Ana I. Olives

Spectrofluorimetric determination of stoichiometry and association constants of the complexes of harmane and harmine with β-cyclodextrin and chemically modified β-cyclodextrins ☆

The association characteristics of the inclusion complexes of the beta-carboline alkaloids harmane and harmine with beta-cyclodextrin (beta-CD) and chemically modified beta-cyclodextrins such as hydroxypropyl-beta-cyclodextrin (HPbeta-CD), 2,3-di-O-methyl-beta-cyclodextrin (DMbeta-CD) and 2,3,6-tri-O-methyl-beta-cyclodextrin (TMbeta-CD) are described. The association constants vary from 112 for harmine/DMbeta-CD to 418 for harmane/HPbeta-CD. The magnitude of the interactions between the host and the guest molecules depends on the chemical and geometrical characteristics of the guest molecules and therefore the association constants vary for the different cyclodextrin complexes. The steric hindrance is higher in the case of harmine due to the presence of methoxy group on the beta-carboline ring. The association obtained for the harmane complexes is stronger than the one observed for harmine complexes except in the case of harmine/TMbeta-CD. Important differences in the association constants were observed depending on the experimental variable used in the calculations (absolute value of fluorescence intensity or the ratio between the fluorescence intensities corresponding to the neutral and cationic forms). When fluorescence intensity values were considered, the association constants were higher than when the ratio of the emission intensity for the cationic and neutral species was used. These differences are a consequence of the co-existence of acid-base equilibria in the ground and in excited states together with the complexation equilibria. The existence of a proton transfer reaction in the excited states of harmane or harmine implies the need for the experimental dialysis procedure for separation of the complexes from free harmane or harmine. Such methodology allows quantitative results for stoichiometry determinations to be obtained, which show the existence of both 1:1 and 1:2 beta-carboline alkaloid:CD complexes with different solubility properties.

An Overview of Analytical Techniques Employed to Evidence Drug-DNA Interactions. Applications to the Design of Genosensors

The demonstration of the existence of non-covalent bonding interactions between different biomolecules (DNA, enzymes, proteins) and drugs or potentially mutagenic agents requires sensitive analytical techniques. Technological advances in key techniques, together with the miniaturization in fluorescence or surface plasmons resonance techniques, have allowed obtaining information in real time concerning the nature, the localization and the strength of drug-DNA interactions. The present chapter describes these analytical techniques and their application to achieve two fundamental goals, namely a deeper knowledge of the nature of the interactions and their application to the design of different devices (aptamers, molecular beacons, DNA-arrays, genosensors) consisting of short single stranded oligonucleotides produced in vitro by well-established methods (polymerase chain reaction, PCR; systematic evolution of ligands by exponential enrichment, SELEX,...) that are capable to detect genetic peculiarities and diseases, biological entities (micro-organisms, genetically modified seeds...) and also genotoxic agents in the environment and in foodstuffs.

Isolation and quantitative methods for analysis of non-steroidal anti-inflammatory drugs.

Non-steroidal anti-inflammatory drugs are widespread employed in both human and animal health care to reduce ongoing inflammation, pain and fever due to their anti-inflammatory, analgesic and, antipyretic actions. Apart from the well-known biological samples, nowadays these compounds are frequently found in the environment, leading to longterm exposure resulting in adverse effects on humans and wildlife. Therefore, it is important to develop analytical methodologies to detect and control the presence of these pharmaceuticals in very different kinds of samples, from urine, serum or plasma, to river and waste water, sediments or sewage sludge, most of them having very complex matrices. Other problems to solve are the low concentration of the target analytes, the presence of a great number of potential interferences and, sometimes, incompatibilities with the detection systems. Consequently, sample pre-treatment is a very important step to take into account in the non-steroidal anti-inflammatory drugs determination. Herein we reviewed the main extraction and clean-up procedures reported in the literature for these substances: ultrasonic extraction, Soxhlet extraction, pressurized- liquid extraction, supercritical fluid extraction, microwave-assisted extraction, dispersive liquid-liquid microextraction, hollow fiber liquid phase microextraction, pressurized hot water extraction, solid-phase extraction, molecularly imprinted solid-phase extraction and solid-phase microextraction. Several analytical methodologies have been developed to quantify non-steroidal anti-inflammatory drugs, including gas chromatography-mass spectrometry, liquid chromatography with ultraviolet detection, diode array detection, fluorescence detection and tandem mass spectrometry.

