6 derivatives of papain bearing coenzyme analogues covalently attached to the active site cysteine have been prepared and characterised. 2 of them, bearing thiazolium groups, were shown to catalyse the dimerisation of 2 ketones apparently following a mechanism similar to that well understood for thiamine dependent enzymes, 4 derivatives, 2 each bearing pyridinium and quinolinium groups, were shown to undergo redox reactions via the 1,4-dihydro form. The 1,4-dihydropyridine derivatives were able to reduce electrophilic carbonyl compounds slowly with small enantiomeric excess.
In each of the 3 groups, the coenzyme analogue bearing a benzyl substituent was superior to that bearing a methyl or ethyl substituent. This result correlated well with the model deduced from the crystal structure of papain which showed that the benzyl group was accommodated in a hydrophobic pocket at the active site. Taking this result together with the experimental studies, it is probable that the reactions are taking place close to the surface of the protein and that the active site pocket is closed by the cofactor analogue.
One of the most important properties of any molecules in living systems is their chirality, that is their existence in one only of a number of stereoisomers. In macromolecules such a proteins, polysaccharides, and DNA, the chirality is also important and in many compounds that are potential drugs or crop protection chemicals, there exist two steroisemers that are non-superimposable mirror images of naturally occuring macromolecules, only one enantiomer of a drug usually contains the required biological activity. At best this means that half of the administered drug is useless and at worst, that half can have catastrophic effects. It is believed that the thalidomide tragedy arose because the drug was sold as a mixture of enantiomers, one of which was safe and useful, but the other was teratogenic. Recently, there as been an increasing awareness of the need for chemicals for use as pharmaceuticals or as crop protection chemical to be manufactured and sold in the correct chiral form. A product with the correct chirality will be intrinsically safer and will thus have a significant market advantage over an inferior product composed of a mixture of isomers.
The demand for synthesis of molecules in the correct chiral from presents a challenge to organic chemistry. There are a great many sophisticated chemical reagents that can be used for such stereoselective synthesis but their high cost is likely to minimise their industrial value. Attention has therefore been turned to using naturally occuring catalysts, namely enzymes, and much recent research has demonstrated the practicability of the use of enzymes in stereoselective chemical synthesis. However naturally occuring enzymes do not satisfy all of the demands of chemical synthesis especially with regard to substrate acceptability and to catalytic activity. There is therefore a need for a radical innovative approach to the production of enzymes with properties ideal for chiral synthesis. This is especially so in the case of carbon-carbon formation which we address in this programme for the first time. A particularly attractive but as yet unexplored way to satisfy the demain for new synthetically useful enzymes is to introduce new catalytic activities into existing enzymes so that the selectivity intrinsic in the natural enzyme ca be applied to new reactions.
Funding SchemeCSC - Cost-sharing contracts