Synthesis and DNA threading properties of quaternary ammonium [Ru(phen)2(dppz)]2+ derivatives

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Abstract

Ruthenium complexes with one dipyrido[3,2-a:2′-3′-c]phenazine (dppz) ligand, e.g. [Ru(phen)2(dppz)]2+ (phen = phenanthroline), shows strong binding to double helical DNA and are well-known DNA “light-switch” molecules. We have here investigated four new [Ru(phen)2(dppz)]2+ derivatives with different bulky quaternary ammonium substituents on the dppz ligand to find relationships between molecular structure and intercalation kinetics, which is considered to be of importance for antitumor applicability. Linear dichroism spectroscopy shows that the enantiomers of the new complexes exhibit very similar binding geometries (intercalation of dppz moiety between adjacent DNA base pairs) as the enantiomers of the parent [Ru(phen)2(dppz)]2+ complex. Absorption spectra and luminescence properties provide further evidence for a final intercalative binding mode which has to be reached by threading of a bulky moiety between the strands of the DNA. Δ-enantiomers of all the new complexes show much slower association and dissociation kinetics than that of a reference complex without a cationic substituent. Kinetics were not very different whether the bulky quaternary group was derived from hexamethylene tetramine or 1,4-diazabicyclo-(2,2,2)octane (DABCO) or whether it had one or two positive charges. However, a complex in which the hexamethylene tetramine substituent is attached via a phenyl group showed a lowered association rate, in addition to an improved quantum yield of luminescence. A second positive charge on the DABCO substituent resulted in a much slower dissociation rate, suggesting that the distance from the Ru-centre and the amount of charge are both important for threading intercalation kinetics.

Introduction

The design and synthesis of transition metal complexes that have selective DNA binding properties are attracting considerable interest in the search of novel conformational probes or for drug leads for chemotherapy [1], [2], [3]. Strong DNA affinity and slow dissociation kinetics are considered to be especially important for antitumor application [4]. We have earlier reported that the binuclear ruthenium complex ([μ-C4(cpdppz)2(phen)4Ru2]4+, C4(cpdppz)2 = N,N′-bis(12-cyano-12,13-dihydro-11H cyclopenta[b] dipyrido [3,2-h:2′,3′-j]phenazine-12-carbonyl)-1,4-diaminobutane, phen = phenanthroline, cpdppz = 12-cyano-12,13-dihydro-11H cyclopenta[b] dipyrido [3,2-h:2′,3′-j]phenazine-12-carbonyl), consisting of two units of the extensively studied mono-intercalator [Ru(phen)2(dppz)]2+, tethered to each other via dppz moieties and a flexible linker, showed reduced dissociation rate by several orders of magnitude due to threading bis-intercalation of the complex into DNA [5], [6]. Even slower kinetics was later found in the semi-rigid dimer [μ-(11,11′-bidppz)(phen)4Ru2]4+ [7], [8] (bidppz = bi(dipyrido-[3,2-a:2′,3′-c]phenazinyl), P, Scheme 1), in which a single bond connects the two dppz moieties. For P intercalation is extremely slow into ct-DNA at room temperature in 10 mM NaCl buffer, but can conveniently be studied at 50 °C and an ionic strength of 150 mM NaCl [9]. The slow kinetics is thought to be due to that the threading of the bulky Ru(phen)2 moiety of the complex through DNA is sterically hindered and requires a transient opening and de-stacking of at least one base pair, which in turn leads to a unprecedented sequence selectivity [10]. The analogue [μ-dtpf(phen)4Ru2]4+ (dtpf = 4,5,9,12,16,17,21,25-octaaza-23H-ditriphenyleno[2,3:b,2,3:h]fluorene) with a totally rigid bridging ligand exhibited three times slower rate of intercalation than P, indicating that the flexibility of the bridging ligand is an important factor for the threading intercalation kinetics [11]. On the other hand, the complex [μ-bidppz([12]aneS4)2Ru2]4+ ([12] aneS4 = 1,4,7,10-tetrathiacyclododecane) with much less bulky and nonaromatic ancillary ligands surprisingly showed two times slower threading kinetics compared to P, and it was concluded that other properties than the size of the ancillary ligand also affect the energy landscape of the threading path [12]. It thus seems that the mechanisms of threading intercalation are complicated and thorough investigations are crucial.