Mineral and Trace Elements Content in 30 Accessions of Tomato Fruits (Solanum lycopersicum L.,) and Wild Relatives (Solanum pimpinellifolium L., Solanum cheesmaniae L. Riley, and Solanum habrochaites S. Knapp & D.M. Spooner)

Tomato quality and its potential health benefits are directly related to its chemical composition. The characterization of nutritional properties of Solanum germplasm is essential to choose suitable donor parents for breeding programs. In this sense, wild species could be very useful for tomato fruit quality genetic improvement. With this objective, in this work, we characterize micronutrients content in Eulycopersicon germplasm (20 cultivars of S. lycopersicum L. and 10 accessions of wild relatives) analyzing mineral (Na, K, Ca, Mg) and trace elements (Cu, Fe, Zn, Mn) and applying multidimensional analysis (principal component and cluster analysis). The classification obtained and the comparison of cultivars performance showed that wild accessions belonging to S. cheesmaniae (L. Riley), S. pimpinellifolium L., and S. habrochaites S. Knapp & D.M. Spooner can be of great usefulness in breeding programs to improve mineral content characteristics of conventional S. lycopersicum varieties due to its higher mineral content.

Eco-friendly liquid chromatographic separations based on the use of cyclodextrins as mobile phase additives

Acetonitrile and methanol are the most popular solvents employed in analytical HPLC, but they suffer from a number of drawbacks from the environmental point of view. Alternative, greener mobile phases employing methanol or the less toxic solvent ethanol as the sole organic solvent are proposed in this paper, and applied to the problem of the separation of β-carbolines on C18-stationary phases. The use of β-cyclodextrin (β-CD) and (2-hydroxypropyl)-β-cyclodextrin (HPβ-CD) as mobile phase additives allowed us to increase the proportion of water in the mobile phases without loss in the resolution or efficiency of the separations, leading initially to a considerable reduction of the proportion of methanol in the mobile phase (from 70% to 50%) and at a later stage, to the development of a mobile phase containing only 30% of ethanol. The β-carboline–cyclodextrin association constants were determined by HPLC, and the inclusion complexes were also characterized by 1H-NMR, 13C-NMR and 2D-ROESY experiments, and these studies were used to explain the chromatographic behaviour. The new chromatographic methodology developed was validated and applied to the quantitation of β-carboline derivatives in spiked human serum samples. For the extraction of β-carboline alkaloids from serum samples, liquid–liquid extraction (LLE) and solid-phase extraction (SPE) procedures were compared. It was concluded that the combination of a pre-treatment procedure (ionic exchange SPE) with a water-enriched chromatographic separation leads to a promising, environmentally friendly new methodology.

Study of non-covalent interactions of luotonin A derivatives and the DNA minor groove as a first step in the study of their analytical potential as DNA probes

