential function convoluted with a Gaussian instrument response function. A two exponential decay indicates the presence of two conformations in solution with different lifetimes. However, the contribution of the long lifetime
component to fluorescence kinetics is very small and counts for around 2 to 8 % of the total decay in water (Table III-1).
Figure III-12: Fluorescence decay of the M, the P and the racemic forms of the nPr2-DMQA and Me2 -DMQA in aqueous buffer solution and acetonitrile after 400 nm excitation.
The black lines represent the best fits.
Solvent System Configuration Φfl τ1 (ns) τ2 (ns)
PBS/EDTA
Me2-DMQA
M 0.019 1.67 (0.92) 5.7 (0.08)
P 0.019 1.74 (0.92) 5.68 (0.08)
racemic 0.023 1.54 (0.98) 6.42 (0.02)
nPr2-DMQA
M 0.036 1.91 (0.96) 7.4 (0.04)
P 0.03 1.96 (0.96) 7.28 (0.04)
racemic 0.03 1.98 (0.93) 6.67 (0.07) PBS/EDTA
with DNA
Me2-DMQA M --a 1.65 7.25
P --a 1.75 7.30
racemic --a 1.57 7.21
nPr2-DMQA M --a 1.95 8.74
P --a 1.97 8.22
racemic --a 1.98 8.39
a data not provided.
Table III-1: Fluorescence quantum yields (Φfl) and time constants τ1 and τ2 (expressed in ns) obtained from multi-exponential analysis of the fluorescence decay of Me2-DMQA and nPr2-DMQA in the
absence and the presence of DNA
76 | P a g e We notice from Table III-1, that the fluorescence quantum yield of the nPr2-DMQA is almost twice the value of its Me2-DMQA analog. Also, with increasing DNA concentration, the quantum yields of both structures increase.18
The fluorescence decay changes dramatically when switching solvents from water to acetonitrile. The time profile becomes monoexponential with a lifetime of 5.6 ns (Table III-2).
Solvent System Configuration Φfl τ1 (ns)
EtOH
nPr2-DMQA
M 0.071 6.90
P 0.059 6.90
racemic --a 6.84
ACNb nPr2-DMQA racemic 0.066 5.63
a data not provided; b acetonitrile
Table III-2: Fluorescence quantum yield (Φfl) variation and time constants obtained for the monoexponential analysis of the fluorescence decay of the nPr2-DMQA in EtOH and ACN
This lifetime is slightly shorter than the long-lived component which was observed in aqueous solution.
This means that in water, the chromophores arrange majoritarily as H-aggregates with a fluorescence lifetime of almost 1.5 ns, with a small amount staying as free unaggregated form. In the case of acetonitrile, the latter form is the only one present in solution.
Another interesting observation was detected in the presence of salmon sperm DNA where the fluorescence decays of the different enantiomers and the racemic mixtures of Me2-DMQA and nPr2 -DMQA change significantly (Figure III-13).
It can be reproduced using a biexponential function (a1*exp(-t/τ1)+ a2*exp(-t/τ2)) convoluted with a Gaussian.
The results show that the contribution of the long-lived decay component a2 to the total decay increases upon DNA addition.
18 Oral communication with Oksana Kel; data not provided.
Figure III-13: Intensity-normalized fluorescence decay of the (-)-M -nPr2-DMQA with increasing DNA concentration after 400 nm excitation.
The black lines represent global fits.
For a global analysis of the fluorescence time profiles, the lifetimes were linked and the coefficients a1 and a2 were registered at various DNA concentrations (Figure III-14).
The decrease of a1 with increasing DNA concentration indicated the disappearance of the aggregated form of the helicenes which have a lifetime of around 2 ns. Global analysis of the fluorescence kinetics with linked lifetimes does not describe well the decay of the DMQA derivatives without DNA. The reason for this is probably that the lifetime of the non-aggregated fluorophore in water is not identical to the lifetime of the fluorophore intercalated in DNA, as suspected from the lifetimes found in organic solvents (Table III-2). The lifetime does not only depend on the aggregation but also on the medium or the environment.
78 | P a g e Figure III-14: Fraction of the nPr2-DMQA and Me2-DMQA bound to DNA determined from global
analysis of the fluorescence profiles.
Considering that no free dyes are present in solution, we can describe the affinity of the dyes to DNA using the following equilibrium:
For this equation, the binding constant K, can be obtained as
K DNADNA (1)
Assuming that the total dye concentration is equal to the free dye + the dye bound to DNA.
[dye]total = [dye] + [dyen*DNA]
And that the total concentration of DNA is equal to the unbound free DNA in solution + the bound DNA to the dye.
