Thesis (Ph. D.)--Hong Kong University of Science and Technology, 2002. / Includes bibliographical references. Also available in electronic version. Access restricted to campus users.

Thesis (Ph.D.) - University of Queensland, 2004. / Includes bibliography.

Thesis (Ph. D.) University of Wisconsin, 1967. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

No description available.

Streptomycetes are Gram positive, soil dwelling filamentous bacteria, known for their production of secondary metabolites. Genome sequencing of Streptomyces coelicolor identified 26 known or predicted secondary metabolite gene clusters ranging from antibiotics to siderophores, lipids, pigments and lantibiotics. Most studies investigating secondary metabolite production in Streptomyces, as well as other bacteria, are undertaken in liquid or on solid media. There is little gene expression data available from in situ studies. This study determined expression in a totally different growth medium, soil, to gain insight into growth and adaptation under more 'normal' habitat conditions and the effect changing environmental factors has on the expression of secondary metabolite gene clusters. In order to do this, S. coelicolor was grown in soil microcosms from which RNA was extracted and amplified using an optimised T7 polymerase-based RNA amplification protocol. The amplified RNA was used to determine gene expression profiles via endpoint RT-PCR and RT-qPCR. In a complementary approach, S. coelicolor sand microcosms were subjected to a novel protein extraction procedure to determine protein expression profiles in soil. This study elucidated how carbon, nitrogen and metal availability, the environmentally bioactive entomopathogenic fungus Metarhizium anisopliae and nematode Steinernema kraussei affect secondary metabolite gene expression in soil. In contrast to the consensus of secondary metabolism commencing after reduction or cessation of growth, this study revealed expression of secondary metabolite biosynthetic genes and proteins related to secondary metabolism before the onset of exponential growth. Some secondary metabolite genes/proteins were even expressed constitutively. Soil microcosms have been shown to be an important tool for gene expression analysis. The results of these novel transcriptomic and proteomic approaches therefore have given new insight into secondary metabolism and its role under natural habitat conditions.

No description available.

This thesis presents an investigation into the chemistry of 6 substituted 5, 6-dihydro-a-pyrone compounds. A comprehensive review of these compounds was published in 1989 and the subsequent literature is covered in an updated review presented below. Eight 6-substituted 5,6-dihydro-a-pyrone metabolites from three different South African plant species Cryptocarya latijolia, Syncolostemon densiflorus, and Syncolostemon argenteus have been the subject of structural and stereochemical investigations. The absolute stereochemistry of the known compound "triacetate" from C. latijolia has been established as 6R-[2R,4S,6S-(triacetyloxy)heptylJ-5,6-dihydro-2H-pyran-2-one (74) using CD and acetonide formation with subsequent application of the modified Moshers method. The absolute stereochemistry of the related metabolite "diacetate", also from C. latijolia, has been assigned as 6R-[2S,4S-diacetyloxypentylJ-5,6-dihydro-2H-pyran-2-one (76). In addition, the outstanding stereochemistry at C-5' in syndenolide, from S. densiflorus, followed from conversion to its diacetonide and subsequent NMR analysis. Syndenolide is therefore 6R-[5S-(acetoxy)-IR,2R,3S-(trihydroxy)-heptylJ-5,6- dihydro-2H -pyran-2-one. The genus Syncolostemon has proved to be a rich source of a-pyrone compounds and the chemistry of S. argenteus, not investigated previously, was examined as part of an ongoing search for new 5,6-dihydro-a-pyrones. The study yielded five new a-pyrone natural products, synargentolide A-E. The structure of synargentolide A (82) has been assigned as 6R[4R,5R,6S-triacetyloxy-lE-heptenylJ-5,6-dihydro-2H-pyran-2-one using CD and NMR techniques. The structures of synargentolide B (87), C (92) and E (94) also followed from a detailed NMR analysis and the stereochemistry tentatively assigned based on CD and NMR data. Synargentolide D (93) was thermally unstable, and a paucity of material prevented stereochemical investigations, however the structure was determined from initial NMR analysis. The marine molluscs of the genus Siphonaria have only become the subject of chemical studies in the last fifteen years. These molluscs characteristically produce polypropionate type natural products. A review of Siphonarian polypropionate metabolites containing a pyrone functionality is presented. Examination of an endemic South African species Siphonaria serrata yielded one novel polypropionate metabolite containing a ),-pyrone functionality, siserrone A (131). The structure of this compound was unambiguously established using standard NMR experiments. The relative stereochemisty of the hemi-ketal moiety was assigned from a careful analysis of the ROESY NMR spectrum and the stereochemisty of the acyclic portion determined from a comparison of the 13C and 'H NMR data of a degradation product with the corresponding data of a synthetic compound. It was also established that the modified Moshers method could not be used to determine the absolute stereochemistry of the secondary hydroxy I substituent at C-11. The absolute stereochemistry of 131 was thus assigned in accordance with the proven stereochemistry of Siphonarian metabolites.

