Petr Táborský

Thermodynamic study of lanthanide(III) complexes with bifunctional monophosphinic acid analogues of H4dota and comparative kinetic study of yttrium(III) complexes.

New bifunctional H(4)dota-like ligands with three acetic acid and one phosphinic acid pendant arms and propionate (H(5)do3ap(PrA)) or 4-aminobenzyl (H(4)do3ap(ABn)) reactive groups bound to the phosphorus atom were investigated. Potentiometric studies showed that the ligands have a similar basicity to the parent H(4)dota and the stability constants of their complexes with sodium(i) and selected lanthanide(III) ions are also similar. Formation and acid-assisted decomplexation kinetics of yttrium(III) complexes with a series of H(4)dota-like ligands (H(4)dota and its phosphinic/phosphonic acid analogues) were studied and the reactions are sensitive to a slight modification of the ligand structure. The (2-carboxyethyl)phosphinic acid derivative H(5)do3ap(PrA) and the phosphonic acid ligand H(5)do3ap form complexes faster than H(4)dota. The most kinetically inert complex is that with H(4)do3ap(ABn). Rates of complexation and decomplexation can depend on the ability to transfer proton(s) outside/inside the complex cavity and, therefore, on the hydrophobicity of the ligands. The results demonstrate that the new bifunctional ligands are suitable for labelling biomolecules with yttrium(iii) radioisotopes for utilization in nuclear medicine.

Fluorescence properties of selected benzo[c]phenantridine alkaloids and studies of their interaction with CT DNA

The spectral, especially fluorescence properties, of seven selected quaternary benzo[c]phenantridine alkaloids (sanguinarine, chelerythrine, chelirubine, sanguirubine, chelilutine, sanguilutine, and macarpine) were studied in presence and in absence of double-stranded DNA. This study has proved dramatic differences in fluorescence emission of all studied alkaloids in presence of calf thymus DNA in comparison to fluorescence of free alkaloids. The most remarkable are changes in emission spectra of macarpine, chelirubine, and sanguirubine. Association constants (logK) for interaction of all studied alkaloids with CT DNA were calculated.

Spectroscopic Characterization of Eu(III) Complexes with New Monophosphorus Acid Derivatives of H4dota

The luminescence lifetimes of europium(III) complexes with new monophosphorus acid derivatives of H4dota were measured by means of time-resolved laser-induced luminescence spectroscopy in H2O and D2O. The hydration numbers of these complexes were estimated using different empirical equations [Horrocks and Sudnick (1979) J. Am. Chem. Soc. 101 (1979) 334; Choppin and Barthelemy(1989) Inorg. Chem. 28, 3354–3357; Choppin and Bünzli Lanthanide probes in life, chemical and earth sciences. Theory and practice (1989); Kimura and Kato J. Alloys Comp. 275–277 (1998) 806; Parker (1999) J. Chem. Soc., Perkin Trans. 2, 493–503; Supkowski and Horroks (2002) Inorg. Chim. Acta. 340, 44–48]. It was shown that all the relationships gave similar results with a satisfactory precision. The hydration numbers of complexes of H3do3a and H4dota agreed with the literature values. One water molecule is coordinated in complexes of the new ligands. The results showed that the Choppin formula based on measurements only in H2O can be satisfactorily used for estimation of the hydration numbers.

Interaction of oligonucleotides with benzo[c]phenanthridine alkaloid sanguilutine

Benzo[c]phenanthridine alkaloid sanguilutine was extracted and purified from the dried roots of Sanguinaria canadensis. The interaction of the positively charged iminium form of alkaloid with double-stranded DNA oligonucleotides was studied using luminescence spectroscopy. The results showed that the interaction with various double-stranded oligonucleotides was not specific to A-T or G-C base pairs; also, no preference was found for either homogeneous or heterogeneous base composition of strands. The association constants were calculated to be in the range of (1.31–14.36) × 105 M−1. The luminescence intensity response at 610 nm to low concentrations of double-stranded DNA was found to be linear and can potentially be used for the fluorometric quantification of DNA. The limit of detection was estimated to be 120 ng mL−1 of DNA (calculated by 3σ method).

Spectroscopic study of protonation of oligonucleotides containing adenine and cytosine

Acidobasic properties of purine and pyrimidine bases (adenine, cytosine) and relevant nucleosides (adenosine, cytidine) were studied by means of glass-electrode potentiometry and the respective dissociation constants were determined under given experimental conditions (I = 0.1 M (NaCl), t = (25.0 ± 0.1) °C): adenine (pKHL = 9.65 ± 0.04, pKH2L = 4.18 ± 0.04), adenosine (pKH2L = 3.59 ± 0.05), cytosine (pKH2L = 4.56 ± 0.01), cytidine (pKH2L = 4.16 ± 0.02). In addition, thermodynamic parameters for bases: adenine (ΔH0 = (−17 ± 4) kJ mol−1, ΔS0 = (23 ± 13) J K−1 mol−1), cytosine (ΔH0 = (−22 ± 1) kJ mol−1, ΔS0 = (13 ± 5) J K−1 mol−1) were calculated. Acidobasic behavior of oligonucleotides (5′CAC-CAC-CAC3′ = (CAC)3, 5′AAA-CCC-CCC3′ = A3C6, 5′CCC-AAA-CCC3′ = C3A3C3) was studied under the same experimental conditions by molecular absorption spectroscopy. pH-dependent spectral datasets were analyzed by means of advanced chemometric techniques (EFA, MCR-ALS) and the presence of hemiprotonated species concerning (C+-C) a non-canonical pair (i-motif) in titled oligonucleotides was proposed in order to explain experimental data obtained according to literature.

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

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.

Study of Fluorescently Labelled Peptides and Small Proteins Using Capillary Electrophoresis Combined with Laser Induced Fluorescence and MALDI MS Detection

In this work, we apply CE-LIF, CE-UV and CE - Matrix Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI TOFMS) to investigate derivatization of model peptides and small proteins with rhodamine-based fluorescent reagents, which are compatible with the wavelength of frequency doubled diode pumped Nd:YAG excitation laser (532 nm). While the LIF detection mode is needed for sensitive detection, the MALDI TOFMS analysis can reveal the mass of analytes. Both derivatized and non-derivatized peptides and proteins, the number of fluorescent labels per peptide/proteins and other product of derivatization process can be detected directly from reaction mixtures or after separation. Thus, MALDI TOFMS serves for additional identification of peaks present in CELIF or CEUV electropherograms. For off-line coupling of CE with MALDI MS, subatmospheric deposition interface was used. An examples of final sensitive CELIF separation of derivatized peptides and small proteins are given. Main advantages and disadvantages of LIF, absorbance and MS detector for capillary electrophoresis of polypeptides are disscused. Attention is paid to detection limits in practical analysis.