Berberrubine Phosphate: A Selective Fluorescent Probe for Quadruplex DNA

Abstract

A phosphate-substituted, zwitterionic berberine derivative was synthesized and its binding properties with duplex DNA and G4-DNA were studied using photometric, fluorimetric and polarimetric titrations and thermal DNA denaturation experiments. The ligand binds with high affinity toward both DNA forms (K_b = 2-7 × 10⁵ M^-1) and induces a slight stabilization of G4-DNA toward thermally induced unfolding, mostly pronounced for the telomeric quadruplex 22AG. The ligand likely binds by aggregation and intercalation with ct DNA and by terminal stacking with G4-DNA. Thus, this compound represents one of the rare examples of phosphate-substituted DNA binders. In an aqueous solution, the title compound has a very weak fluorescence intensity (Φ_fl < 0.01) that increases significantly upon binding to G4-DNA (Φ_fl = 0.01). In contrast, the association with duplex DNA was not accompanied by such a strong fluorescence light-up effect (Φ_fl < 0.01). These different fluorimetric responses upon binding to particular DNA forms are proposed to be caused by the different binding modes and may be used for the selective fluorimetric detection of G4-DNA.

Keywords: DNA ligand; G4-DNA; alkaloids; light-up probe; quadruplex DNA; zwitterion.

Scheme 1

Synthesis of 9-O-Hydrogenphosphonatoberberrubin (2).

Figure 1

Emission spectra of 2 (c = 10 µM, with 0.25% v/v DMSO) in glycerol at 20 °C, 30 °C, 40 °C, 50 °C, 60 °C, and 80 °C; λ_ex = 430 nm. The arrows indicate the changes in the emission intensity with increasing temperature. Inset: Plot of the relative emission intensity at 528 nm versus the temperature of the solution.

Figure 2

Photometric titration of 2 (c = 20 μM) with ct DNA (a) in a BPE buffer (c_Na+ = 16 mM, pH 7.0, with 5% v/v DMSO) and with a2 (b) in a K-phosphate buffer (c_K+ = 110 mM, pH 7.0, with 5% v/v DMSO). The arrows indicate the changes in absorption upon the addition of DNA. Inset: Plot of the absorption at 345 nm versus DNA concentration.

Figure 3

CD spectra (a) and LD spectra (b) of ct DNA (c_DNA = 20 μM) in the absence and presence of 2 at LDR = 0 (black), 0.05 (cyan), 0.2 (orange), 0.5 (green), 1.0 (magenta), 1.5 (blue), and 2.0 (red) in a BPE buffer solution (10 mM, pH 7.0; with 5% v/v DMSO). The arrows indicate the changes in absorption with increasing LDR.

Figure 4

CD spectra of a2 (a, c_DNA = 20 μM) and c-myc (b, c_DNA = 20 μM) in the absence and presence of 2 at LDR = 0 (black), 0.05 (cyan), 0.2 (orange), 0.5 (green), 1.0 (magenta), 1.5 (blue), and 2.0 (red) in a K-phosphate buffer (c_K+ = 110 mM, pH 7.0, with 5% v/v DMSO). The arrows indicate the changes in absorption with increasing LDR.

Figure 5

The proposed association of ligand 2 at the terminal quartet of G4-DNA c-myc and c-kit.

Figure 6

Fluorimetric titration of 2 (c = 20 μM, λ_ex = 430 nm) with ct DNA (a) in a BPE buffer (c_Na+ = 16 mM, pH 7.0, with 5% v/v DMSO) and with a2 (b) in a K-phosphate buffer (c_K+ = 110 mM, pH 7.0, with 5% v/v DMSO); λ_ex = 430 nm. The arrows indicate the changes in absorption upon addition of DNA. Inset: Plot of the relative fluorescence intensity versus c_DNA; (c) pictures of the emission color of 2 in a K-phosphate buffer (i) and in the presence of ct DNA (ii) and 22AG (iii) (λ_ex = 360 nm).