Anna K. F.
Mårtensson
and
Per
Lincoln
*
Department of Chemistry and Chemical Engineering, Chalmers University of Technology, SE-41296 Gothenburg, Sweden. E-mail: lincoln@chalmers.se
First published on 3rd April 2018
Isothermal titration calorimetry (ITC) has been utilized to investigate the effect of methyl substituents on the intercalating dppz ligand of the enantiomers of the parent complex Ru(phen)2dppz2+ (phen = 1,10-phenanthroline; dppz = dipyrido[3,2-a:2′,3′-c]phenazine) on DNA binding thermodynamics. The methylated complexes (10-methyl-dppz and 11,12-dimethyl-dppz) have large, concentration-dependent, positive heats of dilution, and a strong endothermic background is also apparent in the ITC-profiles from titration of methylated complexes into poly(dAdT)2, which make direct comparison between complexes difficult. By augmenting a simple cooperative binding model with one equilibrium for complex self-aggregation in solution and one equilibrium for complex aggregation on saturated DNA, it was possible to find an excellent global fit to the experimental data with DNA affinity parameters restricted to be equal for all Δ-enantiomers as well as for all Λ-enantiomers. In general, enthalpic differences, compared to the unsubstituted complex, were small and less than 4 kJ mol−1, except for the heat of intercalation of Δ-10-methyl-dppz (−11,6 kJ mol−1) and Λ-11,12-dimethyl-dppz (+4.3 kJ mol−1).
By introducing small changes into the molecular structure of the complex, it is possible to modify the photophysical properties and also the DNA binding affinity and specificity. Consequently, there have been numerous reports on ruthenium complexes with substituents on both the ancillary and the intercalating ligands.13–18 By adding methyl groups on the outermost benzene ring of the dppz ligand in Ru(phen)2dppz2+ the steric consequences might alter the binding motifs of the complex. Previous studies observed a significant lengthening of the excited state lifetimes of dppz-based ruthenium complexes with methyl substitutions in the 10-position and in the 11,12-positions of the dppz moiety, which was attributed to a steric interference of the hydration cage around the complex.6,19 The structurally similar complex Ru(TAP)2dppz2+ (TAP = 1,4,5,8-tetraazaphenanthrene) has been reported to bind more strongly to DNA when methyl-substituted in the 11,12-position of the dppz moiety.20 Methyl substituents in the 11,12-positions of the dppz moiety of Cr(phen)2dppz3+ also increased the binding affinity of the parent complex.21 In contrast, methyl substituted dpq (dpq = dipyrido[3,2-f:2′,3′-h]-quinoxaline), a close analogue of dppz, has shown a decrease in DNA binding strength compared to its parent complex Ru(phen)2dpq2+, which was attributed to steric hindrance of the bulky methyl groups.22 Clearly, methyl substituents may alter the binding properties of intercalative complexes, but there are still many questions on the underlying binding mechanism that have been left unanswered. Also, many of the previous studies have limited themselves to unresolved ruthenium complexes. This is unfortunate as it has been demonstrated on numerous occasions by various experimental methods that DNA binding is highly influenced by the chirality of tris-bidentate ruthenium complexes with the right-handed Δ-form generally having a stronger affinity to DNA than the Λ-form.1,18,23–27
Isothermal titration calorimetry (ITC) is a high-precision tool used for the thermodynamic characterization of ligand binding to a macromolecule (e.g. DNA) and is the only thermodynamic method that directly measures all energetics associated with the ligand–macromolecule interaction process.28–30 Aliquots of the ligand are added to a solution of the macromolecule and the resulting heat effects observed can then be analysed for quantitative characterization of the energetic processes associated with the binding reaction. As the titration progresses, the binding sites on the macromolecule become increasingly occupied and at the end of the titration, only a small heat change caused by ligand dilution and other non-specific effects will be registered. In most cases this is corrected for by simply subtracting the average heat of dilution from a blank titration of ligand into buffer. However, if the blank titration shows a large non-constant heat of dilution it cannot simply be dismissed by subtraction. As an effect of the highly concentrated titrant solution, the ligands may self-aggregate to form oligomers, which partly dissociate when added to the macromolecule solution.31,32 Being another equilibrium process involving the free ligand in solution, the self-aggregation needs to be included in the analysis of the ligand–DNA interaction.
Intercalating dppz-based ruthenium complexes have previously been demonstrated to either facilitate or hinder the adjacent binding of neighboring complexes along the DNA polymer.27,33 In addition, the binding sites on a DNA polymer are in such close proximity of each other that each intercalated ruthenium complex covers more than one binding site. Based on the classical McGhee and von Hippel model, where DNA is treated as a one-dimensional lattice of binding sites,34 we have recently developed a general algorithm that can be utilized for the model fitting of binding interactions between ligands and linear biopolymers.35
In this study, we seek to thermodynamically characterize the DNA binding of Ru(phen)2dppz2+ with methyl groups substituted in the 10-position or the 11,12-positions of the dppz moiety (Fig. 1). In order to avoid the effects of DNA sequence heterogeneity, we chose to use poly(dAdT)2 (AT-DNA) for the ligand–DNA interaction. Since the methylated complexes show a pronounced non-constant heat of dilution, this has been included in the analysis.
