Donald S. Thomas

An investigation into the supramolecular structure, solubility, stability and antioxidant activity of rutin/cyclodextrin inclusion complex

The formation of supramolecular inclusion complexes between rutin and four cyclodextrins, namely β-cyclodextrin (β-CD), (2-hydroxypropyl)-α-cyclodextrin (HP-α-CD), (2-hydroxypropyl)-β-cyclodextrin (HP-β-CD) and (2-hydroxypropyl)-γ-cyclodextrin (HP-γ-CD), and the effects of the complexation on the stability and antioxidant activity of rutin were investigated. Results from phase-solubility studies showed that rutin formed 1:1 stoichiometric inclusion complexes with HP-α-CD, β-CD, HP-β-CD and HP-γ-CD; the complexes formed with HP-γ-CD and HP-β-CD had the greatest stability constants, followed by β-CD and HP-α-CD. Thermodynamic studies demonstrate that the inclusion of rutin into HP-β-CD was an exothermic process which occurred spontaneously. Two-dimensional rotating-frame nuclear Overhauser effect spectroscopy (2D ROESY) (1)H NMR analyses show that the A ring of rutin was the part of the molecule that most likely inserted into the cavity of HP-β-CD, thus forming a supramolecular inclusion complex. Formation of such an inclusion complex conferred moderate degrees of protection to rutin from degradation by heat and UV radiation during storage, and significantly enhanced its antioxidant capacity as determined by three different procedures.

Circular dichroism, kinetic and mass spectrometric studies of copper(I) and mercury(II) binding to metallothionein

The metalloprotein metallothionein (MT) is remarkable in its metal binding properties: for the mammalian protein, well-characterized species exist for metal to sulfur ratios of M7S20, M12S20, and M18S20, where M = Cd(II), Zn(II), Hg(II), Ag(I), Au(I), and Cu(I). Optical spectra in general, and circular dichroism (CD) and luminescence spectra in particular, provide rich detail of a complicated metal binding chemistry when metals are added directly to the metal-free or zinc-containing protein. CD spectral data unambiguously identify key metal to protein stoichiometric ratios that result in well-defined structures. Electrospray ionization-mass spectrometry data are reported for reactions in which Hg(II) binds to apo-MT 2A as previously described from CD data. Emission spectra in the 450-750 nm region have been reported for metallothioneins containing Ag(I), Au(I), and Cu(I). The luminescence of Cu-MT can also be detected directly from mammalian and yeast cells. We report both steady-state and new dynamic data for titrations of Zn-MT with Cu(I). Analysis of kinetic data for the addition of the first two Cu(I) atoms to Zn-MT indicates a first-order mechanism over a concentration range of 5-50 microM. Three-dimensional modeling was carried out using the results of the CD and EXAFS studies, model calculations for Zn7-MT, Hg7-MT, and Cu12-MT are described.

Influence of amine ligands on the aquation and cytotoxicity of trans-diamine platinum(II) anticancer complexes

Three (15)N-labelled trans-Pt(ii) amine complexes with isopropylamine ((15)N-ipa), methylamine ((15)N-ma) and dimethylamine ((15)N-dma) have been prepared and characterized. 2D [(1)H,(15)N] HSQC NMR spectroscopy was used to obtain the rate and equilibrium constants for the aquation of trans-[PtCl(2)((15)N-ipa)((15)N-ma)] ((15)N-1), trans-[PtCl(2)((15)N-dma)((15)N-ma)] ((15)N-2) and trans-[PtCl(2)((15)N-dma)((15)N-ipa)] ((15)N-) in 100 mM NaClO(4) solutions at 298 K. New (15)N shift ranges for H(2)N-Pt(II)-N and HN-Pt(II)-N groups are reported. Formation of the diaqua complex was not observed for and accounted for <2% of the species at equilibrium for 1 and 2 . The first aquation step is significantly faster for 2 (k(1) = 14 x 10(-5) s(-1)) than for the two complexes with the bulkier ipa ligand (k(1) = 5.5 x 10(-5) s(-1) (), 6.1 x 10(-5) s(-1) (3)), but 2 is the least aquated of the three complexes at equilibrium. The pK(a) values for the monoaqua adducts of 1-3 are similar (5.98, 5.85 and 5.91, respectively) and 0.4 pH units lower than the related cis complex cis-[PtCl(2)(dma)(2)], indicating a smaller proportion of more reactive aqua species will exist at physiological pH. The pK(a) values for the diaqua adduct of 2 (4.59 and 7.98) are 0.3-0.6 pH units higher than those of 1(4.31 and 7.30) and 3 (4.28 and 7.29), which have very similar values. The speciation profiles of 1-3 , calculated on the basis of the calculated equilibrium and dissociation constants, indicate that <1% hydrolyzed species will exist under physiological conditions in cancer cells. The cytotoxicity of 1-3 (non-(15)N-labelled) was assessed in three cancer lines (SF268, MCF-7 and NCI-H460). The new trans-Pt(ii) diamine complex 2 is more active than 1 and 3 in all cases and is more potent than cisplatin in the MCF-7 adenocarcinoma cell line.

