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Biologically Active Secondary Metabolites from Freshwater and Terrestrial Cyanobacteria

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Title: Biologically Active Secondary Metabolites from Freshwater and Terrestrial Cyanobacteria
Author(s): Kang, Hahk-Soo
Advisor(s): Orjala, Jimmy
Contributor(s): Mankin, Alexander; Murphy, Brian; Wardrop, Duncan; Swanson, Steven
Department / Program: Medicinal Chemistry and Pharmacognosy
Graduate Major: Pharmacognosy
Degree Granting Institution: University of Illinois at Chicago
Degree: PhD, Doctor of Philosophy
Genre: Doctoral
Subject(s): Cyanobacteria Secondary metabolites Anticancer activity Structure determination Lipopeptides Cyclophanes PK-NRP hybrid Depsipeptides
Abstract: Cyanobacteria (blue-green algae) are a prolific source of biologically active secondary metabolites. Although many studies have been done on cyanobacterial secondary metabolites, these studies have been mainly focused on cyanobacterial blooms from both freshwater and marine environments. Non-bloom forming cyanobacteria that appear only as small colonies in the environments still remain under-explored. In this study, we employed culture-dependent approach to explore chemistries of non-bloom forming cyanobacteria that we isolated from field-collected samples (UIC) and also acquired from three commercial culture collections (UTEX, SAG and CCALA). An extract library was prepared and screened for antiproliferative activity against cancer cells and brine shrimp toxicity. Active extracts were subjected to fractionation followed by dereplication using LC-MS and 1H NMR in combination with HPLC-based activity profiling that associated LC chromatogram with activity. This was used to identify active peaks at fraction level. Based on the results of dereplication as well as HPLC-based activity profiling, the strains Anabaena minutissima (UTEX 1613), Cf. Anabaena sp. (UIC 10035), Nostoc sp. (UIC 10062) and Oscillatoria sancta (SAG 74.79) were selected for further chemical investigation. Twelve cyclic lipodecapeptides, named minutissamides A-D and homesteadamides A-H, were isolated from the cultured cyanobacteria Anabaena minutissima (UTEX 1613) and Cf. Anabaena sp. (UIC 10035), respectively. The planar structures were determined using various spectroscopic techniques including HRESIMS, and 1D and 2D NMR experiments. The amino acid sequences determined from 2D NMR analysis was further confirmed by analyzing fragmentation pattern acquired from the tandem MS/MS spectra. The absolute configurations of the α-amino acid residues were assigned using Marfey’s method after acid hydrolysis. The absolute configuration of a β-amino acid residue was assigned by a combination of the advanced Marfey’s method, J-based configurational analysis and ROE spectroscopic analysis. The structures of minutissamides A-D and homesteamides A-H were characterized by the presence of three non-standard α-amino acid residues (α,β-dehydro-α-aminobutyric acids, O-methylated Thr and one N-methylated Asn) and one β-amino acid residue (2-hydroxy-3-amino-4-methyl dodecanoic acid or 2-hydroxy-3-amino-4-methyl hexadecanoic acid). Minutissamides A-D and homesteadamides A-H exhibited antiproliferative activity against cancer cells with low micromolar IC50 values ranging between 1 and 10 μM. Two cyclophanes, named merocyclophanes A and B, were isolated from the cell extract of a cultured terrestrial Nostoc sp. (UIC 10062), obtained from a sample collected at Grand Mere State Park in Michigan. Their structures were determined by various spectroscopic techniques including HRESIMS, and 1D and 2D NMR analyses. The stereoconfiguration was assigned on the basis of X-ray crystallographic and CD analyses. The structures of merocyclophanes A and B established a hitherto unknown [7.7]paracyclophane skeleton in nature, as characterized by α-branched methyls at C-1/14. Merocyclophanes A and B displayed antiproliferative activity against the HT-29 human colon cancer cell line with IC50 values of 3.3 and 1.7 μM, respectively. A 14-membered polyketide-nonribosomal peptide (PK-NRP) hybrid macrolide, named sanctolide A, was isolated from the cultured cyanobacterium Oscillatoria sancta (SAG 74.79). The planar structure was determined by various spectroscopic techniques including HRESIMS, and 1D and 2D NMR analyses. The relative configuration was assigned by J-based configurational analysis in combination with NOE correlations. The absolute configuration was determined by Mosher ester and enantioselective HPLC analyses. The structure of sanctolide A featured a rare N-methyl enamide and a 2-hydroxyisovaleric acid, which were incorporated to form a 14-membered macrolide ring structure, comprising a new type of cyanobacterial macrolides derived from a PKS-NRPS hybrid biosynthetic pathway. Chemical investigation of the four prioritized strains by our dereplication resulted in the isolation of total fifteen new compounds that fell into the three distinct structural groups, cyclic lipodecapeptides (the minutissamides and homesteadamides), [7.7]paracyclophanes (the merocyclophanes) and 14-membered PK-NRP hybrid macrolide (sanctolide A). Except for the homesteadamides, all of the compounds isolated in this study showed quite distant structural relationship to previously isolated compounds belonging to the same structural families. These examples clearly demonstrated that non-bloom forming cyanobacteria are a prolific source of biologically active secondary metabolites and yet remain relatively under-explored. Therefore, using culture-dependent approach to study these non-bloom forming cyanobacteria, which are widespread in the environments, will continue to provide new biologically active natural products. During a collection trip to Highland Lake District of northern Wisconsin in 2010, the occurrence of cyanobacterial bloom was observed in North Nokomis Lake. Microscopic analysis of the bloom sample indicated that the bloom sample contained one major true-branching cyanobacterium belonging to the order Stigonematales. Detailed morphological and 16S rRNA gene sequence analyses of the bloom sample suggested this bloom-forming true branching cyanobacterium to be Stigonema sp. Due to the rarity of reports of this genus, chemical investigation was conducted with the bloom sample and resulted in the isolation of a new Ahp-containing depsipeptide, named stigonemapeptin. The planar structure was determined by 1D and 2D NMR experiments as well as HRESIMS analysis. The absolute configurations of the amino acids were determined using the advanced Marfey’s method after acid hydrolysis. Stigonemapeptin, characterized by the presence of the Ahp residue, also contained the modified amino acids Abu (2-amino-2-butenoic acid) and N-formylated Pro. Stigonemapeptin exhibited in vitro elastase and chymotrypsin inhibitory activity with IC50 values of 0.26 and 2.93 µM, respectively.
Issue Date: 2012-12-13
Genre: thesis
Rights Information: Copyright 2012 Hahk-Soo Kang
Date Available in INDIGO: 2012-12-13
Date Deposited: 2012-08

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