The enzyme creatine kinase (CK) is stabilized by encapsulation in silicate sol-gel monoliths. Its activity is measured as a function of long-term storage time at both room temperature and at elevated temperatures and is compared to that in solution. At room temperature, the activity of the encapsulated enzyme decreases to 50% of its initial value after more than 5 months, whereas in solution it decreases to 50% after 10 days. At 47 °C, the immobilized enzyme retains 50% of its maximum activity after 5 days of constant heating compared to that at 13 h in solution. At 60 °C, the immobilized enzyme retains 50% of its maximum activity after 5 h of heating as compared to that for less than an hour in solution. Surprisingly, a 4-fold increase in activity is observed after short exposures to the elevated temperatures. This increase is explained by structural changes in both the enzyme and the sol-gel matrix. The structural integrity and conformational changes of the encapsulated enzyme are observed by circular dichroism spectroscopy. The spectrum shows that the initially encapsulated enzyme has a structure different from that in solution but that upon heating the enzyme reverts to a conformation similar to that in solution. In addition, the encapsulated enzyme does not completely denature at temperatures up to 90 °C while in solution the midpoint temperature of the unfolding transition is 75 °C. These effects are interpreted in terms of electrostatic interactions between the positively charged patches on the enzyme's surface and the sol-gel matrix and to conformational changes within the pores upon heating. Heat treatment also affects the silica matrix by increasing the pore size as measured by gas absorption/desorption isotherms. The increase may allow small changes in the enzyme's structure, but in general the pore constrains the enzyme and inhibits denaturation.

An enzyme-based, dual working electrode system is described for the sensing of cyanide. Horseradish peroxidase (HRP) is incorporated as the sensing element. A continuous monitoring of oxidative activity by the enzyme results through the generation and regeneration of substrates at the electrode surfaces. Thus, HRP is oxidized by hydrogen peroxide generated from dissolved oxygen, at the primary electrode, and then reduced through the secondary electrode by mediated electron transfer using ferrocene as a carrier. Ferrocene regeneration at this electrode is proportional to the intrinsic activity of HRP. The dynamics of the system are investigated by using a rotating ring-disk electrode. The enzyme is immobilized to provide better control over its catalytic activity and to increase the lifetime of the biosensor. Cyanide inhibition of current can be modeled by reversible binding kinetics. Detection of cyanide is possible in submicromolar (ppb) concentrations, with a half maximal response at 2 microM. The response time for detection of introduced cyanide is within 1 s. The sensor can be operated between 5 and 40 degrees C, and cyanide inhibition is unaffected by pH changes between 5 and 8. The sensor is reproducible for cyanide determination and is stable for over 6 months.

An enzyme-based "electrochemical canary" is described for the detection of cyanide. The sensing system imitates cyanide's site of toxicity in the mitochondria. The terminal sequence of electron transfer in aerobic respiration is mimicked by mediator coupling of tyrosinase catalysis to an electro-chemical system. An enzyme-coupled oxygen electrode is created which is sensitive to selective poisoning. Biocatalytic reduction of oxygen is promoted by electrochemically supplying tyrosinase with electrons. Thus, ferrocyanide is generated at a cathode and mediates the enzymatic reduction of oxygen to water. An enzyme-dependent reductive current can be monitored which is inhibited by cyanide in a concentration-dependent manner. Oxygen depletion in the reaction layer can be minimized by addressing enzyme activity using a potential pulsing routine. Enzyme activity is electrochemically initiated and terminated and the sensor becomes capable of continuous monitoring. Cyanide poisoning of the biological component is reversible, and it can be reused after rinsing. The resulting sensor detects cyanide based on its biological activity rather than its physical or chemical properties.

An approach to the evaluation of carbon catalysts suitable for the industrial manufacture of phosgene is described. Relative reactivity and the oxidative stability of catalysts have been measured in laboratory microreactors. Comparison of the performance of full size catalyst pellets versus that of crushed catalyst allowed catalyst effectiveness factors and therefore effective gas diffusivities to be estimated. These data have been combined with a 2-dimensional model incorporating a description of heat and mass transfer to predict catalyst performance in industrial scale phosgene reactors.

The synthesis of (S)-N-Boc-bis(4-fluorophenyl)alanine, an intermediate in the synthesis of denagliptin, is described from the synthesis of a 12 g proof of principle sample to a >900 kg cGMP manufacturing campaign. The chiral centre was established by the asymmetric hydrogenation of the sterically crowded precursor, ethyl 2-acetamido-3,3-bis(4-fluorophenyl)acrylate. The ability to isolate the various intermediates in a physical form that would readily allow filtration, washing, and ultimately purification underpinned the successful manufacturing campaign.