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Title: Synthetic studies relating to the biosynthesis of erthyromycin
Author: Cutter, A.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 1997
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Erythromycin A (1) is a macrolide antibiotic produced by the soil bacterium, Saccharopolyspora erythraea. It has been shown that erythromycin is biosynthesised from the sequential condensation of one propionyl and six methylmalonyl units on the polyketide synthase (PKS), to give the first enzyme free intermediate, 6-deoxyerythronolide B(2). The polyketide synthase consists of three large multifunctional proteins, DEBS 1, DEBS 2 and DEBS 3. 6-Deoxyerythronolide B (2) is then elaborated by a series of further tailoring enzymes, culminating with the production of erythromycin A (1). Three organisms capable of producing truncated erythromycin PKSs have been constructed; DEBS 1-TE, which contains a copy of the thioesterase from the end of DEBS 3 at the end of module 2, DEBS 2-TE, which contains a thioesterase at the end of module 4 and ΔDH DEBS 2-TE, another truncated PKS with the thioesterase at the end of DEBS 2, but with a non-functional dehydratase in module 4. In addition, a synthetic route was devised for the preparation of the spiroketal lactone pentaketide (7), one of the proposed products from the modified protein DEBS 2-TE. The attempted isolation of 7, or its open form (8) from the modified protein, DEBS 2-TE was unsuccessful and it has been concluded that no pentaketide is being produced by DEBS 2-TE. The attempted synthesis of the hydrated spiroketal lactone pentaketide (9), one of the proposed products from the modified protein ΔDH DEBS 2-TE, is also discussed. The work described in this dissertation deals with the synthesis of diketide intermediates for incubation studies with DEBS 1-TE, the synthesis of some postulated products from the modified proteins, DEBS 1-TE, DEBS 2-TE and the attempted synthesis of the product from ΔDH DEBS 2-TE. It also describes the attempted isolation of products from the modified protein, DEBS 2-TE. The syntheses of some simple analogues of the β-hydroxy diketide and β-keto diketide intermediates of the biosynthesis of erythromycin are described. These contain variations at the C2 and C4 alkyl groups and were used to investigate diketide incorporation by the PKS of DEBS 1-TE. The syntheses of the N-acetylcysteamine (NAC) thioester (3) of the triketide intermediate from the biosynthesis of erythromycin, and the triketide lactone (4), produced from the mutant DEBS 1-TE, are discussed. The route to the triketide lactone (4) enabled incorporation of the deuterium (2H) and carbon-14 (14C) isotopes to give the labelled lactones 5, 6. Triketide lactones containing a variety of C5 alkyl groups were isolated from an array of experiments based around the modified protein DEBS 1-TE. These included heterologous expression of DEBS 1-TE in an unnatural host, purification of the protein to homogeneity and subsequent incubation with natural and unnatural starter acids, extender units and NADPH, and the expression of a chaemeric DEBS 1-TE containing a loading domain from the avermectins producing PKS from Streptomyces avermitilis. The synthetic route to the triketide lactone was modified, by using a variety of aldehydes in the first aldol condensation, to afford synthetic standards of these new lactones, whose syntheses are described. These standards were used to aid isolation and structure elucidation of the produced novel lactones.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available