The interaction between DNA and several newly synthesized derivatives of the natural anticancer compound luotonin A has been studied. The results from our work reveal an effective and selective alkaloid/double-stranded DNA (ds-DNA) interaction. In the presence of increasing amounts of ds-DNA, a noticeable fluorescence quenching of the luotonin A derivatives under study was observed. However, this effect did not take place when single-stranded DNA (ss-DNA) was employed. The association constant alkaloids/ds-DNA was calculated by quantitation of such a quenching effect. The influence of other quenchers, namely Co2+ and Br− on the native fluorescence of luotonin A and derivatives was also studied, and a remarkable quenching effect was observed for both ions. We have also investigated how by binding DNA the alkaloids could get protected from the external Co2+ and Br− quenchers. The Stern–Volmer constants (KSV) for Co2+ and Br− quenching effect on the studied alkaloids were considerably reduced (10–50%) after incubation of the compounds in the presence of DNA with regard to the KSV values in absence of DNA. An increase in the fluorescence anisotropy values of luotonins was also produced only in the presence of ds-DNA but not in the case of ss-DNA. To better characterize the nature of that interaction, viscosimetry assays and ethidium bromide displacement studies were conducted. With regard to DNA reference solutions, the viscosity of solutions containing DNA and luotonin A derivatives was reduced or not significantly increased. It was also observed that the studied compounds were unable to displace the intercalating agent ethidium bromide. All of these results, together with the obtained association constants values (Kass = 2.2 × 102 – 1.3 × 103), support that neither covalent nor intercalating interactions luotonin A derivatives/ds-DNA are produced, leading to the conclusion that these alkaloids bind ds-DNA through the minor groove. The specific changes in the fluorescence behavior of luotonin A and derivatives distinguishing between ss-DNA and ds-DNA binding, lead us to propose these compounds as attractive turn-off probes to detect DNA hybridization.

Influence of the presence of methyl cyclodextrins in high-performance liquid chromatography mobile phases on the separation of β-carboline alkaloids

The presence of cyclodextrins (CDs) in the mobile phase alters the chromatographic equilibria and induces a secondary chemical equilibrium associated to the chromatographic separation by HPLC. In this study the influence of the presence of CDs in the mobile phase as chemically modified beta-CDs, i.e. 2,3-di-O-methyl-beta-cyclodextrin (DMbeta-CD) and 2,3,6-tri-O-methyl-beta-cyclodextrin (TMbeta-CD) on the separation of the alkaloids norharmane, harmane and harmine is described. These beta-carboline alkaloids are chemically and structurally related and their quantitation by RP-HPLC is of interest due to their biological and pharmacological properties. Two stationary phases (methyl-, C(1) and octadecyl-, C(18)) were employed, with methanol:buffered aqueous solution and ethanol:buffered aqueous solution as mobile phases. The role of tert-butyl alcohol as a mobile phase modifier and also as an inclusion complex stabiliser was also considered. The concentrations of DMbeta-CD and TMbeta-CD vary from 0 to 17 mM. The presence of increasing amounts of CDs in the mobile phase reduces the retention factor. The changes observed in the retention factor allow the determination of the alkaloid/CD apparent association constants, whose magnitude is influenced by the chemical and structural properties of the guest molecules but also by the composition of the mobile phase. Assuming a 1:1 stoichiometry for the inclusion complexes, the apparent association constants obtained were higher for norharmane and for both DMbeta-CD and TMbeta-CD. The strength of the complexes is higher for DMbeta-CD than for TMbeta-CD and this behaviour can be explained considering the steric problems associated to the permethylated-beta-CDs. Besides significant differences in the magnitude of the apparent association constants were observed for the two stationary phases employed and thus can be related to the adsorption of CDs on the stationary phase. A significant reduction in the proportion of organic solvent in the mobile phase (50%) without a decrease in the selectivity or resolution of the separation is a favourable consequence of the presence of the CDs in the mobile phase.

Liquid chromatographic analysis of the anticancer alkaloid luotonin A and some new derivatives in human serum samples.

