Stephen J. Haswell

Consequences of manganese replacement of copper for prion protein function and proteinase resistance

The prion protein (PrP) binds copper and has antioxidant activity enhancing the survival of neurones in culture. The ability of the PrP to bind other cations was tested and it was found that only manganese could substitute for copper. Although initially manganese‐loaded PrP exhibited similar structure and activity to copper‐loaded PrP, after aging, manganese‐loaded PrP became proteinase resistant and lost function. It was also found that manganese could be incorporated into PrP expressed by astrocytes and that this PrP was partially proteinase resistant. These results show that it is possible to generate proteinase‐resistant PrP from cells and suggest a possible mechanism for the formation of the scrapie isoform of the PrP as generated in sporadic prion disease.

The application of micro reactors for organic synthesis

This tutorial review describes how micro reactors are being applied to synthetic chemistry covering a wide range of applications, from the preparation of nanograms of material for drug discovery and screening to the multi-tonne production of fine chemicals. This article explores how miniaturisation may revolutionise chemical synthesis and demonstrates that products are generated in higher yield and purity compared to the equivalent bulk reactions, in much shorter periods of time.

Antioxidant activity related to copper binding of native prion protein

We have developed a method to affinity‐purify mouse prion protein (PrPc) from mouse brain and cultured cells. PrPc from mouse brain bound three copper atoms; PrPc from cultured cells bound between one and four copper atoms depending on the availability of copper in the culture medium. Purified PrPc exhibited antioxidant activity, as determined by spectrophotometric assay. Incubation of PrPc with the neurotoxic peptide, PrP106‐126, inactivated the superoxide dismutase‐like activity. Culture experiments showed that PrPc protects cells against oxidative stress relative to the amount of copper it binds. These results suggest that PrPc is a copper‐binding protein which can incorporate varying amounts of copper and exhibit protective antioxidant activity.

Metal imbalance and compromised antioxidant function are early changes in prion disease.

The prion protein (PrP) has been shown to bind copper. In the present study we have investigated whether prion disease in a mouse scrapie model resulted in modification of metal concentrations. We found changes in the levels of copper and manganese in the brains of scrapie-infected mice prior to the onset of clinical symptoms. Interestingly, we noted a major increase in blood manganese in the early stages of disease. Analysis of purified PrP from the brains of scrapie-infected mice also showed a reduction in copper binding to the protein and a proportional decrease in antioxidant activity between 30 and 60 days post-inoculation. We postulate that alterations in trace-element metabolism as a result of changes in metal binding to PrP are central to the pathological modifications in prion disease.

The use of a novel microreactor for high throughput continuous flow organic synthesis

Abstract The aim of this study was to investigate the performance characteristics of a flow injection microreactor with reference to both the chemistry and reactor design using a model system, the established synthesis of 4-cyanobiphenyl based on a modified Suzuki coupling of an aryl halide and an organoboron compound. The catalytic reaction was carried out in micro-channels (300 μm wide and 115 μm deep) etched into glass and sealed with a top plate. The mobility of the reagent solutions was achieved using electroosmotic flow (EOF) assisted by the incorporation of a microporous silica structure within the microreactor channels, which acted as both a micro-pump and an immobilisation technique for the catalyst bed (1.8% palladium on silica). The yield of 4-cyanobiphenyl was determined by GC–MS. The synthesis of 4-cyanobiphenyl at room temperature in a flow injection microreactor, using a supported catalyst, without the addition of a base gave a product yield of 67±7% ( n =6). This was achieved by injecting 4-bromobenzonitrile for 5 s, with a 25-s injection interval, into a continuous stream of phenylboronic acid. A series of injections were performed over a 25-min period and the product collected for analysis. Palladium contamination in the crude product was found to be in the range of 1.2–1.6 ppb, determined using ICP–MS, indicating a low leach rate from the immobilised catalyst. A conventional laboratory batch scale method was also performed for the same synthesis using the identical conditions as those used in the flow injection microreactor, with and without the addition of a base, at both room and elevated temperatures (75–80°C) in an inert atmosphere under reflux for 8 h. The product yield for the non-optimised bulk reaction was 10% (determined by GC–MS), significantly lower than with the flow injection microreactor illustrating the potential of microreactors for clean efficient synthesis.

Green chemistry: synthesis in micro reactors

The importance of minimizing the impact that chemical processing has on the environment is growing, with an increased appreciation of the need to reduce pollution and the depletion of our finite environmental resources. Optimal use of material, energy and consequent waste management can be recognised as important factors for environmental protection. In the case of minimising waste there are two approaches, the traditional approach aims at reducing waste at the end of the pipeline, for example, decreasing emission by catalytic incineration of exhaust fumes. The second approach is based on minimising waste at the source. In this case, innovative procedures have to be employed to change both the method and the technology used throughout the production cycle. The miniaturisation of chemical reactors offers many fundamental and practical advantages of relevance to the pharmaceutical and fine chemicals industry, who are constantly searching for controllable, information rich, high throughput, environmentally friendly methods of producing products with a high degree of chemical selectivity. Indeed, for pharmaceutical companies an informatics-based approach, that micro reactor chemistry can uniquely deliver, may be the trigger for a step change in processes. This review explores how miniaturisation may revolutionise chemical synthesis, highlighting in particular the environmental benefits of this new technology, which include solvent free mixing, in situ reagent generation and integrated separation techniques. Furthermore, the possibility of preparing the chemicals in the required volume at point of use, negates the need to store and transport hazardous materials.

Chemical and biochemical microreactors

Abstract Research into the fundamental and practical advantages of using micrometre scale reactors for chemical and biochemical applications is now growing at a considerable rate. This review tracks such developments, illustrating their inherent strengths and identifying areas where further development of a technology is poised to revolutionise significant areas of synthetic chemistry and biochemistry.

Microfluidic combinatorial chemistry.

Microreactors are finding increasing application in the field of combinatorial chemistry. In the past few years, microreactor chemistry has shown great promise as a novel method on which to build new chemical technology and processes. It has been conclusively demonstrated that reactions performed within microreactors invariably generate relatively pure products in high yield. One of the immediate and obvious applications is therefore in combinatorial chemistry and drug discovery.

On-line microwave digestion of slurry samples with direct flame atomic absorption spectrometric elemental detection

A flow injection (FI) system for on-line microwave digestion of slurried samples with direct elemental determinations by flame atomic absorption spectrometry is described. Organically based elemental reference samples were prepared as slurries in 5% v/v HNO3 and the system was optimized for slurry mass, acid strength and tube and microwave cavity geometry. Bubble formation during digestion was controlled by post-digestion cooling and pressure regulation. Comparison of direct and FI calibrations indicated no apparent loss in sensitivity. Various samples were examined and elemental recoveries for Ca, Fe, Mg and Zn were typically found to be in the range 94–107% with precisions of less than 4.5% relative standard deviation. The major source of error was found to be in the dispersion of solids (<180 µm) as slurries in dilute HNO3. The through put of samples in the system developed was found to be 1–2 min per sample.

The application of micro reactors to synthetic chemistry

A feature article describing the fundamental characteristics and emerging applications of micro technology in the field of synthetic chemistry.