Although chemists often exploit the high stereo selectivity of hydrolytic enzymes to produce pure enantiomers, the difficulty in selecting the best hydrolase from hundreds of commercial hydrolases and microorganisms remains a major deterrent to their wider use. Moreover, the emerging ability to create large libraries of recombinant enzymes demands fast and simple selection methods. The current method of selection is time-consuming because it requires carrying out small-scale reactions and measuring enantiomeric purity. This thesis focuses on the development of methods to speed up this selection process. / We first report the development of empirical substrate models for Aspergillus niger lipase (ANL) to predict which enantiomer of a racemate reacts faster. For secondary alcohols, a rule based upon the size of the substituents at the stereocentre of a substrate previously proposed for other lipases and esterases works for ANL. Surprisingly, a rule based upon charge rather than size of the substituents can predict the preferred enantiomer of alpha-amino acids. These qualitative rules aid in the design of new synthetic applications of ANL. / Next, we report the development of a spectropholometric assay to accurately but rapidly measure the enantioselectivity of a hydrolase towards a target substrate without measuring enantiomeric purity called "quick E". The initial rates of hydrolysis of each enantiomer of a substrate are separately measured relative to a reference compound. The ratio of the two relative rates yields the enantioselectivity. We first developed this method for chromogenic esters. Next, to extend quick E to non-chromogenic esters, we developed a pH indicator assay to quantitatively detect ester hydrolysis. We optimized this method for screening in 96-well plates for speed. / Finally, we apply these spectrophotometric assays to solve a synthetic problem. Dioxolane nucleosides are powerful pharmacological agents used in the treatment of HIV and hepatitis-B virus but their syntheses require expensive and tedious silica gel chromatography to separate mixtures of diastereomers. Using the quick E method, we rapidly identify two hydrolases that produce the desired dioxolane diastereorner in good yield and excellent diastereomeric excess (>98%). / Overall, our screening methods are simpler to perform than traditional methods and require significantly less substrate and hydrolase (mug quantities per measurement) without compromising sensitivity and quantitativeness.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.37580 |
| Date | January 1998 |
| Creators | Janes, Lana Elizabeth. |
| Contributors | Kazlauskas, Romas J. (advisor) |
| Publisher | McGill University |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Format | application/pdf |
| Coverage | Doctor of Philosophy (Department of Chemistry.) |
| Rights | All items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated. |
| Relation | alephsysno: 001762044, proquestno: NQ55398, Theses scanned by UMI/ProQuest. |
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