Stereoselective Fluorination Alters the Geometry of a Cyclic Peptide: Exploration of Backbone‐Fluorinated Analogues of Unguisin A

New methods for enhancing the efficiency of peptide cyclization, and for fine-tuning the conformations of cyclic peptides, are valuable from a drug development perspective. Herein stereoselective fluorination is investigated as a new strategy for achieving these goals. Four vicinal difluorinated analogues of the natural cyclic heptapeptide unguisin A have been efficiently synthesized. The analogues are found to adopt dramatically different secondary structures, controlled by the fluorine stereochemistry.

Macromolecular ruthenium complexes as anti-cancer agents

Ruthenium complexes have shown promise as anti-cancer therapeutics. Among them, RAPTA-C [RuCl2(p-cymene)(PTA)] with the water-soluble 1,3,5-triaza-7-phosphaadamantane (PTA) ligand holds great promise as an alternative to platinum drugs. This paper explores synthetic pathways by which RAPTA-C can be attached to polymer moieties to create a water-soluble macromolecular drug that can potentially display an enhanced permeation and retention (EPR) effect. A reactive scaffold for RAPTA-C conjugation, poly(2-chloroethyl methacrylate) [PCEMA], was prepared via RAFT polymerisation. To increase the reactivity of the substrate, poly(2-iodoethyl methacrylate) [PIEMA] was prepared from PCEMA via the Finkelstein reaction. Two pathways were employed to conjugate RAPTA-C to the polymer, either by attachment of PTA first followed by reaction with the RuCl2(p-cymene) dimer, or by the direct conjugation of RAPTA-C to the polymer. Prior to the study with the polymer, both routes were tested using n-butyl iodide as a model compound. Several NMR experiments, namely 31P, 1H, [1H–31P] HMBC, [1H–13C] HMBC and [1H–15N] HMBC confirmed that both routes lead to the complete reaction of n-butyl iodide and that only the desired product was obtained. However, the direct reaction of RAPTA-C with the polymer required elevated temperature, which led to the loss of the p-cymene ligand. The two-step process at ambient temperature led to the successful formation of the product, albeit the amount of iodide reacted was now only 50% due to steric hindrance. Subsequently, a copolymer based on 2-hydroxypropyl methacrylamide [HPMA] and IEMA was synthesised. The two-step reaction with PTA and RuCl2(p-cymene) dimer led to a water-soluble polymer with an intense orange colour, indicative of the pendant RAPTA-C complex. The monomodal molecular weight distribution monitored using UV-VIS detection of the macromolecular ruthenium complex confirmed drug attachment and indicates the absence of crosslinking events during drug conjugation. A cytotoxicity profile for the copolymer–RAPTA-C product was measured on the ovarian cancer cell line OVCAR-3 and compared with the profile of the lone RAPTA-C drug.

Using Supramolecular Binding Motifs To Provide Precise Control over the Ratio and Distribution of Species in Multiple Component Films Grafted on Surfaces: Demonstration Using Electrochemical Assembly from Aryl Diazonium Salts

Supramolecular interactions between two surface modification species are explored to control the ratio and distribution of these species on the resultant surface. A binary mixture of aryl diazonium salts bearing oppositely charged para-substituents (either -SO3(-) or -N(+)(Me)3), which also reduce at different potentials, has been examined on glassy carbon surfaces using cyclic voltammetry and X-ray photoelectron spectroscopy (XPS). Striking features were observed: (1) the two aryl diazonium salts in the mixed solution undergo reductive adsorption at the same potential which is distinctively less negative than the potential required for the reduction of either of the two aryl diazonium salts alone; (2) the surface ratio of the two phenyl derivatives is consistently 1:1 regardless of the ratio of the two aryl diazonium salts in the modification solutions. Homogeneous distribution of the two oppositely charged phenyl species on the modified surface has also been suggested by XPS survey spectra. Diffusion coefficient measurements by DOSY NMR and DFT based computation have indicated the association of the two aryl diazonium species in the solution, which has led to changes in the molecular orbital energies of the two species. This study highlights the potential of using intermolecular interactions to control the assembly of multicomponent thin layers.

Silent information regulator 1 modulator resveratrol increases brain lactate production and inhibits mitochondrial metabolism, whereas SRT1720 increases oxidative metabolism.

