Biomedical subjects
Publications and source records attributed to Helen C Hailes.
A tetrazolium red-based colorimetric assay has been devised to screen for transketolase activity with a range of aldehyde acceptors. The colorimetric TK assay is able to detect >8% bioconversion using non-alpha-hydroxylated aldehydes as acceptor substrates and is significantly faster and more convenient to use than chromatographic procedures.
In view of the range of properties required from supramolecular materials, there is clearly a need for new strong quadruple hydrogen bonded modules, which can be used in polymer or copolymer synthesis via the self- or hetero-association of complimentary units. A cytosine-based module has been prepared for supramolecular applications using a straightforward synthetic approach. The cytosine module was designed such that it does not undergo tautomeric changes observed with ureidopyrimidinones. The cytosine module was capable of forming quadruple hydrogen bonded assemblies both in solution and in the solid state, and the structure of the dimeric self-assembled unit was confirmed by single-crystal X-ray and solution NMR techniques. The dimerization constant was estimated to be greater than 9 x 106 M-1 in deuterated benzene. The capacity of the cytosine-based module to strongly hetero-associate with the ureidopyrimidinone module was demonstrated, and a supramolecular polymer of a bifunctional unit incorporating the cytosine module and PEG-based linker was described.
Highly stable cyclic dimers have been assembled through a combination of non-covalent interactions, including multiple hydrogen bonding, parallel stacking and hydrophobic shielding.
We have previously described a lipopolyplex formulation comprising a mixture of a cationic peptide with an integrin-targeting motif (K16GACRRETAWACG) and Lipofectin, a liposome consisting of DOTMA and DOPE in a 1:1 ratio. The high transfection efficiency of the mixture involved a synergistic interaction between the lipid/peptide components. The aim of this study was to substitute the lipid component of the lipopolyplex to optimize transfection further and to seek information on the structure-activity relationship of the lipids in the lipopolyplex. Symmetrical cationic lipids with diether linkages that varied in alkyl chain length were formulated into liposomes and then incorporated into a lipopolyplex by mixing with an integrin-targeting peptide and plasmid DNA. Luciferase transfections were performed of airway epithelial cells and fibroblasts in vitro and murine lung airways in vivo. The biophysical properties of lipid structures and liposome formulations and their potential effects on bilayer membrane fluidity were determined by differential scanning calorimetry and calcein-release assays. Shortening the alkyl tail from C18 to C16 or C14 enhanced lipopolyplex and lipoplex transfection in vitro but with differing effects. The addition of DOPE enhanced transfection when formulated into liposomes with saturated lipids but was more variable in its effects with unsaturated lipids. A substantial improvement in transfection efficacy was seen in murine lung transfection with unsaturated lipids with 16 carbon alkyl tails. The optimal liposome components of lipopolyplex and lipoplex vary and represent a likely compromise between their differing structural and functional requirements for complex formation and endosomal membrane destabilization.
[structure: see text] 2-Ureido-4-[1H]-pyrimidinones have been reported to dimerize via quadruple hydrogen bonding systems with dimerization constants >10(6) M(-1) in CDCl3. The dimerization constant, K(dim), is dependent on the solvent as well as the ring-substituents present, where previously alkyl (e.g., R1 = Me) and aromatic moieties (e.g., R1 = p-NO2C6H4, R1 = C6H2(OC13H27)3) have been incorporated at the C-6 position. To assess the influence of alternative, functionalizable, electron-donating groups on the dimerization motif and tautomeric distribution of isomers, the synthesis of compounds possessing aminophenyl functionality at the C-6 position has been achieved. NMR spectroscopy chemical shift analysis revealed that compound 2 (R1 = p-NH2C6H4, R2 = C6H13) existed as the 2-ureido-4-pyrimidinol dimeric DADA array in DMSO-d6, where a dimerization constant of 46 M(-1) was determined. This is the first time that a ureidopyrimidinone quadruple hydrogen bonding DADA array has been observed in pure DMSO, a highly polar solvent. The azo-derivative 5 of compound 2 was prepared which also adopted the pyrimidin-4-ol form in DMSO-d6. Compounds 7, 10 and 11 were then synthesized containing a more hydrophilic PEG unit in the lateral chain and the tautomeric distributions were determined.