Although highly kinetically selective for long AT stretches, the binuclear complex P and its congeners have the drawback of being highly charged molecules with high molecular weights, which are disadvantageous properties for potential drugs. It thus seems motivated to also study smaller systems to find the minimal requirements for selective threading intercalation. As starting minimal systems, we have synthesized four new threading ruthenium intercalators BE with a quaternary ammonium substituent on the dppz ligand and compared them to the 11-methyl substituted complex A (Scheme 1). Firstly, to assess the effect of cationic substituent and its distance from the Ru-centre, two mono-quaternary methylene-tetraazaadamantane derivatives were prepared, one with the substituent directly attached to the dppz ligand, and one in which the substituent is attached via a phenyl group: B and C, respectively. To investigate how the threading intercalation kinetics may depend on the charge of the dppz substituent, we designed D, which has a bis-quaternary methylene-diazabicyclo[2,2,2]octane (DABCO) substituent on the dppz ligand, giving the complex the same total charge (4+) as the slowly threading dimer P, and compared to complex E with a mono-quaternary DABCO. The binding geometries of the Δ- and Λ-enantiomers to DNA were investigated by linear dichroism (LD). Since the quaternary ammonium substituents in several cases decreased the quantum yield of luminescence for the intercalated complexes, we chose to only study the binding kinetics of the more strongly luminescent Δ-enantiomers.

Section snippets

Chemicals

All of the reagents and solvents used in the synthesis of ligands and complexes were purchased from Aldrich and were used without further purification. Homochiral Ru(phen)2(1,10-phenanthrolin-5,6-dione)Cl2 was prepared according to the literature [13].

1H NMR spectra were obtained on a Varian spectrometer at 400 MHz for proton. The splitting of proton resonances in the reported. 1H NMR spectra are defined as s = singlet, m = multiplet, d = doublet, and t = triplet. Chemical shifts are referenced to

Synthesis

Mono-quaternary complexes B, C and E were readily prepared by reaction of an excess of the appropriate tertiary amine with the corresponding chloromethyl derivative, and bis-quaternary complex D was subsequently prepared from E by reaction with iodomethane (Supplementary material, Scheme). The chloromethyl substituted complexes were prepared by reaction of DMF and oxalyl chloride with the corresponding hydroxymethyl analogue according to the method of Collins et al., and the latter complexes

Binding Geometry

The parent complex [Ru(phen)2(dppz)]2+ binds to DNA by intercalation of dppz ligand between the base-pairs, placing metal and ancillary ligands in the groove as proved e.g. by resolution of the linear dichroism spectrum and the strong luminescence upon interaction with DNA [27]. One of the important observations in this study is that both Δ- and Λ-enantiomers of the new complexes A, B, D and E, and to a less extent C, despite their large differences in steric bulk and charge, show very similar

Conclusions

Four new threading ruthenium intercalators with a quaternary ammonium substituent on the dppz ligand have been synthesized and compared to the 11-methyl substituted complex. The binding geometries of the Δ- and Λ-enantiomers to DNA were investigated by linear dichroism (LD), and indicate that all complexes are intercalated with very similar geometries as the corresponding enantiomers of the parent complex. For the present series, charge seems to be the most important determined for slowing down

Abbreviations

Phenphenanthroline
dppzdipyrido[3,2-a:2′-3′-c]phenazine
DABCO1,4-diazabicyclo-(2,2,2)octane
bidppzbi(dipyrido-[3,2-a:2′,3′-c]phenazinyl
dtpf4,5,9,12,16,17,21,25-octaaza-23H-ditriphenyleno[2,3:b,2,3:h]fluorene
C4(cpdppz)2N,N′-bis(12-cyano-12,13-dihydro-11H-cyclopenta[b] dipyrido [3,2-h:2′,3′-j]phenazine-12-carbonyl)-1,4-diaminobutane
[12]aneS41,4,7,10-tetrathiacyclododecane

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