New drugs, particularly antibiotics, are urgently required to combat the increasing problem of antibiotic resistant human pathogens. Due to the scarcity of products available today, the pharmaceutical industry is now under pressure to reassess compounds derived from plants, soil and marine organisms. Pharmaceutical companies are showing renewed interest in marine biotechnology as the oceans represent a rich source of both biological and chemical diversity of novel molecular structures with anti-cancer, anti-inflammatory and antibiotic properties. Formerly unexplored locations, such as deep ocean sediments, show great potential as a source of genetically novel microorganisms producing structurally unique secondary metabolites. In this research, a metabolite producing marine Pseudoalteromonas strain, known as AP5, was initially used to develop methods for the detection, optimisation of production and extraction of bioactive metabolites from other potentially novel marine isolates. Two hundred and seventy six (276) marine isolates from water and sediment samples from the Antarctic Ocean and Marion Island were isolated. Ten visually different isolates were screened for bioactivity against Gram-positive and -negative bacteria, fungi and yeast. Three out of the 10 isolates, WL61 , WL 114 and WL 136, appeared to be novel Streptomyces spp. showing activity against different test organisms. Many of these marine microorganisms are difficult to culture in the laboratory, particularly when they are cultivated continuously in shake flasks as they can stop producing bioactive compounds. The cultivation of marine isolates in bioreactors may be a more beneficial process for the optimisation of metabolite production compared to conventional liquid fermentation techniques whereby the solid-liquid-air interface of membrane bioreactors can imitate the natural environment of microbes. The membrane bioreactor system is a stable growth environment with low shear that supports steady-state biofilm growth consisting of a high cell density due to a high mass transfer of nutrients and oxygen to the cells. This approach was employed and isolates WL61, WL114 and WL136 were immobilised onto ceramic membranes using Quorus single fibre bioreactors (SFR). The SFRs were used to establish the most suitable growth medium for continuous secondary metabolite production. The best growth conditions were applied to the Quorus multifibre bioreactor (MFR) for scale up of biologically active metabolites, highlighting the potential of bioreactor technology for use in bioprospecting for isolating and screening novel and known organisms for new and interesting natural products. Furthermore, the Quorus MFR was shown to be suitable for the production of high yields of antimicrobial metabolites and is an efficient new fermentation production system. Purification by HPLC fractionation was used to characterise four major compounds from isolate WL 114 extracts. NMR structure elucidation identified one of the two primary compounds as Bisphenol A. The complete chemical structure for the second potent bioactive compound could not be determined due to the low concentration and volume of material. / KMBT_363 / Adobe Acrobat 9.54 Paper Capture Plug-in

Seaweeds of the genus Plocamium are known to produce a variety of halogenated monoterpenes. In addition to their ecological role as feeding deterrents, biological activities reported for these compounds include antibacterial, antialgal, antifungal and anticancer activities. An investigation of the non-polar extracts of the seaweed Plocamium corallorhiza resulted in the isolation of six known halogenated monoterpene compounds, 4-bromo-5-bromomethyl-1-chlorovinyl-2, 5-dichloro-methylcyclohexane (2.68), 1,4,8-tribromo-3 ,7-dichloro-3, 7-dimethyl-1,5-octadiene (2.67), 8-bromo-1 ,3,4,7-tetrachloro-3, 7-dimethyl-1,5-octadiene (2.66), 4,6-dibromo-1,1-dichloro-3,7-dimethyl-2,7-octadiene (2.64), 4,8-dibromo-1,1,7-trichloro-3,7-dimethyl-2,5-octadiene (2.65) and 3,4 ,6,7-tetrachloro-3, 7-dimethyl-1-octene (2.63) as well as eight new compounds, including five halogenated monoterpene aldehydes. The new compounds were identified by 1D and 2D NMR spectroscopic techniques as: 8-Bromo-6,7-dichloro-3,7-dimethyl-octa-2,4-dienal (2.72), 8-Bromo-1,1,2,7-tetrachloro-3,7-dimethyl-octa-3,5-diene (2.70), 4,8-Dichloro-3,7-dimethyl-octa-2,4,6-trienal (2.74), 4-Bromo-8-chloro-3, 7-di methyl-octa-2, 6-dienal (2 76), 8-Bromo-4-chloro-3, 7-dimethyl-octa-2,4 ,6-trienaI (2.75), 4-Bromo-1,3,6,7-tetrachloro-3 ,7-dimethyl-octa-1,4-diene (2.71), 8-Bromo-1,3,4,7-tetrachloro-3,7-dimethyl-octa-1,5-diene (2.69), 4,6-Dibromo-3,7 -dimethyl-octa-2,7-dienal (2.73). All compounds were screened for antimicrobial activity, brine shrimp lethality and cytotoxicity towards oesophageal cancer cells. Compound 2.68 was toxic to brine shrimp larvae at a concentration of 50 μ/mL. It also showed promising activity towards oesophageal cancer cells with an IC₅₀, of 2 μg/mL.