The benefits of applying biocatalysts to organic synthesis, such as their high chemo-, regio-, and enantio-specificity and selectivity, must be seriously considered, especially where chemical routes are unavailable, complex or prohibitively expensive. In cases where a potential biocatalytic route is not yet efficient enough to compete with chemical synthesis, directed evolution, and/or process engineering could be implemented for improvements. While directed evolution has demonstrated great potential to enhance enzyme properties, there will always be some aspects of biocatalytic processes that it does not address. Even where it can be successfully applied, the resources required for its implementation must currently be weighed against the feasibility of, and resources available for developing a chemical synthesis route. Here, we review the potential of combining directed evolution with process engineering, and recent developments to improve their implementation. Favourable targets for the directed evolution of new biocatalysts are the syntheses of highly complex molecules, especially where chemistry, metabolic engineering or recombineering provide a partial solution. We also review some of the recent advances in the application of these approaches alongside the directed evolution of biocatalysts.
We used static light scattering to obtain new measurements on the internal structure of aggregated non-viral gene-delivery particles in colloidal suspension. The vector particles are prepared by charge neutralization of plasmid DNA either by poly-L-lysine or by a Lipofectin/integrin-targeting peptide. We use established theories of the stability of colloidal particles and fractal concepts to explain the aggregation processes and demonstrate the existence of a new property (fractal dimension) of the aggregated vector particles. Aggregation is shown to produce particles with fractal dimensions in the range between 1.8 and 2.4; the former suggests a loose three-dimensional structure and the latter characterizes an aggregation process that leads to the formation of particles with tightly packed structures. We show that the fractal dimension of the vector particles is sensitive to changes in physicochemical conditions (ionic strength) of the buffer solution and propose that fractal dimension may provide a useful means of monitoring the physical state of non-viral delivery-vector particles during preparation and storage.
The cationic diether-linked cytofectin DOTMA (available commercially as a mixture, Lipofectin comprised of DOTMA:DOPE, 1:1) and analogues including DIMRIE and DORIE are frequently used for in vitro and in vivo transfections. Despite this wide usage direct synthetic routes to the optical isomers have received little attention to date. Here we describe strategies to synthesize enantiomers of DOTMA and analogues, including an extremely concise procedure to the trimethylammonium salts. One strategy utilized N-protection, as the imine, with concomitant ether formation and deprotection during the workup. Methylation of the 1-amino-2,3-dialkyloxypropane then generated the trimethylammonium cationic lipids directly. This methodology was extended to synthesize a novel headgroup functionalized lipid. A second route was also developed using an alternative chiral synthon.
Human airway epithelial cell targeting peptides were identified by biopanning on 1HAEo-cells, a well characterised epithelial cell line. Bound phage were recovered after three rounds of binding, high stringency washing and elution, leading to the production of an enriched phage peptide population. DNA sequencing of 56 clones revealed 14 unique sequences. Subsequent binding analysis revealed that 13 of these peptides bound 1HAEo-cells with high affinity. Three peptides, SERSMNF, YGLPHKF and PSGAARA were represented at high frequency. Three clearly defined families of peptide were identified on the basis of sequence motifs including (R/K)SM, L(P/Q)HK and PSG(A/T)ARA. Two peptides, LPHKSMP and LQHKSMP contained two motifs. Further detailed sequence analysis by comparison of peptide sequences with the SWISSPROT protein database revealed that some of the peptides closely resembled the cell binding proteins of viral and bacterial pathogens including Herpes Simplex Virus, rotavirus, Mycoplasma pneumoniae and rhinovirus, the latter two being respiratory pathogens, as well as peptide YGLPHKF having similarity to a protein of unknown function from the respiratory pathogen Legionella pneumophila. Peptides were incorporated into gene delivery formulations with the cationic lipid Lipofectin and plasmid DNA and shown to confer a high degree of transfection efficiency and specificity in 1HAEo-cells. Improved transfection efficiency and specificity was also observed in human endothelial cells, fibroblasts and keratinocytes. Therefore, on the basis of clone frequency after biopanning, cell binding affinity, peptide sequence conservation and pathogenic similarity, we have identified 3 novel peptide families and 5 specific peptides that have the potential for gene transfer to respiratory epithelium in vivo as well as providing useful in vitro transfection reagents for primary human cell types of scientific and commercial interest.
