Graduation Year

2022

Document Type

Dissertation

Degree

Ph.D.

Degree Name

Doctor of Philosophy (Ph.D.)

Degree Granting Department

Chemistry

Major Professor

Bill J. Baker, Ph.D.

Committee Member

James W. Leahy, Ph.D.

Committee Member

Theresa Evans-Nguyen, Ph.D.

Committee Member

Sandy D. Westerheide, Ph.D.

Keywords

Natural products, crannenols, tuaimenals, steroids, cervical cancer, respiratory syncytial virus

Abstract

Secondary metabolites produced primarily by terrestrial plants and animals have provided the basis for treatments and cures of human diseases as far back as historians have been able to probe. However, recent technological advances including the self-contained underwater breathing apparatus, and even further remotely operated vehicles, have allowed researchers a unique opportunity for targeted collection of marine organisms at depths previously thought to possess conditions unsustainable for life as was understood. This has afforded a glimpse into the potential for not only life, but the potential for new biochemical adaptations developed over billions of years that have been evolutionarily developed by deep-sea invertebrates to increase their chances of survival in harsh and competitive environments. These defenses often also possess the ability to combat a host of diseases responsible for countless ailments and needless human deaths worldwide each year including the anticancer eribulin (Halaven©) inspired by halichondrin B from a deep-sea sponge, and the antiviral sponge-derived vidarabine (ara-A©), amongst others. However, with such a vast environment left to explore there remains a stark need for investigation into the pharmacological potential of novel compounds from the deep.

The described studies herein are aimed at probing the chemical potential of such metabolites produced by three deep-sea soft corals existing on continental shelves surrounding Ireland. This unique landscape contains waters that rapidly drop from a few hundred meters to a few thousand meters in an environment that has been proven to host a plethora of biological diversity existing in close proximity to related, and competing species, leading to a battle for survival. Such a battle can only be won by sessile organisms that are able to produce a host of potent chemical defenses. Perhaps excitingly, no prior investigation into species belonging to the genus Acanella had been conducted. However, previous research into the species Duva florida had revealed a singular meroterpene in tuaimenal A with biological activity against both cervical cancer and the recently emerging SARS-CoV-2 virus. Further, recent work done on the genus Anthomastus of the Antarctic species bathyproctus had revealed the cytotoxic potential of a series of ring-A-containing dienone steroids. Thus, it was determined that there was still a strong justification for further exploration into the secondary metabolomes of the specimens described in this work.

Major aims of these studies were three-fold and included: 1) isolation and elucidation of new natural products from all three organisms described, 2) comprehensive biological evaluation of any new compounds against a panel of human disease targets, and 3) chemical probing of closely related haplotypes belonging to the same genus to support or contradict species identifications. Polar and nonpolar exhaustive extracts from each organism were subjected to repeat rounds of normal (NP) and reversed (RP) phase chromatography until pure compounds were obtained as determined by 1H and 13C NMR data. Chemical structures were then elucidated using a combination of high-resolution mass spectrometry and 2D NMR experiments. Following this workflow, four novel sesquiterpenes, crannenols A-D (2.15-2.16, 2.19-2.20), were isolated and characterized from the Alcyonacean Acanella arbuscula, for which no biological activity of the series was observed against the currently tested disease targets requiring further investigation into their potential. Similarly, seven new analogs of tuaimenal A (3.5), tuaimenals B-H (3.6-3.12), as well as two known ring-A aromatized steroids (3.13 and 3.14), were discovered from previously unexplored crude fractions of the same Duva florida specimens that yielded 3.5. Of these analogous metabolites, four were shown to possess increased antiproliferative effects against cervical cancer in vitro, with 3.11 representing a potential lead candidate for pharmacological development displaying an EC50 value of 0.04 M against the HPV-negative C33A cell line. From two genetically distinct haplotypes of an Alcyonacean soft coral belonging to the genus Anthomastus five new steroids (4.11-4.15), as well as five known steroids (4.16-4.19, 4.21), and a singular known briarine diterpene (4.20) were isolated and characterized. Following characterization, biological evaluation as well as investigation into the chemical relatedness of the secondary metabolomes of difficult to assign closely related species were conducted. These analyses revealed the potential of 4.11 to inhibit the expression of Renilla in RSV infected cells at a sub 10 g/mL IC50 value without proving to be cytotoxic to the cell itself as determined by evaluation of MTT cell viability. Additionally, cross-haplotype comparisons at the species level were analyzed on the basis of molecular networks prepared from MS/MS data of all ten metabolites.

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