Dioxygenase-catalysed cis-dihydroxylation of meta-substituted phenols to yield cyclohexenone cis-diol and derived enantiopure cis-triol metabolites

cis-Dihydroxylation of meta-substituted phenol (m-phenol) substrates, to yield the corresponding cyclohexenone cis-diol metabolites, was catalysed by arene dioxygenases present in mutant and recombinant bacterial strains. The presence of cyclohexenone cis-diol metabolites and several of their cyclohexene and cyclohexane cis-triol derivatives was detected by LC-TOFMS analysis and confirmed by NMR spectroscopy. Structural and stereochemical analyses of chiral ketodiol bioproducts, was carried out using NMR and CD spectroscopy and stereochemical correlation methods. The formation of enantiopure cyclohexenone cis-diol metabolites is discussed in the context of postulated binding interactions of the m-phenol substrates at the active site of toluene dioxygenase (TDO).     

Observation of the reversibility of a covalent pyrrolobenzodiazepine (PBD) DNA adduct by HPLC/MS and CD spectroscopy

Pyrrolobenzodiazepines (PBDs) are sequence-selective DNA minor-groove binding agents that covalently bond to guanine with a reported preference for Pu-G-Pu sequences (Pu = Purine). Using HPLC/MS and Circular Dichroism (CD) methodologies, we have established for the first time that the aminal bond formed between PBD molecules and DNA is reversible. Furthermore, we have shown that while the rate of aminal bond cleavage does not depend on the sequence preference of a PBD molecule for a particular binding site, the rate of re-formation of the PBD-DNA adduct does. We have also shown that the PBD anthramycin (2) appears to be an exception to this rule in that, during cleavage from the DNA, its C-ring aromatizes and it cannot then re-attach due to a loss of electrophilicity at the C11-position. Although the C-ring aromatization of anthramycin has been previously reported to occur in the absence of DNA and after treatment with trifluoroacetic acid (TFA), in this case no pH lowering was required, with the DNA itself appearing to catalyse the process.     

Spin-crossover in [Fe(3-bpp)2][BF4]2 in different solvents--a dramatic stabilisation of the low-spin state in water

The temperature of spin-crossover in [Fe(3-bpp)(2)][BF(4)](2) (3-bpp = 2,6-di{pyrazol-3-yl}pyridine) tends to increase in associating solvents. In particular, T(?) shifts to 60-70 K higher temperature in water compared to organic solvents.     

Structure inducing ionic liquids-enhancement of alpha helicity in the Abeta(1-40) peptide from Alzheimer's disease

We have studied the impact of ionic liquid solvents on the structure of the Abeta(1-40) peptide from Alzheimer's disease and found that ionic liquid solvents were able to induce a conformational change in the structure of the Abeta(1-40) peptide. This conformational change impacts the self-assembly of the peptide into amyloid fibrils.     

Thienyl directed polyaromatic C-C bond fusions: S-doped hexabenzocoronenes

With a view to combining the desirable electronic and photochemical properties of hexabenzocoronene (HBC) and the C-C bond forming capabilities of thiophenes, 1-(3-thienyl)-2,3,4,5,6-penta(4-tert-butyl-phenyl)benzene (1) was oxidised using FeCl(3). The resulting products, superaromatic thiophene (2) and its 5,5'-dimer (3), are S-HBC systems and provide a new pair of spectral comparators.     

Organic modification and subsequent biofunctionalization of porous anodic alumina using terminal alkynes

Porous anodic alumina (PAA) is a well-defined material that has found many applications. The range of applications toward sensing and recognition can be greatly expanded if the alumina surface is covalently modified with an organic monolayer. Here, we present a new method for the organic modification of PAA based on the reaction of terminal alkynes with the alumina surface. The reaction results in the the formation of a monolayer within several hours at 80 °C and is dependent on both oxygen and light. Characterization with X-ray photoelectron spectroscopy and infrared spectroscopy indicates formation of a well-defined monolayer in which the adsorbed species is an oxidation product of the 1-alkyne, namely, its α-hydroxy carboxylate. The obtained monolayers are fairly stable in water and at elevated temperatures, as was shown by monitoring the water contact angle. Modification with 1,15-hexadecadiyne resulted in a surface that has alkyne end groups available for further reaction, as was demonstrated by the subsequent reaction of N-(11-azido-3,6,9-trioxaundecyl)trifluoroacetamide with the modified surface. Biofunctionalization was explored by coupling 11-azidoundecyl lactoside to the surface and studying the subsequent adsorption of the lectin peanut agglutinin (PNA) and the yeast Candida albicans, respectively. Selective and reversible binding of PNA to the lactosylated surfaces was demonstrated. Moreover, PNA adsorption was higher on surfaces that exposed the β-lactoside than on those that displayed the α anomer, which was attributed to surface-associated steric hindrance. Likewise, the lactosylated surfaces showed increased colonization of C. albicans compared to unmodified surfaces, presumably due to interactions involving the cell wall β-glucan. Thus, this study provides a new modification method for PAA surfaces and shows that it can be used to induce selective adsorption of proteins and microorganisms.     

