Author: David Rudd
Rudd, David, 2016 Mass spectrometric analysis, in situ imaging and preparative tools for the study of marine secondary metabolites: Chemo-ecological and natural products research on the muricid Dicathais orbita, Flinders University, School of Biological Sciences
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Species in the marine environment interact and thrive based on a language of chemical cues composed of secondary metabolites. Involved at the most basic level of organism to organism interaction, secondary metabolites affect a species ability to feed, fight, thrive and reproduce. Through these mechanisms, highly successful secondary metabolites termed ‘molecules of keystone significance,’ can drive resource allocation and shape ecosystem structures and diversity. Beyond an ecological value, some secondary metabolites have been developed into successful biomedical resources, and the marine environment can be considered a relatively untapped resource. Whether for ecological function or biomedical evaluation, secondary metabolites from the marine environment are poorly understood. Few studies have effectively defined function, in part due to the hurdles in method execution associated with demonstrating in situ synthesis, storage and deployment; key pieces of evidence in describing ecological relevance. Recently, analytical tools including mass spectrometry imaging have emerged to enable the spatial detection of chemical entities in biological tissues. Sophisticated approaches, including nanostructured surface-assisted laser desorption/ ionisation mass spectrometry imaging, have enabled the mapping of low molecular weight metabolites directly from tissue or tissue imprints, which is ideal for the analysis of secondary metabolites. Muricidae molluscs, famed for the production of the historically significant dye Tyrian purple, provide a well characterised model system for application of novel analytical and preparative tools to study secondary metabolite distribution. One Australian species in particular, Dicathais orbita, is an extensively studied model organism for muricid chemistry and ecology. D. orbita produce two classes of secondary metabolites that originate in the hypobranchial gland, brominated indoles, which are currently being investigated as chemo-preventative compounds and choline esters, known to be natural muscle relaxants. To investigate the biodistribution and ecological role of brominated indoles and choline esters in D. orbita, novel methods for mass spectrometry imaging were developed. In Chapter 2, a method for tissue imprinting for absorption of small molecular weight compounds onto functionalised porous silicon (pSi) chips was developed, then laser desorption/ionisation on pSi (DIOS) and nanostructure-assisted laser desorption ionisation (NALDI) mass spectrometry imaging were employed to spatially map brominated indoles and choline esters in situ within the hypobranchial gland. These methods were successful in detecting a range of secondary metabolites (molecular weights from 72 to 825 Da), across a broad range of polarities (from Log P -3.37 – 4.47) in D. orbita. In Chapter 3 this approach was further optimised using “on surface” solvent separation directly from mollusc tissue onto nanostructured surfaces, as a mechanism for simplifying the interpretation, annotation and quantification of MS data acquired from the complex mixtures of secondary metabolites found in natural tissue samples. Water, ethanol, chloroform and hexane selectively extracted compounds of different polarity and key metabolites of interest could be quantified by comparison to standard curves on the nanostructured surfaces. In Chapter 4, DIOS-MSI was applied to investigate the chemical ecology of D. orbita over the reproductive cycle. This confirmed the presence of tranquilising choline esters in the egg capsule gland of an adult female during the egg laying phase, and tracked maternally derived choline esters and brominated indole precursors into the fluid content of egg capsules. The chemical constituents of capsules encasing early stage embryos included hydrophilic tyrindoxyl sulfate and tranquilising murexine, whilst analysis of mature capsules accommodating hatching stage veligers detected hydrophobic Tyrian purple, 6,6’-dibromoindigo and 6,6’-dibromoindirubin. Changes in the secondary metabolite profile detected using DIOS-MSI demonstrates a slow process of chemical ripening. A change in the distribution and abundance of choline ester murexine suggests an important role in egg laying and larval development. The effect of murexine on larvae was further investigated by developing a larval mobility bioassay. The choline ester murexine was found to temporarily relax veliger larvae, with full recovery after 60mins exposure. These findings, coupled with the MS data showing the loss of polar compounds from the egg capsules over time, indicate that the secondary metabolite murexine relaxes the vulnerable early stage larvae to achieve developmental maturity inside the capsule environment, potentially influencing survival. Chapter 5 contributes to the potential development of the brominated indoles from D. orbita as medicinal products. A novel method for preparative extraction was designed and employed to selectively capture the bioactive fraction from D. orbita tissue without the need to use toxic chlorinated solvents. Supercritical CO2 extraction was successful in separating the bioactive brominated indoles from the polar non-active precursor and potentially toxic choline esters. Changes in the partial pressure enabled selective concentration of the anti-cancer agent 6-bromoisatin, with high yields that were comparable to solvent extraction. This provides a safe and effective extraction method for future in vivo bioactive testing of brominated indoles as chemo-preventative compounds and for nutraceutical development. Future preclinical studies will analyse the in vivo gastrointestinal absorption and metabolism of these brominated indoles employing the novel mass spectrometry imaging methods developed in this study. The inescapable presence of secondary metabolites in the marine biome is a testament to their value in shaping ecosystems and driving diversity. They are both an ecological resource and a natural product resource, providing humans with chemical structures of medicinal interest. Tools such as mass spectrometry imaging and supercritical fluid extraction that can aid in these dual pursuits will be of broad value to future marine natural products research.
Keywords: Mass Spectrometry Imaging, Chemical Ecology, Molluscs, Dicathais orbita, Brominated Indoles, Choline Esters, Marine Secondary Metabolites, Hypobranchial gland, Australian Whelk
Subject: Biological Sciences thesis
Thesis type: Doctor of Philosophy
School: School of Biological Sciences
Supervisor: Associate Professor James Harris