DNA – ligand interactions gained and lost : light-induced ligand redistribution in a supramolecular cascade †

Controlled delivery and release of drugs in a target organ or tissue are key challenges of modern medicine and pharmacology. Specifically, DNA-targeting chemotherapy requires highly efficient and selective modes of operation to minimize damage to the healthy cells. In this regard, encapsulation of drugs within macrocyclic ‘‘containers’’ is one of the most successful approaches allowing realization of targeted delivery and release with minimal side effects. Cyclodextrins (CD) and cucurbiturils (CB[n]) represent host molecules of major importance due to their low toxicity, chemical stability, and large association constants of the host– guest complexes. On-demand drug release from the host carrier can be realized by activation of the encapsulated drug molecules with various external triggers, e.g. changes in temperature, pH and oxidation states, variations in electric or magnetic fields, displacement by a competing ligand, or irradiation. In this respect, light is a non-invasive stimulus that enables local and temporal control without interfering with physiological media. Several CD-containing systems for photocontrolled drug release have been reported that operate by light-induced isomerization of azobenzene inside the CD cavity. However, in these cases the photoreaction provides just a mechanical output resulting in the release of loaded drug molecules from carriers and does not lead to the formation of drug species from the complexed precursors. Surprisingly, there are only a very few examples of ‘‘nonazobenzene’’ systems with light-triggered guest release directly from the CD or CB[n] cavity; and these ones have not been considered for biological purposes. Although interactions of ligand–CD and ligand–CB[n] complexes with DNA have been reported already, in these cases the ligand distribution is governed by the chemical equilibria between interacting components, i.e. no external stimuli were applied to trigger a DNA-binding event in these systems. Moreover, established CDor CB[n]-comprising supramolecular DNA-binding systems included a maximum of three interactive components, presumably because of the potential ambiguity of the interaction pattern. Herein, we present for the first time a supramolecular reaction cascade consisting of five components that enables the light-controlled in situ generation and redistribution of a DNA-binding ligand between different host systems. The key process that provides a temporal and spatial control of the formation of a DNA-binder is the photocyclization reaction of styrylpyridine derivative 1, followed by aerobic oxidation to give benzo[c]quinolizinium 2 (Scheme 1). In analogy to the properties of a structurally resembling styrylbenzothiazole we proposed that pyridine derivative 1 does not interact with DNA, whereas the photocyclization product 2 is an efficient DNA-intercalator.


Materials and Methods
All reagents and solvents were obtained from commercial sources and used as received.(2-Hydroxypropyl)-β-cyclodextrin with average M w ~1,460 (0.8 molar substitution) was purchased from Sigma Aldrich.Purified water with resistivity ≥ 18 MΩ cm −1 was used for preparation of buffer solutions and spectrometric measurements.Na-phosphate buffer (10 mM, pH 7.0) was used for all measurements.
NMR spectra were recorded on a Varian VNMR-S 600 spectrometer equipped with 3 and 5 mm dual broadband and 3mm triple resonance inverse probes.Solvent signals were used as internal standard (Acetone-d6: δ H = 2.05, δ C = 29.8ppm; CD 3 CN: δ H = 1.93, δ C = 1.28 ppm), all spectra are recorded at T = 25°C.Pulse sequences were taken from Varian pulse sequence library.Spectra in H2O/D2O 9:1 were recorded at T = 25 °C with water suppression using wet sequence.
High-resolution mass spectra were recorded on a time-of-flight mass-spectrometer Bruker MicrOTOF in a positive-ion mode using electrospray ionization method.
Mass spectra (ESI in the positive-ion mode) were recorded with a Finnigan LCQ Deca instrument; only m/z values in the range of 100-2000 units were analyzed.Electronic absorption spectra were recorded using a Varian Cary 100 Bio spectrophotometer.
Fluorescence spectra were recorded on a Varian Cary Eclipse spectrofluorometer.Circular dichroism spectra were measured with an Applied Photophysics Chirascan CD spectrometer.Spectrophotometric measurements were performed in thermostated quartz sample cells of 10 mm pathlength at 20 ± 1 °C.Preparation and handling of the solutions were carried out under red light.
The actual concentrations of DNA samples were determined photometrically using the extinction coefficient ε 260 = 12824 cm −1 M −1 (bp).Photochemical reactions were carried out with a high pressure Hg vapor lamp (145 W) and an immersed Hg photoreactor (125 W).

Synthesis
Cucurbit[7]uril (purity >95%) was obtained according to the published protocol, 1 and its purity was confirmed by NMR and ESI MS data.

Styrylpyridine derivative 1 -HP-β-CD interaction
Absorption, fluorescence and circular dichroism spectroscopy        Protonation of the CB-bound ligand is a consequence of charge-dipole interactions between a cucurbituril host and a guest that result in the shifting of the pK a of the guest towards higher values. 2In contrast, the pK a of a ligand decreases when bound to cyclodextrin because hydrophobic interactions are the dominant interactions in this complex. 3Considering these differences the competition of the two hosts for ligand 1 at the applied pH value of 7.0 was assessed, i. e. when the non-protonated form of 1 prevails.

Figure S4 .
Figure S4.ROESY spectrum of styrylpyridine derivative 1 (c 1 = 0.3 mM) in the presence of a 10fold excess of HP-β-CD in D 2 O.
13C NMR spectrum of 2 in CD 3 CN.