Prevention of the accumulation of undesirable biological material i.e. biofouling upon a solid surface requires the use of antifouling systems. The solid surface may be a contact lens, an off shore oil rig or a living organism. When chemicals are employed as a mechanism of defense against biofouling, the agents involved are known as antifouling agents. Marine algae must protect themselves from fouling organisms and it is thought that one of the mechanisms used by these organisms is the production of secondary metabolites with an array of biological activities. In vitro studies have shown numerous compounds isolated from marine algae to possess antibacterial, antifungal and antimacrofouling activity. The aim of this study was to evaluate the secondary metabolite extracts of selected Southern African marine macro-algae as a potential source of compounds that inhibit biofilm formation and that could be used as antifouling agents. In this project, marine macro-algae were collected from various sites along the South African coastline. Their extracts were screened for antimicrobial activity against four ubiquitous microorganisms, Staphylococcus aureus, Klebsiella pneumoniae, Mycobacterium aurm and Candida albicans. Results of screening assays guided the fractionation of two Rhodophyta, Plocamium corallorhiza and Laurencia flexuosa. The algae were fractionated using silica gel column chromatography and compounds were isolated by semi-preparative normal phase HPLC. Compound characterization was performed using UV, IR and advanced one- and two-dimensional NMR (¹H, ¹³C NMR, COSY, HSQC, HMBC and NOESY) spectroscopy and mass spectrometry. Ten halogenated monoterpenes including four members of the small class of halogenated monoterpene aldehydes were isolated from extracts of P. corallorhiza. The compounds isolated included the known compounds 3,4,6,7-tetrachloro-3,7-dimethyl-1-octene; 4,6-dibromo-1, 1-dichloro-3,7 -dimethyl-2E,7 octadiene; 4,8-d ibromo-1,1,7 -trichloro-3, 7-dimethyl-2,5Eoctadiene;1 ,4,8-tribromo-3, 7 -dichloro-3,7-dimethyl-1 E,5E-octadiene; 8-bremo-6, 7-dichloro-3,7-dimethyl-octa-2E,4E-dienal; 4-Bromo-8-chloro-3,7-dimethyl-octa-2E,6E-dienal; 4,6- Dibromo-3,7-dimethyl-octa-2E,7-dienal; 2,4-dichloro-1-(2-chlorovinyl)-1-methyl-5-methylidene-cyclohexane and two new metabolites 4,8-chloro-3,7-dimethyl-2Z,4,6Z-octatrien-1-al and Compound 3.47. Methodology was developed for the chemical derivatization and mass spectrometric analysis of the aldehydic compounds, The aldehyde trapping reagent 0-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine hydrochloride was used to derivatize the molecules, stabilizing them and allowing for their complete characterization. From Laurencia flexuosa a new cuparene sesquiterpene 4-bremo-2-(5-hydroxy-1,2,2- trimethylcyclopent-3-enyl)-5-methylphenol was isolated along with two geometric isomers of the vinyl acetylene bromofucin , An halogenated monoterpene 3S*,4R*-1-bromo-3,4,8-trichloro-9-dichloromethyl-1-E,5-E,7-Z-octatriene was also isolated but was suspected to be a contaminant and an investigation into its biological source revealed that it originated from Plocamium suhrii. A third alga, Martensia elegans was extracted based on published reports of antimicrobial compounds in related species. A new a-alkyl malate derivative was isolated and characterized. Selected compounds isolated during the course of the study were employed in preliminary assays that tested their ability to inhibit biofilm formation by Pseudomonas aeruginosa. The halogenated monoterpenes isolated from the Plocamium species were the only active compounds. 