A study of the secondary metabolism of two northeastern Pacific sponges and two Sri Lankan nudibranchs has led to the isolation of thirteen new and one previously known natural products. The structures of all of the compounds were determined by a combination of spectroscopic data analysis and chemical interconversions. A study of the chemistry of the northeastern Pacific sponge Hexadella sp. has resulted in the isolation of six new brominated alkaloids. Two of these, hexadellins A (77) and B (78), are derived from dibromotyrosine. The structures of compounds 77 and 78 were determined via their acetylated derivatives 79 and 80. Four compounds possessing novel bis(indole) structures have also been isolated. Topsentin B2 (74) was isolated as a mixture of two slowly interconverting tautomers, 74a and 74b. Methylation of 74 resulted in the production of trimethyltopsentin B2 (75); the structure of 75 was determined spectroscopically. The structures of dragmacidons A (81), B (82), and C (83) were determined by a combination of spectral data interpretation and chemical interconversions. Dragmacidon C (83) was originally incorrectly assigned as 88; the correct structure was determined based upon synthesis of the model compound 94. Compounds 77 and 78 possess antimicrobial activities. Compounds 74 and 81 show considerable cytotoxic and antineoplastic activities. Five new triterpene glycosides have been isolated from the northeastern Pacific sponge Xestospongia vanilla. The structures of isoxestovanin A (125), xestovanin C (127), dehydroxestovanin A (129), epi-dehydroxestovanin A (131), and dehydroxestovanin C (132) were all determined by a combination of spectral data interpretation and chemical interconversions. These compounds all contain the deoxy sugars L-rhamnose and D-fucose. Isoxestovanin A (125) possesses a new carbon skeleton and xestovanin C (127) and dehydroxestovanin C (132) possess linear trisaccharide fragments which have not been previously encountered in triterpene glycosides isolated from X. vanilla. The Sri Lankan nudibranch Chromodoris glenei has yielded the known compound 12-desacetoxyshahamin C (153) and the new metabolite shahamin K (155). Both compounds possess dendrillane diterpene skeletons. The compound chromodorolide B (156) was isolated from specimens of Chromodoris cavae. It is only the second known diterpene natural product possessing the chromodorane skeleton. / Science, Faculty of / Chemistry, Department of / Graduate

