Pritesh Prasad

Cytotoxic and antibacterial substances against multi-drug resistant pathogens from marine sponge symbiont: Citrinin, a secondary metabolite of Penicillium sp.

OBJECTIVE To Isolate, purify, characterize, and evaluate the bioactive compounds from the sponge-derived fungus Penicillium sp. FF001 and to elucidate its structure. METHODS The fungal strain FF001 with an interesting bioactivity profile was isolated from a marine Fijian sponge Melophlus sp. Based on conidiophores aggregation, conidia development and mycelia morphological characteristics, the isolate FF001 was classically identified as a Penicillium sp. The bioactive compound was identified using various spectral analysis of UV, high resolution electrospray ionization mass spectra, 1H and 13C NMR spectral data. Further minimum inhibitory concentrations (MICs) assay and brine shrimp cytotoxicity assay were also carried out to evaluate the biological properties of the purified compound. RESULTS Bioassay guided fractionation of the EtOAc extract of a static culture of this Penicillium sp. by different chromatographic methods led the isolation of an antibacterial, anticryptococcal and cytotoxic active compound, which was identified as citrinin (1). Further, citrinin (1) is reported for its potent antibacterial activity against methicillin-resistant Staphylococcus aureus (S. aureus), rifampicin-resistant S. aureus, wild type S. aureus and vancomycin-resistant Enterococcus faecium showed MICs of 3.90, 0.97, 1.95 and 7.81 µg/mL, respectively. Further citrinin (1) displayed significant activity against the pathogenic yeast Cryptococcus neoformans (MIC 3.90 µg/mL), and exhibited cytotoxicity against brine shrimp larvae LD50 of 96 µg/mL. CONCLUSIONS Citrinin (1) is reported from sponge associated Penicillium sp. from this study and for its strong antibacterial activity against multi-drug resistant human pathogens including cytotoxicity against brine shrimp larvae, which indicated that sponge associated Penicillium spp. are promising sources of natural bioactive metabolites.

C3 and 2D C3 Marfey’s methods for amino acid analysis in natural products

We validate the improved resolution and sensitivity of the C3 Marfeys method, including an ability to resolve all Ile isomers, against an array of amino acids commonly encountered in natural products and by comparison to an existing Marfeys method. We also describe an innovative 2D C3 Marfeys method as an analytical approach for determining the regiochemistry of enantiomeric amino acid residues in natural products. The C3 and 2D C3 Marfeys methods represent valuable tools for probing and defining the stereocomplexity of hydrolytically accessible amino acid residues in natural products.

Callyspongisines A–D: bromopyrrole alkaloids from an Australian marine sponge, Callyspongia sp.

An extract of the Great Australian Bight marine sponge Callyspongia sp. (CMB-01152) displayed inhibitory activity against the neurodegenerative disease kinase targets casein kinase 1 (CK1), cyclin-dependent kinase 5 (CDK5) and glycogen synthase kinase 3 (GSK3β). Chemical investigation, employing HPLC-DAD-MS single ion extraction protocols, facilitated identification of the new bromopyrrole alkaloids, callyspongisines A-D (1-4), and two known co-metabolites, hymenialdisine (5) and 2-bromoaldisine (6). Structure elucidation of 1-6 was supported by detailed spectroscopic analysis and chemical interconversion, as well as biosynthetic and synthetic considerations. Callyspongisine A (1) is only the second reported example of a natural imino-oxazoline, and the first to feature a spiro heterocyclic framework, while callyspongisines B-D (2-4) were speculated to be storage and handling artefacts of 1. The kinase inhibitory activity detected in Callyspongia sp. (CMB-01152) was attributed to 5.

Talarolide A, a Cyclic Heptapeptide Hydroxamate from an Australian Marine Tunicate-Associated Fungus, Talaromyces sp. (CMB-TU011)

A miniaturized 24-well plate microbioreactor approach was used to explore secondary metabolite media dependence in an Australian marine tunicate-associated fungus, Talaromyces sp. (CMB TU011). Detailed chemical investigations of an antifungal M1-saline cultivation yielded talarolide A (1), only the second reported natural cyclic peptide hydroxamate, and the first from a fungus. The antifungal properties of the M1-saline extract were attributed to the known diterpene glycoside sordarin (2). Structure elucidation of 1 and 2 was achieved by detailed spectroscopic analysis, with amino acid configurations in 1 assigned by the C3 and C18 Marfeys methods, and l-Ala and d-Ala regiochemistry by the recently reported 2D C3 Marfeys method.

Asymmetric Total Synthesis of (+)-Dragmacidin D Reveals Unexpected Stereocomplexity

The first asymmetric total synthesis of the bis-indole marine alkaloid (+)-dragmacidin D (1) has been achieved. This synthesis revises an earlier configurational assignment based on biosynthetic considerations and assigns an R absolute configuration to (+)-1. The current studies reveal that natural dragmacidin D is isolated as either a racemate or a scalemic mixture (39% ee).

