Photochemical and oxidative cyclisation of tetraphenylpyrroles.

The photochemical and oxidative cyclodehydrogenation reactions of tetraphenylpyrroles act in a complementary fashion for the cyclisation of N-ethyl and N-benzyl derivatives. In the case of the former, a doubly cyclised product was isolated from cyclisation with solid FeCl3, while the latter gives a rearranged 3H-pyrrole upon irradiation.

Variation of guest selectivity within [Fe4L4](8+) tetrahedral cages through subtle modification of the face-capping ligand.

We report here the host-guest behaviour of two isoelectronic [Fe4L4](8+) tetrahedral cages that differ only in the nature of their face-capping ligand and possess either triazine (L1) or benzene (L2) cores. Crystallography reveals these hosts to be flexible and adaptable, while NMR spectroscopy shows them to be selective and discriminating in their host-guest behaviour.

Identification of uric acid as the redox molecule secreted by the yeast Arxula adeninivorans.

The yeast Arxula adeninivorans has been previously shown to secrete a large amount of an electro-active molecule. The molecule was produced by cells that had been cultivated in a rich medium, harvested, washed and then suspended in phosphate-buffered saline (PBS). The molecule was easily detectable after 60 min of incubation in PBS, and the cells continued to produce the molecule in these conditions for up to 3 days. The peak anodic potential of the oxidation peak was 0.42 V, and it was shown to be a solution species rather than a cell-attached species. We have optimised the production of the molecule, identified it by high-pressure liquid chromatography (HPLC) fractionation and high-resolution mass spectrometric analysis and determined the pathway involved in its synthesis. It has a mass/charge ratio that corresponds to uric acid, and this identification was supported by comparing UV spectra and cyclic voltammograms of the samples to those of uric acid. An A. adeninivorans xanthine oxidase gene disruption mutant failed to produce uric acid, which added further validity to this identification. It also demonstrated that the purine catabolism pathway is involved in its production. A transgenic A. adeninivorans strain with a switchable urate oxidase gene (AUOX) accumulated uric acid when the gene was switched off but did not when the gene was switched on. Cultivation of cells on amino acid and purine-free minimal media with an inorganic nitrogen source suggests that the cells synthesise purines from inorganic nitrogen and proceed to degrade them via the normal purine degradation pathway.

Endo-ß-N-acetylglucosaminidase catalysed glycosylation: tolerance of enzymes to structural variation of the glycosyl amino acid acceptor.

Endo-ß-N-Acetylglucosaminidases (ENGases) are highly useful biocatalysts that can be used to synthetically access a wide variety of defined homogenous N-linked glycoconjugates in a convergent manner. The synthetic efficiency of a selection of family GH85 ENGases was investigated as the structure of the acceptor substrate was varied. Several different GlcNAc-asparagine acceptors were synthesised, and used in conjunction with penta- and decasaccharide oxazoline donors. Different enzymes showed different tolerances of modification of the GlcNAc acceptor. Whilst none tolerated modification of either the 4- or 6-hydroxyl, both Endo M and Endo D tolerated modification of OH-3. For Endo D the achievable synthetic efficiency was increased by a factor of three by the use a 3-O-benzyl protected acceptor. The presence of a fucose at position-3 was not tolerated by any of the enzymes assayed.

A face-capped [Fe4L4]8+ spin crossover tetrahedral cage.

Reported here is a face-capped Fe(ii) molecular tetrahedron, [Fe(4)L(4)](BF(4))(8), . Single crystal X-ray diffraction at 153 and 293 K suggest spin crossover (SCO) and variable temperature magnetic susceptibility measurements confirm displays thermally driven SCO behaviour in the solid state and in dilute acetone solution centred around 284-288 K.

Large amplitude, proton- and cation-activated latch-type mechanical switches: O-protonated amides stabilized by intramolecular, low-barrier hydrogen bonds within macrocycles.

Large amplitude molecular switches have been developed using oxonium ions as the novel switching mechanism. Macrocycles that contain a polyether ring that are preorganized and of optimum geometry such that strong, linear Low-Barrier Hydrogen Bonds (LBHB, 2.4 to 2.6 A in length) are formed between a protonated amide oxygen and a cyclic ether, that lend significant iminol character to the amide. Deprotonation yields a large conformational change between closed and open forms, mindful of a new hinged, latch-type mechanical proton switch. Numerous open and closed forms have been characterized by X-ray crystallography, and the intramolecular hydrogen bond that forms between the protonated amide oxygen and the cyclic polyether oxygen accounts for the stability of these new acids. The open form of the deprotonated adducts persist in solution as indicated by the magnitude of coupling constants and other Nuclear Overhauser Effect experiments. Different saturated and unsaturated solid acids have been characterized including products derived from acetonitrile, propionitrile, caprylonitrile, acrylonitrile and adiponitrile, and also by reaction with primary amides in the case of phenyl and norbornene derivatives. We have also demonstrated that metal cations can replace the proton in the switching mechanism, characteristic of nascent synthetic pores.

Mutating the tight-dimer interface of dihydrodipicolinate synthase disrupts the enzyme quaternary structure: toward a monomeric enzyme.

Dihydrodipicolinate synthase (DHDPS) is a tetrameric enzyme that is the first enzyme unique to the ( S)-lysine biosynthetic pathway in plants and bacteria. Previous studies have looked at the important role of Tyr107, an amino acid residue located at the tight-dimer interface between two monomers, in participating in a catalytic triad of residues during catalysis. In this study, we examine the importance of this residue in determining the quaternary structure of the DHDPS enzyme. The Tyr107 residue was mutated to tryptophan, and structural, biophysical, and kinetic studies were carried out on the mutant enzyme. These revealed that while the solid-state structure of the mutant enzyme was largely unchanged, as judged by X-ray crystallography, it exists as a mixture of primarily monomer and tetramer in solution, as determined by analytical ultracentrifugation, size-exclusion chromatography, and mass spectrometry. The catalytic ability of the DHDPS enzyme was reduced by the mutation, which also allowed the adventitious binding of alpha-ketoglutarate to the active site. A reduction in the apparent melting temperature of the mutant enzyme was observed. Thus, the tetrameric quaternary structure of DHDPS is critical to controlling specificity, heat stability, and intrinsic activity.

Using high-performance liquid chromatography to measure the effects of protein-stabilizing cosolvents on a model protein and fluorescent probes.

The effect of cosolvents on the fluorescence of solutes was measured manually and in an automated high-performance liquid chromatography (HPLC) system that eliminates fluorescent contaminants on-line. The HPLC system was used to show that the effect of cosolvents on the fluorescence spectrum of heated chymotrypsin (a measure of unfolding) correlates with the effect of the solutes on the heat stabilization of catalytic activity; r2=0.73 with 12 example cosolvents. Changes in the fluorescence of model probes showed that known counteracting solutes slightly decrease the polarity of the solvent. Different cosolvents affect the proton transfer indicator, 2-naphthol (a model for tyrosinyl residues) differently, polyhydric alcohols enhance the protonated naphthol emission whereas zwitterionic solutes enhance naphthoxide fluorescence. The results with the automated system are consistent with the known stabilizing effects of the cosolvents and validate it as a tool to explore the development of novel cosolvents and their effects on multiple biological systems.