Marine phytoplankton produce extracellular metabolites which may be important in controlling the interactions among species or the competition for a limiting nutrient. While the absolute amount of these metabolites may be small compared to the primary organics released by the phytoplankton cell, the control of the production of these unique metabolites may be an important factor in the ecology of the producing species. These compounds have not been extensively studied due to the difficulty in isolating these minute quantities from seawater. In this thesis, two externally produced metabolites have been investigated. The concentration of 1 -(2,6,6-trimethyl-4-hydroxycyclohexenyl)- 1,3-butanedione, a nor-caroteniod commonly referred to as the β-diketone, was quantitatively determined during the exponential and senescent stages of growth of Prorocentrum mi n imum in P-, N-, and iron-deficient batch cultures. The β-diketone was released extracellularly in a single 'pulse' during the stationary stage of growth. Several factors such as temperature, irradiance, type of nutrient-deficiency (N, P, or Fe), and the ambient nitrate concentration were important in establishing the amount of the β-diketone produced. The environmental factors did not influence the temporal pattern of production, only the absolute amount of the β-diketone produced. The limits of the range of production of the β-diketone were narrower than the range of maximum growth for any environmental influence. The inhibition of growth and the heterotrophic uptake of glucose by marine bacteria, demonstrated the antibacterial properties of the β-diketone. The second extracellular organic examined was prorocentrin. Prorocentrin is the extracellular siderophore produced by Prorocentrum minimum, P. mariae-lebouriae, and P.gracile. Functionally similar compounds are produced by Thalassiosira pseudonana and Dunaliella tertiolecta. This study is first to characterize this type of high-affinity iron (111)-transport system in marine eukaryotic phytoplankton. The pattern of siderophore production by all species is the same, although the absolute amount of the material produced is species specific. There is no intracellular or extracellular siderophore production under iron-sufficient culture conditions. When iron was deficient there was a short period of rapid extracellular siderophore production during the stationary stage of growth. The intracellular prorocentrin concentration was very low which suggests that de novo synthesis of the prorocentrin occurs just prior to extracellular release. The persistence of the extracellular siderophore in the culture medium was brief. There was an increase in the in vivo fluorescence following the loss of the siderophore from the medium. The increase in in vivo fluorescence was not accompanied by an increase in cell concentration. An hypothesis concerning the mechanism of the iron-uptake system is proposed. Procedures for the isolation and characterization of prorocentrin are presented. Prorocentrin appears to be a tri-hydroxamate siderophore with a molecular weight between 560 and 590 daltons. The iron-prorocentrin complex is stable over a wide pH range. / Science, Faculty of / Earth, Ocean and Atmospheric Sciences, Department of / Graduate

Tandem mass spectrometry (MSMS) has become a powerful tool for the analysis of biomolecules. To reveal molecular identity, experimental mass (m/z) data are either matched to appropriate databases or processed de-novo. Both approaches are essentially one-dimensional, because the m/z values of fragment ions play dominant role, while the intensities (the second dimension) are being neglected, despite their potential to corroborate or contradict the identification. Unlike the trivial case of m/z values, predicting the ion intensities has not been mastered yet beyond empirical observation and statistical treatment. This dissertation presents a fundamental, structure-based algorithm for the prediction of fragment ion intensities in MSMS spectra of peptides and metabolites. The algorithm builds on the central hypothesis that the fragment ion intensities reflect the relative abundances of respective protonated precursor isomers prior to fragmentation. The hypothesis is supported by extensive experimental evidence showing that in ion trap mass detectors, relative ion intensities do not depend on the energy or activation time of fragmentation when commonly used ranges of conditions are explored. The multi-step algorithm developed includes molecular mechanics Monte Carlo conformational space sampling, semi-empirical calculations, and Density Functional Theory (DFT) quantum chemistry computations for structure refinement and energy calculations. A Boltzmann distribution determined from energy values of pertinent precursors accurately corresponded with relative ion intensities in MSMS spectra of three model pentapeptides. Reproducibility of the algorithm was tested, and while substantial differences were revealed among the multiple Monte Carlo samplings started from the same initial structures, the inconsistency was mitigated in following semi-empirical and DFT steps. The algorithm was optimized for efficiency as well. Computational costs were lowered (by more than 50%) by narrowing the energy window in which the conformers were taken to the following steps in the algorithm and finally to the Boltzmann distribution. For metabolites, ion intensity orders for nine out of eleven molecules were predicted correctly. However, the accuracy of the prediction of relative ion intensities was not satisfactory. Nevertheless, predicting the most intensive ion alone could be invaluable for preliminary metabolite identification and selecting good candidate standards for ultimate identification based on matched properties of analyte and standard.