Production and purification of a bioactive substance against multi-drug resistant human pathogens from the marine-sponge-derived Salinispora sp.

ABSTRACT Objective To isolate, purify, characterize, elucidate structure and evaluate bioactive compounds from the sponge-derived Salinispora sp. FS-0034. Methods The symbiotic actinomycete strain FS-0034 with an interesting bioactivity profile was isolated from the Fijian marine sponge Theonella sp. Based on colony morphology and obligatory requirement of seawater for growth, and mycelia morphological characteristics the isolate FS-0034 was identified as a Salinispora sp. The bioactive compound was identified by using various spectral analysis of ultraviolet, high resolution electrospray ionization mass spectroscopy, 1 H nuclear magnetic resonance, correlated spectroscopy and heteronuclear multiple bond coherence spectral data. A minimum inhibitory concentration assay were performed to evaluate the biological properties of the pure compound against multi-drug resistant pathogens. Results Bioassay guided fractionation of the ethyl acetate extract of the culture of Salinispora sp. FS-0034 by different chromatographic methods yielded the isolation of an antibacterial compound, which was identified as rifamycin W (compound 1). Rifamycin W was reported for its potent antibacterial activity against methicillin-resistant Staphylococcus aureus , wild type Staphylococcus aureus and vancomycin-resistant Enterococcus faecium and displayed minimum inhibitory concentrations of 15.62, 7.80 and 250.00 μg/mL, respectively. Conclusions The present study reported the rifamycin W from sponge-associated Salinispora sp. and it exhibited appreciable antibacterial activity against multi-drug resistant human pathogens which indicated that sponge-associated Actinobacteria are significant sources of bioactive metabolites.

Talaropeptides A-D: Structure and Biosynthesis of Extensively N-methylated Linear Peptides From an Australian Marine Tunicate-Derived Talaromyces sp.

An Australian marine tunicate-derived fungus, Talaromyces sp. CMB-TU011 was subjected to a program of analytical microbioreactor (MATRIX) cultivations, supported by UHPLC-QTOF profiling, to reveal conditions for producing a new class of extensively N-methylated 11-12 residue linear peptides, talaropeptides A-D (2-5). The structures for 2-5, inclusive of absolute configurations, were determined by a combination of detailed spectroscopic and chemical (e.g., C3 and C18 Marfeys) analyses. We report on the biological properties of 2-5, including plasma stability, as well as antibacterial, antifungal and cell cytotoxicity. The talaropeptide mega non-ribosomal peptide synthetase (NRPS) is described, as second only in size to that for the fungus-derived immunosuppressant cyclosporine (an 11-residue extensively N-methylated cyclic peptide).