Microbubble-sonosensitiser conjugates as therapeutics in sonodynamic therapy

A Rose Bengal sonosensitiser has been covalently attached to a lipid microbubble and the resulting conjugate shown to produce higher levels of singlet oxygen, enhanced cytotoxicity in a cancer cell line and a greater reduction in tumour growth than the sonosensitiser alone.     

The role of the C8 proton of ATP in the catalysis of shikimate kinase and adenylate kinase

ABSTRACT: BACKGROUND: It has been demonstrated that the adenyl moiety of ATP plays a direct role in the regulation of ATP binding and/or phosphoryl transfer within a range of kinase and synthetase enzymes. The role of the C8-H of ATP in the binding and/or phosphoryl transfer on the enzyme activity of a number of kinase and synthetase enzymes has been elucidated. The intrinsic catalysis rate mediated by each kinase enzyme is complex, yielding apparent KM values ranging from less than 0.4 muM to more than 1 mM for ATP in the various kinases. Using a combination of ATP deuterated at the C8 position (C8D-ATP) as a molecular probe with site directed mutagenesis (SDM) of conserved amino acid residues in shikimate kinase and adenylate kinase active sites, we have elucidated a mechanism by which the ATP C8-H is induced to be labile in the broader kinase family. We have demonstrated the direct role of the C8-H in the rate of ATP consumption, and the direct role played by conserved Thr residues interacting with the C8-H. The mechanism by which the vast range in KM might be achieved is also suggested by these findings. RESULTS: We have demonstrated the mechanism by which the enzyme activities of Group 2 kinases, shikimate kinase (SK) and adenylate kinase 1 (AK1), are controlled by the C8-H of ATP. Mutations of the conserved threonine residues associated with the labile C8-H cause the enzymes to lose their saturation kinetics over the concentration range tested. The relationship between the role C8-H of ATP in the reaction mechanism and the ATP concentration as they influence the saturation kinetics of the enzyme activity is also shown. The SDM clearly identified the amino acid residues involved in both the catalysis and regulation of phosphoryl transfer in SK and AK1 as mediated by C8H-ATP. CONCLUSIONS: The data outlined serves to demonstrate the "push" mechanism associated with the control of the saturation kinetics of Group 2 kinases mediated by ATP C8-H. It is therefore conceivable that kinase enzymes achieve the observed 2,500-fold variation in KM through a combination of the various conserved "push" and "pull" mechanisms associated with the release of C8-H, the proton transfer cascades unique to the class of kinase in question and the resultant/concomitant creation of a pentavalent species from the gamma-phosphate group of ATP. Also demonstrated is the interplay between the role of the C8-H of ATP and the ATP concentration in the observed enzyme activity. The lability of the C8-H mediated by active site residues co-ordinated to the purine ring of ATP therefore plays a significant role in explaining the broad KM range associated with kinase steady state enzyme activities.     

Bacterial dioxygenase- and monooxygenase-catalysed sulfoxidation of benzo[b]thiophenes

Asymmetric heteroatom oxidation of benzo[b]thiophenes to yield the corresponding sulfoxides was catalysed by toluene dioxygenase (TDO), naphthalene dioxygenase (NDO) and styrene monooxygenase (SMO) enzymes present in P. putida mutant and E. coli recombinant whole cells. TDO-catalysed oxidation yielded the relatively unstable benzo[b]thiophene sulfoxide; its dimerization, followed by dehydrogenation, resulted in the isolation of stable tetracyclic sulfoxides as minor products with cis-dihydrodiols being the dominant metabolites. SMO mainly catalysed the formation of enantioenriched benzo[b]thiophene sulfoxide and 2-methyl benzo[b]thiophene sulfoxides which racemized at ambient temperature. The barriers to pyramidal sulfur inversion of 2- and 3-methyl benzo[b]thiophene sulfoxide metabolites, obtained using TDO and NDO as biocatalysts, were found to be ca.: 25-27 kcal mol(-1). The absolute configurations of the benzo[b]thiophene sulfoxides were determined by ECD spectroscopy, X-ray crystallography and stereochemical correlation. A site-directed mutant E. coli strain containing an engineered form of NDO, was found to change the regioselectivity toward preferential oxidation of the thiophene ring rather than the benzene ring.