3S*,4R*-1-bromo-3,4,S-trichloro-g-dichloromethyl-1-E,5-E,7-octatriene from P. suhrii inhibited biofilm formation through antibacterial activity on planktonic cells but could not prevent biofilm formation when employed as a film on the surface of microtitre plate wells. 1,4,8-tribromo-3,7-dichloro-3,7-dimethyl-1E,5E-octadiene and 4,6-dibromo-1,1-dichloro-3,7-dimethyl-2E,7-octadiene inhibited biofilm formation when applied as a film to the microtitre plate wells but had no significant antibacterial activity. No potential antifouling agents were identified in this project but the antimicrobial activity exhibited by the crude algal extracts was highly encouraging and a number of new research areas have been identified. / KMBT_363 / Adobe Acrobat 9.54 Paper Capture Plug-in

Secondary metabolites from natural sources are fast growing as popular drug leads. The structural novelty and favourable biological activity that these compounds display contribute to their popularity as drugs of the future. Examples of such compounds include the potent anticancer drug paclitaxel isolated from the bark of a yew tree as well as the more commonly known analgesic aspirin which stems from the bark of the willow tree. The biological activities exhibited by these secondary metabolites are vast and range from antimicrobial to anticancer activity to mention but a few. As a result, the isolation of novel compounds from natural sources is on the rise. The South African seaboard is home to a wealth of various marine algal species which produce fascinating secondary metabolites. For example, Portierria hornemanii was shown to produce halomon, a halogenated monoterpene which has displayed promising cytotoxic activity. This study thus focused primarily on pursuing novel compounds from three endemic South African marine algal species which have never been analysed previously from a chemical perspective. These are Plocamium rigidum (Bory de Saint-Vincent), Laurencia natalensis (Kylin) and Delisea flaccida (Suhr) Papenfuss. Four known compounds and one new halogenated monoterpene, (2E,5E,7Z)-8-chloro- 7-(dichloromethyl)-4-hydroxy-3-methylocta-2,5,7-trienal, were isolated from Plocamium rigidum. The breast cancer (MCF-7 cell line) inhibitory activity for these compounds was assessed and it was observed that an increase in the lipophilic nature of the compounds produced more favourable IC50 values. A pre-cursor to bromofucin type compounds, cis-laurencenyne, was isolated from Laurencia natalensis, as well as a new acetoxy chamigrane type compound, 4-bromo- 3,10-dichloro-7-hydroxy-3,7,11,11-tetramethylspiro [6.6] undec-1-yl acetate. Delisea flaccida was seen to contain two known bromofuranone type compounds isolated as an isomeric mixture, 1-[(5Z)-4-bromo-5-(bromomethylidene)-2-oxo-2,5- dihydrofuran-3-yl] butyl acetate and 1-[(5E)-4-bromo-5-(bromomethylidene)-2- oxo-2,5-dihydrofuran-3-yl]butyl acetate. These compounds are famous for their ability to inhibit bacterial biofilm production and they have been isolated before from an Australian Delisea spp / Adobe Acrobat 9.53 Paper Capture Plug-in