Design and Application of Probes of Novel Bioactivities

Abstract

Whilst methods for the isolation and optimisation of bacterial enzymes for preparative biotransformations are well established the use of plant biocatalysts is relatively unexplored. Since the plant kingdom contains a greater diversity of potential biocatalysts this represents an untapped resource. Two simple plant enzyme systems were investigated: AtSFGH and AtCXE12. A series of profluorescent esters based on 4 methylumbelliferone and fluorescein were synthesised and used in screening experiments against these enzymes. To produce libraries of recombinant proteins molecular biology techniques such as site-directed mutagenesis and error-prone PCR were utilised and Agrobacterium tumefaciens infiltration methods developed to increase the levels of transformation within plant protoplasts.
In order to isolate specific enzymes probe molecules were developed in which a desired bioactivity resulted in the ‘switching on’ of a fluorophore during enzymatic processes. Several reactivity probes were synthesised which contained a fluorophore, a fluorescent quencher moiety and a functional group which was able to react with a specific protein target. An ester probe was developed which reacted with porcine liver esterase, with the enzymatic response causing the molecule to fragment in such a way as to release the fluorescent quencher and result in a concomitant increase in fluorescence. The modular synthesis and structure of the probe allowed for a variety of reactive groups to be introduced.
Several BODIPY analogues were synthesised that were able to exclusively target the peroxisome organelle, as demonstrated by co-localisation studies. Their proliferation in the presence of external signals, such as the drug clofibrate and biotic stress, were studied. Confocal microscopy enabled the dynamics of the peroxisome to be visualised in a variety of cell types and species, including both plant and mammalian cells. In addition to this computational studies were carried out to enable the rational design of probes based on their fluorescent properties. Systematic alteration of the probe molecule has highlighted areas of the compound which are amenable to adaptation. This includes the ability to extend the conjugation of the pyrrole functionality which enables the emission wavelength, and hence colour, to be changed.