Paul Wormell

Two dimensional reversed-phase-reversed-phase separations isomeric separations incorporating C18 and carbon clad zirconia stationary phases

Informational theory and a geometric approach to factor analysis were employed to evaluate the degree of orthogonality of a two-dimensional reversed-phase-reversed-phase chromatographic system. The system incorporated a C18 column as one dimension and a carbon clad zirconia column as the second dimension. In order to study the resolving power of this system, the separation of a sample matrix containing an artificial mix of 32 isomers (structural and diastereoisomers) was evaluated. Using this system, between 25 and 28 of the 32 isomers could be separated, depending on the mobile phase combinations--with resolution that could not possibly be achieved in a single one dimensional separation. The results from this study indicate that in order to fully evaluate the resolving power of a 2D system multiple methods of analysis are most appropriate. This becomes increasingly important when the sample contains components that are very closely related and the retention of solutes is clustered in one quadrant of the 2D space. Ultimately, the usefulness of the 2D separation is determined by the goals of analyst.

Spectroscopic studies of 9-hydroxyellipticine binding to DNA

The binding of 9-hydroxyellipticine to calf thymus DNA, poly[d(A-T)]2, and poly[d(G-C)]2 has been studied in detail by means of CD, linear dichroism, resonance light scattering, and molecular dynamics. The transition moment polarizations of 9-hydroxyellipticine were determined in polyvinyl alcohol stretched film. Spectroscopic solution studies of the DNA/drug complex are combined with theoretical CD calculations using the final 50 ps of a series of molecular dynamics simulations as input. The spectroscopic data shows 9-hydroxyellipticine to adopt two main binding modes, one intercalative and the other a stacked binding mode involving the formation of drug oligomers in the DNA major groove. Analysis of the intercalated binding mode in poly[d(A-T)]2 suggests the 9-hydroxyellipticine hydroxyl group lies in the minor groove and hydrogen bonds to water with the pyridine ring protruding into the major groove. The stacked binding mode was examined using resonance light scattering and it was concluded that the drug was forming small oligomer stacks rather than extended aggregates. Reduced linear dichroism measurements suggested a binding geometry that precluded a minor groove binding mode where the plane of the drug makes a 45 degrees angle with the plane of the bases. Thus it was concluded that the drug stacks in the major groove. No obvious differences in the mode of binding of 9-hydroxyellipticine were observed between different DNA sequences; however, the stacked binding mode appeared to be more favorable for calf thymus DNA and poly[d(G-C)]2 than for poly[d(A-T)]2, an observation that could be explained by the slightly greater steric hindrance of the poly[d(A-T)]2 major groove. A strong concentration dependence was observed for the two binding modes where intercalation is favored at very low drug load, with stacking interactions becoming more prominent as the drug concentration is increased. Even at DNA: drug mixing ratios of 70:1 the stacked binding mode was still important for GC-rich DNAs.

Improved oxidative stability of sunflower oil in the presence of an oxygen-scavenging film

The oxidative stability of sunflower oil (SFO) was measured during storage at 23 and 37°C in the presence of a novel oxygen-scavenging film that contained polyfuryloxirane (PFO). Commercially refined and deodorized SFO was stored in a lighted room in sealed transparent packages containing either PFO film or an antioxidant, 0.02% butylated hydroxytoluene (BHT). Oxidative stability was evaluated by determination of peroxide values and gas-chromatographic measurement of headspace hexanal. SFO stored in the presence of the oxygen-scavenging film was more stable than oil stored without the film, or than film stored with 0.02% BHT. The PFO film scavenges oxygen through energy-transfer sensitization of singlet oxygen. The film is doped with eosin and the naturally-occurring dye, curcumin, which absorb over a wide range of visible wavelengths. Curcumin transfers its absorbed energy to eosin, which sensitizes the production of singlet oxygen. The singlet oxygen is scavenged by PFO. The use of two dyes increases the efficiency of the sensitization process, reducing the illumination time and intensity required for effective oxygen scavenging.

