Influence of Solvent Polarity and DNA-Binding on Spectral Properties of Quaternary Benzo[c]phenanthridine Alkaloids

Abstract

Quaternary benzo[c]phenanthridine alkaloids are secondary metabolites of the plant families Papaveraceae, Rutaceae, and Ranunculaceae with anti-inflammatory, antifungal, antimicrobial and anticancer activities. Their spectral changes induced by the environment could be used to understand their interaction with biomolecules as well as for analytical purposes. Spectral shifts, quantum yield and changes in lifetime are presented for the free form of alkaloids in solvents of different polarity and for alkaloids bound to DNA. Quantum yields range from 0.098 to 0.345 for the alkanolamine form and are below 0.033 for the iminium form. Rise of fluorescence lifetimes (from 2-5 ns to 3-10 ns) and fluorescence intensity are observed after binding of the iminium form to the DNA for most studied alkaloids. The alkanolamine form does not bind to DNA. Acid-base equilibrium constant of macarpine is determined to be 8.2-8.3. Macarpine is found to have the highest increase of fluorescence upon DNA binding, even under unfavourable pH conditions. This is probably a result of its unique methoxy substitution at C12 a characteristic not shared with other studied alkaloids. Association constant for macarpine-DNA interaction is 700000 M(-1).

Fig 1. Structure, numbering and acid-base equilibrium of studied QBAs.

Fig 2. Acid-base properties of 3 μM macarpine.

Dependence of absorbance at 495 nm (black) and fluorescence at 450 nm (red) on pH, n = 3. Mean values ± SD and fits to Eq (4) are shown. Inset: Absorption (black) and emission (red) spectra of iminium (—) and alkanolamine (…) form. Ordinates are the same as for bigger figure.

Fig 3. Lippert–Mataga plot of QBAs.

Black–macarpine, red–sanguilutine, blue–chelirubine, green–sanguinarine, gold–sanguirubine, violet–chelerythrine; 1 –benzene, 2 –diethyl ether, 3 –octanol, 4 –ethanol, 5 –methanol, 6–0.01M borate buffer, pH 9.45. Samples in borate buffer (dashed box) excluded from fitting.

Fig 4. Absorbance and fluorescence spectra of QBAs in absence and presence of ctDNA.

QBAs (3 μM in 20mM acetate buffer, 200 mM NaCl, 2mM EDTA, pH 5) in absence (—) and presence (…) of ctDNA (DNA base pair-to-drug ratio 15.9:1). a-f–absorption spectra, g-l–emission spectra; a, g–sanguinarine, b, h–chelerythrine, c, i–chelirubine, d, j–sanguilutine, e, k–sanguirubine, f, l–macarpine. Note that intensities are in arbitrary units due to conversion to wavenumber scale using Eq (1).

Fig 5. Representative fit of macarpine-DNA interaction.

Macarpine (10 μM) binding to salmon testes DNA (0–116 μM bp) in 0.05 M citrate buffer, pH 6.15, [Na⁺] = 0.122 M was measured as a fluorescence change at 625 nm. Data were fitted to 1:1 binding model using DynaFit software.