Marine biodiversity: exploring bioactive chemical space

............................................................................................................................................... ii Declaration by author ......................................................................................................................... iv Publications during candidature .......................................................................................................... v Publications included in this thesis ...................................................................................................... v Contributions by others to the thesis .................................................................................................. vi Statement of parts of the thesis submitted to qualify for the award of another degree ...................... ix Acknowledgements ............................................................................................................................. x Keywords ........................................................................................................................................... xii Australian and New Zealand Standard Research Classifications (ANZSRC) ................................... xii Fields of Research (FoR) Classification ............................................................................................ xii Table of Contents .............................................................................................................................. xiii List of Figures ................................................................................................................................... xxi List of Tables .................................................................................................................................. xxix List of non standard abbreviations used in the thesis ..................................................................... xxxi 1 Chapter 1. Literature Review ..................................................................................................... 1 1.1 Marine Natural Products ......................................................................................................... 1 1.2 Trends in Drug Discovery ...................................................................................................... 1 1.3 Natural Products from Marine Sponges ................................................................................. 2 1.4 Australian Marine Sponges – A Treasury of Natural Products .............................................. 2 1.4.1 Terpenes ........................................................................................................................... 5 1.4.1.1 Sesquiterpenes .......................................................................................................... 5 1.4.1.2 Diterpenes ................................................................................................................. 6 1.4.1.3 Sesterterpenes ........................................................................................................... 8 1.4.1.4 Mixed Terpenes/ Sterols ......................................................................................... 10 1.4.2 Lipids ............................................................................................................................. 11 1.4.3 Peptides .......................................................................................................................... 13 1.4.4 Macrolides ..................................................................................................................... 16 xiv 1.4.5 Alkaloids ........................................................................................................................ 17 1.4.6 Miscellaneous ................................................................................................................ 24 1.5 Summary ............................................................................................................................... 25 1.6 Aims and Objectives ............................................................................................................. 25 2 Chapter 2. Chemical and Biological Profiling of Marine Extracts ....................................... 27 2.1 Overview .............................................................................................................................. 27 2.2 Background ........................................................................................................................... 28 2.2.1 Marine Extract Libraries ................................................................................................ 28 2.2.2 Bioassays ....................................................................................................................... 29 2.2.2.1 Bacillus Calmette-Guerin (BCG): Tuberculosis Surrogate .................................... 30 2.2.2.2 Screening for GABAA alpha 5 Modulator .............................................................. 31 2.2.2.3 CspB assay .............................................................................................................. 32 2.3 Results and Discussion ......................................................................................................... 32 2.3.1 Chemical Profiling and UHPLC-DAD Database .......................................................... 32 2.3.2 BCG Screening of Marine BuOH extract Library ......................................................... 32 2.3.2.1 Preliminary BCG screening .................................................................................... 32 2.3.2.2 Secondary Screening and Prioritisation .................................................................. 33 2.3.3 Chemical Investigation of Prioritised BCG Active Extracts ......................................... 35 2.3.3.1 Marine Sponge Acanthella sp. (CMB-01918) ........................................................ 35 2.3.3.1.1 Extraction and Isolation ................................................................................................. 35 2.3.3.1.2 Characterisation of Carduusyne E (2.01) ....................................................................... 36 2.3.3.1.3 Carduusyne E Ethyl Esters (2.02) .................................................................................. 38 2.3.3.1.4 Carduusyne F Ethyl Esters (2.03) .................................................................................. 39 2.3.3.1.5 Biological Evaluation of Carduusynes ........................................................................... 40 2.3.3.2 Chemical investigation of Marine Sponge Axinella sp. (CMB-01945) .................. 41 2.3.3.2.1 Extraction and Isolation ................................................................................................. 41 2.3.3.2.2 Characterisation of 10E-hymenialdisine (2.05) ............................................................. 42 2.3.3.2.3 10Z-hymenialdisine (2.06) ............................................................................................. 43 2.3.3.2.4 2-bromoaldisine (2.07) ................................................................................................... 44 2.3.3.2.5 Biological Evaluation ..................................................................................................... 46 2.3.3.2.6 Tracking BCG Active Metabolites ................................................................................ 46 xv 2.3.3.3 Metabolites from Marine Sponge Homaxinella sp. (CMB-01982) ........................ 47 2.3.3.3.1 Extraction and Isolation ................................................................................................. 47 2.3.3.3.2 5-Bromotubercidin (2.08) .............................................................................................. 49 2.3.3.3.3 5-Bromopyrrole[2,3,d]pyrimidine (2.09) ....................................................................... 50 2.3.3.3.4 Biological Evaluations ................................................................................................... 51 2.3.4 Using Bioassay Guided Fractionation for Prioritisation ................................................ 53 2.3.4.1 Prioritisation of Sub-fractions at Replicating Conditions ....................................... 53 2.3.4.2 Prioritisation of Sub-fractions at Non-replicating Conditions ................................ 54 2.3.4.3 Bioassay-guided Fractionation and Dereplication of Marine Sponge (CMB-01870) Metabolites ............................................................................................................................ 55 2.3.4.3.1 Preliminary Fractionation .............................................................................................. 55 2.3.4.3.2 Chemical Profiling ......................................................................................................... 55 2.3.5 α5β3γ2 GABAA Rs Modulators ...................................................................................... 57 2.3.5.1 Prioritisation of Sub-fractions as GABAA Modulators .......................................... 57 2.3.5.2 Phenethylamine, a GABAA Modulator from Marine Sponge Spongia sp. (CMB-02732) ........................................................................................................................ 58 2.3.5.2.1 Preliminary Fractionation and Biological Profiling ....................................................... 58 2.3.5.2.2 Extraction and Isolation ................................................................................................. 59 2.3.5.2.3 Characterisation of Phenethylamine (2.14) .................................................................... 60 2.3.5.2.4 Biological Evaluations of Phenethylamine and Analogues ........................................... 61 2.3.5.3 Sargassum

Pursuing sesterterpene lactams in Australian Irciniidae sponges

Chemical investigation of two Irciniidae sponges collected by hand (SCUBA) from Australian near shore waters, afforded six new examples of a rare class of sesterterpene lactam; ircinialactams B (1), G (2), H (5), and I (6), and 8-hydroxyircinialactams C (3) and G (4); together with the new biosynthetically related lactone, ircinialactone A (7). Also isolated were seven biosynthetically related known Irciniidae metabolites; ircinialactams A (8) and C (9), (7E,12E,20Z,18S)-variabilin (10), (7Z,12Z,20Z,18S)-variabilin (11), (7E,12Z,20Z,18S)-variabilin (12), (7Z,12E,20Z,18S)-variabilin (13) and irciniafuran A (14). The structure elucidation of 1-14 was achieved by detailed spectroscopic analysis, and consideration of a plausible biosynthetic relationship linking Irciniidae sesterterpene β-furans, lactams and lactones.