Synthesis, binding studies and click modification of diazido acridine intercalators : a versatile two-step approach to functional nanomaterials

Abstract

Two diazido acridine drivatives, N,N'-(acridine-3,6-diyl)bis(2-azidoacetamide), PD and 3,6-bis(2-azidoacetamido)-10-methylacridin-10-ium, PDMe⁺ were prepared from 3,6- diamino acridine, Pf. The azide groups were exploited by click chemistry to synthesise functional intercalators by reaction with the alkyne-bearing groups, ethynyl ferrocene EFc, phenylacetylene, N-pentynyl-2-(2-thienyl)-pyrrole pent-TP and acetylene-PEG4- carboxyrhodamine, APCR. The click reaction products were characterised by ¹H NMR spectroscopy, ¹³C NMR spectroscopy, ES-MS, IR and, where appropriate, cyclic voltammetry. IR studies showed the disappearance of the azide and alkynyl stretching signals at 2114 cm⁻¹ and 2112 cm⁻¹ respectively after the click reaction. Cyclic voltammetry revealed that the click product with EFc was redox active, E = 346 mV compared to 441 m V for the free EFc, a negative shift due to the electron donating effect of the triazole ring. Intercalation of PD and PDMe⁺ into DNA, were studied by UV/vis, fluorescence, circular dichroism (CD), linear dichroism (LD) and DNA denaturation experiments. The titration of Pf and PDMe⁺ with DNA showed bathocromic and hypochromic effects in the absorption spectra. However, for PD an interesting phenomenon, a bathochromic and hyperchromic effect was observed. The pKa of PD (4.3) suggests that as PD approaches DNA it becomes protonated due to the low local pH and so aids binding to DNA. Both UV and fluorescence titrations gave DNA binding constants for Pf, PD and PDMe⁺ of ≥ 3.8 × 10⁶, 5.3 × 10⁵ and 2 × 10⁶ respectively. These findings were consistent with the observed increasing stabilisation of the double helix by the intercalators, PD < PDMe+ < Pf. DNA complexes of PD and PDMe⁺ were click modified with EFc, pent-TP and APCR and characterised by ES-MS, UV/vis, IR, fluorescence microscopy, LD, atomic and electrostatic force microscopy (AFM and EFM), and cyclic voltammetry. As for the free reaction, cyclic voltammetry, IR and UV/vis spectroscopy confirmed the successful click reaction of the DNA complexes, but could not confirm PD or PDMe⁺ remained intercalated after click modification. AFM studies confirmed DNA retained its natural wire-like topology after intercalation and click functionalization. Fluorescence microscopy images of DNA complexes with PD and PDMe⁺ that had been click modified with APCR showed illuminated strands 17 μm in length, comparing well with the dimensions of λ-DNA. LD experiments indicated that PDMe⁺ reamains inserted into the DNA helix after the click reaction was performed in-situ using pent-TP.