The preparation of two compounds designed to mimic the active site of Isopenicillin N Synthase (IPNS) is presented. Both compounds consist of two fragments from the enzyme linked by a lipophilic unit. The first contains a short linker unit namely 11-aminoundecanoic acid and the second contains a longer linker consisting of three of these units. The compounds were prepared by solid phase peptide synthesis and purified using a combination of gel permeation chromatography and Phenyl Sepharose hydrophobic interaction chromatography. These compounds have been examined in a two phase system of <i>n</i>-octane and aqueous buffer, containing the IPNS cofactors and tripeptide substrate. The postulated mimic would involve the lipophilic linker unit residing in the organic phase, thus holding the two peptides in close proximity in the aqueous phase. It was hoped that the addition of the Fe<SUP>2</SUP>+ cofactor might promote the adoption of a structure resembling that of the enzyme active site. Cyclisation of the penicillin precursors ACV and PCV has not been observed under the conditions investigated, however the compound containing the short linker has been observed to bind iron. Work on immunological studies on IPNS fragments has resulted in the preparation of anti-sera to the N-terminal peptide (IPNS(1-21). These anti-sera have been shown to bind to the peptides IPNS(1-21) and Cys-IPNS(1-21) in an ELISA test and have also been used to detect the native enzyme IPNS by the Western blot method.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:652359 |
| Date | January 1991 |
| Creators | Henry, Jennifer A. |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/12076 |
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