Electronic spectra of some pterins and deazapterins

Abstract The electronic absorption spectra of some derivatives of pterin and N5-deazapterin are analysed using the CNDO/S-CI method, including allowance for solvent shifts. These calculations give good agreement with the spectra, which may be assigned to a group of π*←π transitions. There are some differences between calculated values for gas-phase and solution models but their general level of conformity with experiment is similar. Band shifts caused by methyl substitution, protonation and replacement of ring nitrogens are investigated, and a number of structural and spectroscopic problems are addressed. The spectral predictions agree well with experimental assignments for tautomeric forms and protonation sites for known compounds, and predictions are made for the spectra of N8-deazapterin and N5,N8-dideazapterin which have not been reported.

Synchrotron radiation linear dichroism spectroscopy of the antibiotic peptide gramicidin in lipid membranes

We have developed synchrotron radiation linear dichroism (SRLD) to measure the insertion of peptides into lipid bilayers, significantly improving both signal-to-noise and wavelength range over existing methods. Our wavelength cut-off is currently determined by the quality of quartz in the cell, rather than the light source, with signal quality still high at the cut-off. We demonstrate the use of a lipid probe to measure the orientation of the lipid bilayers under flow and describe the way in which this can be used to further interpret SRLD data. The antibiotic peptide gramicidin is shown to exhibit drastically different kinetic and equilibrium behaviour when interacting with lipid membranes with different properties. The charge on the membrane is of interest because of differences in charge between human and bacterial membranes. For this reason we increased the negative charge on the membrane by changing the lipid composition. Increasing negative charge in the gel phase stabilises the liposomes but changes the kinetics of peptide folding. In a gel phase with no negatively charged lipids, gramicidin does not fold well and gives a small signal that indicates a change in orientation of the tryptophan side chains over time. In the fluid phase with no negatively charged lipids, there is initially >10-fold greater peptide signal relative to the gel phase indicating a highly folded and ordered gramicidin backbone. This is followed by liposome disruption. In the gel phase with negatively charged lipids the liposomes are resistant to disruption by gramicidin and exhibit different folding kinetics depending on membrane composition. In the fluid phase with negatively charged lipids there is little signal from either the peptide or the lipid probe indicating that the liposomes have been disrupted by the gramicidin in the time it takes to make the first measurement.

THE ELECTRONIC SPECTRUM OF PHTHALAZINE. THEORY AND EXPERIMENT

Abstract Ab initio configuration interaction-singles (CIS) calculations of the electronic spectra of phthalazine and pyridazine predict two neighbouring singlet ( π ∗ , n) states: 1A2 followed by 1B1 for phthalazine, and the reverse for pyridazine. A comparison of the 3–21G and 6–31G∗ basis sets for pyridazine shows that the 3–21G basis set gives satisfactory results, and may be used in CIS calculations for the larger molecule, phthalazine. The lowest-energy π ∗ ← n transition for phthalazine is predicted to be forbidden, in agreement with experiment, but in conflict with most semiempirical calculations. Ab initio calculations are used to predict the vibrational frequencies of phthalazine and phthalazine-d4 at the HF/6–31G∗ and CIS/3–21G levels for the ground and excited electronic states, respectively. The ground-state calculations for phthalazine are in good agreement with experiment, including the results of several SERS studies. The vapour absorption spectrum of phthalazine is reported for the first time and analysed using ab initio, semiempirical and rotational-contour calculations. The first 6000 cm−1 of the spectrum is assigned to a forbidden π ∗ ← n transition, confirming the findings of crystal-state studies and the CIS/3–21G predictions. The higher-energy regions of this band system show no clear evidence of a second π ∗ ← n transition, despite theoretical predictions and circular-dichroism observations to the contrary.

Synthesis and Analysis of the Structure, Diffusion and Cytotoxicity of Heterocyclic Platinum(IV) Complexes.

