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

<div><p>Quaternary benzo[<i>c</i>]phenanthridine alkaloids are secondary metabolites of the plant families <i>Papaveraceae</i>, <i>Rutaceae</i>, and <i>Ranunculaceae</i> 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.</p></div

Lippert–Mataga plot of QBAs.

<p>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.</p

Representative fit of macarpine-DNA interaction.

<p>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.</p

Acid-base properties of 3 μM macarpine.

<p>Dependence of absorbance at 495 nm (black) and fluorescence at 450 nm (red) on pH, n = 3. Mean values ± SD and fits to Eq (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129925#pone.0129925.e004" target="_blank">4</a>) are shown. Inset: Absorption (black) and emission (red) spectra of iminium (—) and alkanolamine (…) form. Ordinates are the same as for bigger figure.</p

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

<p>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 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129925#pone.0129925.e001" target="_blank">1</a>).</p

Spectroscopic properties of alkanolamine (QOH) and iminium (Q⁺) forms of QBAs.

<p>Where two results were obtained (pK_ROH by two methods, two lifetimes), second value is indicated in brackets.</p><p>a) all QBAs except macarpine ref. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129925#pone.0129925.ref030" target="_blank">30</a>].</p><p>b) this work, spectrophotometry, n = 3, SE = 0.02</p><p>c) this work, spectrofluorometry, n = 3, SE = 0.06</p><p>d) n = 4, estimated RSD ca. 15%</p><p>e) n = 3, SE ≤ 0.1 ns</p><p>f) 3.3 ns (ref. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129925#pone.0129925.ref022" target="_blank">22</a>])</p><p>g) 3.5 ns (610 nm)</p><p>h) 5.8 ns (610 nm)</p><p>i) 2.4 ns (ref. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129925#pone.0129925.ref022" target="_blank">22</a>])</p><p>Spectroscopic properties of alkanolamine (QOH) and iminium (Q⁺) forms of QBAs.</p

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

<p>Structure, numbering and acid-base equilibrium of studied QBAs.</p

Modulatory Effects of Eschscholzia californica Alkaloids on Recombinant GABAA Receptors.

The California poppy (Eschscholzia californica Cham.) contains a variety of natural compounds including several alkaloids found exclusively in this plant. Because of the sedative, anxiolytic, and analgesic effects, this herb is currently sold in pharmacies in many countries. However, our understanding of these biological effects at the molecular level is still lacking. Alkaloids detected in E. californica could be hypothesized to act at GABAA receptors, which are widely expressed in the brain mainly at the inhibitory interneurons. Electrophysiological studies on a recombinant α 1 β 2 γ 2 GABAA receptor showed no effect of N-methyllaurotetanine at concentrations lower than 30 μM. However, (S)-reticuline behaved as positive allosteric modulator at the α 3, α 5, and α 6 isoforms of GABAA receptors. The depressant properties of aerial parts of E. californica are assigned to chloride-current modulation by (S)-reticuline at the α 3 β 2 γ 2 and α 5 β 2 γ 2 GABAA receptors. Interestingly, α 1, α 3, and α 5 were not significantly affected by (R)-reticuline, 1,2-tetrahydroreticuline, codeine, and morphine-suspected (S)-reticuline metabolites in the rodent brain

Modulatory Effects of Eschscholzia californica Alkaloids on Recombinant GABA A Receptors

The California poppy (Eschscholzia californica Cham.) contains a variety of natural compounds including several alkaloids found exclusively in this plant. Because of the sedative, anxiolytic, and analgesic effects, this herb is currently sold in pharmacies in many countries. However, our understanding of these biological effects at the molecular level is still lacking. Alkaloids detected in E. californica could be hypothesized to act at GABA A receptors, which are widely expressed in the brain mainly at the inhibitory interneurons. Electrophysiological studies on a recombinant 1 2 2 GABA A receptor showed no effect of N-methyllaurotetanine at concentrations lower than 30 M. However, ( )-reticuline behaved as positive allosteric modulator at the 3 , 5 , and 6 isoforms of GABA A receptors. The depressant properties of aerial parts of E. californica are assigned to chloride-current modulation by ( )-reticuline at the 3 2 2 and 5 2 2 GABA A receptors. Interestingly, 1 , 3 , and 5 were not significantly affected by ( )-reticuline, 1,2-tetrahydroreticuline, codeine, and morphine-suspected ( )-reticuline metabolites in the rodent brain