Fig. 1 Structures of ruthenium complexes Ru(phen)2dppz2+ (1), Ru(phen)2dppzCH32+ (2) and Ru(phen)2dppz(CH3)22+ (3). |
The enantiopure Δ- and Λ-[Ru(phen)2dppz]Cl2, [Ru(phen)2dppzCH3]Cl2 and [Ru(phen)2dppz(CH3)2]Cl2 (here denoted 1, 2 and 3, respectively) used in this study were synthesized and resolved as previously reported.23,36
Other chemicals were purchased from Sigma-Aldrich and used without purification.
Absorption spectra were measured on a Varian Cary 4000 UV/vis (Agilent Technologies) spectrophotometer (path length = 1 cm).
Calorimetric data were obtained using a MicroCal iTC200 isothermal titration calorimeter (Malvern) controlled by Origin 7.0 software. The ITC profiles of the resolved ruthenium complexes were obtained by a single injection of 1 μl followed by 19 sequential injections of 2 μl aliquots of stock solution (∼590 μM) of the complex from a syringe into the sample cell (206 μl) loaded with AT-DNA in 150 mM NaCl aqueous buffer solution (∼312 μM nucleotides) or with buffer alone. All ITC experiments were performed at 25 °C. The injection spacing was 180 s, the syringe rotation was 750 rpm and there was an initial delay of 120 s prior to the first injection. The raw ITC data peaks were automatically integrated using the Origin 7.0 software. For improved accuracy of the integration, the integration range for the spacing between each heat peak was narrowed, thus reducing the background noise from the baseline.
(1) |
Two different intercalation pockets, TA/TA and AT/AT, alternate in the AT-DNA used for this study. However, for simplicity, the present model considers them to be equal, and thus n is expected to be close to 2 as for classical intercalation.
Furthermore, each intercalated ligand is also assumed to be able to become an external binding site for an additional ligand, characterized by an external binding constant Kext, which we have arbitrarily set as 104 for all ligands. The effect of external binding on the mass balance has been neglected, since Kext used will be at least 100 times less than the effective ligand intercalation binding constant, which is in the range between Ky and Ky2 when the free ligand concentration starts to rise as a consequence of increasing intercalation site saturation.
Linear dichroism studies in solution do not indicate significant binding geometry differences between 1, 2 and 3 for either enantiomer,19 and high resolution X-ray crystal structures of the Λ-enantiomers show virtually identical intercalation geometries irrespective of methyl substituents.20 Thus, it seemed reasonable to attempt a global fit to the ITC data assuming identical intercalation equilibrium constants K and neighbor interaction parameters y for each set of enantiomers, while allowing the binding site coverage parameter n to vary freely.
Aside from the intrinsic () and neighbor interaction () reaction enthalpies, this model also considers the oligomer dissociation () as well as the external DNA association () enthalpy. As can be seen in Fig. 2, it is possible to find a very good global fit to the integrated peaks of the raw data in Fig. S1 and S2 (ESI†) which gave nRMSD = 7.3% (nRMSD: normalized root-mean-square-deviation, the Euclidian norm of the residual divided by the Euclidian norm of the data). In fact, allowing K and y to vary freely gave only a slightly lower nRMSD (6.4%), indicating that the assumption of similar binding affinity parameters for each set of enantiomers was consistent with the data.
Table 1 gives the best global fit binding parameter values, showing that the Δ-enantiomer is anti-cooperative in its nearest-neighbor interactions while the Λ-enantiomer is cooperative. It is worth noting that although the intrinsic binding constant K differs by more than two orders of magnitude between the enantiomers, the effective binding constant close to saturation, Ky2, is rather similar: Ky2 = 5.9 × 106 for Δ and 3.2 × 106 for Λ.
K Δ/106 | y ΔΔ | n Δ | K Λ/106 | y ΛΛ | n Λ | K 1/(m−1) m | m | |
---|---|---|---|---|---|---|---|---|
1 | 41 | 0.38 | 2.17 | 0.28 | 3.4 | 2.29 | 400 | 2.0 |
2 | 41 | 0.38 | 2.25 | 0.28 | 3.4 | 2.99 | 3300 | 3.0 |
3 | 41 | 0.38 | 2.24 | 0.28 | 3.4 | 2.25 | 8900 | 2.7 |
Interestingly, the Λ-enantiomers all have larger binding site coverage parameters n compared to Δ. While the difference is small for complexes 1 and 3, it is prominent for complex 2.
