Ahmad, Mansoor (2011) Investigations into the isolation, structure elucidation and biosynthesis of bioactive natural products. PhD thesis, University of Warwick.

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Abstract

This project is divided into two parts. The first part of the project involved the
investigation of fish toxins produced by Streptomyces species. Samples for fishexposure
experiments were prepared from an actinomycete belonging to the S. griseus
clade isolated from a site of major fish kills, as well as from the type strain,
Streptomyces griseus DSM 40236. Fish-exposure samples were prepared through a
series of consecutive HPLC separations. We were able to narrow down the highest
level of fish toxicity to four fractions, each containing only a handful of compounds.
Comparison of the metabolic profile of the most toxic fractions showed that many of
the compounds were common to all of them e.g. compounds yielding ions with m/z =
213, 241, 239, 301 and 309 were present in all the fractions. Some of these
compounds were purified and analysed by high resolution mass spectrometry to
determine their molecular formulae. A sample preparation and purification protocol
for the fish toxins has been developed in this work. It was shown that the S. griseus
type strain produces ichthyotoxic metabolites in addition to the environmental strain.
This is a novel and unexpected observation.
The second part of the project involved structural and biosynthetic investigations of
the iron-chelator and natural Angiotensin Converting Enzyme (ACE) inhibitor
foroxymithine. The high structural similarity of foroxymithine to the known
siderophore coelichelin and the lack of unambiguous experimental evidence in the
literature to support the proposed structure of foroxymithine provided the impetus for
these investigations. Foroxymithine was purified from a novel source, the culture
supernatant of Streptomyces narbonensis. The gallium complex of purified
foroxymithine was prepared and analysed by one- and two-dimensional high-field
NMR experiments (1H, COSY, HSQC, HMBC, NOESY, TOCSY and Difference
NOE), which allowed the 1H and 13C NMR signals for the complex to be assigned.
Careful inspection of the 1H NMR spectrum of Ga-foroxymithine revealed two signals
(a major and a minor) for several of the protons. The origin of these signals was
investigated using variable temperature and 71Ga NMR experiments, and by LC-MS analyses on a homochiral stationary phase. The duplicate signals were proposed to be
associated with the existence of conformational isomers of Ga-foroxymithine.
The absolute stereochemistry of the four amino acid constituents of Ga-foroxymithine
was determined using Marfey’s method. Authentic standards of two of the anticipated
acid-hydrolysis products of Ga-foroxymithine, D- and L-N5-hydroxyornithine were
chemically synthesized to facilitate the Marfey’s analysis. The results confirmed that
foroxymithine contains L-N5-hydroxyornithine. Similar analysis was performed using
the authentic standards of D- and L-serine and the results confirmed that
foroxymithine contains L-serine. Marfey’s method was also applied to elucidate the
absolute stereochemistry (previously unknown) of coelichelin. Fe-coelichelin was
purified from Streptomyces coelicolor M145. Marfey’s derivatised coelichelin
hydrolysate and Marfey’s derivatives of L-Thr, L-allo-Thr, D-allo-Thr were analyed.
The results showed that coelichelin contains D-allo-Thr. Similarly, analyses were
carried out using the Marfey’s derivatives of chemically synthesised D- and L-N5-
hydroxyornithine, however the results were inconclusive.
The biosynthesis of foroxymithine in S. narbonensis was also investigated. A gene
fragment proposed to be within the putative foroxymithine biosynthetic gene cluster
was amplified by PCR from the genomic DNA of S. narbonensis. The gene fragment
was cloned into a plasmid vector and sequenced. This confirmed that it encodes part
of a putative formyl transferase that could be involved in foroxymithine biosynthesis.
Fosmid libraries of S. narbonensis genomic DNA were prepared. Despite exhaustive
efforts, a fosmid clone containing the putative formyl transferase encoding gene
fragment could not be identified via PCR based screening of the library.

Item Type:	Thesis (PhD)
Subjects:	Q Science > QD Chemistry
Library of Congress Subject Headings (LCSH):	Marine toxins -- Analysis, Streptomyces, Angiotensin converting enzyme -- Inhibitors -- Analysis
Official Date:	October 2011
Dates:	Date Event October 2011 Submitted
Institution:	University of Warwick
Theses Department:	Department of Chemistry
Thesis Type:	PhD
Publication Status:	Unpublished
Supervisor(s)/Advisor:	Challis, Gregory L.
Sponsors:	Great Britain. Environment Agency ; Engineering and Physical Sciences Research Council (EPSRC)
Extent:	xv, 153 leaves : ill., charts
Language:	eng

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