Studies in Marine Natural Products : Onchidoris bilamellata, Nanaimoal and Capnellene

The following thesis is divided into three chapters. The first describes the isolation and identification of a sphingolipid L from the methanolic skin extract of the British Columbia nudibranch Onchidoris bilamellata. The long chain base has been identified as (E)-l,3-dihydroxy-2-amino-16-methyl-4-octadecene (1̲3) and the fatty acid moiety as palmitic acid (1̲2). This ceramide possesses antibacterial activity towards the microorganisms Bacillus subtilis and Staphylococcus aureus. [formula omitted] The second chapter presents our attempt to synthesize a hypothetical structure of nanaimoal (2̲4), a marine sesquiterpenoid, isolated from the British Columbia nudibranch Acanthadoris nanaimoensis. The proposed route envisaged photolysis of a 2,2,10,10-tetrasubstituted cyclodecanone. This work lend to a study of the solution photochemistry of 2,2,10-trimethyl-cyclodecanone (3̲5). [formula omitted] The last chapter outlines studies directed toward the synthesis of capnellene (5̲1), a tricyclopentanoid obtained by Djerassi and co-workers from the soft coral Capnel laimbricata. Starting with an improved synthesis of the known ketone Δ¹̕̕̕⁵-bicyclo[3.3.0] octen-2-one (1̲3̲8), an acylation method was developed for the preparation of Δ¹̕⁵-3-carboethoxybicyclo-[3.3.0] octen-2-one (1̲6̲0). The procedure is of general utility and has been extended to related ketones. This unsaturated keto-ester underwent a Michael addition with methyl vinyl ketone to give 1̲7̲8. Selective Knoevenagal condensation of the methyl ketone produced Δ¹̕⁵-3-carboethoxy-3-(dimethyl-3-methyl-3-butenyl-4,4-dicarboxylate)-bicyclo[3.3.0] octen-2-one (179). Reduction of the unsaturated ketone and dehydration of the allylic alcohol produced a 1,1,3,4-tetrasubstituted cyclopentadiene derivative 1̲8̲1. Intramolecular Diels-Alder cyclization of the latter afforded the tetracyclic tricarboxylic ester 1̲8̲2. Selective hydrolysis of the malonic estertricarboxylic ester 1̲8̲2 followed by decarboxylation led to 1̲8̲3, a key intermediate for the ultimate construction of the capnellane ring system. [formula omitted]Science, Faculty ofChemistry, Department ofGraduat

Terpenoids from two British Columbia nudibranchs

The two British Columbia nudibranchs Cadiina luteomarginata and Acanthadoris nanaimoensis have sweet fragrances. The possible importance of odours in the interactions of marine organisms initially aroused our chemical curiosity. The results of our research concerning the structural investigation of seven terpenoids obtained from the organic extracts of these two opisthobranch molluscs is presented in this thesis. Five of the molecules isolated from luteomarginata have been identified as furodysin (53), furodysinin (54), microcionin-2 (55), albicanyl acetate (51) and albicanol (52). A sixth molecule, luteone (57) which gives the sweet fragrance to C. luteomarginata has been partially characterized . A crystalline derivative of this methyl ketone has been submitted for X-ray diffraction analysis. The sweet fragrance of A. nanaimoensis has been related to the presence of a sesquiterpenoid (existing as two constitutional isomers, in a 5:1 ratio). Three hypothetical structures based on spectral analysis, chemical reactions and biosynthetic reasoning are proposed. The biological origin of the seven terpenoids has also been investigated and is discussed in this thesis.Science, Faculty ofChemistry, Department ofGraduat

Analysis of pyrene metabolites in marine snails by liquid chromatography using \ufb02uorescence and mass spectrometry detection