Tuberculosis remains the leading cause of death among infectious diseases, accounting for more than two million deaths annually. The incidence of the disease is increasing globally, partially because of the resurgence of drug-resistant strains of Mycobacterium tuberculosis. Calixarenes are macrocyclic oligomers, some of which are able to modify the growth of M. tuberculosis in infected cells. Most experimental work has been carried out with Macrocyclon, also known as HOC 12.5EO. In this study, we demonstrate that Macrocyclon is effective in controlling M. tuberculosis infections, and we provide evidence that its effect is partially mediated by an l-arginine-dependent mechanism of macrophage activation that involves the activity of the inducible nitric oxide synthase. We also show that Macrocyclon is effective in athymic and major histocompatibility complex class II-/- mice and synthesized a number of structurally related calixarenes expressing significant antimycobacterial activity.
Methodologies to access water soluble large ringed calixarenes in good yield using efficient synthetic procedures have been investigated. Symmetrical partial functionalisations at the lower rim are described using activated [n]ethylene glycol chains and the addition behaviour contrasted with that of bromoalkanenitriles which proceeds with no observed regioselectivity. Full functionalisations of the calixarenes bearing hydrophilic groups are then investigated and a two-step procedure established which appears to be generally applicable for the addition of different [n]ethylene glycol chains. Furthermore, difunctionalisation under different reaction conditions are described. Throughout, strategies for the characterisation of these high mass compounds are outlined.
Concerns with insertional mutagenesis for retrovirus and immunogenicity for adenovirus have motivated research into development of non-viral vectors that can safely deliver desired gene constructs to target cells in tissues and organs. Many non-viral vectors suffer from unacceptably poor in vivo cell transfection and low transgene expression. Evidence suggests that cell transfection is linked to particle size - vector particles below about 200 nm are considered desirable. Experimental measurements indicate, however, that vector particles are susceptible to significant aggregation under most conditions of pH and ionic strength, including physiological conditions, although there are currently no means of predicting the kinetics of aggregation. The present paper addresses this challenge by presenting a mathematical framework based on the Monte Carlo simulation techniques for modelling the dynamics of aggregation. The approach is used to simulate the evolution of particle-size distribution for an integrin-targeting lipid-peptide-DNA vector system in buffers of different pH and ionic strength. The simulations required two input parameters, including the initial-size distribution of the particles and a fitting parameter (alpha). Comparison of simulations with experimental data showed that alpha was closely related to the zeta potential of the particles in the buffer medium, making simulations fully predictive. The modelling approach may be used in other vector systems.
A series of ferrocenyl conjugates to fatty acids have been designed and synthesized to establish the key properties required for use in biomolecular binding studies. Amperometric detection of the ferrocene conjugates was sought in the region of 0.3 V (vs Ag/AgCl) for use in protein/blood solutions. Different linkers and solubilizing moieties were incorporated to produce a conjugate with optimal electrochemical properties. In electrochemical studies, the linker directly attached to the ferrocene was found to affect significantly the E(1/2) value and the stability of the ferrocenium cation. Ester-linked ferrocene conjugates had E(1/2) ranging from +400 to +410 mV, while amide-linked compounds ranged from +350 to +370 mV and the amines +260 to +270 mV. Folding of long-chain substituents around the ferrocene, also significantly affected by the choice of linker, was inferred as a secondary effect that increased E(1/2). The stability of the ferrocenium cation decreased systematically as E(1/2) increased. Disubstituted ferrocene ester and amide conjugates, with oxidation potentials of +640 and +570 mV, respectively, showed only a barely discernible reduction wave in cyclic voltammetry at 50 mV/s. Electrochemical measurements identified two lead compounds with the common structural characteristics of an amide and carbamate linker (compounds 17 and 21) with a C(11) fatty acid chain attached. It is envisaged that such molecules can be used to mimic and study the biomolecular binding interaction between fatty acids and molecules such as human serum albumin.
The development of electrochemical probes useful for investigating the occupancy by other molecules of sites on complex proteins such as human serum albumin (HSA) is described. Ferrocenyl-(oxoethylene)-fatty acid compounds of different fatty acid chain length probed different binding sites on HSA. The interaction could be changed from one primarily with a drug binding site, when the probe was ferrocene methanol, to one predominantly with medium-chain fatty acid binding sites, by adding an (oxoethylene)-fatty acid substituents. Finally, the interaction could be changed to one interacting primarily with high-affinity long-chain fatty acid binding sites, as the fatty acid chain length in ferrocene-(oxoethylene)-fatty acid molecules increased. These results strongly implied that the binding could be further tailored by relatively simple modifications to the probe, for example, by changing the balance of hydrophobicity and hydrophilicity. The possibility of a procedure using mass-produced electrochemical cells to determine the fractional occupancy of different sites on HSA is demonstrated.