We have developed six dihydroxidoplatinum(IV) compounds with cytotoxic potential. Each derived from active platinum(II) species, these complexes consist of a heterocyclic ligand (HL) and ancillary ligand (AL) in the form [Pt(HL)(AL)(OH)2](2+), where HL is a methyl-functionalised variant of 1,10-phenanthroline and AL is the S,S or R,R isomer of 1,2-diaminocyclohexane. NMR characterisation and X-ray diffraction studies clearly confirmed the coordination geometry of the octahedral platinum(IV) complexes. The self-stacking of these complexes was determined using pulsed gradient stimulated echo nuclear magnetic resonance. The self-association behaviour of square planar platinum(II) complexes is largely dependent on concentration, whereas platinum(IV) complexes do not aggregate under the same conditions, possibly due to the presence of axial ligands. The cytotoxicity of the most active complex, exhibited in several cell lines, has been retained in the platinum(IV) form.

Selectivity and the Separation of Isomers of Low Molecular Weight Polystyrenes

Abstract The separation of diastereoisomers of polystyrene has been investigated on carbon‐clad zirconia (CCZ), C18, and diamond C18 stationary phases using eluents of widely different properties. Eluents were chosen according to their selectivity, solvent polarity index, and polymer solubility constants. Acetonitrile mobile phases in combination with CCZ produced the best diastereoisomer separations, however, when methanol was employed as the mobile phase, the separation profiles were very similar, except that retention was greatly increased. Retention and isomer separation decreased when isopropanol was employed. Surprisingly, some diastereoisomer separation was still apparent using the CCZ column and a 99% hexane/1% isopropanol mobile phase, although none was observed for the same solvent mixture on a C18 column. Indeed, significant diastereoisomer separation was only obtained on the C18 column‐using acetonitrile as the mobile phase, but the resolution was far inferior to that observed on the CCZ column. The diamond C18 surface, which is a hybrid of C18 and CCZ, gave separations that were intermediate between those of the parent surfaces, with enhanced molecular weight selectivity in comparison with CCZ, but slightly less discrimination on the basis of stereochemistry. Polystyrenes eluted unretained if either dichloromethane or tetrahydrofuran were used as the mobile phases on all stationary phases.

Investigation of Spectra and Ionization Constants of 8-Alkyl Pterins by Fluorimetry

Summary Spectrofluorimetric studies of the properties of 8-alkyl-pterins arc reported, together with preliminary results on the use of fluorimetry in studying the binding of these new mechanism-based substrates to dihydrofolate reductase (DHFR). The compounds fluoresce strongly in the 460-490 nm range with emission maxima for the cations being blue-shifted by ~5-20 nm. Quantum yields for the cations of ~0.14-0.23 have been measured. pKa values for the basic ionization of ~5.3-5.7 have been determined from fits to both the emission and excitation spectra using sample concentrations in the 1-2 µM range. Although the UV /vis spectra between pH 0 and 4 show little change. there were significant sigmoidal changes in fluorescence intensity which could he fitted to apparent ionization constant values of ~1.4-1.0 with fluorescence intensity decaying to almost zero at pH 0. Studies of the fluorescence for the cations and neutral forms in organic acids and polar organic solvents respectively indicate little solvent dependence. Spectral modelling using the CNDO/S-CI method confirms that the spectra are due to π→π* transitions. and that the long wavelength absorption hand of ~400 nm is due to transitions involving mostly the pyrazine ring and N1. Significant differences in emission wavelength and intensity between 6-methyl- and 7 -methyl-8-alkyl-pterins have been characterised with the latter exhibiting fluorescence stronger by a factor of ~2 and blue-shifted by ~20 nm which arc readily detectable visually: aqua-blue and green fluorescence respectively for the 7-methyl and 6-methyl isomers. From this understanding of the fluorescence properties of 8-alkyl-pterins and their dependence on charge state and pyrazine-ring substituents, and taking into account the fluorescence characteristics of DHFR and cofactor NADP. we have assessed the potential of fluorimetric experiments for studying both pterin dissociation constants and the form of enzyme-bound pterin

Computer-Aided Design of Mechanism-Based Pterin Analogues and MD/FEP Simulations of Their Binding to Dihydrofolate Reductase

Using a combined theoretical and experimental approach we have been able to predict several chemical properties and the contributions of the many factors which determine the macroscopic binding behaviour of these new mechanism-based compounds with DHFR, and also analyse experimental data to develop structure-activity relationships.