As part of a study of the metabolismof aromatic compounds in marine gastropods, a sensitive and selective method was developed to detect, identify and quantify pyrene (PY) and four of its metabolites in tissues: 1-hydroxypyrene (PYOH), pyrene sulfate (PYOS), pyrene glucuronide (PYOG) and pyrenediol disulfate (PYDS). Liquid chromatography (LC) with \ufb02uorescence detection was \ufb01rst used to detect the PY derivatives in the visceral mass of whelks exposed to PYOH. The identi\ufb01cation of metabolites was accomplished through a combination of retention time and spectral matching with standards, enzymatic hydrolysis, solid phase extraction and LC coupled with electrospray ionization mass spectrometry. In addition to four known PY derivatives, two novel metabolites were identi\ufb01ed as pyrenediol glucuronide sulfate and a second isomer of PYDS. The methanol extraction of metabolites from tissue gave excellent mean recoveries, ranging from 67 to 97%, for the available standards PY, PYOH, PYOS and PYOG spiked in both the muscle and visceral mass of Buccinum spp. The mean recoveries of a surrogate standard, 2-hydroxy\ufb02uorene, spiked in all tissue samples were 100% and 95% for visceral and muscle tissue samples, respectively. The method limits of detection for these compounds were all below 0.2 ng/g of wet tissue, low enough to detect metabolites in reference animals. Results from the application of this method to the quantitative analysis of biotransformation products in the visceral mass of the whelk Neptunia lyrata exposed to PYOH contaminated food are also presented. This method will be useful to apply to the analysis of PY metabolites in soft tissues of other animals.Peer reviewed: YesNRC publication: Ye

Bioaccumulation and biotransformation of pyrene and 1-hydroxypyrene by the marine whelk Buccinum undatum

The fates of a phenolic contaminant and its hydrocarbon precursor have rarely been compared, especially in an invertebrate species. Two groups of Buccinum undatum were exposed to equimolar amounts of pyrene and 1-hydroxypyrene over 15 d through their diets. Tissue extracts from the muscle and visceral mass were analyzed by liquid chromatography with fluorescence and mass spectrometry detection. Nine biotransformation products were detected in animals from both exposures. These included 1-hydroxypyrene, pyrene-1-sulfate, pyrene-1-glucuronide, pyrene glucose sulfate, two isomers each of pyrenediol sulfate and pyrenediol disulfate, and one isomer of pyrenediol glucuronide sulfate. These compounds represent a more complex metabolic pathway for pyrene than is typically reported. Diconjugated metabolites were as important in animals exposed to pyrene as in those exposed to 1-hydroxypyrene. Biotransformation products represented >90% of the material detected in the animals and highlight the importance of analyzing metabolites when assessing exposure. A mean of only 2 to 3%of the body burden was present in muscle compared with the visceral mass of both groups. The analytical methods were sufficiently sensitive to detect biotransformation products both in laboratory control whelks and in those sampled offshore. The tissue distribution of [ 14C]pyrene was also studied by autoradiography. Radioactivity was present primarily in the digestive and excretory system of the whelks and not in the gonads or muscle tissue.Peer reviewed: YesNRC publication: Ye

Up-regulation of hepatic ABCC2, ABCG2, CYP1A1 and GST in multixenobiotic-resistant killifish (Fundulus heteroclitus) from the Sydney Tar Ponds, Nova Scotia, Canada

Cellular defence against accumulation of toxic xenobiotics includes metabolism by phase I and II enzymes and export of toxicants and their metabolites via ATP-binding cassette (ABC) transporters. Liver gene expression of representatives of these three protein groups was examined in a population of multixenobiotic-resistant killi\ufb01sh (Fundulus heteroclitus) from the Sydney Tar Ponds, Nova Scotia, Canada. The Tar Ponds are heavily polluted with polycyclic aromatic hydrocarbons, polychlorinated biphenyls and heavy metals. The relationship among ABC transporters ABCB1, ABCB11, ABCC2, ABCG2, phase I enzyme cytochrome P4501A1 (CYP1A1) and phase II enzyme glutathione-S-transferase (GST-mu) was investigated by quantifying hepatic transcript abundance. In Tar Pond killi\ufb01sh, hepatic mRNA expression levels of ABCC2, ABCG2, CYP1A1 and GST-mu were elevated compared to reference sites, suggesting that hydrophobic contaminants undergo phase I and II metabolism and are then excreted into the bile of these \ufb01sh. Hepatic ABCB1 and ABCB11 mRNA were not up-regulated in Tar Pond \ufb01sh compared to two reference sites, indicating that these two proteins are not involved in conferring multixenobiotic resistance to Tar Pond killi\ufb01sh. The results suggest instead that liver up-regulation of phase I and II enzymes and complementary ABC transporters ABCC2 and ABCG2 may confer contaminant resistance to Tar Pond \ufb01sh.Peer reviewed: YesNRC publication: Ye