[DNA]total = [DNA] + [dyen*DNA]
By substituting the concentrations of the free DNA and the free dye in equation (1), we obtain
K dye DNA
dye dye DNA x DNA dye DNA
The concentration of intercalated dye can be obtained from the fit of the fluorescence decays, supposing that the coefficient a2 is proportional to the concentration of the dye bound to DNA [dyen*DNA]. The binding constants and the parameter n for both studied DMQA and their corresponding enantiomers are displayed in Table III-3.
System Configuration K n
Me2-DMQA
M 0.079 ± 0.006 0.5 ± 0.08 P 0.061 ± 0.005 0.5 ± 0.2 racemic 0.069 ± 0.002 1.02 ± 0.06
nPr2-DMQA
M 0.053 ± 0.001 1.05 ± 0.02 P 0.033 ± 0.001 0.94 ± 0.02 racemic 0.035 ± 0.004 1.17 ± 0.13
Table III-3: Binding constants K and number of bound units n to DNA for the racemic, the M and the P of the Me2-DMQA and nPr2-DMQA
These experiments show that the cationic [4]helicenes Me2-DMQA and nPr2-DMQA are stereoselective in binding DNA. The values indicated clearly that the M enantiomer interacted more efficiently with DNA than its antipode, the P enantiomer, for both systems, regardless of the side chain. This proves that the helicity or the conformation of the M enantiomer is behind such a favorable distinction.
According to the figure III-14, and very interestingly, the racemic mixture possessed the smallest binding profile, since the interaction is less intense than with each of the P and M alone. It also seems to contradict the values in table III-3 that show an intermediate value for the binding constant of the racemic compared to the corresponding enantiomers. This could be possibly explained by the fact that H-aggregation in aqueous solution is stronger for the heterochiral racemic mixture than between the homochiral enantiopure units. There is therefore less of “free”
DMQA in solution prior to DNA binding for the racemic compound than for the enantiopure
80 | P a g e ones and hence the lower induced fluorescence in figure III-4. We also noticed the strongest binding constant of the methyl derivative compared to the propyl analog.
Figure III-15: Suggested DNA binding models for Me2-DMQA and nPr2-DMQA represented respectively as Hel-Me and Hel Pr. The blue forms represent adjoining base pairs of DNA.
The parameter n of the model indicates the number of dye units that interact with one DNA base-pair. Thus, for the M or P enantiomers of the Me2-DMQA, one dye cooperates with two base-pairs of DNA (which is usually considered as the saturation level due to “the nearest-neighbour exclusion principle19) whereas the ratio for nPr2-DMQA is 1:1 (Table III-3). Such a difference in behavior might be due to different binding modes due to the difference in size of the chromophores. Me2-DMQA is probably able to fit between two base-pairs of DNA causing
“intercalation” while the more bulky nPr2-DMQA with longer side chains is prevented from intercalating between two base-pairs, having the only alternative to remain in proximity in a DNA groove (Figure III-15). Interestingly, the parameter n found for the racemic mixture of Me2-DMQA differs from the one found for each of its two enantiomers, which so far remains unexplained.
In conclusion, a series of physical experiments were performed to test whether or not cationic [4]helicenes could bind to DNA. Two derivatives were chosen with short and longer alkyl side chains (Me2-DMQA and nPr2-DMQA). These chromophores have shown the formation of H-aggregates in water, which is not the case when the compounds are dissolved in an organic solvent.
19 McGhee, J. D. and Von Hippel, P. H. J. Mol. Biol. 1974, 86, 469-489.
We were lucky to find that indeed, these chiral structures present a stereoselective binding mode to DNA that is stronger with the M enantiomer. The short side chain Me2-DMQA also showed results that confirmed possible intercalation to DNA compared to nPr2-DMQA which apparently remains in proximity to the polynucleotide.20
III-5.Conclusion
In the end, self-aggregation which is a common feature of dye chemistry; has been observed in the photophysical investigation of DMQA derivatives.
The results of DNA binding to these molecules were interestingly shown to be related to this phenomenon. H-aggregates seemed stronger with the racemic mixtures than in the separated enantiomers which affected a smaller binding constant than the enantiopure form. The amount of
“free” or unbound DMQA in solution may possibly account for this difference in behavior.
Finally, although a large amount of data has already been obtained, more work is still ongoing to assess precisely the behavior of cationic[4]helicenes in water and in the presence of DNA.
20 Another study was done on the diol DMQA derivatives 1b. The first results figured in the Ph.D thesis of Alexandre Fürstenberg, in 2008, and were later persued by Oksana Kel of the same group of Pr. Vauthey.
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