The self-aggregation (expressed as the effective dimerization constant K1/(m−1)m in Table 1) increases with the number of methyl groups, but the effective dimerization enthalpy remains relatively constant as shown in Table 2. The external binding contribution to the ITC-signal becomes more prominent upon methylation, in particular for the Λ-enantiomers, but the weak binding makes it impossible to partition this trend into external binding affinity and external binding enthalpy. However, the distinct diastereomeric differences for 2 and, in particular, for 3 suggest that the distal benzene ring of the dppz of one complex and the phenanthroline of another are important for the external binding mode. By electrostatic arguments, it appears most likely that this interaction occurs in the groove opposite to where the Ru(phen)2-moieties of the intercalated complexes reside, i.e. that it is the phenanthrolines of the externally bound complexes that interacts with the methyl groups of the intercalated ones.
The differences in the intercalation enthalpy () between the enantiomers of the same complex show no clear trend, but the neighbor interaction enthalpy () for Λ is about 2–3 times more exothermic than that of Δ. (As an example, the different enthalpy contributions are illustrated for complex 2 in Fig. S3 of ESI.†)
Table 3 highlights the differences in the enthalpy values due to methyl substitution. The much more exothermic intercalation for the Δ-enantiomer of 2 compared to 1 stands out as the most significant effect, followed by the less exothermic intercalation of the Λ-enantiomer of 3 and the less exothermic neighbor interaction enthalpies of the Δ-enantiomers of 2 and 3.
Table 4 gives the free energy changes and entropy contributions calculated from the data in Tables 1 and 2. Since the free energy changes were assumed to depend on chirality only, differences in TΔS° between different complexes directly reflect the enthalpy changes in Table 3. However, these are small enough not to change the overall pattern: intercalation is in all cases predominantly entropy driven, and most so for Δ-enantiomers; the neighbor interaction on the other hand, in all cases, has a large unfavorable entropy.
For any binding model to be of practical use, a large number of fitting parameters is not acceptable. Here, we demonstrate an algorithm making it possible to fit experimental data of complicated ligand–macromolecule systems using only a minimum of additional parameters. In order not to over-interpret the data, we have limited the binding model to be the simplest possible. Thus effects due to AT/AT and TA/TA-differences, possible different groove-locations and different intercalation geometries are all neglected. Furthermore, we test the hypothesis that methyl substitution does significantly alter K and y for a given enantiomer.
When the self-aggregation and external binding effects had been accounted for, the seemingly disparate ITC-curves of 1, 2 and 3 (Fig. 2) were indeed found to be consistent with the same binding affinity for all Δ and all Λ enantiomers, i.e. all differences due to methylation could satisfactorily be accounted for as differences in the binding site coverage (n) and the binding and the interacting enthalpy values. This suggests that methylation on the distant benzene ring does not dramatically alter the binding affinity characteristics typical for the enantiomers.
The graphs of concentration changes in the total bound ligand and the neighboring bound ligand, respectively, for Δ and Λ are depicted for 1 in Fig. 3. The more cooperative binding of Λ becomes apparent in the initial part of the interaction curve; the convex shape is due to the facilitated binding of additional ligands. Δ-enantiomers, in contrast, have a concave initial shape due to the anti-cooperative behavior of the ligands. Since the fitting model needs to produce a close coincidence of the Δ and Λ-curves at the end of the titrations, the intrinsic binding constant K has to be correspondingly higher for the anti-cooperative Δ-enantiomer than for Λ.
Fig. 3 ITC titrations with complex 1 into poly(dAdT)2 with the changes in the concentration of the total bound ligand (solid line) and the neighboring bound ligand (dotted line) (black: Δ; red: Λ). |
X-ray structures by Hall et al. have demonstrated the Ru(TAP)2dppz analogues of Λ-1, -2 or -3 to intercalate with the same depth and the same angular orientation.20 The X-ray structure further showed that the 10-methyl group on dppz efficiently protects the exposed dppz aza-nitrogen from interaction with water, consistent with the observation by Olofsson et al. that methyl substitution in the 10-position gave longer luminescence lifetimes for both enantiomers, in polyol solution as well as when intercalated to DNA.19Fig. 4 (left) shows a model of three Δ-2 complexes consecutively intercalated (from the minor groove, as per the suggestion from X-ray structures) two base pairs apart from each other. A more hydrophobic environment around the 10-methyl group, as suggested by the model, than for the methyl groups of the 11,12-disubstituted derivative Δ-3 (Fig. 4 (right)) might account for the significantly more exothermic intercalation enthalpy of Δ-2. If this is the case, the environment around the methyl group of the corresponding enantiomer Λ-2 is expected to be different, perhaps due to a different intercalation depth or different angular orientation in the intercalation pocket.
Footnote |
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c8cp01151f |
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