Silent information regulators (SIRTs) have been shown to deacetylate a range of metabolic enzymes, including those in glycolysis and the Krebs cycle, and thus alter their activity. SIRTs require NAD+ for their activity, linking cellular energy status to enzyme activity. To examine the impact of SIRT1 modulation on oxidative metabolism, this study tests the effect of ligands that are either SIRT‐activating compounds (resveratrol and SRT1720) or SIRT inhibitors (EX527) on the metabolism of 13C‐enriched substrates by guinea pig brain cortical tissue slices with 13C and 1H nuclear magnetic resonance spectroscopy. Resveratrol increased lactate labeling but decreased incorporation of 13C into Krebs cycle intermediates, consistent with effects on AMPK and inhibition of the F0/F1‐ATPase. By testing with resveratrol that was directly applied to astrocytes with a Seahorse analyzer, increased glycolytic shift and increased mitochondrial proton leak resulting from interactions of resveratrol with the mitochondrial electron transport chain were revealed. SRT1720, by contrast, stimulated incorporation of 13C into Krebs cycle intermediates and reduced incorporation into lactate, although the inhibitor EX527 paradoxically also increased Krebs cycle 13C incorporation. In summary, the various SIRT1 modulators show distinct acute effects on oxidative metabolism. The strong effects of resveratrol on the mitochondrial respiratory chain and on glycolysis suggest that caution should be used in attempts to increase bioavailability of this compound in the CNS.

Efficient functionalisation of dextran-aldehyde with catechin: potential applications in the treatment of cancer

Here, we report a simple and efficient method to functionalise dextran with catechin – a polyphenol present in red wine, red berries, and green tea. Aldehyde functionalised dextran was reacted with catechin in the presence of acetic acid to yield dextran-aldehyde-catechin conjugates via a condensation reaction. 1D and 2D (DOSY) 1H NMR and UV-Vis analyses were employed to characterise the polymers. The degree of catechin conjugation was easily manipulated by varying the aldehyde amount in the dextran, the catechin concentration and the reaction temperature and conjugates with up to 38 wt% catechin functionalisation were prepared. In particular, the conjugation reaction was found to be especially sensitive to temperature and this was shown to be partially due to the equilibrium between the aldehyde and the hemiacetal in dextran aldehyde shifting to a greater proportion of aldehyde groups as the temperature increased. The stability of the dextran–aldehyde–catechin conjugates was enhanced in comparison with native catechin in both deionised water and phosphate buffer solution. Finally, the anticancer properties of the dextran–aldehyde–catechin conjugates were investigated in vitro, demonstrating cytotoxic efficacy against neuroblastoma cells whilst showing no cytotoxicity towards normal cells.

Analysis of Thiol-sensitive Core-cross-linked Polymeric Micelles Carrying Nucleoside Pendant Groups using ‘On-line’ Methods: Effect of Hydrophobicity on Cross-linking and Degradation

Amphiphilic block copolymers were prepared via reversible–addition fragmentation chain transfer (RAFT) polymerization and their synthesis, cross-linking, and degradation were studied using on-line monitoring. The focus of this work is the systematic alteration of the hydrophobic block using copolymers based on 5′-O-methacryloyluridine (MAU) and styrene at different compositions to determine the effect of the copolymer composition on the properties of the micelle. A poly(poly(ethylene glycol) methyl ether methacrylate) (PEGMA) macroRAFT agent was chain extended with a mixture of styrene and MAU. In both systems, an increasing fraction of styrene was found to reduce the rate of polymerization, but the functionality of the RAFT system was always maintained. The amphiphilic block copolymers were dialyzed against water to generate micelles with sizes between 17 and 25 nm according to dynamic light scattering (DLS). Increasing styrene content lead to smaller micelles (determined by DLS and transmission electron microscopy) and to lower critical micelle concentrations, which was measured using surface tensiometry. The micelles were further stabilized via core-cross-linking using bis(2-methacroyloxyethyl) disulfide as crosslinker. NMR analysis revealed a faster consumption of crosslinker with higher styrene content. These stable cross-linked micelles were investigated regarding their ability to degrade in the presence of dithiothreitol as a model reductant. Increasing the styrene content resulted in a faster degradation of the cross-linked micelles into unimers.

Tuneable catechin functionalisation of carbohydrate polymers.

In this contribution, we present a strategy to functionalise three natural carbohydrate polymers (dextran - a neutral polymer, sodium alginate - an anionic polymer and chitosan - a cationic polymer) with catechin with excellent degrees of functionality. In a first step, the carbohydrate polymers were oxidised by sodium periodate to yield aldehyde functionalised carbohydrate polymers. The presence of aldehyde groups was exploited to attach catechin by an acid catalysed nucleophilic reaction. The degree of catechin functionalisation could be easily tuned by varying the acid concentration in the reaction mixture, achieving catechin functionalisation levels of up to 48% for dextran aldehyde catechin, 35% for chitosan-aldehyde-catechin and 22% for sodium alginate aldehyde catechin. 1H, 1H-13C HSQC and DOSY NMR were performed to elucidate the structural differences between the three aldehyde functionalised polysaccharides and how this affects their reactivity and conjugation behaviour. All three carbohydrate polymer-catechin conjugates showed superior free-radical scavenging activity compared with the non-functionalised polymers.