The quantitation of the natural cytotoxic and anti-inflammatory alkaloid luotonin A and five recently synthesized derivatives is described, constituting the first report of a HPLC method for the analysis of these compounds in human serum samples. The conditions for the chromatographic separation were optimized and the method was validated for the analysis of these compounds in biological samples according to international guidelines. An RP-HPLC method with fluorimetric detection and a C(18) stationary phase was applied. Different ACN/water mobile phases were assayed, including 0-4% of a mobile phase modifier such as tetrahydrofuran, dioxane or tert-butyl methyl ether. Isocratic and gradient elution conditions are compared. The influence of pH on the efficiency and resolution of the separation was also considered. The developed method was applied to the determination of luotonins in pooled human serum samples by gradient elution RP-HPLC using a simple cleanup procedure. The proposed chromatographic method exhibits satisfactory analytical figures of merit, with LOD from 1.0 x 10(-10) to 2.0 x 10(-10) M, intraday and interday precision below 6% except for the concentration level closest to LOD, and good agreement between experimental and theoretical concentrations. Therefore, the developed method is suitable, reliable, rapid, and simple.

B-ring-aryl substituted luotonin A analogues with a new binding mode to the topoisomerase 1-DNA complex show enhanced cytotoxic activity.

Topoisomerase 1 inhibition is an important strategy in targeted cancer chemotherapy. The drugs currently in use acting on this enzyme belong to the family of the camptothecins, and suffer severe limitations because of their low stability, which is associated with the hydrolysis of the δ-lactone moiety in their E ring. Luotonin A is a natural camptothecin analogue that lacks this functional group and therefore shows a much-improved stability, but at the cost of a lower activity. Therefore, the development of luotonin A analogues with an increased potency is important for progress in this area. In the present paper, a small library of luotonin A analogues modified at their A and B rings was generated by cerium(IV) ammonium nitrate-catalyzed Friedländer reactions. All analogues showed an activity similar or higher than the natural luotonin A in terms of topoisomerase 1 inhibition and some compounds had an activity comparable to that of camptothecin. Furthermore, most compounds showed a better activity than luotonin A in cell cytotoxicity assays. In order to rationalize these results, the first docking studies of luotonin-topoisomerase 1-DNA ternary complexes were undertaken. Most compounds bound in a manner similar to luotonin A and to standard topoisomerase poisons such as topotecan but, interestingly, the two most promising analogues, bearing a 3,5-dimethylphenyl substituent at ring B, docked in a different orientation. This binding mode allows the hydrophobic moiety to be shielded from the aqueous environment by being buried between the deoxyribose belonging to the G(+1) guanine and Arg364 in the scissile strand and the surface of the protein and a hydrogen bond between the D-ring carbonyl and the basic amino acid. The discovery of this new binding mode and its associated higher inhibitory potency is a significant advance in the design of new topoisomerase 1 inhibitors.

Challenging core-shell stationary phases with the separation of closely related anti-cancer compounds: performance studies and application to drug quantitation in cell cultures with multi-well plate clean-up.

In the present paper, we compare the chromatographic behaviour of six different stationary phases (5 μm fully porous and 2.6 μm core-shell particles, featuring octadecyl-, pentafluorophenyl-propyl- and phenyl-hexyl- functionalizations) for the separation of camptothecin and a family of closely related analogues of the natural anti-cancer drug luotonin A under different experimental conditions (organic solvent nature and proportion, temperature and presence of modifiers). We found two differentiated behaviours for the phenyl-hexyl-functionalized supports depending on the temperature. The efficiency and kinetic performance of fully porous vs. core-shell particles was compared by means of van Deemter and kinetic plots, using an optimized conventional HPLC system. Columns packing core-shell particles afforded improved efficiencies, permeabilities, analysis times and consumption of solvents. Nevertheless, our optimized system was not able to release the full intrinsic efficiencies of these columns. The replacement of acetonitrile with methanol had a dramatic effect in the behaviour of the pentafluorophenyl-propyl stationary phase. The aggregate of these results led us to select the core-shell C-18 stationary phase along with a H2O:MeCN gradient for the rapid and sustainable quantitation of the anti-tumour agents in cell cultures. A simple and rapid clean-up step was optimized using 96-well deproteinization and delipidation plates. The validation of the proposed analytical method showed excellent figures of merit. The combination of this sample pre-treatment procedure with the separation on core-shell particles yielded a fast methodology able to process a large number of samples with minimal waste generation and environmental impact.