Andrew J. White

Cyclophanes with self-recognising components

High dilution techniques have been employed to construct a series of charged flexible cyclophanes comprised of π-electron-rich hydroquinone and π-electron-deficient bipyridinium residues linked by polyether spacer units of varying length: significant conformational changes, leading to intramolecular and intermolecular interactions between the π-donors and π-acceptors, have been observed—as evidenced by X-ray crystallographic and variable temperature 1H NMR and 2D ROESY spectroscopic investigations—within the series of the cyclophanes both in the solid and solution states as a result of increasing the lengths of the polyether spacer units.

Acyl-Chymotrypsins with Unsaturated Ligands Give Anomalous Vibrational Spectra

The stretching vibration of the unique ester acyl carbonyl of trans-β-aryl-acryloyl-chymotrypsins (aryl-CH=CH-C(=O)-O-chy) have previously been examined by both infrared (ir) difference spectroscopy [1] and resonance Raman (rR) spectroscopy [2]. The former is subject to artefacts arising from perturbation of enzymic amide and carboxyl groups, and the latter is subject to artefacts arising from formation of cis ligand by the exciting laser [3]. Isotopic substitution at the acyl carbonyl has previously successfully been used to eliminate perturbation derived features from ir difference spectra, and cinnamoyl-chymotrypsin (O-CH=CH-C(=O)-O-chy) was studied in this way [4,5]. According to both ir difference spectroscopy combined with isotopic substitution, and to rR spectroscopy disregarding the artefactual cis band, trans-β-aryl-acryloyl-chymotrypsins give two acyl carbonyl stretch bands at ca. 1700cm-1 with ca.5–15cm-1 separation. These were respectively assigned to a non-productive and a productive conformation [1]. Vibrational theory was used to estimate acyl carbonyl enthalpies on the basis of ir stretching frequencies, and this information related to generation of catalytic rate acceleration by the enzyme [1]. Later, the rR acyl carbonyl stretching frequencies for the productive conformations were related to the logarithm of the deacylation rate constants of the acyl-enzymes [6]. This produced a good linear relationship, although the deacylation rates were not corrected for intrinsic reactivity, and moreover, the series of trans-β-aryl-acryloyl ligands used were structurally very similar with a rather dubious inclusion of frequencies for the cis ligands [6].

The pH-Dependence of Conformational Distributions and Protein Perturbation in IR Difference Spectra of Acyl-Chymotrypsins

The serine proteinases have long attracted attention as excellent model systems for the study of enzyme catalysis. Despite an enormous experimental and theoretical knowledge base the underlying features responsible for the generation of the catalytic power of these enzymes has still not been resolved in detail. Such factors as the influence of remote site interaction on transition state stabilisation and the relative importance of the relay system vis a vis the oxyanion hole have not been fully resolved, despite extensive site specific mutagenesis and kinetic studies. It is clear that a combination of structural and dynamic methods will be necessary for further progress to be made. Vibrational spectroscopy offers a fruitful channel towards such progress since structural information en be gleaned in a time resolved fashion.

NMR and IR Studies of the Effect of Calcium on the Binding of Inhibitors to Phospholipase A2

The phospholipases A2 are a ubiqiuitous family of Ca2+-dependent enzymes which catalyse the hydrolysis of fatty acids from phospholipids [1]. Subsequent metabolism results in the formation of leukotrienes and prostaglandins, which play a major role in the inflammatory response. There is thus a strong interest in the design of compounds which may inhibit enzymes of this class for use as drugs. In order to be able to do this efectively it is necessary to have a detailed knowledge of both the mode of substrate binding to the enzyme and the catalytic mechanism.